AN ASSOCIATION BETWEEN low birth weight (LBW)

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1 X/05/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 90(8): Printed in U.S.A. Copyright 2005 by The Endocrine Society doi: /jc Normal Secretion and Action of the Gut Incretin Hormones Glucagon-Like Peptide-1 and Glucose- Dependent Insulinotropic Polypeptide in Young Men with Low Birth Weight Jakob Hagen Schou,* Kasper Pilgaard,* Tina Vilsbøll, Christine B. Jensen, Carolyn F. Deacon, Jens Juul Holst, Aage Vølund, Sten Madsbad, and Allan A. Vaag Steno Diabetes Center (J.H.S., K.P., C.B.J., A.A.V.), 2820 Gentofte, Denmark; Department of Internal Medicine F (T.V.), Gentofte Hospital, DK 2820 Hellerup, Denmark; Department of Medical Physiology (C.F.D., J.J.H.), Panum Institute, University of Copenhagen, DK 2200 Copenhagen, Denmark; Novo Nordisk (A.V.), DK-2880 Bagsværd, Denmark; and Department of Endocrinology (S.M.), Hvidovre University Hospital, DK 2650 Hvidovre, Denmark Context: Low birth weight (LBW) is associated with increased risk of type 2 diabetes mellitus. An impaired incretin effect was reported previously in type 2 diabetic patients. Objective: We studied the secretion and action of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) in young LBW men (n 24) and matched normal birth weight controls (NBW) (n 25). First Published Online May 17, 2005 * J.H.S. and K.P. contributed equally to this study. Abbreviations: AUC, Area under the curve; BG incr, incremental blood glucose; DI, disposition index; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide-1; HDL, high-density lipoprotein; HOMA, homeostasis model assessment; LBW, low birth weight; NBW, normal birth weight; Si, insulin sensitivity; T2DM, type 2 diabetes mellitus. JCEM is published monthly by The Endocrine Society ( endo-society.org), the foremost professional society serving the endocrine community. Results: LBW subjects were 5 cm shorter but had a body mass index similar to NBW. LBW subjects had significantly elevated fasting and postprandial plasma glucose, as well as postprandial (standard meal test) plasma insulin and C-peptide concentrations, suggestive of insulin resistance. Insulin secretion in response to changes in glucose concentration ( -cell responsiveness ) during the meal test was similar in LBW and NBW but inappropriate in LBW relative to insulin sensitivity. Fasting and postprandial plasma GLP-1 and GIP levels were similar in the groups. First- and second-phase insulin responses were similar in LBW and NBW during a hyperglycemic clamp (7 mm) with infusion of GLP-1 or GIP, respectively, demonstrating normal action of these hormones on insulin secretion. Conclusion: Reduced secretion or action of GLP-1 or GIP does not explain a relative reduced -cell responsiveness to glucose or the slightly elevated plasma glucose concentrations observed in young LBW men. (J Clin Endocrinol Metab 90: , 2005) AN ASSOCIATION BETWEEN low birth weight (LBW) and impairment of glucose homeostasis was first proposed by Hales and Barker (1) in Since then, several studies have confirmed and elaborated on these findings, thereby underscoring the importance of the intrauterine environment for the development of later adult diseases, including hypertension, cardiovascular disease (2), and abnormal glucose tolerance (3 8). Because the association between LBW and impaired glucose homeostasis becomes more pronounced with age, most research has been performed in elderly subjects. Nevertheless, recent studies have shown defects of glucose and insulin homeostasis in young adults and children with LBW (9, 10). The incretin effect is defined as an enhanced insulin secretion after oral vs. iv glucose administration due to the insulinotropic effect of the gastrointestinal hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). During the past two decades, evidence of various defects in the enteroinsular axis among type 2 diabetes mellitus (T2DM) patients has accumulated. An impaired incretin effect in type 2 diabetic patients has been showed by Nauck et al. (11). Furthermore, recent studies have reported reduced postprandial secretion of GLP-1 in T2DM patients (12, 13). Animal studies have shown that mice with a targeted deletions of the GLP-1 and GIP receptor become glucose intolerant and develop fasting hyperglycemia (14, 15), supporting the hypothesis that impaired function of the enteroinsular axis contributes to the inappropriate insulin secretion in type 2 diabetes. The primary actions of GLP-1 on glucose homeostasis involve enhancement of glucose-dependent insulin secretion, decreased glucagon secretion, and subsequently reduced hepatic glucose production (16). Furthermore, GLP-1 inhibits gastric emptying and reduces appetite/food intake (17). Recent studies have also provided evidence of a beneficial effect on -cell proliferation (18, 19), islet neogenesis (20, 21), and reduced -cell apoptosis (22). GIP also has an insulinotropic effect on the -cell in healthy subjects and may play an important role in lipid metabolism (23). Together, GLP-1 and GIP account for approximately 50 60% of insulin secretion during a meal (24) We recently found evidence of a reduced insulin secretion (when corrected for insulin resistance) during an oral glucose tolerance test in a population of young men with LBW (25). Insulin secretion per se in response to an iv glucose bolus was 4912

2 Schou et al. Normal Incretin Secretion and Action in LBW Men J Clin Endocrinol Metab, August 2005, 90(8): not reduced, and we therefore proposed that defective secretion and/or action of the gut incretin hormones might explain (or contribute to) the disproportionately reduced insulin secretion and mildly elevated plasma glucose levels in these individuals. The aims of the present study were as follows: 1) to study the endogenous secretion of insulin, GLP-1, and GIP in response to a standard (physiological) breakfast meal test; 2) to quantify -cell responsiveness to glucose and the gut incretins during the breakfast meal in subject with LBW and normal birth weight (NBW); and 3) to estimate both the first- and the late-second-phase insulin response during a hyperglycemic clamp with continuous infusion of GIP or GLP-1. Thus, we hypothesized that defective secretion and/or action of one or both of the two incretin hormones GIP and GLP-1 might explain the inappropriate enhancement of insulin secretion in young men with LBW. Participants Subjects and Methods Forty-nine singleton Caucasian men born at term (39 41 wk) in in Copenhagen County were identified and recruited from the Danish Medical Birth Registry, according to birth weight. Twentyfour men (LBW) had birth weights below the 10th percentile (wk 39, 2800 g; wk 40, 2960 g; wk 41, 3010 g), whereas 25 (NBW) had birth weights in the upper normal range (50th to 90th percentile) (wk 39, g; wk 40, g; wk 41, g). None of the participants had parents, grandparents, or siblings who had any type of diabetes, and none received medication known to interfere with glucose homeostasis. The protocol was approved by the regional ethics committee, and procedures were performed according to the principles of The Helsinki Declaration. After thorough written and oral explanation of the study, all subjects gave their written consent. Experimental protocol Each subject was studied in randomized order on three separate occasions with intervals of a minimum of 7 d. Protocol 1 consisted of a meal test, and protocol 2 consisted of a hyperglycemic clamp in conjunction with continuous infusion of either GLP-1 (d 1) or GIP (d 2). Subjects were instructed to abstain from strenuous physical activity and to consume a diet rich in carbohydrate for a period of at least 48 h before each study day. Smoking was prohibited during experiments, as was any intake of food or drinks besides water. Study days began at 0830 h and were preceded by a 10-h overnight fast. Blood samples were obtained through a polyethylene catheter placed in a dorsal hand vein. Subjects were studied in a recumbent position, and the cannulated hand was kept in a heated box throughout the experiment. Protocol 1 (mixed meal test) All subjects underwent a meal tolerance test with measurement of plasma glucose, insulin, C-peptide, GIP, and GLP-1 in blood samples collected at 15, 0, 15, 30, 45, 60, 75, 90, 105, 120, 135, 165, and 195 min after intake of a standardized breakfast (served at 0800 h; 2370 kj; 47% carbohydrate, 19% protein, and 34% fat). Protocol 2 (hyperglycemic clamps) To quantify insulin secretion and the action of GLP-1 (d 1) and GIP (d 2), we used a hyperglycemic clamp (7 mmol/liter) with primed continuous infusion of GLP-1 or GIP, respectively. A polyethylene catheter was placed in an antecubital vein for test infusions (glucose and GLP-1, or glucose and GIP). At t 30 min, a 20% glucose bolus was infused to raise plasma glucose to 7 mmol/liter, calculated as [(7 mmol/ liter fasting plasma glucose 35 mg glucose weight in kilograms/ 1000) 5]. At t 2 min, a bolus of either GLP-1 or GIP was infused to increase the plasma concentration to approximately 120 and 1000 pmol/liter, respectively. At t 0 min, a continuous infusion of GLP-1 (60 pmol/kg h) or GIP (240 pmol/kg h) was initiated. Plasma glucose concentration was maintained at 7 mmol/liter by continuous infusion of 20% (w/v), glucose and infusion rates of glucose were adjusted every 5 min according to bed-site plasma glucose level. Arterialized blood was sampled at 30, 25, 20, 15, 10, 5, 0, 1, 3, 5, 10, 15, 20, 30, 45, 60, 65, 75, 90, 105, and 120 min. The last 30 min of study time was predefined as steady-state period. Fasting samples for determinations of fasting levels of total cholesterol, high-density lipoprotein (HDL), lowdensity lipoprotein, and triglycerides were also obtained. Analysis Plasma glucose was measured using an automated glucose oxidation method (Glucose Analyzer 2; Beckman Instruments, Fullerton, CA). Plasma insulin and C-peptide samples were centrifuged immediately at 4 C and stored at 80 C. Plasma insulin and C-peptide concentrations were determined by 1235 AutoDELPHIA automatic immunoassay system (Wallac Oy, Turku, Finland). The plasma insulin assay had a detection limit of approximately 3 pmol/liter. Cross-reactivity with intact pro-insulin was 51%, 35% with split pro-insulin and 92% with split pro-insulin (no detectable cross-reactivity with insulin), intraassay variation of 5%, and interassay variation of 8%. Cholesterol, HDL cholesterol, free fatty acids, and triglycerides were measured by enzymatic methods. Blood for analysis of GLP-1 and GIP was sampled in heparin-edta tubes (6 mmol/liter) with aprotinin (500 kiu/ml blood; Trasylol, Bayer, Leverkusen, Germany). Tubes were immediately cooled on ice and centrifuged at 4 C for 20 min. Plasma was stored at 20 C until analysis. Total GIP was measured using the C-terminally directed antiserum R65 (26, 27), which reacts fully with intact GIP and the N-terminally truncated metabolite GIP(3 42). The assay has a detection limit of less than 2 pmol/liter and an intraassay variation of approximately 6%. Intact, biologically active GIP was measured using a newly developed assay. The assay is specific for the intact N terminus of GIP and cross-reacts less than 0.1% with GIP(3 42), or with the structurally related peptides GLP-1(7 36) amide, GLP-1(9 36) amide, GLP-2(1 33), GLP-2(3 33), or glucagon at concentrations of up to 100 nmol/liter. Intraassay variation was less than 6%, and interassay variation was approximately 8 and 12% for 20 and 80 pmol/liter standards, respectively. Plasma samples were assayed for GLP-1 immunoreactivity using RIAs that are specific for each terminus of the GLP-1 molecule: the C-terminal assay measuring the sum of the intact peptide plus the primary metabolite and the N-terminal assay measuring the concentration of intact surviving GLP-1. The C-terminal immunoreactivity of GLP-1 was measured using standards of synthetic GLP-1(7 36) amide and antiserum (28). The assay cross-reacts less than 0.01% with C-terminally truncated fragments and 83% with GLP-1(9 36) amide and has a detection limit less than 1 pmol/liter. N-terminal immunoreactivity was measured using antiserum 93242, which cross-reacts approximately 10% with GLP-1(1 36) amide and less than 0.1% with GLP-1(8 36) amide and GLP-1(9 36) amide. The assay has a detection limit of 2 pmol/liter. For both assays, intraassay and interassay coefficients of variation were less than 6 and 15%, respectively, at 40 pmol/liter. Peptides Synthetic GLP-1(7 36) amide and GIP were purchased from PolyPeptide Laboratories (Wolfenbüttel, Germany). The peptides were dissolved in sterilized water containing 2% human serum albumin [human albumin (Statens Serum Institute, Copenhagen, Denmark), guaranteed to be free of hepatitis-b surface antigen, hepatitis-c virus antibodies, and human immunodeficiency virus antibodies] and subjected to sterile filtration. Appropriate amounts of peptide for each experimental subject were dispensed into glass ampoules and stored frozen under sterile conditions until the day of the experiment. The peptides were more than 97% pure and identical to the natural human peptides by HPLC, mass, and sequence analysis. Calculations Meal test. Areas under the curve (AUCs) for plasma glucose, GLP-1, GIP, and insulin secretion rates were calculated using the trapezoidal method (0 90 min, min). In addition, the incremental AUC for glucose

3 4914 J Clin Endocrinol Metab, August 2005, 90(8): Schou et al. Normal Incretin Secretion and Action in LBW Men TABLE 1. Subject characteristics LBW NBW P Birth weight (g) Weight (kg) NS Height (m) BMI (kg/m 2 ) NS Waist-to-hip ratio Systolic blood pressure NS (mm Hg) Diastolic blood pressure (mm Hg) NS Data are means SD. BMI, Body mass index; NS, not significant. more than 5 mmol/liter was calculated. Insulin secretion rates were calculated by deconvolution of the measured C-peptide concentrations by applying population-based individual parameters for C-peptide kinetics as described previously (29, 30). The estimated secretion rates were represented by cubic splines and expressed as picomoles per kilogram of body weight. To explore the relationship between insulin secretion and glucose, GIP, and GLP-1, respectively, linear correlations between these variables (AUCs) and the artificial variable formed as the product of AUC glucose more than 5 mmol/liter [incremental blood glucose (BG incr )] and the GIP or the GLP-1 AUC were done. The rationale for this variable is the concept that GIP and GLP-1 have no effect on insulin secretion when glucose is below basal levels but stimulates insulin secretion primarily at elevated glucose levels. These analyses were performed in the two groups separately and combined. In addition, both univariate and multivariate analysis using the same variables was performed. Finally, changes in insulin secretion rates in response to changes in glucose during the meal expresses the efficacy by which changes in plasma glucose concentrations and gut factors (i.e. GLP-1 and GIP) stimulate insulin secretion. Therefore, the relationship between plasma glucose concentrations and insulin secretion rates during the meal were evaluated by cross-correlation analyses in each patient. The relationship was linear in all subjects, and the slope of the line was used as an index ( -index) of -cell response to glucose (and incretins). Thus, a change in insulin secretion rates induced by a change in plasma glucose by 1 mmol/liter is expressed as picomoles of insulin secreted per minute per kilogram of body weight. Hyperglycemic clamps. For both study days, d 1 (GLP-1) and d 2 (GIP), the first-phase insulin response was defined as AUC(insulin secretion) 0 10 min and the second-phase response as AUC(insulin secretion) min. Glucose infusion rates (milligram per kilogram per minute) were calculated at clamp steady state (t min). AUC for glucose, insulin, C-peptide, and insulin secretion rates (calculated as above) were calculated (t 0 120, t 0 10, and t min). Insulin sensitivity. Insulin sensitivity (Si) was expressed in two different ways: 1) using the homeostasis model assessment (HOMA) model, an estimate of Si derived from fasting plasma glucose and insulin levels (fasting insulin ( U/ml) fasting glucose (mmol/liter))/22.5) (31), and 2) the ratio between glucose infusion rate and mean plasma insulin (Si, t min) on d 2 (32). It has been suggested that GLP-1 may exert an independent extrahepatic effect on peripheral glucose uptake and metabolism (33). Therefore, we did not estimate Si during GLP-1 infusion. Disposition indices. Finally, to express insulin secretion relative to Si, two disposition indices (DIs) were calculated: 1) DI 1 : Si HOMA meal -index, and 2) DI 2 : Si clamp meal -index. Statistical methods Data are presented as means sem unless otherwise indicated. The nonparametrical Mann-Whitney U test was used, unless data were normally distributed (t test). P 0.05 during two-tailed analysis was considered significant. Correlations between the continuous measured or calculated variables were evaluated by linear regression analysis. Results Subject characteristics (Table 1) LBW subjects were, on average, 5 cm shorter and had borderline significantly higher waist-to-hip ratio than NBW controls. Current weight, body mass index, total cholesterol, low-density lipoprotein, HDL, and triglycerides were similar in the groups. Fifty-four percent of LBW subjects compared with 60% of NBW subjects reported smoking on a regular basis. Meal test (Table 2 and Fig. 1) Figure 1 shows the mean profiles of plasma glucose, insulin, C-peptide, GIP, GLP-1, and insulin secretion. Although the insulin secretion rate closely follows that of glucose concentration and returns to near fasting levels after 90 min, both incretin hormones exhibit prolonged profiles that do not reach fasting levels after 195 min. Fasting plasma glucose, but not insulin or C-peptide concentrations, was significantly higher in LBW compared with NBW subjects. Plasma glucose peaked in both study groups 38 min postprandially, reaching levels of mmol/liter in LBW subjects vs mmol/liter in the control group (P 0.02). In addition, LBW subjects had significantly higher postprandial glucose, insulin, and C-peptide concentrations at all time points, hence also total and incremental AUC glucose and insulin secretion rates, as can be seen in Table 2. The AUCs of GIP and GLP-1 were nearly identical in the two groups. The correlation coefficients between the AUCs of insulin, BG incr, GLP-1, and GIP in the two groups appeared to differ only due to random variation. This indicates that the relationships are similar in the two groups and the groups may therefore be combined. The cross product variable AUC(BG incr ) AUC(GIP) shows the overall correlation with TABLE 2. Comparison of AUC responses Period 0 90 min Total period min LBW (n 24) NBW (n 25) LBW (n 24) NBW (n 25) Insulin secretion (pmol/kg) a , b Glucose concentration (mmol liter 1 min) b , c 1, Glucose concentration 5 mmol/liter (mmol liter 1 min) b c GIP concentration (pmol liter 1 min) 4, , , , GLP-1 concentration (mmol liter 1 min) 1, , , , Data are presented as mean SEM. P P P

4 Schou et al. Normal Incretin Secretion and Action in LBW Men J Clin Endocrinol Metab, August 2005, 90(8): FIG. 1. Mean plasma glucose, insulin, C-peptide, GIP, GLP-1, and insulin secretion rates in NBW (filled circles) and LBW (open circles) during the standard meal test. insulin secretion. Figure 2 illustrates this correlation for the 0 90 min period. The distribution of the points in Fig. 2 indicates that the same regression line can describe the data. This was formally tested by means of analysis of covariance, which showed that the significant difference in insulin secretion between the two groups (Table 2) disappeared when AUC(BG incr ) AUC(GIP) was included as an explanatory variable. In other words, the increased insulin secretion response FIG. 2. The relationship between insulin secretion and the variable formed as the product of AUC for glucose more than 5 mmol/liter and AUC for GIP during the standard meal. (ISR) in the LBW group relative to the control group can be explained by a combined effect of higher glucose and variation in GIP. Although the latter was similar in the two groups, the general correlation with GIP combined with increased glucose may be interpreted as a similar responsiveness or sensitivity of the -cell to glucose and GIP in the two groups. A similar result was seen when the corresponding data for the min period were analyzed (results not shown). Finally, it should be briefly mentioned that multivariate correlation analysis using the data from Table 2 shows that the pair of explanatory variables AUC(BG) and AUC(GIP) gave a slightly better description of the variation in insulin secretion (for both periods) than the univariate correlation with AUC(BG incr ) AUC(GIP). Both AUC(BG) and AUC(GIP) contributed significantly, whereas none of the other variables improved the description (Table 3. The two variables were also capable of explaining the group difference in insulin secretion. Whether it is the single combined variable AUC(BG incr ) AUC(GIP) or the pair AUC(BG) and AUC(GIP) that gives the most correct prediction of insulin secretion cannot be settled by the statistical analysis. Furthermore, -cell responsiveness to changes in glucose ( -index), expressed as the slope of the lines expressing the relationship between the calculated ISR and the concomitant blood glucose concentrations, was not different between the groups [NBW, vs. LBW, (pmol insulin) kg 1 min 1 (mmol glucose) 1 liter; P 0.68]. According to the HOMA index, LBW subjects tended to be insulin resistant ( vs ; P 0.14), and

5 4916 J Clin Endocrinol Metab, August 2005, 90(8): Schou et al. Normal Incretin Secretion and Action in LBW Men TABLE 3. Correlation coefficients between insulin secretion and AUC glucose, GIP, and GLP-1 Explanatory AUC variable Period 0 90 min Total period min LBW (n 24) NBW (n 25) All (n 49) LBW (n 24) NBW (n 25) All (n 49) Glucose b 0.44 a c GIP 0.48 a a 0.48 a b GLP BG incr b 0.44 a c BG incr GIP 0.46 a 0.47 a 0.53 c 0.50 a 0.45 a 0.57 c BG incr GLP a Data are presented as mean SEM. a P b P c P consequently, the disposition index DI 1 tended to be lower in LBW subjects [ vs (pmol insulin) 2 (mmol glucose) 2 kg 1 min 1 liter 1 ; P 0.11]. Hyperglycemic clamps with infusion of GLP-1 and GIP (Table 4 and Fig. 3) Plasma glucose, insulin, and C-peptide concentrations, as well as insulin secretion rates, were similar in the two groups, regardless of study day (Fig. 3). Consequently, neither firstphase nor second-phase insulin responses differed between groups (data not shown). Note that, although the GLP-1- and GIP-induced first-phase insulin responses were of significant difference, this difference in percentage is minimal compared with the difference in second-phase insulin response (t min), being approximately 50% decreased during GIP compared with the GLP- 1 infusion (Table 5). The amount of glucose infused (expressed as AUC glucose 0 120) to maintain plasma glucose at 7 mmol/liter during GLP-1 coinfusion was significantly lower in the LBW subjects throughout the clamp (NBW, ; LBW, mg/kg min; P 0.05) and mean glucose infusion during steady-state (t min) (NBW, vs. LBW, mg/kg min; P 0.04), which is suggestive of insulin resistance. No significant difference was observed with GIPinfusion (AUC glucose 0 120: NBW, vs. LBW, mg/kg min). Nevertheless, when Si was calculated as the glucose infusion rate/insulin (t min) during GIP infusion, LBW subjects tended to dispose less glucose compared with NBW [NBW, vs. LBW, (milligrams of glucose per kilogram per millimolar insulin); P 0.11]. Disposition index, DI 2 (based on glucose infusion rates during GIP coinfusion), was insignificantly lower in LBW ( vs U; P 0.18). Discussion This study has revealed the following findings: young healthy LBW men have 1) normal secretion of both GLP-1 and GIP in response to a standard breakfast meal, 2) normal action of GLP-1 and GIP in terms of -cell stimulation when infused iv during a 7 mm hyperglycemic clamp, and 3) similar -cell responsiveness to changes in glucose during a standard meal test but in the face of significantly elevated plasma glucose concentrations and insulin resistance, suggesting the presence of a subtle -cell defect in LBW subjects. Although defective secretion and action of GLP-1 and GIP were shown previously to contribute in different ways to the disturbed glucose homeostasis in type 2 diabetic patients (11 15, 23, 34), the present very detailed study did not provide evidence for a primary involvement of the gut incretin hormones GLP-1 and GIP in the development of elevated fasting and postprandial plasma glucose concentrations in young, presumably prediabetic men with LBW. Nyholm et al. (35) demonstrated in healthy offspring of type 2 diabetic patients a normal GLP-1 secretion but an increased GIP concentration after a meal. Previously, data from our group has indicated a nongenetic origin of the defective GLP-1 secretion in monozygotic twins discordant for type 2 diabetes (36). Moreover, studies of several diabetic phenotypes (latent autoimmune diabetes of adults, maturity-onset diabetes of the young type 3, chronic pancreatitis, newly diagnosed type 1 diabetes mellitus, and thin and obese T2DM patients) have lead us to conclude that GIP was unable to generate a significant late-phase insulin response in any of those groups (34). Therefore, we propose that impaired secretion and action of gut incretin hormones develop at a later time point, potentially as a consequence of hyperglycemia and/or hyperlipidemia in prediabetic and diabetic states, rather than being a primary abnormality of glucose homeostasis. We TABLE 4. Data obtained during hyperinsulinemic clamp GLP-1 GIP NBW (n 25) LBW (n 24) P NBW (n 25) LBW (n 24) P AUC Glu. total t:0 120 (mg/kg min) NS Infusion rate t: (mg/kg min) NS AUC Ins. total t:0 120 (pmol/min) 42,145 6,362 42,691 5,844 NS 19,094 1,390 22,771 2,286 NS AUC ins. incr t:0 120 (pmol/min) 29,944 6,573 30,691 5,830 NS 9,124 1,988 11,512 1,248 NS AUC ins. t:0 10 (pmol/min) 2, , NS 3, , NS Data are means SEM. NS, Not significant; Glu., glucose; ins., insulin.

6 Schou et al. Normal Incretin Secretion and Action in LBW Men J Clin Endocrinol Metab, August 2005, 90(8): FIG. 3. Plasma glucose (1A, 1B), glucose infusion rates (2A, 2B), plasma insulin (3A, 3B), plasma C-peptide (4A, 4B), and insulin secretion rates (5A, 5B) during the hyperglycemic clamps in men with NBW (black lines) and LBW (stippled lines). Figures labeled A illustrate the results during GLP-1 infusion, and figures labeled B illustrate the results during GIP infusion.

7 4918 J Clin Endocrinol Metab, August 2005, 90(8): Schou et al. Normal Incretin Secretion and Action in LBW Men TABLE 5. First and second phase insulin secretion responses to infusion of GIP and GLP-1 First phase ( 2 to 10 min) (pmol/kg) Second phase (10 to 120 min) (pmol/kg) Infusion NBW (n 25) LBW (n 24) GLP b a GIP GLP c c GIP Data are means SEM. No statistically significant differences between the two groups for any of the four responses. First and second phase responses are significantly higher (P 0.001) in both groups with GLP-1 than GIP. a P b P c P measured the total GLP-1, which is the sum of the biologically active intact molecule GLP-1(7 36) amide and the primary inactive metabolite GLP-1(9 36) amide. The use of this assay rather than an NH-terminal assay measuring only the intact, biologically active GLP-1 is essential to estimate the rate of secretion of GLP-1 because the hormone is metabolized intravascularly and extremely rapidly (with an apparent half-life of 1 15 min and a clearance rate exceeding cardiac output (37). Thus, it is the sum of the concentrations of the primary metabolite and the intact hormone that reflects the secretory rate of GLP-1. The finding of identical plasma GLP-1 and GIP levels during the study days with iv GLP-1 and GIP infusions does not indicate different metabolism of the two peptides in the LBW and control subjects in this study. Another potential source of error could be different gastric emptying rates in the two study groups, which may influence the incretin hormone secretion in a differential manner (38). However, given that the plasma GIP and GLP-1 levels and profiles after the meals were virtually identical in LBW subjects and controls, we do not believe that our results are influenced by differences in gastric emptying. To obtain a well-defined separation and accurate quantification of first- and second-phase insulin responses, we used deconvolution to estimate insulin secretion. Furthermore, to ensure action of the glucose-dependent incretin hormones on insulin secretion, we used hyperglycemic clamps within the physiological glucose range and demonstrated normal action of both GLP-1 and GIP to enhance insulin secretion in the LBW men. Nevertheless, the effect of the two incretin hormones GLP-1 vs. GIP on first- and second-phase insulin responses differed in an interesting manner. Thus, although first-phase responses were near similar during GLP-1 and GIP infusions, second-phase insulin response during GIP infusion was only half of that observed during GLP-1 infusion in both study groups. Whereas first-phase insulin secretion is believed to be primarily determined by the rate of exocytosis of previously docked insulin granula, the second-phase response is believed to rely on several factors, including the rate of de novo insulin synthesis, insulin granula translocation, and membrane fusion (39, 40). Experimental reduction of -cell mass decreases both first- and second-phase insulin secretion (41). One interpretation of the present data may be that GIP primarily amplifies the initial part of insulin secretion by promoting the exocytosis of previously docked insulin granulas, whereas GLP-1 in addition to its enhancing effect on first-phase insulin secretion also stimulates second-phase insulin secretion through the mechanisms outlined above. This is a novel observation contributing to our understanding of the mode of action of the gut incretin hormones in nondiabetic subjects. The LBW subjects had significantly higher fasting glucose as well as elevated plasma glucose, insulin, and C-peptide concentrations during the meal tests. Although within the normal nondiabetic range, the magnitude of the elevation of the fasting plasma glucose concentration in the LBW subjects in this study was virtually identical to that reported in our previous study (25). However, -cell responsiveness to glucose (and incretin hormones) under these physiological conditions (mixed meal test) was normal, which is suggestive of a normal -cell sensitivity to glucose in the absolute sense in LBW subjects at early stages. Nevertheless, insulin secretion was not quite sufficient to overcome the degree of insulin resistance, evident by mild hyperglycemia and nonsignificantly lower disposition indices in the LBW group. Relative -cell failure, despite normal GLP-1 and GIP secretion and action, was also found in our previous study (25), and this subtle defect is likely to be functional rather than due to a decrease in -cell mass, because the first- and second-phase insulin responses were identical during the hyperglycemic clamps. It should be noted that insulin action in the present study, in contrast to our previous study, were assessed using indirect measures such as the HOMA index and glucose infusion during GIP clamps. This may explain the lack of a significant reduction of the disposition index in the LBW subjects in this study. The slightly higher waist-to-hip ratios in the present LBW cohort may explain why the LBW subjects tented to be insulin resistant compared with the control subjects. We have demonstrated previously blunted insulinstimulated forearm glucose uptake (42), impaired insulinstimulated glycolysis, but normal whole-body glucose disposal in 19-yr-old men with LBW (25) who had been selected according to the same criteria as in the present study. In conclusion, the consistent finding of elevated fasting and postprandial glucose levels in young healthy men with LBW support the idea of an important role for the intrauterine environment for the development of type 2 diabetes. We have shown that young LBW men have normal -cell responsiveness to iv glucose and the two major gut incretin hormones GLP-1 and GIP, as well as normal incretin hormone secretion. Nevertheless, insulin secretion was relatively insufficient to overcome a mild insulin resistance in the LBW subjects, resulting in significantly elevated glucose concentrations during meal tests. It remains to be determined whether this is due to an intrinsic functional abnormality of the -cell or whether it may be due to a yet unidentified abnormal signal to the -cell. Acknowledgments We thank Susanne Reimer and Marianne Modest for skillful assistance during meal tests and clamps on Hvidovre Hospital. Also, sincere thanks to Lone Bagger at Panum University for skillful assistance on analyzing GLP-1 and GIP samples.

8 Schou et al. Normal Incretin Secretion and Action in LBW Men J Clin Endocrinol Metab, August 2005, 90(8): Received February 22, Accepted May 6, Address all correspondence and requests for reprints to: Jakob Hagen Schou or Kasper Pilgaard, Steno Diabetes Center, Niels Steensens Vej 2, 2820 Gentofte, Denmark. oranje@tiscali.dk or kasperpilgaard@hotmail.com. This work was supported by grants from the Danish Diabetes Association. References 1. Hales CN, Barker DJ, Clark PM, Cox LJ, Fall C, Osmond C, Winter PD 1991 Fetal and infant growth and impaired glucose tolerance at age 64. BMJ 303: Hales CN, Barker DJ 2001 The thrifty phenotype hypothesis. Br Med Bull 60: Veening MA, Van Weissenbruch MM, Delemarre-Van De Waal HA 2002 Glucose tolerance, insulin sensitivity, and insulin secretion in children born small for gestational age. 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