Intact Glucagon-like Peptide-1 Levels are not Decreased in Japanese Patients with Type 2 Diabetes

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1 Or i g i n a l Advance Publication Intact Glucagon-like Peptide-1 Levels are not Decreased in Japanese Patients with Type 2 Diabetes Soushou Lee*, Daisuke Yabe**, Kyoko Nohtomi*, Michiya Takada*, Ryou Morita*, Yutaka Seino** and Tsutomu Hirano* *Department of Diabetes, Metabolism, and Endocrinology, Showa University, School of Medicine, Tokyo, Japan **Department of Medicine, Kansai Electric Power Hospital, Osaka, Japan Received September 14, 2009; Accepted October 20, 2009; Released online October 31, 2009 Correspondence to: Tsutomu Hirano, M.D., Ph.D., Department of Diabetes, Metabolism, and Endocrinology, Showa University School of Medicine, Hatanodai, Shinagawa-ku, Tokyo , Japan. Abstract. Impaired secretion of glucagon-like peptide 1 (GLP-1) has been suggested to contribute to the deficient incretin effect in patients with type 2 diabetes mellitus (T2DM). Recent studies, however, have not always supported this notion. Since Japanese patients with T2DM usually have severe impairment in the earlyphase of insulin secretion, the measurement of incretin secretions in Japanese T2DM patients would be useful for assessing the association between incretin levels and insulin secretion. We conducted an oral glucose tolerance test (75 g) (OGTT) and meal tolerance test (480 kcal) (MTT) for subjects with normal glucose tolerance (NGT, n=12), subjects with impaired glucose tolerance (IGT, n=7), and T2DM patients (n=21). The tests were carried out over 120-min study periods on separate occasions. Intact GLP-1, GIP, and dipeptidyl peptidase (DPP)-IV were measured by ELISA. T2DM exhibited an impaired early phase of insulin secretion and a reduction in glucagon suppression. There were no significant differences in GLP-1 or GIP levels at each sampling time among NGT, IGT, and T2DM after the ingestions; hence the incremental areas under the curve (IAUC) for the three groups were quite similar. The levels of DPP-IV, a limiting enzyme for the degradation of incretins, were comparable among the three groups. The GLP-1-IAUC was not correlated with IAUCs of insulin, C-peptide, or glucagon determined by the OGTT or the MTT. We concluded that intact GLP-1 levels are comparable between non-diabetics and T2DM, suggesting that impaired insulin secretion in Japanese T2DM is not attributable to defect in GLP-1 secretion. Key words: Incretins, GLP-1, GIP, Japanese, Type 2 diabetes Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), two major incretins secreted from the small intestine, stimulate the production of insulin and suppress the secretion of glucagon after meals. The impairment of these incretin effects is a well known feature in patients with type 2 diabetes mellitus (T2DM) [1-4]. Given that the insulinotropic action of GIP is severely suppressed in patients with T2DM [5,6], only GLP-1 is thought to exert an incretin effect in diabetic populations [7]. Earlier studies have demonstrated an impairment of GLP-1 secretion in T2DM patients after the ingestion of a meal or glucose [8-12]. This suggests that the lack of incretin effects in these patients is at least partly attributable to a GLP-1 deficiency. Yet recent measurements of intact GLP-1 (6-36amide and 6-37) have revealed no decreases in the intact GLP-1 concentrations in T2DM patients [13, 14]. It thus remains debatable whether GLP-1 deficiency truly exists in T2DM [7]. Japanese T2DM patients are rarely obese, and their insulin response to glucose is substantially lower than that of Western diabetics [15-17]. As such, the major pathogenesis for Japanese T2DM may be insulin deficiency rather than insulin resistance. Before the present study, however, there 1

2 had been no attempts to compare incretin levels between Japanese T2DM patients and non-diabetic subjects. Until this data is complete, we will not be able to elucidate whether the defect in incretin secretion actually plays a role in the impaired insulin secretion in Japanese T2DM. In the present study we investigated how incretin levels are associated with the different stages of glucose intolerance by measuring intact GLP-1 and GIP levels after the administration of glucose and mixed meal in subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and T2DM. We also measured dipeptidyl peptidase (DPP)-IV, a rate limiting enzyme for the degradation of incretins, and examined its association with incretin levels. Subjects and Methods Forty subjects participated in the study. Among them, 21 were newly diagnosed with T2DM (World Health Organization criteria) with relatively mild hyperglycemia (HbA1c levels of less than 7.5%), 7 had impaired glucose tolerance (IGT), and 12 had normal glucose tolerance (NGT). IGT was diagnosed during the oral glucose tolerance tests. None of the T2DM patients included in the study were currently receiving any glucose-lowering medications. The profiles of the subjects are shown in Table 1. The Committee on Human Investigation of Showa University approved the study. All participants were informed of the nature of the study and provided written informed consent. Oral glucose tolerance test (OGTT): 75 g glucose was ingested after an overnight fast. Blood samples were drawn in the fasting state (baseline) and at 10, 20, 30, 60, 90, and 120 min after the glucose ingestion. Two to 4 weeks after the OGTT, a meal tolerance test (MTT) was performed in the same individuals. The total caloric content of the test meal was 480 kcal (carbohydrates: protein: fat=2.8:1:1). Blood samples were drawn in the baseline and at 10, 20, 30, 60, 90, and 120 min after meal ingestion. The insulinogenic index [15] was calculated as the ratio of the increment in the plasma insulin level to that of the plasma glucose level during the first 30 min after ingestion of glucose or meal. Beta-cell function and insulin resistance were assessed based on the fasting insulin and plasma glucose values using homeostasis model assessment HOMA-β and HOMA-R models, respectively [18]. Incremental changes were assessed for the postchallenge levels of plasma insulin, C-peptide, GLP-1, and GIP, and for the calculated area under the curve (IAUC) for the same plasma hormone concentrations versus time by the trapezoidal rule. The blood used for the GIP and GLP-1 determinations was collected in EDTA-coated tubes (1.5 µg/ml blood) containing aprotinin (40 µl/ml blood; Trasylol, Serological Proteins, Kankakee, IL) and an inhibitor of DPP-IV (10 µl/ml blood; Linco Research Inc., MO, USA). Plasma samples for insulin, C-peptide, and glucagon were assayed by enzyme-linked immunosorbent assay (ELISA) (Morinaga, Yokohama, Japan ). ELISA was used for plasma intact GLP-1 determinations (GLP-1 Active ELISA kit, Cat#EGLP-35k, Linco Research Inc.). This kit can measure intact GLP-1 without cross-reacting with the inactive form of GLP-1 (9-36). The lowest level that can be detected by the original assay was 2 pm, however, we modified applied sample volume and the standard curve which enabled to measure down to 0.2 pm. GIP was measured by a sandwich enzyme immunoassay using anti-human GIP Rabbit IgG (IBL, Takasaki, Japan). The use of this kit posed some uncertainly, as it remained unknown whether the measurements yielded the total or intact GIP. We could confirm, however, that the values measured by this kit corresponded well with the range of intact GIP levels measured elsewhere [14, 19, 20]. The lowest limit of this assay was 0.6 pm. The intra-assay variation coefficients for intact GLP-1, GIP, insulin, C-peptide, and glucagon were 5, 4.8, 3.6, 3.6, and 4.8%, respectively, and the interassay variation coefficients were 14, 6.8, 2.5, 3.3, and 12%, respectively. DPP-IV was measured by ELISA for CD26 mass [R&D Systems Inc, MN, USA, Cat # DC260]. After finding no significant change in DPP-IV during tolerance tests in a preliminary study, we decided to proceed by measuring only baseline samples. Low-density-lipoprotein(LDL)-cholesterol and high-density-lipoprotein (HDL)-cholesterol were measured by the direct homogenous methods. 2

3 Standard methods were used to compute means and SD. ANOVA and Bonferroni s multiple comparison post hoc test were used to compare the mean values of the three groups. A p value of less than 0.05 was assumed to indicate statistical significance. Results The T2DM patients enrolled in this study were somewhat older and heavier than the NGT patients. However, none were massively obese (BMI>30). Compared with the NGT group, the T2DM groups had significantly higher levels of fasting glucose, HbA1c, HOMA-R, and triglycerides, a lower insulinogenic index, and lower levels of HOMA-β and HDL-cholesterol (Table 1). The values for these parameters in the IGT group were between those in the NGT and T2DM groups (Table 1). Plasma LDL- cholesterol and DPP-IV levels were all comparable among the three groups (Table 1). As expected, glucose concentrations were higher in the T2DM group than in the NGT and IGT groups both after the oral glucose load and after the mixed meal ingestion (Fig. 1, Table 2). The T2DM patients had a significantly reduced insulin secretion from 0 to 30 min after the oral glucose or meal load, compared with the NGT group (Fig. 1). The IGT group had insignificantly reduced insulin secretion from 0 to 30 min after the oral glucose or meal load. Plasma insulin levels tended to be elevated in the T2DM and IGT groups from 60 to 120 min after the glucose or meal load. The insulin-iauc in the OGTT was significantly lower in the T2DM group than in the NGT group, but the insulin-iauc in the MTT was comparable among three groups (Table 2). As observed with the insulin response, the responses of the C-peptide levels to the oral glucose and meal challenges remained lower in the T2DM group than in the NGT group from 0 to 30 min after the loads (Fig. 2). The C-peptide levels from 0 to 30 min after the oral glucose or meal load were lower in the IGT group than in the NGT group, but the difference was not significant (Fig. 2). The C-peptide -IAUCs determined by the OGTT and MTT were significantly lower in the T2DM group than in the NGT group (Table 2). The C-peptide -IAUCs were comparable in the IGT and NGT groups (Table 2). For the entire duration of the OGTT or MTT, the plasma glucagon levels at each sampling point were significantly higher in the T2DM group than in the NGT group (Fig. 2). The levels of glucagon, a hormone suppressed by the subsequent glucose or meal load, were similar in the IGT and NGT groups (Fig. 2). The integrated glucagon levels after glucose or meal ingestion tended to be higher in the T2DM group than in the NGT group (Table 2). Fig. 3 (the upper panel) depicts changes of intact GLP-1 levels in the OGTT and MTT. The baseline levels of GLP-1 were around 1 pmol/l, and peaked at 20 to 30 min after the oral glucose or meal loading in all three groups. The peak levels of GLP-1 were 7-9 pmol/l GLP-1 in the OGTT and 3-5 pmol/l in the MTT. These levels gradually decreased with time, but the levels at 120 min postchallenge were still higher than baseline in all groups. There were no significant differences in the peak height among the NGT, IGT, and T2DM groups in either of the challenge tests, although GLP-1 was lower in the T2DM group than in the NGT group at 120 min in the OGTT. The GLP-1-IAUCs were comparable among the three groups (Table 2). The GLP-1- IAUCs measured by the OGTT were significantly (p<0.01) greater than those measured by the MTT in all three groups (Table 2). Fig. 3 (the lower panel) depicts changes of GIP levels in the OGTT and MTT. The baseline levels of GIP were around 17 pmol/l and peaked at 10 min in the OGTT. The GIP levels from 10 to 30 min after glucose loading were higher in the T2DM group than in the NGT or IGT group, but the difference was not statistically significant. In the OGTT, the peak levels of GIP were maintained up to 120 min in all three groups. The GIP level peaked at 20 min in the MTT, or about 10 min sooner than it had peaked in the OGTT. The GIP levels from 0 to 30 min were higher in the IGT group than in the NGT or T2DM group, but the difference was not statistically significant. The peak levels of GIP were pmol/l in the OGTT and pmol/l in the MTT. There were no significant differences in the peak height among the NGT, IGT, and T2DM groups in either challenge test. The peak levels of GIP gradually decreased in the IGT group, whereas the peak 3

4 values were maintained up to 120 min postchallenge in the NGT and T2DM groups. There were no significant differences in the integrated GIP levels determined by the OGTT and MTT among the three groups (Table 2). Unlike GLP-1, the GLP-1-IAUCs measured by the OGTT were comparable to those measured by the MTT in every group. The GLP-1-IAUCs determined by the OGTT and MTT were not correlated with age, BMI, HbA1c, FPG., HOMA-β, or HOMA-R. Similarly, the GIP-IACUs were also uncorrelated with these parameters. The GLP-1-IAUC was not correlated with any of the IAUCs of glucose, insulin, C-peptide, glucagon, or GLP-1 determined by the OGTT or the MTT. The baselines of GLP-1 or GIP did not predict their increments after glucose or meal loads. The GIP-IACUs were not correlated with any of the IAUCs of glucose, insulin C-peptide, glucagon or GLP-1 determined by the OGTT or MTT. The DPP-IV levels were not associated with the GLP-1-IAUC or GIP-IACU determined by the OGTT or MTT (data not shown). Discussion Defects in GLP-1 secretion have been reported in patients with type 2 diabetes after meal ingestion or glucose [7-12]. Vollmer et al. [14], however, recently found that GLP-1 levels were similar in NGT, IGT, and T2DM groups in both the OGTT and MTT. To explain their finding, they proposed that impaired insulin secretion can develop in the absence of impaired GLP-1 secretion. Similarly, the levels of GLP-1 secretion stimulated by glucose or mixed meal loading in our present study were not lower in the T2DM patients than in the subjects with NGT or IGT. The T2DM patients enrolled in the present study clearly had typical characteristics of T2DM in the Japanese setting; i.e., substantially low insulin responses after the ingestion of glucose or meal without massive obesity or severe insulin resistance. To avoid the influences of diabetic complications affecting the incretins and drugs affecting glucose and insulin levels, we selected mildly diabetic patients who were drug-naïve, free of diabetic complications, and had not poor glycemic control (HbA1c levels less than 7.5%). Therefore, the present results might not apply to severely diabetic patients who receive treatment with hypoglycemic agents or who suffer from serious diabetic complications. On the other hand, there are no apparent data showing that GLP-1 levels are affected by HbA1c or the duration of diabetes. Several studies have shown negative associations between GLP-1 levels and BMI [3, 14]. We found no such association, but our study population did not include any massively obese subjects. We measured the intact GLP-1 with an ELISA kit designed not to cross-react with degraded GLP-1. Thus, the total GLP-1 levels in Japanese T2DM are unknown. Nevertheless, our data do not support the notion that a defect in intact GLP-1 secretion is a pathogenesis for insulinopenia or hyperglucagonemia in Japanese patients with T2DM. The insulinotropic action of GIP is abolished in patients with diabetes [5, 6]. GIP thus differs from GLP-1 in an important respect: a deficiency of the former cannot become a pathogenesis for impaired insulin secretion in diabetes. Yet the actions of these two incretins in regulating each other is of some interest. Several studies comparing T2DM patients with controls have reported that GIP levels after mixed meals were not lower, but somewhat higher, in T2DM [20]. Vollmer et al. [14] reported that the initial GIP response was exaggerated in patients with type 2 diabetes compared with control subjects after mixed meal. They also found that GIP responses were significantly higher after mixed meal ingestion than after oral glucose loading, whereas GLP-1 levels were similar in both challenges. The early responses of GIP levels in the GTT in our study were somewhat higher in the T2DM group than in the NGT or IGT group, but no such difference was observed in the MTT. We also observed that the GLP-1 responses were stronger after the glucose load than after the meal, whereas the GIP responses were comparable between glucose load and meal load. Our test meal contained 480 Kcal, whereas Vollmeret et al. [14] challenged their patients with a load of 820 Kcal. This may help explain the discrepancies in the incretin responses to glucose and mixed meal loads. If DPP-IV activity, a rate-limiting degradation enzyme for incretins, were changed, we would 4

5 not be able to accurately evaluate the secretion of incretin from the intestine. Ryskjaer et al. [13] observed higher DPP-IV activity in T2DM patients, but they found no significant relations between this activity and incretin concentrations. While our measurements of DPP-IV mass could not be readily applied for DPP-IV activity, we did at least confirm an overall similarity in DPP-IV mass between control subjects and T2DM patients. Taken together, these data suggest that increased DPP-IV is unlikely to overestimate the incretin levels in T2DM. Having found no significant associations between GLP-1 and GIP responses to glucose and meal, we postulate that GIP and GLP-1 concentrations may be regulated independently from each other via different factors. The baseline incretin concentrations were not associated with these IAUCs in the OGTT and MTT. This prevented us from estimating the postprandial incretin secretion by the fasting values, as the assessment would require a standard tolerance test for the evaluation of incretin secretion. OGTT is common and widely practiced in the clinical setting, and hence can be applied as a standard methodology for evaluating incretin secretion. We postulate, however, that the meal test has a potential advantage for evaluating incretin secretion. Given that mixed meal contains fat, protein, and carbohydrates, all of which stimulate incretin production [19], it can be expected to elicit an overall incretin secretion comparable to that elicited by general food. The present data did not show a close association between the OGTT and MTT for postprandial incretin concentration. This suggests that different nutrients may modulate incretin secretion in different ways. This study had two important limitations: a small number of subjects and a lack of data on total GLP-1 values. To confirm whether T2DM patients actually secrete GLP-1 at levels similar to nondiabetic subjects, it will be necessary to study a larger number of subjects with different assays for GLP-1. Acknowledgements This study was partly supported by Banyu Pharmaceutical Company. No potential conflicts of interest relevant to this article were reported. References 1. Nauck M, Stöckmann F, Ebert R, Creutzfeldt W: Reduced incretin effect in type 2 (non-insulin-dependent) diabetes. Diabetologia1986; 29: Vilsbøll T, Holst JJ: Incretins, insulin secretion and type 2 diabetes mellitus. Diabetologia 2004;47: Muscelli E, Mari A, Casolaro A, Camastra S, Seghieri G, Gastaldelli A,et al. Separate impact of obesity and glucose tolerance on the incretin effect in normal subjects and type 2 diabetic patients. Diabetes. 2008;57: Knop FK, Vilsboll T, Hojberg PV, Larsen S, Madsbad S, Volund A, et al. Reduced incretin effect in type 2 diabetes: cause or consequence of the diabetic state? Diabetes 2007; 56: Nauck MA, Heimesaat MM, Orskov C, Holst JJ, Ebert R, Creutzfeldt W.Preserved incretin activity of glucagon-like peptide 1[7-36 amide] but not of synthetic human gastric inhibitory polypeptide in patients with type-2 diabetes mellitus. J Clin Invest 1993; 91: Elahi D, McAloon-Dyke M, Fukagawa NK, Meneilly GS, Sclater AL, Minaker KL, et al.the insulinotropic actions of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (7-37) in normal and diabetic subjects. Regul Pept 1994;51: Holst JJ.The physiology of glucagon-like peptide 1. Physiol Rev 2007;87: Vilsbøll T, Krarup T, Deacon CF, Madsbad S, Holst JJ. Reduced postprandial concentrations of intact biologically active glucagon-like peptide 1 in type 2 diabetic patients. Diabetes 2001;50: Toft-Nielsen MB, Damholt MB, Madsbad S, Hilsted LM, Hughes TE, Michelsen BK, et al. Determinants of the impaired secretion of glucagon-like peptide-1 in type 2 diabetic patients. J Clin 5

6 Endocrinol Metab 2001;86: Vaag AA, Holst JJ, Volund A, Beck-Nielsen HB. Gut incretin hormones in identical twins discordant for non-insulin-dependent diabetes mellitus (NIDDM) evidence for decreased glucagon-like peptide 1 secretion during oral glucose ingestion in NIDDM twins. Eur J Endocrinol 1996;135: Vilsboll T, Knop FK, Krarup T, Johansen A, Madsbad S, Larsen S, et al. The pathophysiology of diabetes involves a defective amplification of the late-phase insulin response to glucose by glucose-dependent insulinotropic polypeptide-regardless of etiology and phenotype. J Clin Endocrinol Metab 2003;88: , Vilsbøll T, Krarup T, Sonne J, Madsbad S, Vølund A, Juul AG, et al. Incretin secretion in relation to meal size and body weight in healthy subjects and people with type 1 and type 2 diabetes mellitus. J Clin Endocrinol Metab 2003 ;88: Ryskjaer J, Deacon CF, Carr RD, Krarup T, Madsbad S, Holst J, et al. Plasma dipeptidyl peptidase- IV activity in patients with type-2 diabetes mellitus correlates positively with HbAlc levels, but is not acutely affected by food intake. Eur J Endocrinol 2006;155: Vollmer K, Holst JJ, Baller B, Ellrichmann M, Nauck MA, Schmidt WE, et al. Predictors of incretin concentrations in subjects with normal, impaired, and diabetic glucose tolerance. Diabetes 2008;57: Kosaka K, Kuzuya T, Hagura R.Insulin secretory response in Japanese type 2 (non-insulin-dependent) diabetic patients. Diabetes Res Clin Pract 1994;24:S Fukushima M, Suzuki H, Seino Y. Insulin secretion capacity in the development from normal glucose tolerance to type 2 diabetes. Diabetes Res Clin Pract 2004;66 Suppl 1:S Kuroe A, Fukushima M, Usami M, Ikeda M, Nakai Y, Taniguchi A, et al. Impaired beta-cell function and insulin sensitivity in Japanese subjects with normal glucose tolerance. Diabetes Res Clin Pract 2003;59: 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: Kim W, Egan JM. The role of incretins in glucose homeostasis and diabetes treatment. Pharmacol Rev 2008;60:

7 Fig. 1. Plasma glucose and insulin levels in the 75g-oral glucose tolerance test (OGTT) and meal tolerance test (MTT). NGT=normal glucose tolerance ( ), IGT=impaired glucose tolerance ( ), and T2DM=type 2 diabetes ( ). Data represent the mean plus minus SD. a=vs. NGT; b=vs. IGT at p<0.05. Fig. 2. Plasma C-peptide and glucagon levels in the OGTT and MTT. 7

8 Fig. 3. Plasma GLP-1 and GIP levels in the OGTT and MTT. Table 1. Characteristics of subjects with normal glucose tolerance (NGT), with impaired glucose tolerance (IGT), and patients with type 2 diabetes (T2DM) NGT (12) IGT -7 T2DM (21) Age (y) 54plus minus13 58plus minus10 61plus minus9 a BMI 20plus minus2 22plus minus2 a 23plus minus3 a HbA1c (%) 5.0plus minus plus minus0.5 a 6.4plus minus0.5 ab Fasting Glucose (mg/dl) 90plus minus11 102plus minus14 140plus minus27 ab Triglyceride (mg/dl) 85plus minus40 86plus minus38 133plus minus75 a LDL-C (mg/dl) 127plus minus41 120plus minus28 123plus minus17 HDL-C (mg/dl) 80plus minus22 74plus minus10 62plus minus14 a Insulinogenic index a 0.17 ab HOMA-β 82plus minus41 70plus minus64 48plus minus36 a HOMA-R 1.6plus minus plus minus plus minus3.8 a DPP-IV (ng/ml) 247plus minus plus minus66 247plus minus115 Data represent the mean plus minus SD. a vs. NGT; b vs. IGT at p<0.05 8

9 Table 2. Incremental area under the curves (IAUC) of parameters in OGTT and MTT OGTT NGT IGT T2DM Glucose (mg. min/dl) 492.8plus minus plus minus131.9 a plus minus429.0 ab Insulin (μu. min/ml) 411.2plus minus plus minus plus minus153.8 b C-peptide (ng. min/ml) 52.9plus minus plus minus plus minus13.1 ab Glucagon (pg. min/ml) plus minus plus minus plus minus112.8 GLP-1 (pmol. min/l) 61.8plus minus plus minus plus minus25.1 GIP (pmol. min/l) 250.5plus minus plus minus plus minus240.2 MTT NGT IGT T2DM Glucose (mg. min/dl) 292.0plus minus plus minus182.8 a 928.2plus minus324.2 ab Insulin (μu. min/ml) 280.0plus minus plus minus plus minus109.1 C-peptide (ng. min/ml) 36.8plus minus plus minus plus minus9.4 a Glucagon (pg. min/ml) 13.8plus minus plus minus plus minus148.6 a GLP-1 (pmol. min/l) 13.6plus minus plus minus plus minus21.4 GIP (pmol. min/l) 330.7plus minus plus minus plus minus253.7 Data represent the mean plus minus SD. a vs. NGT; b vs. IGT at p<0.05 9