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THE EFFECT OF WHEY PROTEIN ON SHORT-TERM
FOOD INTAKE AND POST-MEAL GLYCEMIC
REGULATION IN YOUNG ADULTS
by
Tina Akhavan
A thesis submitted in conformity with the requirements
For the degree of Doctor of Philosophy
Graduate Department of Nutritional Sciences
University of Toronto
© Copyright by Tina Akhavan 2012
ii
THE EFFECT OF WHEY PROTEIN ON SHORT-TERM FOOD
INTAKE AND POST-MEAL GLYCEMIC REGULATION IN
YOUNG ADULTS
Doctor of Philosophy Tina Akhavan
Graduate Department of Nutritional Sciences University of Toronto
2012
ABSTRACT
The hypothesis that consumption of whey protein (WP) prior to a meal suppresses short-
term food intake and reduces post-meal glycemia by insulin-dependent and -independent
mechanisms in healthy young adults was explored in three studies. Study one investigated the
effect of solid vs. liquid forms of WP (50 g) and sucrose (75 g) on food intake at 1 h. Whey
protein, whether in solid or liquid form, suppressed food intake more than sucrose. Study two
examined the effect of WP (10-40 g) consumed 30 min prior to a meal on food intake, and pre-
and post-meal blood concentrations of glucose and insulin. Whey protein reduced food intake
and post-meal glycemia in a dose-dependent manner without increased blood insulin
concentrations. In the third study, glycemic control after WP was compared with glucose, at
doses of 10 and 20 g. Both pre-meal WP and glucose consumption reduced post-meal glycemia
similarly. However, WP resulted in lower pre-meal blood glucose and delayed gastric emptying,
lower pre-and post-meal and overall insulin secretion and concentrations and higher GLP-1 and
PYY concentrations compared with glucose. Thus, the results of this research support the
hypothesis that consumption of WP prior to a meal suppresses short-term food intake and
reduces post-meal glycemia by insulin-dependent and -independent mechanisms in healthy
young adults.
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to express my genuine gratitude to my graduate supervisor Dr.
Harvey Anderson for his guidance and support throughout my doctoral program. Thank you Dr.
Anderson for your constant training that allowed me to be an independent and analytical thinker
scientist. I will always remain grateful to you.
My utmost thanks go out to Dr. Peter Brown, Kraft Inc for his compassion and
continuous support. I sincerely appreciate the insight you provided me through my research.
I would like to extend my gratitude to the members of my advisory committee, Drs. Paul
Pencharz, Thomas Wolever, and Ravi Retnakaran for their intellectual contribution and
constructive guidance during my research. I am honored to have such knowledgeable,
experienced mentors during my PhD program. I would like to thank Drs. Vladimir Vuksan and
Angelo Tremblay for serving as my examiners and for providing insightful and positive
appraisals of my thesis. I would also like to thank Drs. Ahmed El- Sohemy and David Irwin for
chairing my thesis defense.
My sincere thank to Dr. Bohdan Luhovyy for his unrestrained assistance through my
research. Bohdan, you have thought me not only to be a better scientist, but also to be a better
person.
My special thanks to all of my friends and colleagues at the Department of Nutritional
Sciences for their delightful friendship and support: Dr. Sandra Reza Lopez, I will always
cherish the memories of our joyful conversations over coffee breaks and your true friendship.
Shirin Panahi, I will never forget all of your efforts in helping and encouraging me to achieve my
goal. I also appreciate Pedro Huot and Chris Smith for their assistance with my computer; Clara
Cho, Sophie Floret and Tanya Mozek for helping me to run the studies; Mariafernanda Nunez
and Ati Hamedani for your moral support. Special thanks also go to my research assistants: Dr.
Ruslan Kubant, Chi Lan Tran Nghiem, Dalena Dang, Armita Dehmoobad, Isabella Branimirova
and
I would like to dedicate this thesis to my husband, parents and sister. It would not have
been possible to complete this thesis without the devoted support of my beloved husband, Dr.
Svitlana Yurchenko.
iv
Kayvan Abbasi, and unconditional love of my parents and sister, who although live across the
ocean and are far away from me, but are always so proud of me. I would not have been able to
accomplish my dream if it was not for your continuing love and strength.
v
TABLE OF CONTENTS
Contents
LIST OF TABLES ......................................................................................................................... xi
LIST OF FIGUURES ................................................................................................................... xii
LIST OF ABBREVIATIONS ...................................................................................................... xiv
LIST OF PUBLICATIONS ARISING FROM THESIS ............................................................. xvi
CHAPTER 1 ................................................................................................................................... 1
CHAPTER 1. INTRODUCTION .................................................................................................. 2
CHAPTER 2. LITERATURE REVIEW ....................................................................................... 5
2.1. Introduction ......................................................................................................................... 5
2.2. Dairy Products, Proteins, Obesity, Metabolic Syndrome and T2D .................................... 5
2.2.1. Milk Proteins ............................................................................................................. 7
2.2.1.1. Physical Properties ..................................................................................... 7
2.2.1.2. Physiological Properties ............................................................................. 8
2.3. Proteins, Satiety and Food Intake ...................................................................................... 10
2.3.1. Solid vs. Liquid Forms of Proteins ......................................................................... 13
2.4. Proteins and Blood Glucose Control ................................................................................. 14
2.4.1. Dairy Products and Proteins and Glycemia ............................................................ 15
2.5. Physiological Control of Satiety, Food Intake and Glycemia ........................................... 16
2.5.1. Regulation of Food Intake ...................................................................................... 17
2.5.1.1. Hormonal Regulation of Satiety and Food Intake ................................... 17
i. Pancreatic Peptides ............................................................................... 17
iii. Gastointestinal Peptides ..................................................................... 18
2.5.1.2. Gastric Distension and Emptying Rate .................................................... 18
2.5.1.3. Post-absorptive Satiety Signals ................................................................ 19
vi
i. Amino Acid and Brain Neurotransmitter ............................................. 20
ii. Amino Acids and Thermogenosis ....................................................... 20
2.5.2. Regulation of Glycemia .......................................................................................... 21
2.5.2.1. Hormonal Regulation of Glycemia .......................................................... 21
i. Pancreatic Peptides ............................................................................... 21
ii. Gastointestinal Peptides ...................................................................... 22
2.5.2.2. Gastric Emptying ..................................................................................... 23
2.6. Whey Protein and the Regulation of Satiety, Food Intake and Glycemia ........................ 23
2.6.1. Satiety and Food Intake .......................................................................................... 23
2.7. Summary and Research Rationale .................................................................................... 26
CHAPTER 3. HYPOTHESES AND OBJECTIVES .................................................................. 29
3.1. General Hypothesis and Objective .................................................................................... 29
Hypothesis ......................................................................................................................... 29
Objectives ......................................................................................................................... 29
3.2. Specific Hypotheses and Objectives ................................................................................. 29
CHAPTER 4. EFFECT OF DRINKING COMPARED TO EATING SUGARS OR WHEY PROTEIN ON SHORT-TERM APPETITE AND FOOD INTAKE ....................................... 32
4.1. Abstract ............................................................................................................................. 33
4.2. Introduction ....................................................................................................................... 34
4.3. Subjects and Methods ....................................................................................................... 35
4.3.1. Subjects ................................................................................................................... 35
4.3.2. Treatments ............................................................................................................... 35
4.3.3. Protocol ................................................................................................................... 37
4.3.4. Statistical Analysis .................................................................................................. 39
4.4. Results ............................................................................................................................... 39
4.4.1. Subjects ................................................................................................................... 39
vii
4.4.2. Food Intake ............................................................................................................. 39
4.4.3. Average Appetite Score .......................................................................................... 40
4.4.4. Average appetite AUC ............................................................................................ 41
4.4.5. Blood Glucose Concentration ................................................................................. 41
4.4.6. Blood Glucose AUC ............................................................................................... 42
4.5. Discussion ......................................................................................................................... 42
4.6. Conclusion ......................................................................................................................... 44
CHATER 5. EFFECT OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN AND ITS HYDROLYSATE ON FOOD INTAKE AND POST-MEAL GLYCEMIA AND INSULIN RESPONSES IN YOUNGE ADULTS ................................................................... 53
5.1. Abstract ............................................................................................................................. 54
5.2. Introduction ....................................................................................................................... 55
5.3. Research Methods and Procedures .................................................................................... 56
5.3.1. Participants .............................................................................................................. 56
5.3.2. Preloads ................................................................................................................... 56
5.3.3. Protocol ................................................................................................................... 57
5.3.4. Statistical Analysis .................................................................................................. 59
5.4. Results ............................................................................................................................... 60
5.4. 1. Participant Characteristics ..................................................................................... 60
5.4. 2. Food and Water Intake ........................................................................................... 60
5.4. 3. Subjective Average Appetite Score ....................................................................... 60
5.4. 4. Subjective Average Appetite AUC ........................................................................ 61
5.4. 5. Blood Glucose Concentration ................................................................................ 61
5.4. 6. Blood Glucose AUC .............................................................................................. 62
5.4. 7. Insulin .................................................................................................................... 63
5.4. 8. Insulin AUC ........................................................................................................... 63
5.4. 9. Relations among Dependent Measures .................................................................. 64
viii
5.5. Discussion ......................................................................................................................... 65
5.6. Conclusion ......................................................................................................................... 67
CHAPTER 6. MECHANISM OF ACTION OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN ON GLYCEMIC CONTROL IN YOUNG ADULTS ........................................... 81
6.1. Abstract ............................................................................................................................. 82
6.2. Introduction ....................................................................................................................... 83
6.3. Participants and Research Methods .................................................................................. 84
6.3.1. Participants .............................................................................................................. 84
6.3.2. Protocol ................................................................................................................... 85
6.3.3. Preloads ................................................................................................................... 86
6.3.4. Blood Parameters .................................................................................................... 86
6.3.5. Meal ........................................................................................................................ 87
6.3.6. Data Analysis and Calculation ................................................................................ 87
6.4. Results ............................................................................................................................... 88
6.4.1. Subjects ................................................................................................................... 88
6.4.2. Plasma Glucose, Insulin, C-peptide and Amylin .................................................... 88
6.4.3. Plasma GLP-1, GIP, PYY, CCK and Ghrelin Concentrations ............................... 90
6.4.4. Gastric Emptying Rate (Plasma Paracetamol Concentrations) ............................... 92
6.4.5. Triglycerides and Free Fatty Acids ......................................................................... 92
6.5. Discussion ......................................................................................................................... 92
CHAPTER 7. GENERAL DISCUSSION ................................................................................. 112
7.1. Study Design: Strengths and Limitations ........................................................................ 116
7.2. Significance and Implications ......................................................................................... 117
CHAPTER 8. GENERAL SUMMARY AND CONCLUSIONS ............................................. 118
CHAPTER 9. FUTURE DIRECTIONS .................................................................................... 118
CHAPTER 10. REFERENCES ................................................................................................. 120
ix
CHAPTER 11. APPENDICES .................................................................................................. 143
APPENDIX 1. Sample Size Calculation ................................................................................ 144
APPENDIX 2. Subjects Characteristics ................................................................................. 145
APPENDIX 3. Pizza Meal Composition ............................................................................... 146
APPENDIX 4. Fixed Pizza Meal Calculation ........................................................................ 147
APPENDIX 5. Amino Acid Profile of Sweet, Acid and Hydrolyzed Whey Proteins ........... 149
APPENDIX 6. Information Sheet and Consent Forms .......................................................... 150
APPENDIX 7. Screening Questionnaires .............................................................................. 162
Phone Screening Questionnaire ...................................................................................... 162
Recruitment Screening Questionnaire ............................................................................ 162
7.1. Phone Screening Questionnaire .......................................................................... 163
7.2. Recruitment Screening Questionnaire ................................................................ 164
7.3. Sleep Habit Questionnaire .................................................................................. 165
7.4. Eating Habit Questionnaire ................................................................................. 166
7.5. Food Acceptability Questionnaire ...................................................................... 167
7.6. Recruitment Advertising ..................................................................................... 168
APPENDIX 8. Study Day Questionnaire ............................................................................... 169
8.1. Sleep Habit and Stress Factor Questionnaire ...................................................... 170
8.2. Recent Food Intake and Activity Questionnaire ................................................. 171
8.3. Motivation to Eat VAS ....................................................................................... 172
8.4. Physical Comfort VAS ....................................................................................... 173
8.5. Energy and Fatigue VAS .................................................................................... 174
8.6. Treatment and Test Palatability .......................................................................... 175
8.7. Test Meal Record ................................................................................................ 176
APPENDIX 9. Blood Glucose and Insulin Record ................................................................ 177
APPENDIX 10. Pizza Test Meal Record ............................................................................... 179
x
APPENDIX 11. Data from Chapter 6 .................................................................................... 180
11.1. Plasma Concentrations of Glucose ..................................................................... 181
11.2. Plasma Concentrations of Insulin ....................................................................... 183
11.3. Plasma Concentrations of C-peptide ................................................................... 185
11.4. Insulin Secretion Rate ......................................................................................... 187
11.5. Plasma Concentrations of Amylin ...................................................................... 189
11.6. Plasma Concentrations of GLP-1 ........................................................................ 191
11.7. Plasma Concentrations of PYY .......................................................................... 193
11.8. Plasma Concentration of Total GIP .................................................................... 195
11.9. Plasma Concentrations of Total Ghrelin ............................................................. 197
11.10. Plasma Concentrations of CCK .......................................................................... 199
11.11. Plasma Concentrations of Free-Fatty Acids ....................................................... 201
11.12. Plasma Concentrations of Triglyceride ............................................................... 203
xi
LIST OF TABLES
Table 4. 1. Experiment 1: effect of gelatin treatments on energy intake, cumulative energy intake,
caloric compensation and water intake a ....................................................................................... 45
Table 4. 2. Experiment 2: effect of sugar treatments on energy intake, cumulative energy intake,
caloric compensation and water intake a ....................................................................................... 46
Table 4. 3. Experiment 3: effect of whey protein treatments on energy intake, cumulative energy
intake, caloric compensation and water intake a ........................................................................... 47
Table 4. 4. Experiment 1 and 2: effect of gelatin and sugars treatments on blood glucose
concentration a and AUC b ............................................................................................................ 48
Table 5. 1. Experiment 1: effect of pre-meal whey protein on energy intake, cumulative energy
intake, caloric compensation and water intake1 ............................................................................ 68
Table 5. 2. Experiment 1: effect of pre-meal whey protein on pre- and post-meal blood glucose
response1 ....................................................................................................................................... 69
Table 5. 3. Experiment 2: effect of pre-meal whey protein on pre- and post-meal blood glucose
response1 ....................................................................................................................................... 70
Table 5. 4. Experiment 2: effect of pre-meal whey protein on pre- and post-meal insulin
response1 ....................................................................................................................................... 71
Table 6. 1. Mean plasma concentrations of glucose, insulin, C-peptide, and amylin after the
preloads1 ........................................................................................................................................ 98
Table 6. 2. Mean plasma concentrations of gastrointestinal hormones after the preloads1 ........ 100
Table 6. 3. Mean plasma concentrations of paracetamol after the preloads1 .............................. 102
Table 6. 4. Mean plasma concentrations of triglycerides and free fatty acids after the preloads1
..................................................................................................................................................... 103
xii
LIST OF FIGUURES
Figure 4. 1. Subjective average appetite scores after treatments to 60 min .................................. 49
Figure 4. 2. Subjective appetite AUC after treatments ................................................................. 50
Figure 4. 3. Supplement. Food intake after treatments at 60 min ................................................ 51
Figure 5. 1. Pre-meal, post-meal and cumulative blood glucose AUC after whey protein preloads
....................................................................................................................................................... 72
Figure 5. 2. Pre-meal and post-meal blood glucose and insulin AUCs after whey protein preloads
....................................................................................................................................................... 73
Figure 5. 3. Cumulative blood glucose and insulin AUCs after whey protein preloads ............... 74
Figure 5. 4. Ratio of cumulative blood glucose/insulin AUC safter whey protein preloads ........ 75
Figure 5. 5. Ratio of blood glucose/insulin concentration after whey protein preloads ............... 76
Figure 5. 6. Supplement 1. Average appetite scores after whey protein preloads ........................ 77
Figure 5. 7. Supplement 2. Experiment 1: average appetite AUC after whey protein preloads ... 78
Figure 5. 8. Supplement 3. Experiment 2: average appetite scores after whey protein preloads . 79
Figure 6. 1. Mean plasma concentrations of glucose and ß-cell hormones ................................ 104
Figure 6. 2. Mean plasma concentrations of the incretins .......................................................... 105
Figure 6. 3. Mean plasma concentrations of gastrointestinal hormones ..................................... 106
Figure 6. 4. Mean plasma concentrations of paracetamol .......................................................... 107
Figure 6. 5. Mean ratios of pre-meal and post-meal plasma concentrations of C-peptide/insulin,
glucose/GLP-1and insulin/GLP-1 ............................................................................................... 108
Figure 6. 6. Mean pre-meal, post-meal and total insulin secretion rate ...................................... 109
xiii
Figure 6. 7. Whey protein induced post-meal hypoglycaemia: contribution of non-insulin
pathways compared with the water control ................................................................................ 110
Figure 6. 8. Whey protein induced post-meal hypoglycaemia: contribution of non-insulin
pathways compared with glucose ............................................................................................... 111
xiv
LIST OF ABBREVIATIONS
α-LA Alpha-Lactalbumin
ANOVA Analysis of Variance
AUC Area Under the Curve
β-LG Beta-Lactoglobulin
BBB Blood–Brain Barrier
BG Blood Glucose
BMI Body Mass Index
BCAA Branched-Chain Amino Acids
CHO Carbohydrate
CCK Cholecystokinin
CNS Central Nervous System
CLA Conjugated Linoleic Acids
CMP Caseinomacropeptide
DPP-IV Dipeptidyl Peptidase IV
FI Food Intake
FFA Free Fatty Acids
H Hour
GI Glycemic Index
GIP Glucose-Dependent Insulinotropic Peptide or Gastrointestinal Peptide
GLP-1 Glucagon-Like Peptide-1
GLP-1R GLP-1 Receptors
G50:F50 50% Glucose: 50% Fructose
GMP Glycomacropeptide
Kcal Kilocalories
Min Minutes
NS Not Statistically Significant
PYY Peptide Tyrosine Tyrosine (3-36)
T2D Type 2 Diabetes
WI Water Intake
WP Whey Protein
xv
WPH Whey Protein Hydrolysate
SEM Standard Error of the Mean
VAS Visual Analogue Scale
xvi
LIST OF PUBLICATIONS ARISING FROM THESIS
Peer-Reviewed Articles:
Akhavan T, Luhovyy BL and Anderson GH. Effect of Drinking Compared with Eating Sugars
or Whey Protein on Short-term Appetite and Food Intake. 2010. Int J Obes (Lond).
Akhavan T, Luhovyy BL, Brown PH, Cho CE and Anderson GH. The effect of pre-meal
consumption of whey protein and its hydrolysate on food intake and post-meal glycemia and
insulin responses in young adults. Am J Clin Nutr. 2010. 91: p. 966-975 (Chapter 5)
2011. 35, p.
562–569 (Chapter 4)
Akhavan T, Luhovyy BL, Brown PH and Anderson GH. The Mechanism of Action of Pre-Meal
Consumption of Whey Protein on Glycemic Control in Young Adults. (Chapter 6)
Review Paper:
Luhovyy BL, Akhavan T and Anderson GH. Whey Proteins in the Regulation of Food Intake
and Satiety. 2007. J Am Coll Nutr. 26(6):704-712
Book Chapters:
Anderson GH, Luhovyy BL, Akhavan T and Panahi S. Milk Proteins in the Regulation of Body
Weight, Satiety, Food Intake and Glycemia. In Milk and Milk Products in Human Nutrition. Eds:
Clemens RA and Michaelsen KF. 2011, Nestec Ltd., Vevey/S. Karger AG: Basel. Vol 67,
pp.147-159
Akhavan T, Panahi S, Anderson GH and Luhovyy BL. Application of Dairy-Derived
Ingredients in Food Intake and Metabolic Regulation in Dairy-Derived Ingredients: Food and
Nutraceutical Uses. Eds: M. Corredig. 2009, Woodhead Publishing Ltd: Cambridge, UK. p.
212-237
xvii
Anderson, GH, Akhavan T and Mendelson R. Food Ingredients Implicated in Obesity: Sugars
and Sweeteners in Novel Food Ingredients for Weight Control. Eds: H. CJK. 2007, Woodhead
Publishing Ltd: Cambridge, UK. p. 104-127
Published Abstracts:
Akhavan T, Luhovyy BL, Brown PH, Panahi S and Anderson GH. Insulin-Independent
Mechanisms of Action of Pre-Meal Consumption of Whey Protein on Post-meal Glycemic
Response in Healthy Adults. Experimental Biology, April 2012, San Diego, US (Abs. # 7225)
Akhavan T, Luhovyy BL, Brown PH, Panahi S and Anderson GH. Mechanism of Whey Protein
Control of Post-meal Glycemia. Canadian Diabetes Association, Oct 2011, Toronto, Canada
(Abs. # 170)
Panahi A, Luhovyy BL, Akhavan T and Anderson GH. The Effect of Preloads of Fluid Milks
and Substitutes on Short-Term Food Intake, Appetite and Glycemic Response in Healthy Young
Men and Women. Experimental Biology, April 2011, Washington, US (Abs. #. 223.8)
Akhavan T, Luhovyy BL and Anderson GH. The Effect of Whey Protein on Post-Meal Blood
Glucose and Insulin. Experimental Biology, April 2009, New Orleans, US (Abs. # 545)
Akhavan T, Mozek T, Luhovyy BL and Anderson GH. Effect of Physical States of Whey
Protein and Sugar Preloads on Subjective Appetite and Short-Term Food Intake. The Obesity
Journal, September 2007, Vol 15, p. 219 (Abs. # 699)
Scholarships and Academic Award:
Natural Science and Engineering Research Council of Canada Postgraduate Scholarship (2007-2011)
Christine Gagnon Memorial Travel Award - Canadian Federation of Biological Sciences (June 2007)
1
CHAPTER 1
INTRODUCTION
2
CHAPTER 1. INTRODUCTION
The prevalence of obesity has reached epidemic proportions. According to the 2007-09
Canadian Health Measures Survey, 24.3% of Canadian adults are obese (1). A comparison
between these data and the 1981 Canada Fitness Survey shows that obesity has roughly doubled
across all studied age groups. Obesity increases the risk of metabolic disorders and medical
complications involving several organ systems including the eyes, kidneys, liver, cardiovascular
system, and brain (2).
Obesity is a major risk factor for the development of type 2 diabetes (T2D), which is the
most common form of diabetes and is characterized by high blood glucose in the presence of
insulin resistance and/or relative insulin deficiency. The prevalence of T2D has been increasing
worldwide over the last few decades (3) and its complications continue to grow in Canada (4).
Based on the 2011 Canadian Diabetes Association report,
4
285 million people are currently
affected directly and indirectly by diabetes and this number is expected to hit 438 million by
2030 ( ). Currently, it is estimated that 9 million people have been diagnosed with diabetes or
pre-diabetes which can contribute to a variety of complications
The reasons for the increased prevalence of obesity and T2D are many, but food and diet
are significant factors. Consistent with the increase in obesity, there have been many changes in
food consumption. One notable pattern has been a decline in the consumption of cow’s milk and
this has coincided with an increased popularity of sugar-sweetened beverages (SSB). Dietary
survey data show that SSB have displaced milk in many meals of children’s (
including heart, kidneys and
eyes.
5-7). Thus, it has
been hypothesized that the decreased consumption of milk and increased consumption of SSB
have contributed to increased prevalence of obesity (8-10)
In support of the hypothesis, several epidemiological studies suggest that the
consumption of milk and dairy products is inversely related to obesity (
.
11, 12), metabolic
syndrome (13, 14) and T2D (13, 15). As a result, it has been proposed that dairy components and
specifically its calcium and protein contents (16) are the primary factors in dairy accounting for
the associations seen in epidemiological data. This has led to many experimental studies aimed at
adding plausibility to these associations by examining the effects of dairy consumption and of
3
dairy proteins and calcium in weight loss diets and short-term experimental studies of the effect
of dairy and dairy proteins on satiety, food intake and metabolic regulation (16). Of the dairy
proteins, the physiologic properties of whey protein (WP) have been the most frequently studied,
because WP is a readily available waste byproduct of making cheese and has a number of
physiological functions beyond providing amino acids for muscle protein synthesis. However, its
potential use in beverages and food formulations for the control of satiety, food intake and
glycemia has received relatively little investigation. Therefore, the focus of this thesis is on the
effect of WP consumption on satiety, food intake and glycemia, and its mechanisms of action in
young adults.
4
CHAPTER 2
LITERATURE REVIEW
5
CHAPTER 2. LITERATURE REVIEW
2.1. Introduction
As background for the thesis research, the following literature review is composed of five
main sections. After a brief introduction to the relationship between consumption of dairy
products and obesity, metabolic syndrome and T2D, the role of dairy components and proteins in
this relationship is examined. This is followed by a review of experimental studies assessing the
effect of dairy, then by a closer examination of the effects of proteins, in particular dairy proteins
on satiety, food intake and glycemic control. A later section provides a brief orientation to
physiological mechanisms of satiety, food intake and glycemic control and is followed by an
examination of what is known about the role of dairy proteins, particularly WP, and their
mechanisms of action on satiety, food intake and glycemic control.
2.2. Dairy Products, Proteins, Obesity, Metabolic Syndrome and T2D
A variety of approaches including pharmacological treatments have been used to prevent,
control and treat obesity and T2D. However, a dietary and lifestyle approach is recommended as
the primary strategy for the prevention and management of obesity. Thus, it is important to
identify dietary practices, foods and food components that contribute to a healthy body weight
and reduce risk of obesity and obesity related co-morbidities (17).
Dairy products and components, particularly dairy proteins, are of interest for several
reasons. First, a growing body of evidence from epidemiological studies suggests a strong
inverse association between higher dairy consumption and lower risk of obesity (11, 12) as well
as lower incidence of the metabolic syndrome (13, 14) and T2D (13, 15). Second, the reduced
consumption of milk and other dairy products in the last few decades has been associated with a
simultaneous rise in the consumption of SSB (5-7) and increased prevalence of metabolic disease
rates such as obesity, dysglycemia and dyslipidemia. Third, milk and dairy products are readily
available, relatively inexpensive, and good sources of high quality nutrients including protein
and calcium. While the beneficial effect of dairy is mainly attributed to its protein content (16-
20), other components in milk and dairy products such as calcium, vitamin D and magnesium
6
(16, 21-23), medium-chain triglycerides (24) and conjugated linoleic acids (CLA) (25) may play
a role in body weight control and metabolic regulation. Several studies both in humans (26-28)
and mice (29) suggest that milk and dairy sources of calcium and vitamin D exert stronger
effects on weight loss than non-dairy sources e.g. supplements (30). Dairy vitamin and minerals
are also factors modulating lipid metabolism and energy expenditure through the reduction of
body fat mass (31) and increased thermogenesis (32), fecal fat excretion (33) and fat oxidation
(34-36). Similarly, dairy medium-chain triglycerides (24) and CLA (25) have also been reported
to increase energy expenditure and increased fatty acid
37-43
beta-oxidation, thus, leading to decrease
adiposity and increase lean body mass. Finally, dairy products and proteins, are known to
enhance satiety, suppress short-term food intake and stimulate the mechanisms that regulate food
intake ( ) and glycemic control (44-46), and the effect is attributed to factors beyond the
energy content of the dairy products alone. Therefore, consumption of dairy products and
proteins have the potential to be an effective part of countermeasures against obesity and related
diseases (17, 37).
To date, however, despite numerous published epidemiological studies, there is no clear
consensus on the role of milk and dairy products in the prevention and treatment of obesity (11,
12, 47-49) and the incidence of T2D (15, 21). Two systematic reviews and meta-analyses
showed an inverse relationship between the consumption of dairy products, particularly low-fat
dairy products, and incidence of T2D (15, 50). Similarly, the dietary patterns characterized by
high intakes of low-fat milk and dairy products, whole grains, fruits and vegetables were
inversely associated with the risk of weight gain and obesity (11, 13, 51) as well as the incidence
of T2D (52-54). In contrast, others did not find any inverse associations between milk and dairy
product consumption and the incidence of obesity (48, 55, 56) and T2D (57, 58).
The inconsistency in observational studies is perhaps due to diverse study populations
(49, 55, 59, 60), ethnicities (49) and methods of intake measurement (61), as well as the wide
range of fat intakes and contents in milk and dairy products (48, 49, 62, 63). In many of these
studies (63-66), the inverse relationship between milk and dairy consumption and the incidence
of obesity and T2D seemed to be mainly resulting from low-fat dairy products (67) including
skim/low-fat milk, yogurt, and cottage/ricotta cheeses, but not from high-fat dairy products
including whole milk, cream, sour cream, butter, ice cream and cream cheese. Thus, in addition
7
to the high calorie content of high-fat dairy, the presence of saturated fat may be a potential
factor for the weak or lack of inverse association between high-fat dairy consumption and the
incidence of obesity and T2D (65). In addition, to assess dietary patterns or exposures, 24-hr
dietary recalls are usually used in these studies, but have several inherent limitations including
dependence on memory and failure to reflect usual intake (68). Furthermore, due to the nature of
observational studies, residual confounding could not be ruled out, and reported associations
cannot determine causality in the associations found between higher milk and dairy product
intake and lower incidence of obesity, metabolic syndrome and T2D (13). Thus, experimental
and mechanistic studies are needed to explain the reported associations found in observational
studies, as well as identify the key component of dairy products that regulates the physiological
mechanisms of satiety and food intake.
However, there is accumulating evidence that the primary effect of milk and dairy
products on both food intake and metabolic regulation can be attributed to milk proteins.
Therefore, the following section provides a review of the proteins in milk and their physical and
physiological properties.
2.2.1. Milk Proteins
The major components of dairy proteins are casein and whey proteins which account for
80% and 20% of total cow’s milk proteins, respectively. Each has unique physical and
physiological properties.
2.2.1.1. Physical Properties
Bovine casein consists of four major protein fractions, including αs1, αs2, β and κ-casein
(18). Casein is a major component of cheese normally produced by acidification of milk and then
coagulation of casein by the addition of rennet, a proteolytic enzyme obtained from the stomachs
of calves. The solids are separated and pressed into final form of cheese. The liquid remaining
after the cheese processing is called whey which is a byproduct of the cheese or casein
manufacturing. Thus, a large amount of casein is consumed in cheese and other dairy products,
but WP is often under-utilized.
8
Casein contains naturally glycosylated proteins, κ-casein, that play physicochemical and
biological roles in the organism (17, 18). During both stomach digestion of dairy proteins and
cheese manufacturing, the macro-bioactive peptide, called caseinomacropeptide (CMP), is
cleaved from casein (69) and goes into the liquid whey. A glycosylated form of CMP, called
glycomacropeptide (GMP), accounts for 50% of the total κ-casein or bovine CMP (70).
Glycomacropeptide is a mixture of different glycoforms due to the carbohydrates sialic acid (N-
acetylneuraminic acid, NeuNAc), galactose, galactosamine and glucosamine attached by O-
glycosidic linkages (71). In vivo studies showed that GMP is released intact from the stomach
and undergoes only partial hydrolysis by pancreatic enzymes (72, 73), although its digestion
depends on the degree of glycosylation in food form (74).
Bovine WP is also a complex mix of proteins and includes the major fractionated proteins
such as beta-lactoglobulin (β-LG), serum albumin, immunoglobulin and GMP, as well as minor
fractionated proteins such as alpha-lactalbumin (α-LA), lactoferrin, and secretory components
including insulin-like growth factor (IGF) and γ- globulin (37, 75).
There are two types of WP produced for human consumption, dependent upon the
process used to manufacture cheese. Sweet WP is a byproduct of solid cheese production, e.g.
cheddar cheese, and typically contains 10-15% GMP as a result of the enzymatic coagulation of
milk. Acid WP is a byproduct of soft cheese production, e.g. cottage cheese, and is obtained
when the coagulation is carried out by acid and is typically GMP-free. Commercial forms of WP
are mainly sweet WP converted into WP concentrate (35-85% protein) and WP isolates (90%
protein) (17).
2.2.1.2. Physiological Properties
Casein and WP are high-quality proteins for meeting the amino acid requirements of
humans. Beyond providing amino acids required for synthesis of new proteins in the body, their
high content of the BCAA and bioactive peptides play an array of metabolic roles in regulating
thermogenesis (76), blood pressure (77), satiety (38-40), short-term food intake (42) and blood
glucose (78, 79).
9
Whey protein has an earlier effect than casein on satiety, food intake and glycemic
regulation (41), perhaps due to its insulinotropic properties (19, 20, 44), but also to its faster
digestion and absorption rates (80) and quicker release of the gastrointestinal hormones affecting
satiety. However, the action of WP on glycemic control is not related simply to the BCAA, as
strong stimulators of insulin release, but is due to the synergistic action of amino acids and
bioactive peptides (45). Healthy subjects ingesting a mixture of free amino acids of WP
including BCAA (leucine, isoleucine and valine), lysine and threonine had similar glycemic and
insulinemic responses to those after ingestion of intact WP (45), suggesting that the BCAA
content in WP is the major determinant of insulinemia and reduced glycemia. However, unlike
WP, the free amino acid mixture failed to stimulate glucose-dependent insulinotropic peptide or
gastrointestinal peptide (GIP). Therefore, the induced hyperinsulinemia by WP occurs by at least
two separate pathways: one connected to certain amino acids, e.g. BCAA, but the other
connected through the gastrointestinal hormones such as glucagon like peptide-1 (GLP-1) and
GIP which are believed to be stimulated by bioactive peptides derived from intact WP (45).
Bioactive peptides are not only naturally present in milk, but are also released during
digestion. At least 26 bioactive peptides are encrypted in the primary structure of milk proteins
and many of them have been isolated from dairy products, including sour milk, yogurts, and
cheeses (81) and shown to be biologically functional. Some of the physiological functions of
dairy bioactive peptides include the modulation of blood pressure (ACE-inhibitory effect) (77),
inflammatory processes, hyperglycemia and systems regulating food intake such as increased
cholecystokinin (CCK) secretion from the gastrointestinal tract (17, 18).
The role of bioactive peptides in food intake regulation and metabolism has received
relatively little investigation. In rats, bioactive peptides released from casein suppress food intake
via peripheral opioid receptors and CCK-A receptors in the gut (82) and stimulate GLP-1 release
(83). Whey protein contains GMP from casein but is also the precursor of many bioactive
peptides, including α-LA and β-LG. This likely explains a greater effect of WP on food intake
and gut hormone responses compared to the other proteins. The GMP content of WP has been
found to stimulate CCK, a satiety hormone that reduces postprandial gastric emptying. Although
the CCK secretagogue effect of GMP may be dependent on the GMP variant (A or B) and on
10
glycosylation (84), it has been suggested that the GMP content of WP is responsible for the
effects of WP on satiety and food intake.
However the present literature is inconsistent on the
85
role of GMP on WP-induced satiety
and food intake suppression. In healthy adults, a breakfast containing either 10% (15 g protein)
or 25% (37.5 g of protein) of energy from WP containing GMP enhanced satiety and reduced
food intake 3 h later at ad libitum lunch compared with breakfast with WP without GMP ( ,
86). The presence or absence of GMP (0.8 g) in WP shakes containing 27-29 g protein did not
have remarkable effects on satiety, CCK concentrations or food intake at 75 min (84).
Additionally, a comparison of the effect of three glycoforms of GMP (50 g, 895 kJ) and a GMP-
depleted WP concentrate showed no difference in CCK response, subjective appetite and food
intake in overweight and obese males (71). In adults, a CMP beverage (0.4 and 2.0 g/ 100 mL,
34 J) had no effect on satiety and food intake 1 h later (87). Consistently, no effect of GMP on
satiety or food intake suppression was shown when healthy subjects received a milkshake
(300 mL) with either maltodextrin CHO (control), WP with no GMP, WP with 21% GMP (8.4 g)
or WP with naturally present 21% GMP plus added GMP (24.2 g GMP) (88). Similarly, meal
replacements containing GMP-enriched WP powder (30 g protein/ day, 90% GMP, 1800 kJ) for
12 months had no additional effect on the weight loss produced by an iso-caloric skim milk
powder supplement in adults (89).
To date, therefore, the mechanisms by which dairy proteins lead to satiety, food intake
and glucose regulation are not fully explained. The majority of short-term studies on WP have
tested the effect of WP either with CHO or as part of a meal on satiety, food intake and
glycemia.
2.3. Proteins, Satiety and Food Intake
Clinical randomized control studies that have used either body weight change or food
intake as outcomes have failed to elucidate a clear role for the epidemiological associations of
diets higher in milk and dairy products with healthier body weights and less adiposity (90-93).
Controlled studies of dairy consumption has been reported to result in reduced (11, 13, 22),
36unchanged ( , 94), or increased (95) body weight.
11
The effects of dairy proteins on short-term satiety, food intake and glycemia support, in
part, the biological plausibility of the inverse associations found between consumption of dairy
products and obesity, metabolic syndrome or T2D. Proteins, in general, have been found to be
the most satiating macronutrients (96, 97), and reduce ad libitum food intake more than CHO
and fat (40, 43, 88, 98, 99).
Although some studies show no effect of protein sources on satiety and food intake, these
can be readily explained by proteins providing only a small percentage of the total calories of test
meals, the quantity of protein in preloads consumed before a meal, or the time interval between
consumption of protein and food intake measurements. If the comparisons of proteins are made
with in meals and the size of the meal is large, the energy content may be the most likely
determinant of satiation in the meal, post-meal satiety and later food intake. For example, in one
study (100), six dietary protein sources (egg albumin, casein, gelatin, soy protein, pea protein,
and wheat gluten) accounted for 22% of 5193 kJ lunches but their effect on food intake was not
measured until 8 h later. In another (101), breakfasts (2520 kJ) containing 10% of energy from
protein (15 g) found WP decreased hunger scores more than breakfasts with casein or soy, but
food intake was not measured until 3 h later. As shown by preload studies of proteins, these
small amounts of the three protein sources could not be expected to cause differences in food
intake at that time (101).
In studies of dairy proteins, pre-meal consumption of WP in relatively large amounts and
with short durations to the meal enhances satiety and suppresses food intake more than other
dietary proteins (41, 42, 102). Consumption of WP (45-50 g) led to greater reduction in satiety
and food intake at an ad libitum meal 60-90 min later than egg albumin (42) and casein (41,
102). Similarly, food intake at 240 min was lower after WP (50 g) compared with tuna, turkey
and egg albumin in lean men (n = 22) (103). However, the magnitude of the effect of protein is
altered, in part, by the protein quantity and timing of consumption (42) as well as bioavailability
of the protein.
When smaller doses are consumed, WP has similar effects on food intake as other
proteins (104, 105). Consumption of two cheese snacks (836 kJ), containing either casein (22 g)
or mixture of WP (16 g) and casein (7 g), similarly reduced food intake at a lunch 60 min later
compared with no snack in healthy normal-weight women (n = 32) (104). In addition, no effect
12
on food intake at 180 min was observed after consumption of preloads containing 15 g of pea
protein hydrolysate, WP, a combination of both proteins, or milk protein (105). In the latter
study, the subjects received each preload 5 times. Thus, it is not clear if these repeated treatment
exposures, designed to induce a learned response behavior in the subjects, influenced the effect
of the protein sources on food intake.
Bioavailability of proteins may alter the effect of a protein on satiety and food intake.
Proteins hydrolyzed to smaller peptides, mainly di, tri and tetra-peptides, by using proteases
similar to those in the body’s digestive system, are
105
digested faster than intact protein. Thus,
protein hydrolysate, as opposed to its intact protein, has been proposed to induce greater satiety
and food intake suppression due to its faster release of free amino acid absorption and
stimulation of gut hormone secretion ( ) in both rats (106) and human (107). An early study
by van Loon et al. reported a faster increase in plasma amino acid concentration and greater
insulin response after preloads of hydrolyzed
108
WP (57.1 g) compared with intact casein protein
( ). However, the use of different sources of protein confounded the interpretation of the
results. In a more appropriate comparison, consumption of casein hydrolysate accelerated protein
digestion and absorption and led to greater postprandial amino acid availability compared to
intact casein (109). To more compare completely hydrolyzed and intact proteins, four iso-
volumetric and iso-caloric treatments of intact WP, intact casein, WP hydrolysate and casein
hydrolysate, in-dependent of the degree of protein fractionation, were given intragastrically to
rats. The WP hydrolysate and intact WP resulted in similar gastric emptying rate and
gastrointestinal satiety hormones of GLP-1 and peptide tyrosine tyrosine (PYY) over 2 h (110).
However, the hydrolysate treatments elicited a greater GIP concentration during the first 20 min
and lower concentration during 60-120 min of the postprandial period. Furthermore, intact WP
and WP hydrolysate preloads which were matched for volume (600 mL), nitrogen content (9.3
g/L) and energy density (1069-1092 kJ/L) resulted in similar rates of gastric emptying and
intestinal absorption of amino acids (110). Unlike WP, hydrolysate compared with intact casein
elicited higher plasma amino acid concentrations during the first 20 min of the postprandial
period (110). This may be due to the physical properties of casein that clots in the stomach (80)
and is released in two phases, first a slow solid phase (the first 20 min) and a faster liquid phase
(20-120 min) (110).
13
In general, however, WP hydrolysate has not been found to be superior over the intact
WP in reducing food intake 110 ( , 111). Hydrolyzed WP has not been found to have a greater
effect over intact WP in reducing food intake and gastric emptying (112). Both intact WP and its
hydrolysate (50 g) suppressed food intake 2 h later similarly in healthy young men (42).
Similarly, consumption of three doses (0.3, 0.4 and 0.6 g/kg) of intact WP and WP hydrolysate
resulted in similar blood glucose, insulin and glucagon responses over 120 min (111).
2.3.1. Solid vs. Liquid Forms of Proteins
As noted, the majority of short-term preload studies have been conducted with liquid
forms of proteins, or if solids are used as in meals, no comparison have been made with liquid
equivalent meals or diets (37). Dairy products are consumed in both liquid and solid forms and
whether this is a factor in determining the relationship between dairy consumption and healthier
body weight has not been explained.
The effect of liquid compared with solid sources of calories has been the focus of many
articles and much debate, primarily because it has been used to explain that the concurrent rise in
obesity associated with higher consumption of SSB. Decreased milk consumption (5-7) has been
concurrent with an increased SSB consumption (113). However, short-term studies comparing
the effect of milk and SSB on satiety and food intake have failed to shed light on the association.
Both cola and chocolate milk (900 kJ/ 500 mL
91
) similarly increased subjective ratings of satiety
and fullness and decreased hunger, prospective consumption and food intake ( ). Due to the
lack of a control in this study, it is unclear if either of the preloads suppressed food intake.
However, skim milk, in comparison with an iso-caloric fruit drink (1,062 kJ/
90
600 mL), increased
perceptions of satiety and decreased food intake at 240 min ( ). The enhanced satiety may be
explained by the protein content of the skim milk.
Furthermore, t
114
hese associations have led to the hypothesis that liquid calories bypass
satiety compared with solid calories and therefore, promote positive energy intake and body
weight ( , 115). However, the evidence that liquids have less impact on satiety than solids
remains inconclusive for two reasons. First, no report has tested directly the hypothesis that
liquid calories are less satiating than solid calories by examining the effect of pure
macronutrients e.g. sugars or proteins with similar tastes, ingredients, volumes and matrices on
14
satiety and food intake. The majority of clinical studies have compared the effect of solid and
liquid forms of fruit (116), soup (117-119) or yogurt with fruits (93) on satiety and food intake.
Secondly, the hypothesis has not been supported by some experimental studies (120-122). Iso-
caloric preloads (300 kcal) of regular cola (24 oz) and fat-free raspberry cookies (3 oz) resulted
in similar food intake at both 20 min and 2 h later (122), suggesting that energy content of liquid
or solid foods consumed prior to a meal may be the primary factor affecting short-term food
intake (122).
If the concept that liquid calories bypass food intake regulatory systems is valid, then
increased milk consumption, or milk proteins in beverages would be expected to be less
beneficial than solid forms of dairy products. This seems unlikely as caloric compensation for
the energy content of WP preloads at a test meal is found to be equal or greater than its energy
content and more than that found for egg protein or glucose or sucrose (42).
2.4. Proteins and Blood Glucose Control
Poor long-term glycemic control results in health complications that contribute to the
morbidity, mortality, and economic burden of T2D (123). Therefore, it is important to identify
dietary approaches that contribute to glycemic control by improving insulin secretion and action
as well as other mechanisms regulating glycemia.
Ingestion of the majority of proteins, either as part of a CHO meal or glucose drink,
reduces blood glucose both in healthy and T2D adults. A comparison of 25 g of 7 protein sources
(lean beef, turkey, gelatin, egg albumin, cottage cheese, fish, and soy) added to 50 g of glucose
and a 50 g glucose meal in T2D adults demonstrated that all protein sources, except egg albumin,
reduced blood glucose, with the lowest blood glucose response over 5 h after the turkey, gelatin
and cottage cheese (mainly casein) meals (124). Insulin concentrations were higher following all
protein meals, with the highest and lowest responses found after cottage cheese and egg albumin,
respectively, compared with the glucose meal. Unlike proteins, glucose decreases glucagon
concentrations (124). The primary mechanism attributed to their glucose lowering effects has
been the stimulation of insulin release, but this effect varies with the protein source. Dairy
proteins (125) and beef (126) have stronger insulinotropic properties than fish and gelatin. Soy
protein is classified as a fast protein, and it is digested in a manner more similar to WP than
15
casein, but has an intermediate insulinotropic effect between WP and casein (125). But, gelatin
has little effect on insulin (127).
The amino acid profile of dietary proteins may account for the different effects of
proteins on insulin and glucagon. The BCAA, arginine, lysine, ornithine and alanine stimulate
insulin (128), whereas aromatic amino acids such as phenylalanine, histidine, and tryptophan
stimulate glucagon secretion (129).
2.4.1. Dairy Products and Proteins and Glycemia
Clinical randomized controlled studies examining the effect of dairy proteins on glycemic
control, in part, support the inverse association found between consumption of dairy products
and metabolic syndrome or T2D (13, 14). Consumption of dairy proteins (5-60 g) with a CHO
drink or as part of meal (25-60 g) offers benefits to metabolic regulation including improved
glycemic control (44, 103, 130). Similar to other proteins, the effect is mainly attributed to the
insulinotrophic effect of dairy proteins (17, 18, 20). A WP meal (50 g) was shown to lower
glycemia compared to a turkey or egg albumin meal and produced higher insulin responses over
240 min compared to turkey, tuna or egg albumin meal (103). Consumption of meals containing
25 g CHO and 18.2 g protein from either reconstituted milk powder or WP led to a lower
postprandial glucose response than a white bread reference meal (44). Whey protein meals had a
substantially higher insulin response than did the cheese, cod or wheat-gluten meal, matched for
CHO and protein content (44).
The insulinotrophic effect of dairy proteins when consumed with large amounts of CHO
is sustained in obese (131) and individuals with T2D (44-46, 132), thus, this may be an effective
dietary approach to control glycemia in these populations. Consumption of 25 g WP with 25 g
fructose resulted in lower blood glucose and higher insulin responses over 240 min than a 50 g
fructose preload in obese individuals (131). Furthermore in T2D adults, consumption of a casein
protein hydrolysate and leucine drink (0.3 g/kg) with a standardized main meal (64% CHO, 25%
fat, and 11% protein) resulted in a 11% decline in the average 24-h blood glucose response
compared to a meal without the protein drink (133). Similarly, consumption of cottage cheese
(25 g) with either fructose (25 g) (20) or glucose (50 g) (124) improved glycemia in individuals
with T2D.
16
However, glycemic control following milk or milk proteins is regulated by mechanisms
beyond their insulinotropic effect, including the stimulation of gastrointestinal peptides and
slower stomach emptying, which may explain the variation in glucose and insulin responses after
dairy proteins as shown by the following. First, the addition of milk (200 or 400 mL) to a low-
glycemic index mixed meal elicited insulinotropic effects, but did not reduce blood glucose
(134). Second, addition of milk and dairy products (250 g, containing 17-19 g protein) to CHO
breakfasts, which had equi-CHO to a white bread reference, resulted in similar insulin response
compared to the white bread, but glycemic responses were markedly lower (44, 135). Third,
consumption of a mixed meal containing 33% of calorie as soy or casein proteins (44 g) resulted
in lower blood glucose area under the curve (AUC) over 90 min compared with the cod protein
meal (125). However, there was no difference in insulin AUC over 90 min between the different
protein meals (125). Fourth, both WP breakfast (containing 27.6 g protein and 50.0 g CHO) and
WP lunch (containing 27.6 g protein and 46 g CHO) stimulated greater plasma insulin
concentrations more than when WP was exchanged for lean ham, but a lower glycemic response
(21%, over 120 min) was produced only after lunch (46). Finally, the hypoglycemic effect of WP
is not blunted by insulin resistance. In non-diabetic adults with low, medium and high fasting
serum insulin concentrations, addition of WP (30 g) to a glucose drink (50 g) improved glycemic
control over 120 min (136).
Taken together these studies suggest that the glycemic control achieved by WP extends
beyond the synergistic release of insulin after protein plus CHO ingestion.
137
Nevertheless, to date
only one study has reported the effect of consumption of WP consumed prior to a meal on post-
meal glycemia. In patients with T2D, consumption of WP (55 g) in a soup before a serving of
mashed potatoes with added glucose (total of 59 g CHO) led to a reduced glycemic response
( ). However, the effect of smaller amounts of WP consumed prior to a meal on short-term
food intake and post-meal glycemia in healthy adults is unclear and is explored in this thesis.
2.5. Physiological Control of Satiety, Food Intake and Glycemia
Both long- and short-term feeding behaviors and glycemic control are regulated by a
complex interactions among neural, metabolic, peripheral and hormonal signals entering the
hypothalamus in the brain (138). Protein ingestion and digestion leads to the stimulation of many
17
signals regulating satiety, food intake and metabolisms. Therefore, a brief review of mechanisms
controlling satiety, food intake and blood glucose is presented here, and then followed by an
examination of the effects of WP on these mechanisms.
2.5.1. Regulation of Food Intake
The physiological mechanisms regulating food intake are modulated by pre- and post-
absorptive signals in response to food consumption. Pre-absorptive signals arise from gastric
distension, gastric emptying and gastrointestinal peptides release in response to food. Post
absorptive signals are generated from increased concentrations of glucose and amino acids in the
blood and brain.
Both long-term and short-term energy intake and blood glucose control rely on
integration of signals in brain. The hypothalamus is the integrator of signals arising from the
gastrointestinal tract, liver and pancreas.
2.5.1.1. Hormonal Regulation of Satiety and Food Intake
The physiological mechanisms of food intake are modulated by the interaction between
pancreatic and gastrointestinal hormones released in response to food ingestion. These peptides
are predominantly synthesized and secreted from three main organs including the pancreas,
stomach and intestine.
i. Pancreatic Peptides
Insulin, a pancreatic peptide released from ß-cells, is a regulator of both long-term and
short-term food intake not only through its effect on blood glucose, but also its direct interaction
on food intake regulation in the brain. Insulin is an important central satiety signal (139), and
there is a strong association between plasma glucose and insulin and increased satiety (140, 141).
Insulin modulates food intake more than any of the gastrointestinal satiety hormones (142, 143).
ii. Gastric Peptides
18
Ghrelin is a single orexigenic (appetite stimulating) hormone that its major effect is
exerted within the central nervous system (CNS) to enhance appetite and food intake and
increase gastric motility and emptying. Little is known about the regulation of ghrelin. There is
some evidence that plasma ghrelin may be suppressed by an elevation in insulin (144), glucagon
(145-147), irrespective of glycemia (148), as well as by increased FFA concentration (149).
However, the relationship between ghrelin secretion, circulating FFA (43, 150) and insulin and
glucagon levels (146) is unclear.
iii. Gastointestinal Peptides
There are more than 20 different regulatory peptides released in the gastrointestinal tract
within minutes of food ingestion, via an extensive neural network, that exert anorexigenic
(appetite suppressing) responses in the brain, particularly in the hypothalamus. These
gastrointestinal hormones also modulate satiety and food intake by their effect on insulin
secretion 151( , 152), and gastric emptying (153). Thus, the role of insulin compared with gut
peptides on the regulation of food intake is not clear. GLP-1, CCK and PYY drive their satiety
and metabolic effect through their action on slowing gastric emptying. CCK, released from the
enteroendocrine I-cells predominantly in the duodenum and jejunum of the small intestine (154,
155), and GLP-1 (156) and PYY (157), secreted from L-cells from the distal parts of the GI tract,
also cross the blood–brain barrier (BBB) and activate the central and peripheral nervous system
to inhibit appetite and gastric emptying.
These endogenous peptides respond differently to various nutrients. CCK (41, 158, 159)
and PYY (110, 157, 160) are released postprandially after fat and protein ingestion, but not
CHO. Although GLP-1 concentrations are increased in response to all three macronutrients,
several studies reported a higher GLP-1 response after protein compared to other nutrients (97,
161, 162).
2.5.1.2. Gastric Distension and Emptying Rate
When large volumes of food are consumed, gastric distension stimulates satiety signals to
the CNS via the vagus nerve (163). However, the effect of small or moderate volumes of food,
on gastric distension is short lived and does not produce a sensation of satiety per se (164).
19
Increasing the volume of the preload affects satiety and short-term food intake due to increase
gastric distension and post-gastric mechanisms. For example, 600-mL iso-caloric milk-based
preloads (499 kcal) suppressed satiety and food intake at 30 min more than the 300-mL preload
in normal-weight men (n= 20) (165). In addition, there is some evidence that gastric distension-
induced satiety can be modulated by gut hormones e.g. CCK (166) and GLP-1(167). Women
consumed 13% less food 30 min after a 400-mL iso-caloric preload (611 kcal) compared with a
200-mL preload infused intragastrically, bypassing cognitive and sensory cues of food (168).
Among several factors affecting gastric emptying, the nutrient content and physical state
of food play major roles. Liquid foods are emptied from the stomach at a faster rate than solid
foods (169, 170), which is why it has been proposed that liquid compared to solid foods fail to
induce satiety and suppress short-term food intake (114, 171, 172). The effect of nutrients on
gastric emptying is regulated by feedback mechanisms from the intestine. Fat and proteins (e.g.
dairy proteins) exert a stronger inhibitory effect on gastric emptying than CHO (43) 173( ).
2.5.1.3. Post-absorptive Satiety Signals
The primary post-absorptive satiety signals after digestion and absorption of nutrients
from the gut are derived from increased concentrations of glucose and amino acids in the blood
and the brain as encompassed in the glucostatic and aminostatic theories.
The glucostatic theory of appetite regulation was proposed by Mayer more than half a
century ago (174), suggesting changes in blood glucose concentrations or arteriovenous glucose
differences detected by glucoreceptors in the hypothalamus regulate appetite and consequently
food intake. Since glucose is the main energy substrate of brain cells, increased availability of
glucose produces more ATP reflecting greater utilization of glucose in neurons, and thus
increasing satiety (175, 176). According to this theory, a drop in blood glucose utilization
stimulates appetite and triggers the onset of feeding; whereas, an increase in blood glucose
enhances satiety and terminates food intake. This means that, glucose uptake and utilization may
play a central and metabolically privileged role in the control of satiety and energy intake
regulation (177). Since peripheral glucose utilization is induced by insulin, therefore increased
insulin concentrations in response to glucose and certain amino acids has also been shown to
promote satiety and suppress short-term food intake (138, 142). Insulin crosses the BBB, by a
20
saturable transport system, to act within the brain to help control appetite (178).
Another metabolic theory that has been suggested to contribute to the perception of
postprandial satiety is the aminostatic theory (179). Post absorptive elevations in blood and brain
concentration of certain amino acids may be accompanied by appetite and food intake
suppression. While the post-absorptive mechanisms regulating satiety and food intake by
proteins and amino acids is unclear, a number of mechanisms have been proposed.
i. Amino Acid and Brain Neurotransmitter
Certain amino acids including tryptophan, tyrosine, and histadine act as precursors for
neurotransmitters including serotonin, norepinephrine, dopamine and histamine, found to be
involved in feeding regulation (180). These neurotransmitters transmit signals via vagal feedback
first to the brainstem nucleus tractus solitaries (nTS)
181
where it regulates satiety responses, and
second to the hypothalamus where it suppresses feelings of hunger ( ). Therefore, the satiety
effect of WP may be induced partially through its amino acid composition such as tryptophan or
phenylalanine that have been found to act as precursors to neurotransmitters regulating satiety
(182, 183).
ii. Amino Acids and Thermogenosis
Dietary protein-induced thermogenesis has been also proposed to indirectly contribute to
satiety effect of proteins (184). Dietary proteins, due to synthesis and oxidation of amino acids,
result in greater diet-induced energy expenditure, thus the net metabolizable energy from
proteins is lower than either CHO or fat. The thermogenic effect of protein arises from a greater
ATP cost of post-prandial protein synthesis, thus increasing peripheral and central temperature
(185). The increase in body temperature may be translated through specific temperature-sensitive
neurons in the brain into greater satiety (186).
The thermogenic effect of protein varies among proteins and depends on protein
metabolism and digestion rate. Whey protein, which is a rapidly digested protein, results in a
stronger increase in post-prandial protein synthesis, amino-acid oxidation and greater
thermogenesis than casein, which is a slowly digested protein (76, 187).
21
2.5.2. Regulation of Glycemia
Plasma glucose concentrations originate from three sources, including exogenous glucose
from post-meal intestinal absorption and endogenous glucose
188
from glycogenolysis and
gluconeogenesis ( ).
During the fasting state, hepatic glucose is produced from glycogenolysis, the breakdown
of glycogen to produce glucose, and gluconeogenesis, glucose production primarily from lactate
and amino acids. It is estimated each gram of amino acids contributes to 0.6 to 0.7 g de novo
glucose production (189, 190). Thus, dairy proteins modulate glycemia through hepatic
gluconeogenesis. Additionally, it is suggested that BCAA contribute to glucose recycling
through the glucose-alanine cycle (191, 192). A continuous flux of BCAA from visceral tissues
through the blood to skeletal muscle provides the amino groups to produce alanine from
pyruvate. The alanine produced in muscle is moved to liver to further support hepatic
gluconeogenesis (191)
2.5.2.1. Hormonal Regulation of Glycemia
i. Pancreatic Peptides
Glucose homeostasis is achieved through many regulatory pathways, primarily in
response to insulin action. During the fed state, glucose appearance and disappearance in the
circulation is regulated primarily by insulin. The rate of glucose disappearance in the peripheral
tissues and endogenous glucose production in the liver is at a constant rate and prevents blood
glucose elevation (191). Insulin provides a signal via the portal vein to the liver to suppress or
stop production and release of glucose via glycogenolysis and gluconeogenesis (193). The
release of insulin acts on the peripheral tissues to stimulate insulin-sensitive GLUT-4 receptors,
primarily in skeletal muscle, to accelerate glucose uptake (194).
Glycogenolysis and gluconeogenesis are mainly controlled by the hormone glucagon
which is synthesized and secreted from the pancreatic α-cells. In healthy humans, insulin and
glucagon function antagonistically. These two hormones are secreted in a coordinated, pulsatile
Within the brain, glucose,
utilized by the partially insulin-sensitive GLUT-1 receptors, is the major fuel. Lactate, a minor
fraction of glucose metabolized, is also a readily used fuel by the neurons (195).
22
manner in a reciprocal fashion at approximately 5-min intervals (196). Unlike CHO, protein
ingestion has been found to stimulate the release of both insulin and glucagon (111, 197).
Although not fully understood, protein-induced glucagon contributes to prevention of insulin-
induced hypoglycemia, thus, proteins may contribute to long-term glycemic control by reducing
and preventing hypoglycemic episodes.
Insulin reduces blood glucose concentrations through increased glucose uptake by liver
and other insulin-dependent tissues such as skeletal muscle, which converts the glucose to
glycogen. Insulin is quickly removed by the liver, thus to estimate the rate of insulin secretion C-
peptide hormone, which is secreted in equimolar concentrations with insulin, but not extracted
by the liver, is often measured (198).
Amylin is produced by the pancreatic β-cell and secreted in a ratio of approximately
1:100 with insulin. It is not known to affect insulin secretion or sensitivity, however, it
contributes to the regulation of glucose homeostasis through two main mechanisms (199, 200).
First, amylin slows gastric emptying, thus, limiting the rate of nutrient uptake from the small
intestine (201-204). Secondly, amylin suppresses nutrient-stimulated glucagon secretion,
thereby, decreasing postprandial glucagon-stimulated-hepatic glucose output (205). It has been
previously shown that ingestion of glucose resulted in an increase in plasma concentrations of
amylin and insulin in lean healthy subjects (206). Thus far, there is no study on the effect of
protein on amylin secretion and blood concentrations, but is probable because feeding cats a
high- protein diet for six weeks resulted in higher amylin concentrations than the high-CHO or
high-fat diets (207).
ii. Gastointestinal Peptides
The gut hormones GLP-1 and GIP, referred to as incretins, via stimulation of vagus nerve
account for 50-70% of the total insulin secretion after a meal 151( , 152). GIP, release from K
cells in the upper small intestinal, is an insulinotropic 208hormone ( , 209). The insulin
secretagogue action of GIP is via activation of the vagus nerve to the ß-cells increasing insulin
secretion and stimulating glucose uptake (210). GIP stimulates lipoprotein lipase activity leading
to increased uptake and incorporation of fatty acids by adipocytes. However, unlike other gut
peptides, GIP does not affect glucagon section and gastric emptying (156). It has been shown
23
that GIP is released after consumption of CHO and fat, but not most proteins (132, 211, 212),
with the exception of dairy protein (44, 46).
The effect of GLP-1 on glycemic control is mainly induced either through its action on
pancreatic hormones e.g. insulin secretion
151
and glucagon suppression, thereby inhibiting hepatic
glucose production and lowering blood glucose levels ( , 152), and/or its effect on inhibition
of gut motility and slowing gastric emptying. While it is also a potent stimulator of insulin, GLP-
1, unlike GIP, stimulates insulin secretion in a glucose-dependent manner. That is, GLP-1
contributes to insulin secretion when blood glucose is elevated. However, due to a rapid cleavage
of these incretins by dipeptidyl peptidase IV (DPP- IV) enzyme, which deactivates the
hormones, only a small percentage of the total of secreted incretins reaches pancreatic β-cells to
stimulate insulin secretion (213).
GLP-1 contributes to the maintenance of blood glucose beyond its effect on insulin
secretion (214, 215). The peripheral activation of GLP-1 receptors (GLP-1R) enhances hepatic
insulin action to replenish hepatic glycogen stores (216) as well as to increase disposal
217
of meal-
derived glucose by activation of neurons with the hypothalamus ( ). GLP-1R expressed in the
gastrointestinal tract exert inhibitory action on gastric emptying (213).
2.5.2.2. Gastric Emptying During the fed state, gastric emptying rate is a major determinant of postprandial
glycemic. Slower rates of gastric emptying are associated with lower glycemia (137, 218-220).
Reduced gastric emptying rate slows the rate at which nutrients enter absorptive sections of the
gut and therefore, reduces the demand for insulin secretion. In the postprandial state, the
regulation of gastric emptying 153by the gut hormones such as GLP-1( ), PYY (157, 160) and
CCK (221) has a major influence on glucose homeostasis (82, 166, 219).
2.6. Whey Protein and the Regulation of Satiety, Food Intake and
Glycemia
2.6.1. Satiety and Food Intake
24
Dairy proteins, particularly WP, increase CCK (84, 105, 158), GLP-1 (41, 44, 105), and
PYY (110) and are associated with enhanced satiety and food intake suppression and improved
glycemic control, without increases in insulin demand (43, 222). A high dairy protein breakfast
(57 g protein and 14 g CHO) reduced blood glucose over 3 h, without an increase in insulin
concentration, compared with a high CHO breakfast in healthy men (173). However, the reduced
glycemia after a dairy protein breakfast was associated with reduced gastric emptying rate and
ghrelin and increased CCK, glucagon and GLP-1. Similarly, high protein preloads of WP (50 g)
increased satiety and improved blood glucose response, despite a lower insulin response,
compared with an iso-caloric glucose preload (161). In addition, improved blood glucose
response associated with elevation of GLP-1 and CCK and prolonged ghrelin suppression (161).
Limited studies on the effect of dairy protein on PYY (110, 160) show that increased PYY
concentration after dairy proteins associates with decreased gastric emptying rate and reduced
glycemia.
The unique physiological properties of WP may be due to its bioactive peptide content as
shown by its different effect on gastrointestinal peptide responses compared to other proteins. A
WP-enriched meal, containing 25 g CHO and 18.2 g protein, improved glycemic response and
increased GIP more than a reference meal of white bread and other protein meals, enriched with
cheese, cod or wheat gluten (44). Addition of WP (27.6 g), compared with the addition of lean
ham, to white bread (50 g CHO) at breakfast and mashed potato with meatballs (46 g CHO) at
lunch reduced blood glucose response, increased insulin and GIP concentrations in subjects with
T2D (46). Although the concentrations of ghrelin and GIP are modulated in response to only
CHO and fat ingestion (211, 212), there is some evidence that WP increases GIP (44, 46) and
suppresses ghrelin (173) concentrations. It has been suggested that an increase in glucagon
concentrations after ingestion of WP, as part of a meal, may be due to ghrelin suppression, but it
seems more likely that ghrelin response is affected by only the calorie content of the meal (145-
147).
The physical properties of dairy proteins may also contribute to its functionality in food
systems. The classification of WP as a fast protein is based on its contribution to protein
synthesis and its effect on plasma amino acid concentrations (80). Whey protein is a soluble
protein and is rapidly digested. In humans, the intake of WP (0.45 g/kg body weight) results in a
25
fast, but transient, increase in plasma amino acids that peak between 40 min to 2 h after its
ingestion and returns to baseline values after 3 to 4 h. Thus, consistent with its physical
properties, WP (48-50 g), compared to casein, reduced short-term food intake at 60-90 min (41,
102) and resulted in greater plasma concentrations of CCK (60%), GLP-1 (65%) and GIP (36%)
(41). However, casein clots in the stomach due to its precipitation by gastric acid and results in
plasma amino acid concentrations that rise more slowly and sustain a lower and prolonged
plateau lasting for at least 7 h after its consumption (80).
Studies examining the effect of WP and casein consumed as part of a mixed
macronutrient beverage or meal on gastric emptying rate have provided inconsistent results.
Plasma concentrations of paracetamol, as an indirect biomarker of gastric emptying rate, in the
Hall et al. study (41) showed a casein meal resulted in a faster initial rise in plasma paracetamol
concentrations and then a prolonged lower paracetamol concentration compared with the WP
meal, suggesting that casein slows gastric emptying more than WP. However, Calbet et al (110),
showed that beverages of intact WP and casein or their matched hydrolysate peptides were
emptied from the stomach at similar rates (110). In addition, both casein- and WP- predominant
infant milk formulas resulted in a similar gastric emptying rate over 2 h in preterm infants (n =
20) (223). Due to the presence of CHO in the beverage and meal and the lack of a control such as
glucose or water control in these studies, the effect of WP and casein consumed alone on gastric
emptying rate remains unclear.
2.6.2. Glycemic Control
Proteins, in particular dairy proteins, unlike sugars, enhance insulin secretion with no or
only a slight increase in blood glucose. The capacity of WP, when consumed with or without
CHO, to act as direct insulin secretagogues, even more than casein (44), has been consistently
reported in both healthy and T2D subjects (46, 130, 136). This may be explained by the amino
acid composition of WP, particularly its BCAA content (44). Leucine is one of the most potent
insulin secretagogues (224, 225). Consumption of a mixture of leucine, arginine and
phenylalanine (0.4 g/kg) in combination with CHO (0.8 g/ kg) produced strong insulinotropic
effects in healthy normal-weight males (108). However, WP induces several physiological
functions, beyond its energy content and its insulinotropic effect, regulating energy and glucose
26
homeostasis, thus, it is unlikely that the satiety, food intake and glycemic effect of WP can be
attributed only to insulin secretion.
Limited research has been published on the effects of WP when consumed alone prior to
a meal on post-meal glycemic control in humans. One study conducted in individuals with T2D
showed that consumption of 55 g WP 30 min before a meal containing 59 g CHO reduced
gastric emptying rate and increased GLP-1, GIP and CCK concentrations, leading to improved
blood glucose response (137). Therefore, it remains to be determined whether small amounts of
WP consumed alone prior to a meal improves glycemia in healthy adults.
2.7. Summary and Research Rationale
A large body of evidence suggests consumption of dairy products and proteins has been
inversely associated with prevalence of obesity, metabolic syndrome and T2D. Dairy proteins,
including casein and WP, have been suggested as primary components driving the association.
Whey protein is of specific interest because it is a readily available byproduct of cheese making
and is well-known to be insulinotropic due to its high content of BCAA and bioactive peptides.
To date, however, the mechanisms by which WP leads to satiety, food intake and glucose
regulation are not fully explained. Additionally, the majority of short-term studies on WP have
tested the effect of WP either with a glucose drink or as part of a CHO meal on satiety, food
intake and glycemia. Thus, no study has examined the effect of WP alone in solid and liquid
forms on satiety, food intake and post-meal glycemia. In addition, there are no reports of the
relationship between the dose of WP consumed before a meal and efficacy for reducing food
intake and post-meal glycemic response in healthy individuals
The known effects of proteins and of WP on gut hormone release and gastric emptying
rate suggest that WP may contribute to satiety and glycemic regulation by mechanisms beyond
its effects on insulin. To date, however, the effect of small amounts of WP consumed alone or in
comparison to CHO prior to a meal on food intake, gastric emptying rate and post-meal
concentrations of blood glucose and gastrointestinal hormones in healthy adults has not yet been
reported. Furthermore, while both proteins and sugars have been shown to suppress appetite and
short-term food intake, there is no study that directly compares the effect of WP alone to sugars,
in solid and liquid forms, on appetite, food intake and glycemia.
27
Therefore, the objective of this thesis is to describe the effect and mechanism of action of
WP consumed before a meal on satiety, food intake and pre- and post-meal blood glucose. The
results may add plausibility to the inverse associations found between dairy intake, obesity and
the metabolic syndrome.
28
CHAPTER 3
HYPOTHESES AND OBJECTIVES
29
CHAPTER 3. HYPOTHESES AND OBJECTIVES
3.1. General Hypothesis and Objective
Hypothesis
• Consumption of WP prior to a meal suppresses short-term food intake and reduces post-
meal glycemic response by both insulin-dependent and -independent mechanisms
Objectives
• To identify the effects and mechanism of action of WP consumed prior to a meal on
short-term food intake and post-meal glycemic response
3.2. Specific Hypotheses and Objectives
Chapter 4: EFFECT OF
Hypothesis:
DRINKING COMPARED TO EATING SUGARS OR WHEY
PROTEIN ON SHORT-TERM APPETITE AND FOOD INTAKE
• Solid, compared with liquid, forms of preloads, sweet WP, compared with acid WP, and
sucrose, compared with glucose 50%: fructose 50% (Glu50: Fru50) suppress short-term
appetite and food intake in a greater extent
Objective:
• To compare the effect of eating solid vs. drinking liquid forms of gelatin, sucrose and its
component mixtures, and WP on subjective appetite and food intake in young men
30
Chapter 5: EFFECT OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN AND ITS
HYDROLYSATE ON FOOD INTAKE AND POST-MEAL GLYCEMIA AND INSULIN
RESPONSES IN YOUNG ADULTS
Hypothesis:
• Whey proteins induce satiety, suppress food intake and reduce post-meal glycemic
responses by its insulinotropic action
Objective:
• To describe the effect of WP or its hydrolysate when consumed before a meal on food
intake and pre- and post-meal concentrations of blood glucose and insulin in healthy
young adults
Chapter 6: MECHANISM OF ACTION OF PRE-MEAL CONSUMPTION OF WHEY
PROTEIN ON GLYCEMIC CONTROL IN YOUNG ADULTS
Hypothesis:
• Whey protein consumed prior to a meal improves post-meal glucose control by both
insulin and insulin-independent mechanisms
Objective:
• To describe and compare the effect of WP and glucose consumed before a fixed meal on
pre- and post-meal gastric emptying rate and plasma concentrations of glucose and
hormones involved in regulating gastric emptying and glycemia
31
CHAPTER 4
EFFECT OF DRINKING COMPARED TO EATING SUGARS OR
WHEY PROTEIN ON SHORT-TERM APPETITE AND FOOD
INTAKE
32
CHAPTER 4. EFFECT OF DRINKING COMPARED TO EATING
SUGARS OR WHEY PROTEIN ON SHORT-TERM APPETITE AND
FOOD INTAKE
Preface:
To address the hypothesis that solid, compared with liquid, forms of preload, sweet whey,
compared with acid whey protein, and sucrose, compared with glucose 50%: fructose 50%
(Glu50: Fru50) suppress short-term appetite and food intake in a greater extent.
This work was published in the International Journal of Obesity (Lond). 2010. 35, p. 562–569;
doi:10.1038/ijo.2010.163; published online 24 August 2010.
This material has been printed with the permission by the International Journal of Obesity
(Lond).
33
4.1. Abstract
Background: It is hypothesized that a solid form of food or food components suppresses
subjective appetite and short-term food intake (FI) more than a liquid form.
Objective: To compare the effect of eating solid vs. drinking liquid forms of gelatin, sucrose and
its component mixtures, and whey protein on subjective appetite and FI in young men.
Design and subjects: A randomized crossover design was used in three experiments in which
the subjects were healthy males of normal-weight. Solid and liquid forms of gelatin (6 g)
(experiment 1, n = 14), sucrose (75 g) and a mixture of 50% glucose/50% fructose (G50:F50)
(experiment 2, n = 15), and acid and sweet whey protein (50 g) (experiment 3, n = 14) were
compared. The controls were water (experiments 1 and 3) and calorie-free sweetened water with
gelatin (sweet gelatin, experiment 1) or calorie-free sweetened water (sweet control, experiment
2). Subjective average appetite was measured by visual analog scales over 1 h and ad libitum FI
was measured 1 h after treatment consumption.
Results: Average appetite area under the curve was not different between solid and liquid forms
of sugars, but was larger, indicating greater satiety for solid compared with liquid forms of
gelatin and sweet, but not acid whey protein. The FI was not different from that of control
because of solid or liquid sugars or gelatin treatments. However, both solid and liquid forms of
whey protein, with no difference among them, suppressed FI compared with control (P < 0.05).
Conclusion: Macronutrient composition is more important than physical state of foods in
determining subjective appetite and FI.
34
4.2. Introduction
There are multiple causes for the prevalence of overweight and obesity, many of which
are diet-dependant (226). In additional to the specific effects of macronutrients, the physical
properties of food influence food intake (FI) regulation (226, 227).
Some epidemiologic and experimental studies have provided evidence that obesity may
be due to the consumption of caloric beverages, because compared with solid foods, they have
been reported to fail to suppress appetite (114, 171, 172) and consequently promote a positive
9energy balance ( , 115, 171). However, other experimental studies provide no link between
energy intake from liquids and body weight change or FI (120, 121).
Many studies have found solid and liquid forms of the same foods lead to similar FI.
More than 25 yrs ago, it was reported that solid and liquid forms of the same food (identical
mixture of yogurt and fruit in blended or chopped forms) led to similar FI at 3 or 6 h later (228).
Similarly, FI was reported to be similar after breakfasts (matched for macronutrient contents)
with different physical forms (liquid, solid with 87 g locust bean gum or solid with 8 g gelatin)
(229). In another study, there was no satiety deficit or difference on FI following the ingestion of
a beverage (regular cola) compared with a solid food (cookies); however, FI was lower at 20 min
than at 2 h following both preloads, suggesting the timing of consumption may be more
important than the physical form of energy consumed (122).
Liquid preloads have been reported to suppress FI both more or less than solid preloads.
A liquid preload in the form of tomato soup suppressed FI more than iso-caloric solid preloads
including cheese on crackers with apple juice (230), cheese on crackers alone or melon (118). In
contrast, other studies have reported weaker satiety and energy compensatory effects of liquid
compared with solid foods (114, 231, 232). Whole apples have been reported to be more
satiating than apple juice (233), and watermelon, cheese, coconut providing greater
compensation than iso-caloric drinks of watermelon juice, milk and coconut milk in lean and
obese adults (115). In a longer-term study, energy intake was lower in subjects after four weeks
of consuming jelly beans than soda, supporting the view that liquid foods lead to a positive
energy balance (114).
35
Thus, it is clear that the hypothesis that energy consumed from solid foods evokes a
greater satiety response and suppresses energy intake at a subsequent meal compared with liquid
foods is unresolved (171, 227). One possible reason for this is that the majority of studies utilized
familiar foods and beverages, and their consumption as well as later food consumption may be
affected by learned behaviors (234). Therefore, the objective of the present studies was to further
test the hypothesized differences in solid compared to liquids on subjective appetite and FI by
eliminating familiarity as a factor in the comparison.
4.3. Subjects and Methods
4.3.1. Subjects
Healthy lean males with BMI between 18- 24.9 kg/m2
235
and an age range of 19 to 28 yrs
were recruited for the three experiments through advertisement postings around St. George
Campus of the University of Toronto. Diabetics (fasting blood glucose ≥ 7.0 mmol/L), smokers,
breakfast skippers or dieters were excluded from the experiments. Individuals under medications
or with a history of liver or kidney disease and major medical or surgical event within the last six
months were excluded. Restrained eaters were identified by a score of 11 or higher on an Eating
Habits Questionnaire ( ) and excluded from the experiments. Fourteen subjects in experiment
1 and 3 and fifteen subjects in experiment 2 completed the sessions. Sample size was calculated
based on previous short-term FI studies on sugar (142) and protein (42, 222). Subjects were
financially compensated for participating in the studies. The Human Subjects Review
Committee, Ethics Review Office of the University of Toronto approved the procedures of the
experiments.
4.3.2. Treatments
To provide the solid state of the treatments, pure gelatin (Pork Skin Gelatin, Nitta Gelatin
Canada, Inc. ON, Canada) was added to water (Crystal Springs, Quebec City, QC, Canada).
Gelatin solubilizes quickly in the stomach and is rapidly digested (236) and does not influence
nutrient absorption in the gut (229). Gelatin has previously been used as vehicle for providing
different physical states in study of satiety and FI (229), but its effects when consumed alone
have not been examined. Therefore, in experiment 1 its suitability as a vehicle for testing the
36
liquid vs. solid food hypothesis was examined. Fourteen subjects consumed five treatments
which included liquid and solid gelatin, calorie-free sweetened water with and without gelatin
and water. To the liquid form, gelatin was added immediately before consumption, whereas the
solid form was prepared by letting the treatment set for 3 h at 4˚C after mixing. Gelatin (6 g) and
sucralose (0.13 g, McNeil Specially Products Company, New Brunswick, NJ) were used to
formulate the iso-volumetric treatments (300 mL). Organic orange extract (1 mL, Flavorganics,
Newark, NJ) was also added to the treatments to improve palatability and aroma and to mask
taste differences among them.
In experiment 2, treatments were 75 g CHO from the disaccharide sucrose (Redpath
Sugar, Tate and Lyle North American Sugars, Toronto, ON, Canada) in solid and liquid forms
and its monosaccharide component mixture of G50:F50, in liquid form. These sugars were
selected as a treatment because the primary assumption of the proposed relationship between
sugars and obesity has focused on the hypothesis that these sugars in liquid form in commonly
consumed beverages enhance caloric overconsumption (237). D-glucose monohydrate (Now
Natural Foods, Bloomingdale, IL) and pure fructose (Grain Process Enterprises LTD.
Scarborough, ON, Canada) were used for formulation of the G50:F50 treatment. Treatments
were iso-caloric (300 kcal), iso-volumetric (300 mL), and all, including the control, contained 6
g of gelatin. Due to the sweet taste of the sugars, the water control was sweetened with sucralose
(0.13g sucralose) as in previous studies (142). Because 75 g of sugars in 300 mL of water is very
sweet, lemon concentrate (Equality, the Great Atlantic and Pacific Company of Canada Ltd,
Toronto, ON, Canada) was added to provide an acceptable level of sweetness and palatability.
In experiment 3, the treatments were 50 g protein from sweet whey protein (NZMP Whey
Protein Concentrate 392, Fonterra Co-operative
42
Group Limited, New Zealand) and acid whey
protein (Kraft Foods, Inc. Chicago, IL) in solid and liquid forms. Whey protein was selected
because whey protein suppresses FI more than sugar and egg-albumin when consumed in liquid
form ( ) and it is often consumed in beverage forms. However, a comparison of its effect when
consumed in liquid and solid forms has not been reported.
Sweet and acid whey protein were compared because they differ in content of CMP and
GMP, a glycosylated form of CMP, which are bioactive peptides naturally present in milk
protein and affect FI regulation (17, 18, 155, 238). Sweet whey protein is a byproduct of solid
37
cheese production (e.g. cheddar cheese) and contains 15% GMP while acid whey protein, a
byproduct of soft cheese production (e.g. cottage cheese), is GMP-free. All treatments were
equalized for calories (300 kcal), nutrients (50 g protein, 10 g CHO and 4 g fat), and volume
(300 mL) and with 6 g gelatin added to provide different physical states. To improve the
palatability, 1 g of orange-flavored energy-free sweetener (1 g Kool-Aid; Kraft Canada Inc.
North York, ON) was added to treatments and water was used as a control (300 mL). All
preloads were served chilled. Solid forms were of a texture similar or slightly harder than cubes
of jello and were served in a plate and eaten with knife and fork. After consuming the treatments,
subjects were given 100 mL of water to eliminate the aftertaste of preloads.
Prior to the experiments, palatability (in experiment 2 and 3) and sweetness (in
experiment 2) of treatments were judged to be similar by a test panel of 10 volunteers, similar to
study participants’ rating.
4.3.3. Protocol
Similar to previous studies (142, 222), participants were provided with an outline of the
studies and requested to complete the initial screening requirements including a baseline
information questionnaire and sign consent forms. Subjects were asked to maintain consistent
levels of activity the day and morning before each session, refrain from alcohol consumption and
unusual activity the night before the session, consume a similar meal the night before each test
session, and be at the same time and the same day of the week for all sessions. The time of the
test session was controlled by asking participants to consume their breakfast and start the session
at the same time for all the sessions. Subjects were provided a standard breakfast consisting of a
single serving of a ready-to-eat breakfast cereal (Honey Nut Cheerios, General Mills,
Mississauga, ON, Canada), a 250 mL box of 2% milk (Sealtest Skim Milk, Markham, ON,
Canada) and a 250 mL box of orange juice (Tropicana Products Inc. Bradenton, Florida). Four hr
after their breakfast consumed at home, subjects were scheduled to come to the Department of
Nutritional Sciences between 10:00 a.m. to 1:00 p.m., one week apart for each session. They
were asked not to consume anything between the breakfast and study session, except for water
up to one hr before the session.
Upon arrival, participants filled out a Sleep Habits and Stress Factors Questionnaire (142,
38
222). If these did not reflect their usual pattern, they were asked to reschedule. Visual Analogue
Scale (VAS) questionnaires measuring subjective average appetite (Motivation to Eat) and
Physical Comfort were completed prior to the treatments. In experiment 1 and 2, capillary blood
glucose sample was obtained (222) prior to treatment. Subjects consumed one of the treatments
over the span of 5 min following by completion of sweetness and palatability VASs. In all
experiments, subjective average appetite and in experiment 1 and 2, blood glucose were
measured at 15, 30, 45, 60 min after treatment. At 60 min, participants were served an ad libitum
pizza lunch (McCain Foods Ltd. Florenceville, NB) and a bottle of water (Canadian Crystal
Spring, Mississauga, ON, Canada). Participants were fed their ranked preference for three
varieties of pizzas (Deluxe, Pepperoni and Three Cheese). These pizzas do not have a crust
extending beyond the filing, which results in a pizza with uniformly distributed energy content
(average, 226 kcal/ 100g) and size (5” diameter). Mean protein, fat and CHO contents of the
three varieties of pizzas were 10.0, 7.6, 26.6 g, respectively. Each cooked pizza (8 min in 430◦
Cumulative energy intake was calculated by adding the energy consumed from treatment to
the energy consumed at the test meal (
F,
and cut in 4) was weighed before serving. Participants were served two pizzas of their first
choice and one each of their second and third choice per tray. A second identical hot tray was
presented in 10 min and the first tray removed. Subjects were instructed to eat until they were
“comfortably full”. Upon termination of the test meal, subjects rated the palatability of the pizza
and completed a post meal Motivation to Eat VAS. Energy intake from the pizza was calculated
from the weight consumed and the compositional information provided by the manufacturer.
Water intake was measured by weight (g).
142). Caloric compensation, expressed as percent, was
calculated by subtracting the calories consumed after the treatment from that after the control,
divided by the calories in the treatment and multiplied by 100. Caloric compensations of <100%
indicate that the subject had low compensation for the preload energy at the test meal, whereas
scores > 100% indicate overcompensation for treatment
A composite score of the four Motivation to Eat VAS was calculated to obtain the average
appetite score as described previously (
energy at the test meal.
142, 143) and was used as a summary measure of
subjective average appetite for analyses.
Blood glucose was measured by a glucose meter (Accu-Chek Compact, Roche
39
Diagnostics Canada, Laval, Quebec, Canada) from capillary blood samples obtained by finger
prick by a Monojector Lancet Devices (Sherwood Medical, St. Louis, MO, U.S.A.) (142, 222).
4.3.4. Statistical Analysis
To conduct the statistical analysis, SAS version 9.1 (Statistical Analysis Systems, SAS
Institute Inc., Carey, NC) was used in all three experiments. Two-way repeated measures
analysis of variance (ANOVA) using the Proc Mixed procedure was used to analyze the effects
of time, treatment and their interaction on outcome variables measured over 60 min. When an
interaction was statistically significant, one-way ANOVA using Proc Mixed procedure was
followed by Tukey’s post-hoc test to identify mean differences among treatments at each time of
measurement.
One-way ANOVA using the Proc Mixed procedure was used to determine the effect of
the treatments on the outcome variables like food and water intakes at 60 min, palatability of
treatments and pizza, physical comfort, perceived sweetness (experiments 1 and 2) and net
incremental area under the curve (AUC) calculated for average appetite and blood glucose in
experiment 1 and 2.
Pearson’s Correlation Coefficients were analyzed on dependent measures. Significance
was set at P < 0.05. Data are presented as mean ± standard error of the mean (SEM).
4.4. Results
4.4.1. Subjects
In experiment 1 (n = 14), experiment 2 (n = 15) and experiment 3 (n = 14), subjects had a
BMI of 22.5 ± 0.4, 22.1 ± 0.5 and 22.7 ± 0.3 kg/m2
4.4.2. Food Intake
, age of 21.9 ± 0.6, 21.4 ± 0.4 and 23.6 ± 0.9
y and weight of 69.7 ± 1.6, 68.5 ± 2.6 and 67.5 ± 1.7 kg, respectively.
In experiment 1, food and water intakes, cumulative energy intake and caloric
compensation were not affected by the gelatin and/or sucralose treatments (Table 4.1);
40
indicating that addition of 6 g gelatin and 0.13 g sucralose to the treatments does not affect FI 1 h
later compared with the water control. Thus, in the subsequent studies, gelatin was used in the
formulation of the physical states of the treatments and sucralose was used as the energy-free
sweetener to mask taste difference among the treatments.
In experiment 2, food and water intakes at the test meal and caloric compensation were
not significantly different among the liquid and solid sugars treatments (Table 4.2). However,
compensation for the energy content of the sugars treatments at the next meal was low, averaging
only 34%. As a result, all sugars treatments resulted in greater cumulative energy intake than
after the energy-free sweet control (P < 0.05) (Table 4.2).
In experiment 3, treatments of acid and sweet whey protein in both solid and liquid
forms suppressed 1 h FI compared with the water control (P < 0.0001) but were not different
from each other (Table 4.3). Water intake at the test meal and caloric compensation were not
different between the treatments (Table 4.3). All whey protein treatments resulted in caloric
compensation of approximately 80%, except liquid acid whey protein for which compensation
was >100%.
4.4.3. Average Appetite Score
In experiment 1 and 2, average appetite scores were affected by time (P < 0.0001), but
not by treatment or a time and treatment interaction (Figure 4.1a and b). Average appetite
responses were lowered at 15 min (in both experiments) and 30 min (in experiment 2) compared
to other measured times.
In experiment 3, average appetite scores were affected by treatment (P < 0.0001), time (P
< 0.0001), and treatment and time interaction (P < 0.0001) (Figure 4.1c). Compared with the
water control, all whey protein treatments reduced average appetite at 15 and 30 min (P <
0.0001) (Figure 4.1C). At 45 min all whey protein treatments, except liquid sweet whey, were
more satiating (P < 0.0001) but at 60 min, only solid sweet whey reduced average appetite
compared to the control (P < 0.001).
41
4.4.4. Average appetite AUC
In experiment 1, average appetite AUC (0-60 min) was reduced by solid compared with
the liquid gelatin treatment (P < 0.05), suggesting a greater satiating effect of solid vs. the liquid
gelatin treatment (Figure 4.2a). In experiment 2, there was no significant difference on average
appetite AUC between the sugars treatments, whether in liquid or solid form and the sweet
control (Figure 4.2b). In experiment 3, the average appetite AUC following all whey protein
treatments was reduced compared with the water control (P < 0.0001) (Figure 4.2c). In addition,
solid compared to liquid sweet whey protein was more satiating as shown by significantly lower
average appetite AUC (P < 0.0001).
4.4.5. Blood Glucose Concentration
In experiment 1, blood glucose concentration was affected by time (P < 0.0001), but not
by treatment or time-by- treatment interaction (Table 4.4). Overall mean blood glucose
concentration was significantly higher at 60 min than at baseline (5.1 ± 0.0 vs. 5.0± 0.0) (Two-
way ANOVA, P < 0.0001).
In experiment 2, blood glucose concentrations were affected by time (P < 0.0001),
treatment (P < 0.0001) and time-by- treatment interaction (P < 0.0001) (Table 4.4). Overall
mean blood glucose concentration was the lowest at baseline (4.8 ± 0.1), followed by at 60 min
(6.3 ± 0.2), which was lower than blood glucose concentration at 15 and 45 min (6.9 ± 0.2 and
7.2 ± 0.2, respectively) and was the highest at 30 min (8.1 ± 0.3) (Two-way ANOVA, P <
0.0001).
At baseline, there was no difference on blood glucose concentration among the
treatments. Compared to the sweet control, all sugars treatments increased blood glucose
concentrations over 60 min (P < 0.0001). There was no difference between the sugars treatments
on blood glucose response at any measured times, except at 15 min, when sucrose solid led to
lower blood glucose response than the G50:F50 liquid treatment, but did not differ from sucrose
liquid.
42
4.4.6. Blood Glucose AUC
In experiment 1, there was no significant difference on blood glucose AUC between the
treatments (Table 4.4). In experiment 2, all sugars treatments resulted in higher and similar blood
glucose AUC compared to the sweet control (P < 0.0001) (Table 4.4).
4.5. Discussion
These studies indicate that consumption of liquid and solid forms of sugars and proteins
elicit similar responses in later FI when the treatments are not familiar food forms and when the
caloric content, volume, macronutrient composition and taste of the treatments are similar.
Subjective appetite was reduced by the solid compared with liquid gelatin and with sweet but not
with acid whey or sugars. Compensation at the test meals for the energy content of the sugars
treatments was only one-third that found after the whey treatments. Thus, macronutrient
composition of a food or beverage may be more important than its liquid or solid states.
The present studies are the first to test the hypothesis that liquid calories are less satiating
than solid calories by using pure macronutrients and similar tastes, ingredients, volumes and
matrices to eliminate the confounding factors of taste, texture, smell, structure and familiarity of
foods. A previous study concluded that a solid breakfast was more satiating than liquid, but the
results were confounded by a design in which gelatin or fiber were added to create solid forms of
treatments but were not added to the liquid control (229). In the present studies, gelatin addition
was deemed appropriate for comparing liquid and solid forms for the following reasons. First, by
adding gelatin to the control beverages immediately before they were consumed, the treatment
would be expected to remain in liquid form because gelatin does not gel in water at room or body
temperature, and therefore in the stomach (236). Second, gelatin alone, whether in liquid or solid
form, had no effect on average appetite scores or FI compared to the water control, its addition to
sugar or whey protein. Finally, gelatin addition had no detectable effect on blood glucose (Table
4.4) as previously shown in even higher doses (218), further supporting it use as a vehicle for
these comparisons. Power analyses, with alfa level of 0.05 and the power of 0.8, from
experiment 2 and 3 suggest a sample size of 5000 and 168 subjects would be required to see a
significant difference between solid and liquid forms of sugars and whey protein, respectively.
43
Unlike sugars, both solid and liquid forms of whey protein suppressed appetite and FI
compared to control consistent with previous reports that protein is more satiating than CHO (18,
42, 239-241). Caloric compensation was 3 times more at a meal following protein consumption
than CHO (Table 4.2 and 4.3).
In the present study, sweet and acid whey proteins were compared because they differ in
GMP content. Based on the information provided by the whey protein manufacturer (NZMP,
Fonterra Co-operative
41
Group Limited, New Zealand), sweet whey contains 15% GMP but acid
whey contains no GMP. Sweet whey suppresses FI more than casein at 60 min ( , 155, 159,
238); and GMP stimulates cholecystokinin (CCK), a gastrointestinal hormone known to suppress
short-term food intake (41, 158, 159). However, no differences in the effect of the source was
found suggesting that the presence or absence of GMP in whey preloads did not influence the
effect of whey protein on satiety or food intake at a test meal in agreement with previous reports
(87) (84).
Blood glucose concentrations were measured because they associate with satiety and
food intake (226) and may have been affected by an insulinotropic effect of gelatin (218) or by
different absorption rates of the liquid and solid sugars, thus confounding interpretation of the
results. However, treatments of gelatin or the disaccharide sucrose and its monosaccharide
equivalent of G50:F50 in both liquid and solid forms did not differentially influence blood
glucose (Table 4.4) except at 15 min when the liquid G50:F50 mixture led to a higher blood
glucose than the solid sucrose. This result indicates that the liquid G50:F50 mixture was more
quickly absorbed, perhaps due to more rapid absorption of the free monosaccharides compared
with the dissaccharide that needs to be digested first. The glucose AUC response to both sucrose
with gelatin and the G50:F50 mixture with gelatin was similar, as found in a comparison of the
effect of these sugars without gelatin on blood glucose and insulin concentrations (142).
These findings indicate there may be a weak effect of food form on subjective feelings of
satiety immediately following the treatment consumption due to the cephalic phase of ingestion
(242), independent of physiologic actions of solids and liquids in the stomach or small intestine.
Unlike sugars and acid whey, sweet whey protein and gelatin in solid form suppressed subjective
appetite AUC more than liquid forms (Figure 4.2). However, the effect of physical state of
44
treatment did not affect FI at 1 h, showing a disconnect between subjective feelings and FI, as is
often observed (243).
It is also possible that this study did not test the effect of familiar foods in solid compared
with liquid forms. Gelatin may not have retained its solid form in the stomach and may not have
slowed stomach emptying as might be expected from a solid food of complex composition (e.g.
an apple) (118, 230). However, a previous report showed that solid and liquid meals of similar
calorie content led to similar gastric emptying rate (244). Therefore, an examination of stomach
emptying rate as an explanation of differences in satiety and energy intakes after liquid and solid
forms of a food with similar macronutrient and energy content may be informative.
4.6. Conclusion
In conclusion, macronutrient composition is a more important factor than physical state
of food in determining appetite, subsequent FI and cumulative energy intake. Future studies are
required to isolate the effects of chewing compared with drinking on cephalic phase and
physiologic origins of satiety and food intake.
45
Table 4. 1. Experiment 1: effect of gelatin treatments on energy intake, cumulative energy
intake, caloric compensation and water intake
a
Energy intake
Treatments Test meal Cumulative b Water intake c
Kcal G Sweet control 1319.1 ± 62.3 d 1319.1 ± 62.3 371.1 ± 31.0
Water control 1417.9 ± 72.2 e 1417.9 ± 72.2 347.7 ± 36.9
Gelatin solid 1323.1 ± 86.1 f 1342.8 ± 86.1 368.6 ± 35.6
Gelatin liquid 1372.8 ± 58.7 g 1392.5 ± 58.7 338.5 ± 30.5
Sweet gelatin liquid h 1272.8 ± 78.7 1292.5 ± 78.7
314.5 ± 31.0
P
NS
NS
NS
Abbreviation: NS, not significant. a Mean ± s.e.m. (kcal); n = 14, (one-way ANOVA for
treatment effect, Tukey’s post hoc). b Energy (kcal) consumed in a test meal 60 min after the
treatments. c Energy in the treatment (kcal)+energy from the test meal (kcal). d Sucralose (0.13 g)
per 300 mL water. e Water (300 mL). f Gelatin (6 g) per 300 mL in solid form. g Gelatin (6 g) per
300 mL in liquid form. h
Gelatin (6 g) + 0.13 g sucralose per 300 mL water).
46
Table 4. 2. Experiment 2: effect of sugar treatments on energy intake, cumulative energy intake,
caloric compensation and water intake
a
Energy intake
Treatments Test meal Cumulative b Caloric compensation c Water intake d
Kcal % G Sweet control 1465.4 ± 111.5 e
1465.4 ± 111.5
f 297.5 ± 44.1
Sucrose solid 1368.9 ± 115.7 g
1668.9 ± 115.7
h 32.2 ± 19.2
316.2 ± 50.8
Sucrose liquid 1359.5 ± 102.8 i
1659.5 ± 102.8
h 35.3 ± 21.6
291.2 ± 43.5
G50:F50 liquid j 1358.0 ± 123.0
1658.0 ± 123.0
h 35.8 ± 23.7
344.2 ± 42.7
P
NS
<0.05
NS
NS
a Mean ± s.e.m. (kcal); n = 15, * Means in the same column with different superscripts are
different (one-way ANOVA for treatment effect, Tukey’s post hoc, P <0.05). b Energy (kcal)
consumed in a test meal 60 min after the treatments. c Energy in the treatment (kcal) + energy
from the test meal (kcal). d ((kcal consumed at the test meal after water control – kcal consumed
at the test meal after sugars treatment)/300 kcal (in sugars treatment)) x 100. e Sucralose (0.13 g)
per 300 mL water. g Sucrose (75 g) per 300 mL in solid form. i Sucrose (75 g) per 300 mL liquid
form. j
Carbohydrate (75 g) from 50% glucose: 50% fructose per 300 mL in liquid form.
47
Table 4. 3. Experiment 3: effect of whey protein treatments on energy intake, cumulative energy
intake, caloric compensation and water intake a
Energy intake
Treatments Test meal Cumulative b Caloric
compensation
c Water intake d
Kcal % g Water control
1460.8 ± 73.2e
f 1460.8 ± 73.2
_ 345.5 ± 32.8
Sweet whey solid
1214.5 ± 77.4g
h 1514.5 ± 77.4
82.1 ± 16.6
382.1 ± 33.3
Acid whey solid
1122.3 ± 86.1g
h 1422.3 ± 86.1
112.8 ± 19.9
354.1 ± 31.0
Sweet whey liquid
i 1224.6 ± 95.9
h 1524.6 ± 95.9
78.7 ± 22.0
349.1 ± 36.5
Acid whey liquid
i 1193.3 ± 92.7
h 1493.3 ± 92.7
89.2 ± 19.9
388.6 ± 31.0
P
<0.0001
NS
NS
NS
a Mean ± s.e.m. (kcal); n=14, * Means in the same column with different superscripts are
different (one-way ANOVA for treatment effect, Tukey’s post hoc, P < 0.05). b Energy (kcal)
consumed in a test meal 60 min after the treatments. c Energy in the treatment (kcal) + energy
from the test meal (kcal). d ((kcal consumed at the test meal after water control – kcal consumed
at the test meal after whey protein treatment)/300 kcal (in whey protein treatment)) x100. e Water
(300 mL). g Whey protein (50 g) per 300 mL in solid form. i
Whey protein (50 g) per 300 mL in
liquid form.
48
Table 4. 4. Experiment 1 and 2: effect of gelatin and sugars treatments on blood glucose
concentration a and AUC
Treatments
b
Baseline
15 min
30 min
45 min
60 min
Blood Glucose
AUC
mmol l -1 mmol * min l
Experiment 1 -1
Sweet
5.0 ±
5.1 ±
5.0 ±
5.1 ±
5.1 ±
3.1 ± control c
0.1
0.1
0.1
0.1
0.1
4.4
Water
5.0 ±
5.0 ±
5.1 ±
5.0 ±
5.1 ±
1.5 ± control
d 0.1
0.1
0.1
0.1
0.1
2.6
Gelatin
5.0 ±
5.1 ±
5.1 ±
5.2 ±
5.2 ±
9.0 ± solid
e 0.1
0.1
0.1
0.1
0.1
4.3
Gelatin
5.0 ±
5.0 ±
5.0 ±
5.1 ±
5.2 ±
5.0 ± liquid
f 0.1
0.1
0.1
0.1
0.1
3.3
Sweet
5.0 ±
5.1 ±
5.1 ±
5.2 ±
5.1 ±
6.8 ± gelatin liquid 0.1 g
0.1
0.1
0.1
0.1
3
P NS NS NS NS NS NS
Experiment 2 Sweet
4.8 ±
0.0 ±
0.0 ±
0.1 ±
0.1 ±
2.4 ±
control
0.1
0.1
h 0.1
i 0.1
i 0.1
i 3.7
i
Sucrose
4.8 ±
2.3 ±
4.1 ±
3.3 ±
1.9 ±
159.5 ± solid
j 0.1
0.2
i 0.2
k 0.3
k 0.3
k 8.4
k
Sucrose
4.7 ±
2.9 ±
4.3 ±
3.2 ±
1.8 ±
169.4 ± liquid
l 0.1
0.3
ki 0.4
k 0.4
k 0.4
k 16.8
k
G50:F50
4.8 ±
3.3 ±
4.7 ±
3.2 ±
2.1 ±
182.6 ± liquid m
0.1
0.3
k 0.4
k 0.4
k 0.3
k 13.6
k
P NS <0.0001 <0.0001 <0.0001 <0.0001 <0.001
a Mean ± s.e.m. (mmol l-1) (experiment 1, n = 14, two-way ANOVA for time effect, P < 0.0001) (experiment 2, n = 15, two-way ANOVA for treatment, time-by-treatment interaction, P < 0.0001). b Mean ± s.e.m. (mmol * min-1) (one-way ANOVA for treatment effect, Tukey’s post hoc, experiment 1, n = 14, NS; experiment 2, n = 15, P < 0.001). c Sweet energy-free control (sucralose (0.13 g) per 300 mL water). d Water (300 mL). e Gelatin (6 g) per 300 mL in solid form. f Gelatin (6 g) per 300 mL in liquid form. g Gelatin (6 g) +0.13 g sucralose per 300 mL water). j Sucrose (75 g) per 300 mL in solid form. l Sucrose (75 g) per 300 mL liquid form. m
Carbohydrate (75 g) from 50% glucose: 50% fructose per 300 mL in liquid form. Means in the same column with different superscripts (h,i,k) are different (one-way ANOVA for treatment effect, Tukey’s post hoc, experiment 1, NS; experiment 2, P < 0.0001).
49
Figure 4. 1. Subjective average appetite scores after treatments to 60 min
c)
ΔA
vera
ge A
ppet
ite (m
m)
Time (min)
-35
-25
-15
-5
5
15
0 15 30 45 60
Sucrose (S)
Sucrose (L)
G50:F50 (L)
Sw eet Control
-35
-25
-15
-5
5
15
0 15 30 45 60
Gelatin (S)Gelatin (L)Sw eet Gelatin (L)Sw eet Control Water Control
b
cc
b
ab
bcbc
b-b-b
ab ab
bcbc
b
aba
a
a a
-35
-25
-15
-5
5
15
0 15 30 45 60
Sw eet Whey (S)Acid Whey (S)Sw eet Whey (L)Acid Whey (L)Water Control
a)
b)
Mean (± s.e.m) change from baselines average appetite scores measured by visual analog scales after consumption of gelatin solid (—▲—), gelatin liquid (--Δ--), sweet gelatin liquid (--□--), sweet control (--X--) and water control (—X—) treatments (A), sucrose solid (—●—), sucrose liquid (--○--), G50:F50 liquid (--●--) and sweet control (--X--) treatments (B), sweet whey solid (—♦—), acid whey solid (—■—), sweet whey liquid (--◊--), acid whey liquid (--□--) and water control (—X—) treatments (C). Baseline average appetite score was 73.7 ± 1.2 mm in experiment 1 (n = 14), 69.7 ± 1.8 mm in experiment 2 (n = 15) and 77.0 ± 1.5 mm in experiment 3 (n = 14). In experiment 1 and 2, average appetite score was affected by time (P < 0.0001), but not with treatment or treatment and time interaction. In experiment 3, treatment (P < 0.0001), time (P < 0.0001) and treatment by time interaction (P < 0.0001) affected average appetite scores (Two-way ANOVA). S, solid form of the treatment; L, liquid form of the treatment.
50
Figure 4. 2. Subjective appetite AUC after treatments
a)
b)
c)
Ave
rage
App
etite
AU
C (m
m●m
in)
-600
-500
-400
-300
-200
-100
0
100
200Water Control
Sw eetControl Gelatin (S) Gelatin (L)
Sw eetGelatin (L)
a
b
ab
ab ab
-600
-500
-400
-300
-200
-100
0
100
200Sw eet Control Sucrose (S) Sucrose (L) G50:F50 (L)
-2000
-1500
-1000
-500
0
500Water Control
Sw eet Whey(S)
Acid Whey(S)
Sw eet Whey(L)
Acid Whey(L)
bc
b
c
bc
a
Mean (± s.e.m) subjective average appetite net area under the curve (AUC) to 60 min (mm *
min) after treatment consumption. Means with different superscripts are significantly different in
experiment 1 (n = 14, P < 0.05) and experiment 3 (n = 14, P < 0.0001) (One-way ANOVA for
treatment effect, Tukey’s post hoc,), but not in experiment 2 (n = 15). S, solid form of the
treatment; L, liquid form of the treatment.
51
Figure 4. 3. Food intake after treatments at 60 min
Mean (± s.e.m) food intake at 60 min (kcal) after treatment consumption. Means with different
superscripts are significantly different in experiment 3 after whey protein (Figure C, n = 14, P <
0.0001), but not in experiment 1 after gelatin (Figure A, n = 14) and experiment 2 after sugars
(Figure B, n = 15) (One-way ANOVA for treatment effect, Tukey’s post hoc,). S, solid form of
the treatment; L, liquid form of the treatment.
52
CHATER 5
EFFECT OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN
AND ITS HYDROLYSATE ON FOOD INTAKE AND POST-MEAL
GLYCEMIA AND INSULIN RESPONSES IN YOUNGE ADULTS
53
CHATER 5. EFFECT OF PRE-MEAL CONSUMPTION OF WHEY
PROTEIN AND ITS HYDROLYSATE ON FOOD INTAKE AND
POST-MEAL GLYCEMIA AND INSULIN RESPONSES IN
YOUNGE ADULTS
Preface:
To address the hypothesis that all doses of whey proteins induce satiety, suppress food intake at
30 min, reduce post-meal glycemic response and increase insulin response.
This work was published in the American Journal of Clinical Nutrition (2010). 91: p. 966-975;
doi:
This material has been reprinted with the permission by the American Journal of Clinical
Nutrition.
10.3945/ajcn.2009.28406.
54
5.1. Abstract
Background: Dairy protein ingestion before a meal reduces food intake and, when consumed
with CHO, reduces blood glucose.
Objective: The objective was to describe the effect of whey protein (WP) or its hydrolysate
(WPH) when consumed before a meal on food intake, pre- and post-meal satiety, and
concentrations of blood glucose and insulin in healthy young adults.
Design: Two randomized crossover studies were conducted. WP (10–40 g) in 300 mL water was
provided in experiment 1, and WP (5–40 g) and WPH (10 g) in 300 mL water were provided in
experiment 2. At 30 min after consumption, the subjects were fed an ad libitum pizza meal
(experiment 1) or a preset pizza meal (12 kcal/kg, experiment 2). Satiety, blood glucose, and
insulin were measured at baseline and at intervals both before and after the meals.
Results: In experiment 1, 20–40 g WP suppressed food intake (P < 0.0001) and 10–40 g WP
reduced post-meal blood glucose concentrations and the area under the curve (AUC) (P < 0.05).
In experiment 2, 10–40 g WP, but not WPH, reduced post-meal blood glucose AUC and insulin
AUC in a dose-dependent manner (P < 0.05). The ratio of cumulative blood glucose to insulin
AUCs (0–170 min) was reduced by ≥10 g WP but not by 10 g WPH.
Conclusions: WP consumed before a meal reduces food intake, post-meal blood glucose and
insulin, and the ratio of cumulative blood glucose to insulin AUCs in a dose-dependent manner.
Intact WP, but not WPH, contributes to blood glucose control by both insulin-dependent and
insulin-independent mechanisms.
55
5.2. Introduction
A role of milk protein consumption and its physiologic functionality beyond the
provision of nutrients in the management of obesity and the metabolic syndrome is of interest
due to strong associations between high dairy consumption and low body weight (95, 245). In
addition, experimental studies show that milk proteins reduce short-term appetite, food intake
(17, 18, 42, 96) and blood glucose response when consumed with CHO (19, 124, 246).
Whey protein accounts for 20% of cow milk protein and is of specific interest because it
is a readily available byproduct of cheese making. It enhances satiety and suppresses food intake
in humans (96) and reduces food intake more than casein (41), egg albumin or soy protein (42).
When WP is consumed with CHO, it reduces the subsequent glycemic response (44-46), as do
other proteins (246). The reduction in blood glucose is suggested to occur because of whey
protein’s rapid digestion (80, 110) and high content of branched-chain amino acids (44),
resulting in rapid insulin release (16-18). Support for the importance of the BCAA in whey
protein as the mediator of its effect has arisen by the similarity of the effect of intact whey,
hydrolyzed whey protein (111) and BCAA (45) on plasma insulin and glucose concentrations.
However, mechanisms other than insulin may be of importance because a branched-chain amino
acid mixture does not reproduce the effect of the intact whey protein on gut peptides involved in
control of glycemia and stomach emptying (45).
There are many reports of the effect of whey protein in large amounts (40-60 g),
consumed alone in beverage form on reducing subsequent food intake (41-43),or when
consumed with CHO on reducing glycemic response (44-46). However, there is only one recent
report suggesting that consuming whey protein alone prior to a meal may be beneficial to
glycemic control (137). Consumption of 55 g whey protein in a soup before a serving of mashed
potato with added glucose (total of 59 g CHO) reduced glycemic excursions in patients with
T2D (137). However, the relationship between the dose of whey protein consumed before a meal
of usual quantities and CHO content on efficacy for reducing food intake and post-meal
glycemic and insulin response in either healthy individuals or patients with T2D has not been
reported.
56
Therefore, the objective of these two experiments was to determine the relationships
among whey protein dose, or its hydrolysate, on satiety, food intake, and pre- and post-meal
glycemic and insulin responses in healthy subject when consumed in beverages 30 min before an
ad libitum pizza meal (experiment 1) or before a preset meal of a fixed quantity (experiment 2).
5.3. Research Methods and Procedures
5.3.1. Participants
Healthy individuals participated in the experiments (experiment 1: 16 men and
experiment 2: 12 men and 10 women). Participants were of normal-weight, characterized by a
BMI between 18-24.9 kg/m², and aged 20-27 yr old. They were recruited through advertisements
posted on the campus of University of Toronto. Breakfast skippers, smokers, dieters or
individuals with diabetes (fasting blood glucose ≥ 7.0 mmol/L) or other metabolic diseases were
excluded. Restrained eaters identified by a score of 11 or higher on the Eating Habits
Questionnaire (235) and those taking medication were also excluded. Upon completion of the
study and analysis of the blood samples, one woman participant was found to be
hyperinsulinemic and her data were excluded from the study. The sample size required was
based on a previous short-term food intake study on protein (42). Participants were financially
compensated for completing the studies. Procedures of the studies were approved by the Human
Subject Review Committee, University of Toronto Ethics Review Office.
5.3.2. Preloads
Pre-meal treatments were control and 10, 20, 30 and 40 g whey protein (NZMP Whey
Protein Concentrate 392, Fonterra Co- operative Group Limited, New Zealand) in experiment 1,
and control and 5, 10, 20 and 40 g whey protein and 10 g whey protein hydrolysate (WPH)
(Hilmar 8350, Hilmar Ingredients, CA, USA) in experiment 2. The whey protein (both
experiments) and WPH (experiment 2) powders contained approximately 80% protein, 5%
lactose, 6% fat, 4% ash and 4% moisture (Appendix 5). Therefore for example, to provide the
equivalent of 10 g protein, 12.5 g either whey protein or WPH was added to the treatment, and
provided approximately 50 kcal energy. Based on the observation that a branched chain amino
57
acid mixture did not result in similar effects on the release of gut peptides as intact protein (45), a
WPH was included in experiment 2 to further explore the role of intact protein, compared with
hydrolyzed protein, on pre- and post-meal glycemic control. The WPH was prepared by
enzymatic hydrolysis and, as reported by the manufacturer, resulted in a distribution profile of
approximately 40% free amino acids and short peptides of up to 10 amino acids, 27% of peptides
of 10-50 amino acids, 16% with 50-200 amino acids and 17% larger than 200 amino acid chains.
All preloads were iso-volumetric (300 mL) and served chilled in beverage form. Iso-
volumetric flavoured water was used as a control in both experiments. The addition of orange
flavored energy-free sweetener (1 g Kool-Aid, Kraft Canada Inc. North York, ON) equalized the
palatability of the preloads, as judged by a test panel of 12 subjects. An additional 100 mL of
water was provided to participants after consumption of the preload to reduce after-taste of the
protein preloads.
5.3.3. Protocol
The experiment protocol was similar to previously published procedures (142). A
standard breakfast (300 kcal) consisted of a single serving of a ready-to-eat breakfast cereal
(Honey Nut Cheerios, General Mills, Mississauga, ON), a 250 mL box of 2% milk (Sealtest
Skim Milk, Markham, ON) and a 250 mL box of orange juice (Tropicana Products Inc.
Bradenton, Florida). Breakfasts were given to subjects to be consumed at their preferred time in
the morning (6:00 - 9:00 A.M.) after a 10 h overnight fast, and were asked not to consume
anything between the breakfast and study session 4 h later (10:00 A.M. to 1:00 P.M.), except
water until 1 h before the session. Each participant was scheduled to arrive at the same time for
each treatment in the Department of Nutritional Sciences at University of Toronto and instructed
to refrain from alcohol consumption and any unusual exercise and activity the night before the
study sessions. Because impaired insulin sensitivity has been observed following an oral glucose
tolerance test in the luteal phase of the menstrual cycle in healthy women (247), women
participants were scheduled for the sessions during the follicular phase.
Upon their arrival, participants completed Visual Analogue Scale (VAS) questionnaires
asking about Sleep Habits, Stress Factors, Food Intake and Activity Level, Feelings of Fatigue
58
and Motivation to Eat. A composite score of the four appetite questions in Motivation to Eat
VAS was calculated to obtain the average appetite score (142, 248) for statistical analysis.
A baseline capillary blood sample was taken by finger prick to measure glucose and
insulin. Blood samples were obtained by a Monojector Lancet Devices (Sherwood Medical, St.
Louis, MO, U.S.A.). Concentrations of blood glucose that correspond to the plasma level were
measured by a glucose meter (Accu-Chek Compact, Roche Diagnostics Canada, Laval, Quebec).
In experiment 2, following measurement of blood glucose, 300 µl of capillary blood was
collected into Microvette 300 blood collection tubes (Sarstedt, Nümbrecht, Germany). Insulin
was measured by enzyme-immunoassay (Insulin EIA, Alpco Diagnostics, Salem, NH).
Men participants were provided the preloads in random order once per week in the first
experiment and twice weekly in the second experiment. The women in the second experiment
were studied twice a week in the first two weeks of their menstrual cycle. They were instructed
to drink the preload within 5 min with a constant pace. Following consumption, palatability,
taste, and texture of the preloads were measured by VAS. Subjective appetite and blood glucose
were measured at 15 and 30 min from the time when subjects started drinking the treatments.
Participants were asked to remain seated throughout the experimental session and were allowed
to read or listen to music.
At 30 min, participants were fed either an ad libitum pizza meal (McCain Foods Ltd.
Florenceville, NB) in experiment 1 (248), or a fixed quantity of pizza based on 12 kcal/kg of
body weight for subjects in experiment 2, with a bottle of spring water (500 mL Crystal Springs,
Mississauga, Canada). In experiment 1, subjects were provided three varieties of pizza (Deluxe,
Pepperoni and Three Cheese) based on their preferences and asked to eat until they were
“comfortably full” and allowed 20 min to eat. In experiment 2, they were provided only the
Deluxe variety and were asked to consume all of the food in 20 min. Immediately following the
meal at 50 min, and again at 65, 80 and 95 min (in both experiments) and 110, 140 and 170 min
(in experiment 2), subjective appetite and blood glucose were measured. In experiment 2, insulin
was also measured at 0, 30, 50, 95, 110, 140 and 170 min.
Detailed information of the nutrient content of the pizza and method of cooking has been
reported previously (142, 248). Test meal consumption was calculated from the weight of the
59
consumed pizza based on the compositional information provided by the manufacturer. Water
intake was measured by weight (g).
Cumulative energy intake was calculated by adding the energy consumed from the whey
preload to the energy consumed at the test meal (142). Caloric compensation, expressed as
percent, was calculated by subtracting the calories consumed after the whey preload from that
after the water control, divided by the calories in the whey preload and multiplied by 100.
Caloric compensations of < 100% indicate that the subject had low compensation for the whey
preload energy at the test meal, whereas scores > 100% indicate overcompensation for the whey
preload
The ratios of blood glucose/insulin concentration (mmol • L
energy at the test meal.
-1/µiU • mL-1) and cumulative
incremental area under the curve (AUC)(mmol • min • L-1/µiU • min • mL-1
249
) were calculated to
provide an evaluation of the efficacy of insulin action as previously used to identify the
relationship between glucose and insulin ratios post-meal ( ). The lower the ratio, the higher
the efficacy of insulin action (250).
5.3.4. Statistical Analysis
All analyses were conducted using SAS version 9.1 (SAS Institute Inc., Carey, NC). Two
and three-way repeated measures analysis of variance (ANOVA; via Proc Mixed procedure)
were performed to analyze the effects of time, sex, preload and their interaction on outcome
variables measured over the study period including average appetite scores, blood glucose and
insulin responses. When a preload and time interaction was statistically significant, one-way
ANOVA using Proc Mixed procedure was followed by Tukey’s post-hoc test to investigate the
effect of preload on absolute and changes from baseline for blood glucose and insulin at each
time of measurement. Pre-meal changes from baseline were calculated from 0 min (immediately
before preload consumption) and post-meal changes from 30 min (before meal consumption).
The effect of preload on food intake, cumulative energy intake and caloric compensation
in experiment 1 and on pre-meal, post-meal and AUC (251) for appetite and blood glucose (in
both experiments) and insulin (in experiment 2) were tested by one-way ANOVA (Proc Mixed
procedure), followed by Tukey’s post-hoc test to identify differences among preloads.
60
Pearson’s Correlation Coefficients were used to detect associations among dependent
measures. Significance was set at P < 0.05. Data are presented as mean ± standard error of the
mean (SEM).
5.4. Results
5.4. 1. Participant Characteristics
In experiment 1, men participants (n = 16) had a mean age of 22.3 ± 0.6 y, body weight
of 69.5 ± 1.6 kg, height of 1.8 ± 0.0 m and BMI of 22.6 ± 0.4 kg/m2. In experiment 2, men (n =
12) and women (n = 9) with a mean age of 21.8 ± 0.6 and 21.8 ± 0.9 y, body weight of 69.7 ± 2.1
and 59.9 ± 2.2 kg, height of 1.8 ± 0.0 and 1.67 ± 0.0 m and BMI of 22.1 ± 0.5 and 21.4 ± 0.5
kg/m2
5.4. 2. Food and Water Intake
, completed the study, respectively.
In experiment 1, treatment affected food intake (P < 0.0001) and all doses of whey
protein, except 10 g, significantly suppressed food intake at 30 min, with the lowest food intake
after 40 g whey protein, compared to control (Table 5.1). Cumulative energy intake from the
preload and pizza meal, caloric compensation and water intake were not significantly different
among the treatments.
5.4. 3. Subjective Average Appetite Score
In experiment 1, average appetite was affected by time (P < 0.0001), being suppressed
more at 15 min after the preloads than at 30 min, and at 50 min (immediately after the meal)
compared to 80 and 95 min. However, average appetite was not affected by preload or time and
preload interaction. Mean average appetite score was 69.6 ± 2.0 and 63.6 ± 1.9 mm at 0 and 30
min, respectively (Figure 5.1).
In experiment 2, average appetite was affected by preload (P < 0.01) and sex (P <
0.0001) .When expressed as mean concentration over all measured times, men had higher
subjective average appetite scores than women (40.2 ± 1.0 vs. 22.9 ± 1.0 mm, two-way ANOVA,
61
P < 0.0001). However, average appetite was not affected by a preload by sex interaction. Thus,
the data are shown as pooled for the sexes.
Mean average appetite score was 64.5 ± 2.1 and 63.8 ± 1.8 mm at 0 and 30 min,
respectively. Average appetite response was affected by time (P < 0.0001), being reduced at 15
min after all preloads compared with 0 min or 30 min, and markedly reduced at 50 min
(immediately after the meal) (Figure 5.6). Preload was a factor (P < 0.0001) with the 20 g and 40
g whey protein preload suppressing average appetite more than the control. However, there was
no time and preload interaction.
5.4. 4. Subjective Average Appetite AUC
In experiment 1, there was no difference among preloads in pre-meal average appetite
AUC (0-30 min) (Figure 5.7). However, post-meal average appetite AUC (30-95 min) was
suppressed less following the 30 and 40 g whey protein compared to control, likely due to the
lower food intake after these preloads (P < 0.01).
In experiment 2, no differences were found in the pre-meal AUC due to treatments. As
expected, due to consumption of the preset meal, post-meal subjective average appetite AUCs
were not affected by the pre-meal treatments (Figure 5.8).
5.4. 5. Blood Glucose Concentration
In both experiments, the baseline data are reported followed by an analysis of the change
from baselines (0 min for pre-meal and 30 min for post-meal responses) because in experiment 2
differences in baseline blood glucose concentration among treatments were observed (P < 0.05).
In experiment 1, blood glucose response was affected by time (P < 0.0001), preload (P <
0.0001) and an interaction between time and preload (P < 0.0001).
At 0 and 30 min, overall mean blood glucose concentrations with both sexes combined were 5.0
± 0.0 and 5.2 ± 0.0 mmol/L, respectively (Table 5.2). At 30 min, the 20, 30 and 40 g whey
resulted in lower blood glucose concentrations compared to the control (P < 0.01), but no
difference was detected among the doses. Post-test meal blood glucose was lower at 50 and 65
62
min after all whey protein doses compared to the control. This effect was sustained at 80 and 95
min for all except the 10 g doses (P < 0.0001).
In experiment 2, blood glucose response was affected by time (P < 0.0001), preload (P <
0.0001) and an interaction between time and preload (P < 0.0001). There was no effect of sex or
sex with preload interaction on blood glucose response (two-way ANOVA). Therefore, the
results shown for blood glucose are the pooled data for the sexes.
At 0 and 30 min, overall mean blood glucose concentrations with both sexes combined
were 4.9 ± 0.0 and 4.9 ± 0.0 mmol/L, respectively (Table 5.3). At 15 min, 10 g WPH and at 30
min, 10 g WPH and 40 g whey protein resulted in higher blood glucose response than the control
(P < 0.001).
Post-meal blood glucose, expressed as change from 30 min, was reduced by the whey
protein preloads, in a dose-dependent manner (Table 5.3). From 50 to 95 min, 20 and 40 g and at
110 min, 40 g whey protein resulted in lower post-meal blood glucose (P < 0.001). Compared to
the control, 10 g of whey protein reduced post-meal blood glucose at 65, 80 and 170 min, but 10
g WPH reduced the post-meal blood glucose increase only at 65 min (P < 0.05). There was no
difference in blood glucose among the preloads at 140 min
5.4. 6. Blood Glucose AUC
In experiment 1, 20 and 30 g whey protein, with no difference among the whey doses, led
to a small, but significantly higher pre-meal blood glucose AUC (0-30 min) compared to the
control (P = 0.01) (Figure 5.1A). Post-meal blood glucose AUC (30-95 min) was reduced by all
whey protein preloads (Figure 5.1). Among the whey protein preloads, 10 and 40 g resulted in
the highest and lowest post-meal blood glucose AUCs (P < 0.0001), respectively, with the 20
and 30 g whey protein preloads resulting in intermediate AUCs. Compared to the control,
cumulative blood glucose AUC (0-95 min) was reduced by all whey protein doses with the
lowest after 40 g (P < 0.0001) (Figure 5.1B).
In experiment 2, 20 and 40 g whey protein and 10 g WPH resulted in higher pre-meal
blood glucose AUC than after 10 g whey or the control (P < 0.01) (Figure 5.2A). Pre-meal blood
63
glucose AUC after the 5 g whey protein preload did not differ from any other preloads. Post-
meal blood glucose AUC was reduced by 10, 20 and 40 g whey protein (P < 0.0001), but not by
5 g whey protein or 10 g WPH, compared with the control (Figure 5.2).
Cumulative blood glucose AUC (0-170 min) was lower after the 10, 20 and 40 g whey
protein preloads compared to 5 g whey protein and 10 g WPH and the control (P < 0.0001) with
no difference among the latter (Figure 5.3A).
5.4. 7. Insulin
In experiment 2, insulin was affected by time (P < 0.0001), preload (P < 0.01) and an
interaction between time and preload (P < 0.01). Insulin was affected by sex (P < 0.002). When
expressed as mean concentration over all measured times, women had higher capillary insulin
concentrations than men (29.5 vs. 17.4 µIU/ mL, two-way ANOVA, P < 0. 002). However, there
was no interaction between the effect of sex and preload. Therefore, the pooled data are
presented for men and women.
There was a trend for a significant difference between the preloads at baseline insulin
concentration (P = 0.07). Therefore, the baseline data are reported followed by an analysis of the
change from baselines.
At 0 and 30 min, overall mean insulin concentration with both sexes combined was 4.5 ±
0.2 and 11.8 ± 0.8 µIU/ mL, respectively (Table 5.4). All whey doses, except 5 g, increased
insulin response to 30 min, immediately prior to the meal, compared to control (P < 0.0001),
with the highest response after 20 and 40 g whey protein, followed by 10 g whey protein and 10
g WPH. At 50 min, right after the meal, 40 g whey protein resulted in less increase in insulin
compared to control (P < 0.05). From 80 to 170 min, all whey doses, except 5 g, reduced post-
meal insulin response (P < 0.0001).
5.4. 8. Insulin AUC
Pre-meal insulin AUC (0-30 min) was higher after all whey protein doses, except 5 g,
compared to the control (P < 0.0001) (Figure 5.2B). The 20 and 40 g whey protein preloads
64
resulted in the highest pre-meal insulin AUC, followed by 10 g WPH and 10 g whey protein (P <
0.0001). Conversely, post-meal insulin AUC (30-170 min) was reduced by all doses of whey
protein, except 5 g, compared to the control (P < 0.001) (Figure 5.2). There was no statistically
significant differences in cumulative insulin AUC (0-170 min) among the protein preloads or
control (Figure 5.3B).
5.4. 9. Relations among Dependent Measures
In experiment 1, food intake was not correlated with pre- or post-meal blood glucose
AUC or subjective average appetite AUC. Pre-meal blood glucose AUC was associated
inversely with post-meal blood glucose AUC (r = - 0.35, P = 0.002). Larger pre-meal and post-
meal blood glucose AUCs were associated with greater suppression of pre-meal (r = - 0.33, P =
0.003) and post-meal (r = -0.22, P < 0.05) average appetite AUCs. The greater suppression of
pre-meal average appetite AUC, the less suppression of post-meal average appetite AUC (r = -
0.30, P = 0.006).
In experiment 2, positive associations were found between pre-meal (r = 0.31, P < 0.001),
post-meal (r = 0.56, P < 0.0001) and cumulative (r = 0.35, P < 0.0001) blood glucose with
insulin AUCs. Pre-meal insulin AUC was inversely correlated with post meal blood glucose
AUC (r = - 0.42, P < 0.0001), but pre-meal blood glucose AUC was not associated with post-
meal blood glucose AUC (r = - 0.1, P = 0.1) or post-meal insulin AUC (r = - 0.7, P = 0.4). Pre-
meal insulin AUC (0-30 min) was inversely associated with post-meal insulin AUC (30-170
min) (r = - 0.22, P < 0.05). The greater suppression of post-meal and cumulative average appetite
AUCs was associated with the greater post-meal blood glucose AUC (r = - 0.22, P < 0.05) and
cumulative blood glucose AUC (r = - 0.20, P < 0.05), respectively. Post-meal and cumulative
average appetite AUCs and insulin AUCs were not associated.
An inverse association was found between the dose of whey protein and ratios of
cumulative blood glucose /insulin AUC (r = - 0.33, P < 0.001) (Figure 5.4) and change from 30
min blood glucose /insulin response at 80 min (r = - 0.41, P < 0.0001) (Figure 5.5).
65
5.5. Discussion
These studies are the first to evaluate the effect of consuming whey protein alone prior to
a meal on blood glucose and insulin responses in healthy young adults. The results of these
studies show that whey protein, in relatively small amounts and when consumed prior to a meal,
reduces food intake and post-meal blood glucose while reducing post-meal insulin response.
Thus, the effects of pre-meal intact whey protein, but not WPH, on post-meal blood glucose
control are not explained in full by its insulinotropic action. Furthermore, because WPH did not
reduce post-meal blood glucose response, it may be suggested that non-insulintropic mechanisms
require stimulation arising from the digestion of intact proteins.
The preload doses were given 30 min before the meal based on the peak insulin response
time after protein and CHO loads (41, 80). Thus it was anticipated that responses in food intake,
blood glucose and insulin would be observed at lower doses than used previously. In experiment
2, a fixed size meal was fed with the objective of isolating the effect of whey protein and WPH
on blood glucose control by insulin, independent of variations in food intake. However, it is clear
that the benefit of pre-meal consumption of whey protein in ad libitum eating patterns resides in
its effect on reducing both food intake and post-meal glycemic and insulin responses.
Because plasma insulin increased similarly after 10 g of both whey protein and WPH
preloads (Table 5.3), the role of branched-chain amino acids in stimulating insulin release and
secretion is supported (44). However, four lines of evidence suggest that insulin alone, as
indicated by plasma insulin concentrations, cannot be the only cause of the lower post-meal
blood glucose after pre-meal consumption of intact whey protein. First, the lower post-meal
blood glucose with increasing doses of whey protein in both experiments (Figures 5.1A and
5.2A) was achieved in the presence of a lower, not higher, post-meal insulin AUC (Figure 5.2B)
and a similar cumulative (0-170 min) insulin AUC (Figures 5.3B) in experiment 2. Second, when
the cumulative AUC for blood glucose was divided by the cumulative AUC for insulin to
evaluate the efficacy of insulin action (249, 250), the ratio was decreased, in a dose-dependent
manner, to 50% of the control after pre-meal consumption of intact whey protein of 40 g (Figure
5.4). Thus, it is clear that lower blood glucose occurred without an overall increase in insulin
requirement. Third, the ratio of blood glucose to insulin response at 80 min was also decreased in
66
a dose-dependent manner to less than 25% of the control (Figure 5.5). Fourth, in contrast to 10 g
of intact protein, WPH at 10 g did not result in a lower cumulative blood glucose than the
control even though it increased the post-meal (Figure 5.2B) and cumulative insulin (Figure
5.3B) AUC similarly.
Although the mechanism by which pre-meal whey protein brings about improved post-
meal glucose control is unclear, the most probable explanation for the insulin-independent
actions of pre-meal consumption of whey protein on blood glucose control resides in the effect
of protein on gastric emptying. Even a modest change in gastric emptying rate affects the
magnitude and timing of postprandial blood glucose and insulin increase (252, 253) and is
decreased by protein ingestion consumed either with CHO (218) or alone (41). Reduced stomach
emptying is suggested in the present study by the lower peak blood glucose at 80 min (30 min
after the meal), after all protein preloads, but the peak rise in blood glucose was much more
attenuated after the 20, 30 and 40 g of whey protein and continued to rise until 140 min (90 min
post-meal), at which time blood glucose concentrations were not different among the treatments
(Table 5.3). Furthermore, slower stomach emptying would be expected because whey protein
and other proteins release cholecystokinin (CCK), glucagon-like peptide 1 (GLP-1) (41),
glucose-dependent insulinotropic polypeptide (GIP) (45) and peptide tyrosine tyrosine (PYY)
from the intestinal enteroendocrine cells (82, 137, 218). The release of these hormones may be
an explanation for the lack of effect of 10 g whey protein hydrolysate on blood glucose, but not
intact whey protein because a branched-chain amino acid mixture produces the effect of the
intact whey protein on insulin but not on gut hormones, including CCK and GLP-1 (45).
The dose of whey protein required to suppress food intake when consumed 30 min prior
to the meal was shown to be in the 20-40 g range in experiment 1 based on a sample size of 16
individuals. However, based on a recalculation of sample size using the variability found in this
study, the reduction of 78 kcal after 10 g whey protein may have been found to be statistically
significant with a sample size of 40 subjects.
The efficacious dose of pre-meal whey protein required to affect insulin and post-meal
glycemic response in healthy young adults was found to be as low as 10 g and possibly 5 g.
Power calculations suggest that an increased sample size of 40 subjects would detect an effect of
5 g whey with a power of 0.8. The efficacy of consumption of 55 g whey protein prior to a meal
67
in controlled type II diabetic patients has been confirmed (137), and suggested to be comparable
to the effect of pharmacological therapy such as sulphonylureas on reduction of postprandial
glycemia, but the subjects were provided only 59 g of CHO at the meal. In our study, the men
and women averaged 103 and 82 g of CHO intake at the fixed size pizza meal, suggesting that
pre-meal administration of whey protein in relatively small amounts is very efficacious in
contributing to glycemic control in healthy subjects. It remains to be determined, however, if
these small doses have a benefit in patients with T2D.
In both experiments, an inverse association was found between post-meal blood glucose
and average appetite AUCs (experiment 1, r = -0.22; experiment 2, r = -0.21, P < 0.05),
suggesting that, independent of either pre-meal treatment or the amount of CHO consumed
(experiment 2), the higher the post-meal blood glucose, the greater the suppression of average
appetite, as observed in previous studies (142). In experiment 2, there was no significant
association between insulin and average appetite AUCs suggesting that, although in the short
term insulin acts as a satiety hormone, blood glucose was a better predictor of satiety (254).
5.6. Conclusion
In conclusion, whey protein consumed prior to a meal reduces food intake and post-meal
blood glucose and insulin and the ratio of the cumulative blood glucose /insulin AUC in a dose-
dependent manner. Intact whey protein, but not WPH, contributes to blood glucose control by
both insulin-dependent and insulin-independent mechanisms. Thus, pre-meal ingestion of whey
protein may be an effective strategy for achieving blood glucose control in healthy and insulin-
resistant humans.
68
Table 5. 1. Experiment 1: effect of pre-meal whey protein on energy intake, cumulative energy
intake, caloric compensation and water intake
Whey Protein
1
Preload
Energy Intake Caloric
Compensation4
%
Water Intake
g
Test Meal2 Cumulative3
kcal
Control5 1142 ± 59a 1142 ± 59 363 ± 40
10 g 1064 ± 55ab 1115 ± 55 153 ± 87 363 ± 34
20 g 989 ± 71b 1091 ± 71 150 ± 59 367 ± 28
30 g 983 ± 50b 1136 ± 50 104 ± 30 351 ± 33
40 g 837 ± 41c 1041 ± 41 150 ± 23 343 ± 33
P* <0.0001 NS NS NS
1 All values are means ± SEMs; n = 16, Values in the same column with different superscript
letters are significantly different, P < 0.0001 (one-factor ANOVA for preload effect followed by
Tukey’s post hoc test) 2 Energy consumed in an ad libitum meal 30 min after the preloads. 3 Energy in preloads + energy from the meal 4 Caloric compensation = [(kcal consumed at the meal after the water control - kcal consumed at
the meal after the whey protein preload)/kcal in the whey protein preload] x 100 5 Water (300 mL)
69
Table 5. 2. Experiment 1: effect of pre-meal whey protein on pre- and post-meal blood glucose
response1
Time Control2 Whey Protein P*
10 g 20 g 30 g 40 g
mmol/L
Absolute concentration
0 min3 5.0 ± 0.1 5.0 ± 0.1 5.2 ± 0.1 5.0 ± 0.1 5.0 ± 0.1 NS
Change from 0 min
15 min 0.0 ± 0.0 0.1 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.1 ± 0.1 NS
30 min 0.0 ± 0.1b 0.1 ± 0.1ab 0.3 ± 0.1a 0.3 ± 0.1a 0.2 ± 0.1a <0.01
Absolute concentration
30 min4 5.0 ± 0.1c 5.2 ± 0.1bc 5.5 ± 0.1a 5.3 ± 0.1ab 5.3 ± 0.1abc <0.001
Change from 30 min
50 min 2.0 ± 0.2a 0.9 ± 0.2b 0.3 ± 0.2c 0.2 ± 0.2c -0.2 ± 0.1c <0.0001
65 min 2.5 ± 0.2a 1.5 ± 0.2b 0.7 ± 0.2c 0.7 ± 0.2c 0.1 ± 0.1c <0.0001
80 min 1.9 ± 0.3a 1.6 ± 0.2a 0.8 ± 0.2b 0.8 ± 0.2b 0.3 ± 0.1b <0.0001
95 min 1.6 ± 0.2a 1.3 ± 0.2ab 0.8 ± 0.2bc 1.0 ± 0.1bc 0.6 ± 0.2c <0.0001
1 All values are means ± SEMs; n = 16. Values at each time of measurement with different
superscript letters are significantly different [2-factor ANOVA, time-by-treatment interaction (P
< 0.0001), followed by one-factor ANOVA for preload effect and Tukey’s post hoc test (P
<0.05)] 2 Water control (300 mL) 3 Prior consumption of preloads (baseline) 4 Prior consumption of ad libitum pizza meal (30 min)
70
Table 5. 3. Experiment 2: effect of pre-meal whey protein on pre- and post-meal blood glucose
response1
Time Control2 Whey Protein P*
5 g 10 g
WPH3
10 g 20 g 40 g
Absolute concentration
0 min4 4.8 ± 0.1ab 4.8 ±
0.1ab
4.7 ±
0.1b
5.0 ±
0.1a
4.9 ±
0.1ab
4.9 ±
0.1ab
<0.05
Change from 0 min
15 min -0.0 ± 0.1b 0.1 ±
0.1ab
0.3 ±
0.1a
-0.1 ±
0.1b
0.2 ±
0.1ab
0.2 ±
0.1ab
<0.002
30 min -0.1 ± 0.1c 0.0 ±
0.1abc
0.2 ±
0.1a
-0.1 ±
0.1bc
0.2 ±
0.1abc
0.2 ±
0.1ab
<0.001
Absolute concentration
30 min5 4.7 ± 0.1c 4.8 ±
0.1bc
5.0 ±
0.1ab
4.9 ±
0.1ab
5.0 ±
0.1ab
5.1 ±
0.1a
<0.0001
Change from 30 min
50 min 0.6 ± 0.1a 0.5 ± 0.1a 0.4 ±
0.1ab
0.2 ±
0.1ab
0.0 ±
0.1bc
-0.3 ±
0.1c
<0.0001
65 min 2.6 ± 0.2a 2.4 ±
0.2ab
2.0 ±
0.1bc
1.6 ±
0.2c
0.7 ±
0.1d
0.1 ±
0.1e
<0.0001
80 min 27 ± 0.3a 2.4 ±
0.3ab
2.2 ±
0.2ab
1.8 ±
0.2b
1.0 ± 0.1c 0.3 ±
0.1d
<0.0001
95 min 2.3 ± 0.3a 1.9 ± 0.3a 2.0 ±
0.3a
1.6 ±
0.2ab
1.0 ±
0.1bc
0.5 ±0.2c <0.0001
110 min 2.0 ± 0.3a 1.6 ±
0.2ab
1.7 ±
0.3ab
1.4 ±
0.2abc
1.2 ±
0.1bc
0.8 ±
0.2c
<0.001
140 min 1.5 ± 0.2 1.6 ± 0.2 1.4 ±
0.2
1.3 ±
0.1
1.3 ± 0.1 1.1 ± 0.2 NS
170 min 1.4 ± 0.1a 1.0 ±
0.1ab
1.3 ±
0.1ab
1.0 ±
0.1b
1.0 ±
0.1ab
1.3
±0.2ab
0.01
1 All values are means ± SEMs; n = 21. Values at each time of measurement with different
superscript letters are significantly different [2-factor ANOVA, time-by-treatment interaction (P
< 0.0001), followed by one-factor ANOVA for preload effect and Tukey’s post hoc test (P <
0.05)] 2 Water control (300 mL) 3 Whey protein hydrolysate (10 g) 4 Prior consumption of
preloads (baseline) 5 Prior consumption of preset fixed pizza meal (30 min)
71
Table 5. 4. Experiment 2: effect of pre-meal whey protein on pre- and post-meal insulin
response1
Time Control2 Whey Protein P*
5 g 10 g
WPH3
10 g 20 g 40 g
Absolute concentration
0 min4 3.6 ± 0.5 4.7 ±
0.5
4.4 ±
0.5
4.5 ±
0.5
4.9 ±
0.5
5.2 ±
0.6
0.07
Change from 0 min
30 min 0.0 ±
0.3c
3.2 ±
0.7bc
6.3 ±
0.8b
7.2 ±
0.9b
12.4 ±
1.7a
14.5 ±
2.2a
<0.0001
Absolute concentration
30 min5 3.6 ±
0.5c
7.9 ±
0.8bc
10.7 ±
1.0b
11.7 ±
1.2b
17.3 ±
2.0a
19.7 ±
2.3a
<0.0001
Change from 30 min
50 min 19.0 ±
3.7a
12.4 ±
3.1ab
13.5 ±
3.0ab
13.7 ±
3.1ab
12.7 ±
2.2ab
8.5 ±
2.4b
<0.05
80 min 38.8 ±
5.6a
30.2 ±
3.5ab
25.9 ±
3.8bc
23.2 ±
3.3bc
18.8 ±
3.7bc
17.0 ±
2.9c
<0.0001
110 min 31.3 ±
5.3a
23.9 ±
3.9ab
18.7 ±
3.3bc
16.0 ±
2.9bc
11.1 ±
2.6bc
13.3 ±
3.9c
<0.0001
140 min 23.5 ±
3.4a
19.6 ±
3.0a
12.1 ±
1.9b
11.5 ±
1.7b
8.3 ±
2.4b
11.9 ±
2.9b
<0.0001
170 min 20.7 ±
3.6a
13.3 ±
2.1ab
8.9 ±
1.4bc
8.8 ±
1.7bc
2.7 ±
1.9c
6.7 ±
2.2bc
<0.0001
1 All values are means ± SEMs; n = 21. Values at each time of measurement with different
superscript letters are significantly different [2-factor ANOVA, time-by-treatment interaction (P
< 0.01), followed by one-factor ANOVA for preload effect and Tukey’s post hoc test (P <
0.05)], 2 Water control (300 mL), 3 Whey protein hydrolysate (10 g), 4 Prior consumption of
preloads (baseline),5 Prior consumption of preset fixed pizza meal (30 min)
72
Figure 5. 1. Experiment 1: pre-meal, post-meal and cumulative blood glucose AUC after whey
protein preloads
Mean (± SEM) blood glucose AUC (mmol • min/L) after the whey protein preload consumption
was calculated for pre-meal (0- 30 min) and post-meal (30-95 min) (A) and cumulative (0-95
min) (B) (n = 16). One-factor repeated-measures ANOVA followed by Tukey’s post hoc was
used to compare the effect of preloads (means with different superscripts at each time are
different, P < 0.0001).
73
Figure 5. 2. Experiment 2: pre-meal and post-meal blood glucose and insulin AUCs after whey
protein preloads
Mean (± SEM) blood glucose AUC (mmol • min/L) (A) and insulin AUC (µiU • min/ mL) (B)
after the whey protein and whey protein hydrolysate (WPH) preload consumption were
calculated for pre-meal (0-30 min) and post-meal (30-170 min) (n = 21). One-factor repeated-
measures ANOVA followed by Tukey’s post hoc was used to compare the effect of preloads
(means with different superscripts at pre- and post-meal are different, P < 0.05).
74
Figure 5. 3. Experiment 2: cumulative blood glucose and insulin AUCs after whey protein
preloads
Mean (± SEM) cumulative (0-170 min) blood glucose AUC (mmol • min/L) (A) and insulin
AUC (µiU • min/mL) (B) after the whey protein and whey protein hydrolysate (WPH) preload
consumption (n = 21). One-factor repeated-measures ANOVA followed by Tukey’s post hoc
was used to compare the effect of preloads (means with different superscripts at pre- and post-
meal are different, P < 0.001).
75
Figure 5. 4. Experiment 2: ratio of cumulative blood glucose/insulin AUC safter whey protein
preloads
Mean (± SEM) ratio of cumulative blood glucose/insulin AUCs (mmol • min • L-1/µiU • min •
mL-1) after the whey protein and whey protein hydrolysate (WPH) preload consumption (A)(n =
21). One-factor repeated-measures ANOVA followed by Tukey’s post hoc was used to compare
the effect of preloads (mean with different superscripts are different, P < 0.05). Association
determined by Pearson’s Correlation Coefficients (r = -0.33, P < 0.001) between whey protein
doses (g) and ratio of cumulative (0-170 min) blood glucose/ insulin AUC (mmol • min • L-1/µiU
• min • mL-1) after the preload consumption (B)
76
Figure 5. 5. Experiment 2: ratio of blood glucose/insulin concentration after whey protein
preloads
Mean (± SEM) ratio of blood glucose/insulin concentration (mmol • L-1/µiU • mL-1) at 80 min
after the whey protein and whey protein hydrolysate (WPH) preload consumption (30 min after
the meal)(A) (n = 21). One-factor repeated-measures ANOVA followed by Tukey’s post hoc was
used to compare the effect of preloads (mean with different superscripts are different, P < 0.05).
Association determined by Pearson’s Correlation Coefficients (r = -0.41, P < 0.0001) between
whey protein dose (g) and the ratio of blood glucose/insulin concentration (mmol • L-1/µiU • mL-
1) at 80 min after the preload consumption (B).
77
Figure 5. 6. Average appetite scores after whey protein preloads
ΔA
vera
ge A
ppet
ite (m
m)
A
B
Time (min)
Ad libitum Meal
-60-50-40-30-20-10
010
0 15 30 50 65 80 95
control10 g 20 g 30 g40 g
-60
-50
-40
-30
-20
-10
0
100 15 30 50 65 80 95 110 140 170
control5 g10 g WPH10 g 20 g 40 g
dd c
Preset Meal
Mean (± SEM) change from baselines average appetite scores measured by visual analog scales
after consumption of whey preloads, 10 g (—□—), 20 g (—◊—), 30 g (--♦--), 40 g (—○—), and
water (····○····) in experiment 1 (A) and 5 g (--Δ--), 0 g (—□—), 20 g (—◊—), 40 g (—○—)
and 10 g whey protein hydrolysate (---X---) and water (····○····) in experiment 2 (B). Average
appetite score in experiment 1 (n = 16) at 0 min was 69.6 ± 2.0 and at 30 min was 63.6 ± 1.9 mm
and in experiment 2 (n = 21) was 64.5 ± 2.1 and 63.8 ± 1.8 mm, respectively. Preload (P < 0.001,
only in experiment 2) and time (P < 0.0001), with no interaction, affected average appetite scores
in both experiments (Two-factor repeated-measures ANOVA).
78
Figure 5. 7. Experiment 1: average appetite AUC after whey protein preloads
Pre-Meal (0-30 min)
Ave
rage
App
etite
AU
C
(mm
* m
in)
-3500-3000-2500-2000-1500-1000-500
0control 10 g 20 g 30 g 40 g control 10 g 20 g 30 g 40 g
b
ab aba a
Post-Meal (30-95 min)
Mean (± SEM) subjective average appetite AUC (mm • min) after the whey protein preload
consumption was calculated for pre-meal (0-30 min) and post-meal (30-95 min)(n = 16). One-
factor repeated-measures ANOVA followed by Tukey’s post hoc was used to compare the effect
of treatments. Different superscripts at post-meal are different between treatments, P < 0.05.
79
Figure 5. 8. Experiment 2: average appetite scores after whey protein preloads
-
-7000-6000-5000-4000-3000-2000-1000
0control 5 g
10 gWPH 10 g 20 g 40 g control 5 g 10 g H 10 g 20 g 40 g
Ave
rage
App
etite
AU
C(m
m *
min
)Pre-Meal (0-30 min) Post-Meal (30-170 min)
Mean (±SEM) subjective average appetite AUC (mm • min) after the whey protein and whey
protein hydrolysate (WPH) preload consumption was calculated for pre-meal (0-30 min) and
post-meal (30-170 min)(n = 21). There was no significant difference in pre- and post-meal
average appetite AUC between treatments (One-factor repeated-measures ANOVA followed by
Tukey’s post hoc).
80
CHAPTER 6
MECHANISM OF ACTION OF PRE-MEAL CONSUMPTION OF
WHEY PROTEIN ON GLYCEMIC CONTROL IN YOUNG ADULTS
81
CHAPTER 6. MECHANISM OF ACTION OF PRE-MEAL
CONSUMPTION OF WHEY PROTEIN ON GLYCEMIC CONTROL
IN YOUNG ADULTS
Preface:
To address the hypothesis that whey protein consumed prior to a meal results in post-
meal glucose control by both insulin and insulin-independent glycemic control mechanisms
This work has not been published.
82
6.1. Abstract
Background: Whey protein (WP), when consumed in small amounts prior to a meal, improves
post-meal glycemic control more than can be explained by insulin-dependent mechanisms (222).
Objective: The mechanism of action of WP on the reduction of post-meal glycemic response
was explored.
Design: In a randomized crossover study, healthy young men received iso-volumetric preloads
(300 mL) of WP (10 and 20 g), glucose (10 and 20 g) or water control. Paracetamol (1.5 g) was
added to the preloads to measure gastric emptying. Plasma concentrations of paracetamol,
glucose, and secreted ß-cell and gastrointestinal hormones were measured before preloads
(baseline) and at intervals before (0-30 min) and after (50-230 min) a preset pizza meal (12
kcal/kg).
Results: Whey protein slowed pre-meal gastric emptying rate compared to the control and 10 g
glucose (P < 0.0001), and increased insulin and C-peptide less than the glucose preloads (P <
0.0001). Glucose, but not WP, increased pre-meal plasma glucose concentrations (P < 0.0001).
Both WP and glucose similarly reduced post-meal glycemia (P = 0.0006) and increased CCK (P
< 0.0001). However, compared with glucose, WP reduced post-meal glycemia despite lower
post-meal insulin secretion and concentrations. This may be due to increased efficacy of insulin
without enhanced secretion, elevated GLP-1 and PYY concentrations, and delayed gastric
emptying.
Conclusion: Pre-meal consumption of WP lowers post-meal glycemia by both insulin-dependent
and insulin-independent mechanisms.
83
6.2. Introduction
When consumed with carbohydrate (CHO), proteins in general (124, 218) and, milk
proteins specifically (46, 130) reduce glycemic response compared with CHO alone. Whey
protein (WP), which accounts for 20% of cow’s milk proteins, is insulinotropic (17, 18). This is
likely due to its rapid digestion (80), resulting in high amino acid bioavailability (197) and
increased plasma concentrations of the branched-chain amino acids (44), that stimulate insulin
secretion. The addition of 20-60 g WP (46, 136) or whey peptides (130) to a glucose drink or
CHO meal reduces glycemic response.
Several studies suggest that WP is more insulinotropic than the other proteins. A
breakfast and lunch, each containing 28 g of WP and 50 g CHO served to adults with T2D,
resulted in higher insulin concentrations after both meals, but lower blood glucose only after the
lunch than when WP was exchanged for lean ham (46). Similarly, 50 g WP in a meal lowered
glycemia compared to a similar amount of turkey or egg albumin, and induced higher insulin
concentrations over 240 min compared to a similar amount of turkey, tuna or egg albumin (103).
However, it is unclear if the insulinotropic effect of WP is the only mechanism by which
it reduces the glycemic response when consumed with CHO. Whey protein ingestion increases
blood concentrations of the gastrointestinal hormones, glucagon-like peptide-1 (GLP-1),
cholecystokinin (CCK), gastric inhibitory polypeptide (GIP), peptide tyrosine-tyrosine (PYY)
(136, 173), and decreases blood concentrations of ghrelin (173). Elevated GLP-1(255), PYY
(157) and CCK (221) concentrations inhibit gut motility and slow gastric emptying, which may
explain why WP consumption reduces post-meal glycemia without proportional increases in
insulin concentrations (43, 222).
Although protein decreases gastric emptying rate and improves glycemia when consumed
with either CHO (218) or alone (41, 80), or before a meal (137), the effect of pre-meals ingestion
of WP may be more efficacious than when consumed with meal (137, 222). Pre-meal
consumption of 55 g WP resulted in higher GLP-1 and delayed gastric emptying than when
consumed with the meal (137), and much smaller amounts (as low as 10 g) of WP consumed
prior to a meal reduced post-meal concentrations of glucose and insulin (222). The latter
suggests that insulin alone, as indicated by reduced plasma insulin concentrations, cannot be the
84
only cause of the lower post-meal blood glucose after pre-meal consumption of WP (222). Thus,
gut-induced gastric emptying has been proposed as the most probable explanation for the insulin-
independent action of WP on post-meal glycemic control (222). Furthermore, it has recently
been reported that 30 g WP, when consumed with 50 g CHO, improves glycemia through either
decreased hepatic insulin extraction or increased C-peptide clearance (136). Whether or not this
is a factor in determining the effect of pre-meal ingestion of WP on post-meal glycemia has not
determined.
Therefore, the hypothesis of this study was that WP consumed prior to a meal improves
post-meal glycemic control by both insulin-dependent and -independent mechanisms. The
objective of the study was to describe and compare the effect of WP and glucose consumed 30
min before a fixed meal in healthy men on pre- and post-meal gastric emptying rate and plasma
concentrations of pancreatic β-cell hormones, including insulin, C-peptide and amylin,
gastrointestinal hormones, including ghrelin, GIP, GLP-1, CCK and PYY, and the important fuel
substrates, including glucose, free fatty acids (FFA) and triglycerides (TG).
6.3. Participants and Research Methods
6.3.1. Participants
Participants were recruited through advertisement posted on the University of Toronto
campus. At initial contact by phone or e-mail, eligibility requirements were described to the
potential subjects and they were asked for their age, body weight, height, if they smoke or were
taking any medications. Breakfast skippers, smokers, dieters and individuals with diabetes or
other metabolic diseases were ineligible to participate in the study. Individuals who fulfilled
eligibility requirements were asked to come to the Department for a second screening to
complete questionnaires regarding food habits, food preference and dietary restraint (235) and to
read and sign the consent and information form. Their height and weight were measured to
calculate their body mass index (BMI). Qualified subjects were invited to participate in the
study. Subjects were financially compensated for completing the study. The procedures of the
study were approved by the Human Subject Review Committee, Ethics Review Office at the
University of Toronto.
85
Based on previous clinical studies on gut hormones with the sample size required for
blood glucose response (111, 130, 142), 10 male subjects, aged 18-29 years with a BMI between
18.5-24.9 kg/m2, were recruited and completed the sessions.
6.3.2. Protocol
Experimental sessions took place at the Department of Nutritional Sciences at University
of Toronto and subjects participated in the study twice per week. Similar to our previous study
(222), a standard breakfast (300 kcal) consisted of a single serving of a ready-to-eat breakfast
cereal (Honey Nut Cheerios; General Mills, Mississauga, Canada), a 250-mL box of 2% milk
(Sealtest Skim Milk, Markham, Canada) and a 250-mL box of orange juice (Tropicana Products
Inc, Bradenton, FL). Breakfasts were provided to subjects to be consumed at their preferred time
in the morning (0600–0900) after a 10-h overnight fast. Subjects were asked not to consume
anything between the breakfast and the study session 4 h later (1000 to 1300). Participants were
permitted to consume water until 1 h before the session. Each subject arrived at the same chosen
time for each session. They were instructed to refrain from alcohol consumption and any unusual
exercise and activity the night before the study sessions.
Upon arrival, subjects filled out the Sleep Habits and Stress Factors Questionnaire and
Food Intake and Activity Questionnaire forms. Visual Analogue Scales (VAS) questionnaires
were then completed to measure Physical Comfort and Fatigue/Energy (142, 248). If they
reported significant deviations from their usual patterns, they were rescheduled.
Following completion of the VAS questionnaires, an indwelling intravenous catheter was
inserted in the antecubital vein by a registered nurse to take a baseline blood sample.
Immediately thereafter, subjects drank one of the five preloads within 5 min, followed by
completion of a palatability VAS (at 5 min). Blood samples were collected at baseline (before
preload consumption), and at 10, 20 and 30 min (pre-meal) and 50, 60, 70, 80, 110, 140, 170,
200 and 230 min (post-meal).
Salivary cortisol was measured at baseline and 30 min time intervals pre- and post-meal
to assess stress and anxiety (256). To collect saliva, participants chewed on a roll-shaped
synthetic saliva collector for 1 min, which was placed in the Salivette tubes (Starstedt, Germany)
86
and centrifuged for 2 min at 2000 rpm. A clear, fluid sample was obtained for analysis of cortisol
by an enzyme immunoassay kit (Salimetrics LLC, PA).
To present variation in post-meal glycemic response among individual treatments as done
previously (222), subjects were served a preset fixed pizza meal (12 kcal/ kg body weight of
subjects) at 30 min and were allowed 15 min to eat. Subjects remained seated throughout the
experimental sessions and were allowed to read or listen to music.
6.3.3. Preloads
Pre-meal drinks included iso-volumetric amounts (300 mL) of 10 and 20 g intact WP
(NZMP Whey Protein Concentration 392, Fonterra Co- operative
6.3.4. Blood Parameters
Group Limited, New
Zealand), and 10 and 20 g glucose control (D-Glucose Monohydrate , Grain Process Enterprises
LTD. Scarborough, ON) and a water control. Paracetamol (1.5 g, Panadol; GlaxoSmithKline)
was also dissolved in each of the five preloads. Preloads were served chilled with an additional
100 mL of water (in a separate glass) to be consumed upon completion of the drinks to reduce
aftertaste. Lemon flavor (0.5 mL, Flavorganics, Newark, NJ), lemon juice (2 tsp, Equality; The
Great Atlantic and Pacific Company of Canada Ltd, Toronto, Canada) and sucralose (0.15 g,
McNeil Specialty Products Company, New Brunswick, NJ) were added to the drinks to equalize
palatability and sweetness and to blind the subjects to the preloads.
Blood was collected in 8.5 mL BD™ P800 tubes (BD Diagnostics, Franklin Lakes, NJ)
containing spray-dried K2EDTA anticoagulant and proprietary additives to prevent their
immediate proteolytic degradation for measurement of plasma concentrations of glucose,
paracetamol, TG, FFA, pancreatic β-cell hormones including insulin, C-peptide and amylin, the
gastric hormone ghrelin, and intestinal hormones including GLP-1, GIP, CCK and PYY. The
tubes were centrifuged at 1300 RCF for 20 min at 4°C
Plasma glucose was measured using the enzymatic hexokinase method (Roche
Diagnostic, Laval, QC, Canada). Insulin and C-peptide were assessed with
. Collected plasma samples were aliquoted
in Eppendorf tubes and stored at -70 C for analyses.
87
electrochemiluminescence assays “ECLIA” (Roche Diagnostic, Laval, QC, Canada). Free fatty
acids (mEq/L) were analyzed colorimetrically with the enzymatic assay (Wako Chemicals USA
Inc, Richmond, VA). These analyses were performed by the Pathology and Laboratory Medicine
at Mount Sinai Hospital (Toronto, ON, Canada). However, the remaining biomarkers were
measured in our laboratory at the Department of Nutritional Sciences, University of Toronto.
A commercially-available paracetamol (acetaminophen) enzymatic assay was used to
detect and quantify free paracetamol in human plasma (Cambridge Life Sciences, Ely,
Cambridge, UK). Triglycerides were measured by the enzymatic hydrolysis method of the TG by
lipase to glycerol and FFA (# 10010303, Cayman chemical company, Ann Arbor, MI). Human
active GLP-1 (# EGLP-35K), total
Plasma concentrations of glucose, insulin and C-peptide were measured at all sampling
times, however, due to the high cost of the kits and measurements, plasma concentrations of
hormones were measured at only baseline, 20 and 30 min (pre-meal) and 60, 80, 140 and 230
min (post-meal).
ghrelin (# EZGRT-89K), total GIP (# EZHGIP-54K), total
PYY (# EZHPYYT66K) and total amylin (# EZHAT-51K) were measured with ELISA kits
(Millipore, Billerica, MA). Human CCK was measured with enzyme immunoassay kit (# EIA-
CCK-1, RayBiotech Inc, Norcross, GA).
6.3.5. Meal
The primary objective of the study was to determine the mechanisms of pre-meal
consumption of WP on post-meal glycemic control, thus, similar to our previous study (222),
subjects were served a preset pizza meal with a bottled spring water (500 mL Crystal Springs) to
eliminate variability in food intake on post-meal glycemic response. Subjects were asked to
consume the pizza meal within 20 min. Pizza (5” diameter deluxe pizza, McCain Foods Ltd.
Florenceville, NB) was served at all sessions after cooking from frozen according to
manufacturer’s directions. Subjects completed a VAS questionnaire on the palatability of the
pizza after finishing the pizza meal.
6.3.6. Data Analysis and Calculation
88
A two-way repeated measures analysis of variance (ANOVA), using SAS Proc Mixed
model followed by Tukey’s post hoc test, was conducted on pre-meal (0-30 min), post-meal (50-
230 min) and total (0-230 min) plasma concentrations of the dependent measures to test for time
and preload effects, thus, pre-meal, post-meal and total means of concentrations were reported.
When an interaction was found, one-way ANOVA was performed to test for the effect of preload
at each time of sampling. Because there were no differences at baseline glucose, hormone and
paracetamol concentrations, only the absolute concentrations are reported. Significance was set
at P < 0.05. Data were presented as mean ± standard error of the mean (SEM).
Pre-hepatic insulin secretion was calculated from deconvolution of C-peptide
concentration by using ISEC computer program (257). The results are presented in Figure 6.6
and Appendix Table 11.4.
6.4. Results
6.4.1. Subjects
Ten subjects completed the study. Two were excluded from the analyses due to high
mean insulin (704 ± 341 vs. 197 ± 5 pmol/L) and C-peptide (2293 ± 797 vs. 1455 ± 26 pmol/L)
concentrations compared to other subjects both at baseline and throughout the sessions, most
possibly due to either consumption of food before starting the sessions, or physiological
differences such as hyperinsulinemia. Therefore, 8 healthy males with a mean age of 22.9 ± 1.2
y, body weight of 66.4 ± 2.9 kg, height of 1.7 ± 0.0 m, and BMI of 21.8 ± 0.6 kg/m2 completed
the study.
6.4.2. Plasma Glucose, Insulin, C-peptide and Amylin
Pre-meal, post-meal and total mean concentrations of plasma glucose, insulin, C-peptide
and amylin, followed by p-values for the preload, time and time by preload interaction effects are
shown in Table 6.1.
Pre-meal Mean Plasma Concentrations
89
Over the entire pre-meal period (0-30 min), mean plasma concentrations of glucose,
insulin, C-peptide and amylin were higher (P < 0.0001) after glucose preloads compared with
WP and the control (Table 6.1). Both doses of WP increased insulin and C-peptide, however
only the 20 g WP increased amylin concentrations compared with the control (P < 0.0001).
Pre-meal concentrations of glucose, insulin, C-peptide (P < 0.0001) and amylin (P =
0.002) were affected by a time by preload interaction (Table 6.1). Thus, one-way ANOVA at
each time showed that glucose, but not WP, preloads increased plasma glucose concentrations in
a dose-dependent manner (P < 0.0001) (Figure 6.1 A). Higher concentrations of insulin and C-
peptide after both doses of glucose were observed at 10 min (P < 0.05), 20 min (P < 0.0001) and
30 min (P < 0.0001) (Figure 6.1 B). After WP, insulin concentrations were increased only after
20 g WP (P < 0.0001), but C-peptide concentrations were higher after both doses of WP (P <
0.0001) at 20 and 30 min compared to the control, but less than the equal doses of glucose
(Figure 6.1 C). Amylin concentrations were higher after 20 g WP and glucose preloads than 10
g WP and the control (Table 6.1). At 20 min, glucose preloads (P = 0.0008) and at 30 min,
glucose preloads and 20 g WP (P < 0.0001) increased amylin concentrations compared to the
control (Figure 6.1 D).
Post-meal Mean Plasma Concentrations
Over the entire post-meal period (50-230 min), mean plasma glucose concentrations were
lower (P = 0.0006) than control after WP and glucose preloads, but were not different from each
other (Table 6.1). Mean insulin concentrations were lower (P = 0.0003) after the 20 g WP than
the glucose preloads, and lower after 10 g WP than 20 g glucose. However, there was no
difference between the control and WP preloads. Mean C-peptide concentrations were higher (P
< 0.0001) after glucose than after WP, and after 20 g glucose than the control. Post-meal amylin
concentrations were not affected by preload, but were affected by time (P = 0.006), that is, post-
meal concentrations tended to return to baseline (Table 6.1).
Post-meal plasma concentrations of glucose (P < 0.005) and C-peptide (P = 0.03), but not
insulin, were affected by a time by preload interaction (Table 6.1). Thus, one-way ANOVA at
each time showed that plasma glucose concentrations were lower after both WP doses and 10 g
glucose preloads at 60 min (P = 0.003), and after 20 g WP at 70 min (P < 0.05) compared to the
90
control (Figure 6.1 A). Plasma glucose concentrations at 200 min were lower after 10 g
compared to 20 g glucose (P < 0.04). Plasma insulin concentrations, after an initial post-meal
rise from 50-70 min decreased to 230 min (Figure 6.1 B). While there was no difference
between WP and the control at any of the post-meal times, C-peptide concentrations were higher
after 20 g glucose compared with the control and WP at 50 min (P < 0.0001), 60 min (P < 0.002)
and 70 min (P = 0.0002), with WP at 80 min (P = 0.0009), with the control and 20 g WP at 110
min (P = 0.002), and with 10 g glucose and 10 g WP at170 min (P = 0.01) (Figure 6.1 C). The 10
g glucose preload led to a greater C-peptide concentration compared with the control at 50 min
(P < 0.0001) and compared with 20 g WP at 70 min (P = 0.0002) (Figure 6.1 C).
Total Mean Plasma Concentrations
Over the entire pre- and post-meal period (0-230 min), mean plasma glucose
concentrations were lower after WP, but higher after glucose, compared to the control (P <
0.0001) (Table 6.1). Mean plasma concentrations of insulin and C-peptide were higher (P <
0.0001) after the glucose preloads than the control. Mean plasma amylin concentrations were
higher (P = 0.0002) after glucose preloads than the control and 10 g WP. However, 20 g WP
result in a higher total amylin concentration compared with the control (Table 6.1).
6.4.3. Plasma GLP-1, GIP, PYY, CCK and Ghrelin Concentrations
Pre-meal, post-meal and total mean concentrations of plasma concentrations of
gastrointestinal hormones, followed by p-values for the preload, time and time by preload
interaction effects are shown in Table 6.2.
Pre-meal Mean Plasma Concentrations
Over the entire pre-meal period (0-30 min), each dose of WP led to higher mean plasma
GLP-1 concentrations than the equivalent dose of glucose and was higher than the control (P <
0.0001) (Table 6.2). Mean plasma GIP concentrations were higher (P = 0.0002) after the
preloads than the control, but did not differ from each other. Mean plasma PYY concentrations
(P = 0.01) were higher only after 20 g WP compared to the control. Pre-meal CCK
concentrations were not affected by preloads. Mean plasma concentrations of ghrelin did not
91
change after the preloads than the control, however, 20 g glucose suppressed (P < 0.05) mean
ghrelin concentrations compared with 10 g WP (Table 6.2).
Pre-meal plasma concentrations of GLP-1 (P = 0.01), GIP and CCK (P < 0.04) were
affected by a time by preload interaction (Table 6.2), but pre-meal concentrations of PYY and
ghrelin were not. Plasma concentrations of GLP-1 were higher after 20 g WP and 20 g glucose at
20 min (P = 0.0004), and after 20 g WP at 30 min (P = 0.0001) than the control (Figure 6.2 A).
Plasma GIP concentrations were higher after all preloads at 20 min (P < 0.0001), after all
preloads except 10 g glucose at 30 min (P = 0.004) than the control (Figure 6.2 B). Plasma
concentrations of PYY (Figure 6.3 A) and ghrelin (Figure 6.3 C) were not affected by time and
a time by preload interaction. Although pre-meal mean CCK concentrations were not affected by
time or preload, however, there was an interaction (Table 6.2), showing that response to the
preloads changed over time (Figure 6.3 B).
Post-meal Concentrations
Over the post-meal period (60-230 min), mean plasma concentrations of GLP-1 and PYY
were higher (P < 0.0001) after WP than glucose preloads (Table 6.2). However, compared to the
control, the 20 g WP resulted in higher concentrations of PYY (P < 0.0001) and lower GIP (P =
0.03). Mean plasma CCK concentrations were higher (P < 0.0001) after all preload than the
control. Mean plasma ghrelin concentrations were suppressed (P < 0.002) only after 20 g glucose
compared with 10 g WP (Table 2).
Post-meal plasma concentrations of GLP-1 (P < 0.0001) and PYY (P = 0.0009) were
affected only by time, after rising at 80 min the post-meal concentrations decreased to 230 min
(Figure 6.2 and 6.3 A). However at post-meal, there was no time by preload interaction effects
on these hormones (Table 6.2).
Total Mean Plasma Concentrations
Over the entire pre- and post-meal period (0-230 min), mean concentrations of GLP-1
and PYY were higher (P < 0.0001) after both doses of WP compared with glucose preloads and
the control (Table 6.2). However, mean GIP was not affected by preload. Mean concentrations of
92
CCK were higher (P < 0.0001) after both glucose and WP than the control. Mean ghrelin
concentrations were lower (P = 0.0001) after 20 g glucose than 10 g WP (Table 6.2).
6.4.4. Gastric Emptying Rate (Plasma Paracetamol Concentrations)
Pre-meal (0-30 min) concentrations of paracetamol were affected by preload (P < 0.0001)
and time (P < 0.0001), but there was no interaction (Table 6.3). Mean plasma concentrations of
paracetamol were lower after both doses of WP compared to the control and 10 g glucose (Table
6.3).
Post-meal (50-230 min) concentrations of paracetamol decreased with time (P < 0.0001),
but were not affected by preloads (Figure 6.4).
Over the entire pre- and post-meal period (0-230 min), mean plasma paracetamol
concentrations were lower after WP compared to 10 g glucose (Table 6.3).
6.4.5. Triglycerides and Free Fatty Acids
Pre-meal (0-30 min) plasma TG and FFA concentrations were not affected by preloads;
but post-meal (60-230 min), the 10 g WP preload resulted in higher TG concentrations than 20 g
glucose and 20 g WP (P < 0.02). In addition, post-meal FFA concentrations were higher after 10
g glucose than the control (P < 0.04). However, there was no difference in post-meal FFA
concentrations among the other preloads.
While total (0-230 min) FFA concentrations were not affected by the preloads, TG
concentrations were higher after the 10 g WP preload compared with 20 g glucose, 20 g WP and
the control (P < 0.01).
6.5. Discussion
The results of this study support the hypothesis that the post-meal plasma glucose
concentrations after pre-meal consumption of WP are affected in part by mechanisms beyond
insulin secretion or action alone. Both WP and glucose preloads similarly reduce post-meal
glycemia. The results provide several lines of evidence suggesting that lower post-meal glycemia
93
after WP, unlike glucose, occurs in part by insulin-independent mechanisms. First, WP,
compared to glucose, led to lower plasma insulin and C-peptide concentrations (Table 6.1) and
insulin secretion rate (Figure 6.6). Secondly, WP, compared to the water control, did not increase
post-meal concentrations of insulin and C-peptide, but reduced post-meal glycemia. Third, WP
led to higher plasma concentrations of GLP-1 and PYY (Table 6.2). Finally, WP delayed pre-
meal gastric emptying (Table 6.3).
In the present study, following pre-meal consumption of WP, three possible mechanisms
of post-meal glucose control were investigated, beyond the insulinotropic effect. These included
reduced hepatic insulin extraction (136), increased gut hormone secretion or action (136, 137,
173) and reduced gastric emptying (137, 218).
Reduced hepatic insulin extraction after WP consumed with CHO compared with CHO
alone was shown recently to lead to a higher sustained insulin concentration, but had no effect on
C-peptide concentration or insulin secretion rate (136). C-peptide is secreted in equimolar
concentrations with insulin from pancreatic ß cells, but unlike insulin is not extracted by the
liver. Hence, the relationship between the two is used as an indication of hepatic insulin
extraction. However in the present study, altered hepatic insulin extraction does not appear to
provide an explanation for the lower post-meal glycemic effect of WP for two reasons. First,
plasma concentrations of both insulin and C-peptide were affected similarly after pre-meal
consumption of WP (Table 6.1). While pre-meal concentrations of insulin and C-peptide were
increased similarly after WP, their relative post-meal concentrations did not change compared to
the control. This finding suggests that insulin secretion and extraction were at similar rates after
WP. Secondly, the pre-meal ratio of C-peptide/insulin (Figure 6.5 A), which is used to estimate
insulin removal by the liver and reflect hepatic insulin extraction (258), was lower after both WP
and glucose, compared to the control. However, the post-meal ratios did not differ among the
preloads. Thus, it seems unlikely that pre-meal consumption of WP affected post-meal hepatic
insulin extraction.
The results of this study indicate that elevated gut hormone secretion or action may be a
more likely explanation for mechanisms beyond insulin secretion of glycemic control after pre-
meal consumption of WP. While the ratios of plasma glucose/GLP-1 (Figure 6.5 B) and
insulin/GLP-1 (Figure 6.5 C) were lower after WP and the control than after glucose, the post-
94
meal ratios remained lower after WP compared to both glucose and the control (Figure 6.5 B and
C). Thus, the physiologic regulation of glycemia after WP may not be explained in full by
insulin, but also by GLP-1 concentration.
The higher plasma concentrations of GLP-1 and PYY after WP may more likely indicate
a differentiating WP mechanism of post-meal glycemia compared to glucose and the control.
Increased concentrations of these gut hormones after pre-meal consumption of WP may be
explained by both increased synthesis from the enteroendocrine L-cells and by reduced
breakdown through an inhibitory action of WP on dipeptidyl peptidase IV (DPP-IV), an enzyme
that rapidly deactivates several endogenous peptides including the L-cell derived hormones
GLP-1 (259) and PYY (260). Higher sustained concentrations of GLP-1 and PYY have been
shown in rodents after reduced activity of DPP-IV following WP gavage (132).
Higher plasma concentrations of GLP-1 and PYY may contribute to reduced post-meal
glycemia independent of and beyond insulin secretion through two gut-brain-liver neural
pathways (156, 261). First, 156GLP-1 ( ) and PYY (262) are small peptides that rapidly cross the
blood-brain barrier (BBB) and directly access the central nervous system (CNS) to transmit
signals to inhibit gastric emptying. Additionally, GLP-1 receptors (GLP-1R) are expressed in the
stomach and regulate gastric acid secretion and motility through ascending vagal afferent signals
to the CNS (263, 264). Another insulin-independent mechanism is via increased GLP-1 156( ,
265) and PYY (266, 267) concentrations in the portal vein which stimulate hepatic vagal
afferents and pancreatic vagal afferents. These activate peripheral sensors linked to enhanced
glucose disposal, thereby, augmenting portal-mediated glucose clearance.
In addition, WP is digested and absorbed quickly and results in a more rapid gastric
emptying rate compared to casein (
Therefore, these
enhanced gut-mediated effects of WP on augmented glucose usage may reduce demand for
insulin secretion to regulate post-meal glucose response (14-16).
41), this study shows that WP consumption slowed gastric
emptying, and resulted in 27% lower pre-meal gastric empting rate compared to glucose and the
water control.
Moreover, the post-meal elevated GLP-1 concentration after WP did not contribute to
higher insulin concentrations compared to the control. This may be supported by the glucose-
95
dependent insulin secretion of GLP-1 (188). While both GLP-1 and GIP are known
This is the first report showing that 20 g WP stimulates amylin release, similar to glucose
(Figure 6.1D). The effect of glucose on amylin secretion, a pancreatic ß-cell hormone secreted
simultaneously with a ratio of approximately 1:100 to insulin, in healthy subjects is consistent
with the literature (
to have
potent insulinotropic function (156), GLP-1, unlike GIP, enhances insulin secretion only when
plasma glucose concentrations are high (188). Thus, the increased post-meal GLP-1, in the
presence of reduced post-meal glycemia (Figure 6.1 A), after pre-meal consumption of WP did
not increase insulin concentration compared to the control, as would be predicted (188).
206). However, it is unlikely that reduced gastric emptying after WP can be
attributed solely to amylin, because high amylin concentrations after glucose were not reflected
in lower gastric emptying rates (Figure 6.1 D). While elevated plasma concentrations of pre-meal
amylin and GIP and post-meal CCK after both WP and glucose may have contributed to
improved post-meal glycemia, the responses were similar. Thus, it is unlikely that these
hormones accounted for the slower gastric emptying after only WP. Amylin not only affects
gastric emptying (201-204), but also regulates glucose homeostasis (199, 200) by acting centrally
to suppress nutrient-stimulated glucagon secretion from the α-cells (205), which in turn
suppresses the release of endogenous hepatic glucose production (201-204).
The plasma concentrations of ghrelin decreased with time by 30% after the preloads and
meal, with the highest suppression at 140 min (34%). However, neither WP nor glucose
suppressed pre-meal ghrelin concentrations compared with the control, possibly due to the small
total energy content of the WP or glucose preloads (40-80 kcal). The majority of previous studies
reporting a suppression of ghrelin examined the effect of these nutrients either as part of a meal
(173, 268) or at higher doses (43, 269).
Free fatty acids were measured in this study for several reasons. First, slowing the rate of
meal ingestion reduces postprandial FFA response and the blood glucose response elicited by a
subsequent meal (270). Second, FFA may be a potential cofactor linking insulin and ghrelin
blood glucose concentrations (271). That is, for a similar insulin level, ghrelin concentrations are
higher if FFA are elevated. This is supported by the results of our study that 10 g WP resulted in
higher FFA and ghrelin compared to 20 g glucose. Third, since insulin inhibits lipolysis (271), it
was unclear if a lower post-meal insulin after WP compared to glucose results in higher FFA
96
concentrations. Finally, high FFA interfere with the access of insulin to skeletal muscle or
interfere with insulin signaling resulting in reduced glucose transport into muscle, and thus may
cause acute peripheral insulin resistance (272). However, plasma concentrations of FFA were not
different among the WP and glucose preloads, suggesting that a reduced insulin secretion after
WP did not trigger the release of the FFA from adipose tissues which is normally associated with
enhancing liver glucose production.
The rationale behind comparing WP to the equal doses of glucose was that both rapidly
leave the stomach and stimulate insulin and gut hormones that directly effect glycemia and
gastric emptying rate. However, there are some limitations in the study. First, the sample size of
the study is relatively small, thus, making it difficult to find significant differences between the
lower dose of WP and the control. Based on a calculation of sample size using the variability
found in this study, post-meal PYY concentrations after 10 g WP and the control may have been
found to be statistically significant with a sample size of 32 subjects. Secondly, the paracetamol
absorption test is an indirect method used to measure of liquid gastric emptying rate in humans
(41, 273, 274). While the use of plasma concentrations of paracetamol in liquid preloads in the
present study provides a reliable and reasonably accurate estimate for gastric emptying rate and
digestion rate (275), it may not identify gastric emptying rate per se. The gold standard method
for measuring gastric emptying is scintigraphy which is technically challenging method and
requires expensive equipment and special licensing for radioactive substances (276). Third, total
ghrelin accounting for both active (acyl ghrelin) and inactive forms of ghrelin (des-acyl ghrelin)
was measured in this study and measure of active ghrelin is now the accepted standard of
measure as it relates more clearly to functionality. Fourth, this study assessed the acute and
short-term effect of WP on glycemia and hormonal responses, however, the effectiveness of WP
consumption on long-term glycemic control is unclear. Finally, since the study was conducted on
healthy young men, it is unclear whether they are representative of diabetes. Thus, the
application to T2D needs to be explored.
A previous study suggested that the effect of WP is comparable to pharmacological
therapies such as sulphonylureas on the reduction of postprandial glycemia (137). However, the
effect was observed when participants were provided a very large amount of WP (55 g) to a meal
containing 59 g of CHO. As shown in this study and a previous study (222), pre-meal
97
administration of WP in relatively small amounts (≥ 10 g or 0.15 g/kg body weight) contributes
to glycemic control following a pizza meal. Therefore, it remains to be determined if these small
doses will have a benefit in individuals with T2D.
In conclusion, WP consumption prior to a meal results in post-meal glucose control by
both insulin-dependent and –independent mechanisms.
98
Table 6. 1. Mean plasma concentrations of glucose, insulin, C-peptide, and amylin after the
preloads1
Biomarkers
Control 10 g
Glucose
20 g
Glucose
10 g
Whey
20 g
Whey
P*
Preload Time Interaction
Glucose
(mmol/ L)
Pre-
meal2
4.98 ±
0.06c
6.49 ±
0.19b
6.84 ±
0.27a
5.05 ±
0.05c
5.26 ±
0.11c <0.0001 <0.0001 <0.0001
Post-
meal3
6.04 ±
0.09a
5.72 ±
0.10b
5.59 ±
0.09b
5.68 ±
0.09b
5.59 ±
0.08b 0.0006 <0.0001 <0.005
Total4
5.71 ±
0.08b
5.96 ±
0.09a
5.98 ±
0.12a
5.49 ±
0.07c
5.49 ±
0.06c <0.0001 <0.0001 <0.0001
Insulin
(pmol/ L)
Pre-
meal
36.44 ±
3.83d
153.12 ±
16.32b
207.94 ±
24.29a
82.78 ±
9.12c
106 ±
13.55c <0.0001 <0.0001 <0.0001
Post-
meal
229.82 ±
11.53bc
240.46 ±
11.84ab
266.62 ±
13.4a
218.13 ±
11.26bc
208.5 ±
9.43c 0.0003 <0.0001 NS
Total
170.32 ±
11.92c
213.58 ±
10.36b
248.57 ±
12.14a
176.48 ±
10.3c
176.96 ±
9.0c <0.0001 <0.0001 <0.0001
C-peptide
(pmol/ L)
Pre-
meal
530.72 ±
24.21d
1081.75
± 73.78b
1280.32
± 112.27a
772.44 ±
50.13c
847.84 ±
48.92c <0.0001 <0.0001 <0.0001
Post-
meal
1604.69 ±
47.71bc
1725.82
± 45.73b
2024.92
± 63.33a
1589.76 ±
44.4c
1558.21
± 40.65c <0.0001 <0.0001 0.03
Total
1274.24 ±
59.38c
1527.64
± 48.58b
1795.81
± 65.02a
1338.28 ±
50.55c
1339.63
± 45.32c <0.0001 <0.0001 <0.0001
Amylin
(pM)
Pre-
meal
18.46 ±
2.58b
27.26 ±
3.45a
28.67 ±
3.61a
21.28 ±
2.21b
26.71 ±
3.53a <0.0001 0.0004 0.002
Post-
meal
35.65 ±
2.84
38.18 ±
3.14
39.51 ±
3.52
36.36 ±
2.55
35.84 ±
3.27 NS 0.006 NS
Total
28.28 ±
2.26c
33.50 ±
2.41a
34.87 ±
2.62a
29.89 ±
1.998bc
31.93 ±
2.46ab 0.0002 <0.0001 <0.005
99
1 All values are ± SEM. n = 8. Data were analyzed for pre-meal, post-meal and total for preload ,
time, and preload x time interaction by 2-factor ANOVA (Proc Mixed) and significance was
assessed using Tukey's post hoc, P < 0.05 for all, NS (not significant) 2 Pre-meal values are means of all plasma concentrations before the test meal and calculated from
0-30 min 3 Post-meal values are means of all plasma concentrations after the test meal and calculated from
50-230 min for plasma glucose, insulin and C-peptide and from 60-230 for plasma amylin 4 Total values are means of all plasma concentrations before and after the test meal and
calculated from 0-230 min
100
Table 6. 2. Mean plasma concentrations of gastrointestinal hormones after the preloads1
P*
Biomarkers
Control 10 g
Glucose
20 g
Glucose
10 g
Whey
20 g
Whey Preload Time Interaction
GLP-1
(pg/ mL)
Pre-
meal2
4.89 ±
0.36c
5.24 ±
0.36c
5.81 ±
0.44bc
5.86 ±
0.34b
7.21 ±
0.43a <0.0001 <0.0001 0.01
Post-
meal3
6.43 ±
0.42b
5.92 ±
0.25b
6.28 ±
0.38b
7.99 ±
0.43a
8.47 ±
0.46a <0.0001 <0.0001 NS
Total4
5.77 ±
0.30c
5.63 ±
0.21c
6.07 ±
0.29c
7.07 ±
0.32b
7.93 ±
0.33a <0.0001 <0.0001 NS
GIP
(pg/ mL)
Pre-
meal
82.72 ±
9.66b
157.56 ±
14.54a
139.26 ±
14.83a
152.9 ±
15.12a
166.5 ±
13.54a 0.0002 0.002 <0.04
Post-
meal
393.91 ±
21.06a
372.53 ±
17.74ab
367.37 ±
20.32ab
364.46 ±
16.05ab
319.61 ±
18.62b 0.03 <0.0001 NS
Total
260.54 ±
24.31
280.4 ±
18.57
269.61 ±
20.1
273.79 ±
17.98
253.99 ±
15.78 NS <0.0001 0.0021
PYY
(pg/ mL)
Pre-
meal
199.64 ±
11.20b
202.13 ±
8.08ab
204.94 ±
8.97ab
222.95 ±
7.81ab
230.28 ±
11.33a 0.01 NS NS
Post-
meal
230.99 ±
10.33bc
223.19 ±
6.47c
220.43 ±
6.52c
249.61 ±
8.60ab
270.40 ±
12.71a <0.0001 0.0009 NS
Total
217.55 ±
7.83b
214.17 ±
5.21b
213.79 ±
5.40b
238.19 ±
6.15a
253.20 ±
9.07a <0.0001 <0.0001 NS
CCK
(ng/ mL)
Pre-
meal
100.15 ±
8.76
103.47 ±
9.07
101.51 ±
7.77
121.97 ±
9.07
113.99 ±
8.08 NS NS <0.04
Post-
meal
77.77 ±
8.07b
127.46 ±
8.33a
113.84 ±
6.38a
123.53 ±
8.96a
122.95 ±
8.02a <0.0001 NS NS
Total
87.36 ±
6.08b
117.18 ±
6.30a
108.55 ±
4.96a
122.86 ±
6.37a
119.10 ±
5.73a <0.0001 NS 0.04
Ghrelin
(pg/ mL)
Pre-
meal
405.00 ±
42.22ab
443.87 ±
64.62ab
322.70 ±
27.03b
512.25 ±
95.66a
407.37 ±
61.84ab <0.05 NS NS
Post-
meal
304.12 ±
30.27ab
340.52 ±
32.04ab
215.55 ±
21.58b
431.72 ±
78.06a
328.01 ±
35.08ab <0.002 NS NS
Total 347.36 ±
25.69bc
384.81 ±
33.56ab
261.47 ±
18.22c
466.23 ±
60.26a
362.02 ±
33.31abc 0.0001 0.0006 NS
101
1 All values are ± SEM. n = 8. Data were analyzed for pre-meal, post-meal and total for preload ,
time, and preload x time interaction by 2-factor ANOVA (Proc Mixed) and significance was
assessed using Tukey's post hoc, P < 0.05 for all, NS (not significant) 2 Pre-meal values are means of all plasma concentrations before the test meal and calculated from
0-30 min 3 Post-meal values are means of all plasma concentrations after the test meal and calculated from
60-230 min 4 Total values are means of all plasma concentrations before and after the test meal and
calculated from 0-230 min
102
Table 6. 3. Mean plasma concentrations of paracetamol after the preloads1
Biomarkers
Control 10 g
Glucose
20 g
Glucose
10 g
Whey
20 g
Whey
P*
Preload Time Interaction
Paracetamol
(mmol/ L)
Pre-
meal2
13.36 ±
1.60a
14.75 ±
1.57a
12.24 ±
1.35ab
10.02 ±
1.14b
10.48 ±
1.09b <0.0001 <0.0001
NS
Post-
meal3
12.19 ±
0.55
12.47 ±
0.52
13.21 ±
0.50
12.34±
0.48
12.24 ±
0.51 NS <0.0001 NS
Total4
12.55 ±
0.62ab
13.18 ±
0.61a
12.91 ±
0.54ab
11.63 ±
0.49b
11.7 ±
0.49b <0.004 <0.0001 0.004
1 All values are ± SEM. n = 8. Data were analyzed for pre-meal, post-meal and total for preload ,
time, and preload x time interaction by 2-factor ANOVA (Proc Mixed) and significance was
assessed using Tukey's post hoc, P < 0.05 for all, NS (not significant) 2 Pre-meal values are means of all plasma concentrations before the test meal and calculated from
0-30 min 3 Post-meal values are means of all plasma concentrations after the test meal and calculated from
50-230 min 4 Total values are means of all plasma concentrations before and after the test meal and
calculated from 0-230 min
103
Table 6. 4. Mean plasma concentrations of triglycerides and free fatty acids after the preloads1
Variables
Control
10 g
Glucose
20 g
Glucose
10 g
Whey
20 g
Whey
P*
Preload Time Interaction
Triglycerides
(mg/ dL)
Pre-
meal1
23.96 ±
1.90
30.84 ±
3.78
27.25 ±
2.62
29.34 ±
2.65
27.41 ±
2.53 NS NS NS
Post-
meal2
36.91 ±
3.11ab
37.34 ±
2.86ab
36.5 ±
3.02b
44.31 ±
3.79a
36.0 ±
3.02b <0.02 0.0004 NS
Total3
31.36 ±
2.12b
34.55 ±
2.32ab
32.54 ±
2.13b
37.9 ±
2.62a
32.32 ±
2.1b
<0.01 <0.0001 NS
Free Fatty
Acids
(mEQ/ L)
Pre-
meal 0.14 ±
0.03
0.11 ±
0.01
0.12 ±
0.01
0.13 ±
0.02
0.12 ±
0.03
NS NS NS
Post-
meal
0.07 ±
0.01b
0.1 ±
0.01a
0.08 ±
0.01ab
0.1 ±
0.01ab
0.08 ±
0.01ab <0.04 <0.002 NS
Total
0.09 ±
0.01
0.1 ±
0.01
0.09 ±
0.01
0.11 ±
0.01
0.09 ±
0.01 NS <0.0001 NS
1 All values are ± SEM. n = 8. Data were analyzed for pre-meal, post-meal and total for preload ,
time, and preload x time interaction by 2-factor ANOVA (Proc Mixed) and significance was
assessed using Tukey's post hoc, P < 0.05 for all, NS (not significant) 2 Pre-meal values are means of all plasma concentrations before the test meal and calculated from
0-30 min 3 Post-meal values are means of all plasma concentrations after the test meal and calculated from
60-230 min 4 Total values are means of all plasma concentrations before and after the test meal and
calculated from 0-230 min
104
Figure 6. 1. Mean plasma concentrations of glucose and ß-cell hormones
Mean (±SEM) plasma concentrations of glucose (A), insulin (B), C-peptide (C) and amylin (D)
in 8 healthy men after intake of water (····Ӂ····), 10 g glucose (····Δ····), 20 g glucose
(····▲····), 10 g whey protein (—○—), and 20 g whey protein (—●—). Preload, time and a
preload by time interaction affected the variables as presented in Table 6.1 (Two-way ANOVA,
Proc Mixed, followed by Tukey’s post hoc, P < 0.05). Different superscripts at each measured
time are different between preloads (one-way ANOVA, Proc Mixed, followed by Tukey’s post
hoc, P < 0.05).
105
Figure 6. 2. Mean plasma concentrations of the incretins
Mean (± SEM) plasma concentrations of GLP-1 (A) and GIP (B) in 8 healthy men after intake of
water (····Ӂ····), 10 g glucose (····Δ····), 20 g glucose (····▲····), 10 g whey protein (—○—),
and 20 g whey protein (—●—). Preload, time and a preload by time interaction affected the
variables as presented in Table 6.2 (Two-way ANOVA, Proc Mixed, followed by Tukey’s post
hoc, P < 0.05). Different superscripts at each measured time are different between preloads (one-
way ANOVA, Proc Mixed, followed by Tukey’s post hoc, P < 0.05).
106
Figure 6. 3. Mean plasma concentrations of gastrointestinal hormones
Mean (± SEM) plasma concentrations of PYY (B), CCK (C) and ghrelin (C) in 8 healthy men
after intake of water (····Ӂ····), 10 g glucose (····Δ····), 20 g glucose (····▲····), 10 g whey
protein (—○—), and 20 g whey protein (—●—). Preload, time and a preload by time interaction
affected the variables as presented in Table 6.2 (Two-way ANOVA, Proc Mixed, followed by
Tukey’s post hoc, P < 0.05). Different superscripts at each measured time are different between
preloads (one-way ANOVA, Proc Mixed, followed by Tukey’s post hoc, P < 0.05).
107
Figure 6. 4. Mean plasma concentrations of paracetamol
Mean (± SEM) plasma concentrations of paracetamol, as an indirect marker of liquid gastric
emptying rate, in 8 healthy men after intake of water (····Ӂ····), 10 g glucose (····Δ····), 20 g
glucose (····▲····), 10 g whey protein (—○—), and 20 g whey protein (—●—). Preload, time
and a preload by time interaction affected the variables as presented in Table 6.3 (Two-way
ANOVA, Proc Mixed, followed by Tukey’s post hoc, P < 0.05). Different superscripts at each
measured time are different between preloads (one-way ANOVA, Proc Mixed, followed by
Tukey’s post hoc, P < 0.05).
108
Figure 6. 5. Mean ratios of pre-meal and post-meal plasma concentrations of C-peptide/insulin,
glucose/GLP-1and insulin/GLP-1
Mean (± SEM) ratios of pre-meal and post-meal plasma concentrations of C-peptide/insulin,
glucose/GLP-1 (mmol • pg −1) and insulin/GLP-1 (pmol • mL • pg −1 • L −1) and after the whey
protein and glucose preload consumption (n = 8). Two-factor repeated-measures ANOVA
followed by Tukey’s post hoc was used to compare the effect of preloads (means with different
superscripts at pre-meal (0-30 min) and post-meal (60-230 min) are different, P < 0.05).
109
Figure 6. 6. Mean pre-meal, post-meal and total insulin secretion rate
Mean (± SEM) pre-mea, post-meal and total insulin secretion rate (pmol/kg/min) calculated from
plasma C-peptide concentrations (pmol/L) after the whey protein and glucose preload
consumption (n = 8). Two-factor repeated-measures ANOVA followed by Tukey’s post hoc was
used to compare the effect of preloads (means with different superscripts at pre-meal (0-30 min),
post-meal (50-230 min) and total (0-230 min) are different, P < 0.0001).
110
Figure 6. 7. Whey protein induced post-meal hypoglycaemia: contribution of non-insulin
pathways compared with the water control
GLP-1
PYY
Whey Protein
vs.
Control
Hepatic glucose production (↓) Inhibition
Activation
Inhibition
Blood Glucose (↓)
Muscle glucose uptake (↑)
Delayed gastric
emptying
AMPKactivation
Insulin
C-peptideAmylin
CCK
Amino acids
GIP (↓)
(↑)
(↓)
(↑) ↑
(-)
(-)
111
Figure 6. 8. Whey protein induced post-meal hypoglycaemia: contribution of non-insulin
pathways compared with glucose
GLP-1
PYY
Whey Protein
vs.
Glucose
Hepatic glucose production (↓) Inhibition
Activation
Inhibition
Blood Glucose (similarly ↓)
Muscle glucose uptake (↑)
Delayed gastric
emptying
AMPKactivation
Insulin (↓)
C-peptideAmylin
CCK
Amino acids vs. Glucose
GIP (-)
(↑)
(↓*)
(-)
(↓)
↑
(-)
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CHAPTER 7. GENERAL DISCUSSION
The results of this research support the overall hypothesis that consumption of WP prior
to a meal suppresses short-term food intake and reduces post-meal glycemia by insulin-
dependent and -independent mechanisms in healthy young adults. Whey protein, whether in solid
or liquid form, enhanced satiety and suppressed food intake more than sugars (Chapter 4),
reduced post-meal glycemia in a dose-dependent manner without increased insulin concentration
(Chapter 5), and when compared with glucose led to similar reductions in post-meal blood
glucose, but with lower pre-meal blood glucose, lower pre-and post-meal and total insulin
secretion and concentrations and higher GLP-1 and PYY concentrations, as well as delayed
gastric emptying (Study 6).
These studies further an understanding of the role of WP when consumed prior to a meal
on the regulation of food intake and post-meal glycemia. A novel finding of this research is that
consumption of WP prior to a meal improves post-meal glycemia by both insulin-dependent and
-independent mechanisms. The reproducibility and validity of reduced post-meal glycemic and
insulin responses after WP consumption was confirmed by not only the consistent results
between the last two studies (Chapter 5 and 6), but also by corresponding physiological
responses from the metabolic study (Chapter 6).
As discussed in the previous chapter, there are several lines of evidence suggesting that
lower post-meal glycemia after pre-meal ingestion of WP occurs in part by insulin-independent
mechanisms. The first and most important evidence is that WP led to lower pre- and post-meal
and total plasma concentrations of insulin and C-peptide, or insulin secretion rate, than the
glucose preloads, even though post-meal glycemic responses were similar after both WP and
glucose. Thus, results suggest that overall, less insulin is required to control blood glucose after
WP than glucose, supporting that improved post-meal glycemia may have occurred by increased
efficacy of insulin without enhanced secretion. Secondly, WP led to higher pre- and post-meal
and overall plasma concentrations of GLP-1 and PYY, known to be involved in neural regulation
of energy and glucose homeostasis. The increased and prolonged GLP-1 and PYY responses
independent of post-meal insulin secretion and concentrations after protein compared to CHO
may be due to the BCAA and bioactive contents of protein increasing the secretion of these
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hormones from intestinal L-cells as well as their inhibitory effect on DPP-IV activity (132). The
higher concentrations of these gut hormones, may account for the delayed gastric emptying
(Chapter 6) and therefore, have reduced the demand for insulin secretion to regulate post-meal
glucose response (82, 153, 166, 219, 221).
The elevation of these gut hormones in the presence of lower insulin concentrations after
WP are consistent with previous studies comparing the effect WP and CHO on pre-meal insulin
and gut responses. Consumption of WP (55 g) compared to the iso-caloric glucose preload led to
lower glycemia, with lower insulin, but higher GLP-1 and CCK concentrations (161). Similarly,
WP (55 g) compared with a glucose drink resulted in lower post meal blood glucose
concentrations in the presence of similar concentrations of insulin and ghrelin, but higher CCK
and reduced gastric emptying rate (43). Although amylin (201-204) and CCK (41), respectively
secreted from pancreas and small intestine, contribute to the regulation of glucose and energy
homeostasis through reduced gastric emptying, in the metabolic study, both WP and glucose
increased concentrations of pre-meal amylin and post-meal CCK (Chapter 6). Thus, the higher
concentrations of these hormones do not offer an explanation for the stronger gastric emptying
rate reduction after WP. Nevertheless, to our knowledge, this is the first study examining the
effect of protein on amylin secretion, suggesting that WP, like glucose, is a stimulator of amylin.
Whether dairy proteins have unique physiological properties among other proteins to stimulate
secretion of amylin, similar to their distinctive effects on ghrelin (173) and GIP (41, 45, 46),
requires further investigation.
However, elevated concentrations of GLP-1 and PYY after WP may explain the different
physiologic regulation of post-meal glycemia between the WP and glucose. Among several
factors, the regulation of gastric emptying by GLP-1(153) and PYY (157, 160) has a major
influence on glucose homeostasis (82, 166, 219). The 26% reduction in pre-meal gastric
emptying after WP compared to other preloads
Whey protein-induced GLP-1 and PYY may also modulate energy and glucose
homeostasis independent of and beyond insulin secretion through two gut-brain-liver neural
pathways
slows the speed at which proteins appear in the
absorptive sections of the gut and therefore, limits the rate of nutrient uptake by the gut and
reduces the demand for insulin secretion.
(156). First, GLP-1 and PYY are small peptides that rapidly cross the BBB and
114
directly access the CNS, and GLP-1R, expressed in the stomach ascending vagal afferent signals
to the CNS, directly inhibit gastric emptying (263, 264). Secondly, GLP-1 and PYY secreted into
the portal vein transmits signals, via hepatic vagal afferents and pancreatic vagal afferents, to the
CNS. These activate peripheral sensors linked to enhanced satiety and glucose disposal, t
156
hereby,
augmenting portal-mediated glucose clearance independent of insulin elevation ( , 265).
The higher concentrations of GLP-1 and PYY, strong satiety hormones, after WP may
also account for the stronger appetite and food intake suppression of WP compared with sugars
observed in Chapter 4. Preloads of WP (50 g) containing lower energy content, both in solid and
liquid forms, suppressed appetite and food intake at 60 min more than sugars (75 g), consistent
with prior evidence that proteins are more satiating than CHO (
Therefore, although the current research does not fully elucidate the mechanism of action of WP
on post-meal glycemia, it indicates that improved post-meal blood glucose after pre-meal
consumption of WP associates with increased efficacy of insulin without increased insulin
concentration (Chapter 5 and 6), increased concentrations of GLP-1 and PYY and reduced
gastric emptying (Chapter 6).
96). The average compensation
for the energy content of the preloads of WP and sugars at the next meal was 91% and 34%,
respectively in Chapter 4, suggesting that while the regulation of food intake relies on
physiological signals arising from the gut to the CNS in response to macronutrients, the failure to
fully compensate for the energy content of glucose preloads is another determinant of positive
energy intake.
Furthermore, the higher concentration of these gut hormones after intact WP in Chapter 6
may explain the results of Chapter 5 where 10 g of both intact and hydrolyzed WP preloads
resulted in similar post-meal insulin concentrations, but lower post-meal blood glucose after only
intact WP. While the BCAA contents of intact and hydrolyzed WP contributed to similar insulin
responses, the gut hormone-induced post-meal hypoglycemia after intact WP may be attributed
to the bioactive peptides content of intact WP. This is consistent with previous research showing
that a BCAA mixture increased insulin, but failed to reproduce the effects of the intact WP on
gut peptides involved in control of glycemia and stomach emptying (45).
To date, with the exception of one study (137), none have focused on the effect of pre-
meal ingestion of protein on post-meal glycemic control. Thus, these are novel results for two
115
reasons. First, this is the first study to describe physiologic mechanisms of post-meal glycemic
control after WP. Second the results suggest that proteins and perhaps WP specifically, when
consumed before a meal may be more efficacious than when consumed with a meal on reducing
food intake and blood glucose (137, 222).
There are many reports of the effect of large amount of proteins (30-60 g) when
consumed with CHO drink or in a meal on reduced gastric emptying (137, 218), increased gut
hormone secretion or action (136, 137, 173), food intake suppression (41, 42, 102) and improved
glycemia (44, 46, 103, 131). However, the present studies identify the relationship between dose
of WP, short-term food intake and pre- and post-meal glycemic responses, and show for the first
time that pre-meal consumption of small amounts of WP contributes to food intake suppression
(≥ 20 g, or 0.3 g/kg body weight) in Chapter 4 and 5, and reduced post-meal glycemia (≥ 10 g, or
0.15 g/kg body weight) in Chapter 5 and 6.
The results of this research, in part, provide plausibility to the associations between
increased dairy consumption and healthier body weights and less T2D. The dairy proteins, WP
and casein are most often consumed together in fluid milk and the most solid forms are cheeses
containing only casein, which accounts for 80% of milk proteins. When consumed alone in a
pre-meal beverage, WP, as a fast protein, exerts a stronger effect on food intake suppression 60-
90 min later (41, 102) than casein. The contribution of cheese, containing primarily casein, in
diets associated with healthier body weights and less T2D has not been identified as an
independent factor, nor are there any reports of the effect of cheeses, whether consumed prior to
a meal or within a meal on post-meal glycemia. However, casein, due to its precipitation by
gastric acid (80) results in a longer gastric emptying rate and similar or stronger food intake
suppression effect at later time (about 150 min) (102). The results of current research (Chapter 4)
show that physical state of WP was not a major factor in suppressing food intake. However, the
effects of solid and liquid forms of dairy proteins on gastric emptying and satiety hormones are
unclear from this study, as both treatments were in a liquid form in the stomach. A recent study
by Mattes and colleagues shows that the cognitive, sensory and physiological effects are
different for gastric-solid compared to gastric-liquid treatments matched for macronutrients and
calories (277). Thus, the combination of WP and casein in milk and solid milk products may be
expected to stimulate satiety and glycemic control in a synergistic manner and contribute to the
116
favorable associations seen between increased dairy consumption and healthier body weights and
less T2D.
Pre-meal consumption of 2% milk (260 kcal/500 mL), compared to soy beverage, infant
formula and juice, prior to a meal improves post-meal glycemia in young adults (278). The
attenuated post-meal glycemia has been attributed to milk’s protein content (18 g). The results
also indicate that the dairy sources of protein play an important role in post-meal glucose control,
as the soy beverage containing 14 g of soy protein did not (278).
Overall, the results of this research lead to the hypothesis that pre-meal consumption of
proteins rather than CHO in beverages may provide a strategy for management of healthy body-
weight and blood glucose concentrations by suppressing pre-meal appetite and mealtime food
intake (Chapter 4 and 5) and reducing post-meal glycemia without a higher demand for insulin
(Chapter 5 and 6). In addition, proteins have physiological functions that contribute to healthier
body weights and composition including increased muscle protein synthesis (187), and
regulation of blood pressure (77). However, much longer term studies are required to evaluate
the role of pre-meal consumption of proteins on these parameters.
7.1. Study Design: Strengths and Limitations
Strengths
A major strength of the design was that these studies isolated the effect of WP on food
intake and post-meal glycemia as it was not consumed in combination with any other nutrients
which could interfere with the outcomes. Many studies on WP failed to demonstrate the
magnitude of the effect of WP on gastric emptying and glycemic regulation, most commonly due
to addition of WP to other nutrients such as CHO and fat in the form of a preload (43, 136, 218)
or as part of a meal (46, 103) or due to the lack of a control (110).
In addition, the conclusions of the last two studies (Chapter 5 and 6) on the effect of pre-
meal consumption of WP on post-meal glycemic control were strengthened by utilizing a preset
fixed meal design to eliminate variability in food intake on post-meal glycemic response.
117
Limitations
One of the limitations of these studies was due to the design of the experiments where
subjects were scheduled to start the study after their breakfast consumption. Although they were
provided the standardized breakfast and instructed to consume 4 h before arriving to the
laboratory to start the sessions, a few subjects had dramatically higher baseline plasma
concentrations of glucose and insulin, therefore, they were excluded from the analyses in the
metabolic study and resulted in the small sample size in the last study. Moreover, due to the
design of these studies, it is difficult to compare the effect of WP or glucose on food intake and
gastrointestinal hormones with the literature, as in most of the studies, examining the effects of
proteins and glucose, subjects were fasting at baseline.
In addition, the acute and short-term effects of WP on food intake, glycemia and
hormonal responses was assessed, however, the effectiveness of WP consumption on long-term
satiety and food intake regulation as well as glycemic control is unclear from the results of these
studies.
The use of plasma concentrations of paracetamol (41, 273, 274) in liquid preloads in the
present study provides a reliable and reasonably accurate estimate for gastric emptying rate and
digestion rate (275), however it may not identify gastric emptying rate per se. The gold standard
method for measuring gastric emptying is scintigraphy which is technically challenging method
and requires expensive equipment and special licensing for radioactive substances (276).
7.2. Significance and Implications
These novel experiments shed new insight into an understanding of insulin-independent
mechanisms of glycemic, satiety and food intake control originating in the gastrointestinal tract
from pre-meal consumption of WP. Thus, in contrast to previous suggestions, other proteins may
also assist with glycemic control without only increasing insulin demand
This research provides evidence for the efficacy of WP as a value-added ingredient
incorporated into foods and diets to suppress short-term food intake and improve blood glucose.
118
The results of these studies may lead to new beverage or food formulations and supplements and
to dietary strategies for the prevention and treatment of obesity, hyperglycemia and T2D.
CHAPTER 8. GENERAL SUMMARY AND CONCLUSIONS
Consumption of WP, both in solid and liquid forms, with or without GMP content, prior
to a meal reduces short-term food intake more than sugars. Pre-meal consumption of liquid WP,
similar to glucose preload, reduces post-meal glycemia, however, post-meal lower of glycemia
after WP is achieved in the presence of reduced requirement for insulin due to alternative
mechanisms including increased plasma concentrations of GLP-1 and PYY and delayed gastric
emptying rate.
In conclusion, WP consumed prior to a meal reduced short-term food intake and pre- and
post-meal glycemia by both insulin-dependent and -independent mechanisms in healthy young
adults.
CHAPTER 9. FUTURE DIRECTIONS
This research provided physiological explanations for the role of dairy protein
consumption on satiety, suppression of short-term food intake and improved glycemic control in
healthy adults. Thus, the results add some biological plausibility to the inverse associations
found between consumption of dairy products and obesity, metabolic syndrome or T2D.
However, further investigation is now required to determine the long-term effects of WP and its
role in the management of body weight and metabolic regulation. It has recently been reported
that 56 g WP supplementation over 6 months resulted in lower body weight and fat mass in free-
living overweight and obese adults, without imposed energy restriction, compared with those
who consumed iso-energetic supplemental CHO (279). However, future research should examine
the smaller doses of WP as well as other proteins consumed prior to a meal on body weight and
metabolic regulation following an energy restricted diet.
Additionally, previous short-term studies have reported different results on appetite and
food intake regulation in lean versus obese adults (43, 131, 280). Whey protein also failed to
suppress appetite and food intake in children (281). Thus, it remains to be investigated if WP
119
would suppress food intake and improve glycemic response in obese, similar to the lean adults in
our studies.
There is only one published study that reported the effect of consumption of 55 g WP prior
to a meal on post-meal glycemia in individuals with T2D (137). However, the effect of smaller
amounts of WP consumed prior to a meal on short-term food intake and post-meal glycemia in
patients with T2D needs to be examined.
Furthermore, the short- and long-term effects of consumption of other proteins prior to a
meal on post-meal glycemic control in both healthy and T2D individuals need to be investigated.
120
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CHAPTER 11. APPENDICES
144
APPENDIX 1. Sample Size Calculation Sample size calculation when testing for the mean of a normal distribution (two-sided alternative), for within subject designs, is:
n = [(z1-α/2 + z1-β
) · σ/Δ]2 α = 0.05, probability of Type 1 error β = 0.20, probability of type II error Z 0.975 = 1.96 Z 0.80 = 0.84 σ = 186.2 kcal Δ = 157.0 kcal n = 15
Values were taken from my M.Sc thesis, 2007 (142). σ represents standard deviation, Δ represents the minimal difference in food intake between sugar treatment and control. n is the number of subjects required.
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APPENDIX 2. Subjects Characteristics Chapter 4
Subject Age Weight Height BMI Age Weight Height BMI Age Weight Height BMINo. (y) (kg) (m) (kg/m2) (y) (kg) (m) (kg/m2) (y) (kg) (m) (kg/m2)
Gelatin Study Sugar Study Whey Protein Study1 22.0 61.0 1.7 20.1 21.0 72.5 1.8 21.7 23.0 66.0 1.7 21.82 20.0 60.0 1.7 20.5 22.0 56.0 1.7 19.2 30.0 73.5 1.7 24.63 27.0 72.8 1.8 22.2 21.0 66.0 1.7 22.0 21.0 71.0 1.8 22.94 21.0 63.8 1.7 22.6 21.0 80.0 1.8 24.4 21.0 65.2 1.7 22.35 20.0 81.0 1.8 24.5 19.0 60.0 1.7 21.1 26.0 68.6 1.7 23.56 22.0 76.0 1.8 23.7 21.0 79.0 1.9 22.6 20.0 72.6 1.8 22.77 24.0 68.7 1.8 21.0 21.0 64.5 1.7 22.3 28.0 69.0 1.7 23.68 20.0 73.5 1.7 24.8 21.0 74.0 1.8 22.0 25.0 54.4 1.7 20.09 24.0 75.0 1.7 24.8 20.0 51.0 1.6 19.7 28.0 60.5 1.7 22.2
10 23.0 70.8 1.7 23.4 25.0 70.0 1.8 22.8 23.0 78.5 1.8 24.811 20.0 67.0 1.8 20.9 19.0 70.5 1.8 21.8 22.0 67.4 1.7 22.312 25.0 67.5 1.8 21.5 22.0 75.0 1.8 24.4 22.0 59.8 1.7 21.413 20.0 69.5 1.8 22.4 23.0 54.0 1.7 19.2 21.0 65.5 1.7 22.714 19.0 69.5 1.7 23.0 23.0 70.0 1.7 23.1 21.0 72.6 1.8 23.215 22.0 85.0 1.9 24.8
Mean 21.9 69.7 1.8 22.5 21.4 68.5 1.8 22.1 23.6 67.5 1.7 22.7SEM 0.6 1.6 0.0 0.4 0.4 2.6 0.0 0.5 0.9 1.7 0.0 0.3
Chapter 5Subject Age Weight Height BMI Age Weight Height BMI Age Weight Height BMI
No. (y) (kg) (m) (kg/m2) (y) (kg) (m) (kg/m2) (y) (kg) (m) (kg/m2)Study 1 Study 2 Study 2Male Male Female
1 21.0 54.2 1.7 19.4 20.0 61.5 1.8 20.1 21.0 54.2 1.7 19.42 21.0 68.5 1.7 23.7 21.0 83.7 1.9 24.5 21.0 68.5 1.7 23.73 23.0 56.4 1.7 20.7 21.0 69.7 1.8 22.5 23.0 56.4 1.7 20.74 20.0 55.4 1.6 21.0 20.0 65.5 1.8 20.9 20.0 55.4 1.6 21.05 20.0 65.8 1.7 23.3 20.0 71.1 1.8 22.4 20.0 65.8 1.7 23.36 22.0 55.2 1.6 20.9 26.0 62.3 1.7 22.9 22.0 55.2 1.6 20.97 27.0 60.5 1.7 21.7 23.0 73.0 1.9 20.9 27.0 60.5 1.7 21.78 18.0 53.0 1.6 19.9 22.0 80.0 1.8 25.0 18.0 53.0 1.6 19.99 24.0 69.8 1.8 22.3 23.0 75.1 1.9 21.9 24.0 69.8 1.8 22.310 25.0 64.0 1.7 21.411 22.0 67.0 1.8 20.012 19.0 63.3 1.7 22.4
Mean 21.8 59.9 1.7 21.4 21.8 69.7 1.8 22.1 21.8 59.9 1.7 21.4SEM 0.9 2.2 0.0 0.5 0.6 2.1 0.0 0.4 0.9 2.2 0.0 0.5
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Chapter 6Subject Age Weight Height BMI
No. (y) (kg) (m) (kg/m2)1 29.0 75.8 1.8 23.92 21.0 59.7 1.7 20.43 27.0 65.1 1.8 21.04 21.0 54.2 1.6 20.75 20.0 64.3 1.7 21.56 19.0 66.6 1.8 21.07 24.0 65.2 1.8 20.68 22.0 80.0 1.8 25.2
Mean 22.9 66.4 1.7 21.8SEM 1.2 2.9 0.0 0.6
APPENDIX 3. Pizza Meal Composition
NutritionalInformation Pepperoni Deluxe Three
Per 100g Cheese
Protein (g) 11 9.1 13
Total Fat (g) 7.7 6.2 8.4
Carbohydrate (g) 28 27 29
Energy (kcal) 219 195 237
McCain Foods: Deep and Delicious, 5” Pizza
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APPENDIX 4. Fixed Pizza Meal Calculation Chapter 5
Subject kg/BW Food Deluxe Pizza CHO Protein Fat
No. kcal g g g g Male
1 74.0 888.0 400.0 108.0 36.4 28.0 2 70.5 846.0 381.1 102.9 34.7 26.7 3 61.5 738.0 332.4 89.8 30.3 23.3 4 83.7 1004.4 452.4 122.2 41.2 31.7 5 69.7 836.4 376.8 101.7 34.3 26.4 6 66.0 792.0 356.8 96.3 32.5 25.0 7 65.5 786.0 354.1 95.6 32.2 24.8 8 71.1 853.2 384.3 103.8 35.0 26.9 9 62.3 747.6 336.8 90.9 30.6 23.6 10 73.0 876.0 394.6 106.5 35.9 27.6 11 80.0 960.0 432.4 116.8 39.4 30.3 12 75.1 901.2 405.9 109.6 36.9 28.4 13 64.0 768.0 345.9 93.4 31.5 24.2 14 67.4 808.8 364.3 98.4 33.2 25.5 15 61.8 741.6 334.1 90.2 30.4 23.4 16 66.9 802.8 361.6 97.6 32.9 25.3
Mean 69.5 834.4 375.8 101.5 34.2 26.3 SEM 1.6 19.4 8.7 2.4 0.8 0.6
Female 1 54.2 650.4 293.0 79.1 26.7 20.5
2 68.5 822.0 370.3 100.0 33.7 25.9 3 56.4 676.8 304.9 82.3 27.7 21.3 4 55.4 664.8 299.5 80.9 27.3 21.0 5 65.8 789.6 355.7 96.0 32.4 24.9 6 55.2 662.4 298.4 80.6 27.2 20.9 7 60.5 726.0 327.0 88.3 29.8 22.9 8 53.0 636.0 286.5 77.4 26.1 20.1 9 69.8 837.6 377.3 101.9 34.3 26.4
Mean 59.9 718.4 323.6 87.4 29.4 22.7 SEM 2.2 26.1 11.8 3.2 1.1 0.8
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Chapter 6
Subject Kg/BW Pizza Meal
Pizza Meal CHO Protein Fat
No. kcal G g g g
1 75.8 909.6 409.7 110.6 37.3 28.7 2 59.7 716.4 322.7 87.1 29.4 22.6 3 65.1 781.2 351.9 95.0 32.0 24.6 4 54.2 650.4 293.0 79.1 26.7 20.5 5 64.3 771.6 347.6 93.8 31.6 24.3 6 66.6 799.2 360.0 97.2 32.8 25.2 7 65.2 782.4 352.4 95.2 32.1 24.7 8 80.0 960 432.4 116.8 39.4 30.3
Mean 66.4 796.4 358.7 96.9 32.6 25.1 SEM 2.7 32.7 14.7 4.0 1.3 1.0
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APPENDIX 5. Amino Acid Profile of Sweet, Acid and Hydrolyzed Whey Proteins
Sweet
Hydrolyzed
Acid
Whey Protein
Whey Protein
Whey Protein
Energy (kcal/ 100 g) 410.0
386.0
393.0
Protein (%) 80.3
81.1
75.7
Carbohydrate (%) 7.0
3.0
15.8
Fat (%)
6.2
5.1
3.0
Moisture (%) 3.7
4.8
2.9
Ash (%)
2.8
5.2
2.7
Amino acids (g/ 100 g)
Alanine
3.5
3.6
3.5
Arginine
1.9
2.5
2.0
Aspartic acid 7.9
8.6
8.2
Cysteine
1.7
1.7
1.9
Glutamic acid 13.1
13.4
12.7
Glycine
1.4
1.5
1.3
Histidine
1.4
1.5
1.6
Isoleucine 4.7
4.8
4.2
Leucine
7.9
9.0
9.2
Lysine
7.0
7.0
7.7
Methionine 1.6
1.7
1.7
Phenylalanine 2.4
2.6
2.6
Proline
9.5
5.7
8.1
Serine
3.7
4.8
3.1
Threonine 4.8
5.0
3.6
Tryptophan 1.3
1.3
1.6
Tyrosine
2.1
2.3
2.4
Valine
4.6
4.5
4.0
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APPENDIX 6. Information Sheet and Consent Forms
Chapter 4. EFFECT OF
DRINKING COMPARED TO EATING SUGARS OR WHEY PROTEIN ON SHORT-TERM APPETITE AND FOOD INTAKE
Investigators: Tina Akhavan, MSc Department of Nutritional Sciences, University of Toronto Phone: (416) 978-3700 Email: [email protected] Dr. G. Harvey Anderson, PhD Primary Investigator, Department of Nutritional Sciences, U of T Phone: (416) 978-1832 Email: [email protected] Funded by a Kraft Canada Inc. and NSERC Collaborative Research and Development Grant to Dr. Anderson The objective of this research project is to determine the effect whey protein, sugars and gelatin on blood glucose and appetite. The results from this study will be submitted for publication. All of your personal information will remain confidential and will be locked in a filing cabinet to which only the two above investigators have access. Upon completion of the study your name and address will be removed from all documents and only a number will remain for organizational purposes. If the results are published, only average values will be used. To participate in this study you must be healthy and be between the ages of 20 and 30 y. You must be a nonsmoker and you cannot be taking any medications. Approximately 16 people will participate in this study. Before agreeing to participate in this research study, it is important that you read and understand this research consent form. You waive no legal rights by participating in this study. If you have any questions or concerns about your rights as a subject you can contact Dr. Thomas Wolever in the Department of Nutritional Sciences at (416) 978-5556. If you have any questions after you read through this information please do not hesitate to ask the investigators for further clarification.
Initial Screening Interview: Participant will provide the interviewer with basic information (height, weight, health status, etc.) and answer questionnaires pertaining to food habits, in addition to completing a food acceptability list.
Outline of Participant’s Role
Sessions: 5 in total During Study: Participants are asked to adhere to their typical routine, including exercise, thought the study and to eat a similar meal the night before each session. Morning of Each Session: Fast for 12 hours except for water, subjects will eat provided breakfast containing a single serving of a ready-to-eat cereal, a box of 2% milk, and a 250 mL
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box of juice and tea or coffee without sugar four hours before arrival. Water may be consumed up to one hour before the session. Day of Each Session: Note
: Participants will choose a start time between 11 and 2 pm, and must arrive at this time for all sessions. Below is and example of a session schedule for an 11 am arrival.
11 Participants will arrive at the Department of Nutritional Sciences, Rm. 329. Participants are expected to stay within the department for the duration of the experiment (11 a.m. to 1:00 p.m.).
11:05 Participants will complete a sleep habits and stress factors questionnaire. In
addition, motivation to eat and physical comfort will be measured by Visual Analogue Scales (VAS). A finger-prick blood sample will be taken to determine baseline levels of blood parameters including glucose.
Participants must comply with the following precautions:
Use your own finger-prick gun for each test, never share with someone
else Swab your finger with alcohol and use a new sterile lancet Dispose of needles immediately into the sharps container provided
To avoid accidentally sticking two people with the same needle, participants must put their own lancet into the finger-prick gun before each blood sample and discard it in the sharps box immediately afterwards. Once the lancet is in the gun, there is little chance of accidentally being stuck. Participants can do their own finger-pricks, or have an investigator assist them.
11:10 Participants will be given one of 5 treatments. Participants will have five minutes
to consume the treatments and will then rate the treatment’s palatability using VAS.
11:14 – 12:34 All participants will complete motivation to eat (VAS) every 15 minutes for 60
min. 12:35-12:50 Participants are given a pizza meal. After completion of the meal, participants
will be asked to rate the palatability of the meal and complete motivation to eat VAS.
Consent Form
The objective of this study is to determine the effect of protein on appetite and blood glucose. I have been fully informed of what is expected of me as a participant in this research project and I have been provided with a typewritten copy of these expectations as outlined in the attachment to this consent form.
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I am aware that my participation will not involve any health risk to me; that personal information will remain confidential; and that my name will not appear in any publication. I understand that for the purposes of this research project, it is hoped that I will complete all five (5) sessions. However, I may choose to withdraw at any time without prejudice, whereupon I will receive the prorated portion of the total payment of ($75). Should I complete all 5 sessions, I will also receive a bonus in the amount of ($15). Upon completion of the study, a summary of the results will be made available to me for pickup from the Department of Nutritional Sciences. DATE: ____________________ PARTICIPANT’S NAME: _____________________________ PARTICIPANT’S SIGNATURE: ________________________ WITNESS’ SIGNATURE: _____________________________
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Chapter 5. EFFECT OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN AND ITS HYDROLYSATE ON FOOD INTAKE AND POST-MEAL GLYCEMIA AND INSULIN RESPONSES IN YOUNGE ADULTS Investigators: Tina Akhavan (MSc) Department of Nutritional Sciences, University of Toronto Phone: (416) 978-3700 Email: [email protected]
Dr. G. Harvey Anderson, PhD Primary Investigator, Department of Nutritional Sciences, U of T Phone: (416) 978-1832 Email: [email protected]
Funding Source
:
Funding for this project is provided by the Kraft Canada Inc/NSERC Collaborative Research and Development Grant. The project has been peer-reviewed and approved for its scientific merits. The objective of this research project is to determine the effect whey protein on blood glucose and appetite. The results from this study will be submitted for publication. All of your personal information will remain confidential and will be locked in a filing cabinet to which only the two above investigators have access. Upon completion of the study your name and address will be removed from all documents and only a number will remain for organizational purposes. If the results are published, only average values will be used. To participate in this study you must be healthy and be between the ages of 20 and 30 y. You must be a nonsmoker and you cannot be taking any medications. Approximately 16 people will participate in this study. Before agreeing to participate in this research study, it is important that you read and understand this research consent form. You waive no legal rights by participating in this study. If you have any questions or concerns about your rights as a subject you can contact Dr. Thomas Wolever in the Department of Nutritional Sciences at (416) 978-5556. If you have any questions after you read through this information please do not hesitate to ask the investigators for further clarification.
Outline of Participant’s Role
Initial Screening Interview: Participant will provide the interviewer with basic information (height, weight, health status, etc.) and answer questionnaires pertaining to food habits, in addition to completing a food acceptability list. Sessions: 5 in total During Study: Participants are asked to adhere to their typical routine, including exercise, thought the study and to eat a similar meal the night before each session.
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Morning of Each Session: Fast for 10-12 hours except for water, subjects will eat provided breakfast containing a single serving of a ready-to-eat cereal, a box of 2% milk, and a 250 mL box of orange juice and tea or coffee without sugar four hours before arrival. Water may be consumed up to one hour before the session. Day of Each Session: Note
: Participants will choose a start time between 11 and 1 pm, and must arrive at this time for all sessions. At each session, you will be asked to drink a treatment, give blood samples and to fill out questionnaires at the times outlined in the table below. Blood will be sampled before the treatment and 15, 30, 50, 65, 80, 95, 110, 140 and 170 minutes after the treatment.
You will be provided breakfast including; milk, cereal and orange juice to eat and drink on the morning of each session. Before meeting with us, you will be asked to eat the provided breakfast between 6:00 to 9:00 am following a 10 hour fast (do not eat 10 hours before eating breakfast). Four hours later, you will start the study session at the FitzGerald Building (between 10:00 a.m. and 1:00 pm). You will be asked to stick to your normal routine, including exercise and to eat a similar meal the night before each session. You will be asked not to eat any food in the period between the breakfast and meeting with us at the Fitzgerald Building. You can drink water until one hour before meeting with us.
This study includes 6 sessions. During each session, blood will be sampled 10 times by finger prick over 3 hours. Over the study period, a total of 60 finger pricks will be taken.
You will be asked to complete visual analog scales (VAS) questionnaires measuring your appetite, physical comfort and energy/fatigue as well as the palatability (pleasantness) of the treatment and pizza during the study sessions. You will be served a pizza meal 30 minutes after you eat the treatment. Each session will take up to 3.5 hours of your time.
The detailed procedure for each session is shown below in an example of a session schedule for 10 a.m.
Time Activity 6:00 Eat standard breakfast 9:45 Arrive at the FitzGerald Building (University of Toronto) 9:50 Fill in Sleep, Stress, and VAS questionnaires and take baseline blood
sample 10:00 - 10:05 Drink treatment or water (through out 5 min) 10:15 & 10:30 Blood Sampling and VAS questionnaires (15 and 30 min) 10:30 - 10:45 Pizza served and eaten 10:50- 12:50 Blood sampling and VAS questionnaire (50, 65, 80, 95, 110, 140 and
170 min) Voluntary Participation and Early Withdrawal: It is hoped that you will complete all six sessions. However, you may choose to stop being in the study at any time without any problems.
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Risks All of the treatments and pizza are prepared hygienically in the kitchen and present minimal risk.
The risks and discomfort will come from the blood sampling procedure. Great care will be taken when taking your finger prick blood samples. The investigator will help you. To make sure that you are not exposed to another person’s needle, we will ask you to sit away from other study participants. We will put a lancet into the finger prick gun before taking each blood sample and then put it into the safety container. We will swab your finger with alcohol before and after each finger prick and will use a new sterile needle each time.
Some discomfort will be felt as a result of a sharp momentary pain caused as the needle enters the skin. However, because the lancet needle is very small the pain felt is usually less than you might feel from a skin puncture during vaccination or if a blood sample is taken by a needle inserted in a vein.
There is very little chance of infection. Before the finger is pricked the area is cleaned with an alcohol swab. There might be slight bruising under the skin, but this will be minimized by applying pressure after the finger is pricked and blood sugar is measured.
Benefits: You will not benefit directly from taking part in this study. You will be shown your blood sugar results and if they are not normal you will be told and advised to talk to your doctor. All foods and drinks will be free.
Confidentiality and Privacy: Confidentiality will be respected and no information that discloses your identity will be released or published without your permission unless required by law. Your name, medical history and signed consent form will be kept in a locked filing cabinet in the investigator’s office. Your results will not be kept in the same place as your name. Your results will be recorded on data sheets and in computer records that have an ID number for identification, but will not include your name. Your results, identified only by an ID number, will be made available to the study sponsor if requested. Only study investigators will have access to your individual results.
Publication of Results: The results of the study may be presented at scientific meetings and published in a scientific journal. If the results are published, only average and not individual values of the subjects will be reported.
Possible Commercialization of Findings: Results from this study may lead to commercialization of a product, new product formulation, changes in the labeling of a product and/or changes in the marketing of a product; you will not share in any way from the possible gains or money made by commercial application of findings.
New Findings: If anything is found during the course of this research which may have an effect on your decision to continue taking part in this study, you will be told about it.
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Compensation: You will be paid $36 per session. You will also be reimbursed $6 per session for travel (bus, subway). If you withdraw from the study before finishing or asked to withdraw, you will be paid on the basis of the sessions already completed.
Rights of Subjects: Before agreeing to take part in this research study, it is important that you read and understand your role as described here in this study information sheet and consent form. You waive no legal rights by taking part in this study. If you have any questions or concerns about your rights as a participant you can contact the Ethics Review Office at [email protected] or call 416-946-3273.
If you have any questions after you read through this information please do not hesitate to ask the investigators for further explanation.
Consent Form
I acknowledge that the research study described above has been explained to me and that my questions have been answered to my satisfaction. I have been informed of the alternatives to participation in this study, including the right not to participate and the right to withdraw. As well, the potential risks, harms and discomforts have been explained to me. I understand that I will receive compensation for my time spent participating in the study.
As part of my participation in this study, I understand that I may come in contact with certain confidential information. I agree to keep the confidentiality of such, if any, information unless it is necessary to disclose it to my health care provider(s), or to my legal representative(s).
I hereby agree and give my authorized consent to participate in the study and to treat confidential information in a restrictive manner as described above. I have been given a copy of the consent form to keep for my own records. DATE: ____________________ PARTICIPANT’S NAME: _____________________________ PARTICIPANT’S SIGNATURE: ________________________ WITNESS’ SIGNATURE: _____________________________
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Chapter 6. MECHANISM OF ACTION OF PRE-MEAL CONSUMPTION OF WHEY PROTEIN ON GLYCEMIC CONTROL IN YOUNG ADULTS
Investigators Department of Nutritional Sciences, University of Toronto
: Dr. G. Harvey Anderson, PhD (Principle investigator)
Phone: (416) 978-1832 Email: [email protected] Dr. Bohdan Luhovyy, PhD (Research Associate) Department of Nutritional Sciences, University of Toronto Phone: (416) 978-6894 Email: [email protected]
Tina Akhavan, MSc, PhD candidate
Department of Nutritional Sciences, University of Toronto Phone: (416) 978-3700
Email: [email protected]
Funding Source Funding for this project is provided by the Kraft Inc/NSERC Collaborative Research and Development Grant. The project has been peer-reviewed and approved for its scientific merits.
:
Background and Purpose of Research In 2004, almost 60% of adult Canadians were overweight or obese. This is a serious health problem because both are related to many common health risks, including increased blood sugar, blood lipids and blood pressure. It is important to find food-based answers for the prevention and treatment of overweight and obesity.
:
There are two types of protein found in milk; whey protein and casein. Whey protein comes from making cheese and is commonly used by athletes to increase muscle mass. There are studies that show that whey protein may reduce appetite and improve health by decreasing blood sugar. Therefore, the purpose of this study is to test the effect of whey protein (10 and 20 g), and glucose (10 and 20 g) drinks on appetite, blood glucose, insulin and hormonal responses before and after a pizza meal. The information gathered from this study will be used to find out whether the addition of whey protein or glucose into a diet can help to control blood sugar and appetite. This study will have 24 participants. Invitation to Participate You are being invited to take part in this study. If you chose to take part, you will be given a treatment (whey protein, glucose or water) in five separate sessions over 2 weeks. Your appetite will be assessed by filling out the Visual Analogue Scales questionnaires and your blood
:
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will be sampled by a registered nurse to be measured for blood glucose, insulin and hormonal responses after consumption of the treatment and the pizza meal. Eligibility To take part in this study you must be healthy and between the ages of 18 and 29 years. You must be a nonsmoker and you cannot be taking any medications. The study will take place in the Department of Nutritional Sciences, Room 305, 334, 331 and 331A, FitzGerald Building, 150 College Street, Toronto, ON.
:
Procedure To determine if you can take part, you will be asked to fill out questionnaires, which ask questions about your age, your health, if you smoke, exercise, if you are on any medications and your eating habits. Your height and weight will be measured.
:
If you can take part, you will be asked to fill out questionnaires about the foods you like. You will be scheduled to meet with us five times (5 sessions) over a three week period. You will be provided breakfast including; milk, cereal and orange juice to eat and drink on the morning of each session. Before meeting with us, you will be asked to eat the provided breakfast between 6:00 to 9:00 am following a 10 hour fast (do not eat 10 hours before eating breakfast). Four hours later, you will start the study session at the FitzGerald Building (between 10:00 a.m. and 1:00 pm). You will be asked to stick to your normal routine, including exercise and to eat a similar meal the night before each session. You will be asked not to eat any food in the period between the breakfast and meeting with us at the Fitzgerald Building. You can drink water until one hour before meeting with us. At each session, an indwelling intravenous catheter will be inserted in your vein by a registered nurse to collect blood samples. The catheter will remain in your arm over the study session and be used to sample blood in small amounts during the test. Blood will be sampled before the treatment and after the treatment at 10, 20, 30, 50, 60, 70, 80, 110, 140, 170, 200 and 230 minutes. You will be asked to drink a whey protein, glucose or water. Each drink will contain 1.5 g of paracetamol, which is an over-the-counter analgesic that is found in numerous cold medications and is used to measure gastric emptying. During each session, blood will be sampled 11 times over 4 hours. Over the study period, a total of 450 mL blood will be collected. You will be asked to complete visual analog scales (VAS) questionnaires, measuring your appetite and physical comfort as well as the palatability (pleasantness) of the treatment and pizza during the study sessions. You will be served a pizza meal 30 minutes after you eat the treatment. Each session will take up to 4 hours of your time. In addition, salivary cortisol is a useful measure of stress and will be measured every 30 minutes. Each session will last up to 4 hours. The detailed procedure for each session is shown below in an example of a session schedule for 10 a.m. Time and Activity Schedule for Each Session
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Time Activity 6:00 Eat standard breakfast 9:45 Arrive at the FitzGerald Building (University of Toronto) 9:50 Fill in Sleep, Stress, and appetite measurement and take baseline
blood sample 10:00 - 10:05 Drink treatment or water (through out 5 min) 10:10 & 10:30 Blood sampling and appetite measurement (10, 20 and 30 min) 10:30 - 10:45 Pizza served and eaten 10:50- 13:50 Blood sampling and appetite measurement (50, 60, 70, 80, 110, 140,
170, 200 and 230 min) Voluntary Participation and Early Withdrawal: It is hoped that you will complete all five sessions. However, you may choose to stop being in the study at any time without any problems. Risks:
The risks and discomfort will come from the blood sampling procedure. During intravenous blood sampling, the volume of blood taken presents a minimal risk to subjects. At each session, blood will be sampled over a 4 hour period, representing 1/4 of the amount of blood required for a donation (450 mL). Over the study period (2.5 weeks), 450 mL will be taken, which amounts to a single donation. Subjects will be advised to refrain from donating blood during or within one month of the end of the study. There is minimal risk of infection from insertion of the catheter or venous puncture, as the area will be swabbed with alcohol. Subjects will feel a small pinch (no more than that felt when having blood taken or receiving vaccinations). There may be a small amount of bruising (blood under the surface of the skin) following the procedure, which will be minimized by pressing on the site after the catheter or syringe needle is removed.
There are no risks from measuring salivary cortisol. Saliva collection is clean and hygienic, carried out by chewing new cotton wool swabs at the indicated time points to obtain fluid samples. Sampling is also painless and can be repeated without distress. No specialised training is required. The paracetamol absorption test for the determination of gastric emptying is a safe test. The amount used in this study is below the recommended daily limit for adults of 4 g and no studies have reported adverse reactions to weekly administration of this dose. However, acute overdoses of paracetamol can lead to toxic liver damage and renal impairment. Hence, you will be asked to refrain from using paracetamol or any analgesic drugs containing it during the study. There is always a possibility that you will become ill following consumption of food, but that is very unlikely in this study. All treatments as well as pizza are hygienically and freshly prepared at the time of your session. The pizzas are stored frozen and cooked accordingly to the manufacturer’s instructions immediately before you are served.
Although no feelings of gastrointestinal discomfort are anticipated because of the treatments, physical comfort, energy/fatigue and stress will be monitored during each session using VAS questionnaires. Benefits:
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You will not benefit directly from taking part in this study. You will be shown your blood sugar results and if they are not normal you will be told and advised to talk to your doctor. All foods and drinks will be free. Confidentiality and Privacy: Confidentiality will be respected and no information that discloses your identity will be released or published without your permission unless required by law. Your name, medical history and signed consent form will be kept in a locked filing cabinet in the investigator’s office. Your results will not be kept in the same place as your name. Your results will be recorded on data sheets and in computer records that have an ID number for identification, but will not include your name. Your results, identified only by an ID number, will be made available to the study sponsor if requested. Only study investigators will have access to your individual results. Publication of Results: The results of the study may be presented at scientific meetings and published in a scientific journal. If the results are published, only average and not individual values of the subjects will be reported. Possible Commercialization of Findings: Results from this study may lead to commercialization of a product, new product formulation, changes in the labeling of a product and/or changes in the marketing of a product; you will not share in any way from the possible gains or money made by commercial application of findings. New Findings:
If anything is found during the course of this research which may have an effect on your decision to continue taking part in this study, you will be told about it. Compensation:
You will be paid $39 per session. You will also be reimbursed $6 per session for travel (bus, subway), therefore you will be paid a total of $45 per session. If you withdraw from the study before finishing or asked to withdraw, you will be paid on the basis of the sessions already completed. Rights of Subjects: Before agreeing to take part in this research study, it is important that you read and understand your role as described here in this study information sheet and consent form. You waive no legal rights by taking part in this study. If you have any questions or concerns about your rights as a participant you can contact the Ethics Review Office at [email protected] or call 416-946-3273. If you have any questions after you read through this information please do not hesitate to ask the investigators for further explanation. Dissemination of findings:
161
A summary of results will be made available to you to pick up after the study is done Copy of informed consent for participant: You are given a copy of this informed consent to keep for your own records.
Consent:
I acknowledge that the research study described above has been explained to me and that my questions have been answered to my satisfaction. I have been informed of the alternatives to participation in this study, including the right not to participate and the right to withdraw. As well, the potential risks, harms and discomforts have been explained to me. I understand that I will receive compensation for my time spent participating in the study. As part of my participation in this study, I understand that I may come in contact with certain confidential information. I agree to keep the confidentiality of such, if any, information unless it is necessary to disclose it to my health care provider(s), or to my legal representative(s). I hereby agree and give my authorized consent to participate in the study and to treat confidential information in a restrictive manner as described above. I have been given a copy of the consent form to keep for my own records. ___________________ ___________________ _____________ Participant Name Signature Date __________________ ___________________ _____________ Witness Name Signature Date ___________________ ___________________ _____________ Investigator Name Signature Date
162
APPENDIX 7. Screening Questionnaires
Phone Screening Questionnaire
Recruitment Screening Questionnaire
Sleep Habit Questionnaire
Eating Habit Questionnaire
Food Acceptability Questionnaire
Recruitment Advertising
163
7.1. Phone Screening Questionnaire Name: ________________________
Phone: ________________________ Age: _________ (20-30 years) Weight: ____________ Height: ___________ BMI: __________ Are you lactose intolerance? Yes Excluded
No _____
Do you have diabetes? Yes Excluded
No _____
What time do you normally wake up? Weekday ___________ Weekend ______ Do you usually eat breakfast? Yes ________ No Excluded
Are you currently on a diet? Yes Excluded
No _____
Are you taking any medications? Yes Excluded
No _____
Do have any major gastrointestinal liver or kidney diseases? Yes Excluded
No _____
Have you had any major surgery, medical condition within the last 6 month? Yes Excluded
No _____
Screening schedule? Yes _____ No ______ Day: __________ Time: ______ Researcher: _________________ Date: _______________
164
7.2. Recruitment Screening Questionnaire NAME: _____________________________________ AGE: _______ ADDRESS: ________________________________________________ PHONE #: _ (_____) ___________________________ HEIGHT: _________________ WEIGHT: _______________ BMI: ________ PARTICIPATION IN ATHLETICS/ EXERCISE:
ACTIVITY HOW OFTEN? HOW LONG? (HOURS)
_____________________________________________________________________ Do you usually eat breakfast? YES _______ NO _______ If YES, what do you usually eat for breakfast? _____________________________ _____________________________________________________________________ Health Status: Do you have diabetes? YES _______ NO _______ Do you have any other major disease? YES _______ NO _______ If YES, please specify _____________________________________________________________________
Are you taking any medications? YES _______ NO _______ Do you have reactions to any foods? YES _____ NO ________ If YES, please specify _____________________________________________________________________ Are you on a special diet? YES _______ NO _______ If YES, please specify _____________________________________________________________________ Do you smoke? YES _______ NO _______
165
7.3. Sleep Habit Questionnaire
1. What time do you normally wake up in the morning? During the week: ________ Weekends/days off: ________ 2. What time do you normally get out of bed? (if different from above) During the week: _________ Weekends/days off: _________ 3. What is the earliest you would get up in a normal week?
During the week: _________
Weekends/days off: _________
4. What is the latest you would get up in a normal week? During the week: _________ Weekends/days off: _________ 5. How long do you wait to eat after rising? During the week: _________
Weekends/days off: _________
166
7.4. Eating Habit Questionnaire Choose the appropriate answer to best describe your personal situation. 1. How often are you dieting?
Never ____ rarely _____ sometimes _____ often _____ always _____
2. What is the maximum amount of weight (in pounds) that you have ever lost within one month? 1 - 4 _____ 5 - 9 _____ 10 - 14 _____ 15 - 19 _____ 20+ _____
3. What is your maximum weight gain within one week?
0 – 1 ____ 1.1 - 2 _____ 2.1 – 3 _____ 3.1 - 5 _____ 5.1+ _____ 4. In a typical week, how much does your weight fluctuate?
0 – 1 _____ 1.1 – 2 _____ 2.1 - 3 _____ 3.1 - 5 _____ 5.1+ _____ 5. Would a weight fluctuation of 5lbs affect the way you live your life?
Not at all _____ slightly _____ moderately _____ very much _____ 6. Do you eat sensibly in front of others and splurge alone?
Never _____ rarely _____ often _____ always _____
7. Do you give too much time and thought to food?
Never _____ rarely _____ often _____ always _____
8. Do you have feelings of guilt after overeating?
Never _____ rarely _____ often _____ always _____
9. How conscious are you of what you are eating?
Not at all _____ slightly _____ moderately _____ extremely _____ 10. How many pounds over your desired weight were you at your maximum weight?
0-1 _____ 2 - 5 _____ 6 - 10 _____ 11 - 20 _____ 21+ _____
167
7.5. Food Acceptability Questionnaire
Please indicate with a rating between 1 and 10 how much you enjoy the following foods (1 = not at all, 10 = very much) and how often you eat them (never, daily, weekly, monthly). Enjoyment? How often?
1. Pasta __________ __________ 2. Rice __________ __________
3. Potatoes (mashed, roasted) __________ __________
4. French fries __________ __________
5. Pizza __________ __________
6. Bread, bagels, dinner rolls __________ __________
7. Sandwiches, subs __________ __________
8. Cereal __________ __________
9. Cake, donuts, cookies __________ __________
10. Tomato/vegetable juice __________ __________
Will you be able to drink a protein beverage? YES NO
At the end of each session, you will be provided with pizza. In order to provide you with a meal that you will enjoy, we ask that you rank the following pizzas according to your personal preferences (i.e. 1st, 2nd, 3rd choice)
in the space provided. If you do NOT like a particular type of pizza, then do not rank it but instead place an “X” in the space provided.
Pepperoni (cheese, pepperoni) __________ Deluxe (cheese, pepperoni, peppers, mushrooms) __________ Three-cheese (mozzarella, cheddar, parmesan) __________
168
7.6. Recruitment Advertising
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
University of TorontoDepartment of Nutritional Sciences
Adults Participates Needed!
Requirements: age 18-29 years, non-smoking
Involves: 5 sessions (4 hour session)
$ Compensation and Food are provided $
Please contact Tina at 416-978-3700 or [email protected]
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
Nutritional
Study at the UofT.
Tina
416-978-
3700 or
shorttermFI@yahoo.
ca
169
APPENDIX 8. Study Day Questionnaire
Sleep Habit and Stress Factor Questionnaire
Recent Food Intake and Activity Questionnaire
Motivation to Eat VAS
Physical Comfort VAS
Energy and Fatigue VAS
Treatment and Test Palatability
Test Meal Record
170
8.1. Sleep Habit and Stress Factor Questionnaire DATE: ________________________ ID: ____________________________ Session: ________________________
1. Did you have a normal night’s sleep last night? Yes______ No______ 2. How many hours of sleep did you have? ___________ 3. What time did you go to bed last night? ___________ 4. What time did you wake up this morning? _________ 5. Recount your activities since waking: Time Activity ________ _____________________ ________ _____________________ ________ _____________________ ________ _____________________ ________ _____________________ 6. Are you experiencing any feelings of illness or discomfort, other than those from hunger? Today: Yes ____ No_____ Past 24 hours: Yes ____ No_____ If yes, please describe briefly: ___________________________________
7. Are you under any unusual stress? Exams/reports/work deadlines, personal, etc. Today: Yes ____ No_____ Past 24 hours: Yes ____ No_____ If yes, please describe briefly: ___________________________________ ___________________________________ 8. Have you been involved in any physical activity within the past 24 hours that is unusual to your normal routine? Yes______ No______ If yes, please describe briefly: ___________________________________ ___________________________________ 9. Have you had anything to eat or drink, other than water and provided breakfast, for the past 11-12 hours? Yes______ No______ If yes, please describe briefly: ___________________________________
171
8.2. Recent Food Intake and Activity Questionnaire DATE: ________________________ ID: ________________________ Please indicate your dinner last night (list all food and drink and give an estimate of the portion size and time eaten):_______________________________________________ ________________________________________________________________________________________________________________________________________________ ________________________________________________________________________ The following three questions relate to your food intake, activity and stress over the past 24 hours. Please rate yourself by placing a small “x” across the horizontal line at the point which reflects your present feelings. 1. How would you describe your food intake over the past 24 hours? Much Much more less than usual than usual 2. How would you describe your level of activity over the last 24 hours? Much Much more less than usual than usual 3. How would you describe your level of stress over the last 24 hours? Much Much more less than usual
172
8.3. Motivation to Eat VAS DATE: ________________________ ID: ________________________ These questions relate to your “motivation to eat” at this time. Please rate yourself by placing a small “x” across the horizontal line at the point which best reflects your present feelings. 1. How strong if your desire to eat? Very Very weak strong 2. How hungry do you feel? Not As hungry hungry as I have ever felt at all 3. How full do you feel? Not full Very at all full 4. How much food do you think you could eat? Nothing A large at all amount
173
8.4. Physical Comfort VAS DATE: ________________________ ID: ________________________ These questions relate to your physical comfort at this time. Please rate yourself by placing a small “x” across the horizontal line at the point which best reflects your present feelings. 1. Do you feel nauseous? Not Very at all much 2. Does your stomach hurt? Not Very at all much 3. How well do you feel? Not well Very at all well
174
8.5. Energy and Fatigue VAS DATE: ________________________ ID: ________________________ These questions relate to your energy level and fatigue at this time. Please rate yourself by placing a small “x” across the horizontal line at the point which best reflects your present feelings. 1. How energetic do you feel right now? Not Very at all energetic 2. How tired do you feel right now? Not Very at all tired
175
8.6. Treatment and Test Palatability DATE: ________________________ ID: ________________________ These questions relate to the palatability of the food you just consumed. Please rate the pleasantness of the beverage by placing a small “x” across the horizontal line at the point which best reflects your present feelings. 1. How pleasant have you found the treatment? Not Very at all pleasant pleasant 2. How tasty have you found the treatment? Not Very at all tasty tasty 3. How did you like the texture of the treatment? Not Very at all much 4. How sweet have you found the treatment? Not Extremely at all Sweet
176
8.7. Test Meal Record
DATE: ________________________ ID: ________________________ These questions relate to the palatability of the food you just consumed. Please rate the pleasantness of the beverage by placing a small “x” across the horizontal line at the point which best reflects your present feelings. 1. How pleasant have you found the food? Not Very at all pleasant pleasant 2. How tasty have you found the food? Not Very at all tasty tasty
177
APPENDIX 9. Blood Glucose and Insulin Record ID: _______________ Treatment: _______________ Date/Time: _______________ Session #: ________________ Monitor: _________________ Standards: high __________ Low _________
Chapter 4
Baseline Blood Glucose
Preload (5 min) 15 min
30 min Blood Glucose
45 min Blood Glucose
60 min Blood Glucose
178
Chapter 5
Baseline Insulin and Blood Glucose
Preload (5 min) 15 min
30 min Insulin and Blood Glucose
Food Intake (20 min)
0 min (50 min) Insulin and Blood Glucose
15 min (65 min)Insulin and Blood Glucose
30 min (80 min) Insulin and Blood Glucose
45 min (95 min) Insulin and Blood Glucose
60 min (110 min) Insulin and Blood Glucose
90 min (140 min) Insulin and Blood Glucose
120 min (170 min) Insulin and Blood Glucose
179
APPENDIX 10. Pizza Test Meal Record
180
APPENDIX 11. Data from Chapter 6
Plasma Concentrations of Glucose
Plasma Concentrations of Insulin
Plasma Concentrations of C-Peptide
Insulin Secretion Rate
Plasma Concentrations of Amylin
Plasma Concentrations of GLP-1
Plasma Concentrations of PYY
Plasma Concentrations of GIP
Plasma Concentrations of Grelin
Plasma Concentrations of CCK
Plasma Concentrations of Free Fatty Acids
Plasma Concentrations of Triglyceride
181
11.1. Plasma Concentrations of Glucose
Time
Control Glucose
Whey Protein
10 g 20 g 10 g 20 g P*
(mmol/L)
0 min
4.93 ± 0.16
5.21± 0.13
4.90 ± 0.06
5.01 ± 0.14
4.75 ± 0.10 NS
10 min
4.93 ± 0.12b
6.03 ± 0.21a
6.09 ± 0.15a
4.89 ± 0.07b
5.05 ± 0.14b <0.0001
20 min
5.01 ± 0.1c
7.38 ± 0.21b
8.00 ± 0.15a
5.08 ± 0.07c
5.51 ± 0.16c <0.0001
30 min
5.06 ± 0.13c
7.34 ± 0.21b
8.39 ± 0.23a
5.23 ± 0.08c
5.74 ± 0.29c <0.0001
50 min
5.91 ± 0.27
5.39 ± 0.22
5.90 ± 0.33
5.08 ± 0.18
5.20 ± 0.18 NS
60 min
7.14 ± 0.25a
6.03 ± 0.15b
6.14 ± 0.32ab
5.89 ± 0.30b
5.55 ± 0.18b 0.003
70 min
6.88 ± 0.27a
6.58± 0.32ab
6.01 ± 0.21ab
6.30 ± 0.33ab
5.78 ± 0.23b <0.05
80 min
6.54 ± 0.17
6.14 ± 0.42
5.80 ± 0.27
6.24 ± 0.28
5.73 ± 0.33 NS
110 min
5.63 ± 0.1
5.41 ± 0.24
5.51 ± 0.25
5.65 ± 0.23
5.50 ± 0.27 NS
140 min
5.68 ± 0.18
5.49 ± 0.28
5.46 ± 0.22
5.63 ± 0.22
5.96 ± 0.21 NS
170 min
5.56 ± 0.13
5.56 ± 0.20
5.46 ± 0.24
5.65 ± 0.23
5.49 ± 0.21 NS
200 min
5.49 ± 0.15ab
5.59 ± 0.29a
4.86 ± 0.13b
5.51 ± 0.22ab
5.54 ± 0.19ab 0.04
230 min
5.49 ± 0.13
5.33 ± 0.19
5.19 ± 0.27
5.21 ± 0.22
5.53 ± 0.25 NS
182
4
4.5
5
5.5
6
6.5
7
7.5
8
8.5
9
0 10 20 30 50 60 70 80 110 140 170 200 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
bb
aa
b
ab
ab
a
c
cc
c
b
a
cc
bbb
b
babab
aab
abababa
Fixed MealP
lasm
a G
luco
se (m
mol
/ L)
∆ P
lasm
a G
luco
se (m
mol
/ L)
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
0 10 20 30 50 60 70 80 110 140 170 200 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
a
bbb
a
a
dcd
b
dd
c
b
a
c
bbbab
a
b
ababab
a
Fixed Meal
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
183
11.2. Plasma Concentrations of Insulin
Time
Control Glucose
Whey Protein
10 g 20 g 10 g 20 g P* (pmol/ L)
0 min
37.9 ± 11.9
55.6 ± 16.6
34.4 ± 3.6
58.6 ± 21.9
31.8 ± 2.6 NS
10 min
35.8 ± 7.0c
140.9 ± 30.3ab
168.7 ± 19.3a
68.1 ± 22.9bc
74.9 ± 10.0bc 0.0004
20
min
39.0 ± 6.7c
238.1 ± 29.1a
297.3 ± 32.2a
104.4 ±
12.8bc 142.8 ± 19.9b <0.0001
30 min
33.1 ± 4.6c
177.9 ± 13.6b
331.3 ± 34.6a
100.0 ± 9.1bc
174.6 ± 31.0b <0.0001
50 min
140.1 ± 23.0
180.8 ± 17.5
174.3 ± 24.7
160.0 ± 28.7
209.5 ± 30.8 NS
60 min
308.6 ± 31.7
260.4 ± 29.3
320.6 ± 32.1
283.6 ± 26.9
260.0 ± 25.6 NS
70 min
346.1 ± 30.2
371.3 ± 24.2
372.1 ± 33.5
333.0 ± 35.6
267.6 ± 27.4 NS
80 min
315.9 ± 34.2
355.5 ± 38.0
352.7 ± 42.6
314.3 ± 35.1
240.8 ± 31.9 NS
110 min
209.3 ± 12.5b
236.5 ± 24.0ab
311.6 ± 35.7a
218.7 ± 17.0b
200.3 ± 27.6b 0.004
140 min
213.9 ± 26.7
211.4 ± 29.4
251.3 ± 41.6
192.6 ± 12.0
193.6 ± 21.1 NS
170 min
202.3 ± 22.4
197.3 ± 18.8
262.4 ± 20.9
190.9 ± 12.9
185.5 ± 24.6 NS
200 min
190.1 ± 19.8
207.1 ± 27.8
169.6 ± 11.1
151.1 ± 13.3
167.9 ± 21.8 NS
230 min
142.1 ± 14.3
144.0 ± 16.2
185.0 ± 36.8
118.9 ± 14.2
151.4 ± 25.0 NS
184
Insu
lin (p
mol
/L)
∆ In
sulin
(pm
ol/L
)
-50
0
50
100
150
200
250
300
350
400
0 10 20 30 50 60 70 80 110 140 170 200 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
d
cd
bcb
a
d
cd
bc
b
a
ccbcab
a
bababab
abbba
a
bbbab
a
Fixed Meal
0
50
100
150
200
250
300
350
400
450
0 10 20 30 50 60 70 80 110 140 170 200 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
ab
a
cbcbc
c
bc
bb
a
c
bc
b
a
a
Fixed Meal
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
185
11.3. Plasma Concentrations of C-peptide
Time
Control Glucose
Whey Protein
10 g 20 g 10 g 20 g P*
(pmol/ L)
0 min
552.88 ± 70.52
649.38 ± 65.84
558.86 ± 37.84
678.13 ± 117.68
576.25 ± 40.25 NS
10 min
518.13 ± 50.55b
897.38 ± 110.06a
930.22 ± 86.02a
685.75 ± 117.42ab
711.38 ± 47.05ab 0.01
20 min
542.50 ± 39.18c
1391.25 ± 115.30a
1667.30 ± 132.10a
873.63 ± 84.36b
978.13 ±55.44b <0.0001
30 min
509.38 ± 33.77d
1389.00 ± 89.15b
1964.91 ± 129.58a
852.25 ± 71.02c
1125.63 ± 94.00bc <0.0001
50 min
868.25 ± 77.08c
1285.86 ± 75.67ab
1616.88 ± 134.17a
1014.00 ± 92.16bc
1170.50 ± 84.59bc <0.0001
60 min
1548.25± 95.42b
1643.38 ± 102.67ab
2096.28 ± 146.72a
1460.50 ± 113.85b
1454.38 ± 105.76b 0.002
70 min
1887.75 ± 108.64bc
2070.00 ± 73.15ab
2346.16 ± 162.12a
1822.75 ± 132.66bc
1566.88 ± 85.30c 0.0002
80 min
1971.88 ± 138.07ab
2176.00 ± 137.60ab
2380.25 ± 205.01a
1898.50 ± 126.04b
1592.25 ± 101.41b 0.0009
110 min
1711.13 ± 95.05b
1905.88 ± 132.09ab
2347.45 ± 221.40a
1825.88 ± 106.41ab
1600.38 ± 129.64b 0.002
140 min
1686.75 ± 85.07
1722.25 ± 86.67
2138.92 ± 238.52
1718.38 ± 70.68
1839.50 ± 132.49 NS
170 min
1721.00 ± 114.14ab
1659.38± 120.00b
2043.34 ± 122.88a
1687.00 ± 88.87b
1722.25 ± 128.97ab 0.01
200 min
1613.13 ± 112.35
1668.38 ± 106.25
1662.75 ± 76.91
1546.75 ± 59.75
1608.00 ± 91.42 NS
230 min
1434.13 ± 51.26
1401.25 ± 66.40
1592.23 ± 108.92
1334.13 ± 69.57
1469.75 ± 124.53 NS
186
P
lasm
a C
-Pep
tide
(pm
ol/L
) ∆
Pla
sma
C-P
eptid
e (p
mol
/L)
0
500
1000
1500
2000
2500
3000
0 10 20 30 50 60 70 80 110 140 170 200 230
Control10 g Glucose20 g Glucose10 g Whey20 g Whey
b
a
ababa
b
a
c
bb
a
a
bc bcab
a
d
c
b
bc
bab
a
c
cbcbcab
b
a
bbabab
a
bb
ab
aab
bb
ababa
Fixed Meal
-500
0
500
1000
1500
2000
2500
0 10 20 30 50 60 70 80 110 140 170 200 230
Control10 g Glucose20 g Glucose10 g Whey20 g Whey
a
d
cdc
b
a
ccbcab
a
bb
abab
a
cc
b
b
a
a
bb
abab
bbbb
a
b
a
babab ab
bbb
bbababa
Fixed Meal
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
187
11.4. Insulin Secretion Rate
Time
Control Glucose
Whey Protein
10 g 20 g 10 g 20 g P*
(pmol/ kg/ min)
0 min
2.1 ± 0.3
2.0 ± 0.2
2.1 ± 0.2
2.5 ± 0.4
2.2 ± 0.2 NS
10 min
1.8 ± 0.1d
5.1 ± 0.7ab
7.1 ± 0.7a
3.0 ± 0.4cd
3.9 ± 0.4bc <0.0001
20 min
1.9 ± 0.2d
7.4 ± 0.6b
10.7 ± 0.8a
3.9 ± 0.3c
5.3 ± 0.5c <0.0001
30 min
1.9 ± 0.2d
6.5 ± 0.9b
9.9 ± 0.9a
3.6 ± 0.3bc
5.7 ± 0.7cd <0.0001
50 min
6.1 ± 0.6
5.9 ± 0.5
7.4 ± 1.0
5.5 ± 0.5
5.4 ± 0.3 NS
60 min
9.4 ± 0.7ab
8.3 ± 0.5ab
9.9 ± 1.0a
8.0 ± 0.6ab
6.9 ± 0.7b <0.02
70 min
10.4 ± 0.9a
10.2 ± 0.8ab
10.8 ± 1.1a
9.3 ± 0.9ab
7.0 ± 0.5b 0.002
80 min
9.6 ± 0.9ab
10.3 ± 1.1ab
10.5 ± 1.3a
9.3 ± 1.0ab
6.6 ± 0.7b <0.002
110 min
5.9 ± 0.3b
6.3 ± 0.6ab
8.7 ± 1.3a
6.7 ± 0.6ab
6.6 ± 0.7ab <0.04
140 min
6.2 ± 0.6
5.8 ± 0.6
7.4 ± 1.0
6.1 ± 0.3
7.2 ± 0.7 NS
170 min
6.4 ± 0.6
6.0 ± 0.6
6.9 ± 0.5
5.9 ± 0.4
6.1 ± 0.7 NS
200 min
5.5 ± 0.7
6.0 ± 0.7
5.0 ± 0.3
5.2 ± 0.5
5.6 ± 0.5 NS
230 min
4.7 ± 0.5
4.2 ± 0.5
5.8 ± 0.9
4.1 ± 0.6
5.0 ± 0.8 NS
188
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
189
11.5. Plasma Concentrations of Amylin
Time
Control Glucose
Whey Protein
10 g 20 g 10 g 20 g P*
(pM)
0 min
18.58 ± 4.78
22.77 ± 5.58
17.74 ± 5.23
19.15 ± 3.76
23.51 ± 5.82 NS
20 min
18.42 ± 4.55c
32.2 ± 6.81ab
32.57 ± 6.28a
22.21 ± 4.15bc
27.98 ± 6.62abc 0.0008
30 min
18.36 ± 4.67c
26.8 ± 5.76ab
35.71 ± 5.94a
22.47 ± 4.01bc
28.64 ± 6.56ab <0.0001
60 min
33.11 ± 5.83
35.36 ± 6.31
34.62 ± 6.23
35.25 ± 5.39
34.04 ± 6.45 NS
80 min
37.94 ± 6.13
44.77 ± 6.82
41.89 ± 7.2
38.6 ± 5.22
34.33 ± 5.9 NS
140 min
35.84 ± 5.95
41.14 ± 6.24
43.72 ± 7.95
37.55 ± 4.75
37.47 ± 7.42 NS
230 min
35.7 ± 5.87
31.47 ± 5.85
37.82 ± 7.62
34.03 ± 5.89
37.51 ± 7.53 NS
190
0
10
20
30
40
50
60
0 20 30 60 80 140 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
a
c
abc
bc
ab
c
abab
bc
a
Fixed Meal
∆ A
myl
in(p
M)
Am
ylin
(pM
)
10
15
20
25
30
35
40
45
50
55
0 20 30 60 80 140 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
a
c
abc
bc
ab
c
ababbc
a
Fixed Meal
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
191
11.6. Plasma Concentrations of GLP-1
Time
Control Glucose
Whey Protein
10 g 20 g 10 g 20 g P*
(pg/ mL)
0 min
5.02 ± 0.47
5.40 ± 0.63
4.62 ± 0.60
4.96 ± 0.47
5.91 ± 0.45 NS
20 min
4.99 ± 0.84c
5.97 ± 0.61bc
7.25 ± 0.78ab
6.82 ± 0.66abc
8.67 ± 0.80a 0.0004
30 min
4.66 ± 0.56b
4.36 ± 0.55b
5.55 ± 0.66b
5.80 ± 0.52ab
7.05 ± 0.69a 0.0001
60 min
7.94 ± 0.93
6.72 ± 0.64
6.92 ±0.96
9.43 ± 0.78
9.72 ± 1.17 NS
80 min
6.68 ± 0.92
6.01 ± 0.30
6.81 ± 0.94
8.98 ± 0.98
9.61 ± 0.74 NS
140 min
5.99 ± 0.63
5.84 ± 0.37
6.05 ± 0.57
7.48 ± 0.47
8.25 ± 0.67 NS
230 min
5.09 ± 0.59
5.13 ± 0.56
5.32 ± 0.46
6.07 ± 0.63
6.29 ± 0.61 NS
192
-2
-1
0
1
2
3
4
5
6
0 20 30 60 80 140 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
babab
aba
b
ab
aaa
cbc
abab
a
Fixed Meal
2
3
4
5
6
7
8
9
10
11
12
0 20 30 60 80 140 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Wheya
cbcbcab
bb
ababa
Fixed Meal
Pla
sma
GLP
-1
(pg/
mL)
∆ P
lasm
a G
LP-1
(p
g/m
L)
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
193
11.7. Plasma Concentrations of PYY
Time Control Glucose Whey Protein
10 g 20 g
10 g 20 g P*
(pg/ mL)
0
min 211.06 ±
19.67 205.31 ±
13.14 199.90 ± 18.25
216.56 ± 13.15
215.98 ± 17.28 NS
20 min
198.75 ± 19.87
211.97 ± 15.58
212.37 ± 15.31
223.24 ± 12.48
234.26 ± 21.16 NS
30 min
189.11 ± 20.48
189.12 ± 13.75
202.56 ± 14.61
229.05 ± 16.15
240.60 ± 21.71 NS
60 min
231.69 ± 24.89
232.47 ± 11.30
217.06 ± 15.73
259.22 ± 19.92
254.18 ± 25.62 NS
80 min
242.57 ± 23.49
222.08 ± 13.23
234.13 ± 13.97
266.65 ± 16.89
298.62 ± 26.69 NS
140 min
235.01 ± 18.99
231.54 ± 15.56
231.13 ± 12.51
256.51 ± 12.78
284.11 ± 23.90 NS
230 min
214.67 ± 17.33
206.66 ± 11.63
199.38 ± 6.7
216.08 ± 15.53
244.69 ± 25.47 NS
194
-60
-40
-20
0
20
40
60
80
100
120
0 20 30 60 80 140 230
Control10 g Glucose20 g Glucose10 g Whey20 g Whey
babababa
bababab
a
Fixed Meal
100
140
180
220
260
300
340
0 20 30 60 80 140 230
Control10 g Glucose20 g Glucose10 g Whey20 g Whey
Fixed Meal
Pla
sma
PY
Y(p
g/m
L)∆
Pla
sma
PY
Y
(pg/
mL)
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
195
11.8. Plasma Concentration of Total GIP
Time
Control Glucose
Whey Protein
10 g 20 g
10 g 20 g P*
(pg/ mL)
0
min
98.39 ± 23.97
128.27 ± 25.17
61.72 ± 7.76
105.18 ± 26.89
140.79 ± 33.93 NS
20 min
78.56 ± 13.3b
188.24 ± 9.15a
181.68 ± 17.61a
166.41 ± 11.77a
182.66 ± 17.44a <0.0001
30 min
71.22 ± 10.57b
156.17 ± 33.3ab
174.38 ± 22.5a
187.1 ± 29.54a
176.06 ± 14.43a 0.004
60 min
303.62 ± 36.04
302.94 ± 36.37
299.85 ± 29.41
278.65 ± 27.37
217.21 ± 22.79 NS
80 min
401.83 ± 30.81
392.65 ± 34.63
412.47 ± 50.5
364.42 ± 27.15
299.00 ± 31.69 NS
140 min
475.36 ± 34.01
459.68 ± 20.78
420.73 ± 33.21
444.19 ± 17.55
383.41 ± 34.58 NS
230 min
394.83 ± 48.28
334.85 ± 24.13
336.42 ± 36.22
370.59 ± 28.34
378.84 ± 27.95 NS
196
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
197
11.9. Plasma Concentrations of Total Ghrelin
Time
Control Glucose
Whey Protein
10 g
20 g
10 g
20 g
P* (pg/ mL)
0
min
385.97 ± 71.32
538.34 ± 132.04
345.54 ± 42.42
524.98 ± 181.56
480.57 ± 140.45
NS
20 min
401.17 ± 77.14
442.19 ± 114.79
289.80 ± 38.56
545.22 ± 180.38
368.26 ± 98.86
NS
30 min
427.86 ± 80.06
351.07 ± 89.66
332.75 ± 60.60
466.56 ± 155.67
373.29 ± 83.92
NS
60 min
381.55 ± 59.19
359.38 ± 55.69
256.01 ± 52.19
457.81 ± 161.90
338.44 ± 62.88
NS
80 min
289.73 ± 67.73
326.49 ± 57.29
216.97 ± 44.98
414.06 ± 158.52
363.03 ± 74.21
NS
140 min
279.12 ± 56.46
334.16 ± 55.33
180.54 ± 38.20
386.69 ± 168.02
299.70 ± 75.11
NS
230 min
266.09 ± 61.40
342.05 ± 93.03
208.66 ± 40.38
468.32 ± 166.55
310.87 ± 79.71
NS
198
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
199
11.10. Plasma Concentrations of CCK
Time
Control
Glucose
Whey Protein
10 g
20 g
10 g
20 g
P*
(ng/ mL)
0 min
114.75 ± 13.63
106.39 ± 14.82
102.04 ± 12.28
118.49 ± 14.89
96.16 ± 12.66
NS
20 min
100.50 ± 15.29
81.81 ± 14.31
115.65 ± 14.06
117.26 ± 13.79
135.75 ± 14.16
NS
30 min
85.20 ± 16.60
122.21 ± 16.37
86.84 ± 13.66
130.17 ± 19.61
110.07 ± 12.97
NS
60 min
66.93 ± 13.63
111.18 ± 16.57
101.34 ± 10.88
132.56 ± 22.02
123.43 ± 20.16
NS
80 min
67.53 ± 12.28
124.08 ± 18.42
103.31 ± 14.46
130.46 ± 20.46
128.24 ± 18.01
NS
140 min
84.56 ± 20.02
138.44 ± 15.52
122.30 ± 9.74
115.09 ± 13.15
122.39 ± 16.05
NS
230 min
92.09 ± 18.70
136.13 ± 17.52
128.41 ± 14.83
116.0 ± 17.61
117.73 ± 11.97
NS
200
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
201
11.11. Plasma Concentrations of Free-Fatty Acids
Time
Control Glucose Whey Protein
10 g
20 g
10 g
20 g
P*
(mEQ/ L)
0 min
0.15 ± 0.06
0.12 ± 0.02
0.14 ± 0.03
0.15 ± 0.02
0.15 ± 0.05
NS
30 min
0.13 ± 0.03
0.10 ± 0.02
0.09 ± 0.01
0.10 ± 0.02
0.10 ± 0.02
NS
60 min
0.07 ± 0.01
0.09 ± 0.02
0.06 ± 0.01
0.11 ± 0.02
0.09 ± 0.02
NS
80 min
0.04 ± 0.01
0.08 ± 0.02
0.05 ± 0.01
0.09 ± 0.01
0.05 ± 0.01
NS
140 min
0.08 ± 0.01
0.12 ± 0.01
0.11 ± 0.02
0.09 ± 0.02
0.08 ± 0.01
NS
230 min
0.09 ± 0.01
0.11 ± 0.02
0.11 ± 0.02
0.10 ± 0.01
0.10 ± 0.01
NS
202
FFA
, mE
Q/L
∆ FF
A, m
EQ
/L
Fixed Meal
0
0.05
0.1
0.15
0.2
0 30 60 80 140 230
Control10 g Glucose20 g Glucose10 g Whey20 g Whey
-0.2
-0.15
-0.1
-0.05
0
0.05
0 30 60 80 140 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
Fixed Meal
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
203
11.12. Plasma Concentrations of Triglyceride
Time
Control
Glucose
Whey Protein
10 g
20 g
10 g
20 g
P*
(Mg/ dL)
0
min
23.26 ± 2.16
28.34 ± 7.41
26.05 ± 4.58
29.48 ± 4.28
25.87 ± 3.86
NS
20 min
24.86 ± 3.97
31.07 ± 6.65
25.68 ± 4.25
28.64 ± 4.64
28.27 ± 4.59
NS
30 min
23.75 ± 3.86
33.1 ± 6.36
30.02 ± 5.21
29.92 ± 5.4
28.08 ± 5.15
NS
60 min
27.96 ± 4.07
35.75 ± 6.14
29.46 ± 3.66
40.69 ± 7.65
35.95 ± 6.36
NS
80 min
31.14 ± 4.79
35.11 ± 6.43
29.15 ± 3.83
41.18 ± 8.42
33.22 ± 5.8
NS
140 min
44.33 ± 6.11
44.66 ± 6.05
42.56 ± 7.29
46.58 ± 8.6
34.6 ± 5.79
NS
230 min
44.21 ± 7.9
33.85 ± 4.33
44.83 ± 7.12
48.81 ± 6.64
40.23 ± 7.09
NS
204
∆ P
lasm
a TG
, Mg/
dLP
lasm
a TG
, Mg/
dL
-5
0
5
10
15
20
25
30
0 20 30 60 80 140 230
Control
10 g Glucose
20 g Glucose
10 g Whey
20 g Whey
Fixed Meal
15
20
25
30
35
40
45
50
55
60
0 20 30 60 80 140 230
Control10 g Glucose20 g Glucose10 g Whey20 g Whey
Fixed Meal
All values are means ± SEMs; n = 8. Values at each time of measurement with different
superscript letters are significantly different [One-way ANOVA (proc Mixed) for preload effect,
followed by Tukey’s post hoc (P < 0.05)]
