Amylin secretion from the perfused pancreas: dissociation from insulin and abnormal elevation in insulin-resistant diabetic rats

Amylin has been co-secreted from pancreatic islet beta-cells in constant proportion with insulin in some studies. We measured basal and glucose-stimulated amylin and insulin secretion from isolated perfused pancreases of normal and diabetic fatty Zucker rats. Glucose concentrations in the perfusion buffer were increased then decreased in small steps to mimic physiologic changes occurring after a meal. The absolute rate of amylin secretion and the molar ratio of amylin to insulin secreted from diabetic pancreases increased dramatically when infused glucose concentrations fell. Similar changes also occurred in normal pancreases, although the absolute change in amylin secretion was smaller. These studies provide the first evidence that (i) there is a mechanism within the pancreas whereby independent secretion of amylin and insulin can occur; (ii) the molar ratio of amylin to insulin secreted from both normal and diabetic pancreases can vary over a wide range; and (iii) there are important differences in the kinetics of amylin and insulin secretion or their coupling to stimulation by glucose between the isolated pancreases of normal rats and those with genetically transmitted insulin resistance and diabetes mellitus.     

Effects of exogenous somatostatin and insulin on islet amyloid polypeptide (amylin) release from perfused rat pancreas.

This study examined the effects of exogenous somatostatin and insulin on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas. Somatostatin inhibited the release of both amylin and insulin from the perfused pancreas to the same extent. The infusion of 10 nM somatostatin resulted in 40% inhibition of the secretion of both amylin and insulin induced by 11.1 mM glucose and 10 mM arginine, and this inhibition was significantly increased to 70% by the infusion of 100 nM somatostatin (p less than 0.05). The amylin/insulin molar ratios remained constant at 0.8% and were not changed by the infusion of somatostatin. On the other hand exogenous insulin at a concentration of 1.8 nM did not affect the release of amylin induced by 11.1 mM glucose and 10 mM arginine, whereas 180 nM insulin slightly, although not significantly, inhibited the release of amylin by 15%. These findings suggest that the release of amylin may be negatively regulated by somatostatin and that circulating insulin may have no direct effect on the release of amylin at least at a physiological concentration.     

Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas

Amylin, a 37-amino acid polypeptide, is the main component of amyloid deposits in the islets of Langerhans, and has been identified in the B-cell secretory granules. We have investigated the effect of rat amylin on the insulin and glucagon release by the isolated, perfused rat pancreas. Amylin infusion at 750 nM, markedly reduced unstimulated insulin release (ca. 50%, P less than 0.025), whereas it did not modify glucagon output. At the same concentration, amylin also blocked the insulin response to 9 mM glucose (ca. 80%, P less than 0.025) without affecting the suppressor effect of glucose on glucagon release. The inhibitory effect of amylin on glucose-induced insulin secretion was confirmed by lowering the amylin concentration (500 nM) and increasing the glucose stimulus (11 mM); again, no effect of amylin on glucagon release was observed. Finally, amylin, at 500 nM, reduced the insulin response to 3.5 mM arginine (ca. 40%, P less than 0.025) without modifying the secretion of glucagon elicited by this amino acid. It can be concluded that, in the rat pancreas, the inhibitory effect of homologous amylin on unstimulated insulin secretion, as well as on the insulin responses to metabolic substrates (glucose and arginine), favours the concept of this novel peptide as a potential diabetogenic agent.     

Effects of ciglitazone on endogenous plasma islet amyloid polypeptide and insulin sensitivity in obese-diabetic viable yellow mice

The role of islet amyloid polypeptide, also known as amylin, in insulin resistance and in the etiology of diabetes has been a subject of debate. Increased plasma amylin levels have been observed in both obese and type II diabetic patients. However, data on endogenous amylin levels with relation to pharmacological interventions have not been reported. In this study, chronic treatment of obese-diabetic viable yellow mice with ciglitazone was shown to significantly alter various parameters. Blood glucose and plasma insulin, triglyceride, and amylin levels were reduced and glucose tolerance in the presence of exogenous insulin was improved. Insulin/amylin ratios which were found to be significantly elevated in diabetic mice as compared to normal controls, were decreased after ciglitazone treatment. However, observed decreases in both amylin and insulin concentrations due to ciglitazone treatment and their subsequent increases upon withdrawal of treatment were correlated, suggesting cosecretion.     

Evidence that amylin stimulates lipolysis in vivo: a possible mediator of induced insulin resistance

The present study investigated the role of amylin in lipid metabolism and its possible implications for insulin resistance. In 5- to 7-h-fasted conscious rats, infusion of rat amylin (5 nmol/h for 4 h) elevated plasma glucose, lactate, and insulin (P <0.05 vs. control, repeated-measures ANOVA) with peak values occurring within 60 min. Despite the insulin rise, plasma nonesterified fatty acids (NEFA) and glycerol were also elevated (P < 0.001 vs. control), and these elevations (80% above basal) were sustained over the 4-h infusion period. Although unaltered in plasma, triglyceride content in liver was increased by 28% (P < 0.001) with a similar tendency in muscle (18%, P = 0.1). Infusion of the rat amylin antagonist amylin-(8-37) (125 nmol/h) induced opposite basal plasma changes to amylin, i.e., lowered plasma NEFA, glycerol, glucose, and insulin levels (all P < 0.05 vs. control); additionally, amylin-(8-37) blocked amylin-induced elevations of these parameters (P < 0.01). Treatment with acipimox (10 mg/kg), an anti-lipolytic agent, before or after amylin infusion blocked amylin's effects on plasma NEFA, glycerol, and insulin but not on glucose and lactate. We conclude that amylin could exert a lipolytic-like action in vivo that is blocked by and is opposite to effects of its antagonist amylin-(8-37). Further studies are warranted to examine the physiological implications of lipid mobilization for amylin-induced insulin resistance.     

Effects of glucagon-like peptide 1 (7-36) amide and glucagon on amylin release from perfused rat pancreas.

The effects of glucagon-like peptide 1 (7-36) amide [GLP-1 (7-36) amide] and glucagon on the release of islet amyloid polypeptide (IAPP), or amylin, from the isolated perfused rat pancreas were studied. In the presence of 5.6 mM glucose, GLP-1 (7-36) amide and glucagon stimulated the release of amylin from the perfused pancreas. The infusion of GLP-1 (7-36) amide at a concentration of 10(-9) M elicited a biphasic release of amylin similar to that of insulin. The cumulative output of amylin induced by 10(-9)M GLP-1 (7-36) amide was significantly higher than that by 10(-9)M glucagon (p less than 0.01). The amylin/insulin molar ratios induced by GLP-1 (7-36) amide and glucagon were about 1% and did not differ significantly. These findings suggest that GLP-1 (7-36) amide and glucagon stimulate the release of amylin from the pancreas and that the concomitant secretion of amylin and insulin might contribute to glucose homeostasis.     

Associations of amylin with inflammatory markers and metabolic syndrome in apparently healthy Chinese

Background

Cellular and animal studies implicate multiple roles of amylin in regulating insulin action, glucose and lipid metabolisms. However, the role of amylin in obesity related metabolic disorders has not been thoroughly investigated in humans. Therefore, we aimed to evaluate the distribution of circulating amylin and its association with metabolic syndrome (MetS) and explore if this association is influenced by obesity, inflammatory markers or insulin resistance in apparently healthy Chinese.

Methods

A population-based sample of 1,011 Chinese men and women aged 35–54 years was employed to measure plasma amylin, inflammatory markers (C-reactive protein [CRP] and interleukin-6 [IL-6]), insulin, glucose and lipid profiles. MetS was defined according to the updated National Cholesterol Education Program Adult Treatment Panel III criteria for Asian-Americans.

Results

Plasma amylin concentrations were higher in overweight/obese participants than normal-weight counterparts (P<0.001) without sex difference. Circulating amylin was positively associated with CRP, IL-6, BMI, waist circumference, blood pressure, fasting glucose, insulin, amylin/insulin ratio, HOMA-IR, LDL cholesterol and triglycerides, while negatively associated with HDL cholesterol (all P<0.001). After multiple adjustments, the risk of MetS was significantly higher (odds ratio 3.71; 95% confidence interval: 2.53 to 5.46) comparing the highest with the lowest amylin quartile. The association remained significant even further controlling for BMI, inflammatory markers, insulin or HOMA-IR.

Conclusions

Our study suggests that amylin is strongly associated with inflammatory markers and MetS. The amylin-MetS association is independent of established risk factors of MetS, including obesity, inflammatory markers and insulin resistance. The causal role of hyperamylinemia in the development of MetS needs to be confirmed prospectively.     

Differential Effect of Amylin on Endothelial-Dependent Vasodilation in Mesenteric Arteries from Control and Insulin Resistant Rats

Insulin resistance (IR) is frequently associated with endothelial dysfunction and has been proposed to play a major role in cardiovascular disease (CVD). On the other hand, amylin has long been related to IR. However the role of amylin in the vascular dysfunction associated to IR is not well addressed. Therefore, the aim of the study was to assess the effect of acute treatment with amylin on endothelium-dependent vasodilation of isolated mesenteric arteries from control (CR) and insulin resistant (IRR) rats and to evaluate the possible mechanisms involved. Five week-old male Wistar rats received 20% D-fructose dissolved in drinking water for 8 weeks and were compared with age-matched CR. Plasmatic levels of glucose, insulin and amylin were measured. Mesenteric microvessels were dissected and mounted in wire myographs to evaluate endothelium-dependent vasodilation to acetylcholine. IRR displayed a significant increase in plasmatic levels of glucose, insulin and amylin and reduced endothelium-dependent relaxation when compared to CR. Acute treatment of mesenteric arteries with r-amylin (40 pM) deteriorated endothelium-dependent responses in CR. Amylin-induced reduction of endothelial responses was unaffected by the H₂O₂ scavenger, catalase, but was prevented by the extracellular superoxide scavenger, superoxide dismutase (SOD) or the NADPH oxidase inhibitor (VAS2870). By opposite, amylin failed to further inhibit the impaired relaxation in mesenteric arteries of IRR. SOD, or VAS2870, but not catalase, ameliorated the impairment of endothelium-dependent relaxation in IRR. At concentrations present in insulin resistance conditions, amylin impairs endothelium-dependent vasodilation in mircrovessels from rats with preserved vascular function and low levels of endogenous amylin. In IRR with established endothelial dysfunction and elevated levels of amylin, additional exposure to this peptide has no effect on endothelial vasodilation. Increased superoxide generation through NADPH oxidase activity may be a common link involved in the endothelial dysfunction associated to insulin resistance and to amylin exposure in CR.     

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.     

GSK3 involvement in amylin signaling in isolated rat soleus muscle

Amylin can evoke insulin resistance by antagonizing insulin in a non-competitive manner. Here, we investigated the glycogenolytic effect of amylin in isolated skeletal muscle and compared it to the effects of a calcitonin gene-related peptide (CGRP). Amylin alone had no statistically significant effect on glucose transport. However, amylin decreased insulin-stimulated glucose transport by about 30%. The involvement of cAMP could not be detected at the concentrations shown to promote glycogenolysis. Previously, it has been shown that increased glycogen synthase kinase 3 (GSK3) activity plays a role in insulin resistance. Here, the ratio of GSK3 :β isoforms in rat soleus was found to be 1.2:1. We found that amylin increased GSK3 activity, which in turn led to increased phosphorylation of glycogen synthase and decreased glycogen synthesis de novo.     

High-frequency oscillations in circulating amylin concentrations in healthy humans

Amylin is stored in the pancreatic beta-cell granules and cosecreted with insulin in response to nutrient stimuli. To gain further insight into control of hormonal release in beta-cell physiology, we examined whether amylin, like insulin, circulates in a high-frequency oscillatory pattern, and if it does, to compare the secretory patterns of the two hormones. Eight overnight-fasted healthy individuals were studied during intravenous glucose infusion (2.0 mg. kg(-1). min(-1)). Blood was collected every minute for 90 min and analyzed in triplicate for amylin, total amylin immunoreactivity (TAI), and insulin. Mean plasma concentrations of amylin (nonglycosylated), TAI (nonglycosylated plus glycosylated), insulin, and glucose were 2.77 +/- 1.21 pmol/l, 7.60 +/- 1.73 pmol/l, 50.4 +/- 17.5 pmol/l, and 5.9 +/- 0.3 mmol/l, respectively. The 90-min time series of amylin, TAI, and insulin were analyzed for periodicity (by spectral analysis, autocorrelation analysis, and deconvolution analysis) and regularity [by approximate entropy (ApEn)]. Significant spectral density peaks were demonstrated by a random shuffling technique in 7 (out of 7), 8 (out of 8), and 8 (out of 8) time series, respectively, whereas autocorrelation analysis revealed significant pulsatility in 5 (out of 7), 7 (out of 8), and 5 (out of 8), respectively. The dominant periodicity of oscillations determined by spectral analysis was 4.6 +/- 0.3, 4.6 +/- 0.4, and 6. 5 +/- 1.1 min/pulse, respectively (amylin vs. insulin, P = 0.017, TAI vs. insulin, P = 0.018). By deconvolution analysis, amylin and insulin periodicities were, respectively, 6.3 +/- 1.0 and 5.5 +/- 0. 6 min. By application of the regularity statistic, ApEn, 6 (out of 7), 7 (out of 8), and 6 (out of 8), respectively, were found to be significantly different from random. In conclusion, like several other hormones, circulating amylin concentrations exhibit oscillations in the secretory patterns for nonglycosylated as well as glycosylated forms. Whether the high-frequency pulsatile release of amylin is disturbed in diabetes is not known.     

Agouti signaling protein stimulates islet amyloid polypeptide (amylin) secretion in pancreatic beta-cells

Ectopic overexpression of the murine agouti gene results in yellow coat color, obesity, hyperinsulinemia, and type II diabetes. We have shown the human homologue of agouti (agouti signaling protein; ASP) to regulate human adipocyte metabolism and lipid storage via a Ca(2+)-dependent mechanism. We have also demonstrated agouti expression in human pancreas, and that ASP stimulates insulin release via a similar Ca(2+)-dependent mechanism. Plasma amylin is also elevated in agouti mutant mice. Amylin is cosecreted with insulin from beta-cells, and overexpression of human amylin in beta-cells in yellow agouti mutant mice resulted in accelerated pancreatic amyloid deposition, severely impaired beta-cell function, and a diabetic phenotype. We report here that ASP stimulates amylin release in both the HIT-T15 beta-cell line and human pancreatic islets in the presence of a wide range of glucose concentrations (0-16.7 mmol/L), similar to its effect on insulin release; this effect was blocked by 30 mumol/L nitrendipine, confirming a Ca(2+)-dependent mechanism. Accordingly, ASP stimulation of amylin release may serve as a compensatory system to regulate blood glucose in yellow agouti mutants.     

Amylin evokes protein p20 phosphorylation and insulin resistance in rat skeletal muscle extensor digitorum longus

In the present study, we investigate effect of amylin on the insulin sensitivity of rat skeletal muscle extensor digitorum longus (EDL) using in vitro intact muscle incubation in combination with metabolic radioactive labeling. The molecular basis of the amylin action was further examined using proteomic analysis. In particular, proteins of interest were characterized using an integrated microcharacterization procedure that involved in-gel trypsin digestion, organic solvent extraction, high performance liquid chromatography separation, microsequencing and microse-quence analysis. We found that amylin significantly decreased the insulin-stimulated glucose incorporation into glycogen (p < 0.01) and produced a protein spot of approximately 20 ku in size. This amylin responsive protein (hereby designated as amylin responsive protein 1, APR1) was identified to be protein p20. Moreover, ARP1 spots on gels were found to consistently produce a corresponding radioactive spot on X-ray films in 32Pi but not in 35S-     

Amylin effect in extrapancreatic tissues participating in glucose homeostasis, in normal, insulin-resistant and type 2 diabetic state

Amylin is co-secreted with insulin, responds to the same stimuli, is anorectic, lowers body weight by reducing fat mass, and is proposed for diabetes treatment. We examined the effect of a 3-day constant infusion of close to physiological doses of amylin in Wistar rats, on glucotransporter expression, glycogen content (G), glycogen synthase a activity (GSa) and glucose transport (GT), in liver, muscle and fat from insulin resistant (IR) and type 2 diabetic (T2D) models, compared to normal (N) animals; plasma glucose and insulin were measured. Plasma insulin in IR was higher than in N or T2D, and amylin normalized the value. In both, IR and T2D, liver G was lower than normal, accompanied by GLUT-2, mRNA and protein, higher and lower, respectively, than in N; amylin normalized G in both groups, without changes in GLUT-2, except for an mRNA increase in T2D. In IR and T2D, muscle GSa was reduced, together with respective over- and under-GLUT-4 expression; amylin induced only a trend toward GSa normalization in both groups. In isolated adipocytes, GT and GLUT-4 in IR and T2D were lower and higher, respectively, than in N; after amylin, not only GT was normalized in both groups but also the response to insulin was much more pronounced, including that in N, without major changes in GLUT-4. This suggests that the beneficial effect of amylin in states running with altered glucose homeostasis could occur by partially acting on the hexose metabolism of the liver and mainly on that of the adipose tissue.     

The effects of amylin on carbohydrate metabolism in skeletal muscle in vitro and in vivo.

1. The effects of synthetic human amylin on basal and insulin-stimulated (100 and 1000 microunits/ml) rates of lactate formation, glucose oxidation and glycogen synthesis were measured in the isolated rat soleus muscle preparation incubated in the presence of various concentrations of glucose (5, 11 and 22 mM). 2. The rate of glucose utilization was increased by about 2-fold by increasing the glucose concentration from 5 to 22 mM. 3. Synthetic human amylin (10 nM) significantly inhibited (by 46-56%) glycogen synthesis, irrespective of the concentration of insulin or glucose present in the incubation medium. 4. Amylin (10 nM) did not affect insulin-stimulated rates of 2-deoxy[3H]glucose transport and phosphorylation. 5. Intraperitoneal administration of insulin (100 micrograms/kg) to rats in vivo stimulated the rate of [U-14C]glucose incorporation into glycogen in the diaphragm by about 80-fold. This rate was decreased (by 28%) by co-administration of amylin (66 micrograms/kg).     

Syntheses, structures and anorectic effects of human and rat amylin.

Amylin, a 37-residue polypeptide with a single disulfide bond originally isolated from the pancreas of type-II diabetic patients, has been shown to cause peripheral insulin resistance and to attenuate the inhibition of hepatic glucose output by insulin. We have also shown that amylin is present in the rat hypothalamus and that it inhibits food intake by rats. In order to further investigate the anorectic properties we synthesized both human and rat amylin by the solid phase method and purified to homogeneity in an overall yield of 10-20%. Structural analyses indicated that human amylin exhibited predominantly a beta-sheet structure at both acidic and alkaline pH, whereas no ordered structure was evident in the case of rat amylin. Intrahypothalamic injection of rat amylin resulted in a potent dose-dependent inhibitory effect on the food intake by rats adapted to eat their daily ration of food in an eight-hour period. Human amylin was less effective as an anorectic agent. Furthermore, rat amylin completely blocked the potent orexigenic effect of neuropeptide Y (NPY). These investigations show that there is a fundamental difference in the secondary structures of human and rat amylin and that rat amylin is a potent inhibitor of both basal and NPY-induced feeding by rats.     

Amylin evokes protein p20 phosphorylation and insulin resistance in rat skeletal muscle extensor digitorum longus

In the present study, we investigate effect of amylin on the insulin sensitivity of rat skeletal muscle extensor digitorum longus (EDL) using in vitro intact muscle incubation in combination with metabolic radioactive labeling. The molecular basis of the amylin action was further examined using proteomic analysis. In particular, proteins of interest were characterized using an integrated microcharacterization procedure that involved in-gel trypsin digestion, organic solvent extraction, high performance liquid chromatography separation, microsequencing and microsequence analysis. We found that amylin significantly decreased the insulin-stimulated glucose incorporation into glycogen (p < 0.01) and produced a protein spot of approximately 20 ku in size. This amylin responsive protein (hereby designated as amylin responsive protein 1, APR1) was identified to be protein p20. Moreover, ARP1 spots on gels were found to consistently produce a corresponding radioactive spot on X-ray films in ³²Pi but not in ³⁵S-methionine labeling experiments. In conclusion, our results showed that in vitro amylin concomitantly evoked the production of ARP1 and caused insulin resistance in EDL muscle. It is suggested that protein p20 may be involved in amylin signal transduction and the appearance of ARP1 may be a step in a molecular pathway leading to the development of insulin resistance. ARP1 might therefore be a useful molecular marker for amylin action, insulin resistance and Type 2 diabetes.     

Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo

Insulin-degrading enzyme (IDE, insulysin) is the best characterized catabolic enzyme implicated in proteolysis of insulin. Recently, a peptide inhibitor of IDE has been shown to affect levels of insulin, amylin, and glucagon in vivo. However, IDE^−/− mice display variable phenotypes relating to fasting plasma insulin levels, glucose tolerance, and insulin sensitivity depending on the cohort and age of animals. Here, we interrogated the importance of IDE-mediated catabolism on insulin clearance in vivo. Using a structure-based design, we linked two newly identified ligands binding at unique IDE exosites together to construct a potent series of novel inhibitors. These compounds do not interact with the catalytic zinc of the protease. Because one of these inhibitors (NTE-1) was determined to have pharmacokinetic properties sufficient to sustain plasma levels >50 times its IDE IC₅₀ value, studies in rodents were conducted. In oral glucose tolerance tests with diet-induced obese mice, NTE-1 treatment improved the glucose excursion. Yet in insulin tolerance tests and euglycemic clamp experiments, NTE-1 did not enhance insulin action or increase plasma insulin levels. Importantly, IDE inhibition with NTE-1 did result in elevated plasma amylin levels, suggesting the in vivo role of IDE action on amylin may be more significant than an effect on insulin. Furthermore, using the inhibitors described in this report, we demonstrate that in HEK cells IDE has little impact on insulin clearance. In total, evidence from our studies supports a minimal role for IDE in insulin metabolism in vivo and suggests IDE may be more important in helping regulate amylin clearance.     

Role of endogenous amylin in glucagon secretion and gastric emptying in rats demonstrated with the selective antagonist, AC187

Amylin is a 37-amino acid polypeptide co-secreted with insulin from the pancreatic beta-cells. It complements insulin's stimulation of the rate of glucose disappearance (Rd) by slowing the rate of glucose appearance (Ra) through several mechanisms, including an inhibition of mealtime glucagon secretion and a slowing of gastric emptying. To determine if endogenous amylin tonically inhibits these processes, we studied the effects of the amylin receptor blocker AC187 upon glucagon secretion during euglycemic, hyperinsulinemic clamps in Sprague-Dawley (HSD) rats, upon gastric emptying in HSD rats, and upon gastric emptying and plasma glucose profile in hyperamylinemic, and genetically obese, Lister Albany/NIH rats during a glucose challenge. Amylin blockade increased glucagon concentration, accelerated gastric emptying of liquids, and resulted in an exaggerated post-challenge glycemia. These data collectively indicate a physiologic role for amylin in glucose homeostasis via mechanisms that include regulation of glucagon secretion and gastric emptying.