Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that is synthesized and secreted in 2 major molecular forms with equipotent biological activity—GLP-1(7-36)amide and GLP-1(7-37)—from enteroendocrine L cells of the small bowel and large bowel (1). Endogenous concentration is low in the fasting state and increases in response to a meal (1). GLP-1 is believed to be the most potent insulinotropic hormone known to date (1,2). Furthermore, GLP-1 suppresses glucagon secretion and inhibits gastric emptying and acid secretion (1,2).
An attenuated GLP-1 response is suggested to contribute to the impaired insulin response in patients with type 2 diabetes mellitus (1,3). Insulin resistance is associated with an impaired GLP-1 response to a mixed meal (4). GLP-1 receptor—null mice demonstrated glucose intolerance and an attenuated insulin response to oral glucose loading (5). GLP-1 was found to normalize both the fasting and the postprandial glucose levels in humans with type 2 diabetes (6). The insulinotropic effect of GLP-1 has been demonstrated in animal models (7,8): GLP-1 stimulated insulin secretion in a glucose-dependent manner, and it stimulated β-cell proliferation and differentiation (7).
Like many other peptides of the brain-gut axis, GLP-1 is involved in control of food intake (1,2). Recently, GLP-1 has been shown to reduce energy intake and enhance satiety, most likely via delay of gastric emptying and via specific GLP-1 receptors within the central nervous system (1,9). However, the role of GLP-1 in obesity is poorly understood. Some authors have reported an attenuated response of GLP-1 to meals in obese individuals (10,11), whereas others have found no differences in fasting and stimulated GLP-1 levels between normal-weight and obese individuals (12,13). In obese humans losing weight increasing (10,14), decreasing (15), and unchanged fasting and stimulated GLP-1 levels (16) have been reported.
Since the development of GLP-1 analogues as new therapeutic approaches in diabetes and obesity (1,8), it has been interesting to study the relationships between GLP-1, weight status, insulin, and insulin resistance not only postprandially but also in the fasting state. To our knowledge, no studies have been published concerning the relationship between GLP-1, weight status, and insulin levels in children. The advantage of examining children is that potential confusion with further chronic diseases or medication is limited. Therefore, and because studies in obese adults are conflicting, we analyzed fasting GLP-1 and insulin concentrations, insulin resistance, and their changes during 1 year in obese children participating in an obesity intervention program. We compared these parameters with those of lean children. The aim of this study was to analyze whether fasting GLP-1 concentrations are related to weight status, insulin, and/or insulin resistance.
PATIENTS AND METHODS
We examined anthropometric markers, GLP-1, glucose, and insulin concentrations in 42 obese and 16 lean, healthy white children (Table 1). Furthermore, the same parameters were analyzed in the obese children before and after they participated in a 1-year obesity intervention program. Children with endocrine disorders or syndromal obesity were excluded from the study. Obesity was defined according to the definition of the International Obesity Taskforce (17).
The degree of overweight was quantified by Cole's least mean squares method, which normalized the body mass index (BMI), skewed distribution in childhood, and expressed BMI as a standard deviation score (SDS-BMI) (18). Reference data for German children were used (19). Triceps and subscapularis skinfold thickness was measured in duplicate with a caliper and averaged to calculate the percentage of body fat by use of a skinfold thickness equation with the following formulas (20): boys: body fat % = 0.783 × (subscapularis skinfold thickness + triceps skinfold thickness in mm) + 1.6; girls: body fat % = 0.546 × (subscapularis skinfold thickness + triceps skinfold thickness in mm) + 9.7.
The pubertal developmental stage was determined according to Marshall and Tanner and categorized into 2 groups (prepubertal: boys with pubic hair and gonadal stage I, girls with pubic hair stage and breast stage I; pubertal: boys with pubic hair or gonadal stage ≥II, girls with pubic hair stage or breast stage ≥II).
Blood sampling was performed while participants were in the fasting state at 8 AM. Serum GLP-1 concentrations were measured by a high-specific enzyme-linked immunosorbent assay (human active GLP-1 ELISA Kit, Linco Research, St. Charles, Missouri, USA) for GLP-1(7-36)amide and GLP-1(7-37) without cross-reactions to glucagon or to other forms of GLP-1. All values of GLP-1 are composed of GLP-1(7-36)amide and GLP-1(7-37). The sensitivity stated by the manufacturer was 2 pmol/L, whereas we determined a sensitivity of 0.5 pmol/L. Insulin concentrations were measured by microparticle enhanced immunometric assay (MEIA, Abbott, Wiesbaden, Germany). Glucose levels were determined by colorimetric test with a Vitros analyzer (Ortho Clinical Diagnostics, Neckargemuend, Germany). Intraassay and interassay coefficients of variation were <5% in all methods apart from GLP-1 (<13%). Homeostasis model assessment (HOMA) was used to detect the degree of insulin resistance (21): The resistance can be assessed from the fasting glucose and insulin concentrations by the formula: resistance (HOMA) = (insulin [mU/L] × glucose [mmol/L]/22.5).
The obesity intervention program, Obeldicks, has been described in detail elsewhere (22). Briefly, the program was based on physical exercise, nutrition education, and behavior therapy, including individual psychological care of the child and the family. The nutritional course was based on a fat-reduced and sugar-reduced diet compared with the everyday nutrition of German children (22): The diet contained 30% fat, 15% protein, and 55% carbohydrates, including 5% sugar. Substantial weight loss during the 1-year intervention was defined by a reduction in SDS-BMI of 0.5 or more because with a reduction of <0.5 SDS-BMI, no improvement of insulin resistance and cardiovascular risk factors could be measured in obese children (23,24).
Statistical analysis was performed with the Winstat software package. Apart from GLP-1, all variables were normally distributed tested by the Kolmogorov-Smirnov test. Therefore, GLP-1 was log transformed. Differences were tested with χ2 tests, t tests, Mann-Whitney U tests, or Wilcoxon test as appropriate. Pearson and partial correlations adjusted for SDS-BMI were used. Direct multivariate linear regression analyses were conducted for the dependent variable GLP-1, including age, sex, pubertal stage, BMI, glucose, and insulin or HOMA instead of glucose and insulin as independent variables. Sex and pubertal stage were used as classified variables in these models. P < 0.05 was considered significant. Written informed consent was obtained from all of the children and their parents. The study was approved by the local ethics committee of the University of Witten/Herdecke.
The age, stage of puberty, sex, degree of overweight, insulin resistance index (HOMA), insulin, and glucose concentrations of the 42 obese and 16 lean children are shown in Table 1. The obese children demonstrated significantly higher insulin resistance index (HOMA) and insulin than did the lean children, whereas the lean and obese children did not significantly differ in terms of age, sex, pubertal status, glucose, or GLP-1 concentrations.
In the 58 obese and normal-weight children, log transformed GLP-1 did not significantly correlate to the degree of overweight (SDS-BMI) (r = 0.17; P = 0.097) or percentage body fat (r = 0.01; P = 0.464). In partial correlation adjusted to SDS-BMI, log transformed GLP-1 correlated significantly to insulin (r = 0.30; P = 0.011) but not to glucose (r = 0.01; P = 0.476), HOMA (r = 0.12; P = 0.168), or age (r = −0.10; P = 0.236). Insulin correlated significantly to SDS-BMI (r = 0.47; P < 0.001) and percentage body fat (r = 0.44; P = 0.001). In direct multivariate linear regression analysis (r2 = 0.33), GLP-1 correlated significantly to insulin (coefficient 0.37, 95% CI 0.05–0.70; P = 0.027) but not to BMI (P = 0.179), glucose (P = 0.479), age (P = 0.679), sex (P = 0.988), and pubertal stage (P = 0.628). The use of HOMA instead of insulin and glucose in the multivariate linear regression analysis also demonstrated a significant relationship between HOMA and GLP-1 (coefficient 1.8, 95% CI 0.3–3.3; P = 0.019).
The changes in insulin resistance index (HOMA), insulin, glucose, and GLP-1 concentrations during the 1-year period in the 15 obese children with substantial weight loss and the 27 obese children without substantial weight loss are shown in Table 2. Substantial weight loss led to a significant decrease in percentage body fat, insulin concentration, insulin resistance index (HOMA), and GLP-1 levels. We found no significant changes in the obese children without change of weight status. Five (33%) children with substantial weight loss and 9 (33%) children without substantial weight loss entered into puberty. At baseline, there were no significant differences in sex (P = 0.750), percentage body fat (P = 0.443), and SDS-BMI (P = 0.944) between the obese children with and without substantial weight loss, whereas the obese children with substantial weight loss were significantly (P = 0.001) younger and more frequently prepubertal (P = 0.027). Glucose (P = 0.246), insulin (P = 0.216), HOMA (P = 0.143), and GLP-1 concentrations (P = 0.589) did not significantly differ between the children with and without substantial weight loss at baseline.
In the 1-year follow-up, changes in GLP-1 concentrations were significantly correlated to changes in insulin (r = 0.46, P = 0.001) (Fig. 1) and HOMA (r = 0.28; P = 0.036), whereas there were no significant correlations between glucose and GLP-1 (r = 0.14; P = 0.180) by use of partial correlations adjusted to changes in SDS-BMI. Furthermore, changes in GLP-1 were not significantly correlated to changes in percentage body fat (r = −0.13; P = 0.277) or SDS-BMI (r = −0.01; P = 0.476).
At baseline, GLP-1 levels in the 29 girls (median 2.9 interquartile range [IQR] 1.5–5.8 pmol/L) did not significantly (P = 0.681) differ from the GLP-1 concentrations in the 29 boys (median 3.0 IQR 1.3–7.5 pmol/L). Boys and girls did not differ with respect to age (P = 0.626), pubertal stage (P = 0.174), or SDS-BMI (P = 0.648).
The GLP-1 levels in the 33 prepubertal children (median 3.4 IQR 1.4–6.7 pmol/L) did not significantly (P = 0.345) differ from the GLP-1 concentrations in the 25 pubertal children (median 2.2 IQR 1.2–7.4 pmol/L). Prepubertal and pubertal children did not differ with respect to sex (P = 0.189) or SDS-BMI (P = 0.899).
To our knowledge, this is the first study analyzing the cross-sectional and longitudinal relationships between fasting GLP-1, insulin, insulin resistance index, and weight status in childhood. In both cross-sectional and longitudinal analyses GLP-1 was significantly correlated to insulin in the fasting state. Furthermore, the longitudinal relationship between GLP-1 and insulin was stronger than that between GLP-1 and the insulin resistance index (HOMA), and GLP-1 was not related to glucose as a parameter of the insulin resistance index. Therefore, we hypothesize a direct relationship between GLP-1 and insulin concentrations in the fasting state.
The mechanism of this relationship between fasting insulin and GLP-1 seems to be different from that in the postprandial state. GLP-1 stimulated postprandial insulin secretion in a glucose-dependent manner (7,8), whereas fasting GLP-1 was not related to glucose. Furthermore, postprandial GLP-1 concentrations were reported to be impaired in insulin resistance (4), whereas insulin resistance index was not negatively associated with GLP-1 in our fasting study. Probably other factors like leptin, which stimulates GLP-1 secretion from intestinal L cells (25), are involved in the regulation of fasting GLP-1.
However, our findings do not support a direct relationship between GLP-1 and weight status. GLP-1 concentrations did not differ between obese and lean children, in accord with other studies (12,13). Conversely, the relationship between GLP-1 and weight status seems to be more complicated than was anticipated. In our study weight loss was associated with a decrease of GLP-1 concentrations, in accord with a study based on a low–energy intake diet (15). Inasmuch as GLP-1 is a satiety signal, the decrease of GLP-1 concentrations in weight loss could probably, at least in part, explain some of the difficulties in long-term maintenance of weight loss based on dieting.
The decrease of GLP-1 in obese children losing weight substantially may be explained in part by decreasing leptin concentration in weight loss (26). Leptin stimulates GLP-1 secretion from rodents and human intestinal L cells (25). Furthermore, the decrease of GLP-1 in obese children losing weight substantially was probably a consequence of the intervention program. GLP-1 concentrations are reported to be influenced by the manner of dieting and by physical exercise (1,2,15,27). Because exercise therapy and dieting were performed together in our intervention program, we cannot distinguish the possible effects of these 2 factors on GLP-1 concentrations.
The conflicting findings with increasing GLP-1 levels in obese humans losing weight in other studies (28–30) can probably be explained by the small sample sizes and by the effects of gastric surgery in these studies. It is difficult to distinguish between the effects of surgery and weight reduction on the levels of gastrointestinal hormones because the change of GLP-1 release after surgery on the gastrointestinal tract may also be in part due to the absence of gastric and duodenal food stimuli, vagal injury, or lower blood glucose, which mainly regulate GLP-1 secretion (1).
This study has a few potential limitations. First, BMI percentiles and skinfold measurements were used to classify overweight. Although these are good measurements of overweight, one needs to be aware of their limitations as an indirect measure of fat mass. Second, the children with substantial weight loss were younger and more frequently prepubertal than were the obese children without change of weight status. Inasmuch as GLP-1 concentrations were independent of age, sex, and pubertal stage, it seems unlikely that these factors could have influenced our findings. Third, the HOMA model is only an assessment of insulin resistance, and clamp studies are the gold standard for the analysis of insulin resistance. Given that the HOMA model correlated to clamp studies, it is a good method to study insulin resistance in field studies (31). Finally, our study design did not permit us to distinguish between causes and consequences. Therefore, it remains unclear whether fasting GLP-1 stimulates insulin secretion or vice versa.
In summary, fasting GLP-1 levels were independent of weight status, age, sex, and pubertal stage, whereas weight loss was associated with a decrease of GLP-1 concentrations in childhood. Inasmuch as fasting GLP-1 concentrations were related to fasting insulin levels both in cross-sectional and in longitudinal analyses, we hypothesize a relationship between GLP-1 and insulin in the fasting state. Further prospective research is required to examine the relationship between GLP-1 and insulin concentrations in humans.
The authors thank Ms R. Maslak, Children's Hospital, University of Bonn; Ms P. Niklowitz, Children's Hospital, Datteln, University of Witten/Herdecke; and Ms M. Schmidt, Department of Clinical Biochemistry, University of Bonn, for assistance in the laboratory, and Dr R. Reinehr, University of Düsseldorf, for assistance with data analysis.
1. Bojanowska E. Physiology and pathophysiology of glucagon-like peptide-1
(GLP-1): the role of GLP-1 in the pathogenesis of diabetes mellitus, obesity
, and stress. Med Sci Monit 2005; 11:RA271–RA278.
2. Gutzwiller JP, Degen L, Heuss L, et al
. Glucagon-like peptide 1 (GLP-1) and eating. Physiol Behav 2004; 82:17–19.
3. Cancelas J, Sancho V, Villanueva-Penacarrillo ML, et al
. Glucagon-like peptide-1
content of intestinal tract in adult rats injected with streptozotocin either during neonatal period or 7 d before sacrifice. Endocrine 2002; 19:279–286.
4. Rask E, Olsson T, Soderberg S, et al
. Impaired incretin response after a mixed meal is associated with insulin
resistance in nondiabetic men. Diabetes Care 2001; 24:1640–1645.
5. Scrocchi LA, Brown TJ, MaClusky N, et al
. Glucose intolerance but normal satiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene. Nat Med 1996; 2:1254–1258.
6. Ehlers MR, Klaff LJ, D'Alessio DA, et al
. Recombinant glucagon-like peptide-1
(7-36) amide lowers fasting serum glucose in a broad spectrum of patients with type 2 diabetes. Horm Metab Res 2003; 35:611–616.
7. Egan JM, Bulotta A, Hui H, et al
. GLP-1 receptor agonists are growth and differentiation factors for pancreatic islet b-cells. Diabetes Metab Res Rev 2003; 19:115–123.
8. Meier JJ, Gallwitz B, Schmidt WE, et al
. Glucagon-like peptide 1 as a regulator of food intake and body weight: therapeutic perspectives. Eur J Pharmacol 2002; 440:269–279.
9. Verdich C, Flint A, Gutzwiller JP, et al
. A meta-analysis of the effect of glucagons-like-peptide 1 (7-36) amide on ad libitum energy fat intake in humans. J Clin Endocrinol Metab 2001; 86:4382–4389.
10. Lugari R, Dei Cas A, Ugolotti D, et al
. Glucagon-like peptide 1 (GLP-1) secretion and plasma dipeptidyl peptidase IV (DPP-IV) activity in morbidly obese patients undergoing biliopancreatic diversion. Horm Metab Res 2004; 36:111–115.
11. Verdich C, Toubro S, Buemann B, et al
. The role of postprandial releases of insulin
and incretin hormones in meal-induced satiety: effect of obesity
and weight reduction. Int J Obes Relat Metab Disord 2001; 25:1206–1214.
12. Kim BJ, Carlson OD, Jang HJ, et al
. Peptide YY is secreted after oral glucose administration in a gender-specific manner. J Clin Endocrinol Metab 2005; 90:6665–6671.
13. Feinle C, Chapman IM, Wishart J, et al
. Plasma glucagon-like peptide-1
(GLP-1) responses to duodenal fat and glucose infusions in lean and obese men. Peptides 2002; 23:1491–1495.
14. Lugari R, Dei Cas A, Ugolotti D, et al
. Glucagon-like peptide 1 (GLP-1) secretion and plasma dipeptidyl peptidase IV (DPP-IV) activity in morbidly obese patients undergoing biliopancreatic diversion. Horm Metab Res 2004; 36:111–115.
15. Adam TC, Jocken J, Westerterp-Plantenga MS. Decreased glucagon-like peptide 1 release after weight loss
in overweight/obese subjects. Obes Res 2005; 13:710–716.
16. Rubino F, Gagner M, Gentileschi P, et al
. The early effect of the Roux-en-Y gastric bypass on hormones involved in body weight regulation and glucose metabolism. Ann Surg 2004; 240:236–242.
17. Cole TJ, Bellizzi MC, Flegal KM, et al
. Establishing a standard definition for child overweight and obesity
worldwide: international survey. BMJ 2000; 320:1240–1243.
18. Cole TJ. The LMS method for constructing normalized growth standards. Eur J Clin Nutr 1990; 44:45–60.
19. Kromeyer-Hauschild K, Wabitsch M, Geller F, et al
. Percentiles of body mass index in children
and adolescents evaluated from different regional German studies. Monatsschr Kinderheilkd 2001; 149:807–808.
20. Slaughter MH, Lohman TG, Boileau RA, et al
. Skinfold equations for estimation of body fatness in children
and youth. Hum Biol 1988; 60:709–723.
21. Matthews DR, Hosker JP, Rudenski AS, et al
. Homeostasis model assessment: insulin
resistance and beta-cell function from fasting plasma glucose and insulin
concentrations in man. Diabetologia 1985; 28:412–419.
22. Reinehr T, Kersting M, Alexy U, et al
. Long-term follow-up of overweight children
: after training, after a single consultation session and without treatment. J Pediatr Gastroenterol Nutr 2003; 37:72–74.
23. Reinehr T, Kiess W, Kapellen T, et al
sensitivity in obese children
and adolescents according to degree of weight loss
. Pediatrics 2004; 114:1569–1573.
24. Reinehr T, de Sousa G, Andler W. Longitudinal analyses between overweight, insulin
resistance, and cardiovascular risk factors in children
. Obes Res 2005; 13:1824–1833.
25. Anini Y, Brubaker PL. Role of leptin in the regulation of glucagon-like peptide-1
secretion. Diabetes 2003; 52:252–259.
26. Reinehr T, Kratzsch J, Kiess W, et al
. Circulating soluble leptin receptor, leptin, and insulin
resistance before and after weight loss
in obese children
. Int J Obes 2005; 29:1230–1235.
27. Adam TC, Westerterp-Plantenga MS. Activity-induced GLP-1 release in lean and obese subjects. Physiol Behav 2004; 83:459–466.
28. Valverde I, Puente J, Martin-Duce A, et al
. Changes in glucagon-like peptide-1
(GLP-1) secretion after biliopancreatic diversion or vertical banded gastroplasty in obese subjects. Obes Surg 2005; 15:387–397.
29. Clements RH, Gonzalez QH, Long CI, et al
. Hormonal changes after Roux-en Y gastric bypass for morbid obesity
and the control of type-II diabetes mellitus. Am Surg 2004; 70:1–4.
30. Naslund E, Gryback P, Hellstrom PM, et al
. Gastrointestinal hormones and gastric emptying 20 years after jejunoileal bypass for massive obesity
. Int J Obes Relat Metab Disord 1997; 21:387–392.
31. Wallace TM, Matthews DR. The assessment of insulin
resistance in man. Diabet Med 2002; 19:527–534.