Jugoslov Med Biohem 2005; 24 (1) 35

Introduction

The concentration of glucose in the blood is re-gulated by a complex network of various biochemicalprocesses including glycolysis, glyconeogenesis, gly-cogenesis, glycogenolysis and many others. Thesemetabolic pathways are controlled and modulated bya number of hormones, in order to maintain the con-centration of glucose within relatively narrow range.Insulin and, to a lesser extent, insulin-like growth fac-tors are responsible for a decrease in blood glucose,while glucagon, cortisol, epinephrine and growth hor-

mone have the opposite role (1). Numerous diseasestates that involve impaired glucose metabolism havebeen discovered. A disease with the highest incidenceis diabetes mellitus (2). Diabetes mellitus can be re-garded as a group of metabolic disorders of carbohy-drate metabolism in which glucose is underutilized,producing hyperglycemia, and often followed by life-threatening episodes that include ketoacidosis andhyperosmolar coma (1). Prolonged disease causesspecific complications, many of which are related topathological glycosylation (glycation) of various essen-tial molecules and cellular structures. In general, struc-tural modifications fall into two categories: (i) nonen-zymatic glycation per se, which refers to the attach-ment of free carbohydrate to proteins in the Amadoriconstruct, and (ii) generation of advanced glycationendproducts (AGE), which refers to a heterogeneousgroup of carbohydrate-modified products derivedfrom the Amadori adduct by oxidation, polymerizationand other spontaneous reactions (3). Complications,

UC 577,1; 61 ISSN 0354-3447

Jugoslov Med Biohem 24: 35–39, 2005 Originalni nau~ni radOriginal paper

INSULIN AND C-PEPTIDE RESPONSE IN HEALTHY PERSONS AND INDIVIDUALS WITH IMPAIRED GLUCOSE METABOLISM

DURING ORAL GLUCOSE TOLERANCE TEST

Olgica Nedi}, Jasminka Miloradovi}, Marija Ratkovi}, Romana Masnikosa

INEP ’ Institute for the Application of Nuclear Energy, 11080 Belgrade-Zemun, Banatska 31b, Serbia and Montenegro

Summary: Serial measurements of blood glucose concentration before and after giving a specific amountof glucose orally provide a standard method to evaluate glucose metabolism. Although the reference ranges forglucose concentration in various disease states that are based on impaired glucose homeostasis have been estab-lished, the reference values are not clearly defined for insulin and C-peptide concentrations. The aim of this workwas to study the insulin and C-peptide response during OGTT. Healthy individuals exhibited the following profileof insulin and C-peptide levels: 15 ± 4.9 mU/L and 0.5 ± 0.19 nmol/L (0 h), 116 ± 52.8 mU/L and 2.3 ± 0.79nmol/L (1 h) and 59 ± 26.7 mU/L and 2.0 ± 0.67 nmol/L (2 h). Persons with impaired glucose tolerance hadhigher C-peptide levels at 0 h, 0.6 ± 0.17 nmol/L, and significantly higher insulin and C-peptide concentrationsafter 1 h, 209 ± 63.8 mU/L and 3.5 ± 1.00, nmol/L and 2 h, 188 ± 48.8 mU/L and 3.6 ± 0.92 nmol/L. Diabeticpatients had higher basal levels of C-peptide, 0.8 ± 0.23 nmol/L, insulin and C-peptide increased after 1 h simi-larly as in healthy people, but further continued to increase significantly, 181 ± 137.6 mU/L and 3.7 ± 1.49nmol/L. Subjects that exhibited low blood glucose levels demonstrated lower insulin concentrations at all timeintervals, 11 ± 2.5 mU/L (0 h), 63 ± 31.1 mU/L (1 h) and 44 ± 22.9 mU/L (2 h), but the concentration of C-pep-tide leveled with that of the healthy ones. The results of this work may be useful in establishing reference rangesfor insulin and C-peptide concentrations for defined time intervals during OGTT, in health and disease.

Key words: insulin, C-peptide, OGTT

Address for correspondence:

Olgica Nedi}INEP ’ Institute for the Application of Nuclear EnergyBanatska 31b, 11080 Belgrade-ZemunSerbia and MontenegroTel.: (+) 381 11 617 252Fax.: (+) 381 11 618 724e-mail: olgica@inep.co.yu

36 Jugoslov Med Biohem 2005; 24 (1)

including cardiovascular disease, neuropathy, nephro-pathy and others, are frequently diagnosed in patientswith diabetes mellitus (4’6). The opposite, hypo-glycemia, is defined as a state in which blood glucoseconcentration is bellow the fasting range. A rapiddecrease in glucose level produces weakness, shaki-ness, rapid pulse, headache etc. (1).

Insulin is a hormone that stimulates the uptakeof glucose into cells, promotes intracellular transportand conversion into the other metabolic molecules.Proinsulin is produced from preproinsulin in pancrea-tic beta cells. Proinsulin is stored in secretory granulesand is cleaved into insulin and C-peptide upon stimu-lation. A physiological role of insulin is well known,while C-peptide, being more stable than insulin in thecirculation, can be regarded as a reliable monitor ofinsulin secretion (1).

Serial measurements of blood glucose levelsbefore and after giving a specific amount of glucoseorally provide a standard method to evaluate glucosemetabolism. Oral glucose tolerance test (OGTT), rec-ommended by the World Health Organization (WHO),is performed with 75 g of glucose for non-pregnantadults and the blood samples are collected at definedtime intervals: 0 h (before glucose intake), 1 h and 2 h(after glucose consumption). Variations of the methodinclude sampling every 30 min for 2 h, as well as after3h (1). The WHO criteria that differentiated healthypersons from diabetic were adopted in the Nationalguide for clinical practice in Serbia (7). Metabolic res-ponse in healthy individuals exposed to OGTT shouldincrease the concentration of blood glucose at most to7.8 mmol/L and return to normal after 2 h. Impairedglucose tolerance results in higher levels of blood glu-cose and slower clearance. Although the referenceranges for glucose concentration in various diseasestates have been established, the reference values arenot clearly defined for insulin and C-peptide con-centration. The aim of this work was to study insulinand C-peptide response during OGTT and to obtainranges for each hour of the test for healthy personsand individuals with impaired glucose metabolism.

Material and Methods

Eighty seven non-pregnant adult persons (18males and 69 females) were subjected to OGTT. Theywere given orally 75 g of glucose in 200 mL of waterafter the fast blood sampling. The blood was collectedafter 1 h, 2 h and, upon the request of a physician,after 3 h. Each time the serum was separated by cen-trifugation within 45 min. Samples were assayed im-mediately for glucose and within a week for insulin andC-peptide. Meanwhile, sera were kept frozen at ’20 °C,as C-peptide is unstable in serum at higher tempera-tures.

Glucose concentration was measured usingRANDOX commercial reagent (GOD-PAP method);the reference levels according to the producer are4.2’6.4 mmol/L. Insulin and C-peptide levels weredetermined by INEP commercial RIA kits; the referen-ce values are established as 5’25 mU/L for insulin and0.3’0.7 nmol/L for C-peptide. Data were statisticallyanalyzed using the Student t-test for the significance ofdifferences between the groups (patients vs. healthypersons).

Results

According to the results obtained for the con-centration of blood glucose during OGTT and clinicalhistory, the examined persons were divided into fourgroups. The first group (I, n = 21) was characterizedas having the referent blood glucose concentration at0 h (the lowest value was 4.0 and the highest 5.7mmol/L), after 1 h the concentration increased (6.8’8.3 mmol/L) and returned to normal after 2 h (4.8’6.0mmol/L). These individuals were assumed to be he-althy in respect to glucose metabolism. Subjects col-lected as a second group (II, n = 25) exhibited delayedglucose clearance and most of them were monitoredfor 3 h. The fasting blood glucose concentration wasreferent (4.0’5.7 mmol/L), then increased after 1 h(7.4’10.3 mmol/L), decreased after 2 h (6.6’8.3mmol/L), and returned to normal after 3 h (4.0 ’5.5mmol/L). Patients in group three (III, n = 25) satisfied

Table I Blood insulin and C-peptide concentrations (⎯x ± SD) during OGTT in healthy persons (I) and individuals with impaired glucose metabolism (II: impaired glucose tolerance, III: diabetes mellitus, IV: low blood glucose levels).

Data were analyzed and compared (patients vs. healthy people) using t-test (ns: non significant)

Group

III

III

IV

0h 1h 2h15 ± 4.9 116 ± 52.8 59 ± 26.7 17 ± 4.7 209 ± 63.8 188 ± 48.8

ns p = 0.0004 p < 0.000118 ± 8.1 131 ± 75.1 181 ± 137.6

ns ns p = 0.000811 ± 2.5 63 ± 31.1 44 ± 22.9p = 0.009 p = 0.0004 p = 0.08

0h 1h 2h0.5 ± 0.19 2.3 ± 0.79 2.0 ± 0.670.6 ± 0.17 3.5 ± 1.00 3.6 ± 0.92p = 0.007 p = 0.002 p < 0.00010.8 ± 0.23 2.7 ± 1.48 3.7 ± 1.49p = 0.0008 ns p = 0.017 0.5 ± 0.17 2.7 ± 0.93 2.0 ± 0.58

ns ns ns

Insulin (mU/L) C-peptide (nmol/L)

Jugoslov Med Biohem 2005; 24 (1) 37

the OGTT criteria that classified them as diabetics,they also had other signs of diabetes mellitus confir-med biochemically and/or clinically. Their fasting bloodglucose was normal to elevated (5.4’13.9 mmol/L),increased after 1 h (8.8’22.4 mmol/L), then mode-rately dropped or continued to increase after 2 h (7.5’24.0 mmol/L). Finally, subjects with low blood glucoselevels were established as a fourth group (IV, n = 16).At 0 h their concentration of glucose was low to nor-mal (3.1’5.1 mmol/L), it remained almost unchangedafter 1 h (4.0’5.7 mmol/L) and most often decreasedto very low after 2 h (2.2’4.2 mmol/L).

The concentrations of insulin and C-peptide weremeasured in each sample and the results (mean ±standard deviation) are presented in Table I. Althoughthere were significant interindividual variations, which

can be seen from the SD values, specific patternsof insulin and C-peptide alterations during OGTTemerged. Figures 1a and 1b present typical insulinand C-peptide responses in four groups of people du-ring OGTT.

Healthy individuals exhibited increase in insulinand C-peptide levels 1 h after the stimulation and theydecreased after 2 h. Persons with delayed glucoseclearance had slightly higher C-peptide levels at 0 hand significantly higher insulin and C-peptide concen-trations compared to healthy subjects after 1 h and 2h. Concentrations of insulin and C-peptide remainedvery high after 2 h and dropped after 3 h (data notshown). Diabetic patients had higher than normalbasal levels of C-peptide. After 1 h insulin and C-pep-tide increased, on average, as in healthy people butfurther continued to increase significantly. After 2 h theconcentration of insulin and C-peptide in groups II andIII were within the same range. Subjects with low bloodglucose levels demonstrated lower insulin concent-rations at all time intervals, but the concentration ofC-peptide leveled with that of the healthy ones.

Discussion

The results for insulin concentration duringOGTT in healthy persons are in accordance with thedata reported in Tietz Textbook of Clinical Chemistry,0 h: 6’21 mU/L, 1 h: 20 ’120 mU/L and 2 h: 18’56mU/L (8) and Human Clinical Endocrinology, 0 h: 20± 2 mU/L, 1 h: 75 ± 10 mU/L and 2 h: 45 ± 6 mU/L(9). In obese persons both basal and stimulated levelsmay be high, 0 h: 80 ± 5 mU/L, 1 h: 130 ± 7 mU/Land 2 h: 80 ± 5 mU/L (9). Impaired glucose tolerancewas diagnosed in individuals who had fasting bloodglucose levels less than those required for a diagnosisof diabetes mellitus, but had a glucose response du-ring OGTT between normal and diabetic (1). Insulinand C-peptide response, on the other hand, did notfollow that pattern. The initial response after 1 h was,on average, more intense than in diabetics, suggestinggreater pancreatic sensitivity to glucose stimulationand hormonal secretion which, unfortunately, was lesssuccessful in decreasing the blood glucose. The stateof relative insulin resistance at cellular level is mostoften responsible for this insensitivity (10). Mathema-tical analysis of the relationship between insulin sensi-tivity and secretion has revealed a hyperbolic function,such that the product of two variables is constant (11).Measurements of insulin sensitivity provide cliniciansand clinical researchers with valuable tool to evaluatethe efficiency of both current and potentially usefultherapeutic preparations. Although several methodshad been developed and validated to evaluate insulinsensitivity, none of these methods can be universallyused in all patients (12). Evenmore, a method suitablefor clinical or basic research may not necessarily be apractical method for use in regular practice or for epi-demiological studies. Insulin activity may be also influ-

Figure 1. Typical examples of (a) insulin and(b) C-peptide responses during OGTT in healthy persons

(I) and individuals with impaired glucose metabolism(II: impaired glucose tolerance, III: diabetes mellitus,

IV: low blood glucose levels).

IIIIIIIV

300

250

200

150

100

50

0

3.53

2.52

1.51

0.50

(a)

(b)

Insulin response during OGTT

C-peptide response during OGTT

0 0.5 1 1.5 2 2.5Time intervals (h)

0 0.5 1 1.5 2 2.5Time intervals (h)

Insu

lin c

once

ntra

tion,

mU

/LC

-pep

tide

conc

entr

atio

n, n

mol

/L

IIIIIIIV

38 Jugoslov Med Biohem 2005; 24 (1)

enced by the presence of anti-insulin or anti-insulinreceptor antibodies (13, 14). Two hours after the glu-cose intake, insulin/C-peptide status in both groups ofpatients (II and III), on average, became the same.These results should be, however, interpreted withcaution due to very high standard deviation values ingroup III. All examined diabetic persons had normal tohigh insulin levels upon stimulation, which classifiedthem as insulin-independent cases of diabetes mellitus(1). Subtyping of these patients would, possibly, nar-row insulin and C-peptide ranges for each subgroup.

Blood glucose concentration limits are hard todefine for hypoglycemia, as glucose levels are often bel-low fasting range and transient decline may occur (1).OGTT is not a test regularly recommended for diagno-sis of hypoglycemia. Blood insulin concentration in per-sons with low blood glucose levels was found to be sig-nificantly lower than in healthy people, both after fasting

and during OGTT. C-peptide levels, on the other hand,were within reference range at all sampling intervals.These data suggested that pancreatic stimulation waspreserved, but secreted insulin was either cleared fromthe circulation faster than normal or it was present in aform that was not detected by the applied analyticalmethod (immune complexes?).

Although OGTT and blood glucose determina-tions are routinely performed for diagnosis of diabetesmellitus and impaired glucose tolerance (15, 16), pa-rallel measurement of insulin and C-peptide levels, inall cases of impaired glucose metabolism, certainlyhelps in diagnosis and management of these disor-ders. The results of this work may be useful in estab-lishing reference ranges for insulin and C-peptide con-centrations for defined time intervals during OGTT, inhealth and disease.

PROMENA KONCENTRACIJE INSULINA I C-PEPTIDA KOD ZDRAVIH LJUDI I OSOBA SA POREME]ENIM METABOLIZMOM GLUKOZE TOKOM

ORALNOG TESTA TOLERANCIJE GLUKOZE

Olgica Nedi}, Jasminka Miloradovi}, Marija Ratkovi}, Romana Masnikosa

INEP-Institut za primenu nuklearne energije, 11080 Beograd-Zemun, Banatska 31b, Srbija i Crna Gora

Kratak sadr`aj: Metabolizam glukoze se uobi~ajeno procenjuje serijskim odre|ivanjem koncentracije glu-koze u krvi pre i posle oralnog uno{enja odre|ene koli~ine glukoze. Iako je referentni opseg za koncentracijuglukoze utvr|en kod razli~itih stadijuma bolesti u ~ijoj osnovi je poreme}ena homeostaza glukoze, referentne vred-nosti za koncentraciju insulina i C-peptida nisu jasno definisane. Cilj ovog rada je bio da ispita promenu koncen-tracije insulina i C-peptida tokom OGTT. Zdrave osobe su ispoljile slede}i profil promene koncentracije insulina iC-peptida: 15 ± 4,9 mU/L i 0,5 ± 0,19 nmol/L (0 h), 116 ± 52,8 mU/L i 2,3 ± 0,79 nmol/L (1 h) i 59 ± 26,7mU/L i 2,0 ± 0,67 nmol/L (2 h). Osobe sa smanjenom tolerancijom glukoze su imale vi{i nulti nivo C-peptida,0,6 ± 0,17 nmol/L i zna~ajno vi{e koncentracije insulina i C-peptida posle 1 h, 209 ± 63,8 mU/L i 3,5 ± 1,00nmol/L i 2 h, 188 ± 48,8 mU/L i 3,6 ± 0,92 nmol/L. Dijabeti~arima je izmeren vi{i bazalni nivo C-peptida, 0,8 ±0,23 nmol/L, posle 1 h koncentracije insulina i C-peptida su pove}ane sli~no kao kod zdravih ljudi, ali su nastavilei dalje da zna~ajno rastu, 181 ± 137,6 mU/L i 3,7 ± 1,49 nmol/L. Kod osoba sa niskom koncentracijom glukozeu krvi utvr|ena je niska koncentracija insulina u svim testiranim vremenskim intervalima, 11 ± 2,5 mU/L (0 h),63 ± 31,1 mU/L (1 h) i 44 ± 22,9 mU/L (2 h), dok je nivo C-peptida bio sli~an kao kod zdravih. Rezultati ovograda bi mogli biti korisni za odre|ivanje referentnih opsega za koncentraciju insulina i C-peptida, za utvr|ene vre-menske intervale tokom OGTT, u zdravom stanju i tokom bolesti.

Klju~ne re~i: insulin, C-peptid, OGTT

Jugoslov Med Biohem 2005; 24 (1) 39

References

1. Sacks DB. Carbohydrates. In: Burtis CA, Ashwood ER.eds. Tietz Textbook of Clinical Chemistry. Philadelphia:Saunders Company, 1994: 928’1001.

2. Majki}-Singh N. Instructions and recommendations foruse of the laboratory methods in diabetes mellitus diag-nosis and monitoring. Jugoslov Med Biohem 2003; 22(Suppl 1): 1’ 4.

3. Cohen MP. Intervention strategies to prevent pathogeniceffect of glycated albumin. Arch Biochem Biophys2003; 419: 25’30.

4. Podwall D, Gooch C. Diabetic neuropathy: clinical fea-tures, etiology and therapy. Curr Neurol Neurosci Rep2004; 4: 55’61.

5. Khamaisi M, Wexler ID, Skrha J, Strojek K, Raz I, Mili-cevic Z. Cardiovascular disease in type 2 diabetes: epi-demiology, risk factors and therapeutic modalities. IsrMed Assoc J 2003; 5: 801’6.

6. Jerums G, Panagiotopoulos S, Forbes J, Osticka T,Cooper M. Evolving concepts in advanced glycation,diabetic nephropathy and diabetic vascular disease.Arch Biochem Biophys 2003; 419: 55’62.

7. Zamlakar M, Bajovi} Lj, Stefanovi} M. Definicija, dijag-noza i podela dijabetesa. In: Nacionalni vodi~ klini~keprakse, diabetes mellitus. Ministarstvo zdravlja RepublikeSrbije, 2002: 3’10.

8. Painter PC, Cope JY, Smith JL. Appendix ’ ReferenceIntervals. In: Burtis CA, Ashwood ER. eds. Tietz Text-

book of Clinical Chemistry. Philadelphia: Saunders Com-pany, 1994: 2161’217.

9. Slijep~evi} D, Vujovi} S, Nestorovi} Z. Humana klini~kaendokrinologija. Beograd: Obele`ja, 2002: 829’901.

10. Pirola L, Johnson AM, Van Obberghen E. Modulators ofinsulin action and their role in insulin resistance. Int JObes Relat Metab Disord 2003; 27 (Suppl 3): 61’ 4.

11. Ahren B, Pacini G. Importance of quantifying insulinsecretion in relation to insulin sensitivity to accuratelyasses beta cell function in clinical studies. Eur J Endo-crinol 2004; 150: 97’104.

12. Monzillo LU, Hamdy O. Evaluation of insulin sensitivity inclinical practice and in research settings. Nutr Rev 2003;61: 397’ 412.

13. Eisenbarth GS. Insulin autoimmunity: immunogene-tics/immunopathogenesis of type 1A diabetes. Ann N YAcad Sci 2003; 1005: 109’18.

14. Yamasaki H, Yamaguchi Y, Fujita N, Kato C, KuwaharaH, Yamauchi MD et al. Anti-insulin receptor autoanti-bodies in a patient with type B insulin resistance andfasting hypoglycemia. Acta Diabetol 2000; 37: 189’96.

15. Luyckx FH, Scheen AJ. How I explore… the controversyconcerning the role of oral glucose tolerance test in cli-nical practice. Rev Med Liege 2003; 58: 701’5.

16. Ignjatovi} S. Diagnosis of diabetes mellitus. JugoslovMed Biohem 2003; 22 (Suppl 1): 13’19.

Received: April 2, 2004

Accepted: August 5, 2004