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.     

Human and rat amylin have no effects on insulin secretion in isolated rat pancreatic islets

Amylin, an islet amyloid peptide secreted by the pancreatic beta cell, has been proposed as a humoral regulator of islet insulin secretion. Four separate preparations of amylin were tested for effects on hormone secretion in both freshly isolated and cultured rat islets and in HIT-T15, hamster insulinoma cells. With all three experimental models, exposure to human amylin acid and human and rat amylin at concentrations as high as 100 nM had no significant effect on rates of insulin or glucagon secretion. These observations suggest that amylin, even at concentrations appreciably higher than those measured in peripheral plasma, is not a significant humoral regulator of islet hormone secretion.     

The amylin superfamily: A novel grouping of biologically active polypeptides related to the insulin A-chain

The peptide amylin (previously termed Diabetes Associated Peptide) has recently been isolated and characterised from the amyloid of the pancreatic islets of Langerhans from human type 2 diabetics [1]. Amylin shows about 46% identity in amino acid sequence on comparison with the calcitonin gene-related peptides (CGRPs) and also shows some similarity to insulin [1]. Recent studies have also shown that both amylin and CGRP are potent inhibitors of insulin-stimulated glycogen synthesis in skeletal muscle in vitro [2,3]. Hormones may be arranged into families, therefore a degree of order exists even though hormone-mediated effects are complex [4]. The polypeptides insulin, insulin-like growth factors (IGFs) and relaxins have been grouped into such a family with similarities both at the protein-structural and genetic levels [4,5]. We now demonstrate that this insulin-related family, along with amylin and the CGRPs, are members of a peptide superfamily defined by structural similarity in the region corresponding to the A-chain of insulin. In order to distinguish this grouping of small biologically active peptides from the previous one, we have designated it the amylin superfamily. All the members of the previously defined insulin family have a region homologous to the insulin B-chain. Insulin, the IGFs, the relaxins, the CGRPs and amylin are all involved in carbohydrate metabolism and therefore these peptides are functionally as well as structurally related. This grouping of peptides may have important implications for the study of human metabolic disease.     

Prevention and promotion effects of apolipoprotein E4 on amylin aggregation

The misfolding of islet amyloid polypeptide (IAPP, amylin) results in the formation of islet amyloid, which is one of the most common pathological features of type 2 diabetes (T2D). Amylin, a 37-amino-acid peptide co-secreted with insulin and apolipoprotein E (ApoE) from the β-cells of pancreatic islets, is thought to be responsible for the reduced mass of insulin-producing β-cells. However, neither the relationship between amylin and ApoE nor the biological consequence of amylin misfolding is known. Here we have characterized the interaction between ApoE4 and amylin in vitro. We found that ApoE4 can strongly bind to amylin, and insulin can hardly inhibit amylin-ApoE binding. We further found that amylin fibrillization can be prevented by low concentration of ApoE4 and promoted by high concentration of ApoE4. Taken together, we propose that under physiological conditions ApoE4 efficiently binds and sequesters amylin, preventing its aggregation, and in T2D the enhanced ApoE4-amylin binding leads to the critical accumulation of amylin, facilitating islet amyloid formation.     

Islet amyloid polypeptide (IAPP;amylin) influences the endocrine but not the exocrine rat pancreas.

The effect of synthetic rat amylin (10,100,1000 pmol/l) on glucose (10 mmol/) and arginine (10 mmol/l) -stimulated islet hormone release from the isolated perfused rat pancreas and on amylase release from isolated pancreatic acini was investigated. Amylin stimulated the insulin release during the first (+76%) and the second secretion period (+42%) at 1 nmol/l. The first phase of the glucagon release was inhibited concentration dependently by amylin and completely suppressed during the second phase. Amylin diminished the somatostatin release in a concentration dependent manner. This effect was more pronounced at the first than the second secretion period (1 nmol amylin: 1 phase: -60%, 2.phase: -22%). Amylin was without any effect on basal and CCK stimulated amylase release from isolated rat pancreatic acini. Our data suggest amylin, a secretory product of pancreatic B-cells, as a peptide with approximately strong paracrine effects within the Langerhans islet. Therefore, amylin might be involved in the regulation of glucose homeostasis.     

Degradation of amylin by insulin-degrading enzyme

A pathological feature of Type 2 diabetes is deposits in the pancreatic islets primarily composed of amylin (islet amyloid polypeptide). Although much attention has been paid to the expression and secretion of amylin, little is known about the enzymes involved in amylin turnover. Recent reports suggest that insulin-degrading enzyme (IDE) may have specificity for amyloidogenic proteins, and therefore we sought to determine whether amylin is an IDE substrate. Amylin-degrading activity co-purified with IDE from rat muscle through several chromatographic steps. Metalloproteinase inhibitors inactivated amylin-degrading activity with a pattern consistent with the enzymatic properties of IDE, whereas inhibitors of acid and serine proteases, calpains, and the proteasome were ineffective. Amylin degradation was inhibited by insulin in a dose-dependent manner, whereas insulin degradation was inhibited by amylin. Other substrates of IDE such as atrial natriuretic peptide and glucagon also competitively inhibited amylin degradation. Radiolabeled amylin and insulin were both covalently cross-linked to a protein of 110 kDa, and the binding was competitively inhibited by either unlabeled insulin or amylin. Finally, a monoclonal anti-IDE antibody immunoprecipitated both insulin- and amylin-degrading activities. The data strongly suggest that IDE is an amylin-degrading enzyme and plays an important role in the clearance of amylin and the prevention of islet amyloid formation.     

Amylin.

Amylin is a 37 amino-acid peptide which is secreted from the pancreatic islets of Langerhans. It has major sequence homology with calcitonin gene related peptide. Amylin can precipitate out in these cells to form amyloid. Amylin is secreted by similar stimuli to those that secrete insulin. Amylin has a number of effects that may counteract the effect of secreted insulin, i.e., decreased second phase insulin secretion, increased hepatic glucose output, and inhibition of insulin effects on skeletal muscle. It must, however, be recognized that in many cases the doses necessary to produce these effects appear to be supraphysiological. The putative role of amylin in the hyperglycemia of aging and Type II diabetes mellitus therefore remains controversial. Amylin has a number of other effects including inhibition of osteoclastic activity, vasodilatation, anorectic effects and enhanced memory retention. This review postulates a role for amylin in the pathogenesis of a number of age-related changes.     

Molecular model-building of amylin and alpha-calcitonin gene-related polypeptide hormones using a combination of knowledge sources

Amylin is the major component of the amyloid found in the pancreases of noninsulin-dependent diabetics (type 2 diabetes). It is a 37 amino acid polypeptide and has been shown to have 46% sequence identity with the neuropeptide alpha-calcitonin gene-related peptide (alpha-CGRP). Both amylin and alpha-CGRP are known to be potent inhibitors of glycogen synthesis in stripped rat soleus muscle. Secondary structure prediction and tertiary structure model-building show the two polypeptides to have an alpha-helix/beta-strand motif similar to that observed in the insulin B-chain. The results have been supported by CD spectroscopy, although there is no sequence similarity between insulin and amylin/alpha-CGRP. Aggregation states have been predicted based on the dimeric and hexameric arrangements seen in porcine insulin. Rat and hamster amylin have a changed sequence motif in the beta-strand which results in lack of amyloid formation and type 2 diabetes. This, we propose, is caused by disruption of hydrogen bonding which prevents the formation of the dimer.     

Central amylin expression and its induction in rat dams

Amylin, or islet amyloid polypeptide, is known as a satiating signal expressed in pancreatic β‐cells but not in the brain. In this study, regulations of postpartum mRNA expressions were investigated in the preoptic area of the hypothalamus. mRNA levels of lactating dams and mothers whose pups were removed immediately after delivery were compared in microarray experiments. There were 20 genes identified with significantly increased and 14 with decreased expression 9 days postpartum. Amylin mRNA level demonstrated the largest change, a 25.7 times increase. Quantitative RT‐PCR measurements validated the increase in the mRNA level of amylin in the preoptic area of lactating dams while the expression level of other members of the calcitonin gene‐related peptide family did not change. In situ hybridization histochemistry for amylin further verified its induction in lactating mothers and demonstrated the distribution of amylin mRNA in the medial preoptic nucleus, parts of the medial preoptic area, and the ventral part of the bed nucleus of the stria terminalis but nowhere else in the rat brain. Immunolabeling verified the postpartum induction of amylin in the preoptic area at the peptide level, as well. The results suggest that amylin may play a part in maternal regulations.     

Selective amylin inhibition of the glucagon response to arginine is extrinsic to the pancreas

Amylin, a peptide hormone from pancreatic beta-cells, is reported to inhibit insulin secretion in vitro and in vivo and to inhibit nutrient-stimulated glucagon secretion in vivo. However, it has been reported not to affect arginine-stimulated glucagon secretion in vitro. To resolve if the latter resulted from inactive peptide (a problem in the early literature), those experiments were repeated here with well-characterized peptide and found to be valid. In isolated perfused rat pancreas preparations, coperfusion with 1 nM amylin had no effect on arginine-, carbachol-, or vasoactive intestinal peptide-stimulated glucagon secretion. Amylin also had no effect on glucagon output stimulated by decreasing glucose concentration from 11 to 3.2 mM or on glucagon suppression caused by increasing glucose from 3.2 to 7 mM. Amylin at 100 nM had no effect in isolated islets in which glucagon secretion was stimulated by exposure to 10 mM arginine, even though glucagon secretion in the same preparation was inhibited by somatostatin. In anesthetized rats, amylin coinfusion had no effect on glucagon secretion stimulated by insulin-induced hypoglycemia. To reconcile reports of glucagon inhibition with the absence of effect in the experiments just described, anesthetized rats coinfused with rat amylin or with saline were exposed sequentially to intravenous L-arginine (during a euglycemic clamp) and then to hypoglycemia. Amylin inhibited arginine-induced, but not hypoglycemia-induced, glucagon secretion in the same animal. In conclusion, we newly identify a selective glucagonostatic effect of amylin that appears to be extrinsic to the isolated pancreas and may be centrally mediated.     

Amylin is more potent and more effective than glucagon in raising plasma glucose concentration in fasted, anesthetized rats.

Amylin is a 37 amino-acid peptide secreted from the pancreatic beta-cells. It has actions on carbohydrate metabolism in vivo, including elevation of blood glucose. In this study, the hyperglycemic effect of intravenous bolus injections of amylin was compared with similar injections of glucagon in 20-hour fasted rats lightly anesthetized with halothane. Administered doses ranged from 0.01 micrograms to 1000 micrograms (about 7 pmol/kg--750 nmol/kg for amylin and 8 pmol/kg--800 pmol/kg for glucagon). Control animals received an equal volume of saline. A single intravenous injection of amylin or glucagon led to an increase of plasma glucose levels, which peaked approximately at 1 hour after treatment. The calculated ED50 for amylin was 1.48 nmol whereas that for glucagon was 7.46 nmol; the maximum glucose increment was 4.3 mM for amylin, and 2.9 mM for glucagon. These results show that amylin is a more potent and more effective hyperglycemic agent than glucagon under these experimental conditions.     

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.     

Hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities are increased in obese (fa/fa) hyperinsulinemic Zucker rats: effects of glyburide administration

The chronically hyperinsulinemic Zucker fatty rat, with peripheral insulin resistance and glucose intolerance, represents a model of noninsulin dependent diabetes mellitus (NIDDM). These animals have elevated hepatic glycogen levels. Hepatic levels of synthase phosphatase and phosphorylase phosphatase, which are diminished in the IDDM rat, were markedly increased in the obese rats. Glyburide, a sulfonylurea used in treatment of NIDDM, resulted in reduced levels of glycemia and increased insulin levels in Zucker rats. Hepatic glycogen levels were increased, as was the activation of glycogen synthase, although there were no effects of drug administration on synthase phosphatase or phosphorylase phosphatase activities. G6P levels were increased by glyburide in lean rats but not in obese animals. These effects of glyburide on liver glycogen metabolism are accounted for via potentiation of the glycogenic effects of insulin.     

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.     

Cosecretion of amylin and insulin from isolated rat pancreas

Amylin, a 37 amino acid C-terminal amidated peptide is an integral part of secretory granules of pancreatic beta-cells. Utilizing a specific radioimmunoassay system we demonstrate in the present study a cosecretion of amylin and insulin from the isolated rat pancreas. The secretion pattern of both peptides during glucose or glucose plus arginine stimulation is identical. The molar ratio of amylin amounts to 10% of that of insulin. The biological significance of amylin is still unknown, but a paracrine/endocrine role in glucose homeostasis is speculated.     

Non-Aggregating Amylin Fragments as an Inhibitors of the Aggregation Process of Susceptible to Aggregation Fragments 18–22, 23–27, and 33–37 of Hormone

Amylin aggregation is one of the factors in the development of diabetes mellitus, which is classified as a civilization disease. The aim of this research was to find whether non-aggregating fragments 1–7, 8–12, 13–17 and 28–32 of amylin would inhibit the aggregation of the amyloidogenic cores 18–22, 23–27, 33–37 of hormone. In the study of the inhibitory potential of non-aggregating amylin fragments, a set of independent methods were used to study aggregation properties (spectroscopic and fluorescence studies with the use of indicators, microscopic studies, circular dichroism studies) and the method of prediction of aggregation properties. The performed research allowed to select the cyclic fragment (1–7) H-KCNTATC-OH with disulfide bond as an inhibitor capable of inhibiting the aggregation of all amyloidogenic cores 18–22, 23–27, 33–37 of the hormone. Additionally, it was found that this peptide inhibits insulin hot spot aggregation, which may indicate its universal utility in inhibiting the process of aggregation of hormones regulating carbohydrate metabolism directly related to the development of diabetes. Research on the possibility of the extensive use of the cyclic fragment (1–7) of H-KCNTATC-OH as a peptide inhibitor of the polypeptide/protein aggregation process is ongoing.     

Effects of amylin and adrenomedullin on the skeleton

Amylin and adrenomedullin are related peptides with some homology to both calcitonin and calcitonin gene-related peptide (CGRP). All these peptides have in common a 6-amino acid ring structure at the amino-terminus created by a disulfide bond. In addition, the carboxy-termini are amidated. Both amylin and adrenomedullin have recently been found to stimulate the proliferation of osteoblasts in vitro, and to increase indices of bone formation in vivo when administered either locally or systemically. Both amylin and adrenomedullin have also been found to act on chondrocytes (Cornish et al., submitted for publication), stimulating their proliferation in culture and increasing tibial growth plate thickness when administered systemically to adult mice. Studies of structure-activity relationships have demonstrated that osteotropic effects of amylin and adrenomedullin can be retained in peptide fragments of the molecules. The full-length peptide of amylin has known effects on fuel metabolism, and systemic administration of amylin is also associated with increased fat mass. However, the octapeptide fragment of the molecule, amylin-(1-8), is osteotropic and yet has no activity on fuel metabolism. Similar fragments of adrenomedullin have also been defined, which retain activity on bone but lack the parent peptide's vasodilator properties. Both amylin-(1-8) and adrenomedullin-(27-52) act as anabolic agents on bone, increasing bone strength when administered systemically. Thus, these small peptides, or analogues of it, are potential candidates as anabolic therapies for osteoporosis. Both amylin and adrenomedullin may have effects on bone metabolism. Amylin is secreted following eating and may direct calcium and protein absorbed from the meal into new bone synthesis. Amylin circulates in high concentrations in obese individuals, and might contribute to the association between bone mass and fat mass. Our recent findings demonstrating the co-expression of adrenomedullin and adrenomedullin receptors in osteoblasts, along with the findings that the peptide and its receptor are easily detectable during rodent embryogenesis, suggest that this peptide is a local regulator of bone growth. Thus, the findings reviewed in this paper illustrate that amylin and adrenomedullin may be relevant to the normal regulation of bone mass and to the design of agents for the treatment of osteoporosis.     

The effect of pramlintide (amylin analogue) treatment on bone metabolism and bone density in patients with type 1 diabetes mellitus.

Amylin is a 37-amino-acid peptide related to CGRP and calcitonin. It is co-secreted with insulin from pancreatic beta-cells. Amylin is deficient with type 1 diabetes mellitus. To study the in vivo effects of amylin in humans, diabetic patients are an adequate model of chronic amylin deficiency. We investigated the effect of a 12 months pramlintide therapy (amylin analogue) on bone metabolism in patients with type 1 diabetes mellitus. 23 patients with type 1 diabetes mellitus (age 45.2 +/- 10.3 years, duration of diabetes mellitus 20.7 +/- 9.8 years, 13 male, 10 female) injected themselves 0.1 ml pramlintide, a human amylin analogue, four times per day for a period of 12 months. Bone mineral density measurements of the lumbar spine by dual-energy X-ray absorptiometry (DXA), and biochemical markers of bone metabolism (serum-calcium, PTH, osteocalcin, urinary pyridinium cross-links) were obtained before and one year after starting pramlintide therapy. None of the following parameters changed significantly: bone density, serum calcium, PTH, osteocalcin or pyridinium cross-links. Only osteocalcin decreased from 7.205 ng/ml to 5.825 ng/ml, but this change was not statistically significant. We conclude that a one-year pramlintide therapy does not affect bone density or bone metabolism in patients with type 1 diabetes mellitus without osteopenia (based on the markers used).