No correlation between insulin and islet amyloid polypeptide after stimulation with glucagon-like peptide-1 in type 2 diabetes

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1 European Journal of Endocrinology (1997) ISSN No correlation between insulin and islet amyloid polypeptide after stimulation with glucagon-like peptide-1 in type 2 diabetes Bo Ahrén and Mark Gutniak 1 Department of Medicine, Lund University, Malmö and 1 Vällingby Medical Center, Karolinska Institute, Stockholm, Sweden (Correspondence should be addressed to B Ahrén, Department of Medicine, Malmö University Hospital, S Malmö, Sweden) Abstract Objectives: To examine whether glucagon-like peptide-1 (GLP-1), which has been suggested as a new therapeutic agent in type 2 diabetes, affects circulating islet amyloid polypeptide (IAPP), a B-cell peptide of potential importance for diabetes pathophysiology. Design: GLP-1 was administered in a buccal tablet (400 mg) to seven healthy subjects and nine subjects with type 2 diabetes. Serum IAPP and insulin levels were measured before and after GLP-1 administration. Results: In the fasting state, serum IAPP was pmol/l in the controls vs pmol/l in the subjects with type 2 diabetes (P < 0.001). IAPP correlated with insulin only in controls (r=0.74, P ¼ 0.002) but not in type 2 diabetes (r ¼ 0.26, NS). At 15 min after GLP-1, circulating IAPP increased to pmol/l in controls (P ¼ 0.009) and to pmol/l in type 2 diabetes (P ¼ 0.021). In both groups, serum insulin increased and blood glucose decreased compared with placebo. In controls serum IAPP increased in parallel with insulin (r ¼ 0.79, P ¼ 0.032), whereas in type 2 diabetes the increase in IAPP did not correlate with the increase in insulin. Conclusion: Type 2 diabetes is associated with elevated circulating IAPP; GLP-1 stimulates IAPP secretion both in healthy human subjects and in type 2 diabetes; IAPP secretion correlates with insulin secretion only in healthy subjects and not in type 2 diabetes. European Journal of Endocrinology Introduction Islet amyloid polypeptide (IAPP) is a 37 amino acid polypeptide which is synthesized and co-stored with insulin in the islet B cells (1). The peptide is thought to be involved in the pathophysiology or metabolic perturbations of diabetes, since it forms islet amyloid which accompanies type 2 diabetes and inhibits the action and secretion of insulin under experimental conditions (2 4). The peptide might also, however, be antidiabetogenic, since it reduces gastric emptying (5, 6). Thus the relation between IAPP and diabetes is still not established. To gain further insight into the issue of IAPP s involvement in the pathophysiology or metabolic perturbations of diabetes, it is important to establish the regulation of its circulating levels under normal conditions and in subjects with type 2 diabetes. Previous studies have shown that in normal subjects, the peptide is released simultaneously with insulin in response to oral and intravenous glucose, ingestion of a mixed meal and intravenous arginine (7 14). It has also been shown that in non-diabetic obese subjects, circulating IAPP levels are elevated and the IAPP response to glucose is exaggerated compared with non-obese subjects (15, 16). This would suggest that increased secretion of IAPP evolves before the onset of type 2 diabetes, for example in the state of reduced insulin sensitivity which often accompanies obesity (17). Studies in animal models would support this assumption. Thus in insulin resistance induced experimentally in the rat with dexamethasone, overexpression of IAPP in relation to insulin has been documented (18, 19), and in the spontaneous diabetes of the Goto Kakizaki rat a relatively exaggerated secretion of IAPP in comparison with that of insulin has been demonstrated (20). In contrast, it appears that the IAPP response to glucose or glucagon is impaired in subjects with fully developed type 2 diabetes (15, 21). This would suggest that following development of type 2 diabetes, IAPP secretion fails. However, more studies in human diabetes are required on the relative secretion of IAPP and insulin. An important stimulator of insulin secretion after oral ingestion of glucose or a mixed meal is the gut hormone, glucagon-like peptide-1 (7 36) amide or GLP-1 (22 24). This peptide, which is produced in the intestinal glucagon cells through processing from proglucagon (23), is therefore considered a main 1997 Society of the European Journal of Endocrinology

2 644 B Ahrén and M Gutniak EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137 incretin hormone (22 25). GLP-1 is of interest also as a potential future treatment for diabetes, since it exerts antidiabetogenic action both in normal subjects and in patients with type 2 diabetes (26 29). The antidiabetogenic action of the peptide has been thought to be due to stimulation of insulin secretion, inhibition of glucagon secretion and inhibition of gastric emptying (26 29). Whether GLP-1 in addition affects circulating IAPP is not known. In the perfused rat pancreas, GLP-1 has been shown to stimulate IAPP secretion (30), but whether GLP-1 affects IAPP levels in humans is not known. In the present study, we therefore examined whether GLP-1, in parallel with its stimulation of insulin secretion, stimulates also IAPP secretion in healthy humans as well as in humans with type 2 diabetes. We analyzed serum insulin and IAPP levels after application of a 400 mg GLP-1 buccal tablet in seven healthy volunteers and in nine subjects with well-controlled type 2 diabetes. Buccal GLP-1 application has recently been shown to transiently increase plasma levels of GLP-1 to a peak level of pmol/l, which is reached after 30 min followed by a decline with a circulating half-life of 26 min (31). This level of GLP-1 has been considered a potential therapeutic level of the peptide (31, 32). Subjects and methods Subjects Seven healthy volunteers (three females, four males) free from any medication comprised the control group. The subjects were 57 4 years of age (mean S.D.), range years, and their body mass index (BMI) was kg/m 2. All subjects had normal glucose tolerance as judged from a 75 g oral glucose tolerance test according to the WHO criteria. The diabetic group consisted of nine subjects with type 2 diabetes (four females, five males). The diabetic subjects were 61 3 years of age (mean S.D., NS vs controls), range years, and their BMI was kg/m 2 (P ¼ vs controls). The subjects were included in the study on the basis of a 2 h glucose level following a 75 g oral glucose load exceeding 11.1 mmol/l and a C-peptide greater than 0.5 nmol/l at blood glucose greater than 7 mmol/l. All subjects were in good metabolic control, as judged from their fasting levels of blood glucose ( mmol/l) and haemoglobin A 1c ( %). The duration of diabetes was between 1 and 20 years. Six of the subjects were treated with dietary regulations alone, whereas three were also given the sulfonylurea glipizide. Dietary advice was given according to the clinical practice in the hospital, including advice that a third of energy intake should be derived from fat and 50 55% from carbohydrates with a high fiber intake. Except for type 2 diabetes, the patients were healthy, and none was taking any other drugs known to affect carbohydrate metabolism. All studies were undertaken after an overnight fast. The subjects treated with sulfonylurea withheld the medication during 24 h before the study. The study was approved by the Ethics Committee at Lund University, and all subjects gave informed written consent to take part before the study. Study design After an overnight fast, an indwelling catheter was inserted in the brachial vein on one side. Two baseline samples were taken, at ¹5 and ¹2 min. Then a buccal tablet of either GLP-1 or of placebo was applied, and the subject was placed in a rest position. After insertion of the tablet, new samples were taken at various time points. The application of either placebo or GLP-1 was performed randomly with at least 4 days in between. Buccal tablet The tablet was bi-layered and prepared from two separate granulations. The diameter was 9.5 mm. The active layer consisted of 400 mg (119 nmol) human synthetic GLP-1 (Saxons Biochemicals, Hannover, Germany). The placebo tablet consisted of the same tablet devoid of GLP-1. The detailed composition of the tablet has been described previously (31). Assays The samples were immediately centrifuged and serum was stored at ¹20 C until analysis, which was performed less than 3 months after the experiment. Serum insulin concentration was determined by RIA with the use of a guinea pig antihuman insulin antibody, human insulin standard and, as tracer, mono- 125 I- human insulin (Linco Res., St Charles, MO, USA). Separation of free and bound radioactivity was performed by the double antibody technique. Serum IAPP concentrations were analyzed by RIA after reversed-phase extraction. Before the RIA, IAPP was extracted from the serum (3 ml) by Sep-Pak C18 cartridges (Scand. GeneTec, Kungsbacka, Sweden) with acetonitrile (HPLC grade, Labscand Ltd, Dublin, Ireland)/trifluoroacetic acid (TFA, E. Merck, Darmstadt, Germany). The Sep-Pak C18 columns (containing 200 mg C18 cartridges) were first equilibrated by elution with 1 ml 60% acetonitrile in 0.1% TFA, followed by three elutions of 3 ml 0.1% TFA. Thereafter, the columns were loaded with acidified serum (supernatant of 3 ml serum þ 3 ml 0.1% TFA centrifuged at g for 20 min at 4 C) and washed twice with 3 ml 0.1% TFA before elution with 3 ml acetonitrile/tfa (60%/0.1%) without any pressure. The eluates were evaporated to dryness and then reconstituted in 250 ml of the phosphate-based RIA buffer (Peninsula Labs, Belmonte, CA, USA). The recovery of this extraction procedure was 70%. Thereafter, the RIA was performed by the use of rabbit antihuman IAPP, human IAPP as

3 EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137 GLP-1 and IAPP secretion 645 standard and 125 I-labeled human IAPP as tracer (Peninsula Labs). The separation of bound and free radioactivity was performed by the double antibody separation technique. The intra-assay coefficient of variation (CV) in the IAPP assay was <9% and the interassay CV <12% at both low and high values. Finally, the blood glucose concentrations were determined by the glucose oxidase technique. Statistical analyses All data are presented as mean S.E.M. unless otherwise stated. Statistical analysis of the effect of the GLP-1 tablet was performed by the Student s paired or unpaired t-test, and the linear relationships between variables were analyzed by Pearson s product moment correlation. significantly affected by placebo (Fig. 1). The peak IAPP and insulin levels were seen after 15 min, when the IAPP levels had increased to pmol/l or by pmol/l (P ¼ 0.04), whereas the insulin levels had increased to pmol/l or by pmol/l (P ¼ 0.012). IAPP levels after GLP-1 in type 2 diabetes In the diabetic group also, GLP-1 increased circulating IAPP and insulin, whereas circulating glucose was reduced (Fig. 2). No significant alterations in these Results Fasting IAPP and insulin in healthy subjects and in type 2 diabetes In the healthy subjects (n ¼ 7), fasting levels of IAPP were pmol/l in the GLP-1 experiments and pmol/l in the series with placebo (NS). The corresponding levels of serum insulin were and pmol/l (NS). There was a significant correlation between fasting insulin and fasting IAPP levels (r ¼ 0.74, P ¼ 0.002). The circulating insulin/iapp ratio ((pmol insulin/l)/(pmol IAPP/l)) in the fasting condition was in the GLP-1 experiments and in the placebo experiments (NS). In the group with type 2 diabetes (n ¼ 9), fasting IAPP was pmol/l in the experiments with GLP-1 and pmol/l in the placebo experiments (NS). Thus, the subjects with type 2 diabetes had higher fasting IAPP levels than the control group (P < 0.001). The fasting serum insulin in the diabetic group was pmol/l in the GLP-1 vs pmol/l in the placebo experiments (NS). Again, these levels were higher in the group with type 2 diabetes than in the control group (P ¼ 0.025). In the diabetic group, no significant correlation existed between fasting insulin and fasting IAPP levels (r ¼ 0.26, NS). The circulating insulin/iapp ratio in the fasting condition was in the series with GLP-1 and in the series with placebo (NS). These ratios were significantly lower than in the control subjects (P ¼ 0.039). IAPP levels after GLP-1 in healthy subjects In controls, GLP-1 increased the serum levels of both IAPP and insulin in parallel, whereas the levels were not significantly altered by placebo. Concomitantly, blood glucose was transiently lowered by GLP-1, but not Figure 1 Serum levels of IAPP and insulin, and blood glucose levels, in seven healthy subjects before and at various time points after the administration of a buccal tablet containing 400 mg GLP-1 or placebo. Means S.E.M. are shown.

4 646 B Ahrén and M Gutniak EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137 parameters were observed after addition of the placebo tablet. At 15 min after GLP-1, serum IAPP had increased to or by pmol/l (P ¼ 0.021) whereas insulin at the same time point had increased to pmol/l or by pmol/l (P ¼ 0.004). Correlation between insulin and IAPP after GLP-1 stimulation In the healthy subjects there was a significant correlation between the absolute 15 min values after GLP-1 administration of circulating IAPP and insulin (r ¼ 0.68, P ¼ 0.038), and also between the increase in serum IAPP and the increase in serum insulin (r ¼ 0.79, P ¼ 0.032) (Fig. 3). The ratio of the absolute values for circulating insulin and IAPP at 15 min after GLP-1 administration was 40 5 ((pmol insulin/l)/(pmol IAPP/l)), and the ratio between the increase in insulin and the increase in IAPP was In contrast, in the subjects with type 2 diabetes, there was no significant correlation between the 15 min increase in IAPP and the 15 min increase in insulin after administration of GLP-1 (r ¼¹0.26, NS) (Fig. 3), and there was no significant correlation between the absolute values of circulating IAPP and insulin at 15 min Figure 2 Serum levels of IAPP and insulin, and blood glucose levels, in nine subjects with type 2 diabetes before and at various time points after the administration of a buccal tablet containing 400 mg GLP-1 or placebo. Means S.E.M. are shown. Figure 3 Correlation between the increase in insulin and the increase in IAPP at 15 min after the administration of a buccal tablet containing 400 mg GLP-1 in control healthy subjects and in subjects with type 2 diabetes.

5 EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137 GLP-1 and IAPP secretion 647 (r ¼¹0.12, NS). The ratio of the absolute values of insulin and IAPP at 15 min after GLP-1 was and the ratio of the increase in insulin to increase in IAPP was Discussion It has previously been shown that GLP-1 stimulates the secretion of IAPP from the perfused rat pancreas (30). We have shown here that GLP-1 also stimulates the secretion of IAPP in humans. To raise circulating GLP-1, we used a buccal tablet consisting of 400 mg GLP-1, which has been shown to increase plasma GLP-1 levels to approximately pmol/l within 30 min (31). These levels of GLP-1 are those required for its antidiabetogenic action (31, 32) and, therefore, if GLP-1 were to be used in the treatment of type 2 diabetes, an accompanying increase in circulating IAPP would occur. In the normal subjects, fasting circulating IAPP was approximately 4 pmol/l, which is in the range of 2 10 pmol/l found in previous studies in healthy subjects (7 16, 21). Furthermore, in the normal subjects, there was a close correlation between circulating IAPP and circulating insulin in the fasting state, the regression coefficient being 0.74, which confirms previous studies (7, 12, 14). The subjects with type 2 diabetes had elevated circulating IAPP, this being approximately 10 pmol/l. In the subjects with type 2 diabetes, the circulating levels of insulin were also elevated in spite of persisting fasting hyperglycemia, as a sign of reduced insulin sensitivity. Nevertheless, the ratio of circulating insulin to IAPP was reduced in type 2 diabetes. These findings are in accord with previous results that subjects with obesity, exhibiting low insulin sensitivity (18), have elevated IAPP levels (15, 16) and increased IAPP secretion (33, 34). Also, dexamethasoneinduced insulin resistance and streptozotocin-induced diabetes in rodents are accompanied by overexpression of IAPP vs insulin (18, 19). Hence, it seems as if insulin resistance is accompanied by exaggerated increase in IAPP vs the increase in insulin. Previous studies in type 2 diabetes have reported reduced circulating IAPP in subjects with insulin treated, i.e. more severe, diabetes (15), whereas other studies have reported levels not different from a control group (21). Taken together, this would suggest that circulating IAPP is different in different groups of diabetic and pre-diabetic subjects, probably related to different degrees of severity of the disease. Thus, it might be speculated that during insulin resistance, which usually precedes the development of type 2 diabetes, there is an increased circulating IAPP, which is more exaggerated relative to the hyperinsulinemia, whereas during the progression to severe forms of type 2 diabetes, IAPP levels progressively decline (15). The potential contribution of this hyperiappemia for the progression of the disease remains to be established. IAPP is co-stored with insulin in the pancreatic B cells and previous studies have shown that IAPP and insulin are co-released under a variety of experimental conditions (7 16). However, it has also been suggested that under certain conditions IAPP and insulin are released in a non-parallel fashion. For example, in rat cell monolayers, adrenaline and removal of extracellular calcium abolish insulin but not IAPP secretion (35) and, furthermore, glucose increases the insulin/iapp ratio in the perfusate from the rat pancreas (36). Also, in experimental models of hyperglycemia, reduced insulin sensitivity or spontaneous diabetes, overexpression or oversecretion of IAPP vs insulin has been documented (18, 19, 36 41). Hence, the relation between release of IAPP vs insulin might differ under different conditions and remains to be clearly established in relation to type 2 diabetes. We found that in normal subjects GLP-1 increased serum IAPP levels in parallel with an increase in serum insulin, which is similar to our previous result that arginine too stimulated a parallel secretion of insulin and IAPP (14). On a molar basis, circulating insulin increased more than did circulating IAPP, resulting in an increased ratio of circulating insulin to IAPP following GLP-1 administration. This is most likely due to the ratio of insulin to IAPP being much higher in the secretory granules than in the circulation (42) which overrides the differences in the extraction rate between the two peptides in the circulation (the half-life is approximately 12 min for IAPP (43) vs 4 min for insulin (44)). In contrast to what we observed in the healthy subjects, circulating insulin and IAPP did not correlate with each other in the subjects with type 2 diabetes, either in the fasting state or after administration of GLP-1. The mechanism of this imbalance between insulin and IAPP in type 2 diabetes needs to be studied in more detail. It may be speculated that the insulin resistance in the subjects with diabetes, as evident from the fasting hyperinsulinemia in spite of hyperglycemia, has adaptively altered the relative ratio of IAPP vs insulin expression in the B cell secretory granules. It is also possible that the subjects with type 2 diabetes exhibit altered clearance of insulin and/or IAPP which could contribute to the altered relative circulating levels of these peptides. Hypothetically, the imbalance between IAPP and insulin in type 2 diabetes might be of importance for the metabolic perturbations in some of the patients. In conclusion, we have shown that in normal subjects GLP-1 stimulates IAPP secretion in parallel with insulin, emphasizing the co-secretion of these two hormones in man. We have also shown that in type 2 diabetes, fasting IAPP is elevated, and that GLP-1 stimulates the secretion of IAPP in both normal subjects and in type 2 diabetes. Finally, we have shown that in type 2 diabetes, the correlation between IAPP and insulin in the fasting state and after stimulation with GLP-1 is lost, which may be of

6 648 B Ahrén and M Gutniak EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137 pathophysiological importance in some subjects for the metabolic perturbations of type 2 diabetes. Acknowledgements The authors are grateful to Lilian Bengtsson, Ulrika Gustavsson, Eva Holmström and Margaretha Persson for expert technical assistance. The study was supported by the Swedish Medical Research Council (Grant No. 14X-6834), the Ernhold Lundström, Albert Påhlsson and Novo Nordic Foundations, the Swedish Diabetes Association and the Faculty of Medicine, Lund University. The authors thank TheraTech Inc., Salt Lake City, Utah, USA, for supporting the study. References 1 Lukinius A, Wilander E, Westermark GT, Engström U & Westermark P. Colocalization of islet amyloid polypeptide and insulin in the B cell secretory granules of the human pancreatic islets. Diabetologia Ahrén B & Pettersson M. Calcitonin gene-related peptide (CGRP) and amylin in the endocrine pancreas. 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7 EUROPEAN JOURNAL OF ENDOCRINOLOGY (1997) 137 GLP-1 and IAPP secretion Kautzky-Willer A, Thomaseth K, Pacini G, Clodi M, Ludvik B, Streli C, Waldhäusl W & Prager R. Role of islet amyloid polypeptide secretion in insulin-resistant humans. Diabetologia Kautzky-Willer A, Thomaseth K, Clodi M, Ludvik B, Waldhäusl W, Prager R & Pacini G. b-cell activity and hepatic insulin extraction following dexamethasone administration in healthy subjects. Metabolism Kahn SE, Verchere CB, D Alessio DA, Cook DL & Fujimoto WY. Evidence for selective release of rodent islet amyloid polypeptide through the constitutive secretory pathway. Diabetologia O Brien TD, Westermark P & Johnson KH. Islet amyloid polypeptide and insulin secretion from isolated perfused pancreas of fed, fasted, glucose-treated, and dexamethasone-treated rats. Diabetes Pieber TR, Stein DT, Ogawa A, Alam T, Ohneda M, McCorkle K, Chen L, McGarry JD & Unger RH. Amylin insulin relationships in insulin resistance with and without diabetic hyperglycemia. American Journal of Physiology E446 E Novials A, Sarri Y, Casamitjana R, Rivera F & Gomis R. Regulation of islet amyloid polypeptide in human pancreatic islets. Diabetes Mulder H, Ahrén B & Sundler F. Differential expression of islet amyloid polypeptide (amylin) and insulin in experimental diabetes in rodents. Molecular and Cellular Endocrinology Mulder H, Ahrén B & Sundler F. Islet amyloid polypeptide and insulin gene expression are regulated in parallel by glucose in vivo in rats. American Journal of Physiology E1008 E Hiramatsu S, Inoue K, Sako Y, Umeda F & Nawata H. Insulin treatment improves relative hypersecretion of amylin to insulin in rats with non-insulin-dependent diabetes mellitus induced by neonatal streptozotocin injection. Metabolism Kahn SE, D Alessio DA, Schwartz MW, Fujimoto WY, Ensinck JW, Taborsky Jr GJ & Porte Jr D. Evidence of cosecretion of islet amyloid polypeptide and insulin by B cells. Diabetes Bretherton-Watt D, Gilbey SG, Ghatei MA, Beacham J & Bloom SR. Failure to establish islet amyloid polypeptide (amylin) as a circulating beta-cell inhibiting hormone in man. Diabetologia Sönksen PH, Tompkins CV, Srivastava NC & Nabarro JDN. A comparative study on the metabolism of human insulin and porcine proinsulin in man. Clinical Science of Molecular Medicine Received 21 May 1997 Accepted 24 July 1997