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In vivo estimation of gluconeogenesisChacko, Shaji Kurian

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IN VIVO ESTIMATION OF GLUCONEOGENESIS

Shaji Kurian Chacko

Rijksuniversiteit Groningen

In Vivo Estimation of Gluconeogenesis

Proefschrift

ter verkrijging van het doctoraat in de Medische Wetenschappen

aan de Rijksuniversiteit Groningen op gezag van de

Rector Magnificus, dr. E. Sterken, in het openbaar te verdedigen op

woensdag 25 mei 2011 om 11:00 uur

door

Shaji Kurian Chacko

geboren op 15 mei 1969

te Kerala, India

Promotores : Prof. dr. P.J.J. Sauer Prof. dr. A.L. Sunehag Beoordelingscommissie : Prof. dr. H.N. Lafeber

Prof. dr. J.B. van Goudoever Prof. dr. A.K. Groen

Table of Contents

Chapter 1 General introduction and outline of the thesis 7

Chapter 2 Measurement of gluconeogenesis using glucose 23

fragments/mass spectrometry following ingestion

of deuterium oxide

Chapter 3 Gluconeogenesis continues in premature infants 49

receiving TPN

Chapter 4 Gluconeogenesis is not regulated by either glucose 73

or insulin in extremely low birth weight (ELBW)

infants receiving total parenteral nutrition

Chapter 5 Mechanisms to conserve glucose in lactating women 95

during a 42 h fast

Chapter 6 Effect of ghrelin on glucose regulation in mice 129

Chapter 7 Subcutaneous infusion and capillary “Finger Stick” 153

sampling of stable isotope tracer in metabolic studies

Chapter 8 Summary, general discussion and future perspectives 175

Dankwoord 187

Curriculum Vitae 193

List of Publications 195

Chapter 1

General Introduction

and Outline of the Thesis

Glucose is the most important metabolic fuel that sustains the continuous

processes in all living organisms. Thus, maintaining a constant supply of glucose is

essential for their normal functions and survival. Under normal conditions the blood

glucose concentration is maintained within a very narrow range in mammals both during

absorptive and post absorptive states. This is accomplished by maintaining a balance

between the enzyme controlled glucose synthesis and hormone regulated glucose uptake

through their mutual and complex interactions.

The liver plays a predominant role in glucose homeostasis because of its ability to

produce glucose via gluconeogenesis and glycogenolysis during post absorptive

conditions and remove glucose from the blood stream during absorptive conditions.

Since the amount of glucose produced and utilized in the kidneys are more or less equal

except during acidotic and prolonged fasting conditions, they do not or contribute only

negligible amounts to the total whole body glucose production (1; 2). Both hepatic and

muscle glycogen stores are exhausted within a few hours of food deprivation in infants

and several hours in adults. As a result glucose production in the liver via

gluconeogenesis is crucial for extra-hepatic tissues during post absorptive conditions.

Gluconeogenesis plays an important role in glucose homeostasis by producing

glucose from gluconeogenic substrates either mobilized from endogenous sources via

lipolysis and proteolysis or made available from ingested food. Gluconeogenesis also

facilitates the utilization of gluconeogenic precursors such as pyruvate and lactate

generated during glucose utilization (glycolysis). The gluconeogenic pathway is the

functional reversal of the glycolytic pathway regulated by the activation/deactivation of

certain key enzymes via complex mechanisms involving glucose regulatory hormones

8

such as insulin, glucagon, cortisol, growth hormone and epinephrine, and blood glucose

concentration.

Lack of ability to control blood glucose concentration within a narrow range is an

indication of an imbalance between the rates of glucose production and glucose

utilization and is associated with conditions such as diabetes, infection, post surgery etc

(3-10). Therefore, methods and techniques that enable us to obtain quantitative estimates

of the rates of gluconeogenesis and glycogenolysis are essential to investigating glucose

metabolism during various physiological and pathological conditions. The quantitative in

vivo estimates of total gluconeogenesis were difficult because of the complexity of the

gluconeogenic pathway until various tracer methods were developed.

In vivo estimation of Gluconeogenesis

Various approaches such as estimation based on arterial-venous differences,

radioactive or stable isotope labeling of individual substrates have been employed to

determine the proportion of glucose release attributable to gluconeogenesis. About 15

years ago three approaches using compounds labeled with stable isotopes with the

potential to accurately estimate total gluconeogenesis were published (11-15). However,

each of them has both strengths and limitations. These methods are either based on mass

isotopomer distribution analysis during the infusion of uniformly 13C labeled stable

isotope tracers of glucose/ glycerol or on the deuterium labeling in glucose carbon after

the ingestion of deuterium oxide. The approach developed by Landau et al.(15) is widely

considered as the golden standard to estimate gluconeogenesis.

9

Among the various approaches to measure gluconeogenesis using compounds

labeled with stable isotopes, methods using deuterated water are simple and

straightforward (11-15). The principle of this model is based on the hydrogen/deuterium

labeling of new glucose from body water during gluconeogenesis (15). Body water is the

precursor pool for the hydrogen/deuterium on the glucose carbon skeleton. This method

first reported by Landau et al. is based on the deuterium labeling at glucose carbon 5

accounting for all the substrates entering the gluconeogenic pathway and thus, gives an

estimate of total gluconeogenesis (15). However, the analytical difficulty and large

plasma volume requirement have resulted in a limited use of this method. Therefore, a

simple alternative method requiring less sample and expertise is necessary and can

potentially promote research in the field of glucose metabolism.

There are many observations suggesting comparable deuterium labeling on

various glucose carbons during the gluconeogenic pathway. The 2H-NMR spectra of

glucose synthesized from fructose-6-phosphate and dihydroxyacetone phosphate in 2H2O

appears to have comparable deuterium labeling in glucose C-1,3,4,5,6 and C-3,4,5

respectively (16). The amount of deuterium labeling at glucose C-3 and 5 are reportedly

comparable because of the similarity in deuterium labeling at the trios phosphate level of

the gluconeogenic pathway (17). It has been reported that the incorporation of 3H on C-1

of glucose formed by hepatocytes incubated with 3H2O using different substrates was

essentially half that on C-6 (two hydrogen on C-6) suggesting that the primary

mechanism of labeling is similar between C-1 and C-6 (18). The extensive tritium

incorporation in C-3, 4 and 5 of glucose when incubated with a variety of gluconeogenic

substrates indicates that the labeling occurs throughout the gluconeogenic process (18).

10

Several investigators have reported measurements of gluconeogenesis based on

2H-enrichment at C-3, C-5 or C-6 (14; 15; 19-21). Deuterium labeling at the same or

different carbons by a single or multiple substrates (15; 19; 21-23), repeated cycling of

substrates and a series of isomerization/equilibration reactions during the gluconeogenic

pathway should result in a nearly equal distribution of deuterium labeling on glucose C-

1,3,4,5 and 6. Deuterium labeling at glucose C-2 is complete due to the extensive

glucose-6-phosphate to fructose-6-phosphate isomerization process and is not a reflection

of gluconeogenic process (14; 18). Therefore, measurement based on the average

deuterium enrichment at glucose C-1, 3, 4, 5 and 6 should provide an accurate estimate of

gluconeogenesis.

In our preliminary experiments, we studied two children with glycogen storage

disease Type-I using 2H2O to demonstrate that the average enrichment method does not

include any labeling due to non-gluconeogenic exchange reactions. The glycogen storage

disease Type-I patients lack the glucose-6-phosphatase enzyme, and therefore, are unable

to release free glucose into the plasma pool via gluconeogenesis. We observed that

fractional gluconeogenesis was essentially zero. This demonstrated that non-

gluconeogenic exchange reactions or isotope effects do not result in detectable amounts

of deuterium labeling on glucose at the level of deuterium enrichment in body water used

in our studies.

Thus, a new easy and straightforward method based on the average deuterium

enrichment on glucose C-1,3,4,5 and 6 could potentially provide a reasonable estimate of

total gluconeogenesis (including the contribution from all substrates) and that would be

useful to many investigators.

11

Measurement of gluconeogenesis in preterm infants

Disturbed glucose homeostasis is associated with increased morbidity and

mortality in very premature infants during the early weeks of life (24-28). Because of low

tolerance for enteral feeding, preterm infants are dependent on total parenteral nutrition

(TPN). However, these infants also have a reduced tolerance for parenteral glucose,

which often results in hyperglycemia. Detailed knowledge about the physiology of the

glucose metabolism in these infants is necessary to reduce the risk of

hypo/hyperglycemia and to optimize their energy intake for normal growth during this

crucial period of life.

According to recent clinical routines, very premature infants often receive TPN

providing glucose at rates exceeding normal infant glucose turnover rate. However, there

are no data on the effect of this nutritional regimen on total gluconeogenesis and

glycogenolysis in very premature infants. Thus, it is not known if gluconeogenesis is

affected by the increased amount of glucose given as a part of TPN or if it contributes to

the disturbed glucose homeostasis. Furthermore, information on potential hormonal

factors regulating gluconeogenesis under these conditions is crucial to optimize

nutritional strategies in this population. These issues are addressed in this thesis.

Gluconeogenesis during Lactation in humans

Lactating women have increased glucose demands during fasting to meet the

substrate needs for lactose synthesis. Previously it was shown that during a 24 h fast, this

higher demand was met by increased glycogenolysis (29). However, it is not known how

lactating women adapt to the increased glucose demands during extended fasting periods.

12

As a part of this thesis, we sought to determine whether lactating women conserve

plasma glucose by increasing gluconeogenesis during extended periods of fasting.

Effect of Ghrelin on glucose regulation in mice

Obesity is a global epidemic and is associated with significant risk for metabolic

diseases (30-32). Metabolic abnormalities such as type 2 diabetes are of great concern

and their economic burden on society is alarming. Diabetes is associated with disturbed

glucose homeostasis resulting from imprecise control of glucose production and

utilization (3-5). More research is needed to find effective alternatives to treat diabetes

and to control this worldwide health crisis.

In addition to weight loss, remission of type 2 diabetes has been recently reported

after bariatric surgery procedures. Both short term and long term effectiveness of these

procedures in treating diabetes in the obese population has received immense interest (33-

35). Although lowering of plasma ghrelin concentrations after bariatric surgery

procedures has been suggested as the potential mechanism for this altered glucose

metabolism [21], the association between improvement of glucose metabolism and lower

ghrelin concentration remains to be determined. Glucose kinetic studies in the absence of

ghrelin or its receptor using transgenic mice models (36-39) provide an opportunity to

investigate the effects of ghrelin on glucose metabolism and have been used in this thesis.

13

Application of subcutaneous infusion technique to perform in vivo human metabolic

studies

Metabolic studies utilizing stable isotope tracers in humans have typically utilized

intravenous tracer infusions and venous blood sampling and therefore, are always carried

out in an inpatient or outpatient clinical research setting. However, such study conditions

dramatically decrease the subject’s normal activity during the period of study and thus,

do not represent real-life conditions. The use of a subcutaneous tracer infusion and a

“finger stick” blood sampling method is a possible alternative to study glucose kinetics

under more real-life conditions. We have used a simultaneous intravenous and

subcutaneous infusion of two glucose tracers and corresponding blood sampling which

provided an opportunity to evaluate the application of this method in future metabolic

studies in real life situations.

Collectively, the objective of this thesis is to develop a simple, yet accurate and

reproducible method to measure gluconeogenesis, and estimate in vivo rates of

gluconeogenesis applying the new and other methods in different populations under

various conditions to address different issues in the field of glucose metabolism.

Outline of the thesis

The first aim of this thesis is to develop a method to estimate rates of

gluconeogenesis that is straightforward, cost-effective and simple and that requires

minimal sample volumes (i.e. applicable to all subject populations) using deuterated

water. The following questions are addressed:

14

• What is the suitable derivative and gas chromatography-mass spectrometry (GC-

MS) fragment of glucose that carries the exchangeable hydrogen that best

represent the gluconeogenic process?

• How does the new method compare with the hexamethylenetetramine (HMT)

method reported by Landau et al. (15) when measured under different

physiological conditions in different populations? (Chapter 2)

• What is the reproducibility of data on gluconeogenesis obtained by the new

method? (Chapter 2)

The second aim of this thesis is to measure gluconeogenesis in very premature

infants receiving glucose as a part of routine total parenteral nutrition (TPN) i.e. at rates

exceeding their normal glucose turnover rate. Further, this thesis seeks to determine

potential factors regulating gluconeogenesis in Extremely Low Birth Weight infants

receiving routine total parenteral nutrition. The following questions are specifically

addressed:

• Is gluconeogenesis sustained in very preterm infants receiving routine total

parenteral nutrition providing glucose at rates exceeding normal infant glucose

turnover rate? (Chapter 3)

• What is the contribution of total gluconeogenesis to glucose production in very

preterm infants receiving total parenteral nutrition providing glucose exceeding

normal infant glucose turnover rate? (Chapter 3)

• Is gluconeogenesis affected by glucose infusion rate, blood glucose

concentrations, birth weight or gestational age? (Chapter 3 and 4)

15

• Is gluconeogenesis affected by a substantial reduction in glucose supply and

subsequent changes in insulin or any other potential insulin counter regulatory

hormone concentrations? (Chapter 4)

The higher glucose demand in lactating women during a 24 h fast was met by

increased glycogenolysis while gluconeogenesis continued at similar rates to that in

controls (29). The third aim of this thesis is to determine whether or how lactating women

meet the demand of lactose synthesis during an extended period of fasting (without

becoming hypoglycemic). The following questions are addressed:

• How do women adapt to their own higher glucose requirement during lactation?

And does gluconeogenesis increase to meet the substrate needs for lactose

synthesis during an extended period of fasting in lactating women as compared to

controls? (Chapter 5)

The fourth aim of this thesis is to determine the effect of the absence of the

hormone ghrelin or its purported receptor on insulin sensitivity and glucose production

from gluconeogenesis and glycogenolysis in mice. The following questions are addressed:

• What is the effect of the absence of ghrelin or its purported receptor on hepatic

and peripheral insulin sensitivity in mice? (Chapter 6)

• Does gluconeogenesis and glycogenolysis change in the absence of ghrelin or its

purported receptor during post absorptive conditions in mice? (Chapter 6)

16

The fifth aim of the thesis is to develop a metabolic study design using subcutaneous

tracer infusion in humans that minimally interfere with the normal activity of the subject,

such that glucose appearance rate can be measured in real life situations. The following

question is addressed:

• Does the data from subcutaneous infusion followed by” finger stick” blood

sampling compare well with the established intravenous tracer infusion and

venous blood sampling technique? (Chapter 7)

The final section of this thesis (chapter 8) provides a summary, discussion of the

main findings of the thesis and future directions of research in continuum to the works

accomplished as a part of this thesis.

17

References

1. McGuinness OP, Fugiwara T, Murrell S, Bracy D, Neal D, O'Connor D, Cherrington

AD: Impact of chronic stress hormone infusion on hepatic carbohydrate metabolism in

the conscious dog. Am J Physiol 1993;265:E314-322

2. Stumvoll M, Meyer C, Mitrakou A, Nadkarni V, Gerich JE: Renal glucose production

and utilization: new aspects in humans. Diabetologia 1997;40:749-757

3. Gastaldelli A, Toschi E, Pettiti M, Frascerra S, Quinones-Galvan A, Sironi AM, Natali

A, Ferrannini E: Effect of physiological hyperinsulinemia on gluconeogenesis in

nondiabetic subjects and in type 2 diabetic patients. Diabetes 2001;50:1807-1812

4. Iozzo P, Hallsten K, Oikonen V, Virtanen KA, Kemppainen J, Solin O, Ferrannini E,

Knuuti J, Nuutila P: Insulin-mediated hepatic glucose uptake is impaired in type 2

diabetes: evidence for a relationship with glycemic control. J Clin Endocrinol Metab

2003;88:2055-2060

5. DeFronzo RA: Lilly lecture 1987. The triumvirate: beta-cell, muscle, liver. A collusion

responsible for NIDDM. Diabetes 1988;37:667-687

6. van Thien H, Ackermans MT, Dekker E, Thanh Chien VO, Le T, Endert E, Kager PA,

Romijn JA, Sauerwein HP: Glucose production and gluconeogenesis in adults with

cerebral malaria. QJM 2001;94:709-715

7. Dekker E, Hellerstein MK, Romijn JA, Neese RA, Peshu N, Endert E, Marsh K,

Sauerwein HP: Glucose homeostasis in children with falciparum malaria: precursor

supply limits gluconeogenesis and glucose production. J Clin Endocrinol Metab

1997;82:2514-2521

18

8. Gandhi GY, Nuttall GA, Abel MD, Mullany CJ, Schaff HV, Williams BA, Schrader

LM, Rizza RA, McMahon MM: Intraoperative hyperglycemia and perioperative

outcomes in cardiac surgery patients. Mayo Clin Proc 2005;80:862-866

9. McCowen KC, Malhotra A, Bistrian BR: Stress-induced hyperglycemia. Crit Care Clin

2001;17:107-124

10. Faustino EV, Apkon M: Persistent hyperglycemia in critically ill children. J Pediatr

2005;146:30-34

11. Katz J, Tayek JA: Gluconeogenesis and the Cori cycle in 12-, 20-, and 40-h-fasted

humans. Am J Physiol 1998;275:E537-542

12. Haymond MW, Sunehag AL: The reciprocal pool model for the measurement of

gluconeogenesis by use of [U-(13)C]glucose. Am J Physiol Endocrinol Metab

2000;278:E140-145

13. Hellerstein MK, Neese RA, Linfoot P, Christiansen M, Turner S, Letscher A: Hepatic

gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope

study. J Clin Invest 1997;100:1305-1319

14. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC: Use

of 2H2O for estimating rates of gluconeogenesis. Application to the fasted state. J Clin

Invest 1995;95:172-178

15. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC:

Contributions of gluconeogenesis to glucose production in the fasted state. J Clin Invest

1996;98:378-385

16. Schleucher J, Vanderveer PJ, Sharkey TD: Export of carbon from chloroplasts at

night. Plant Physiol 1998;118:1439-1445

19

17. Jones JG, Carvalho RA, Sherry AD, Malloy CR: Quantitation of gluconeogenesis by

(2)H nuclear magnetic resonance analysis of plasma glucose following ingestion of

(2)H(2)O. Anal Biochem 2000;277:121-126

18. Rognstad R, Clark G, Katz J: Glucose synthesis in tritiated water. Eur J Biochem

1974;47:383-388

19. Jin ES, Jones JG, Merritt M, Burgess SC, Malloy CR, Sherry AD: Glucose

production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by

nuclear magnetic resonance analysis of a single glucose derivative. Anal Biochem

2004;327:149-155

20. Chandramouli V, Ekberg K, Schumann WC, Wahren J, Landau BR: Origins of the

hydrogen bound to carbon 1 of glucose in fasting: significance in gluconeogenesis

quantitation. Am J Physiol 1999;277:E717-723

21. Jones JG, Solomon MA, Cole SM, Sherry AD, Malloy CR: An integrated (2)H and

(13)C NMR study of gluconeogenesis and TCA cycle flux in humans. Am J Physiol

Endocrinol Metab 2001;281:E848-856

22. Jones JG, Perdigoto R, Rodrigues TB, Geraldes CF: Quantitation of absolute 2H

enrichment of plasma glucose by 2H NMR analysis of its monoacetone derivative. Magn

Reson Med 2002;48:535-539

23. Leadlay PF, Albery WJ, Knowles JR: Energetics of triosephosphate isomerase:

deuterium isotope effects in the enzyme-catalyzed reaction. Biochemistry 1976;15:5617-

5620

24. Hays SP, Smith EO, Sunehag AL: Hyperglycemia is a risk factor for early death and

morbidity in extremely low birth-weight infants. Pediatrics 2006;118:1811-1818

20

25. Alsweiler JM, Kuschel CA, Bloomfield FH: Survey of the management of neonatal

hyperglycaemia in Australasia. J Paediatr Child Health 2007;43:632-635

26. Beardsall K, Vanhaesebrouck S, Ogilvy-Stuart AL, Vanhole C, Palmer CR, van

Weissenbruch M, Midgley P, Thompson M, Thio M, Cornette L, Ossuetta I, Iglesias I,

Theyskens C, de Jong M, Ahluwalia JS, de Zegher F, Dunger DB: Early insulin therapy

in very-low-birth-weight infants. N Engl J Med 2008;359:1873-1884

27. Hall NJ, Peters M, Eaton S, Pierro A: Hyperglycemia is associated with increased

morbidity and mortality rates in neonates with necrotizing enterocolitis. J Pediatr Surg

2004;39:898-901; discussion 898-901

28. Alaedeen DI, Walsh MC, Chwals WJ: Total parenteral nutrition-associated

hyperglycemia correlates with prolonged mechanical ventilation and hospital stay in

septic infants. J Pediatr Surg 2006;41:239-244; discussion 239-244

29. Tigas S, Sunehag A, Haymond MW: Metabolic adaptation to feeding and fasting

during lactation in humans. J Clin Endocrinol Metab 2002;87:302-307

30. Taubes G: As obesity rates rise, experts struggle to explain why. Science

1998;280:1367-1368

31. Kopelman PG: Obesity as a medical problem. Nature 2000;404:635-643

32. Must A, Spadano J, Coakley EH, Field AE, Colditz G, Dietz WH: The disease burden

associated with overweight and obesity. JAMA 1999;282:1523-1529

33. Abbatini F, Rizzello M, Casella G, Alessandri G, Capoccia D, Leonetti F, Basso N:

Long-term effects of laparoscopic sleeve gastrectomy, gastric bypass, and adjustable

gastric banding on type 2 diabetes. Surg Endosc 2010;24:1005-1010

21

34. Vidal J, Ibarzabal A, Nicolau J, Vidov M, Delgado S, Martinez G, Balust J, Morinigo

R, Lacy A: Short-term effects of sleeve gastrectomy on type 2 diabetes mellitus in

severely obese subjects. Obes Surg 2007;17:1069-1074

35. Miguel GP, Azevedo JL, Gicovate Neto C, Moreira CL, Viana EC, Carvalho PS:

Glucose homeostasis and weight loss in morbidly obese patients undergoing banded

sleeve gastrectomy: a prospective clinical study. Clinics (Sao Paulo) 2009;64:1093-1098

36. Sun Y, Wang P, Zheng H, Smith RG: Ghrelin stimulation of growth hormone release

and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl

Acad Sci U S A 2004;101:4679-4684

37. Ukkola O: Ghrelin and insulin metabolism. Eur J Clin Invest 2003;33:183-185

38. Sun Y, Ahmed S, Smith RG: Deletion of ghrelin impairs neither growth nor appetite.

Mol Cell Biol 2003;23:7973-7981

39. Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M,

Thabet K, Cox HJ, Yancopoulos GD, Wiegand SJ, Sleeman MW: Genetic deletion of

ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl

Acad Sci U S A 2004;101:8227-8232

22

Chapter 2

Measurement of gluconeogenesis using glucose fragments/mass

spectrometry following ingestion of deuterium oxide

Shaji K. Chacko

Agneta L. Sunehag

Susan Sharma

Pieter J. J. Sauer

Morey W. Haymond

J Appl Physiol 104: 944-951, 2008.

23

Abstract: We report a new method to measure the fraction of glucose derived from

gluconeogenesis using gas chromatography mass spectrometry and positive chemical

ionization. Following ingestion of deuterium oxide, glucose derived from

gluconeogenesis is labeled with deuterium. Our calculations of gluconeogenesis are

based on measurement of the average enrichment of deuterium on the 1, 3, 4, 5 and 6

carbons of glucose and the deuterium enrichment in body water. In a sample from an

adult volunteer following ingestion of deuterium oxide, fractional gluconeogenesis using

the “average deuterium enrichment method” was 48.3±0.5% (Mean ± SD) and that of the

C-5 HMT method by Landau et al. was 46.9±5.4%. The coefficient of variation (CV) of

10 replicate analyses using the new method was 1.0% compared to 11.5% for the C-5

HMT method. In samples derived from an infant receiving total parenteral nutrition,

fractional gluconeogenesis was 13.3±0.3% using the new method and 13.7±0.8 % using

the C-5 HMT method. Fractional gluconeogenesis measured in six adult volunteers after

66 h of continuous fasting was 83.7±2.3% using the new method and 84.2±5.0% using

the C-5 HMT method. In conclusion, the “average deuterium enrichment method” is

simple, highly reproducible and cost effective. Further, it requires only small blood

sample volumes. Using an additional tracer, glucose rate of appearance can also be

measured during the same analysis. Thus, the new method makes measurements of

gluconeogenesis available and affordable to large numbers of investigators under

conditions of low and high fractional gluconeogenesis (~10 to ~90) in all subject

populations.

24

Introduction

Until recently it has been difficult to obtain quantitative in vivo estimates of

gluconeogenesis and it still remains a challenging problem. Over the past decade, several

methods to measure gluconeogenesis using compounds labeled with stable isotopes e.g.

[U-13C]glucose, [2-13C]glycerol and deuterium oxide (2H2O) have been published [3-5,9-

12,20,21]. Each of them has its own advantages and disadvantages. The 2H2O method

[10] is based on the measurement of deuterium in glucose carbon 5 which is purported to

represent total gluconeogenesis. The degree of analytical difficulty and high costs of the

isotopes has limited a wide use of these methods. The necessity of a simple, yet accurate

and reproducible method to measure gluconeogenesis is a pre-requisite for conducting

quality research under various physiological and pathological conditions. We have

developed a simple, straightforward and cost effective method to estimate

gluconeogenesis using the average deuterium enrichment on glucose carbons in a specific

glucose fragment measured by gas chromatography mass spectrometry (GCMS), thus,

making the measurement of gluconeogenesis available to large numbers of investigators.

This required identifying a glucose derivative with a mass fragment that carries all

the hydrogens except that on glucose carbon two. It is because of the fact that the

deuterium labeling at glucose carbon two is reported to be due to the isomerization

process and glycogenolysis [7, 11]. The careful analysis of the mass spectrum of the

pentaacetate derivative of a variety of deuterium labeled glucose isotopes using positive

chemical ionization has revealed a simple approach for measuring fractional

gluconeogenesis. Among the various fragments of glucose observed, m/z 169 is of

25

specific interest due to the presence of six covalently bonded H/D to the carbon chain

skeleton of glucose except that on carbon two [2].

The development of this method required: 1) Identifying the carbons and

hydrogens in the m/z 169 GCMS fragment of the pentaacetate derivative of glucose; 2)

Determining the average deuterium enrichment on the glucose carbons in a specific

fragment (m/z 170/169) of the glucose molecule; 3) Evaluating the reproducibility of the

deuterium enrichment measured over a wide range of abundances using m/z 169 fragment

and 4) Comparing fractional gluconeogenesis obtained by the new method with those of

the C-5 HMT method [10] under different physiological conditions. We demonstrate that

the average deuterium enrichment method will provide estimates of gluconeogenesis

equal to those obtained by the C-5 HMT method [10] under conditions of overnight fast,

three day fast and total parenteral nutrition.

Method

a) Materials:

[1-2H]glucose (99 atom % 2H), [3-2H]glucose (98 atom % 2H), [6,6-2H2]glucose

(99 atom % 2H), [1,2,3,4,5,6,6-2H7]glucose (99 atom % 2H), [U-13C]glucose, 2H2O (99

atom % 2H) were obtained from Cambridge Isotope laboratories (Andover, MA); [2-

2H]glucose (97 atom % 2H) was obtained from Isotec (Miamisburg, OH) and [5-2H]glucose

(98 atom % 2H) from Omicron Biochemicals (South Bent, IN). Reagent grade acetic

anhydride, pyridine and ethyl acetate were purchased from Sigma (St. Louis, MO).

b) Derivatization, GCMS and GC-C-IRMS analyses:

26

Enrichments of glucose labeled with deuterium and carbon 13 (13C) were

measured by GCMS and gas chromatography combustion isotope ratio mass

spectrometry (GC-C-IRMS), respectively, using the pentaacetate derivative [1]. A

volume of 25 µl of each standard and deproteinized plasma samples (deproteinized with

100 µl cold acetone) were aliquotted into 4 ml vials and taken to dryness under nitrogen

at room temperature. Fifty micro liters (50 µl) acetic anhydride/pyridine (2:1) were added

to each sample, after which the samples were heated to 60ºC for 10 minutes or allowed to

sit overnight at room temperature. Then, the samples were dried under nitrogen at room

temperature, reconstituted in 50 µl ethyl acetate, and transferred to autosampler vials. The

derivatized standards and samples were analyzed using GCMS (GC 6890, MS 5973N,

Agilent Technologies, Wilmington, DE) with an RTX-1701 column (30m X 0.25mm I.D

X 0.5 um film; Restek, Inc.,Bellefonte, PA). The GC conditions were as follows; injector:

250ºC (splitless injection); oven: initial 70ºC for 1.0 min; ramp 30ºC / min to 275ºC for 7

min. The positive chemical ionization mode of GCMS (Source at 250ºC, Quadrupole at

106ºC) was employed using methane as the reagent gas. Deuterium enrichment in plasma

water was measured using isotope ratio mass spectrometry (Delta+XL IRMS Thermo

Finnigan, Bremen, Germany) as an average of multiple measurements.

Determining the rate of gluconeogenesis requires the use of an additional

glucose tracer (to measure glucose appearance rate), which could potentially interfere

with the measurement of the deuterium enrichment of glucose. The most commonly used

stable isotope glucose tracers to measure plasma glucose appearance rate are [1-

13C]glucose and [6,6-2H2]glucose. The [1-13C]glucose enrichment can be measured by

GC-C-IRMS as previously described [6]. This technique provides a measure of

27

enrichment specifically reflecting the 13C tracer. Alternatively using [6,6-2H2]glucose, the

M+2 enrichment is measured using either m/z 171/169 or m/z 333/331 in the positive

chemical ionization mode during the same run used to measure deuterium labeling from

ingested deuterated water or m/z 244/242 in the electron impact ionization mode of

GCMS [6,15-19] . The slope of standard curves are always used to correct instrument

deviations. We have evaluated whether these tracers would interfere with our approach

measuring gluconeogenesis using 2H2O (See section- “Potential interference of using [1-

13C]glucose or [6,6-2H2]glucose to measure glucose appearance rate”). In the studies

described in this manuscript, we have used [6,6-2H2]glucose for the measurement of

glucose rate of appearance.

The C-5 HMT method of Landau et. al [10], which was used for method

comparison involves HPLC purification of glucose from other plasma components,

conversion of glucose to xylose, HPLC purification of xylose, distillation of the

formaldehyde, and subsequent derivatization to hexamethylenetetramine (HMT). Then

the resulting product (HMT) is analyzed by GCMS using the fragment m/z 141/140 to

measure deuterium incorporation at carbon 5 of glucose.

c) Determination of sites of labeling on glucose fragments and measurement of

enrichments on the carbons of specific fragments of glucose:

For determination of the deuterium/hydrogen labeling sites on the m/z 169 GCMS

fragment of glucose pentaacetate, specific deuterium labeled glucose compounds were

derivatized and analyzed in the positive chemical ionization scan mode of GCMS

(Fig.1a-1h) with subsequent comparison of the mass spectral data [2]. Selective ion

28

Fig 1a-1h

100 200 300 4000

1

2

3

4

5

6

m/z-->

Abu

ndan

ce X

105

61

169

331

109

27121183139 229

289187 419247 393 467309 375352

Fig 1a. Natural glucose

100 200 300 400100 200 300 4000

1

2

3

4

5

6

0

1

2

3

4

5

6

m/z-->

Abu

ndan

ce X

105

61

169

331

109

27121183139 229

289187 419247 393 467309 375352

Fig 1a. Natural glucose Fig 1b. [1,2,3,4,5,6,6-2H7]glucose

100 200 300 4000

4

8

12

16

20

24

28

m/z-->

Abu

ndan

ce X

104

61

338

175

114

89 236 280216143194 393254 426298 467365

Fig 1b. [1,2,3,4,5,6,6-2H7]glucose

100 200 300 400100 200 300 4000

4

8

12

16

20

24

28

0

4

8

12

16

20

24

28

m/z-->

Abu

ndan

ce X

104

61

338

175

114

89 236 280216143194 393254 426298 467365

Fig 1c. [1-2H]glucose

100 200 300 400

0

6

12

18

24

30

m/z-->

Abu

ndan

ce X

104

61

170

332110

83 212 272140 230290188 420393248 467309

Fig 1c. [1-2H]glucose

100 200 300 400100 200 300 400

0

6

12

18

24

30

m/z-->

Abu

ndan

ce X

104

61

170

332110

83 212 272140 230290188 420393248 467309

Fig 1d. [2-2H]glucose

100 200 300 400 5000

10

20

30

m/z-->

Abu

ndan

ce X

105

332

169

109

61 211 271145 24389 291 420189 374 500394 458311

Fig 1d. [2-2H]glucose

100 200 300 400 500100 200 300 400 5000

10

20

30

0

10

20

30

m/z-->

Abu

ndan

ce X

105

332

169

109

61 211 271145 24389 291 420189 374 500394 458311

Fig 1e. [3-2H]glucose

100 200 300 400 5000

4

8

12

16

20

24

28

m/z-->

Abu

ndan

ce X

105

170

332

110

27261

212

14089 243 420292 360 390190 501441 467

Fig 1e. [3-2H]glucose

100 200 300 400 500100 200 300 400 5000

4

8

12

16

20

24

28

m/z-->

Abu

ndan

ce X

105

170

332

110

27261

212

14089 243 420292 360 390190 501441 467

Fig 1f. [5-2H]glucose

100 200 300 4000

4

8

12

m/z-->

Abu

ndan

ce X

105

170

61

332

109

272

212140230

89 290188 420248 393360311 467

Fig 1f. [5-2H]glucose

100 200 300 400100 200 300 4000

4

8

12

m/z-->

Abu

ndan

ce X

105

170

61

332

109

272

212140230

89 290188 420248 393360311 467

Fig 1g. [6,6-2H2]glucose

100 200 300 400

0

2

4

6

8

m/z-->

Abu

ndan

ce X

105

61

171

333

111

213 273

85 231139 291189 421249 393 467375309 357

Fig 1g. [6,6-2H2]glucose

100 200 300 400100 200 300 400

0

2

4

6

8

m/z-->

Abu

ndan

ce X

105

61

171

333

111

213 273

85 231139 291189 421249 393 467375309 357

Fig h. [U-13C]glucose

100 200 300 400

61

175

337

115

277217

14523589

295193 425253 393365 467315

Abu

ndan

ce X

105

m/z-->

0

2

4

6

8

10

12

Fig h. [U-13C]glucose

100 200 300 400

61

175

337

115

277217

14523589

295193 425253 393365 467315

Abu

ndan

ce X

105

m/z-->

0

2

4

6

8

10

12

Figure (1a-h). Mass spectra of various deuterium and carbon labeled glucose compounds

(as pentaacetate derivative) obtained in the positive chemical ionization mode of GCMS

for the determination of the deuterium/hydrogen labeling sites on different GCMS

fragments.

29

monitoring of m/z 170/169 was performed to determine the M+1 enrichment of deuterium

in the circulating glucose carbons (C-1,3,4,5,6,6). To measure accurately the deuterium

labeling in glucose from 2H2O, the enrichment of M+1 resulting from the natural

abundance is subtracted. The average enrichment of deuterium on a gluconeogenic

glucose carbon is then calculated from this M+1 data. Measurement of the enrichment at

M+1 gives the deuterium incorporation resulting from the exchange of deuterium from

2H2O with covalently bonded hydrogens on glucose carbons during the gluconeogenic

pathway when no other tracers are infused or ingested.

d) Calculation;

As evident from the mass spectrum of glucose (Fig 1a-1h), the fragment m/z 169

carries hydrogens at C-1,3,4,5,6,6 (see details in the result section).

1) Assuming that the 2H labeling of exchangeable hydrogens is identical on all glucose

carbons except that on carbon 2 that is in complete exchange with body water, the

average enrichment of 2H on each glucose carbon is calculated using the following

equation:

Average (M+1) d = (M+1) d (m/z 169) / 6 ….….. .........Equ (1)

where (M+1) d (m/z 169) is the M+1 enrichment of deuterium of glucose measured using

m/z 170/169 and ‘6’ is the number of 2H labeling sites on the m/z 169 fragment of

glucose.

2) Since the body water is the precursor pool for the H/D, the extent of deuterium

labeling of glucose during the gluconeogenic process when 2H2O is ingested or infused is

a measure of fractional gluconeogenesis. This is based on the assumption that the

30

deuterium enrichment measurement is performed under near steady state condition.

Therefore, using the average deuterium enrichment in m/z 170/169 for calculating

fractional gluconeogenesis, the equation is:

Fractional gluconeogenesis = Average (M+1 ) d / E H2O …….Equ (2)

where E H2O is the deuterium enrichment in body water.

e) Experiments using human plasma following ingestion of 2H2O;

Samples were utilized from studies intended to measure gluconeogenesis. The

studies were approved by the Institutional Review Board for human research at Baylor

College of Medicine and were performed after written consent had been obtained. We

have compared gluconeogenic measurements using our method based on average

deuterium enrichment with those of the C-5 HMT method [10] under conditions when

fractional gluconeogenesis is low, intermediate and high.

In an adult volunteer, after overnight fasting, five doses of 99.8% 2H2O were

ingested at 2h intervals, (a total of 5g/Kg). Plasma samples were obtained prior to isotope

administration and 6h following the last dose representing approximate steady state

[17,18]. Pentaacetate derivatization of plasma glucose was performed and the deuterium

enrichment in plasma water was measured as described above.

To determine the validity of the new method in samples with low fractional

gluconeogenesis, samples were used from an infant (Birth Weight 880 g, Gestational Age

25 weeks, Postnatal Age 5 days, Blood Glucose 227 mg/dL (12.6 mM)). The infant

received total parenteral nutrition consisting of glucose at 16 g/Kg.day (11.13

mg/Kg.min), lipid (Intralipid 20%) at 4 g/Kg.day (2.65 mg/Kg.min), and protein

31

(TrophAmine) at 3.1 g/Kg.day (2.14 mg/Kg.min). The infant also received sterile 2H2O (4

g/Kg over a period of 2h) dissolved in isotonic saline intravenously via an umbilical

venous catheter to measure gluconeogenesis. Plasma samples were obtained prior to the

2H2O administration, at 9.5h and 10h (“steady state”) after the completion of the 2H2O

infusion.

For validation of the new method under conditions of high fractional

gluconeogenesis, samples from 6 healthy adult male human volunteers (26 ± 3 years,

Weight 69 ± 5 kg and BMI 23 ±1 kg/m2) were used. The volunteers had consumed a

normal diet (50% carbohydrate, 15% protein and 35% fat) for three days prior to the

study after which they were admitted to the Metabolic Research Unit at the Children’s

Nutrition Research Center. In the afternoon of day-1 of the study, a standard meal was

served, after which they were fasted continuously for 66 h except for water ad libitum.

On day-3, 46 h into the fast, a baseline plasma sample was obtained before 5 oral doses

of 99.8% 2H2O (3g/Kg) were administered at 2 h intervals. Blood samples were collected

on day-4 at 15 min intervals between 65 – 66 h of the fast. These samples had previously

been analyzed using the C-5 HMT method [10] to measure fractional gluconeogenesis.

Due to the higher volume requirement of C-5 HMT method [10], not all samples were

available for reanalysis with the average deuterium enrichment method (new method) for

comparative analysis. All five samples were available in two subjects, four samples in

one subject, two samples in one subject and one sample in two subjects for analysis with

the new method (see table-4).

32

f) Potential interference of [1-13C]glucose or [6,6-2H2]glucose during glucose appearance

rate measurement:

To determine the rate of gluconeogenesis, the glucose appearance rate must also be

measured [17-19]. Therefore, it is crucial that the infused glucose tracers do not interfere

with the primary measure of gluconeogenesis. To assess the effect of potential glucose

tracers on the measurements of fractional gluconeogenesis when 2H2O is used, the adult

overnight fasting plasma sample was spiked with either [1-13C]glucose or [6,6-2H2]glucose

to achieve enrichments of 0 to 5%, which covers the range of enrichment used in human

studies. To calculate the M+1 enrichment representing the deuterium label derived from

2H2O, the 13C enrichment (obtained by GC-C-IRMS) was subtracted from the total M+1

enrichment of glucose (the sum of the 13C and 2H labeling of circulating glucose obtained

by GCMS). Measurements of fractional gluconeogenesis were performed employing the

new method to the unspiked sample and the sample spiked with either [1-13C]glucose or

[6,6-2H2]glucose.

Statistics: Coefficient of variation (CV) was calculated by dividing standard deviation (SD)

by the mean. The data obtained by the new method was compared to those of the C-5 HMT

method [10] using paired t- test (data obtained from ten analyses of one adult sample,

triplicate analyses of two infant samples and data obtained in six long term fasting adults).

A p- value <0.05 was used to define significance. In addition, Bland-Altman test was

applied to the 34 paired measurements obtained by the two methods.

33

Results

Glucose fragments and calculation of deuterium enrichment at glucose carbon 5:

Figures (1a-1h) depict the mass spectra of natural glucose, [2H7]glucose, [1-

2H]glucose, [2-2H]glucose, [3-2H]glucose, [5-2H]glucose, [6,6-2H2]glucose and [U-

13C]glucose, respectively, obtained in the positive chemical ionization scan mode. The

mass spectrum of glucose pentaacetate provides three important fragments at m/z 109,

m/z 169 and m/z 331. The hydrogen/deuterium labeling sites in these ion fragments of

different deuterium labeled glucose pentaacetate compounds are depicted in Table-1. The

fragment m/z 169 carries the hydrogen at C-1, C-3, C-4, C-5 and C-6, 6 of glucose, but

not at C-2. The fragment m/z 331 carries all the hydrogen at C-1, C-2, C-3, C-4, C-5 and

C-6, 6 while the fragment m/z 109 has the hydrogen at C-1, C-3, C-4 and C-6, 6 of

glucose. The presence of deuterium label at carbon 4 in the fragment m/z 169 and m/z 109

was confirmed by comparing mass spectrum obtained from [2H7]glucose with the mass

spectra of all the other deuterium labeled glucose compounds. Comparison of mass

spectra (Fig.1a-1h) and mass shifts in m/z 169 of various deuterium labeled glucose

compounds confirms that fragment m/z 169 carries all covalently bonded hydrogens in

the glucose carbon chain except that on carbon 2. Therefore, we selected m/z 169

fragment to calculate the average deuterium enrichment on a glucose carbon.

Measurement of average enrichment (analysis of reproducibility):

Our approach is based on the average deuterium enrichment in the glucose

carbons of the m/z 170/169 fragment. The CV of the M+1/M ratio in a [1-2H]glucose

standard at various levels of enrichment from 0 to 3% mole percent enrichment (MPE)

34

(10 replicates each) was 0.39%. The M+1/M ratio of the adult human baseline plasma

sample was 9.048 ± 0.006 (Mean ± SD) (10 replicate analyses) (CV = 0.1%) (Table-2).

The M+1/M ratio of the adult human plasma sample (6h) obtained after an overnight fast

(following 2H2O ingestion) was 10.424 ± 0.010 (Mean ± SD) (10 replicate analyses) with

a CV of 0.1%. The average MPE of deuterium was 1.376 ± 0.012 (Mean ± SD) with a

CV of 0.9%. The M+1/M ratio of the 9.5 h and 10h infant plasma samples following

2H2O ingestion measured with GCMS (3 replicate analysis each) were 9.641 ± 0.003

(Mean ± SD) and 9.639 ± 0.010 (see table-3). The average MPE of deuterium measured

for the 9.5 h sample was 0.314 ± 0.005 (Mean ± SD) (CV = 1.6%) and that for the 10 h

sample 0.311 ± 0.006 (Mean ± SD) (CV = 1.9%). The mean M+1/M ratio of all the

baseline samples from six adult male subjects was 9.239 ± 0.042 (Mean ± SD) (3

replicate analysis each) and of all the samples between 65 and 66 h 11.373 ± 0.081 (Mean

± SD) with a CV of 0.45% and 0.71%, respectively (3 replicates each). The average MPE

of deuterium was 2.134 ± 0.099 (Mean ± SD) with a CV of 4.6%. The precision of the

average enrichment method is evident from the statistics of the above GCMS

measurements. Due to the lower number of manipulations involved, the propagation of

errors is less in measuring fractional gluconeogenesis using average enrichment method

compared to the C-5 HMT method [10].

As anticipated, no significant increase in the enrichment of M+2 glucose in the

plasma sample was observed at the low 2H enrichment in body water used in human

studies. Using the singly labeled glucose standards, the enrichments measured using the

fragment m/z 170/169 was extremely stable and reproducible over a wide abundance

range (2 to 30 million area counts) (fig-3) as demonstrated by the slope of 0.0024. This

35

Table-1

Sites of labeling on major ion fragments of deuterium labeled glucose & mass shift

Fragment Labeled positions in reference compounds

m/z [1-2H]

glucose

[2-2H]

glucose

[3-2H]

glucose

[5-2H]

glucose

[6,6-2H2]

glucose

[1,2,3,4,5,6,6-2H7]

glucose

109amu +1 +0 +1 +0 +2 +5

169amu +1 +0 +1 +1 +2 +6

331amu +1 +1 +1 +1 +2 +7

Table-2

Comparison of fractional gluconeogenesis of adult human plasma (overnight fasting

study) applying the new method using average deuterium enrichment of glucose and the

C-5 HMT method.

Fractional Gluconeogenesis

No. M+1 Ratio (m/z 170/169) Baseline sample

M+1 Ratio (m/z 170/169)

Adult 6h sample

(Overnight fasting)

Total MPE

Average 2H Enr.

per glucose carbon

Deuterium Enr. in

body H2O Average Enr.

Method (%)

C-5 HMT

method (%)

#1 9.054 10.436 1.382 0.230 0.4736 48.6 44.7 #2 9.043 10.421 1.378 0.230 0.4743 48.4 39.2 #3 9.056 10.419 1.363 0.227 0.4769 47.6 47.4 #4 9.055 10.415 1.360 0.227 0.4756 47.7 46.0 #5 9.040 10.432 1.392 0.232 0.4765 48.7 40.7 #6 9.042 10.433 1.391 0.232 0.4766 48.6 55.6 #7 9.046 10.407 1.361 0.227 0.4764 47.6 46.6 #8 9.051 10.422 1.371 0.229 0.4720 48.4 44.1 #9 9.043 10.414 1.371 0.229 0.4714 48.5 48.8 #10 9.048 10.433 1.385 0.231 0.4726 48.9 55.4

Mean ± SD

9.048 ± 0.006

10.424 ± 0.010

1.376 ± 0.012

0.229 ± 0.002

0.4746 ± 0.0020

48.3 ± 0.5

46.9 ± 5.4

% CV 0.1 0.1 0.9 0.9 0.4 1.0 11.5

36

Table-3

Gluconeogenesis as a fraction of total plasma glucose appearance rate based on two

“steady state” plasma samples from an infant who received total parenteral nutrition and

comparison of the new method using average deuterium enrichment of glucose and the

C-5 HMT method.

Fractional gluconeogenesis

Sample M+1 Ratio (m/z

170/169)

Total MPE deuterium

Average 2H Enr. per glucose carbon

Average Enr.

Method (%)

C-5 HMT method

(%) Baseline # 1 9.331 -- -- -- -- Baseline # 2 9.322 -- -- -- -- Baseline # 3 9.330 -- -- -- -- Mean ± SD 9.328±0.005 -- -- -- --

Infant 9.5h #1 9.639 0.308 0.051 12.9 12.7 Infant 9.5h #2 9.640 0.318 0.053 13.3 13.2 Infant 9.5h #3 9.645 0.315 0.053 13.2 13.0

Mean ± SD 9.641±0.003 0.314±0.005 0.052±0.001 13.1±0.2 13.0±0.3 Infant 10h #1 9.640 0.309 0.051 13.3 15.0 Infant 10h #2 9.629 0.307 0.051 13.2 14.9 Infant 10h #3 9.648 0.318 0.053 13.7 12.9 Mean ± SD 9.639±0.010 0.311±0.006 0.052±0.001 13.4±0.3 14.3±1.2

37

Table-4

Fractional gluconeogenesis measured in six adult male human volunteers following 66 h

of continuous fasting using the average deuterium enrichment method and the C-5 HMT

method. Baseline and steady state sample values represent an average of three replicate

measurements.

Fractional gluconeogenesis

Subject Time M+1 ratio (m/z

170/169)

Total MPE 2H above baseline

Average 2H Enr. (New

method)

2H Enr. on glucose carbon 5 (HMT

method)

2H Enr. of body water Average

Enr. method

(%)

C-5 HMT

method (%)

Baseline 9.253 -- -- -- -- -- -- 7h 11.310 2.057 0.343 0.353 0.3933 87.2 89.8

7h15m 11.295 2.042 0.340 0.352 0.3861 88.2 91.2 7h30m 11.338 2.085 0.348 0.384 0.3894 89.3 98.6 7h45m 11.317 2.064 0.344 0.344 0.3971 86.6 86.6

S # 1

8h 11.295 2.042 0.340 0.337 0.3898 87.3 86.5 Mean -- -- -- -- -- -- 87.7 90.5

Baseline 9.267 -- -- -- -- -- -- 7h 11.368 2.101 0.350 0.360 0.4066 86.1 88.5

7h15m 11.358 2.091 0.348 0.324 0.4148 84.0 78.1 7h30m 11.341 2.074 0.346 0.329 0.4090 84.5 80.4 7h45m 11.358 2.091 0.348 0.369 0.4127 84.4 89.4

S # 2

8h 11.340 2.073 0.345 0.318 0.4175 82.7 76.2 Mean -- -- -- -- -- -- 84.4 82.5

Baseline 9.292 -- -- -- -- -- -- 7h 11.323 2.031 0.338 0.331 0.4161 81.3 79.5

7h30m 11.344 2.052 0.342 0.410 0.4079 83.8 100.5 7h45m 11.301 2.009 0.335 0.380 0.4178 80.1 91.0

S # 3

8h 11.307 2.015 0.336 0.376 0.4253 79.0 88.4 Mean -- -- -- -- -- -- 81.2 89.9

Baseline 9.242 -- -- -- -- -- -- 7h45m 11.532 2.290 0.382 0.360 0.4658 81.9 77.3

S # 4

8h 11.530 2.288 0.381 0.392 0.4681 81.4 83.7 Mean -- -- -- -- -- -- 81.7 80.5

Baseline 9.183 -- -- -- -- -- -- S # 5 8h 11.378 2.195 0.366 0.343 0.4375 83.6 78.4

Baseline 9.197 -- -- -- -- -- -- S # 6 8h 11.352 2.155 0.359 0.360 0.4313 83.3 83.5

38

observation further demonstrates the robust nature of this method using the m/z 170/169

fragment to measure deuterium enrichment of gluconeogenic glucose molecules.

Measurement of fractional gluconeogenesis:

The measurements of fractional gluconeogenesis using ten aliquots of the same

adult human plasma sample (overnight fasting) applying the new method and C-5 HMT

method [10], respectively, are provided in table-2. Fractional gluconeogenesis measured

using the average enrichment in the adult plasma sample was 48.3% ± 0.5 (Mean ± SD)

and that using the C-5 HMT method [10] 46.9% ± 5.4 (Mean ± SD). These measures

were not significantly different (p=0.35). Thus, the CV of 10 replicate analyses for the

new method was 1.0%, whereas that of the C-5 HMT method [10] was 11.5%. The

deuterium enrichment of body water in the adult plasma was 0.4746 ± 0.0021 MPE

(Mean ± SD) (average of ten measurements). Thus, measurements of gluconeogenesis

using the new method compare extremely well with those obtained by the C-5 HMT

method [10] supporting the validity of this new method. Ten independent measures of

fractional gluconeogenesis were calculated from ten replicate analyses of the same

sample using four different instruments with the new method. Fractional gluconeogenesis

for Instrument-A was 48.3 ± 0.5% (Mean ± SD), for Instrument-B 48.6 ± 0.3%, for

instrument-C 46.5 ± 1.5% and for instrument-D 49.4 ± 1.5% with the intra assay CV’s of

1.0, 0.6, 3.2 and 3.0%, respectively. The average fractional gluconeogenesis was 48.2 ±

1.2%. Using these four values, the inter assay CV was 2.5%. .

Fractional gluconeogenesis based on three replicate analyses of two steady state

samples (9.5 and 10 h) from the infant study was 13.3 ± 0.3% (Mean ± SD) for the new

39

method as compared to 13.7 ± 0.8% (Mean ± SD) (Table-3) applying the C-5 HMT

method [10]. These measures were not significantly different (p=0.46). The CV of 3

replicate fractional gluconeogenesis measurements of the 9.5 h sample was 1.5% and that

of the 10 h sample 2.2% using the new method and 2.3% and 8.4% respectively using the

C-5 HMT method [10]. The deuterium enrichment of body water in the infant study was

calculated from the mean of two separate measurements for each steady state sample (9.5

h: 0.3978, 10 h: 0.3877).

The fractional gluconeogenesis measurements performed in six adult male

subjects was 83.7 ± 2.3% (Mean ± SD) using the new method and 84.2 ± 5.0% (Mean ±

SD) using the C-5 HMT method [10] (see table-4). These data were not significantly

different (p=0.78). In addition, the new method was compared with the C-5 HMT method

[10] using the Bland-Altman test (fig-4) including all fractional gluconeogenic

measurements (n=34 pair). The results indicate that new method, on average,

overestimated fractional gluconeogenesis by 0.5% (mean difference between the

methods). The two methods provide similar measurements of fractional gluconeogenesis

(p=0.59). The mean differences between the methods for infant, overnight fasting and 66

h fasting study conditions were 0.4%, -1.7% and 1.8% respectively. All data points

except for one (97%) were within ±2SD and 24/34 data points (71%) were within ±1SD.

Isotope interference from infused glucose tracers:

When [6,6-2H2]glucose was added to the plasma sample, no contamination of the

M+1 fragment was observed and, thus, fractional gluconeogenesis measurements was

unaffected by a plasma enrichment up to 5% MPE of the [6,6-2H2]glucose (See Fig-2).

40

0

20

40

60

80

100

-1 0 1 2 3 4 5 6

Molar ratio

Frac

tiona

l GN

G (%

)[1-13C]glucose

[6,6-2H2]glucose

Unspiked

Fig-2

0

20

40

60

80

100

-1 0 1 2 3 4 5 6

Molar ratio

Frac

tiona

l GN

G (%

)[1-13C]glucose

[6,6-2H2]glucose

Unspiked

Fig-2

Figure 2. Effect of added [1-13C] and [6,6-2H2]glucose tracers to a plasma sample on

percent gluconeogenesis measurement. A cluster of data points (n=5) representing

unspiked samples are in the 47% to 51% range.

1.70

1.90

2.10

2.30

2.50

0 5 10 15 20 25 30 35

Abundance in millions

Enric

hmen

t

Fig-3

1.70

1.90

2.10

2.30

2.50

0 5 10 15 20 25 30 35

Abundance in millions

Enric

hmen

t

Fig-3

Figure 3. Stability of deuterium enrichment measured over a wide abundance range

using a 2.1% [5-2H]glucose standard.

41

0 20 40 60 80

Average Fractional GNG (%)

-15

-10

-5

0

5

10

15

20

Frac

tiona

l GN

G (%

) new

met

hod

-Fra

ctio

nal G

NG

(%)C

-5 H

MT

met

hod

100

Fig-4

0 20 40 60 80Average Fractional GNG (%)

-15

-10

-5

0

5

10

15

20

Frac

tiona

l GN

G (%

) new

met

hod

-Fra

ctio

nal G

NG

(%)C

-5 H

MT

met

hod

100

Fig-4

Figure 4. Plot of the differences between the % fractional GNG measured by the new

method and the % fractional GNG measured by the C-5 HMT method versus the average

of % fractional GNG by the two methods. Results were obtained by the Bland-Altman

procedure. The solid line represents the bias between the methods (0.5%). The dotted

lines represent the upper and lower limits of agreement ( 11.7 and -10.7 respectively),

calculated as bias ± (2 X the SD of the differences)

42

When [1-13C]glucose was added to the adult plasma sample to assess the interference of

an additional tracer in the measurement of gluconeogenesis using 2H2O, fractional

gluconeogenesis was slightly overestimated when the [1-13C]glucose enrichment

exceeded 2.5% MPE (See fig-2). However, at an enrichment of 0.5 to 1.0% MPE of the

[1-13C]glucose, this error was essentially eliminated. Accurate measurement of 13C

enrichment at these levels can easily be obtained by GC-C-IRMS. Although [6,6-

2H2]glucose is the preferred tracer for this purpose due to the potential recycling of 13C

glucose tracers [1], [1-13C]glucose at low enrichment can be used and might be the only

alternative in complex studies requiring more than one glucose tracer.

Discussion

After the ingestion or infusion of deuterium oxide and equilibration in the total

body water pool, deuterium is incorporated into intermediary substrates along the

glycolytic/gluconeogenic pathway. Hence, the degree of deuterium labeling in plasma

glucose is a measure of gluconeogenesis. The isomerization of glyceraldehyde-3-phosphate

to dihydroxyacetone phosphate by triose phosphate isomerase and a series of equilibration

reactions between phosphoenolpyruvate and dihydroxyacetone phosphate is consistent with

deuterium incorporation in C-1, 3, 4, 5 and 6 of glucose during gluconeogenesis. Our

proposed method using average deuterium enrichment is based on the assumption that the

labeling of 2H in glucose C-1,3,4,5,6,6 are all essentially equal. The 2H enrichment at C-2

is purported to be due to complete 2H exchange with body water during the extensive

glucose-6-phosphate to fructose-6-phosphate isomerization. Therefore, the 2H enrichment

in glucose carbon 2 represents body water enrichment and is not a reflection of the

43

gluconeogenic process [11]. This is supported by the studies of Rognstad et. al. [13], who

observed that the incorporation of tritium on C-2 of glucose was higher than in all the other

glucose carbons. Therefore, the ratio of the average enrichment of deuterium at the glucose

carbons in the m/z 170/169 fragment (which does not carry the hydrogen of glucose C-2) to

that of deuterium in body water represents an estimate of fractional gluconeogenesis.

Rognstad et. al [13] reported that the incorporation of 3H on C-1 of glucose formed

by hepatocytes incubated with 3H2O using different substrates is essentially half that on C-6

(two tritium on C-6) suggesting that the primary mechanism of labeling is similar between

C-1 and C-6. The extensive tritium incorporation in C-3, 4 and 5 of glucose when

incubated with various gluconeogenic substrates indicate that the labeling occurs

throughout the gluconeogenic process [13]. Thus, with the exception of C-2, these series of

isomerization and equilibration reactions throughout the gluconeogenic pathway should

result in equal deuterium labeling on each glucose carbon. We have provided data

supporting that the average 2H enrichment (M+1) in carbon 1,3,4,5,6,6 of glucose provides

measures of fractional gluconeogenesis equivalent to the C-5 HMT method [10].

We have observed that multideuterated tracers (d7- glucose) exhibited isotope

discrimination when analyzed in the GCMS positive chemical ionization mode using m/z

169 fragment as reported by Guo et. al [2] during his studies of quantitation of positional

isomers of deuterium labeled glucose. Our studies have shown that such discrimination is

below the level of detection when singly labeled deuterated glucose compounds are used

(also reported by Guo et. al [2]). The average enrichment method used in our studies only

measures singly labeled glucose since the probability of multiple deuteriums getting

labeled on the same glucose molecule is negligible at a lower level of deuterium

44

enrichment in body water. Unlike studies performed by Guo et. al [2] in rats, the deuterium

enrichment in body water used in our human studies to measure gluconeogenesis is

substantially lower.

The ²H Nuclear magnetic resonance data from the studies of Jin et. al [7] carried out

in rats receiving 2H2O suggest relatively lower exchange of deuterium at carbon 3 of

glucose. However, these studies were performed using high deuterium enrichment in body

water resulting in deuterium labeling at multiple positions of the same glucose molecule.

This could potentially affect the accuracy of the deuterium enrichment measurements on

different glucose carbons. Due to the toxicity of 2H2O given at high doses, human studies

must be performed at several fold lower levels of deuterium enrichment in body water.

Jones et. al [8] reported in humans unequal deuterium labeling at various glucose carbons

using nuclear magnetic resonance at ~0.5% deuterium enrichment in body water. However,

no precision or accuracy data were provided at this level of enrichment to determine

whether these differences were significant.

Our method using the average deuterium enrichment to measure fractional

gluconeogenesis is analytically easy to perform and highly reproducible compared to the C-

5 HMT method [10]. Further, our method is robust in that the M+1 ratio measured using

the m/z 170/169 fragment does not fluctuate over a wide range of abundances (fig-3). The

low tracer cost and ease of analysis make this method affordable and accessible to a wide

number of investigators. In addition, the small sample volume requirement makes the

method applicable to studies in infants and children. Although the C-5 HMT method [10] is

conceptually straightforward, it is a very tedious, time consuming analysis (and thus

expensive) requiring a high level of expertise. In addition, the C-5 HMT procedure requires

45

a minimum of 0.5 mL of plasma per sample limiting its use in infants and children. We

have compared measurements obtained by our method based on average deuterium

enrichment with those of the C-5 HMT method [10] under conditions when fractional

gluconeogenesis is low, intermediate and high (~10 to ~90) demonstrating that the two

methods provide nearly identical values. This supports the validity of the method at

different levels of fractional gluconeogenesis. Because it is extremely accurate and

reproducible even when fractional gluconeogenesis is low, it can be used in subjects

receiving parenteral or enteral feedings and during insulin clamp studies.

In summary, our method based on the average deuterium enrichment has a number

of advantages: it is simple; straightforward; it requires only 25 µl of plasma to

accomplish the analysis and it can be completed in a few hours. Further, this method has

high reproducibility and accuracy even when fractional gluconeogenesis is low.

Therefore, the measurement of gluconeogenesis using the average deuterium labeling on

a glucose carbon using GCMS can be performed in most laboratories and can be applied

to all populations including very low birth weight infants. In addition, the high sample

throughput will provide speedy results in studies including large numbers of subjects.

46

References

1. Argoud GM, Schade DS, Eaton RP. Underestimation of hepatic glucose production by radioactive and stable tracers. Am. J. Physiol. 252: E606-E615, 1987.

2. Guo ZK, Lee PW, Katz J, Bergner AE. Quantitation of positional isomers of

deuterium-labelled glucose by gas chromatography/mass spectrometry. Anal. Biochem. 204;273-282, 1992.

3. Haymond MW, Sunehag AL. The reciprocal pool model for the measurement of

gluconeogenesis by use of [U-13C]glucose. Am. J. Physiol. Endocrinol. Metab. 278: E140-E145, 2000.

4. Hellerstein MK, Neese RA. Mass isotopomer distribution analysis: a technique for

measuring biosynthesis and turnover of polymers. Am. J. Physiol. 263: E988-E10001, 1992.

5. Hellerstein MK, Neese RA, Linfoot P, Christiansen M, Turner S, Letscher A,

Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope study. J. Clin. Invest. 100 (5): 1305-1319, 1997.

6. Hertel PM, Chacko SK, Pal S, Sunehag AL, Haymond MW. Subcutaneous infusion

and capillary “finger stick” sampling of stable isotope tracer in metabolic studies. Paediat. Res. 60 (5): 597-601, 2006.

7. Jin ES, Jones JG, Mathew M, Merritt M, Burgess SC, Malloy CR, Sherry AD.

Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative. Anal. Biochem. 327; 149-155, 2004.

8. Jones JG, Perdigoto R, Rodrigues TB, Geraldes CF. Quantitation of absolute 2H

enrichment of plasma glucose by 2H NMR analysis of its monoacetone derivative. Magn. Reson. Med. 48: 535-539, 2002.

9. Katz J, Tayek JA. Gluconeogenesis and Cori cycle in 12, 20 and 40 hour fasted

humans. Am. J. Physiol. 275: E537-E542, 1998. 10. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC.

Contributions of gluconeogenesis to glucose production in the fasted state. J. Clin. Invest 98 (No.2): 378-385, 1996.

11. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC.

Use of 2H2O for Estimating Rates of Gluconeogenesis. J. Clin. Invest 95: 172-178, 1995.

47

12. Neese RA, Schwarz JM, Faix D, Turner S, Letscher A, Vu D, Hellerstein MK. Gluconeogenesis and intrahepatic triose phosphate flux in response to fasting or substrate loads. J. Biol. Chem. 270(24): 14452-14466, 1995.

13. Rognstad R, Clark DG, Katz J. Glucose synthesis in Tritiated Water. Eur. J.

Biochem. 47, 383-388, 1974. 14. Schumann WC, Gastaldelli A, Chandramouli V, Previs SF, Pettiti M, Ferrannini E,

Landau BR. Determination of the Enrichment of the Hydrogen Bound to Carbon 5 of Glucose on 2H2O administration. Anal. Biochem. 297, 195-197, 2001.

15. Sunehag AL, Ewald U, Larsson A, Gustafsson J. Glucose production rate in

extremely immature neonates (<28 w) studied by use of deuterated glucose. Pediatr. Res. 33:97-100, 1993.

16. Sunehag AL, Gustafsson J, Ewald U. Glycerol carbon contributes to hepatic

glucose production during the first eight hours in healthy, term infants. Acta. Paediatr. 85:1339-1343, 1996.

17. Sunehag AL, Toffolo G, Campioni M, Bier DM, Haymond MW. Effects of Dietary

Macronutrient Intake on Insulin Sensitivity and Secretion and Glucose and Lipid Metabolism in Healthy, Obese Adolescents. J. Clin. Endocrinol. Metab. 90: 4496-4502, 2005.

18. Sunehag AL., Toffolo G, Treuth MS, Butte NF, Cobelli C, Bier DM, Haymond

MW. Effects of Dietary Macronutrient Content on Glucose Metabolism in Children. J. Clin. Endocrinol. Metab. 87:5168-5178, 2002.

19. Sunehag AL, Treuth MS, Toffolo G, Butte NF, Cobelli C, Bier DM, Haymond

MW. Glucose production, Gluconeogenesis, and Insulin Sensitivity in Children and adolescents: An evaluation of their reproducibility. Pediatr. Res. 50: 115-123, 2001.

20. Tayek JA, Katz J. Glucose production, recycling, Cori cycle, and gluconeogenesis

in humans: relationship to serum cortisol. Am. J. Physiol. 272: E476-E484, 1997.

21. Tayek JA, Katz J. Glucose production, recycling, and gluconeogenesis in normals and diabetics: a mass isotopomer [U-13C]glucose study. Am. J. Physiol. 270 (Endocrinol. Metab. 33) E709-E717, 1996.

48

Chapter 3

Gluconeogenesis Continues in Premature

Infants Receiving TPN

Shaji K. Chacko

Agneta L. Sunehag

Arch Dis Child Fetal Neonatal Ed 2010;95: F413-F418.

49

Abstract

Objective: To determine the contribution of total gluconeogenesis to glucose production

in preterm infants receiving total parenteral nutrition (TPN) providing glucose exceeding

normal infant glucose turnover rate.

Study design: Eight infants (0.955±0.066 kg, 26.5±0.5 wks, 4±1 d) were studied while

receiving routine TPN. Glucose appearance rate (Ra) (the sum of rates of glucose

infusion and residual glucose production) and gluconeogenesis were measured by stable

isotope-GCMS techniques using deuterated water and applying the Chacko et. al as well

as the Landau et. al. methods.

Results: Blood glucose ranged from 5.2 to 14.3 mmol/L (94 to 257 mg/dl) and glucose

infusion rate from 7.4 to 11.4 mg/kg.min, thus exceeding the normal glucose production

rates of newborn infants in most of the babies. Glucose Ra was 12.4±0.6 and GPR

2.1±0.3 mg/kg.min. Gluconeogenesis as a fraction of glucose Ra was 11.2±1.1% (Chacko

et. al) and 10.5±1.2% (Landau et. al) (NS) and the rate of gluconeogenesis 1.35±0.15

mg/kg.min (Chacko et. al) and 1.29±0.14 mg/kg.min (Landau et. al) (NS).

Gluconeogenesis accounted for 73±11% and 68±10 (NS) of the glucose production rate

for the two methods, respectively. Gluconeogenesis and glycogenolysis were not affected

by total glucose infusion rate, glucose concentration, gestational age or birth weight.

Glucose concentration correlated with total glucose infusion rate and gestational age

(combined R2=0.79, p=0.02).

Conclusions: Gluconeogenesis is sustained in preterm infants receiving routine TPN

providing glucose at rates exceeding normal infant glucose turnover rate and accounts for

50

the major part of residual glucose production. Gluconeogenesis is not affected by glucose

infusion rate or blood glucose concentration.

Abbreviations: Glucose Ra = Appearance rate of glucose; GPR = Glucose Production

Rate; GNG = Gluconeogenesis; GCMS = Gas Chromatography-Mass Spectrometry;

IPPV = Intermittent Positive Pressure Ventilation; CPAP = Continuous Positive Airway

Pressure; TPN = Total Parenteral Nutrition; C5 = Glucose carbon 5; HMT =

Hexamethylenetetramine.

51

Introduction

During their first week of life, very premature infants are at high risk of disturbed

glucose homeostasis, which might result in increased morbidity and mortality [1-5].

Since very premature newborns have a low tolerance for enteral feeding, they are

dependent on total parenteral nutrition. However, parenteral glucose frequently results in

hyperglycemia when the glucose infusion rate exceeds the glucose production rate of

healthy newborn infants (6-8 mg/kg min) [6-12]. Thus, providing an optimal energy

intake, yet maintaining normal glucose homeostasis is a challenge requiring detailed

knowledge about the physiology of the glucose metabolism of these infants.

We have previously demonstrated that very premature infants receiving total

parenteral nutrition providing glucose at rates corresponding to only half the glucose

production rate of healthy newborn infants maintained normoglycemia by glucose

produced primarily via the gluconeogenic pathway [13]. Under these conditions, the

components of the parenteral lipid emulsion (particularly glycerol) was a more important

substrate for gluconeogenesis than the amino acids [14-15].

Studies in adults have demonstrated that glucose production is turned off when

glucose is infused at a rate corresponding to their normal glucose production [16-17]. In

contrast, during the same conditions, very premature infants do not completely suppress

glucose production [10, 16, 18]. In the referenced studies, the infants did not receive

parenteral lipid and amino acids and the contribution from gluconeogenesis and

glycogenolysis were not measured.

The question remains whether very premature infants suppress glucose production

and its components during routine nutritional supply, which most often includes glucose

52

at rates exceeding that of their normal glucose production plus lipids and amino acids. To

our knowledge this issue has not previously been addressed in very premature infants.

The primary aim of the present study was to determine whether gluconeogenesis

is sustained in very premature infants receiving standard nutritional care, and whether it

correlates with glucose infusion rate and/or blood glucose concentration. To assure the

accuracy of the measurements of gluconeogenesis under conditions of high exogenous

glucose infusion rates, two different approaches to measure gluconeogenesis were

applied [19-20].

We hypothesized that in very premature infants receiving routine total parenteral

nutrition, gluconeogenesis accounts for the major part of residual glucose production.

Further, that gluconeogenesis is an ongoing process that is not affected by either glucose

infusion rate or blood glucose concentration.

Patients and Methods

Subjects: The study was approved by the Institutional Review Board for Human

Research at Baylor College of Medicine and the Advisory Board of the General Clinical

Research Center at Texas Children’s Hospital, Houston TX. The infants were recruited

from the Neonatal Intensive Care Unit at Texas Children’s Hospital and they were

enrolled after at least one parent had provided written informed consent.

Eight consecutive premature infants (4 boys and 4 girls) fulfilling our inclusion criteria

i.e. gestational age ≤ 29 wks; AGA (appropriate for gestational age); and absence of

syndromes, anomalies and sepsis were studied. Further, the infants must be clinically

53

stable on IPPV (Intermittent Positive Pressure Ventilation) or CPAP (Continuous Positive

Airway Pressure) and not have a mother with diabetes or substance abuse.

All mothers had received antenatal steroids (Celestone). Three infants were delivered

vaginally and five by cesarean section. The average Apgar score at 5 min was 7 with no

infant having less than 5. Subject characteristics are shown in table 1.

Table 1. Subject Characteristics Subject #

Gender Gestational Age wks

Birth Weight kg

Postnatal Age days

1

m 25 0.880 5

2

f 26 0.780 5

3

f 29 1.250 4

4

f 26 0.805 5

5

m 26 0.900 4

6

f 27 1.050 4

7

m 25 0.780 3

8

m 28 1.191 4

Mean ± SE

26.5 ± 0.5 0.955 ± 0.066 4.3 ± 0.3

At time of the study, 6/8 infants were receiving caffeine and 3/8 were receiving

dopamine (two at 6.4 µg/kg.min and one at 2.8 µg/kg.min). Two infants were still

receiving antibiotics (Ampicillin + Gentamicin). In the remaining six infants, the

antibiotics had been discontinued before start of the study. No infant had any positive

cultures or clinical signs of sepsis. None of the infants had received insulin. The infants

54

were clinically stable with oxygen saturation between 85 and 95% either on Intermittent

Positive Pressure Ventilation (IPPV) (n=5) (19 ± 1/5 ± 1 cm H2O and FiO2 0.25 ± 0.03)

or Continuous Positive Airway Pressure (CPAP) (n=3) (9 ± 1cm H2O and FiO2 averaging

0.29 ± 0.04).

Parenteral Nutrition: None of the infants had received any enteral feedings.

During the study, the parenteral nutrition (TPN) was continued at the pre-study rates as

ordered by the attending physician. These rates had been maintained for about 12 h at

start of the study. When the measurements of metabolic parameters were obtained i.e. at

study hours 9.5 and 10, the TPN had been administered at unchanged rates for more than

20 h. Thus, the TPN provided glucose at 10.12 ± 0.50 mg/kg.min (range 7.37 -11.44

mg/kg.min = 10.61 – 16.48 g/kg.d). In addition, the infants received a tracer dose of [6,6-

2H2]glucose to measure glucose appearance rate [7, 11, 21-22] (see details below under

“Compounds labeled with isotopes”) resulting in a total glucose infusion rate of 10.29 ±

0.50 mg/kg.min (range 7.54-11.60 mg/kg.min = 10.86 -16.70 g/kg.d). Lipid (represented

by 20% Intralipid, Kabivitrum, Stockholm Sweden) were given at a rate of 1.91 ± 0.25

mg/kg.min (range 0.81-3.85 g/kg.d) and amino acids (TrophAmine, Braun Medical Inc.

Bethlehem, PA) at 2.06 ± 0.05 mg/kg.min (range 1.83-2.18 mg/kg.min = 2.63-3.13

g/kg.d) . The fluid volume averaged 119 ± 4 mL/kg.d (range 100-130 ml/kg.d).

Compounds labeled with stable isotopes: Sterile and pyrogen free deuterium

oxide (2H2O), 99 atom percent 2H and [6,6-2H2]glucose, 99 atom% 2H were purchased

from Cambridge Isotopes Laboratories (Andover, MA). The labeled compounds were

tested again for sterility and pyrogenicity, dissolved in isotonic saline, and prepared for

55

intravenous infusion by the Investigation Pharmacy at Texas Children’s Hospital,

Houston, TX.

Study design: The infusions of glucose, Intralipid and TrophAmine were

continued at the pre-study rates throughout the 10 h study period. In addition, during the

first two study hours, a total of 4 g/kg of sterile deuterated water (1 g/mL, made isotonic

by addition of sodium chloride) was given intravenously at a constant rate of 0.033 ±

0.000 g/kg.min. At completion of the infusion of deuterated water (at study hour two), a

constant rate infusion of [6,6- 2H2]glucose (10 mg/mL) was initiated and continued for 8

h at 0.165 ± 0.005 mg/kg.min to measure glucose appearance rate [7, 11, 21-22]. The

TPN solution and the compounds labeled with stable isotopes were infused via umbilical

venous catheters already in place for clinical care purposes.

Blood sampling: Three blood samples (a total of 3 ml/kg) were obtained during

the study: one just prior to start of the infusion of deuterated water, one at study hour 9.5

and one at study hour 10. The blood samples were obtained via umbilical artery catheters,

already in place for clinical care purposes.

Analyses: The blood samples were analyzed for blood glucose concentration

using a glucose analyzer (YSI 2300 Stat Plus, YSI Inc. Yellow Springs, OH, USA).

Isotopic enrichment of [6,6-2H2]glucose was measured by gas chromatography – mass

spectrometry (GCMS) (6890/5973 Agilent Technologies, Wilmington, DE) using the

pentaacetate derivative [21-22]. The incorporation of deuterium in glucose was

determined according to two different methods: 1) Using the average deuterium

enrichment in glucose carbons 1,3,4,5 and 6 according to Chacko et. al [19]; and 2) using

56

the hexamethylenetetramine (HMT) derivative followed by analysis by GCMS in the

electron impact mode according to Landau et. al [20, 23].

Briefly, the average deuterium method [19] involves preparation of the

pentaacetate derivative of glucose, followed by sample analysis using GCMS in the

positive chemical ionization mode. Selective ion monitoring of m/z 170/169 was

performed to determine the M+1 enrichment of deuterium in the circulating glucose

carbons (C-1,3,4,5,6,6) (M is the base mass, 169, representing unlabeled glucose) [19].

After subtracting the enrichment of M+1 resulting from the natural abundance, the

average enrichment of deuterium on a gluconeogenic carbon was calculated from these

M+1 data [19]. Deuterium enrichment in plasma water was determined by Isotope Ratio

Mass Spectrometry (Delta+XL IRMS Thermo Finnigan, Bremen, Germany). For

simplicity, from now on we are referring to the average deuterium enrichment method as

the “Chacko method” and the C5/HMT method as the “Landau method”.

Calculations: All kinetic measurements were performed under steady state

conditions and based on the mean isotopic enrichments obtained at study hours 9.5 and

10 (no values for isotopic enrichments or blood glucose concentration differed by more

than 5% between the two time points). Rates of glucose appearance (Ra) were calculated

using established isotope dilution equations [7, 22].

Rate of glucose production (mg/kg.min) (GPR) = glucose Ra – exogenous glucose

(labeled and unlabeled).

Fractional gluconeogenesis (i.e. gluconeogenesis as a fraction of glucose Ra) was

calculated according to Chacko et. al [19] as follows:

Fractional gluconeogenesis (GNG % Ra) = [(M+1) (2H) (m/z 170/169) /6] / E 2H2O

57

where (M+1)(2H) (m/z 170/169) is the M+1 enrichment of deuterium in glucose measured

using m/z 170/169 and ‘6’ is the number of 2H labeling sites on the m/z 170/169 fragment

of glucose (i.e the average M+1 enrichment derived from deuterated water) and E 2H2O is

the deuterium enrichment in plasma water.

Fractional gluconeogenesis was also calculated from the deuterium enrichment in

glucose carbon 5 derived from deuterated water (product) and the deuterium enrichment

in plasma water (precursor) according to the Landau method as previously described [20,

23].

Fractional gluconeogenesis (GNG % Ra) = E 2H C5/E 2H2O

where E 2H C5 is the deuterium enrichment in glucose carbon 5 by the deuterium

incorporation from deuterated water using the Hexamethylenetetramine (HMT)

derivative [20] and E 2H2O deuterium enrichment in plasma water.

For both methods, rates of gluconeogenesis were calculated as the product of total

glucose appearance rate [19-20, 23] and fractional gluconeogenesis. Glycogenolysis was

calculated by subtracting the rate of gluconeogenesis from the glucose production rate.

Rate of Gluconeogenesis (mg/kg.min) (GNG rate) = gluc Ra × GNG % Ra

where gluc Ra is the glucose rate of appearance and GNG % Ra is gluconeogenesis as a

fraction of Ra.

Rate of glycogenolysis (mg/kg.min) = GPR - GNG rate

where GPR is the glucose production rate and GNG Rate is the rate of gluconeogenesis.

Statistical analyses: All results are provided as mean ± SE. A p value <0.05 was

used to define significance. Linear regression analysis was applied to analyze

relationships between measured variables i.e. glucose appearance rate, glucose

58

concentration, gluconeogenesis, gestational age and birth weight. Multiple regression

analysis was used to assess the effects of individual substrate infusion rates (glucose,

lipids and amino acids) and interactions among them on gluconeogenesis and blood

glucose concentration. The two methods to measure gluconeogenesis [19-20] were

compared using paired t-test and Bland and Altman’s test.

Results

Blood Glucose Concentration averaged 8.9 ± 1.2 mmol/L (160 ± 21 mg/dL) (range 5.2-

14.3 mmol/L; 94-257 mg/dL).

Glucose Appearance Rate averaged 12.4 ± 0.6 mg/kg.min (68.8 ± 3.3 µmol/kg.min).

Glucose Production Rate averaged 2.1 ± 0.3 mg/kg.min (11.7 ± 1.7 µmol/kg.min).

Gluconeogenesis and Glycogenolysis: Fractional gluconeogenesis (i.e. gluconeogenesis

as a fraction of glucose appearance rate) was 11.0 ± 1.1 % (Chacko method) [19] and

10.5 ± 1.2% (Landau method) [20, 23] (NS, p=0.46) (Fig. 1). The rate of gluconeogenesis

was 1.35 ± 0.15 mg/ kg.min (7.44 ± 0.78 µmol/kg.min) (Chacko method) [19] and 1.29 ±

0.14 mg/ kg.min (7.17 ± 0.78 µmol/kg.min) (Landau method) [20], respectively (NS,

p=0.33) [Table 2]. Gluconeogenesis accounted for 73 ± 11% (Chacko method) [19] and

68 ± 10 % of glucose production (Landau method) [20] (NS, p=0.36). Rate of

glycogenolysis was 0.71 ± 0.27 and 0.81 ± 0.27 mg/ kg.min for the Chacko [19]

and,Landau method [20], respectively (NS, p=0.32).

59

0

2

4

6

8

10

12

14

0

0.5

1

1.5

2

2.5

3Glucose Appearance Rate Glucose Production Rate (GPR)

Glycogenolysis

Gluconeogenesis

Chacko Method

[19]

Landau Method

[20]

mg/

kg. m

in

mg/

kg. m

inIV

Glucose

GPR0

2

4

6

8

10

12

14

0

0.5

1

1.5

2

2.5

3Glucose Appearance Rate Glucose Production Rate (GPR)

Glycogenolysis

Gluconeogenesis

Chacko Method

[19]

Landau Method

[20]

mg/

kg. m

in

mg/

kg. m

inIV

Glucose

GPR

Figure 1. Left Panel: Glucose appearance rate = Glucose Production Rate (GPR) ■ +

Intravenous (IV) glucose (labeled and unlabeled) .

Right panel: Glucose production (GPR) = Gluconeogenesis + Glycogenolysis □.

Gluconeogenesis was measured by the Chacko et. al [19] and Landau et. al [20] methods,

respectively. There was no significant difference between the estimates of

gluconeogenesis obtained by the two methods.

60

Table 2. Comparison of fractional gluconeogenesis and rate of gluconeogenesis

determined by the Chacko et. al method [19] vs. the Landau et. al method [20].

Subject # Fractional GNG

Chacko method

(%)

Fractional GNG

Landau method

(%)

Rate of

gluconeogenesis

(Chacko method)

mg/kg.min

Rate of

gluconeogenesis

(Landau method)

mg/kg.min

1 13.1 13.9 1.70 1.80

2 11.3 7.5 1.48 0.98

3 12.8 12.0 1.69 1.58

4 6.9 5.3 0.88 0.68

5 7.9 9.3 1.20 1.41

6 11.3 9.4 1.19 0.99

7 16.8 15.3 1.91 1.74

8 7.5 11.4 0.74 1.13

Mean ± SE 11.0 ± 1.1 10.5 ± 1.2 1.35 ± 0.15 1.29 ± 0.14

Bland and Altman’s test showed a mean difference of 0.46% between the methods for

fractional gluconeogenesis (Fig. 2) and 0.1mg/kg.min for the rate of gluconeogenesis. All

data points were within ± 2 SD.

Correlation Analyses: Gluconeogenesis did not correlate with glucose infusion rate

(R2=0.06) (Fig. 3); glucose concentration (R2=0.02); gestational age (R2=0.03) or birth

weight (R2=0.05). After controlling for glucose infusion rate, gluconeogenesis did also

61

not correlate with infusion rates of lipid or amino acids. Similarly, these factors did not

affect glycogenolysis.

-8

-4

0

4

8

4 6 8 10 12 14 16 18Diff

. Fra

ctio

nal g

luco

neog

enes

is (

%) C

hack

o an

d La

ndau

met

hods

, re

spec

tivel

y.

Average of fractional gluconeogenesis (%) measured by the two methods.

-8

-4

0

4

8

4 6 8 10 12 14 16 18Diff

. Fra

ctio

nal g

luco

neog

enes

is (

%) C

hack

o an

d La

ndau

met

hods

, re

spec

tivel

y.

Average of fractional gluconeogenesis (%) measured by the two methods.

Figure 2. Comparison of fractional gluconeogenesis obtained by the Chacko and Landau

methods [19, 20], respectively according to Bland and Altman procedure. The solid line

represents the bias between the methods (0.46%). The dotted line represent the upper and

lower limits of agreement (5.2 and -4.3, respectively) calculated as bias ± (2 X the SD of

the differences).

62

R2 = 0.43

R2 = 0.06

0

4

8

12

16

8 9 10 11 12Total Glucose Infusion Rate (mg/kg.min)

Pla

sma

gluc

ose

(mM

) and

Glu

cone

ogen

esis

(mg/

kg. m

in)

Plasma Glucose Concentration.Rate of Gluconeogenesis

R2 = 0.43R2 = 0.43

R2 = 0.062 = 0.06

0

4

8

12

16

8 9 10 11 12Total Glucose Infusion Rate (mg/kg.min)

Pla

sma

gluc

ose

(mM

) and

Glu

cone

ogen

esis

(mg/

kg. m

in)

Pla

sma

gluc

ose

(mM

) and

Glu

cone

ogen

esis

(mg/

kg. m

in)

Plasma Glucose Concentration.Rate of Gluconeogenesis

Figure 3. Effect of increased glucose infusion rates on blood glucose concentration and

gluconeogenesis, respectively, in very preterm infants receiving routine total parenteral

nutrition.

Blood glucose concentration was directly correlated with glucose appearance rate,

R2 = 0.51, p = 0.047. Of the two components of glucose Ra (exogenous glucose +

glucose production), the glucose infusion rate accounted for the major part of the

variance in blood glucose concentration, R2 = 0.43 as compared to glucose production, R2

=0.09. Adding gestational age to the regression analysis, increased the R2 value to 0.79 (p

= 0.02) (individual p values were 0.036 for glucose appearance rate and 0.047 for

gestational age). Thus, collectively, glucose appearance rate and gestational age

explained 79% of the variance in blood glucose concentration. After controlling for the

effect of the glucose infusion rate, the amino acid and/or lipid infusion rate did not have

any significant effect on the blood glucose concentration.

63

Discussion

In contrast to healthy adults [16-17], premature infants do not completely turn off

their glucose production when receiving parenteral glucose corresponding to their normal

glucose production rate [10, 16, 18]. The present study demonstrates that in very

premature infants, glucose production is not completely suppressed even when glucose is

supplied at rates exceeding their normal glucose production rate as part of TPN. Further,

gluconeogenesis is sustained and accounts for the major part of residual glucose

production in these infants. Rates of gluconeogenesis were not affected by infusion rates

of glucose, lipid and amino acids or blood glucose concentration, gestational age and

birth weight.

We and others demonstrated several years ago that premature infants receiving no

or very small amounts of intravenous glucose are capable of producing glucose at rates

comparable to those of term newborns within a few hours of birth [7, 11-12, 24-25]. It

was also shown that the gluconeogenic pathway was activated in the immediate neonatal

period in both term and preterm infants [26-29]. Despite their minimal body fuel stores,

very premature infants were capable of using endogenous glycerol for glucose production

to maintain normoglycemia at least for shorter period of times [28]. Since the activity of

gluconeogenic enzymes is very low during fetal life, this indicates that it is the birth

process itself rather than gestational age that activates key gluconeogenic enzymes.

In a subsequent series of studies, we explored whether very preterm infants were

also capable of producing glucose from parenterally supplied lipid and amino acids [13-

15]. We demonstrated that newborn very premature infants receiving parenteral nutrition

with the glucose supply reduced to half their normal glucose production rate (3

64

mg/kg.min) maintained normoglycemia over periods of at least 10-12 h by producing

glucose via the gluconeogenic pathway [13]. Further, the parenteral lipid emulsion

(primarily the glycerol part) was more important than the amino acids as substrate for

gluconeogenesis [13-15].

These previous results led up to the question addressed in the present study i.e.

whether gluconeogenesis is suppressed when very premature infants receive parenteral

nutrition providing glucose at rates exceeding normal glucose turnover rates? Under these

conditions, the infants’ glucose and energy needs are supplied by the parenteral nutrition

and, theoretically, there would be no need for glucose produced via gluconeogenesis.

However, rates of gluconeogenesis and the proportion of glucose production found in the

present study (during infusion of parenteral nutrition providing glucose at an average of

10.3 mg/kg.min) were virtually identical to those obtained in infants receiving parenteral

nutrition providing glucose at only 3 mg/kg.min [13]. Further, rates of gluconeogenesis

were not affected by blood glucose concentration (8.9 mmol/L = 160 mg/dL in the

present study as compared to 3 mmol/L = 54 mg/dL in the previous studies) [13]. These

results clearly demonstrate that in newborn very premature infants receiving routine total

parenteral nutrition, gluconeogenesis is an ongoing process that is not affected by either

glucose infusion rate or blood glucose concentration. Thus, the incomplete suppression of

glucose production is primarily due to the contribution from gluconeogenesis.

The incomplete suppression of glucose production and its components in

premature newborns might be a result of hepatic insulin resistance and/or insufficient

insulin secretion. A potential mechanism might also be hyper glucagonemia , which has

65

been shown to contribute to increased glucose production from gluconeogenesis and

glycogenolysis in type 2 diabetics [30].

After controlling for the glucose infusion rate, infusion rates of lipids and amino

acids did not affect rates of gluconeogenesis. In this study, the infusion rates of lipid

varied within a wide range (0.8 – 3.9 g/kg.d), while amino acid infusion rates were tight

(2.6-3.1 g/kg.d). Multiple regression analyses did not show any significant interaction

among substrates (glucose, lipids and amino acids) that could potentially affect

gluconeogenesis.

We also investigated which factors had an impact on blood glucose concentration

in these infants. Glucose appearance rate i.e. exogenous glucose + glucose production,

explained 51% of the variation in blood glucose concentration. The glucose infusion rate

was the major component contributing 43% of this variation, while residual glucose

production played a minor role. Blood glucose concentration also correlated with

gestational age. Collectively, glucose appearance rate and gestational age explained 79%

of the variation in blood glucose concentration. After controlling for the glucose infusion

rate, lipid and amino acid infusion rates had no effect on blood glucose concentration.

In very preterm infants, the blood volume that can be safely withdrawn is limited.

The method of Chacko et. al [19] requires only 25 µL plasma/sample, thus, providing a

valuable tool to study glucose metabolism and its regulation in newborn infants,

particularly those born prematurely. Since this method was not available when we

designed the study and performed the initial measurements of gluconeogenesis, we used

the Landau method, [20] which requires large sample volumes (~500 µL/sample). This

66

limited the number of blood samples that could be obtained and precluded analysis of

glucose regulating hormones e.g. insulin and glucagon.

In the present study, the infants received substantial amounts of intravenous

glucose, resulting in low fractional gluconeogenesis (∼ 11%). To confirm the accuracy of

our measurements of gluconeogenesis, we used both the Landau method [20] and the

method by Chacko et. al.[19] We previously demonstrated that this method compared

very well with the Landau method in overnight and 3 d fasting adults, in whom

gluconeogenesis as a fraction of glucose Ra (fractional gluconeogenesis) ranged between

40 and 100%. The results of the present study demonstrated that the Chacko method

compares very well with the Landau method also when fractional gluconeogenesis is low.

The mean difference between the methods was only 0.46% for fractional gluconeogenesis

corresponding to 0.1 mg/ kg.min for the gluconeogenic rate.

In conclusion, in very preterm infants receiving routine total parenteral nutrition

providing glucose at rates exceeding their normal glucose production rate,

gluconeogenesis accounts for the major part of residual glucose production. Rates of

gluconeogenesis were not different from those reported earlier in similar infants receiving

parenteral nutrition with the glucose supply reduced to half their normal glucose

production rate with resultant decreased blood glucose concentrations. These results

demonstrate that gluconeogenesis is an ongoing process that is independent of both

glucose infusion rate and blood glucose concentration in very premature infants. Thus,

collectively our present and previous results suggest that a potential strategy to prevent

hyperglycemia without increasing the risk of hypoglycemia or insufficient energy intake

would be to provide a TPN solution supplying glucose at a rate corresponding to the

67

normal glucose production rate of newborn infants and lipid and amino acids according

to current clinical routines during the first days of life.

Future studies using the Chacko method will allow us to determine whether

insulin, glucagon and/or other factors regulate gluconeogenesis.

68

References:

1. Hays SP, Smith E O’Brian, Sunehag AL. Hyperglycemia is a risk factor for early

death and morbidity in Extremely Low Birth Weight Infants. Pediatrics

2006;118:1811-1818.

2. Alsweiler JM, Kuschel CA, Bloomfield FH. Survey of the management of neonatal

hyperglycemia in Australasia. J Paediatr Child Health 2007;43(9):632-635.

3. Beardsall K, Vanhaesebrouck S, Ogilvy-Stuart AL, Vanhole C, Palmer CR, van

Weissenbruck M, Midgley P, Thompson M, Thio M, Cornette L, Ossuetta I, Iglesias

I, Theyskens C, de Jong M, Ahluwalia JS, de Zegher F, Dunger DB. Early insulin

therapy in very low-birth-weight infants. N Eng J Med 2008;359:1873-84.

4. Hall NJ, Peters M, Eaton S, Pierro A. Hyperglycemia is associated with increased

morbidity and mortality rates in neonates with necrotizing enterocolitis. J Pediatr

Surg 2004;39(6):898-901.

5. Alaedeen DI, Walsh MC, Chwals WJ. Total parenteral nutrition-associated

hyperglycemia correlates with prolonged mechanical ventilation and hospital stay in

septic infants. J Pediatr Surg 2006;41(1):239-44.

6. Adam PAJ, King K, Schwartz R. Model for the investigation of intractable

hypoglycemia: Insulin-Glucose interrelationships during steady state infusions.

Pediatrics 1968;41:91-105.

7. Bier DM, Leake RD, Haymond MW, Arnold KJ, Gruenke LD, Sperling MA.

Measurement of “true” glucose production rates with 6,6-dideuteroglucose. Diabetes

1977;26:1016-23.

69

8. Cowett AA, Farrag HM, Gelardi NL, Cowett RM. Hyperglycemia in the

micropremie: Evaluation of the metabolic disequilibrium during the neonatal period.

Prenat Neonat Med 1997;2:360-365.

9. Cowett RM, Oh W, Pollak A, Schwartz R, Stonestreet BS. Glucose disposal of low

birth weight infants: steady state hyperglycemia produced by constant intravenous

glucose infusion. Pediatrics 1979;63:389-396.

10. Kalhan SC, Oliven A, King KC, Lucero C Role of glucose in the regulation of

endogenous glucose production in the human newborn. Pediatr Res 1986;20:49-52.

11. Sunehag AL, Ewald U, Larsson A, Gustafsson J. Glucose production rate in

extremely Immature Neonates (<28 weeks) studied by use of deuterated glucose.

Pediatr Res 1993;33:97-100.

12. Tyrala EE, Chen X, Boden G. Glucose metabolism in the infant weighing less than

1100 grams. J Pediatr 1994;125:283-287.

13. Sunehag AL, Haymond MW, Schanler RJ, Reeds PJ, Bier DM Gluconeogenesis in

very low birth weight infants receiving total parenteral nutrition. Diabetes

1999;48:791-800.

14. Sunehag AL. Parenteral glycerol enhances gluconeogenesis in very premature

infants. Pediatr Res 2003;53:635-641.

15. Sunehag AL The role of parenteral lipids in supporting gluconeogenesis in very

premature infants. Pediatr Res 2003;54:480-486.

16. Cowett RM, Oh W, Schwartz R. Persistent glucose production during glucose

infusion in the neonate. J Clin Invest 1983;71:467-475.

70

17. Wolfe RR, Allsop JR, Burke JF. Glucose metabolism in man: Responses to

intravenous glucose infusion. Metabolism 1979;28(3):210-220.

18. Sunehag AL, Gustafsson J, Ewald U. Very Immature infants (≤ 30 Wk) respond to

glucose infusion with incomplete suppression of glucose production. Pediatr Res

1994;36:550-555.

19. Chacko SK, Sunehag AL, Sharma S, Sauer PJJ, Haymond MW. Measurement of

gluconeogenesis using glucose fragments/mass spectrometry following ingestion of

deuterium oxide. J Appl Physiol 2008;104(4):944-951.

20. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC.

Contributions of gluconeogenesis to glucose production in the fasted state. J Clin

Invest 1996;98:378-385.

21. Argoud GM, Shade DS, Eaton RP. Underestimation of hepatic glucose production by

radioactive and stable tracers. Am J Physiol 1987;273:E192-E201.

22. Tigas S, Sunehag AL, Haymond MW. Impact of duration of infusion and choice of

isotope label on isotope recycling in glucose homeostasis. Diabetes 2002;51(11):

3170-3175.

23. Sunehag AL, Toffolo G, Treuth MS, Butte NF, Cobelli C, Bier DM, Haymond MW.

Effects of dietary macronutrient content on glucose metabolism in children. J Clin

Endocrinol Metab 2002;87(11):5168-5178.

24. Kalhan SC, Bier DM, Savin SM, Adam PA. Estimation of glucose turnover and 13C

recycling in the human newborn by simultaneous [1-13C]glucose and [6,6-2H2]glucose

tracers. J Clin Endocrinol Metab 1980;50(3):456-60.

71

25. Kalhan SC, Savin SM, Adam PAJ. Measurement of glucose turnover in the human

newborn with glucose-1-13C. J Clin Endocrinol Metab 1976;43(3):704-707.

26. Bougneres PF, Karl IE, Hillman LS, Bier DM. Lipid transport in the human newborn.

Palmitate and glycerol turnover and the contribution of glycerol to neonatal hepatic

glucose output. J Clin Invest 1982;70(2):262-70.

27. Frazer TE, Karl IE, Hillman LS, Bier DM. Direct measurement of gluconeogenesis

from [2,3]13C2 alanine in the human neonate. Am J Physiol 1981;240(6):E615-21.

28. Sunehag AL, Ewald U, Gustafsson J. Extremely preterm infants (< 28 weeks) are

capable of gluconeogenesis from glycerol on their first day of life. Pediatr Res

1996;40(4):553-557.

29. Kalhan SC, Parimi P, Van Beek R, Gilfillan C, Saker F, Gruca L, Sauer PJ

Estimation of gluconeogenesis in newborn infants. Am J Physiol Endocrinol Metab

2001;281(5):E991-E997.

30. Gastadelli A, Baldi S, Pettiti M, Toschi E, Camastra S, Natali A, Landau B,

Ferrannini E. Influence of obesity and type 2 diabetes on gluconeogenesis and

glucose output in humans. Diabetes 2000;49:1367-1373.

72

Chapter 4

Gluconeogenesis is Not Regulated by Either Glucose or Insulin

in Extremely Low Birth Weight (ELBW) Infants Receiving

Total Parenteral Nutrition

Shaji K. Chacko

Jorge Ordonez

Pieter J. J. Sauer

Agneta L. Sunehag

The Journal of Pediatrics (E-pub ahead of print). 2011 PMID 21324479

73

Abstract

Objective: To determine potential factors regulating gluconeogenesis in Extremely Low

Birth Weight (ELBW) infants receiving total parenteral nutrition (TPN).

Study Design: Seven infants (0.824±0.068 kg; 25.4±0.5 wks; 3.3±0.2 d) were studied for

11 h during which parenteral lipid and amino acids were continued at pre-study rates.

Glucose was supplied at pre-study rates for the first 5 h (period 1), was then reduced to 6

mg/kg.min for 1 h and further to ∼3mg/kg.min for 5 h (period 2); 2.5 mg/kg.min of the

glucose was replaced by [U-13C]glucose throughout the study for measurements of

glucose production (GPR) and gluconeogenesis (GNG). Concentrations of glucose,

insulin, glucagon and cortisol were determined. Data obtained during periods 1 and 2

were compared using paired t-test.

Results: Gluconeogenesis and GPR remained unchanged, 2.12±0.23 vs. 1.84±0.25

mg/kg.min (NS) and 2.44±0.27 vs. 2.51±0.31 mg/kg.min (NS), respectively, despite a

60% reduction of the glucose infusion rate and subsequent 30% (124.7±10.8 to 82.6±8.9

mg/dL (p=0.009) and 70% (26.9±4.7 to 6.6±0.4 µU/mL (p=0.002)) decreases in glucose

and insulin concentrations, respectively. Cortisol and glucagon concentrations remained

unchanged.

Conclusion: In ELBW infants receiving TPN, gluconeogenesis is a continuous process

that is not affected by infusion rates of glucose or concentrations of glucose or insulin.

74

Introduction

The fine regulation of glucose metabolism in adults is primarily exerted by insulin

and secondarily by its counter regulatory hormones glucagon, epinephrine, cortisol and

growth hormone [1]. In contrast, infants born prematurely are at high risk of disturbed

glucose homeostasis resulting from limited substrate availability and potentially

immature regulation of glucose metabolism. To prevent hypoglycemia and promote

growth and development, infants born prematurely are routinely receiving total parenteral

nutrition with a glucose supply exceeding the normal infant glucose turnover rates [2-8]

from their first days of life. However, their tolerance for parenteral glucose is low

resulting in a frequent occurrence of hyperglycemia [2-3, 9]. There is a paucity of data

on which factors, substrate and hormones, regulate glucose production from its two

sources, gluconeogenesis and glycogenolysis, during total parenteral nutrition (TPN) in

preterm infants. This information is crucial to optimize nutritional strategies in this

population.

We and others have demonstrated that preterm infants initiate gluconeogenesis

within the first days of life both from endogenous body fuel stores and exogenous lipid

and amino acid substrate [10-13]. Further, a variable suppression of glucose production

has been demonstrated in preterm infants receiving parenteral glucose [4, 14-16]. In these

studies, the infants received only glucose (i.e. no lipid and amino acids) and the partition

of glucose production into its components gluconeogenesis and glycogenolysis were not

measured.

Cowett et. al addressed the impact of insulin and its counterregualtory hormones

on glucose production in a systematic fashion in newborn lambs [17]. They conclude that

75

insulin has a greater effect on glucose uptake than on glucose production and, glucagon,

cortisol and growth hormone has no major effects on glucose production even during

hyperinsulinemic hypoglycemia.

In a recent study, we demonstrated that in very low birth weight infants receiving

routine TPN, glucose production was not completely suppressed and that

gluconeogenesis constituted the major part of residual glucose production [18]. Building

on these results, the present study was conducted during routine TPN providing glucose

at high rates and in response to reducing the glucose infusion (as part of TPN) to half

normal newborn glucose turnover rate. This approach enabled us to investigate potential

factors regulating gluconeogenesis in extremely low birth weight (ELBW) infants.

We hypothesized that gluconeogenesis would remain unchanged in response to

reduction in the glucose supply and subsequent decreases in concentrations of glucose

and insulin in ELBW infants receiving TPN due to imprecise hormonal regulation of

glucose metabolism. In addition, since normoglycemia had to be maintained in all

infants, glucagon and cortisol, which have their effects under hypoglycemic conditions

[19-21], was not expected to change. Therefore, these hormones would not affect

gluconeogenesis during administration of TPN providing gluconeogenic substrates.

Patients and Methods

Subjects: The protocol was reviewed and approved by the Institutional Review

Board for Human Research at Baylor College of Medicine and the Advisory Board of the

General Clinical Research Center at Texas Children’s Hospital, Houston TX. The infants

76

were recruited from the Neonatal Intensive Care Unit at Texas Children’s Hospital and

they were enrolled after at least one parent had provided written informed consent.

Seven premature infants (5 boys and 2 girls) fulfilling our inclusion criteria i.e.

gestational age ≤ 29 wks; AGA (appropriate for gestational age); and absence of

syndromes, anomalies and sepsis were studied. Further, the infants must be clinically

stable on IPPV (Intermittent Positive Pressure Ventilation) or CPAP (Continuous Positive

Airway Pressure) and not have a mother with diabetes or substance abuse.

All mothers except one had received antenatal steroids. Four infants were delivered

vaginally and three by cesarean section. The average Apgar score at 5 min was 7 with no

infant having less than 5. Subject characteristics are shown in table 1.

Table 1. Subject Characteristics

Subject #

Gender Gestational Age

wks

Birth Weight

kg

Postnatal Age

days 1

m 27 0.930 3

2

m 27 1.085 3

3

m 25 0.962 3

4

m 24 0.572 4

5

m 26 0.808 4

6

f 25 0.747 3

7

f 24 0.605 3

Mean ± SE

25.4 ± 0.5 0.824 ± 0.068 3.3 ± 0.2

77

At time of the study, 6/7 infants were receiving caffeine and 2/7 dopamine (7.5

µg/kg.min and 10.0 µg/kg.min, respectively). The infants had received antibiotics

(Ampicillin + Gentamicin) prior to study, but in all of them antibiotics had been

discontinued before start of the study. No infant had any positive cultures or clinical

signs of sepsis. This was further verified by measurements of C-reactive protein (CRP).

None of the infants had received insulin. The infants were clinically stable with oxygen

saturation between 85 and 95% either on Intermittent Positive Pressure Ventilation

(IPPV) (n=2) (20/5 cm H2O and FiO2 0.28 ± 0.03) or Continuous Positive Airway

Pressure (CPAP) (n=5) (8 ± 1 cm H2O and FiO2 0.27 ± 0.04).

Study Design

Parenteral nutrition: The infants were studied during a period of 11 h.

Amino acids (TrophAmine, Braun Medical Inc. Bethlehem, PA) were administered at the

pre-study rates as ordered by the attending physician throughout the 11 h study (2.04 ±

0.10 mg/kg.min; range 1.67-2.36 mg/kg.min corresponding to 2.4-3.4 g/kg.d).

Lipid (20% Intralipid, Kabivitrum, Stockholm, Sweden) was also administered at the pre-

study rates throughout the 11 h study (1.12 ± 0.21 mg/kg.min; range 0.50-2.08 mg/kg.min

= 0.72-3.00 g/kg.d).

Glucose: To test the effect of a change in the glucose infusion rate on gluconeogenesis,

we used two significantly different glucose infusion rates. During the first 5 h of the

study, glucose was given at the pre-study rates (8.44 ± 0.47 mg/kg.min; range 6.93-9.97

mg/kg.min corresponding to 9.98-14.36 g/kg.d) (denoted period 1). At study hour 5

(directly after the 5 h blood sample; see below), the glucose infusion rate was reduced

step wise (to minimize counter regulatory responses) first to 6 mg/kg.min (i.e.

78

corresponding to the normal glucose turn over rate of newborn infants [2-8]) for one hour

and then further to 3.41 ± 0.18 mg/kg.min (range 2.94-4.10 mg/kg.min) for the remaining

5 h of the 11 h study period (denoted period 2).

Compounds labeled with stable isotopes: During the entire 11 h study period, 2.48 ± 0.11

mg/kg.min of the above glucose infusion rates was replaced by [U-13C]glucose

(metabolically equivalent to unlabelled natural glucose and used to measure glucose

appearance rate and glucose production from gluconeogenesis and glycogenolysis; see

calculations below). In addition, throughout the entire 11 h study, [1-13C]leucine was

given at a constant rate of 0.010 ± 0.000 mg/kg.min (0.595 ± 0.000 mg/kg.hr) to measure

leucine turn over. The [U-13C]glucose (99 atom % 13C) and [1-13C]leucine (99 atom %

13C) were purchased sterile and pyrogen free from Cambridge Isotope Laboratories

(Andover, MA) and were mixed and dissolved in normal saline at 12.5 and 0.5 mg/ml,

respectively, by the Investigation Pharmacy at Texas Children’s Hospital, Houston, TX.

The prepared tracer solutions were again tested for sterility and pyrogenicity and placed

in sealed vials under sterile conditions by the investigational pharmacy. The TPN

solution and the compounds labeled with stable isotopes were infused via umbilical

venous catheters already in place for clinical care purposes.

Blood sampling: Blood samples (a total of 3 ml/kg) were obtained at start of the study

and at study hours 4.5, 5 (representing period 1), and 10.5 and 11 (representing period 2).

The blood samples were obtained via umbilical artery catheters already in place for

clinical care purposes.

Analyses: The isotopic enrichments and the mass isotopomer distribution of glucose and

lactate during the [U-13C]glucose infusion were determined by gas chromatography –

79

mass spectrometry (GCMS) (6890/5973 Agilent Technologies, Wilmington, DE). The

pentaacetate and acetyl-pentafluorobenzyl derivative of glucose and lactate, respectively,

were prepared as previously described [10]. The blood samples were analyzed for blood

glucose concentration using a glucose analyzer (YSI 2300 Stat Plus, YSI Inc. Yellow

Springs, OH, USA). The 13C isotopic enrichments of α-ketoisocaproic acid (KIC), the

intracellular transamination product of leucine, was measured by GCMS using the oxime-

terbutyldimethylsilyl derivative as reported earlier [10]

Insulin and Glucagon concentrations were determined by radioimmunoassays

(Millipore, Billerica, MA); Cortisol, Adiponectin and C-reactive protein concentrations

by non radioactive human ELISA kits; Cortisol kit from IBL Transatlantic, Toronto, ON

and, Adiponectin and CRP from Millipore Corporation, Billerica, MA.

Calculations: All kinetic measurements were performed under steady state

conditions. Rates of total glucose appearance in plasma, glucose production and

gluconeogenesis were measured at study hours 4.5 and 5 (period 1) and 10.5 and 11

(period 2). Total plasma glucose appearance rate (glucose Ra) was calculated from the

M+6 enrichment of [U-13C]glucose in plasma using established isotope dilution equations

[6,10].

Rate of glucose production (mg/kg.min) (GPR) = glucose Ra – exogenous glucose

(labeled and unlabeled).

Fractional gluconeogenesis (GNG %) (i.e. gluconeogenesis as a fraction of glucose

Ra) was calculated using [U-13C]glucose mass isotopomers distribution analyses (MIDA)

as previously described [10-12, 22].

80

Rates of gluconeogenesis (GNG rate) were calculated as the product of total

glucose appearance rate (glucose Ra) [10] and fractional gluconeogenesis (GNG %).

Rate of Gluconeogenesis (mg/kg.min) (GNG rate) = gluc Ra × GNG % Ra

Glycogenolysis was calculated by subtracting the rate of gluconeogenesis from the

glucose production rate.

Rate of glycogenolysis (mg/kg.min) = GPR - GNG rate

Total leucine rate of appearance was calculated from the [13C]KIC enrichment using

the “reciprocal pool” model [23]. Rate of appearance of endogenous leucine was

calculated by subtracting the rate of infusion of exogenous leucine (TPN) from total

leucine Ra and was considered an indicator of proteolysis. Leucine turnover values were

converted to protein turnover assuming the content of leucine in body proteins is ~8%

[24].

Statistical analyses: Absolute rates of gluconeogenesis, concentration of glucose, insulin,

adiponectin, cortisol and glucagon obtained during the first 5 h period (period 1

representing high glucose infusion rate) were compared to those obtained during the last

5 h period (period 2 representing low glucose infusion rate) using paired t-test. To

account for multiple testing a p value <0.01 was used to define significance. Linear

regression analysis was used to analyze relationships between measured variables i.e.

gluconeogenesis, glucose appearance rate, glucose production rate, glucose

concentration, and the various hormone concentrations. All results are provided as mean

± SE.

81

Results

Concentrations of glucose and insulin are depicted in Fig.1 and glucagon, cortisol,

adiponectin and CRP in Table.2.

High glucose infusionLow glucose infusion

0

5

10

15

20

25

30

35

Insulin ConcentrationµU

/mL

P<0.01

mM

0

1

2

3

4

5

6

7

8

Glucose Concentration

P<0.01

0123456789

10

Rate of Glucose Infusion

mg/

kg. m

in

P<0.01

High glucose infusionLow glucose infusionHigh glucose infusionLow glucose infusion

0

5

10

15

20

25

30

35

Insulin ConcentrationµU

/mL

P<0.01

0

5

10

15

20

25

30

35

Insulin ConcentrationµU

/mL

P<0.01

mM

0

1

2

3

4

5

6

7

8

Glucose Concentration

P<0.01

mM

0

1

2

3

4

5

6

7

8

Glucose Concentration

P<0.01

0123456789

10

Rate of Glucose Infusion

mg/

kg. m

in

P<0.01

0123456789

10

Rate of Glucose Infusion

mg/

kg. m

in

P<0.01

Figure 1. Rates of glucose infusion and concentrations of insulin and glucose during the

high and low glucose infusion rate periods.

Table 2. Concentrations of Glucagon, Cortisol, Adiponectin and CRP during high and low glucose infusion

Period 1 High glucose infusion

period

Period 2 Low glucose infusion

period

p Value

Glucagon (pg/mL)

85.8 ± 8.2 94.8 ± 11.5 0.29

Cortisol (ng/mL)

150.5 ± 28.5 183.1 ± 39.0 0.09

Adiponectin (ng/mL)

9576 ± 1794 9686 ± 1383 0.92

CRP (µg/mL)

1.26 ± 0.46 1.37 ± 0.55 0.45

82

Glucose Appearance Rate averaged 10.88 ± 0.61 mg/kg.min (60.39 ± 3.39 µmol/kg.min)

during period 1 and 5.92 ± 0.24 mg/kg.min (32.86 ± 1.33 µmol/kg.min) during period 2

(p=<0.001).

Glucose Production Rates did not differ significantly between periods 1 and 2, 2.44 ±

0.27 mg/kg.min (13.54 ± 1.50 µmol/kg.min) vs. 2.51 ± 0.31 mg/kg.min (13.93 ± 1.72

µmol/kg.min) (NS). This shows that the difference in glucose appearance rate between

periods 1 and 2 was solely a result of the difference in the glucose infusion rates.

Rates of Gluconeogenesis and Glycogenolysis are depicted in Fig 2 demonstrating that

there was no difference in the rates between the two infusion rate periods (NS).

Gluconeogenesis accounted for 89 ± 5% and 75 ± 5% of glucose production during

periods 1 and 2, respectively (NS).

Leucine Rate of Appearance and Proteolysis: Total leucine rate of appearance was 0.73 ±

0.04 mg/kg.min (5.59 ± 0.33 µmol/kg.min) during period 1 and 0.71 ± 0.03 mg/kg.min

(5.38 ± 0.22 µmol/kg.min) for period 2 (NS). Thus, the total rate of proteolysis was 13.2

± 0.8 g/kg.day during period 1 and 12.7 ± 1.4 g/kg.day during period 2 (NS). Endogenous

leucine Ra from proteolysis averaged 0.45 ± 0.04 mg/kg.min (3.40 ± 0.27 µmol/kg.min)

during period 1 and 0.42 ± 0.02 mg/kg.min (3.20 ± 0.16 µmol/kg.min) for period 2, which

corresponds to a total rate of endogenous proteolysis of 8.04 ± 0.65 g/kg.day during

period 1 and 7.55 ± 0.37 g/kg.day during period 2 (NS).

83

Gluconeogenesis

Glycogenolysis

mg/

Kg.

min

GPRNS

0

0.5

1

1.5

2

2.5

3

Glucose ProductionRate (GPR)

NS

High Glucose Infusion

Low Glucose Infusion

NS

Gluconeogenesis

Glycogenolysis

mg/

Kg.

min

GPRNS

0

0.5

1

1.5

2

2.5

3

Glucose ProductionRate (GPR)

NSNS

High Glucose Infusion

Low Glucose Infusion

NSNS

Figure 2. Rates of glucose production, gluconeogenesis and glycogenolysis during the

high and low glucose infusion rate periods. Error bars refer to gluconeogenesis and

glycogenolysis, respectively.

Linear Regression Analyses did not show any effect of glucose infusion rate or glucose,

insulin, glucagon, cortisol, adiponectin, and CRP concentrations on gluconeogenesis

during either of the glucose infusion rate periods.

During period 1, linear regression analysis demonstrated that glucose infusion rate

explained 54% of the variation in glucose concentration, (R2=0.54; p=0.04). Adding

84

glucose production to the regression analysis, increased the R2 value to 0.83 (p=0.013).

During period 2, the glucose infusion rate was the same in all subjects by design (~3 mg/

kg.min). Thus, as expected there was no relationship between glucose infusion rate and

glucose concentration. However, blood glucose concentration was significantly related to

the glucose production rate, R2=0.60; p=0.026.

The plasma glucose concentration was also not affected by glucagon, cortisol,

adiponectin or CRP concentrations during either period 1 or 2.

Leucine Ra from proteolysis and thus, total protein turn over was not affected by

either glucose infusion rate, plasma glucose, insulin or cortisol concentration during the

two glucose infusion rate periods.

Discussion

We recently reported that gluconeogenesis continues in preterm infants receiving

routine TPN providing glucose at rates exceeding normal infant glucose turnover rates

[18]. However, there are no reports on the regulation of gluconeogenesis in preterm

infants. The primary purpose of the present study was to determine whether insulin or

glucose concentration regulates gluconeogenesis in ELBW infants receiving TPN. We

demonstrated that gluconeogenesis remained unchanged and accounted for the major

contribution to glucose production whether the infants received TPN with a glucose

supply exceeding normal infant glucose turnover rate or the glucose infusion rate was

reduced by 60%. In response to the reduction of the glucose infusion rate, glucose and

insulin concentrations decreased by 30% and 70%, respectively (Fig 1). This clearly

demonstrates that gluconeogenesis is not acutely affected by either insulin or glucose

85

concentrations in ELBW infants receiving TPN. There was a strong relationship between

the decreases in glucose and insulin concentrations between periods 1 and 2 further

showing that these immature infants were able to adjust insulin in response to the lower

glucose concentrations.

The secondary objective was to investigate potential effects of the insulin counter

regulatory hormones, glucagon and cortisol (limitations in the blood volumes that can be

safely withdrawn in ELBW infants precluded measurement of epinephrine and growth

hormone) on gluconeogenesis. We did not observe any changes in these hormones in

response to the reduction in the glucose infusion rate and subsequent decreases in glucose

and insulin concentrations. Glucagon has been shown to increase plasma free fatty acid

and ketone body concentrations in humans even under conditions of elevated plasma

insulin concentrations and to stimulate synthesis of phosphoenolpyruvate, a key step of

the gluconeogenic pathway [20, 25]. Cortisol is known to be a counter regulatory

hormone during prolonged hypoglycemia and stimulates gluconeogenesis by increasing

the delivery of gluconeogenic substrates via lipolysis and proteolysis, and by enhancing

the activity of key gluconeogenic enzymes [26-31]. Since our infants received lipid and

amino acid substrate and remained normoglycemic, the lack of changes in glucagon and

cortisol concentrations is not surprising. Under these conditions, the importance of these

hormones in the regulation of gluconeogenesis is limited. In neonatal lambs, there was an

imprecise response in the concentrations of glucagon and cortisol to hyperinsulinemic

hypoglycemia and these hormones did not affect glucose production [17].

Incomplete suppression of glucose production in preterm infants has been reported

previously [4, 14-15]. However, in these earlier studies the infants received only glucose

86

i.e. no lipid and amino acids. This is an important difference since TPN is introduced

within the first couple of days according to current nutritional guidelines. Further, the

contribution from gluconeogenesis and glycogenolysis were not measured. Kalhan et al.

[13] did not observe any differences in gluconeogenesis between preterm infants

receiving glucose alone or glucose plus lipid and amino acids using a method that

included only the gluconeogenic contribution from pyruvate (i.e. glycerol is not

included). In contrast, the method used in the present study provides an estimate of total

gluconeogenesis i.e. new glucose generated from all non-carbohydrate sources including

glycerol, which accounts for the major part of gluconeogenesis in preterm infants

receiving TPN [10-11]. Thus, as expected, estimates of gluconeogenesis were lower in

their study [13].

Gluconeogenesis remained unchanged in the infants despite the substantial

reduction in glucose infusion rate. However, the infants were able to maintain

normoglycemia by producing glucose primarily via the gluconeogenic pathway in

addition to the low glucose supply confirming our previous reports [10-12]. This

indicates that the rates of gluconeogenesis were already appropriately high during the low

glucose infusion period but was not suppressed during the high glucose infusion rate

period, despite exogenous glucose was supplied at rates exceeding the estimated needs of

the infants.

Studies in human adults have shown fine regulation of glucose metabolism in

response to intravenous glucose administration with complete or nearly complete

suppression of glucose production [14, 32-33]. Similarly, Cowett et. al reported that in

adult sheep, glucose production was suppressed at relatively low glucose infusion rates

87

(~6 mg/kg.min), while it was sustained in newborn lambs until the glucose infusion

reached very high rates (~22 mg/kg.min) [34]. In addition, there is evidence for the

continuation of gluconeogenesis from lactate during a wide range of glucose and insulin

infusion rates in newborn lambs [35]. Further, Farrag et. al showed that suppression of

glucose production in preterm infants (32-33 wks) was only minimally affected by the

insulin concentration during infusion of insulin at various rates [36]. In contrast, they

observed a near complete suppression of glucose production in adults (~89%) during

insulin infusion [36]. In the present study, gluconeogenesis accounted for ~80% of

glucose production regardless of glucose infusion rate and resulting glucose

concentration, suggesting persistent glucose production in extremely low birth weight

infants is primarily a result of ongoing gluconeogenesis.

Hepatic insulin resistance could be a potential reason for the lack of suppression of

gluconeogenesis in extremely low birth weight infants. However, insulin sensitivity is

difficult if not impossible to measure in these infants. Some studies have demonstrated

correlation between adiponectin and insulin sensitivity [37-38]. Therefore, we determined

adiponectin as a potential indicator of insulin sensitivity. We found that adiponectin

concentrations were similar during the two glucose infusion periods and were not related

to rates of gluconeogenesis. The adiponectin concentrations found in our infants were of

the same magnitude as those of the lean insulin sensitive adolescents we have previously

reported [39]. This might indicate that sustained gluconeogenesis in these infants is not

due to hepatic insulin resistance.

In summary, the results of the present study confirm that in extremely low birth

weight infants receiving TPN, gluconeogenesis is not affected by reduction of the glucose

88

infusion to a rate corresponding to half normal infant glucose turnover rate or subsequent

decrease in glucose and insulin concentrations.

Our data also demonstrate that despite substantially higher insulin concentrations

during infusion of glucose at high rates, blood glucose concentrations were elevated. We

speculate that further increase in insulin would not have any major effects on the blood

glucose concentration which would support the reports of minimal clinical benefits and

negative outcome of early insulin therapy in extremely low birth weight infants [40-41].

Additionally, similar protein turnover data during the two periods demonstrates that

reduced supply of glucose as a part of TPN does not increase proteolysis in extremely

low birth weight infants. This might imply that ongoing gluconeogenesis during TPN

providing gluconeogenic substrate prevented a potential need of increased proteolysis to

sustain gluconeogenesis to meet glucose demands even when the glucose supply is low.

In the absence of TPN, Hertz et al. [16] showed that glucose supplied at or above normal

infant glucose turnover rates did not affect proteolysis in extremely premature infants.

Since gluconeogenesis is an ongoing process enabling preterm infants receiving

TPN to remain normoglycemic even during a glucose supply corresponding to half

normal turnover rate, the glucose infusion rate is the primary factor that can be optimized

to reduce the risk of hyperglycemia. Maintaining a glucose infusion rate corresponding to

normal infant glucose turnover rates as part of total parenteral nutrition might be a

feasible approach to prevent both hypo- and hyperglycemia and yet provide sufficient

energy for growth in ELBW infants during their first days of life.

89

References:

1. Cryer PE. Glucose counterregulation: prevention and correction of hypoglycemia in

humans. Am J Physiol Endocrinol Metab 1993; 264(27):E149-E155.

2. Cowett AA, Farrag HM, Gelardi NL, Cowett RM. Hyperglycemia in the

micropremie: Evaluation of the metabolic disequilibrium during the neonatal period.

Prenat Neonat Med 1997;2:360-365.

3. Cowett RM, Oh W, Pollak A, Schwartz R, Stonestreet BS. Glucose disposal of low

birth weight infants: steady state hyperglycemia produced by constant intravenous

glucose infusion. Pediatrics 1979;63:389-396.

4. Kalhan SC, Oliven A, King KC, Lucero C Role of glucose in the regulation of

endogenous glucose production in the human newborn. Pediatr Res 1986;20:49-52.

5. Adam PAJ, King K, Schwartz R. Model for the investigation of intractable

hypoglycemia: Insulin-Glucose interrelationships during steady state infusions.

Pediatrics 1968;41:91-105.

6. Bier DM, Leake RD, Haymond MW, Arnold KJ, Gruenke LD, Sperling MA.

Measurement of “true” glucose production rates with 6,6-dideuteroglucose. Diabetes

1977;26:1016-23.

7. Sunehag AL, Ewald U, Larsson A, Gustafsson J. Glucose production rate in

extremely Immature Neonates (<28 weeks) studied by use of deuterated glucose.

Pediatr Res 1993; 33:97-100.

8. Tyrala EE, Chen X, Boden G. Glucose metabolism in the infant weighing less than

1100 grams. J Pediatr 1994;125:283-287.

90

9. Hays SP, Smith E O’Brian, Sunehag AL. Hyperglycemia is a risk factor for early

death and morbidity in Extremely Low Birth Weight Infants. Pediatrics

2006;118:1811-1818.

10. Sunehag AL, Haymond MW, Schanler RJ, Reeds PJ, Bier DM. Gluconeogenesis in

very low birth weight infants receiving total parenteral nutrition. Diabetes

1999;48:791-800.

11. Sunehag AL. Parenteral glycerol enhances gluconeogenesis in very premature

infants. Pediatr Res 2003;53:635-641.

12. Sunehag AL The role of parenteral lipids in supporting gluconeogenesis in very

premature infants. Pediatr Res 2003;54:480-486.

13. Kalhan SC, Parimi P, Beek RV, Gilfillan C, Saker F, Gruca L, Sauer PJJ. Estimation

of gluconeogenesis in newborn infants. Am J Physiol Endocrinol Metab 2001; 281:

E991-E997.

14. Cowett RM, Oh W, Schwartz R. Persistent glucose production during glucose

infusion in the neonate. J Clin Invest 1983;71:467-475.

15. Sunehag AL, Gustafsson J, Ewald U. Very Immature infants (≤ 30 Wk) respond to

glucose infusion with incomplete suppression of glucose production. Pediatr Res

1994;36:550-555.

16. Hertz DE, Karn CA, Liu YM, Liechty EA, Denne SC. Intravenous glucose suppresses

glucose production but not proteolysis in extremely premature newborns. J. Clin.

Invest 1993; 92:1752-1758.

91

17. Cowett RM, Rapoza RE, Gelardi NL. Insulin counter regulatory hormones are

ineffective in neonatal hyperinsulinemic hypoglycemia. Metabolism 1999; 48(5):568-

574.

18. Chacko SK, Sunehag AL. Gluconeogenesis continues in premature infants receiving

TPN. Arch Dis Child-Fetal Neonatal Ed. 2010:95:F413-418.

19. Gerich JE, Schneider V, Dippe SE, Langlois M, Noacco C, Karam JH, Forsham PH.

Characterization of the glucagon response to hypoglycemia in man. J Clin Endocrinol

Metab. 1974;38:77-82.

20. Gerich JE, Cryer PE. Contrainsulin hormones: Biochemical and Physiological

aspects. In: Cowett RM, ed. Principles of Perinatal-Neonatal Metabolism. New York:

Springer-Verlag, 1991. p.103-127.

21. Jackson L, Williams FLR, Burchell A, Coughtrie MWH, Hume R. Plasma

Catecholamines and the counterregulatory responses to hypoglycemia in infants: A

critical role for epinephrine and cortisol. JCEM 2004; 89(12):6251-6256.

22. Tayek JA, Katz J. Glucose production, recycling, Cori cycle, and gluconeogenesis in

humans: relation to serum cortisol. Am J Physiol 1997; 272:E476-E484.

23. Mathews DE, Motil KJ, Rohrbaugh DK, Burke JF, Young VR, Bier DM.

Measurement of leucine metabolism in man from a primed, continuous infusion of L-

[1-13C] Leucine. Am J Physiol Endocrinol. Metab. 238:E473-E479, 1980.

24. Sunehag AL, Haymond MW. Maternal protein homeostasis and milk protein

synthesis during feeding and fasting in humans. Am J Physiol Endocrinol Metab

285:E420-E426, 2003.

92

25. Schade D, Eaton R. Modulation of fatty acid metabolism by glucagon in man. I.

Effects in normal subjects. Diabetes 1975;24:502-509.

26. Goldstein RE, Wasserman DH, McGuinness OP, Brooks LD, Cherrington AD and

Abumrad NN. Effects of chronic elevation in plasma cortisol on hepatic carbohydrate

metabolism. Am. J. Physiol 1993; 264(Endocrinol. Metab. 27):E119-E127.

27. Divertie GD, Jensen MD, Miles JM. Stimulation of lipolysis in humans by

physiological hypercortisolemia. Diabetes 1991; 40(10):1228-32.

28. Darmaun D, Matthews DE, Bier DM. Physiological hypercortisolemia increases

proteolysis, glutamine, and alanine production. Am J Physiol Endocrinol Metab 1988;

255(18):E366-373.

29. Simmons PS, Miles JM, Gerich JE, Haymond MW. Increased proteolysis; an effect of

increases in plasma cortisol within the physiologic range. JCI 1984; (73), 412-420.

30. McMahon M, Gerich J, Rizza R. Effects of glucocorticoids on carbohydrate

metabolism. Diabetes Metab Rev 1988;4:17-30.

31. Khani S, Tayek JA. Cortisol increases gluconeogenesis in humans: its role in the

metabolic syndrome. Clin. Sci 2001;101,739-747.

32. Wolfe RR, Allsop JR, Burke JF. Glucose metabolism in man: Responses to

intravenous glucose infusion. Metabolism 1979;28(3):210-220.

33. Long CL, Spencer JL, Kinney JM and Geiger JW. Carbohydrate metabolism in

normal man and effect of glucose infusion. J Appl Physiol 1971; 31:102-109.

34. Cowett RM, Susa JB, Oh W, Schwartz R. Endogenous glucose production during

constant glucose infusion in the newborn lamb. Pediat. Res 1978; 12:853-857.

93

35. Susa JB, Cowett RM, Oh W, Schwartz R. Suppression of gluconeogenesis and

endogenous glucose production by exogenous insulin administration in the newborn

lamb. Pediat. Res 1979; 13:594-598.

36. Farrag HM, Nawrath LM, Healey JE, Dorcus EJ, Rapoza RE, Oh W, Cowett RM.

Persistent glucose production and greater peripheral sensitivity to insulin in the

neonate vs. the adult. Am.J.Physiol.Endocrinol.Metab 1997; 272 (35):E86-E93.

37. Weiss R, Dufour S, Groszmann A, Petersen K, Dziura J, Taksali SE, Shulman G,

Caprio S. Low adiponectin levels in adolescent obesity: A marker of increased

intramyocellular lipid accumulation. J Clin Endocrinol Metab 2003; 88(5):2014-2018.

38. Hotta K, Funahashi T, Bodkin NL, Ortmeyer HK, Arita Y, Hansen BC, Matsuzawa

Y. Circulating concentrations of adipocyte protein adiponectin are decreased in

parallel with decreased insulin sensitivity during the progression to type-2 diabetes in

rhesus monkeys. Diabetes 2001; 50: 1126-1133.

39. Sunehag AL, Toffolo G, Campioni M, Bier DM, Haymond MW. Effects of dietary

macronutrient intake on insulin sensitivity and secretion and glucose and lipid

metabolism in healthy, obese adolescents. J Clin Endocrinol Metab 2005; 90(8):4496-

4502.

40. Beardsall K, Vanhaesebrouck S, Ogilvy-Stuart AL, Vanhole C, Palmer CR, van

Weissenbruch M, et al. Early insulin therapy in Very-Low-Birth-Weight Infants. N

Eng J Med 2008; 359(18):1873-1884.

41. Poindexter BB, Karn CA, Denne SC. Exogenous insulin reduces proteolysis and

protein synthesis in extremely low birth weight infants J Pediatr 1998; 132:948-53.

94

Chapter 5

Mechanisms to conserve glucose in lactating women

during a 42 h fast

Mahmoud A Mohammad

Agneta L. Sunehag

Shaji K. Chacko

Amy S. Pontius

Patricia D. Maningat

Morey W. Haymond

Am J Physiol Endocrinol Metab. 2009;297(4):E879-888.

95

Abstract

Little is known about how lactating women accommodate for their increased

glucose demands during fasting to avoid maternal hypoglycemia. The objective of this

study is to determine whether lactating women conserve plasma glucose by reducing

maternal glucose utilization by increasing utilization of FFA and ketone bodies and/or

increasing gluconeogenesis and mammary gland hexoneogenesis. Six healthy exclusively

breastfeeding women and six non-lactating controls were studied during a 42h of fast and

during 6h of re-feeding. Glucose and protein kinetic parameters were measured using

stable isotopes and GCMS and energy expenditure and substrate oxidation using indirect

calorimetry. After 42h of fasting, milk production decreased by 16% but remained within

normal range. Glucose, insulin and C-peptide concentrations decreased with the duration

of fasting in both groups but were lower (P<0.05) in lactating women. Glucagon, FFA

and β-hydroxybutyrate concentrations increased with fasting time (P<0.001) and were

higher (P<0.0001) in lactating women during both fasting and re-feeding. During 42h of

fasting, gluconeogenesis was higher in lactating women when compared to non-lactating

controls (7.7±0.4 vs. 6.5±0.2 µmol⋅kg-1⋅min-1, P<0.05), while glycogenolysis was

suppressed to similar value (0.4±0.1 vs. 0.9±0.2 µmol⋅kg-1⋅min-1, respectively).

Mammary hexoneogenesis did not increase with the duration of fasting. Carbohydrate

oxidation was lower and fat and protein oxidations higher (P<0.05) in lactating women.

In summary, lactating women are at risk for hypoglycemia if fasting is extended beyond

30h. The extra glucose demands of extended fasting during lactation appear to be

compensated by increasing gluconeogenesis associated with ketosis, decreasing

carbohydrate oxidation, and by increasing protein and FFA oxidations.

96

Introduction

Using new techniques to measure gluconeogenesis in vivo, we (4, 12, 18, 30) and

others (13, 15) demonstrated that, gluconeogenesis accounts for approximately 50%

(ranging from 35 to 75%) of glucose production following an overnight fast. During

carbohydrate absorption, plasma glucose and insulin concentrations increase

simultaneously, but plasma glucose concentrations remain in a very narrow range in

normal individuals. This fine control is mediated by glucose induced increase in plasma

insulin, leading to increasing peripheral glucose utilization and glycogen synthesis, while

hepatic glucose production is decreasing as a result of inhibited glycogenolysis (22). In

contrast, during fasting, plasma glucose and insulin concentrations decrease and plasma

FFA, ketone bodies and glucagon concentrations increase (22) resulting in decreased

glucose utilization and/or potentially increased glucose production to maintain

euglycemia.

During a 24 h fast, glucose production in lactating women was ~35% higher than

in non-lactating women presumably to meet the substrate needs for lactose synthesis (30).

The increase in glucose production was the result of increased glycogenolysis since

gluconeogenesis was essentially identical to that of the non-lactating controls. Over the

course of the 24 h fast, rates of lipolysis and β- oxidation of fatty acids, as reflected by

glycerol flux and ketonemia, were similar to those of non-lactating controls suggesting a

lack of “mechanism(s)” to reduce glucose utilization in lactating women. However, the

contribution of plasma glucose to lactose production decreased from 80% to 60% as a

result of an increase in hexoneogenesis within the breast (28).

97

Humans have limited hepatic glycogen storage capacity. After 40-42 h of fasting

in men, the rate of glycogenolysis contributed only about 8% of total glucose production

rate (13, 15). In our previous study, we did not see a difference in glucose storage rate

between lactating and non-lactating women in response to feeding (30). By adding the

extra demands imposed by lactation, we assume that beyond a 24 h fast, hepatic glycogen

stores would be depleted more rapidly in the lactating than in the non-lactating women.

Despite these new insights, we still do not know if or how lactating women adapt to their

increased glucose demands during extended fasting to avoid maternal hypoglycemia.

Therefore, we undertook the present studies to determine whether increasing the duration

of fasting from 24 to 42 h in lactating women would result in: 1) increased glucose

production via an increase in gluconeogenesis together with an increase in

hexoneogenesis; 2) decreased glucose utilization by reducing milk production and/or

maternal glucose utilization; and 3) increased utilization of FFA and ketone bodies.

Materials and Methods

Tracers. Sterile and pyrogen free [1-13C]glucose (99 atom%), [15N2]urea, [6,6-

2H2]glucose, [1-13C]leucine and 2H2O were purchased from Cambridge Isotope

Laboratories (Andover, MA). 2H2O was administered orally without additional

preparation. The other isotopes except the 2H2O were tested again for sterility and

pyrogenicity in the investigation pharmacy of Texas Children’s Hospital. The isotopes

were dissolved in isotonic saline, and the solutions filtered through a 0.2 μm filter

(Millipore Corp. Bedford, MA) into sterile syringes. The sterile solutions were prepared

98

less than 48 h before the study, and maintained at 4°C until just prior to their use, as

previously described (30).

Subjects. Following thorough review and approval by the IRB for Human Subjects and

the General Clinical Research Center (GCRC) Advisory Committee at Baylor College of

Medicine, written consent was obtained from each of the subjects. All subjects were

determined healthy if they had a normal physical examination, fasting blood glucose,

hemoglobin (Hb), HbA1c and liver function tests. None of the women had a history of

gestational diabetes or had children, parents, siblings or grandparents with diabetes.

Except for routine postpartum vitamins and mineral supplements prescribed by their

physician, none was on any medications including birth control pills. All women studied

had a negative pregnancy test at the time of study. To exclude any pregnancy related

changes in body composition and hormones and to ensure a stable physiologic model, all

lactating women were studied between 6 and 12 wk post partum. No attempt was made to

study the non-lactating women at a specific time in their menstrual cycle.

No attempt was made to select subjects on the basis of ethnicity. As a result, six

healthy lactating women [age 26.8 ± 1.2 y, (BMI) 22.7 ± 0.9 kg/m2, height 159.8 ± 2.8

cm, weight 58.8 ± 3.2 kg, and lean body mass (LBM) 38.8 ± 2.1 kg, 4 Caucasian and 2

Hispanic American and their healthy infants] and six (6) healthy, non-pregnant, non-

lactating controls [age 27.9 ± 2.1 y, BMI 21.8 ± 0.7 kg/m2 , Height 162.4 ± 1.4 cm,

Weight 56.6 ± 1.9 kg, and LBM 39.0 ± 1.4 kg, 2 Caucasian, 2 African-American, 1

American-Hispanic and 1 Asian American] were recruited for the study. The infants of

the lactating women weighed 5.69 ± 0.36 kg and were 11.0 ± 0.5 wk of age at the time of

study.

99

Study design. Women were instructed to consume a diet providing approximately 50%

carbohydrate, 15% protein and 35% fat for the week prior to admission. Lactating women

were asked to bring 6 to 10 oz of breast milk to supplement their infants feeding should

they have technical difficulty with breastfeeding and/or decreased milk production. Each

woman and her infant were admitted to the GCRC before 4 PM on the afternoon prior to

study (day 1). At 5 PM, one intravenous (IV) catheter was introduced in the woman’s

antecubital fossa or forearm vein under Emla® cream (Astra Pharmaceuticals, Wayne,

PA) analgesia for blood sampling. Subjects were fed a supper meal of 10 kcal/kg at 6 PM

and were subsequently fasted except for water ad libitum until the re-feeding portion of

the protocol. Blood and milk samples were obtained at 6 PM and subsequently every 6 h

until midnight on the last day of the study (day 3). Glucose concentrations were measured

immediately at each blood draw. If the glucose decreased to < 60 mg/dL, an investigator

was contacted and the frequency of glucose monitoring was increased. In addition, the

mothers were carefully observed and at any signs and/or symptoms consistent with

hypoglycemia, plasma glucose was immediately measured. If the glucose concentration

decreased to < 40 mg/dL on a quantitative laboratory measure, the fast was terminated.

The mothers were requested to breast-feed their infants every 3 h. At each feeding, the

infants were fed from both breasts. Infants were weighed before and after each breast-

feeding to determine the volume of milk consumed (2, 19). Following each feeding the

mothers emptied their breasts completely using a standard electric breast pump (Playtex

Embrace™, Dover, DE). The weight of the resulting milk was measured and an aliquot

frozen for subsequent analysis. This volume of milk was added to that consumed by the

infant at each breast-feeding to determine total milk production (2).

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At 8 PM day1, a second IV was established in the opposite arm under Emla®

cream (Astra Pharmaceuticals, Wayne, PA) analgesia for infusions. Between 8 PM and 9

AM, a primed-constant rate intravenous infusion of [15N2]urea (8 μmol/kg, 0.13

µmol/kg.min) was administered to measure urea rate of appearance. Between 6 AM and 9

AM, the subjects received a primed - constant rate intravenous infusion of [1-13C]leucine

(4 μmol/kg, 0.06 µmol/kg.min) to measure leucine rate of appearance. Between 6 AM

and 1 PM, the subjects received a primed - constant rate intravenous infusion of [1-

13C]glucose (20 μmol/kg and 0.33 µmol/kg.min) to measure glucose rates of appearance

and production and the fractions of milk lactose sugars derived from plasma and

mammary gland synthesis (25, 29). Blood samples (2.5 mL each) were obtained every 10

min during the last half-hour of the initial three-hour glucose tracer infusion period (t = -

30 to t = 0 min). At 9 AM, following the 0 min blood sample, an iv bolus of glucose,

0.30 g/kg containing 10% [6,6-2H2]glucose (SLIVGTT) was administered over 90–120

seconds and blood samples (2.5 mL each) were obtained at +2, 3, 4, 5, 8, 10, 18, 20, 28,

32, 40, 60, 120, 180, 240 min. Indirect calorimetry was performed 3 times between 6 AM

and 9 AM (0 time for the SLIVGTT). Following completion of the SLIVGTT, one of the

venous access sites was removed while the second one was maintained for the duration of

the study.

Beginning at 11 PM of the 2nd day of admission (at 28 h of fasting), the women

consumed 4 doses of 2H2O at 2 h intervals corresponding to a total dose of 3 g/kg. At 4

AM, 2 additional intravenous catheters were introduced in the mother’s antecubital fossa

or forearm vein under Emla® cream analgesia, for isotope infusion and a back-up line for

blood sampling, if needed. At 6 AM, the subjects received primed constant rate infusions

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of [6,6-2H2]glucose (9 μmol/kg, 0.14 μmol·kg-1·min-1), [15N2]urea (8 μmol/kg , 0.13

μmol·kg-1·min-1) and [1-13C]leucine (4 μmol/kg , 0.06 μmol·kg-1·min-1). Blood samples

(6-10 ml) were collected at 60 min intervals beginning at 6 AM until 6 PM. Milk samples

were collected every 3 h from 6 AM through 6 PM. From noon until 6 PM, subjects were

re-fed a ~70 kcal (~70 ml) non-galactose containing nutrient drink, Boost HPl® (Mead

Johnson) (formally Sustacal®) every 30 min. This provided ~840 kcal or approximately

50% of their daily requirements over the 6 h of feeding (assuming 12 h of feeding and 12

h of fasting per day, thus, the adult 60 kg subject received ~1700 kcal/day). The purpose

of this feeding strategy was to evaluate the subject’s acute response to re-feeding not to

provide full caloric requirements. To avoid problems in data interpretation, both lactating

and control subjects received the same caloric load of Boost HP®. CO2 production and

O2 consumption were measured every 3 h from 6 AM through 6 PM using indirect

calorimetry for calculation of substrate oxidations. 12 h urine collections were obtained

during the first 36 h of the study followed by 6 h collections during the last 12 h. During

collection, the urine was kept refrigerated and then frozen at -80 °C until nitrogen

analyses and pH measurements were performed. Non-lactating women were studied

using an identical protocol except no breast milk samples were obviously obtained.

Substrate and hormones determinations. Plasma glucose and lactate concentrations were

determined using enzyme specific methods (YSI Glucose Analyzer, Yellow Springs,

OH). Plasma insulin and C-peptide concentrations were measured using commercially

available radioimmunoassay kits (Linco Research, Inc, St. Charles, MO). Plasma FFA

and β-hydroxybutyrate concentrations were determined by microfluorometric enzyme

analyses using Cobas Fara II Analyzer (Roche Diagnostic Systems, Inc., Montclair, NJ).

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Blood urea nitrogen (BUN) concentration was measured calorimetrically using the Vitros

analyzer (Ortho-Clinical Diagnostics, Rochester, NY). Plasma α-ketoisocaproate (α-

KIC) concentrations were determined by GCMS and reverse isotope dilution using

[2H7]KIC as an internal standard (27). Lactose was hydrolyzed to glucose and galactose

enzymatically and the resultant sugars analyzed individually as previously described (25,

28). Milk lactose concentration was determined using an enzyme specific method (YSI

Glucose Analyzer, Yellow Springs, OH). The concentration of protein was determined

using Bicinchoninic Acid (BCA) protein assay kit (Novagen, Madison, USA) (14, 18).

Urine pH was measured using a pH meter (Accumet Basic pH meter, Fisher Scientific,

USA).

Determination of isotope enrichments. [1-13C]glucose and [6, 6-2H2]glucose were

converted to the pentaacetate derivative and the enrichments of 13C and 2H2 were

measured using GC-C-IRMS and GCMS, respectively (29). The [15N2]urea enrichment

was determined using the 2-pyrimidinol-tert-butyldimethylsilyl derivative as previously

described (11, 27). Plasma α-[1-13C]KIC enrichment was measured after derivatization

to the oxime-tert-butyldimethylsilyl (TBDMS) derivative of α-KIC as previously

described (27). During the intravenous infusion of compounds labeled with stable

isotopes (36-48 h), the fraction of glucose derived from gluconeogenesis was measured

as previously described (4). Briefly, 2H2O enrichment was measured using IRMS (4). The

2H enrichment of glucose excluding C-2 was determined using the recently described

method of Chacko, et al, (4).

Indirect calorimetry. Indirect calorimetry was performed using a MedGraphic Model

Indirect Calorimeter (MedGraphics Inc., Minneapolis, Minnesota). Substrate oxidation

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rates for protein, glucose, lipid and resting energy expenditure were calculated using the

gaseous exchange equations previously described (7). The substrate oxidation rates were

calculated as:

Lipid oxidation = 1.67 (VCO2 - VO2) - 1.92N

Glucose oxidation = 4.55 VCO2 - 3.21 VO2 - 2.87N

Protein oxidation = N / 6.25

where VCO2 and VO2 are the L/min of gaseous exchange obtained from the calorimeter;

N represents total nitrogen excretion (g/min) estimated from the urea Ra.

Calculations:

Total glucose entry and glucose production. The total rate of appearance (Ra) of

glucose into the systemic circulation was calculated under near steady-state condition

using the standard isotope dilution equation:

Ra-total = [Ei/Ep] × I

where Ei and Ep are the infusate and plasma enrichments, respectively, of the labeled

glucose ([1-13C] following 14 h fasting and [6, 6-2H2] following 42 h fasting and re-

feeding) and I is the rate of infusion of the labeled glucose.

In the fasting state, glucose production rate (GPR) was calculated by subtracting the

amount of infused labeled glucose from the glucose Ra:

GPR = Ra-total - I

GPR is the sum of the rates of gluconeogenesis (GNG) and glycogenolysis:

GPR = GNG + Glycogenolysis

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The fraction of glucose derived from gluconeogenesis (GNG) was determined using

2H2O and the average 2H enrichments of carbons 1,3,4,5,and 6 of glucose as recently

described using the following formula (4):

Fractional GNG = [(M+1)2H (m/z 169)/6] / 2H2O

where (M+1)2H (m/z 169) is the M+1 enrichment of 2H glucose measured using the m/z

170/169 fragment of glucose divided by 6, the number of 2H labeling sites on the m/z 169

fragment of glucose and 2H2O is the enrichment of deuterium in body water (4).

The rate of gluconeogenesis is:

GNG = Fractional GNG × Ra-total

The rate of glycogenolysis in the post absorptive state was calculated as the difference

between glucose production and gluconeogenesis:

Glycogenolysisfasting = GPR – GNG.

During meal ingestion, the rate of appearance of dietary glucose into the plasma

pool (Ra-meal) was estimated from the carbohydrate content in the Boost® drink. We

assumed that ~ 85% of orally ingested glucose entered the systemic circulation in the

normal volunteer adults based on our previous studies (i.e. ~14g × 0.85/100 ml of drink

consumed) (30).

The rate of glycogenolysis during meal absorption was estimated as:

Glycogenolysis feed = Ra-total – (GNG + Ra-meal + I)

The fraction of glucose and galactose (product pool) in milk lactose that was derived

from plasma glucose (precursor pool) and the fraction derived from de novo synthesis

were calculated as previously described (25, 30).

105

Urea production rate. The rate of appearance of urea into the systemic circulation was

calculated under near steady-state condition using the standard isotope dilution equation:

Ra-urea = [(Ei/Ep) -1] × I

where Ei and Ep are the infusate and plasma enrichments, respectively, of the labeled urea

and I is the rate of infusion of the labeled urea (27).

Rates of protein oxidation. Based on urea Ra, protein oxidation was calculated using the

following equation:

Protein oxidation (g⋅kg-1⋅d-1) = Urea Ra × 0.47 × 6.25

Where Urea Ra is expressed in g⋅kg-1⋅d-1 and 0.47 is the fraction of urea that is composed

of nitrogen and 6.25 is the inverse of the fraction of nitrogen in protein.

Rate of proteolysis. Leucine appearance rate (Ra-Leu) was used as an indicator of

proteolysis and was calculated using the reciprocal pool model using the [1-13C]KIC

enrichment according to the following equation.

Ra-Leu = [(Ei/Ep) -1] × I

where Ei and Ep are the enrichments of the leucine infusate and plasma KIC, respectively,

and I is the rate of infusion of the labeled leucine. The rate of proteolysis (g⋅kg-1⋅d-1) was

calculated using the following equation as previously described (27) :

Proteolysis = Ra-Leu × 131 × 1440 × 10-6 × 12.5

where Ra-Leu is leucine rate of appearance (µmol⋅kg-1⋅min-1), 131 is its molecular weight,

1440 is to convert min to day, 10-6 is to convert µg to g and 12.5 is the inverse of leucine

content in body protein (~8%).

During fasting, proteolysis is derived solely from endogenous sources, while

during feeding, it is the sum of the rate of entry of leucine from both endogenous and

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exogenous sources. Endogenous leucine entry can be calculated by subtracting

exogenous leucine entry from total leucine Ra. The exogenous leucine entry was

calculated based on the content of protein in the Sustacal drink as provided by

manufacturer (27).

The rate of protein synthesis was calculated using the following equation:

Proteolysis – Protein oxidation = Protein synthesis.

Insulin sensitivity. Insulin sensitivity in the fasting condition was calculated using the

averaged baseline insulin and glucose values using the HOMAR and QUICKI methods

(5) according to the following formulae:

HOMAR = [glucose (mM) × insulin (μU/mL)]/22.5

QUICKI = 1/[log glucose (mg/dL) + log insulin (μU/mL)]

Insulin sensitivity and the first and second phase insulin secretory indices were calculated

from the stable label intravenous glucose tolerance test (SLIVGTT) as previously

described (29).

Statistical analysis. Values obtained during the steady state periods were averaged for

each subject and are presented as mean ± SE. Data between groups were compared using

non-paired Student’s t-test, while data within the same group were compared using paired

Student’s t-test. Changes in substrate and hormone concentrations and urine pH over time

within and between groups were assessed using repeated-measures analysis of variance

(ANOVA). Software programs SPSS (version 16; SPSS Inc, Chicago, IL) and Graph Pad

Prism (version 4; GraphPad Software Inc, CA, USA) were used for all statistical

analyses. Significance was defined as P < 0.05.

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Results

Changes in substrate and hormone concentrations over time. Statistics were carried out

using repeated measures ANOVA to demonstrate differences over time, differences

between groups (non-lactating vs. lactating), and differences due to interaction of group

and time (Figures 1-2). Repeated measures ANOVA revealed a progressive decrease in

the plasma glucose concentration in both groups (P < 0.001) with fasting time. The

decrease was greater (P < 0.0001) in the lactating as compared to the non-lactating

women (Figure 1). During re-feeding, plasma glucose rose in both groups (P < 0.001) but

was not different between the two groups (P = 0.09).

Insulin and C-peptide concentrations followed the same pattern as glucose i.e.

decreasing with the duration of the fasting and increasing (P < 0.0001) in the re-feeding

period. However, only insulin was lower in the lactating (P < 0.01) compared to the non-

lactating women during re-feeding (but not during fasting). In contrast, plasma glucagon

concentrations increased with fasting (P < 0.0001) and were higher (P < 0.0001) in the

lactating when compared to the non-lactating women. Re-feeding lowered glucagon

concentration (P < 0.0001) but, the lactating women continued to have higher values (P <

0.001) than the non-lactating controls (Figure 1).

Lactate concentration fell with time (P < 0.0001) in both non-lactating controls

and lactating women (Figure 2). During re-feeding, the plasma lactate concentration

increased (P < 0.01) in the non-lactating while it decreased (P < 0.01) in the lactating

women. BUN concentrations were higher (P < 0.0001) in the lactating group during both

fasting and re-feeding. FFA concentrations and β-hydroxybutyrate were higher (P <

108

0.0001) in the lactating group over the time course of fasting and re-feeding (P < 0.0001)

(Figure 2).

Figure 1. Mean ± SEM concentrations of plasma glucose, insulin, C-peptide and

glucagon in the non-lactating ( ) (n = 6) and lactating (n = 6) ( ) women during the

fasting (left panel) and re-feeding (right panel) states. Statistics were carried out using

repeated measures ANOVA denoting differences overtime (A) and differences between

groups (B) and difference due to interaction of group and time (AB).

109

Figure 2. Mean ± SEM plasma concentrations of lactate; BUN = blood urea nitrogen;

FFA = free fatty acids; β-OH-BT = β-hydroxybutyrate in the non-lactating ( ) (n = 6)

and lactating ( ) (n = 6) women during fasting and re-feeding. Statistics were carried

out using repeated measures ANOVA denoting differences over time (A), differences

between groups (B) and difference due to interaction of group and time (AB).

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Plasma substrate and hormone concentrations at specific time points:

Plasma substrate concentrations. After 14 h of fasting, the plasma glucose

concentrations were slightly lower in the lactating as compared to the non-lactating

women (4.3 ± 0.1 vs. 4.6 ± 0.1, P = 0.04). Plasma lactate concentrations were also lower

in the lactating group, whereas FFA, β-hydroxybutyrate, KIC and BUN were not

different (Table 1).

Table 1. Average substrate and hormone concentrations during near steady states.

14 h-Fast 42 h-Fast Re-feeding

Non-lactating Lactating Non-lactating Lactating Non-lactating Lactating

Glucose (mM) 4.6 ± 0.1 4.3 ± 0.1* 3.6 ± 0.2 2.6 ± 0.1** 5.8 ± 0.3 6.7 ± 0.3

Insulin (µU/mL) 5.0 ± 0.6 3.8 ± 0.7 2.0 ± 0.3 2.3 ± 0.5 40.8 ± 6.1 31.7 ± 4.8

C-peptide (ng /mL) 1.4 ± 0.1 1.1 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 6.5 ± 0.7 8.4 ± 1.3

Glucagon (pg/ mL) 47.7 ± 4.3 53.9 ± 4.3 70.7 ± 3.3 118.6 ± 18.5* 50.6 ± 7.2 57.4 ± 2.7

Lactate (mM) 1.0 ± 0.1 0.7 ± 0.0* 1.1 ± 0.1 1.2 ± 0.1 1.9 ± 0.2 1.0 ± 0.0**

β-OH-BT (mM) 0.1 ± 0.1 0.3 ± 0.1 2.0 ± 0.4 4.3 ± 0.4** 0.1 ± 0.0 0.5 ± 0.3

FFA (mM) 0.5 ± 0.1 0.7 ± 0.1 1.5 ± 0.3 2.0 ± 0.2* 0.2 ± 0.0 0.6 ± 0.1**

BUN (mg/dL) 11.0 ± 1.0 13.0 ± 1.1 10.4 ± 0.7 14.3 ± 1.1* 13.2 ± 0.5 16.8 ± 0.6**

α-KIC (µM) 30.7 ± 1.6 33.2 ± 3.1 50.9 ± 3.7 46.3 ± 7.3 47.9 ± 3.1 58.5 ± 7.9

n = 6 subjects in each group. Values are mean ± SEM. β-OH-BT = β-hydroxybutyrate; FFA = free fatty acids; BUN = blood urea nitrogen; KIC = ketoisocaproic acid. * P (non- paired) < 0.05, ** P (non- paired) < 0.01, non-lactating (n = 6) vs. lactating (n = 6) women. Statistics related to the effect of fasting or feeding time on different parameters are presented in Figures 1-2.

After 42 h of fasting, plasma glucose concentrations in lactating women remained

lower than in non-lactating women (2.6 ± 0.1 vs. 3.6 ± 0.2 mM, P = 0.0002),

respectively. No difference in plasma lactate or KIC concentrations was observed

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between the two groups, whereas both plasma β-hydroxybutyrate (4.3 ± 0.4 vs. 2.0 ± 0.4

mM, P = 0.007) and FFA (2.0 ± 0.2 vs. 1.5 ± 0.3, P = 0.04) were higher in lactating

women. BUN concentrations were also higher in the lactating group (14.3 ± 1.1 vs. 10.4

± 0.7 mg/dl, P < 0.05) (Table 1).

After 6 h of re-feeding, plasma concentration of glucose were not different

between lactating and non-lactating women while plasma lactate concentrations were

nearly twice as high (P < 0.01) in the non-lactating as compared to the lactating women.

Although plasma β-hydroxybutyrate and FFA concentrations decreased after re-feeding,

FFA concentrations were three times higher in the lactating group (0.6 ± 0.1 vs. 0.2 ± 0.0

mM, P = 0.005) by the end of the study. Plasma KIC concentrations were not different,

but BUN concentrations were higher in the lactating group (16.8 ± 0.6 vs. 13.2 ± 0.5

mg/dL, P < 0.01) (Table 1).

Hormone concentrations and insulin sensitivity. After the overnight fast, plasma insulin,

C-peptide and glucagon were not different between the two groups (Table 1). After 42 h

of fasting, insulin and C-peptide concentrations were not different between lactating and

non-lactating groups. However, the plasma glucagon concentration was higher in the

lactating women (119 ± 19 vs. 71 ± 3 pg/ml, P < 0.05). After 6 h of re-feeding, plasma

concentration of insulin, C-peptide and glucagon concentrations were not different

between the two groups.

After the overnight fast, insulin sensitivity as measured by HOMA (1.04 ± 0.13

vs. 0.73 ± 0.15, P = 0.16) and QUICKI (0.38 ± 0.06 vs. 0.42 ± 0.1, P = 0.12) were not

different between the non-lactating and lactating groups, respectively. Using SLIVGTT,

insulin sensitivity (min-1)/ (µU·ml-1) (5.5 ± 1.1×10-4 vs. 8.9 ± 1.6×10-4) did not differ (P =

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0.10 two sided, P = 0.05 one sided) between the non-lactating and the lactating groups.

First phase (ф1 × 10-9) (335 ± 54 vs. 356 ± 74, P = 0.80) and second phase (ф2 × 10-9)

(13.9 ± 1.7 vs. 10.9 ± 1.8, P = 0.26) insulin secretory indices also were not different

between the lactating and non-lactating groups.

Glucose kinetics. After the overnight fast, GPR was higher in the lactating as compared

to the non-lactating women, respectively (13.3 ± 0.8 vs. 10.5 ± 0.3 µmol⋅kg-1⋅min-1, P =

0.002) (Figure 3). After 42 h of fast, GPR decreased (P < 0.01) to 8.1 ± 0.5 and 7.4 ±

0.2 µmol⋅kg-1⋅min-1 in the lactating and non-lactating groups, respectively, but was no

longer different between the two groups. The rates of gluconeogenesis were higher in

lactating than non-lactating women (7.7 ± 0.5 vs. 6.6 ± 0.1 µmol⋅kg-1⋅min-1, P = 0.04).

However, rates of glycogenolysis were not different between the two groups (0.4 ± 0.2

vs. 0.8 ± 0.2 µmol⋅kg-1⋅min-1, P = 0.06). During re-feeding, rates of total glucose

appearance, glucose production, gluconeogenesis and glycogenolysis were not different

between the two groups.

113

Figure 3. Rates of glucose appearance, glucose production, gluconeogenesis and

glycogenolysis in the non-lactating ( ) and the lactating ( ) women following 14 h

and 42 h of the fasting and 6 h of re-feeding. Values are mean ± SEM. All parameters are

expressed as µmol·kg-1·min-1. All calculations are based on total body weight (kg). * P

(non- paired) < 0.05, ** P (non- paired) < 0.01, non-lactating (n = 6) vs. lactating (n = 6)

women.

114

Figure 4. Rates of appearance of leucine and urea and rates of proteolysis, protein

oxidation and synthesis in the non-lactating ( ) and the lactating ( ) women

following 14 h and 42 h of the fasting and 6 h of re-feeding. Values are mean ± SEM.

All calculations are based on total body weight (kg). * P (non- paired) < 0.05, ** P (non-

paired) < 0.01, controls (n = 6) vs. lactating (n = 6) women.

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Protein kinetics. After an overnight fast, leucine Ra and accordingly, rates of proteolysis

were not different between the two groups. Similarly, urea Ra and, thus protein oxidation

rates, were not different between the non-lactating and the lactating women (Figure 4).

After 42 h of fasting, rate of leucine appearance was higher in the lactating group (2.1 ±

0.2 vs. 1.7 ± 0.1 µmol⋅kg-1⋅min-1, P < 0.05) and accordingly, the corresponding rate of

proteolysis was higher in the lactating group (5.0 ± 0.4 vs. 4.0 ± 0.1g·kg-1·d-1, P < 0.05).

Similarly, urea Ra (0.44 ± 0.02 vs. 0.33 ± 0.03 mg·kg-1·min-1, P < 0.05) and thus, rate of

protein oxidation (1.9 ± 0.1 vs. 1.3 ± 0.1 g·kg-1·d-1, P < 0.05) were higher in the lactating

group (Figure 4). During re-feeding, proteolysis, protein oxidation and synthesis were not

different between the two groups. During the re-feeding period, proteolysis and protein

synthesis rates increased (P < 0.05) when compared to the 42 h fasting in both the

lactating and non-lactating groups, while, protein oxidation increased (P < 0.05) in the

non-lactating group only (Figure 4).

Energy expenditure and substrate oxidations. After an overnight fast, there was no

difference in energy expenditure between the non-lactating and the lactating women.

However, RQ and CHO oxidation were lower and fat oxidation was higher in the

lactating group (Table 2).

After 42 h of fast, energy expenditure was not different between the lactating and

the non-lactating women. However, RQ was lower (0.73 ± 0.01 vs. 0.79 ± 0.02, P < 0.01)

and fat oxidation was higher (1.8 ± 0.2 vs. 1.2 ± 0.2 g·kg-1·d-1, P < 0.05) whereas that of

CHO was lower (-0.2 ± 0.2 vs. 1.4 ± 0.5 g·kg-1·d-1, P < 0.01) in the lactating as compared

to the non-lactating group. During the re-feeding, energy expenditure was also not

116

different between the two groups but fat oxidation remained higher (P < 0.05) and CHO

oxidation remained lower (P < 0.05) in the lactating women.

Table 2. Energy expenditure and substrate oxidation rates during fasting and re-feeding.

14 h-Fast 42 h-Fast Re-feeding

Non-lactating Lactating Non-

lactating Lactating Non-lactating Lactating

RQ 0.85 ± 0.0 0.78 ± 0.0** 0.79 ± 0.0 0.73 ± 0.1** 0.81 ± 0.0 0.75 ± 0.0*

EE 21 ± 1 21 ± 1 22 ± 1 24 ± 2 26 ± 1 27 ± 2

Substrate oxidation rates (g·kg-1·d-1)

Fat 0.8 ± 0.1 1.4 ± 0.1** 1.2 ± 0.2 1.8 ± 0.2* 1.2 ± 0.2 1.9 ± 0.24*

CHO 2.3 ± 0.1 1.1 ± 0.3 ** 1.4 ± 0.5 -0.2 ± 0.2** 1.8 ± 0.3 0.3 ± 0.5*

Protein 1.1 ± 0.1 1.20 ± 0.1 1.1 ± 0.1 1.9 ± 0.2* 1.9 ± 0.1 1.8 ± 0.1

Values are mean ± SEM. RQ = respiratory quotient; EE = Energy expenditure expressed as kcal·kg-1·d-1. * P (non- paired) < 0.05, ** P (non- paired) < 0.01, non-lactating (n = 5) vs. lactating (n = 5) women. Urine pH. Repeated measures ANOVA revealed differences over time (P < 0.001),

differences between groups (non-lactating vs. lactating) (P < 0.001), and differences due

to interaction of group and time (P < 0.001) (Table 3). Using Post Hoc comparisons, the

pH of the urine collected during the first 24 h of fasting was not different between non-

lactating and lactating women. However, following 24 h of fasting, the pH was

consistently lower in the lactating compared to the non-lactating (P < 0.01) women. From

24 h to 42 h of fasting, the pH decreased (6.5 ± 0.2 vs. 4.7 ± 0.1, P < 0.01) in lactating

women; while, it remained unchanged in the non-lactating women (6.7 ± 0.2 vs. 6.1 ±

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0.3, P > 0.05). After re-feeding, the pH did not increase in either the lactating or the non-

lactating. However, it remained lower (P < 0.01) in lactating women.

Table 3. Urine pH in non-lactating and lactating women during fasting and re-feeding. Fasting time Non-lactating Lactating

0-12 h 6.7 ± 0.2 a 6.5 ± 0.2a

12-24 h 6.5 ± 0.1 a 5.9 ± 0.3a

24-36 h 6.4 ± 0.3 a 5.3 ± 0.2 b 36-42 h 6.1 ± 0.3 a 4.7 ± 0.1 c Re-feeding 6.3 ± 0.2 a 4.7 ± 0.1 c Values are mean ± SEM. Repeated measures ANOVA revealed differences over time (P < 0.001), differences between groups (non-lactating (n = 4) vs. lactating (n = 5)) (P < 0.001), and differences due to interaction of group and time (P < 0.001). Different alphabets (based on Post Hoc comparisons) indicate significant difference (P < 0.05).

Milk production and composition. After an overnight fast, milk volume was 99 ± 7

ml/feeding (feeding represents 3h). The average milk productions reduced (P < 0.05) by

14% (86 ± 8 ml/feeding) following 42 h fasting and was not increased after 6 h of re-

feeding (88 ± 8 ml/feeding) (Table 4). Milk protein and lactose concentrations were not

different between fasting and re-feeding. The percentage of lactose hexoses derived from

mammary gland synthesis after 14 h and 42 h of fasting were not different (45 ± 3 and 46

± 5%, P > 0.05). During re-feeding, hexoneogenesis of lactose moieties was entirely

abolished.

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Table 4. Milk production, composition and lactose hexoses derived from hexoneogenesis and plasma glucose during fasting and re-feeding. 14 h-Fast 42 h-Fast Re-feeding Milk volume (ml/feed) (ml/d)$

99 ± 7a

793 ± 55a 86 ± 8 b

684 ± 68b 88 ± 8ab

704 ± 68ab Protein Concentration (g/dl) 1.7 ± 0.2 1.9 ± 0.1 1.7 ± 0.3 Lactose concentration (g/dl) 7.4 ± 0.3 6.5 ± 0.2 6.3 ± 0.2 Milk glucose from Hexoneogenesis (%) 41 ± 4a 40 ± 8a -16 ± 5

Milk galactose from Hexoneogenesis (%) 50 ± 3a 52 ± 7a -1 ±3 b

Milk lactose from Hexoneogenesis (%) 45 ± 3a 46 ± 8a -8 ± 3 b

Milk lactose from plasma glucose (%) 55 ± 3a 54 ± 8a 109 ± 3b

Milk lactose from Hexoneogenesis (µmol·kg-1·min-1) # 1.7 ± 0.1a 1.4 ± 0.1b -0.2 ± 0.1c

Milk lactose from plasma glucose (µmol·kg-1·min-1) ## 2.1 ± 0.1a 1.6 ± 0.1b 3.2 ± 0.2c

n = 6. Values are mean ± SEM. Paired t-test was used. Different alphabets within the same row indicate difference. Significance was set as P (paired) < 0.05. $ calculated by multiplying the milk volume (ml/feed) times 8 feeding/d. # based on lactose derived from hexoneogenesis (%), milk volume and lactose concentration. ## based on lactose derived from plasma glucose (%), milk volume and lactose concentration.

119

Discussion

In our previous studies of 14-24 h fasting lactating women, the plasma substrate

and hormone concentrations were similar in lactating and non-lactating women despite a

30% increase in glucose demands in the lactating subjects. The increased glucose

demands were met by increased rates of glycogenolysis (12, 30). In the present study, we

demonstrated that if the fast was merely extended by 18 h, the lactating women

developed relative hypoglycemia, hyperketonemia and hyper free fatty acidemia when

compared to the non-lactating controls.

Plasma insulin and C-peptide followed the same pattern as glucose (i.e. decreased

with the duration of fasting) in both groups whereas plasma glucagon concentrations

increased progressively with the time of fasting plateauing after 30 h at twice the basal

concentrations in the lactating women. In the non-lactating women in the present study as

well as in previously studied 3-day fasted women (9), plasma glucagon increased by only

~50% from 14 h to 42 h of the fast. It is interesting to speculate, as has been done

previously (9, 16), that the low plasma insulin and elevated glucagon may be the primary

hormonal triggers to orchestrate the controlled mobilization of substrates, regulation of

hepatic glucose production and substrate oxidation.

In the present study, glucose production rate was 30% higher in the lactating

compared to the non-lactating women after 14 h of fasting, results similar to those

obtained following an overnight fasting (12) and 24 h of fasting (30). Extension of fasting

to 42 h decreased glucose production rate by 30 and 40% in the non-lactating and

lactating women, respectively, and was no longer different between the two groups. Rates

of gluconeogenesis were, however, higher at 42 h of fasting in the lactating women

120

contributing to 96 ± 1% of the glucose production as compared to 87 ± 3 % in the non-

lactating women. Thus, glycogenolysis accounted for a very minor part of the glucose

production rate after 42 h fasting with no difference between non-lactating and lactating

women. Our findings agree with those reported by Katz and Tayek (13), who reported

that fractional gluconeogenesis increased from 41 to 92% between 12 and 40 h in fasting

men. Similar results were reported by Landau et al (15). These observations suggest that

after 42 h of fasting, hepatic glycogen stores are essentially depleted. This also would

suggest that the higher rates of glycogenolysis in the lactating women resulted in earlier

depletion of hepatic glycogen stores than in the non-lactating women. Therefore, after 42

h of fasting, lactating women were relying nearly exclusively on gluconeogenesis for

their glucose requirements.

One potential mechanism to conserve maternal glucose during fasting is to

increase the production of lactose from other substrates within the mammary gland.

However, at 42 h of fasting, rates of mammary hexoneogenesis were not different from

those observed following 14 h of fasting in the present study or 14-24 h of fasting in our

previous studies (18, 28, 30). Calculating the minimum amount of lactose derived

directly from plasma glucose, i.e. the amount that could not be contributed from

hexoneogenesis (1.6 µmol·kg-1·min-1) (Table 4), we can calculate the approximate

endogenous rate of glucose utilization in the lactating women. Thus, we estimated the

rate of glucose utilization by maternal body tissues was lower in the lactating women (6.4

± 0.4 vs. 7.4 ± 0.2 µmol·kg-1·min-1, P < 0.05). Even after 42 h of fasting, milk volume

remained within the normal range reported previously (600-800 ml/d) (20) as did the

composition of the aqueous macronutrients constituents. Therefore, even during extended

121

fasting, priority may be given to the delivery of nutrients to the mammary gland to

support milk synthesis

Concurrent to the increased gluconeogenesis in lactating women, following the 42

h of fasting, β-hydroxybutyrate and FFA concentrations were higher than those of the

non-lactating women reflecting higher rates of lipolysis and fat oxidation. Both the

lactating and non-lactating women developed metabolic acidosis based on their ketosis.

However, the lactating women were more acidotic as indicated by their higher plasma

ketone body and FFA concentrations. This supposition is supported by the lower urine

pH values in the lactating women. Metabolic acidosis increases renal ammoniagenesis as

a mechanism to correct the metabolic acidosis (3, 6). Glutamine is the primary substrate

for renal ammonia production while the carbon skeleton is incorporated into renal

glucose production (6, 8, 21, 23). With extended fasting, the rate of proteolysis increased

and, it is reasonable to speculate that the increased gluconeogenesis in the lactating

compared to the non-lactating women may be attributed to the active contribution of

renal gluconeogenesis. However, the increase in urea production in the lactating women

might also indicate an increase in hepatic gluconeogenesis. Presumably both glycerol (24,

26) (a product of lipolysis) and amino acids (8, 17) provide the carbon needed for the

hepatic gluconeogenesis. However, it is impossible to determine the veracity of this

argument without considerably more invasive studies. Furthermore, indirect calorimetry

data revealed that the lactating women had lower CHO and higher fat oxidation rates

following both 42 h of fasting and re-feeding compared to the non-lactating women.

Thus, the increased rates of gluconeogenesis and decreased rates of glucose utilization

122

and oxidation by maternal tissues, presumably by the higher availability of FFA and

ketone bodies, conserved maternal glucose for milk lactose synthesis.

After re-feeding, glucose concentrations increased and glucose production rates

decreased in both groups with no difference between lactating and non-lactating women.

Glucose production was almost entirely represented by gluconeogenesis, which was

apparently decreased and corresponded to the 24 h fasting values previously reported in

lactating and non-lactating women (30). FFA and β-hydroxybutyrate decreased with re-

feeding, but their concentrations remained higher in the lactating group. Despite re-

feeding for 6 h (a total of 50% of daily requirement), sources of energy utilization did not

return to normal (i.e. after 14 h fast). The non-lactating women recovered more rapidly

and shifted to CHO oxidation, whereas, lactating women continued to rely on both fat

and protein as energy sources. Thus, all dietary CHO was most likely delivered to the

mammary gland for milk production.

With the ingestion of High Protein Boost® during the re-feeding period, the Ra of

leucine (and thus the estimate of proteolysis) increased in both the lactating and non-

lactating women. This most likely does not reflect an increased rate of endogenous

proteolysis since in our previous studies endogenous proteolysis decreases with feeding

(1, 10, 27). The increase in urea production rate (and thus protein oxidation) could be the

result of oxidation of the protein consumed since 25% of the calories are provided as

protein and/or this could be due to the incomplete equilibration of the urea tracer over the

6 h period of the study. In the non- lactating women, the increase in protein oxidation

rate was consistent with an increase in the rate of proteolysis of the dietary protein.

However, the increase in proteolysis in the lactating women during re-feeding was not

123

associated with a further increase in protein oxidation suggesting that the dietary amino

acids might be more readily utilized for protein synthesis, e.g. body tissue and/or milk

proteins.

In summary, both the non-lactating and lactating women adapted to a 42 h fast by

reducing endogenous glucose needs by similar mechanisms. However, due to the added

demands imposed by lactation, the metabolic stress was greater in the lactating women as

indicated by lowered glucose concentration, increased glucagon concentrations,

decreased utilization of CHO, higher plasma concentrations of FFA and ketone bodies

(presumed greater metabolic acidosis), and higher plasma concentrations and rates of

urea production, which presumably reflect increased rates of amino acid deamination and

availability of carbon to support the increased rate of gluconeogenesis.

The present study concluded that the lactating women as compared to the non-

lactating women are at risk for fasting hypoglycemia if fasting is extended. Although

milk production and composition were preserved, fasted lactating women were more

acidotic and subjected to protein loss as reflected by increased proteolysis and protein

oxidation and fat loss as indicated by increased fat mobilization and oxidation.

124

References:

1. Beaufrere B, Horber FF, Schwenk WF, Marsh HM, Matthews D, Gerich JE, and

Haymond MW. Glucocorticosteroids increase leucine oxidation and impair leucine

balance in humans. The American journal of physiology 257: E712-721, 1989.

2. Borschel MW, Kirksey A, and Hannemann RE. Evaluation of test-weighing for the

assessment of milk volume intake of formula-fed infants and its application to breast-fed

infants. Am J Clin Nutr 43: 367-373, 1986.

3. Brosnan JT, McPhee P, Hall B, and Parry DM. Renal glutamine metabolism in rats fed

high-protein diets. The American journal of physiology 235: E261-265, 1978.

4. Chacko SK, Sunehag AL, Sharma S, Sauer PJ, and Haymond MW. Measurement of

gluconeogenesis using glucose fragments and mass spectrometry after ingestion of

deuterium oxide. J Appl Physiol 104: 944-951, 2008.

5. Conwell LS, Trost SG, Brown WJ, and Batch JA. Indexes of insulin resistance and

secretion in obese children and adolescents: a validation study. Diabetes care 27: 314-

319, 2004.

6. Fine A. The effects of chronic metabolic acidosis on liver and muscle glutamine

metabolism in the dog in vivo. Biochem J 202: 271-273, 1982.

7. Frayn KN. Calculation of substrate oxidation rates in vivo from gaseous exchange. J

Appl Physiol 55: 628-634, 1983.

8. Hankard RG, Haymond MW, and Darmaun D. Role of glutamine as a glucose

precursor in fasting humans. Diabetes 46: 1535-1541, 1997.

125

9. Haymond MW, Karl IE, Clarke WL, Pagliara AS, and Santiago JV. Differences in

circulating gluconeogenic substrates during short-term fasting in men, women, and

children. Metabolism: clinical and experimental 31: 33-42, 1982.

10. Horber FF and Haymond MW. Human growth hormone prevents the protein

catabolic side effects of prednisone in humans. J Clin Invest 86: 265-272, 1990.

11. Kalhan SC. Rates of urea synthesis in the human newborn: effect of maternal diabetes

and small size for gestational age. Pediatric research 34: 801-804, 1993.

12. Kaplan W, Sunehag AL, Dao H, and Haymond MW. Short-term effects of

recombinant human growth hormone and feeding on gluconeogenesis in humans.

Metabolism: clinical and experimental 57: 725-732, 2008.

13. Katz J and Tayek JA. Gluconeogenesis and the Cori cycle in 12-, 20-, and 40-h-fasted

humans. The American journal of physiology 275: E537-542, 1998.

14. Keller RP and Neville MC. Determination of total protein in human milk: comparison

of methods. Clinical chemistry 32: 120-123, 1986.

15. Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, and Kalhan SC.

Contributions of gluconeogenesis to glucose production in the fasted state. J Clin Invest

98: 378-385, 1996.

16. Marliss EB, Aoki TT, Unger RH, Soeldner JS, and Cahill GF, Jr. Glucagon levels and

metabolic effects in fasting man. J Clin Invest 49: 2256-2270, 1970.

17. Meyer C, Stumvoll M, Dostou J, Welle S, Haymond M, and Gerich J. Renal substrate

exchange and gluconeogenesis in normal postabsorptive humans. American journal of

physiology 282: E428-434, 2002.

126

18. Mohammad MA, Sunehag AL, and Haymond MW. Effect of dietary macronutrient

composition under moderate hypocaloric intake on maternal adaptation during lactation.

Am J Clin Nutr, 2009.

19. Motil KJ, Kertz B, and Thotathuchery M. Lactational performance of adolescent

mothers shows preliminary differences from that of adult women. J Adolesc Health 20:

442-449, 1997.

20. Neville MC, Keller R, Seacat J, Lutes V, Neifert M, Casey C, Allen J, and Archer P.

Studies in human lactation: milk volumes in lactating women during the onset of

lactation and full lactation. Am J Clin Nutr 48: 1375-1386, 1988.

21. Pagliara AS and Goodman AD. Relation of renal cortical gluconeogenesis, glutamate

content, and production of ammonia. J Clin Invest 49: 1967-1974, 1970.

22. Studer RK, Snowdowne KW, and Borle AB. Regulation of hepatic glycogenolysis by

glucagon in male and female rats. Role of cAMP and Ca2+ and interactions between

epinephrine and glucagon. The Journal of biological chemistry 259: 3596-3604, 1984.

23. Stumvoll M, Meyer C, Perriello G, Kreider M, Welle S, and Gerich J. Human kidney

and liver gluconeogenesis: evidence for organ substrate selectivity. The American journal

of physiology 274: E817-826, 1998.

24. Sunehag A, Gustafsson J, and Ewald U. Glycerol carbon contributes to hepatic

glucose production during the first eight hours in healthy term infants. Acta Paediatr 85:

1339-1343, 1996.

25. Sunehag A, Tigas S, and Haymond MW. Contribution of plasma galactose and

glucose to milk lactose synthesis during galactose ingestion. The Journal of clinical

endocrinology and metabolism 88: 225-229, 2003.

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26. Sunehag AL. Parenteral glycerol enhances gluconeogenesis in very premature infants.

Pediatric research 53: 635-641, 2003.

27. Sunehag AL and Haymond MW. Maternal protein homeostasis and milk protein

synthesis during feeding and fasting in humans. American journal of physiology 285:

E420-426, 2003.

28. Sunehag AL, Louie K, Bier JL, Tigas S, and Haymond MW. Hexoneogenesis in the

human breast during lactation. The Journal of clinical endocrinology and metabolism 87:

297-301, 2002.

29. Sunehag AL, Treuth MS, Toffolo G, Butte NF, Cobelli C, Bier DM, and Haymond

MW. Glucose production, gluconeogenesis, and insulin sensitivity in children and

adolescents: an evaluation of their reproducibility. Pediatric research 50: 115-123, 2001.

30. Tigas S, Sunehag A, and Haymond MW. Metabolic adaptation to feeding and fasting

during lactation in humans. The Journal of clinical endocrinology and metabolism 87:

302-307, 2002.

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Chapter 6

Effect of ghrelin on glucose regulation in mice

Shaji K. Chacko

Morey W. Haymond

Yuxiang Sun

Juan C. Marini

Pieter J. J. Sauer

Agneta L. Sunehag

Submitted for publication.

129

Abstract

Objective: To determine whether ghrelin has an impact on gluconeogenesis,

glycogenolysis and insulin sensitivity using a mice model.

Research Design and Methods: Rate of appearance of glucose, gluconeogenesis and

glycogenolysis were measured in a set of five animals in each group of wild type (WT),

ghrelin knockout (ghrelin-/-) and growth hormone secretagogue receptor knockout (Ghsr-/-

) mice following a short term (8h) and prolonged fast (18h). Concentrations of glucose

and insulin were measured, and insulin resistance and hepatic insulin sensitivity were

calculated.

Results: Glucose concentrations were not different among the groups either following

the 8 or 18h fast; however, they were lower after the 18h fast. Plasma insulin

concentrations were lower in the ghrelin-/- and Ghsr-/- than WT animals following the 8h

fast, but were not different after the 18h fast. The rates of gluconeogenesis,

glycogenolysis and glucose clearance, and indices of insulin sensitivity were higher in the

ghrelin-/- and Ghsr-/- than WT animals after the 8h fast, but were not different between the

ghrelin-/- and Ghsr-/- groups. Following an 18h fast, gluconeogenic rates were lower in

the ghrelin-/- and Ghsr-/- than WT animals and gluconeogenesis accounted for nearly all

glucose production in all groups.

Conclusions: This study demonstrates that gluconeogenesis and glycogenolysis are

increased and insulin sensitivity is improved by the ablation of ghrelin or growth

hormone secretagogue receptor in mice.

130

Introduction The improvement in insulin sensitivity and in many cases reversal of Type-2

diabetes after bariatric surgery procedures are well established (1-5). However, the

underlying reason(s) for this improvement is not clear. Improved glycemic control

achieved immediately after surgery but before weight loss suggests a hormonal

mechanism as the explanation for improved glucose homeostasis (6). The plasma ghrelin

concentration was decreased by ~ 70 % following gastric bypass surgery as compared to

matched obese and normal-weight controls (6). The relationship between the

improvement of glucose metabolism and reduced ghrelin concentration remains to be

determined.

The stomach is the major source of circulating ghrelin (7). Further, ghrelin-

containing cells are abundant in the fundus region of the stomach (7-10). Sleeve

gastrectomy, which involves the complete resection of the gastric fundus, resulted in

better glycemic control when compared to other forms of bariatric surgery (3).

Ghrelin stimulates the release of growth hormone via the growth hormone

secretagogue receptor (11-13). Consequently, studying the effects of ghrelin on glucose

metabolism using ghrelin infusion can potentially be confounded by the effects of growth

hormone. Moreover, ghrelin is produced by cells scattered in various tissues (14-17).

Therefore, studies of glucose kinetics in the absence of ghrelin or its receptor using

transgenic mice models (13; 18-20) provide an opportunity to investigate the effects of

ghrelin on glucose metabolism independent of any action of growth hormone.

Ghrelin infusion in growth hormone deficient and normal mice (21) and in humans

(22) indicated that the effects of ghrelin on glucose metabolism also occur in the absence

131

of growth hormone. Another adipocytokine, leptin, also influences glucose metabolism.

Intracerebral infusion of leptin stimulated gene expression of hepatic glucose-6-

phosphatase and PEPCK, and increased gluconeogenesis (23). Similarly infusion of

leptin increased both hepatic and peripheral insulin sensitivity (24-26) and inhibited

insulin secretion (27; 28). Antagonizing effects of ghrelin on leptin demonstrated by co-

administration of leptin and ghrelin (28) indicate that leptin’s influence on

gluconeogenesis and insulin secretion may be counteracted by the presence of ghrelin.

Insulin has been shown to decrease glycogenolysis in a dose dependent manner (29; 30).

The present study was designed to address the following hypotheses; 1)

gluconeogenesis and insulin sensitivity are higher in the absence of ghrelin, 2)

glycogenolysis is higher during post absorptive conditions due to a lower insulin

concentration in the absence of ghrelin. To test these hypotheses, we compared glucose

kinetics among wild type (WT), ghrelin (ghrelin-/-) and growth hormone secretagogue

receptor (Ghsr -/-) knockout mice.

Materials and Methods

Animals and housing: Four to five months old adult male WT, ghrelin-/-, and Ghsr -/- mice

were used for all the experiments. Mice were kept in a standard housing facility and had

access to standard chow diet (Harlan teklad rodent diet 2920x) with ad-lib access to

autoclaved reverse osmosis water. Mice were maintained under a 12 h light cycle (600-

1800h) and constant temperature (75 ± 2ºF). Glucose kinetic measurements were

performed in a set of five animals (n=5) in each group. Body composition was measured

in another set of animals; WT (n=15), ghrelin-/- (n=9), and Ghsr -/- mice (n=7). Glucose

132

and insulin concentrations were measured in identically fasted WT (n=10), ghrelin-/-

(n=5), and Ghsr -/- mice (n=5). All procedures used in the animal experiments were

approved by the Baylor College of Medicine Institutional Animal Care and Use

Committee.

Body Composition: Body composition parameters such as total body fat and total lean

body mass were measured by magnetic resonance imaging (MRI) using EchoMRI-100

(QNMR systems, Houston, TX).

Stable isotopes: Sterile and pyrogen free deuterium oxide (2H2O), 99 atom percent 2H and

[6,6-2H2]glucose, 99 atom% 2H were purchased from Cambridge Isotopes Laboratories

(Andover, MA). [6,6-2H2]glucose was dissolved in 0.5% 2H2O (made isotonic by addition

of sodium chloride), filtered and prepared for intravenous infusion.

Study design: Rate of appearance of glucose and gluconeogenesis were measured in each

group at the end of a short term fast of 8 h and a prolonged fast of 18 h, respectively. On

the day of the infusion, all mice were weighed and received an IP dose of 99% 2H2O

(4mg/g body weight) resulting in a deuterium enrichment of (~) 0.5% in body water.

After this 2H2O dose, the animals were given ad libitum access to water (0.5% 2H2O) for

the rest of the study to maintain the body water deuterium enrichment at ~0.5%. The

mice were then restrained in an infusion box and a tail vein catheter was inserted as

described previously (31). Two hours following the 2H2O dose, a primed constant rate

infusion of [6,6-2H2]glucose at (~) 0.75 mg/kg.min (150 µL/hour) was started and

continued for 4 h.

After 4 h of infusion, blood was drawn from the submaxibular bundle and

centrifuged for 15 min at 4º C. Plasma was separated and kept frozen at -80º C until

133

analyzed. Blood samples from five mice were collected by lateral vein bleeding before

start of the tracer infusion to determine baseline enrichments. A pilot experiment in 3

animals was initially performed to ascertain that the isotopic enrichment had reached

steady state between 3 and 4 hrs. During the pilot study, one sample was collected before

the start of the infusion from the lateral tail vein and two samples at 3h, and 4h

(60µl/sample) by submaxibular bleeding technique.

Analyses: The Isotopic enrichment of [6,6-2H2]glucose was measured by gas

chromatography – mass spectrometry (GCMS) (6890/5973 Agilent Technologies,

Wilmington, DE) using the pentaacetate derivative. The incorporation of deuterium in

glucose was determined using the average deuterium enrichment in glucose carbons

1,3,4,5 and 6 as previously described (32; 33).

Briefly, this method (32; 33) involves preparation of the pentaacetate derivative of

glucose, followed by sample analysis using GCMS in the positive chemical ionization

mode. Selective ion monitoring of m/z 170/169 is performed to determine the M+1

enrichment of deuterium in the circulating glucose carbons (C-1,3,4,5,6,6) (M is the base

mass, 169, representing unlabeled glucose). After subtracting the enrichment of M+1

resulting from the natural abundance, the average enrichment of deuterium on a

gluconeogenic carbon is calculated from these M+1 data (32; 33). Deuterium enrichment

in plasma water is determined by Isotope Ratio Mass Spectrometry (Delta+XL IRMS

Thermo Finnigan, Bremen, Germany).

Insulin concentrations were determined by radioimmunoassay (Millipore,

Billerica, MA) and plasma glucose concentrations using the Precision Xtra blood glucose

monitoring system (Abbott Inc, Alameda, CA).

134

Calculations: All kinetic measurements were performed under steady state conditions.

Total plasma glucose appearance rate (glucose Ra) was calculated from the M+2

enrichment of [6,6-2H2]glucose in plasma using established isotope dilution equations

(34).

Rate of glucose production (mg/kg.min) (GPR) = glucose Ra – exogenous glucose

(i.e only the tracer since the animals were fasting).

Fractional gluconeogenesis (i.e. gluconeogenesis as a fraction of glucose Ra) was

calculated according to Chacko et. al (32; 33) as follows:

Fractional gluconeogenesis (GNG % Ra) = [(M+1) (2H) (m/z 170/169) /6] / E 2H2O

where (M+1)(2H) (m/z 170/169) is the M+1 enrichment of deuterium in glucose measured

using m/z 170/169 and ‘6’ is the number of 2H labeling sites on the m/z 170/169 fragment

of glucose (i.e the average M+1 enrichment derived from deuterated water) and E 2H2O is

the deuterium enrichment in plasma water.

Rate of gluconeogenesis was calculated as the product of total glucose appearance

rate and fractional gluconeogenesis.

Rate of Gluconeogenesis (mg/kg.min) (GNG rate) = gluc Ra × GNG % Ra

Glycogenolysis was calculated by subtracting the rate of gluconeogenesis from the

glucose production rate.

Rate of glycogenolysis (mg/kg.min) = GPR - GNG rate

Glucose Clearance (ml/kg.min) = gluc Ra / C

where gluc Ra is the rate of appearance, which equals the rate of disappearance of

glucose (mg/kg.min) under steady state conditions and C is the plasma glucose

concentration in mg/mL.

135

Insulin resistance was calculated by the homeostasis model assessment, HOMA-IR

(fasting insulin μU/ml x fasting glucose mM /22.5) (35; 36).

Hepatic insulin sensitivity was calculated in the fasting state by the hepatic insulin

sensitivity index (HISI): 1000/ (GPR [µmol/kg · min) x fasting plasma insulin (µU/mL)],

where 1000 is a constant that results in numbers between 1 and 10, as described by

Matsuda et al. (37; 38).

Statistical analyses: ANOVA was used to test significance among groups. ANOVA was

followed by unpaired t-test to compare significant differences between groups. A p value

<0.05 was used to define significance. All results are provided as mean ± SE.

Results

Body Composition (Table 1): Lean body mass was similar in all groups, however, total

body fat was lower in ghrelin-/-, and Ghsr -/- as compared to WT mice.

Table 1. Body composition measurements in WT, ghrelin-/- and Ghsr -/- mice.

WT

ghrelin-/- Ghsr -/- p Value WT vs.

ghrelin-/-

p Value WT vs. Ghsr -/-

p Value ghrelin-/- vs.

Ghsr -/- Lean body mass (g)

23.0 ± 0.3 22.0 ± 0.6 22.4 ± 0.4 NS NS NS

Total body fat (g)

6.7 ± 0.7 4.0 ± 0.3 3.1 ± 0.5 0.006 0.002 NS

Body fat (%)

19.8 ± 1.5 13.7 ± 0.9 10.5 ± 1.4 0.007 0.001 NS

Measurements following a short term fast (8 h): Glucose concentrations were similar

among the groups (Table 2). Plasma insulin concentrations were significantly lower in

ghrelin-/- and Ghsr -/- mice than WT mice (p=0.012 and 0.009, respectively), however,

136

were not different between ghrelin -/- and Ghsr -/- mice (Table 2). The glucose production

rates in ghrelin-/- and Ghsr -/- mice were nearly 60% higher when compared to the WT

group, p=0.008 and 0.0004, respectively (Table 3). The rates were similar (NS) in

ghrelin-/- and Ghsr -/- animals. The rates of gluconeogenesis were higher in ghrelin-/-, and

Ghsr -/- mice than WT (p=0.014 and 0.002, respectively), however, no difference was

observed between the ghrelin-/- and Ghsr -/- groups (Fig 1). Gluconeogenesis accounted

for ~ 70% of glucose production in all three groups. Rates of glycogenolysis (Fig 2) were

higher in ghrelin-/- and Ghsr -/- mice than in WT mice (p=0.017 and 0.003, respectively),

but no difference was observed between the ghrelin-/- and Ghsr -/- groups. Glucose

clearance rate was significantly higher in ghrelin-/-, and Ghsr -/- mice as compared to WT

(p=0.002 and 0.0003, respectively) and no difference was observed between the ghrelin-/-

and Ghsr -/- groups (Table 3). HOMA-IR was lower in ghrelin-/- and Ghsr -/- mice than

WT (p=0.02 and p=0.02, respectively) (Table 2) and HISI was higher in ghrelin-/- and

Ghsr -/- mice than WT (p=0.005 and p=0.0002, respectively).

Table 2. Concentrations of glucose and insulin in WT, ghrelin-/- and Ghsr -/- mice at the end of 8 h and 18 h fast.

8 h Fast 18 h Fast WT

ghrelin-/- Ghsr -/- WT

ghrelin-/- Ghsr -/-

Glucose (mM)

14.0 ± 0.6 12.0 ± 0.9 13.7 ± 0.4 8.2 ± 0.4 7.3 ± 0.6 8.6 ± 0.7

Insulin (ng/mL)

2.07 ± 0.25 0.98 ± 0.14* 0.88 ± 0.21* 0.86 ± 0.18 0.51 ± 0.10 0.53 ± 0.13

Data expressed Mean ± SE and * denotes p<0.05 ghrelin-/- vs. WT group and Ghsr -/- vs. WT group.

137

Table 3. Rates of glucose appearance (Ra), glucose production (GPR) and glucose clearance, and Indices of HOMA-Insulin resistance (HOMA-IR) and Hepatic insulin sensitivity (HISI) in WT, ghrelin-/- and Ghsr -/- mice at the end of 8 h and 18 h fast, respectively.

8 h Fast 18 h Fast

WT

ghrelin-/- Ghsr -/- WT

ghrelin-/- Ghsr -/-

Glucose Ra (mg/Kg.min)

12.89 ± 0.82 20.12 ± 1.89* 19.66 ± 0.82* 13.83 ± 0.51 9.15 ± 0.67* 11.83 ± 1.34

GPR (mg/Kg.min)

12.17 ± 0.81 19.34 ± 1.89* 18.93 ± 0.81* 12.33 ± 0.50 8.32 ± 0.65* 11.03 ± 1.34

Glucose clearance rate (ml/kg.min)

5.13 ± 0.33 9.33 ± 0.88* 7.98 ± 0.33* 9.41 ± 0.35 6.94 ± 0.51* 7.68 ± 0.87

HOMA-IR

32.3 ± 6.6 13.1 ± 2.6* 13.1 ± 3.5* 8.3 ± 3.3 4.0 ± 0.7 5.0 ± 1.3

HISI

0.29 ± 0.01 0.40 ± 0.04* 0.44 ± 0.02* 0.77 ± 0.10 1.76 ± 0.17* 1.21 ± 0.13*

Data expressed Mean ± SE and * denotes p<0.05 ghrelin-/- vs. WT group and Ghsr -/- vs. WT group.

138

0

4

8

12

16

WT Ghrelin -/- Ghsr -/-

GluconeogenesisGlycogenolysis

mg/

Kg. m

in

P<0.05

P<0.05

P<0.05P<0.05

0

4

8

12

16

WT Ghrelin -/- Ghsr -/-

GluconeogenesisGlycogenolysisGluconeogenesisGlycogenolysis

mg/

Kg. m

in

P<0.05P<0.05

P<0.05P<0.05

P<0.05P<0.05P<0.05

Figure 1. Rate of gluconeogenesis and glycogenolysis was significantly higher in ghrelin-

/- and Ghsr -/- mice at the end of the 8 h fast as compared to those in WT group.

0

4

8

12

16

WT Ghrelin -/- Ghsr -/-

mg/

kg. m

in

P<0.05

P<0.05

0

4

8

12

16

WT Ghrelin -/- Ghsr -/-

mg/

kg. m

in

0

4

8

12

16

WT Ghrelin -/- Ghsr -/-

mg/

kg. m

in

P<0.05P<0.05

P<0.05P<0.05

Figure 2. Rate of gluconeogenesis was significantly lower in ghrelin-/- and Ghsr -/- mice

at the end of the 8 h of fast as compared to that in WT group.

139

Measurements following a prolonged fast (18 h): Both glucose and insulin concentrations

were not different among the three groups (Table 2). The glucose production rates were

lower in ghrelin-/- when compared to the WT group, (p=0.001) (Table 3), however, the

differences between the Ghsr -/- and WT groups were not significant. The rates of

gluconeogenesis (Fig 2) were lower in the ghrelin-/- and Ghsr -/- as compared to the WT

group, (p=0.0001 and 0.024, respectively), but were not different between the ghrelin-/-

and Ghsr -/-groups. At the end of the 18 h fast, glycogenolysis was essentially zero in all

groups and gluconeogenesis accounted for nearly all glucose production in all the groups.

The glucose clearance rate was lower in the ghrelin-/- knockout group than WT,

(p=0.004), but the difference did not reach significance between WT and the Ghsr -/-

mice. Following the prolonged fast, HOMA-IR was not different among groups (Table 2)

while HISI was higher in ghrelin-/- and Ghsr -/- mice as compared to WT (p=0.0002 and

p=0.026, respectively).

Glucose concentrations were significantly lower after the 18 h as compared to 8 h

fast in all groups (p<0.004 in all groups). Glucose production rates were lower in both

ghrelin-/- and Ghsr -/- groups following the18 h as compared to the 8 h fast, while no

difference was observed in the WT group.

Discussion

Changes in gut hormones involved in the regulation of glucose metabolism are well

established (39; 40). However, the effect of ghrelin, an endogenous ligand for the growth

hormone secretagogue receptor (13; 15), on endogenous glucose synthesis remains

unclear. In the present study, we demonstrated that ablation of ghrelin or its receptor in

8h fasted mice resulted in increase of gluconeogenesis and glycogenolysis (Fig 1).

140

Interestingly, rates of gluconeogenesis and glycogenolysis were not different between

ghrelin-/- and Ghsr -/- mice. These data demonstrate that ghrelin plays a role in the

regulation of gluconeogenesis and glycogenolysis, and that at least part (if not all) of

those effects occurs via the growth hormone secretagogue receptor.

A potential mechanism for increased gluconeogenesis and glycogenolysis could be

secondary to the opposing effects of ghrelin on leptin action (28). Intracerebral infusion

of leptin stimulated gene expression of the hepatic enzymes glucose-6-phosphatase and

PEPCK with subsequent increase in gluconeogenesis (23). At the end of the 8 h fast in

our study, in the absence of ghrelin (ghrelin-/-) or its mediation through its purported

receptor (Ghsr -/-), gluconeogenesis was higher than in WT mice. The increased rate of

gluconeogenesis might be because of the absence of the inhibiting effect of ghrelin on

leptin since we previously reported that the plasma leptin concentrations were similar in

ghrelin-/-, Ghsr -/- and WT mice (13; 41).

It has also been reported that leptin inhibits insulin secretion and ghrelin reverses

leptin’s inhibiting effect on insulin secretion (27; 28). This suggests reduced insulin

concentration in the absence of ghrelin. Consistent with this we observed that insulin

concentrations were lower in both the ghrelin-/- and Ghsr -/- mice as compared to WT

mice following the 8 h fast (Table 1). Previous studies have shown that insulin decreases

glycogenolysis in a dose dependent manner (29; 30). In the present study, in response to

lower insulin concentration in the knockout groups than in the WT, glycogenolysis was

significantly higher following 8 h of fasting (Fig 1).

The significantly higher hepatic insulin sensitivity index and improved insulin

resistance in ghrelin-/- and Ghsr -/- as compared to WT mice demonstrate increased insulin

141

sensitivity in the absence of ghrelin action on the purported ghrelin receptor. Ghrelin

infusion has been reported to induce insulin resistance and stimulate lipolysis (22; 42;

43), whereas, leptin increased both hepatic and peripheral insulin sensitivity (24-26). This

indicates that leptin unopposed by ghrelin action might be the reason for this increased

insulin sensitivity. Both ghrelin -/- and Ghsr -/- knockout mice are more insulin sensitive

than WT mice (41; 44-46). Sun et al. reported that glucose production was more

suppressed in ghrelin-/- than WT mice during a low-dose insulin clamp suggesting

increased hepatic insulin sensitivity in the absence of ghrelin (41).

Enhanced hepatic insulin sensitivity can potentially stimulate glycogen synthesis

during glucose availability resulting in increased glycogen stores. This might explain our

observation of significantly higher rates of glycogenolysis in the ghrelin-/- and Ghsr -/- as

compared to WT mice at the end of the 8 h fast. A previous report that ghrelin down-

regulates markers of glycogen synthesis (47) is consistent with this observation.

Blood glucose concentrations were similar in all groups of animals at the end of the

8 h fast despite significantly higher rates of gluconeogenesis and glycogenolysis in

ghrelin-/- and Ghsr -/- mice indicating increased glucose uptake in the absence of ghrelin

or its receptor. Higher glucose clearance observed at lower insulin concentration during

short term fast in ghrelin-/- and Ghsr -/- as compared to WT mice in our study might

suggest that less insulin is required for peripheral glucose uptake in the absence of ghrelin

or its receptor under normal physiologic conditions. This is in line with the improved

peripheral insulin sensitivity reported in these knockout mice (41; 44-46). Consistent with

our observation, continuous ghrelin infusion was demonstrated to induce insulin

resistance in muscle and to stimulate lipolysis (22; 42; 43). Reduction of plasma ghrelin

142

below physiological levels by insulin infusion during glucose clamp resulted in a sharp

increase of insulin sensitivity in humans (48).

In agreement with other reports (45; 49; 50), the measurements of body

composition (table 2) revealed that both knockout groups had significantly smaller fat

mass as compared to WT. Lower fat accretion stimulated by the absence of ghrelin or its

receptor might be another reason for improved insulin sensitivity. In contrast, we found

that the lean body mass was not different among the three groups.

During prolonged fasting (18 h), rates of glycogenolysis were essentially zero and

insulin concentrations were appropriately decreased and similar in all groups as reported

previously (19). In WT mice, gluconeogenesis was significantly increased. However, no

increase in gluconeogenesis was observed in the knockout groups (Fig 2). The plasma

glucose concentrations remained similar in all groups. We observed that glucose

clearance was significantly lower in the ghrelin-/- and Ghsr -/- groups suggesting lower

demand for glucose via gluconeogenesis in the knockout groups. Alternatively, smaller

lipid stores in the knockout groups and thus, less mobilization of gluconeogenic

substrates might be another reason for this lower rate of gluconeogenesis.

Thus, our study demonstrates that gluconeogenesis and glycogenolysis are

increased, and insulin sensitivity is improved in mice by the absence of ghrelin action.

This could be a potential explanation for the improvement of glucose metabolism

observed in patients after bariatric surgery. We speculate that improved glucose

metabolism in the absence of ghrelin in mice might be associated with leptin. A recent

report demonstrated that antidiabetic actions of leptin are mediated via the central

nervous system dependent mechanisms (51). Further studies are required to determine the

143

mechanisms underlying the association between leptin and ghrelin in the improvement of

glucose metabolism. Our present data supports a potential prospect of improving insulin

sensitivity in the diabetic condition by the use of ghrelin receptor antagonists.

144

References

1. Vidal J, Ibarzabal A, Nicolau J, Vidov M, Delgado S, Martinez G, Balust J, Morinigo

R, Lacy A: Short-term effects of sleeve gastrectomy on type 2 diabetes mellitus in

severely obese subjects. Obes Surg 2007;17:1069-1074

2. Shah PS, Todkar JS, Shah SS: Effectiveness of laparoscopic sleeve gastrectomy on

glycemic control in obese Indians with type 2 diabetes mellitus. Surg Obes Relat Dis

2010;6:138-141

3. Frezza EE, Wozniak SE, Gee L, Wacthel M: Is there any role of resecting the stomach

to ameliorate weight loss and sugar control in morbidly obese diabetic patients? Obes

Surg 2009;19:1139-1142

4. Buchwald H, Avidor Y, Braunwald E, Jensen MD, Pories W, Fahrbach K, Schoelles

K: Bariatric surgery: a systematic review and meta-analysis. JAMA 2004;292:1724-1737

5. Dixon JB, O'Brien PE, Playfair J, Chapman L, Schachter LM, Skinner S, Proietto J,

Bailey M, Anderson M: Adjustable gastric banding and conventional therapy for type 2

diabetes: a randomized controlled trial. JAMA 2008;299:316-323

6. Cummings DE, Weigle DS, Frayo RS, Breen PA, Ma MK, Dellinger EP, Purnell JQ:

Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. N Engl J

Med 2002;346:1623-1630

7. Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, Suda M, Koh T,

Natsui K, Toyooka S, Shirakami G, Usui T, Shimatsu A, Doi K, Hosoda H, Kojima M,

Kangawa K, Nakao K: Stomach is a major source of circulating ghrelin, and feeding state

determines plasma ghrelin-like immunoreactivity levels in humans. J Clin Endocrinol

Metab 2001;86:4753-4758

145

8. Tomasetto C, Wendling C, Rio MC, Poitras P: Identification of cDNA encoding

motilin related peptide/ghrelin precursor from dog fundus. Peptides 2001;22:2055-2059

9. Yabuki A, Ojima T, Kojima M, Nishi Y, Mifune H, Matsumoto M, Kamimura R,

Masuyama T, Suzuki S: Characterization and species differences in gastric ghrelin cells

from mice, rats and hamsters. J Anat 2004;205:239-246

10. Hayashida T, Nakahara K, Mondal MS, Date Y, Nakazato M, Kojima M, Kangawa

K, Murakami N: Ghrelin in neonatal rats: distribution in stomach and its possible role. J

Endocrinol 2002;173:239-245

11. Korbonits M, Grossman AB: Ghrelin: update on a novel hormonal system. Eur J

Endocrinol 2004;151 Suppl 1:S67-70

12. Howard AD, Feighner SD, Cully DF, Arena JP, Liberator PA, Rosenblum CI,

Hamelin M, Hreniuk DL, Palyha OC, Anderson J, Paress PS, Diaz C, Chou M, Liu KK,

McKee KK, Pong SS, Chaung LY, Elbrecht A, Dashkevicz M, Heavens R, Rigby M,

Sirinathsinghji DJ, Dean DC, Melillo DG, Patchett AA, Nargund R, Griffin PR,

DeMartino JA, Gupta SK, Schaeffer JM, Smith RG, Van der Ploeg LH: A receptor in

pituitary and hypothalamus that functions in growth hormone release. Science

1996;273:974-977

13. Sun Y, Wang P, Zheng H, Smith RG: Ghrelin stimulation of growth hormone release

and appetite is mediated through the growth hormone secretagogue receptor. Proc Natl

Acad Sci U S A 2004;101:4679-4684

14. Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura

S, Kangawa K, Nakazato M: Ghrelin, a novel growth hormone-releasing acylated

146

peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats

and humans. Endocrinology 2000;141:4255-4261

15. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K: Ghrelin is a

growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656-660

16. Kojima M, Hosoda H, Matsuo H, Kangawa K: Ghrelin: discovery of the natural

endogenous ligand for the growth hormone secretagogue receptor. Trends Endocrinol

Metab 2001;12:118-122

17. Rindi G, Necchi V, Savio A, Torsello A, Zoli M, Locatelli V, Raimondo F, Cocchi D,

Solcia E: Characterisation of gastric ghrelin cells in man and other mammals: studies in

adult and fetal tissues. Histochem Cell Biol 2002;117:511-519

18. Ukkola O: Ghrelin and insulin metabolism. Eur J Clin Invest 2003;33:183-185

19. Sun Y, Ahmed S, Smith RG: Deletion of ghrelin impairs neither growth nor appetite.

Mol Cell Biol 2003;23:7973-7981

20. Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M,

Thabet K, Cox HJ, Yancopoulos GD, Wiegand SJ, Sleeman MW: Genetic deletion of

ghrelin does not decrease food intake but influences metabolic fuel preference. Proc Natl

Acad Sci U S A 2004;101:8227-8232

21. Dezaki K, Hosoda H, Kakei M, Hashiguchi S, Watanabe M, Kangawa K, Yada T:

Endogenous ghrelin in pancreatic islets restricts insulin release by attenuating Ca2+

signaling in beta-cells: implication in the glycemic control in rodents. Diabetes

2004;53:3142-3151

147

22. Vestergaard ET, Gormsen LC, Jessen N, Lund S, Hansen TK, Moller N, Jorgensen

JO: Ghrelin infusion in humans induces acute insulin resistance and lipolysis independent

of growth hormone signaling. Diabetes 2008;57:3205-3210

23. Gutierrez-Juarez R, Obici S, Rossetti L: Melanocortin-independent effects of leptin

on hepatic glucose fluxes. J Biol Chem 2004;279:49704-49715

24. Shimomura I, Hammer RE, Ikemoto S, Brown MS, Goldstein JL: Leptin reverses

insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature

1999;401:73-76

25. Pocai A, Morgan K, Buettner C, Gutierrez-Juarez R, Obici S, Rossetti L: Central

leptin acutely reverses diet-induced hepatic insulin resistance. Diabetes 2005;54:3182-

3189

26. Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ: Acute stimulation of

glucose metabolism in mice by leptin treatment. Nature 1997;389:374-377

27. Cases JA, Gabriely I, Ma XH, Yang XM, Michaeli T, Fleischer N, Rossetti L,

Barzilai N: Physiological increase in plasma leptin markedly inhibits insulin secretion in

vivo. Diabetes 2001;50:348-352

28. Kim MS, Namkoong C, Kim HS, Jang PG, Kim Pak YM, Katakami H, Park JY, Lee

KU: Chronic central administration of ghrelin reverses the effects of leptin. Int J Obes

Relat Metab Disord 2004;28:1264-1271

29. Hartmann H, Ebert R, Creutzfeldt W: Insulin-dependent inhibition of hepatic

glycogenolysis by gastric inhibitory polypeptide (GIP) in perfused rat liver. Diabetologia

1986;29:112-114

148

30. Rossetti L, Hu M: Skeletal muscle glycogenolysis is more sensitive to insulin than is

glucose transport/phosphorylation. Relation to the insulin-mediated inhibition of hepatic

glucose production. J Clin Invest 1993;92:2963-2974

31. Marini JC, Lee B, Garlick PJ: Non-surgical alternatives to invasive procedures in

mice. Lab Anim 2006;40:275-281

32. Chacko SK, Sunehag AL, Sharma S, Sauer PJ, Haymond MW: Measurement of

gluconeogenesis using glucose fragments and mass spectrometry after ingestion of

deuterium oxide. J Appl Physiol 2008;104:944-951

33. Chacko SK, Sunehag AL: Gluconeogenesis continues in premature infants receiving

total parenteral nutrition. Arch Dis Child Fetal Neonatal Ed 2010;95:F413-418

34. Bier DM, Leake RD, Haymond MW, Arnold KJ, Gruenke LD, Sperling MA, Kipnis

DM: Measurement of "true" glucose production rates in infancy and childhood with 6,6-

dideuteroglucose. Diabetes 1977;26:1016-1023

35. Matthews DR, Hosker JP, Rudenski AS, Naylor BA, Treacher DF, Turner RC:

Homeostasis model assessment: insulin resistance and beta-cell function from fasting

plasma glucose and insulin concentrations in man. Diabetologia 1985;28:412-419

36. van der Heijden GJ, Wang ZJ, Chu ZD, Sauer PJ, Haymond MW, Rodriguez LM,

Sunehag AL: A 12-week aerobic exercise program reduces hepatic fat accumulation and

insulin resistance in obese, Hispanic adolescents. Obesity (Silver Spring) 2010;18:384-

390

37. Matsuda M, DeFronzo RA: Insulin sensitivity indices obtained from oral glucose

tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care

1999;22:1462-1470

149

38. van der Heijden GJ, Toffolo G, Manesso E, Sauer PJ, Sunehag AL: Aerobic exercise

increases peripheral and hepatic insulin sensitivity in sedentary adolescents. J Clin

Endocrinol Metab 2009;94:4292-4299

39. Drucker DJ, Nauck MA: The incretin system: glucagon-like peptide-1 receptor

agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 2006;368:1696-

1705

40. Holst JJ, Gromada J: Role of incretin hormones in the regulation of insulin secretion

in diabetic and nondiabetic humans. Am J Physiol Endocrinol Metab 2004;287:E199-206

41. Sun Y, Asnicar M, Saha PK, Chan L, Smith RG: Ablation of ghrelin improves the

diabetic but not obese phenotype of ob/ob mice. Cell Metab 2006;3:379-386

42. Broglio F, Prodam F, Riganti F, Gottero C, Destefanis S, Granata R, Muccioli G,

Abribat T, van der Lely AJ, Ghigo E: The continuous infusion of acylated ghrelin

enhances growth hormone secretion and worsens glucose metabolism in humans. J

Endocrinol Invest 2008;31:788-794

43. Vestergaard ET, Djurhuus CB, Gjedsted J, Nielsen S, Moller N, Holst JJ, Jorgensen

JO, Schmitz O: Acute effects of ghrelin administration on glucose and lipid metabolism.

J Clin Endocrinol Metab 2008;93:438-444

44. Dezaki K, Sone H, Koizumi M, Nakata M, Kakei M, Nagai H, Hosoda H, Kangawa

K, Yada T: Blockade of pancreatic islet-derived ghrelin enhances insulin secretion to

prevent high-fat diet-induced glucose intolerance. Diabetes 2006;55:3486-3493

45. Longo KA, Charoenthongtrakul S, Giuliana DJ, Govek EK, McDonagh T, Qi Y,

DiStefano PS, Geddes BJ: Improved insulin sensitivity and metabolic flexibility in

ghrelin receptor knockout mice. Regul Pept 2008;150:55-61

150

46. Sun Y, Butte NF, Garcia JM, Smith RG: Characterization of adult ghrelin and ghrelin

receptor knockout mice under positive and negative energy balance. Endocrinology

2008;149:843-850

47. Murata M, Okimura Y, Iida K, Matsumoto M, Sowa H, Kaji H, Kojima M, Kangawa

K, Chihara K: Ghrelin modulates the downstream molecules of insulin signaling in

hepatoma cells. J Biol Chem 2002;277:5667-5674

48. Lucidi P, Murdolo G, Di Loreto C, De Cicco A, Parlanti N, Fanelli C, Santeusanio F,

Bolli GB, De Feo P: Ghrelin is not necessary for adequate hormonal counterregulation of

insulin-induced hypoglycemia. Diabetes 2002;51:2911-2914

49. Wortley KE, del Rincon JP, Murray JD, Garcia K, Iida K, Thorner MO, Sleeman

MW: Absence of ghrelin protects against early-onset obesity. J Clin Invest

2005;115:3573-3578

50. Zigman JM, Nakano Y, Coppari R, Balthasar N, Marcus JN, Lee CE, Jones JE,

Deysher AE, Waxman AR, White RD, Williams TD, Lachey JL, Seeley RJ, Lowell BB,

Elmquist JK: Mice lacking ghrelin receptors resist the development of diet-induced

obesity. J Clin Invest 2005;115:3564-3572

51. Fujikawa T, Chuang JC, Sakata I, Ramadori G, Coppari R: Leptin therapy improves

insulin-deficient type 1 diabetes by CNS-dependent mechanisms in mice. Proc Natl Acad

Sci U S A 2010;107:17391-17396

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152

Chapter 7

Subcutaneous Infusion and Capillary “Finger Stick” Sampling

of Stable Isotope Tracer in Metabolic Studies

Paula M. Hertel

Shaji K. Chacko

Sunita Pal

Agneta L. Sunehag

Morey W. Haymond

Pediatr Res 60: 597-601, 2006.

153

Abstract

Metabolic studies utilizing stable isotope tracer in humans have typically utilized

intravenous tracer infusions and venous blood sampling. These studies explore

subcutaneous infusion of isotope and “finger stick” capillary blood sampling to measure

glucose turnover. Five subjects received simultaneous eight-hour infusions of glucose

labeled with isotope: [1-13C]glucose subcutaneously, and [6,6-2H2]glucose intravenously.

At regular intervals, venous and finger stick blood specimens were obtained. Finger stick

blood was applied to filter paper. Substrate and isotopic steady state was reached after ~

7.0 h with both routes of infusion. The isotopic enrichments of finger stick and venous

specimens did not differ significantly for the subcutaneously infused [1-13C]glucose (p=

0.33, p =0.23) but the finger stick [6,6-2H2]glucose enrichment was slightly higher (p <

0.03) than that of the venous sample. Using [6,6-2H2]glucose infusion and venous plasma

sampling as the reference method, the [1-13C]glucose gave estimates of glucose Ra which

were 13% (plasma) and 17% (finger stick) lower (p < 0.001 and p < 0.02, respectively).

This difference could be attributed to recycling of 13C label. In conclusion, subcutaneous

infusion and finger stick specimen collection onto filter paper represent a potential

method of conducting in-vivo studies of substrate metabolism outside of a hospital-based

research unit.

154

Introduction

Traditionally, isotope dilution kinetic studies of glucose, free fatty acids, and

amino acid metabolism in humans have primarily utilized intravenous infusion (and oral

administration when tracing enteral absorption) of compounds labeled with stable

isotopes and measurement of substrate enrichment in venous blood (1,2). Intravenous

infusion and sampling enables rapid and direct introduction of labeled substrate into the

intravascular mixing pool and easy blood sampling. Intravenous access, however,

requires that subjects be studied in an inpatient or outpatient clinical research setting to

monitor and manage the I.V. infusion and sampling sites. Additionally, such study

conditions dramatically decrease the subject’s normal activity during the period of study.

The purpose of this study was to evaluate the validity of the use of subcutaneous

infusion of tracer and a “finger stick” blood sampling method to measure glucose kinetics

by comparing these results with data obtained using a simultaneous intravenous infusion

and blood sampling of a second glucose tracer, the current “gold standard” for this type

of study. Were such a model using subcutaneous infusion and finger stick sampling

successful, it would provide a methodology to study substrate kinetics outside of the

metabolic research unit under more real-life conditions.

Methods:

Subjects: The protocol was reviewed and approved by the Institutional Review Board for

Human Subject Research at Baylor College of Medicine in Houston, Texas. Twelve

healthy subjects, 5 females and 7 males, were recruited by newspaper advertisement or

by word of mouth. The mean subject age was 25.1 ± 1.2 years, and mean BMI 22.9 ± 0.8

155

kg/m2. All subjects were healthy, had a normal physical examination and screening

laboratory studies, including hemoglobin and chemistry panel, and a negative pregnancy

test in all women. Written consent was obtained at the screening visit or on study day

prior to beginning the protocol.

Tracers: Sterile and pyrogen free [6,6-2H2]glucose (99% atom% 2H) and [1-13C]glucose

(99% atom % 13C) were purchased from Cambridge Isotope Laboratories (Andover,

MA). The stable isotopes were retested for sterility and pyrogenicity by the

Investigational Pharmacy at Texas Children’s Hospital. For each subject, [6,6-

2H2]glucose was mixed with sterile 0.9% saline to deliver isotope at a rate of 0.04 mg/kg⋅

min (0.22 μmol.kg-1⋅min-1). [1-13C]glucose was mixed with sterile water to a

concentration of 10%. The glucose concentration of all infusates was verified using a

glucose oxidase method (YSI glucose analyzer; YSI, Yellow Springs, OH). The stable

isotope solutions for intravenous or subcutaneous infusion were prepared using sterile

technique, following which they were filtered (0.01 µm, Millipore Corporation, Inc.),

stored in sterile syringes, and kept refrigerated at 4°C until used within 36 hours of

preparation.

Subcutaneous Infusion Pumps: The [1-13C]glucose tracer was infused subcutaneously

using MiniMed® 508 insulin pumps (MiniMed®, Sylmar CA) with sterile, disposable

MiniMed® Sof-set® infusion sets consisting of syringe connection hub, tubing, and 1cm

subcutaneous infusion delivery needle; and sterile, disposable 3.0 mL MiniMed®

reservoirs.

Glucose extraction from filter paper: Before performing studies on human subjects, we

tested a number of extraction techniques from Whatman filter paper of [1- 13C]glucose

156

spiked blood samples. Filter paper was prepared with 2.5% NaF solution as described in

the methods section of this paper. Heparinized blood with a 7% [1- 13C]glucose

enrichment was dropped on Whatman paper previously treated with NaF as well as on

untreated paper. The specimens were allowed to dry and then processed and derivatized

as described below.

NaF-Treated Whatman Filter Paper: Whatman® 7.0 cm type 1 filter papers were

obtained from Sigma Chemical, St. Louis, MO and sodium fluoride powder (NaF),

99.1% pure, obtained from J.T. Baker, Phillipsburg, NJ. The filter papers were dipped

into a 2.5% NaF (wt/vol) solution mixed with Millipore-filtered water and allowed to air-

dry overnight on a clean surface. The NaF inhibits glycolysis in blood cells and would

preserve the glucose collected on the filter paper during drying and sample preparation.

A NaF concentration of 2.5% was chosen because it is comparable to the NaF

concentration attained when blood specimens (of specified volume as indicated by

manufacturer) are placed in commercially sold NaF-containing blood specimen tubes.

Differences in enrichments between the specimens placed on NaF-treated as compared

with untreated paper were not significant, but the amount of ethyl acetate required to

dilute the derivatized specimens prior to the GC/MS procedure was significantly greater

for the specimens extracted from the NaF-containing filter paper (p=0.04) than for those

extracted from the plain filter paper, indicating that a greater amount of substrate was

extracted from these papers. We did not find a significant difference in extractability of

[1- 13C]glucose from Whatman paper types 1, 4, 5, and 42.

In the first 7 subjects studied, we sought to determine both the sensitivity and

reproducibility to carry out a subcutaneous infusion/sampling method. In all of the early

157

subjects, we observed one or more values from the finger stick samples which clearly

reflected contamination (Fig. 1).

0

0.4

0.8

1.2

1.6

0 2 4 6 8 10

Hours

Enric

hmen

t

1-13C Glucose Fingerstick

1-13C Glucose Venous

0

0.4

0.8

1.2

1.6

0 2 4 6 8 10

Hours

Enric

hmen

t

1-13C Glucose Fingerstick

1-13C Glucose Venous

Figure 1. Contaminated finger stick glucose enrichments. The figure depicts the

enrichment in finger stick and venous specimens from a single subject receiving a

subcutaneous infusion of [1-13C]glucose and demonstrates the obvious contamination of

the finger stick samples.

Many, but not all, of these spikes were noted (data not shown) at times corresponding to

discontinuation of the isotope infusions and removal of isotope infusion line at the 8h

time point, suggesting investigator-induced contamination. Subsequently, measures were

taken to prevent contamination including: a) cleaning of the paper puncher between

specimens with ethyl acetate, b) assaying prepared filter paper samples for isotope prior

158

to using them on study day (to verify no contamination was present), c) preparing the

filter papers using a very stringent, “clean” technique. This “clean” technique included

frequent glove changes during the preparation of the paper when the glove may come into

contact with other objects, drying filter paper on clean disposable laboratory paper in

areas where isotopes are not used, and taking great care on study days to keep filter

papers out of contact with any surfaces. Finally, we observed a dramatic reduction in

contamination after the filter papers were enveloped in laminated paper sheaths after

initial drying and using warm-water soaks of subjects’ hands prior to performing each

finger stick. The traditional method of conducting in vivo stable isotope studies employs

intravenous isotope infusion of [6,6-2H2]glucose and blood sampling from the

contralateral arm. Since [6,6-2H2]glucose trace is not recycled back into the sampling

space (1-3), it is considered to be the most valid tracer for calculating total glucose Ra.

Therefore [6,6-2H2]glucose plasma enrichments were the “gold standard” to which the

results of the new methods were compared.

Study Design and Protocol: Subjects were instructed to consume a normal diet during

the three days preceding the study day, to ingest only water beginning at midnight the

night before the study (8 hours prior to beginning of study period), and to remain in the

fasted state until completion of the study.

On the morning of the study day, subjects arrived in the Metabolic Research Unit

(MRU) at the Children’s Nutrition Research Center, Houston, TX by 7:00 AM. Two

antecubital intravenous catheters were placed: One for infusion of [6,6-2H2]glucose, and

one in the contralateral arm for venous blood sampling. Baseline venous and finger stick

blood samples were obtained, and the MiniMed® pump was set up for infusion according

159

to the product manual for insulin infusion. Briefly, a Sof-set® needle was used in the skin

of the lateral part of the abdomen. Subjects remained in bed for the duration of the 11 h

study period. Most subjects briefly left the bed to use the nearby bathroom once or twice

during the study period during which special care was taken to assure continuous isotope

infusion.

After baseline blood sampling, isotope infusions were initiated. Continuous

infusion of [6,6-2H2]glucose (Razel Syringe Pump, Razel Scientific Instruments

Incorporated, Stamford, CT) at a rate of 0.2 μmol.kg-1.min-1 (0.0364 mg· kg-1·min-1) was

“piggy backed” into 0.45% saline infusion (20 mL/h). At the same time, a 10% [1-

13C]glucose was infused subcutaneously at a rate of 350 μL/h, which averaged 0.045 μ

mol·kg-1·min-1 (0.009 mg·kg-1

·min-1) for the five subjects used in the final analyses. Both

isotopes were infused for eight hours. At the end of the eighth hour of isotope infusion,

both infusions were discontinued and infusion needles immediately removed to avoid any

further entry of isotope into the tissues during the isotope decay portion of the study.

Blood sampling was continued for an additional 3h isotope decay period.

Both finger stick and venous blood were obtained at 0 h (immediately prior to

starting infusions), 1h, 2h, 3h, 4h, 5h, 6h, 6h30min, 7h, 7h30min, 8h30min, 9h, 9h30min,

10h, 10h30min, and 11h. Our subjects tolerated the repetitive finger sticks without

difficulty. A several-minute hand soak in fresh warm water in some subjects facilitated

blood flow, and obviated a need for a second stick. Two of our 12 subjects expressed

mild discomfort with the finger sticks toward the end of the study period. Thus, in

prolonged studies with repetitive finger sticks, it may be worthwhile to consider a topical

anesthetic such as Emla® cream to reduce discomfort.

160

Venous blood specimens (5 mL each) were placed immediately into EDTA tubes,

placed on ice, and centrifuged (3000 rpm for 10 minutes at 4°C). The plasma was

separated and stored at -70°C prior to analysis. Finger sampling sites were prepared by

soaking the subject’s hand in fresh, lukewarm tap water (~3-5 min) and dried with a clean

paper towel. The fingertip was then gently scrubbed with isopropyl alcohol, and pricked

with a standard disposable lancet device, as is used for self-glucose monitoring. Blood

was dropped onto NaF-impregnated filter paper (see “materials”) of a quantity sufficient

to saturate an area approximately 2 cm in diameter. The filter papers were enclosed and

allowed to dry thoroughly (for a minimum of 20 minutes).

Sample Preparation: Plasma samples were prepared as described above and analyzed as

described below. A conventional, hand-held office paper puncher with the plastic

reservoir removed was used to punch four 6.5 mm circles from each finger stick

specimen. The paper puncher was cleaned with an ethyl acetate-saturated cotton swab

between specimens to avoid cross-contamination of isotope from one specimen to the

next. Using latex-gloves, each set of four hole-punches was placed into a plastic

microcentrifuge tube for storage at -70°C until analyzed.

Analyses: One hundred and fifty (150) μL of sterile deionized water was placed into each

tube containing a filter paper blood specimen. The tubes were refrigerated at 4°C for 1 h,

then briefly vortexed. The water solution was transferred into 1.5 mL microcentrifuge

tubes. Two hundred (200) μL of ice cold acetone was added to deproteinize the sample,

and each tube briefly vortexed and capped before being refrigerated again for 10 min.

Specimens were centrifuged (3000 rpm at 4°C) for 10 min, and the supernatant decanted

into 4 mL vials and evaporated completely under nitrogen gas. Fifty (50) μL acetic

161

anhydride: pyridine (2:1) was added to each tube. The tubes were sealed tightly with a

Teflon-faced cap, vortexed briefly, and heated at 60°C for 10 min. The contents were

then dried under nitrogen gas and subsequently reconstituted in 30 μL ethyl acetate.

The enrichment of [6,6-2H2]glucose was measured by gas chromatography-mass

spectrometry (Agilent Technologies 6890/5973 GC/MS; GC column, SPB-1701: 30m x

0.25 μm x 0.25μm; Supelco), using the electron impact mode with selective monitoring

of m/z 242, unlabeled glucose; and 244, [6,6-2H2]glucose. Enrichments of ≥0.7% are

detectable using this method, with a coefficient of variation (C.V.) of 1-3% on samples

with enrichments ≥1%. The [1- 13C]glucose enrichment was determined with gas

chromatography-combustion-isotope ratio mass spectrometry (Thermo Finnigan Deltaplus

XL GC-C-IRMS) coupled with a 6890 GC (Agilent Technologies). GC-C-IRMS

measures the 13CO2/12CO2 ratio, thus excluding any interference from the [6,6-

2H2]glucose. Enrichments less than 0.1% are detectable using this method, with a C.V. of

0.3% on samples with enrichments of 0.3-0.9%.

Enrichment data were separated into four groups based on infusion route and

sampling method: 1) [6,6-2H2]glucose finger stick, 2) [6,6-2H2]glucose venous, 3) [1-

13C]glucose finger stick, and 4) [1-13C]glucose venous. Steady-state was achieved

between study hours 7.0 and 8.0. Steady state enrichments were calculated for each

subject by averaging enrichments from time points 7h, 7.5h, and 8h, and mean steady

state enrichments for all subjects within each of the four groups were determined based

on these values.

Calculations: Steady state was defined by C.V. < 10% and curve slope not significantly

different from zero during the 7th hour of isotope infusion. Coefficient of Variation

162

(C.V.) was calculated for each subject for each isotope/sampling method (four C.V.

calculations per subject) using the enrichments of samples taken at 7h, 7.5h, and 8h using

the following equation:

CV = SD of enrichments/mean of enrichments.

Mean CV was then determined for each isotope for each blood sampling method (four

data groups). Slopes during the 7th hour of infusion were also calculated using a

regression plot (Microsoft® Excel v. 9.0.2720), and Student’s t-test (see “statistics”) was

used to determine if slopes were significantly different from zero. Rate of appearance

(Ra) of glucose was calculated using enrichments of each of the tracers ([6,6-2H2]glucose

and [1-13C]glucose) in specimens obtained by each sampling method based on mean

steady state enrichments using the following formula (3):

Glucose Ra =[(Ei/Ep)-1] x I

where Ei is the enrichment of the infusate (99% for [1-13C]glucose and 98% for [6,6-

2H2]glucose), Ep is the enrichment of the tracer in plasma, and I is the infusion rate of the

tracer (μmol·kg-1·min-1).

To compare graphically the rise and fall of the plasma enrichments of the two

isotopes, enrichments at each time point for each subject were converted to percentage of

the calculated mean steady state enrichment. Tracer half-life (T1/2) was calculated using

standard formulas from 8h to 11h (4), based on ln of % mean steady state enrichments.

Regression analysis was used to calculate the rate constant, which was defined as the

slope of the decay curve converted to a linear format by using natural logarithm of each

enrichment value. The half-life was calculated using the following formula derived as

shown, where k = - rate constant (in hours):

163

T1/2 = ln 2/k = 0.693/k

Statistics: All comparisons were made assuming that the traditional intravenous

infusion of [6,6-2H2]glucose and venous sampling is the correct value for glucose

turnover. This is based on the facts that all labels in [6,6-2H2]glucose are lost

during glycolysis and/or gluconeogenesis and as such do not re-enter the

circulation (recycle) (1-3). Paired t tests were used to evaluate the new methods of

infusion/sampling by comparing mean steady-state enrichments, mean rates of

appearance (Ra), mean half-lives for decay curves, and the difference between

steady state slopes (mean enrichments at sample times 7h, 7.5h, and 8h) and a line

with slope zero. P ≤ 0.05 was considered to indicate statistical significance.

Bland-Altman analysis was used to compare methods based on mean Ra. All

statistical analyses were performed on a personal computer using Microsoft®

Excel 2000 (v. 9.0.2720).

Results

Following refinement of the technical aspect of this technique, we studied 5

subjects. Average enrichments are plotted for each isotope with each blood sampling

method; ([6,6-2H2]glucose finger stick, [6,6-2H2]glucose venous, [1-13C]glucose finger

stick, and [1-13C]glucose venous).

Plasma enrichments expressed as % steady state enrichment values are plotted

based on the infused isotope (i.e. intravenous [6,6-2H2]glucose vs. subcutaneous [1-

164

13C]glucose) and blood sampling method (i.e. venous vs. finger stick blood sampling)

(Fig. 2).

[1-13C]Glucose

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

Finger StickSpecimens

Plasma Specimens

Plasma Specimens

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

[6,6-2H2]Glucose

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

Finger Stick Specimens

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

[6,6-2H2]glucose

[1-13C]glucose

Finger StickSpecimens

Plasma Specimens

[6,6-2H2]glucose

[1-13C]glucose

[1-13C]Glucose

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

Finger StickSpecimens

Plasma Specimens

Finger StickSpecimens

Plasma Specimens

Plasma Specimens

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

[6,6-2H2]Glucose

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

Finger Stick Specimens

0

50

100

0 2 4 6 8 10Hours

% S

tead

y St

ate

[6,6-2H2]glucose

[1-13C]glucose

[6,6-2H2]glucose

[1-13C]glucose

[6,6-2H2]glucose

[1-13C]glucose

[6,6-2H2]glucose

[1-13C]glucose

Finger StickSpecimens

Plasma Specimens

Finger StickSpecimens

Plasma Specimens

[6,6-2H2]glucose

[1-13C]glucose

[6,6-2H2]glucose

[1-13C]glucose

[6,6-2H2]glucose

[1-13C]glucose

[6,6-2H2]glucose

[1-13C]glucose

Figure 2. Plasma and finger stick glucose enrichment. Percent steady-state enrichments of

finger stick and venous specimens plotted for subcutaneous infusate ([1-13C]glucose) and

for intravenous infusate ([6,6-2H2]glucose). Rate of rise and fall of the enrichment of

tracer infused subcutaneously ([1-13C]glucose) was less rapid than it for the tracer infused

intravenously ([6,6-2H2]glucose).

Plasma and finger stick blood glucose enrichments of the intravenous and

subcutaneous infused isotopes both reach a steady state plateau by 6 to 7 hours (Fig. 2).

The rate of rise and fall (of % SS enrichment) of tracer infused subcutaneously ([1-

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13C]glucose) was slower than for tracer infused intravenously ([6,6-2H2]glucose), but

steady state was still achieved by ~ 7 hours for both. The coefficient of variation (CV)

was 4.2% or less for all curves shown in Fig. 2, and slopes during this time interval were

not significantly different from zero for all four curves (p≥0.07); i.e. steady state was

reached in all cases.

Mean steady state enrichments of [6,6-2H2]glucose were only slightly but

significantly higher when measured using finger stick specimens than when measured

using plasma specimens (2.15 ± 0.10 vs. 2.08 ± 0.14 mol% E, p=0.03). No significant

difference between finger stick and plasma mean steady state enrichment of [1-

13C]glucose was observed. (Table 1, Fig. 4)

Table 1 Rate of Appearance of Glucose

Intravenous [6,6-2H2]glucose

Subcutaneous [1-13C]glucose

Isotope Infusion rate (μmol.kg-1.min-1) 0.219 ± 0.004 0.054 ± 0.006* Venous Blood Data (μmol.kg-1.min-1) 10.30 ± 0.73 ab 8.18 ± 0.88 a Finger-stick Blood Data (μmol.kg-1.min-1) 9.89 ± 0.58 c 8.59 ± 1.11 bc

a p < 0.001 (21 % difference) b p = 0.02 (17% difference) c p = 0.102 (13 % difference)

*The absolute [1-13C]glucose infusion rate was identical for each subject but since the

subjects weight was similar, the SE is the result of the variance of the weights.

166

0.0

0.5

1.0

7.0 7.5 8.0

[1- 13C]Glucose Infusion

Hours

0

2

4

0

2

4

0

2

4

7.0 7.5 8.0

.

[6,6-2H2]Glucose Infusion

Mol

% E

nric

hmen

t

Hours

Mol

% E

nric

hmen

t

Figure 3. Steady state CV and slope. Enrichment curves during steady state (7h, 7.5h, 8h)

for [1-13C]glucose and [6,6-2H2]glucose. CV is ≤ 4.3% for all curves, and slopes are not

significantly different from zero (p≥0.07). Plasma (■), Finger sticks (♦).

Plasma

Fingerstick

Rate of Appearance

0

4

8

12

[1-13C]Glucose [6,6-2H2]Glucose

Steady State Enrichment

0

1

2

Mol

es %

μmol

·kg-

1 ·min

-1

Plasma

Fingerstick

Rate of Appearance

0

4

8

12

[1-13C]Glucose [6,6-2H2]Glucose

Steady State Enrichment

0

1

2

Mol

es %

μmol

·kg-

1 ·min

-1

Figure 4. Steady state enrichments and turnover rates. Mean steady state enrichments

and rates of appearance for [1-13C]glucose and [6,6- 2H2]glucose. Comparison of results

obtained with each sampling method.

167

Table 2 Mean Decay Half-Life (T1/2 in hours) Plasma Fingerstick

[1-13C]glucose (subcutaneous infusion)

2.50 ± 0.89 1.89 ± 0.79 p= 0.34

[6,6 - 2H2]glucose (intravenous infusion)

1.40 ± 0.39 1.37 ± 0.40 p = 0.80

p = 0.14 p = 0.38

Fingerstick [1-13C]glucose vs plasma [6,6-2H2]glucose T1/2: p = 0.37 Half life (T1/2) calculations. [6,6 - 2H2]glucose (I.V. infusate) has a slightly shorter half-

life than does [1-13C]glucose (subcutaneous infusate), but this does not reach statistical

significance.

Rates of appearance calculated based on the [6,6-2H2]glucose tracer and using

finger stick vs. plasma specimens were not significantly different. Similarly, no

difference was observed in the rates of appearance of glucose using [1-13C]glucose based

on finger stick vs. plasma specimens. However, the rate of appearance of glucose using

exclusively the venous plasma samples was ~ 21% lower (p<0.001) using the [1-

13C]glucose as compared to the [6,6-2H2]glucose data. Rate of appearance of glucose

using the [1-13C]glucose was approximately 13% lower than [6,6-2H2]glucose calculated

based on finger stick specimens, but these values were not statistically different. There

was a 17% difference (p<0.02) in glucose Ra when comparing the data derived from the

[6,62H2]glucose and intravenous sampling and that of [1-13C]glucose with finger stick

blood (Table 1).

168

Rate of decay of each isotope in the blood did not differ significantly when

calculated using finger stick versus plasma enrichments (Fig. 2, Table 2). Rate of decay

of isotope appears to be slightly slower for [1-13C]glucose (subcutaneous administration)

than for [6,6-2H2]glucose (intravenous administration), but half-life calculations are not

significantly different (Fig. 2, Table 2).

Bland-Altman analysis of mean Ra was carried out comparing the result of the

conventional method of intravenous tracer infusion with venous blood specimens to that

using subcutaneous tracer infusion with finger stick blood specimens. Differences in Ra

all fall within one standard deviation of the mean difference, with the exception of a

single subject, for whom the difference in Ras as determined by the different methods

falls within 2 standard deviations from the mean. Although a correct statistical technique,

we recognize that the number of comparisons is very small.

Discussion

The present data demonstrate the utility of subcutaneous infusions of stable

isotope in human subjects to estimate substrate flux when compared to the traditional

intravenous tracer infusions. In addition, blood sampling via finger stick using NaF-

impregnated filter paper is a suitable alternative to venous blood sampling.

Near steady state was achieved by ~7 h for both infusion methods and using either

blood sampling method. However, the intravenously infused tracer approaches steady

state somewhat more rapidly than the subcutaneously administered tracer. Although, the

rate of decay was not significantly different between the two routes of tracer

administration regardless of the blood sampling site (venous vs. finger stick blood), the

169

decay of the IV administered [6,6-2H2]glucose appeared more rapid; A larger sample size

might demonstrate a significant difference.

A significant difference in the rate of appearance of glucose was observed

between the two isotopes employed ([1-13C]glucose and [6,6-2H2]glucose). The rate of

appearance using the [1-13C]glucose data was ~17% lower (specifically, 13% lower by

finger stick measurement, and 21% lower by plasma measurement) than for [6,6-

2H2]glucose. An error in pump infusion rates was ruled out by verification of both the

isotope concentration and the infusion rate in each study. Our finding of an

approximately 17% difference in rates of appearance for [1-13C]glucose and [6,6-

2H2]glucose is in agreement with results reported by Tigas, et al (3). In this latter study,

glucose Ra was underestimation by ~14% after a 14.5 hour infusion of [1-13C]glucose. It

is, therefore, reasonable to assume that this difference is secondary to reappearance of 13C

glucose into the circulation due to recycling of trace presumably via the Cori cycle (3).

The slight delay in decay of [1-13C]glucose compared with [6,6-2H2]glucose might also

be explained by recycling of tracer via the Cori cycle, although a continued entry of

subcutaneously infused tracer into the circulation via lymphatics even after the actual

infusion has stopped might contribute.

Ideally, this study would have been designed such that half of our subjects

received [1-13C]glucose subcutaneously and [6,6-2H2]glucose intravenously, and the other

half with the reverse in site of isotope infusion ([1-13C]glucose intravenously and [6,6-

2H2]glucose subcutaneously) as we have previously done (3). This was not possible in

this study. The maximum infusion rate of the subcutaneous pump used was 0.35 ml per

hour and we used a concentration of glucose of 10% maximizing the rate that could be

170

achieved for the subcutaneous tracer to 35 mg/h. Thus, we were limited by both maximal

pump infusion rate and concern about tissue irritation, which might have occurred at

glucose concentrations above 10%. At this rate of isotope infusion, the levels of

sensitivity and accuracy of measurements of [6,6-2H2]glucose using GC/MS analysis

would have precluded the use of this tracer for subcutaneous infusion. This is not the

case for [1-13C]glucose, which can be easily analyzed with great precision and accuracy

using gas chromatography, combustion isotope ration mass spectroscopy (GC - C -

IRMS).

From a practical point of view, subcutaneous infusion of isotope is very easy to

establish. Placement of the infusion device is done with minimal discomfort and does not

limit mobility during the study. One potential limitation of using an insulin infusion

pump for delivery is reservoir volume. In our Mini-Med pumps, reservoir volume was

slightly greater than 3 mL which, in an adult subject, was sufficient for a maximum of ~9

h infusion. In smaller (pediatric) subjects, however, lower infusion rates could be

employed still producing accurate estimates of enrichments while allowing for infusion

times of up to, perhaps, 18 to 20 h.

Using finger sticks for blood sampling and application of the sample to NaF-

impregnated filter paper is also attractive for human studies. Our primary initial problem

was identifying a methodology which avoids contamination of the sample, which we

believe we have done. Thus, appropriate precautions must be taken in the preparation of

the NaF impregnated filter paper and the application of the sample to the filter paper must

be contamination free. Presumably, if subjects were to be sent out of the unit for the

study period (e.g. at home), simple hand washing would make contamination less likely,

171

as the home environment would not contain highly enriched isotope “fomites” which

plagued our early efforts. In addition, the use of a paper cover for the filter paper, much

in the same manner as a stool guiac test, further avoids direct contact with the filter paper

prior to analysis.

In summary, we have demonstrated both the utility and limitation of the use of

subcutaneous isotope infusion and finger stick sampling. Utilizing this technique could

open the opportunity to study subjects under far more natural circumstances of normal

life than that provided in a metabolic research unit.

172

References

1. Bier DM, Arnold KJ, Sherman WR, Holland WH, Holmes WF, Kipnis DM 1977 In-

vivo measurement of glucose and alanine metabolism with stable isotopic tracers.

Diabetes 26:1005-1015.

2. Bier DM, Leake RD, Haymond MW, Arnold KJ, Gruenke LD, Sperling MA, Kipnis

DM 1977 Measurement of "true" glucose production rates in infancy and childhood with

6,6-dideuteroglucose. Diabetes 26:1016-1023.

3. Tigas SK, Sunehag AL, Haymond MW 2002 Impact of duration of infusion and

choice of isotope label on isotope recycling in glucose homeostasis. Diabetes 51:3170-

3175.

4. Shipley RA, Clark RE 2005 Tracer methods for in vivo kinetics: theory and

applications. Academic Press, New York, pp 3-6.

173

174

Chapter 8

Summary, General Discussion and

Future Perspectives

175

A simple yet accurate and reproducible measurement technique is often a

prerequisite for quality research without which no worthwhile finding is possible within a

reasonable amount of time. The complexity, tediousness and high costs have limited the

broad use of stable isotope methods to determine the quantitative in vivo estimates of

gluconeogenesis in glucose metabolic research.

The primary objectives of this thesis were to develop a simple, accurate and

reproducible method with high sample through-put to measure gluconeogenesis and

further, to estimate in vivo rates of gluconeogenesis in different populations under various

conditions applying the new and other methods to address various metabolic issues.

In Chapter 2 we report a highly reproducible and simple/sensitive method

requiring only small sample volumes, thus, providing a tool to study the details of

glucose kinetics that can be used by many investigators, thereby forwarding research on

glucose metabolism.

After the ingestion or infusion of deuterium oxide and equilibration of deuterium

in the total body water pool, deuterium is incorporated into intermediary substrates along

the glycolytic/gluconeogenic pathway. The isomerization of glyceraldehyde-3-phosphate

to dihydroxyacetone phosphate by triose phosphate isomerase and a series of

equilibration reactions between phosphoenolpyruvate and dihydroxyacetone phosphate is

consistent with deuterium incorporation in C-1, 3, 4, 5 and 6 of glucose during the

gluconeogenic process. The 2H enrichment at C-2 is purported to be due to complete 2H

exchange with body water during the extensive glucose-6-phosphate to fructose-6-

phosphate isomerization and therefore, does not specifically reflect the gluconeogenic

process. Thus, the degree of deuterium labeling in plasma glucose C-1, 3, 4, 5 and 6 is a

176

measure of gluconeogenesis. Among the various GC-MS fragments of glucose

derivatives considered, the m/z 170/169 fragment of the pentaacetate derivative was

selected because of the presence of all the exchangeable hydrogen in C-1, 3, 4, 5 and 6 of

glucose during the gluconeogenic process. We showed that this method is robust in that

the enrichments measured using the m/z 170/169 fragment does not fluctuate over a wide

abundance range.

Despite differences in substrate availability under conditions of overnight and

extended fasting (66 h), and total parenteral nutrition providing glucose at high infusion

rates (and various substrates at high concentration), the average enrichment method

provided results comparable with the C-5 HMT method with a CV of <3%. Thus, the

results obtained at high, intermediate and low fractional gluconeogenesis compared very

well between our average enrichment method and the C-5 HMT method. Because it is

very accurate and reproducible even when fractional gluconeogenesis is low, this new

method can be potentially used in subjects receiving parenteral or enteral feedings and

during insulin clamp studies.

The low tracer cost and simplicity of analysis make this method affordable and

accessible to a wide number of investigators and it can be completed in a few hours. In

addition, the small sample volume requirement makes the method applicable to studies in

infants, children and small laboratory animals.

There are no published reports on total gluconeogenesis in very premature infants

receiving routine total parenteral nutrition providing glucose at rates exceeding normal

infant glucose turnover rate, which often results in hyperglycemia. In the study described

177

in Chapter 3, we determined whether gluconeogenesis is sustained in very premature

infants receiving standard nutritional care, and if it correlates with glucose infusion rate

and/or blood glucose concentration. To ascertain and to compare the accuracy of the

measurements of gluconeogenesis under conditions of high exogenous glucose infusion

rates, we measured gluconeogenesis applying both our new average deuterium

enrichment method and the C-5 HMT method reported by Landau et al.

The study demonstrated that in very premature infants, gluconeogenesis is

an ongoing process even when glucose is supplied at rates exceeding their normal

glucose turnover as part of TPN. Further, gluconeogenesis accounts for the major part of

residual glucose production and thus, the incomplete suppression of glucose production

observed in preterm infants is primarily due to the contribution from gluconeogenesis. In

very premature infants, gluconeogenesis was not affected by infusion rates of glucose,

lipid and amino acids or blood glucose concentration, gestational age and birth weight.

Our studies showed that despite the infants’ glucose and energy needs are

supplied by the parenteral nutrition, gluconeogenesis was sustained indicating a lack of

ability to regulate this process. During our investigations on factors affecting blood

glucose concentration, we found that glucose appearance rate (i.e. glucose infusion +

glucose production) and gestational age explained ~ 79% of the variation in blood

glucose concentration in these infants. Additionally, the agreement between the estimates

of gluconeogenesis obtained by the two methods applied in the present study

demonstrated that the new approach compare very well with the published Landau

method, thus, validating the new method even under conditions of low fractional

gluconeogenesis.

178

The results from this study suggest that a potential strategy to prevent

hyperglycemia without increasing the risk of hypoglycemia or insufficient energy intake

would be to provide a TPN solution supplying glucose at a rate equivalent to the normal

infant glucose production rate in addition to parenteral lipids and amino acids during the

first days of life.

There are no reports on the hormonal regulation of gluconeogenesis in preterm

infants. In the study reported in Chapter 4, we investigated potential factors regulating

gluconeogenesis in Extremely Low Birth Weight (ELBW) infants receiving TPN. This

was achieved by measuring gluconeogenesis during routine TPN providing glucose at

high rates and also in response to reducing the glucose infusion to half normal newborn

glucose turnover rate.

We evaluated the impact of the subsequent changes of insulin and glucose

concentrations occurring in response to the change in the glucose infusion rate on

gluconeogenesis. Lack of changes in gluconeogenesis clearly demonstrated that

gluconeogenesis is not acutely affected by either insulin or glucose concentrations in

ELBW infants receiving TPN. However, a strong relationship between the decreases in

glucose and insulin concentrations between high and low glucose infusion periods

demonstrated that these immature infants are capable of adjusting insulin in response to

the lower glucose concentrations.

Insulin counterregulatory hormones, glucagon and cortisol, which primarily

function during hypoglycemic conditions remained unchanged and this might be because

of the normoglycemia maintained in the infants and availability of gluconeogenic

179

substrates via TPN. This is the first report in which both constituents of glucose

production; gluconeogenesis and glycogenolysis are measured during routine TPN

providing glucose at high and at reduced infusion rates (half the infant glucose turnover

rate) in addition to parenteral lipids and amino acids.

In this study we demonstrated that gluconeogenesis is an ongoing process enabling

preterm infants receiving TPN to remain normoglycemic even during a glucose supply

corresponding to half normal turnover rate. This further supports our earlier report

(Chapter 3) recommending that maintaining a glucose infusion rate equivalent to normal

infant glucose turnover rates as part of total parenteral nutrition is a potential approach to

prevent both hypo- and hyperglycemia and yet provide sufficient energy for growth in

ELBW infants during their first days of life. The results also confirm our previous report

showing that the glucose infusion rate is the primary factor that can be optimized to

reduce the risk of hyperglycemia.

Additionally, our data demonstrated that despite substantially higher insulin

concentrations during infusion of glucose at high rates, blood glucose concentrations

were elevated. This indicates that the use of early insulin therapy might be a questionable

approach to control glucose concentration in extremely low birth weight infants.

Further, similar protein turnover data during the two glucose infusion periods

demonstrated that reduced supply of glucose as a part of TPN does not increase

proteolysis in extremely low birth weight infants. This might imply that providing

gluconeogenic substrates via TPN prevented a potential need of increased proteolysis to

sustain gluconeogenesis to meet glucose demands even when the glucose supply is low.

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Collectively, the data from chapters 3 and 4 indicates that supplying glucose at rates

corresponding to normal infant glucose turnover rate in addition to glucose produced via

gluconeogenesis (utilizing the parenteral lipid and amino acid substrates) is a potential

strategy to prevent hypo/hyperglycemia in infants during their early days of life.

In Chapter 5, we established how lactating women manage their increased glucose

demands during extended fasting periods without compromising the lactation

significantly.

Our study in six healthy exclusively breastfeeding women and six non-lactating

controls during a 42 h of fast, demonstrated that extra glucose demands of lactation

during extended fasting are met by increased gluconeogenesis. After 42 h of fasting, we

observed that milk production remained within the normal range with only 16% reduction

in milk volume. Glucose, insulin and C-peptide concentrations decreased with the

duration of fasting in both groups but were lower in lactating women. Glucagon, FFA and

β-hydroxybutyrate concentrations increased with fasting time and were higher in lactating

women during both fasting and re-feeding. Although gluconeogenesis was higher in

lactating women when compared to non-lactating controls during 42h of fasting,

glycogenolysis was not different.

Interestingly, we found that mammary hexoneogenesis remained unchanged

throughout the entire duration of fasting despite increased risk of hypoglycemia in

lactating women. Further, our data demonstrated that carbohydrate oxidation was lower

and fat and protein oxidation higher in lactating women.

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This study revealed that the extra glucose demands of lactation during the

extended fasting were met by increasing gluconeogenesis. Nonetheless, lactating women

are at risk for hypoglycemia beyond 30h of fasting.

Although reduced plasma ghrelin concentration has been suggested as a potential

mechanism for the improvement of glucose metabolism in patients after bariatric surgery

procedures, the association between the improvement of glucose metabolism and ghrelin

concentration is not yet defined. In Chapter 6 we report a study measuring glucose

kinetics in ghrelin and growth hormone secretagogue receptor deficient mice models to

assess the beneficial effects of the absence of ghrelin action on glucose metabolism. We

demonstrated that ablation of ghrelin or its receptor resulted in increase of

gluconeogenesis and glycogenolysis, following an 8 h of fasting. These data suggest that

ghrelin plays a role in the regulation of gluconeogenesis and glycogenolysis, and that at

least part of these effects occurs via the growth hormone secretagogue receptor.

The increased rate of gluconeogenesis and glycogenolysis might be because of the

absence of the inhibiting effect of ghrelin on leptin. Leptin was previously demonstrated

to enhance gluconeogenesis and diminish insulin secretion; however, antagonizing effects

of ghrelin on leptin action have also been reported.

Significantly higher hepatic insulin sensitivity index and improved insulin

resistance calculated by the HOMA-IR in ghrelin-/- and Ghsr -/- as compared to WT mice

demonstrate increased insulin sensitivity in the absence of ghrelin action on the purported

ghrelin receptor. Higher glucose clearance observed at lower insulin concentration

during short term fast in ghrelin-/- and Ghsr -/- as compared to WT mice might suggest

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that less insulin is required for peripheral glucose uptake in the absence of ghrelin or its

receptor under normal physiologic conditions. During prolonged fasting (18 h), glucose

clearance was reduced in ghrelin deficient mice.

Thus, our study demonstrates that gluconeogenesis and glycogenolysis are

increased, and insulin sensitivity is improved by the ablation of ghrelin or its receptor in

mice. Thus, reduced ghrelin might be an explanation for the improvement of glucose

metabolism seen in patients after bariatric surgery procedures. In addition, the data from

this study in transgenic mice models support a potential prospect of improving insulin

sensitivity in the diabetic condition by the use of ghrelin receptor antagonists.

Finally in Chapter 7, we demonstrated a study technique that can be used to

facilitate metabolic studies under real-life conditions without interfering with the normal

activity of human subjects. We employed subcutaneous tracer infusion and “finger stick”

blood sampling methods to validate this technique. The comparison of data from

simultaneous use of intravenous and subcutaneous infusion using two glucose tracers ([1-

13C]glucose and [6,6-2H2]glucose) and corresponding venous and “finger stick” blood

sampling demonstrated that subcutaneous infusion and “finger stick” blood sampling in

humans is a potential alternative to measure appearance rate of glucose under real life

circumstances.

As anticipated the intravenously infused tracer achieved near steady state faster

than the subcutaneously infused tracer. However, near steady state was achieved by both

infusion methods within a reasonable period of ~7 hours. The rate of decay was not

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significantly different between the intravenous and subcutaneous tracer infusions

regardless of the blood sampling site (venous vs. finger stick).

The rate of appearance using the [1-13C]glucose data was ~17% lower than for

[6,6-2H2]glucose. This difference is most likely a result of the recycling of 13C glucose

into the circulation via the Cori cycle. The slight delay in decay of [1-13C]glucose

compared with [6,6-2H2]glucose observed may also be explained by the recycling of

tracer via the Cori cycle and also by the delayed entry of subcutaneously infused tracer

into the circulation via the lymphatic system. This study technique could be potentially

utilized to execute large studies in a home setting, which would otherwise not be

economically feasible to carry out in a clinical research setting.

Future perspectives

• There are no data on the effect of exogenous insulin (or early insulin therapy)

on gluconeogenesis and glycogenolysis in preterm infants under routine total

parenteral nutrition providing glucose at rates exceeding the normal infant

glucose turn over. Studies addressing this issue are necessary to evaluate

potential benefits of early insulin therapy. Simultaneous measurement of

protein kinetics would provide an opportunity to assess potential benefits of

early insulin therapy on protein metabolism in addition to glucose kinetics.

• Using the mice models to evaluate the utilization of ghrelin receptor

antagonists to improve insulin sensitivity in diabetic state.

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• Ghrelin infusion in ghrelin transgenic mice models should be tested to

investigate whether ghrelin induces insulin resistance. Thus, the role of

ghrelin in the improvement of insulin sensitivity can be further evaluated.

• Ghrelin infusion in growth hormone secretagogue receptor knockout mice

should be performed to confirm the absence of any supplementary relevant

receptors for the hormone ghrelin in mice (other than ghrelin’s purported

growth hormone secretagogue receptor).

• The association of leptin in the improvement of glucose metabolism during

the ablation of ghrelin or the growth hormone secretagogue receptor in mice

should be tested by conducting glucose kinetic measurements in mice where

both ghrelin and leptin or the growth hormone secretagogue receptor and

leptin are ablated. Thus, reverse genetics should be used to investigate the

interplay between leptin and ghrelin or leptin and the growth hormone

secretagogue receptor.

• Glucose metabolic parameters such as gluconeogenesis, glycogenolysis and

insulin sensitivity as well as ghrelin concentration should be measured in

morbidly obese patients before bariatric surgery, immediately after surgery

and a few months after surgery. This would facilitate to explore potential

mechanisms for the metabolic improvements observed immediately after

bariatric surgery, i.e. before any weight loss has occurred.

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