Canine IAPP cDNA sequence provides important clues regarding diabetogenesis and amyloidogenesis in type 2 diabetes

Islet amyloid polypeptide (IAPP) is a recently discovered pancreatic islet hormone which is stored with insulin in beta cell granules. IAPP may have a significant role in the development of Type 2 diabetes mellitus due to its propensity to form islet cell-disrupting amyloid deposits, and by opposing the action of insulin in peripheral tissues. Most evidence to-date suggests that an intrinsic structural motif of IAPP is linked to the amyloidogenicity of IAPP, and that this motif occurs only in those species (e.g., humans and cats) that also develop age-associated or Type 2 diabetes We utilized polymerase chain reaction methodology in this study to obtain the IAPP nucleotide and protein sequences of the dog, a species not known to develop islet amyloid. We show that dog IAPP contains the same putative amyloidogenic sequence (GAILS) at residues 24-28 as human and cat IAPP, and that although dogs do not develop islet amyloid they do develop IAPP-derived amyloid in association with neoplastic beta cells (i.e., insulinomas). These results provide strong evidence that the amyloidogenicity of IAPP is linked to at least two prerequisites: a species-specific amyloidogenic structural motif, and aberrations in the synthesis (or processing) of IAPP which leads to increased concentration of IAPP in the local milieau.     

Secretion of islet amyloid polypeptide in response to glucose

The content of islet amyloid polypeptide (IAPP) in isolated rat pancreatic islets was determined by a radioimmunoassay. Reverse-phase high-performance liquid chromatography analysis revealed that a main peak of IAPP immunoreactivity in the extracts from the islets corresponded to a synthetic rat IAPP. Secretion of IAPP from the cells is regulated by the extracellular glucose concentration. Thus, IAPP may be a novel regulator for glucose homeostasis and changes in the secretion perhaps relate to insular amyloid deposits and impaired glucose tolerance in type 2 diabetes mellitus.     

The beta2-adrenergic modulator celiprolol reduces insulin resistance in obese Zucker rats.

Essential hypertension is associated with an increased incidence of insulin resistance of skeletal muscle glucose transport. The present study determined if celiprolol, an antihypertensive agent with selective beta1-adrenoceptor antagonist and additional beta2-agonistic properties, administered by gavage either acutely (3 hr) or chronically (14 d), had a direct effect on improving glucose tolerance and insulin-stimulated glucose transport activity (using 2-deoxyglucose (2-DG) uptake) in isolated epitrochlearis muscles of the insulin-resistant obese Zucker rat. The effects of a selective beta1-blocker, metoprolol, were also assessed. Acute administration of celiprolol, but not metoprolol, increased insulin-stimulated 2-DG uptake in muscle by 22% (p<0.05). Chronic celiprolol treatment significantly lowered fasting plasma insulin (22%) and free fatty acids (40%) in comparison to obese control values. Moreover, chronic celiprolol administration decreased the glucose-insulin index (calculated as the product of the glucose and insulin areas under the curve during an oral glucose tolerance test), by 32% (p<0.05) compared to obese controls, indicating that peripheral insulin action was increased. Indeed, insulin-stimulated skeletal muscle 2-DG uptake was enhanced by 49% (p<0.05) in these celiprolol-treated obese animals. Metoprolol was without significant effect on any of these variables following chronic administration. These findings indicate that, in this animal model of insulin resistance, the beta1-antagonist/beta2-agonist celiprolol has a specific effect of improving insulin-stimulated skeletal muscle glucose transport that is independent of any hemodynamic alterations.     

Peripheral insulin insensitivity in the hyperglycemic athymic nude mouse: similarity to noninsulin-dependent diabetes mellitus

Previous studies in the homozygous athymic nude mouse (USC colony) have indicated a diabetic state characterized by spontaneous hyperglycemia, abnormal glucose tolerance, and normal or relatively decreased plasma insulin levels. Pancreatic islet cell population assessed by morphometric and immunohistochemical studies demonstrated normal insulin-secreting cells in the hyperglycemic nude mouse. To further elucidate the pathogenesis of the hyperglycemic state in the athymic nude mouse, we have studied the effects of insulin on the transport of glucose in skeletal muscle by measuring basal and insulin-stimulated uptake of a nonmetabolizable glucose analogue, 2-deoxy-D-glucose by using the perfused hindquarter preparation. Although basal 2-deoxy-D-glucose uptake by peripheral skeletal muscle was similar in the hyperglycemic and control mice, the insulin-stimulated uptake of 2-deoxy-D-glucose was significantly decreased in the athymic nude mouse at both 0.1 milliunits/ml and supraphysiologic concentrations of insulin (1 milliunit/ml) when compared with control mice (P less than 0.05 and P less than 0.001, respectively). This form of peripheral insulin insensitivity with normal pancreatic beta cell reserve, in addition to the lean body mass of the diabetic animal, mimics in part the peripheral insulin insensitivity seen in non-insulin-dependent diabetes mellitus.     

High glucose inhibits insulin-stimulated nitric oxide production without reducing endothelial nitric-oxide synthase Ser1177 phosphorylation in human aortic endothelial cells

Recent studies have indicated that insulin activates endothelial nitric-oxide synthase (eNOS) by protein kinase B (PKB)-mediated phosphorylation at Ser1177 in endothelial cells. Because hyperglycemia contributes to endothelial dysfunction and decreased NO availability in types 1 and 2 diabetes mellitus, we have studied the effects of high glucose (25 mM, 48 h) on insulin signaling pathways that regulate NO production in human aortic endothelial cells. High glucose inhibited insulin-stimulated NO synthesis but was without effect on NO synthesis stimulated by increasing intracellular Ca2+ concentration. This was accompanied by reduced expression of IRS-2 and attenuated insulin-stimulated recruitment of PI3K to IRS-1 and IRS-2, yet insulin-stimulated PKB activity and phosphorylation of eNOS at Ser1177 were unaffected. Inhibition of insulin-stimulated NO synthesis by high glucose was unaffected by an inhibitor of PKC. Furthermore, high glucose down-regulated the expression of CAP and Cbl, and insulin-stimulated Cbl phosphorylation, components of an insulin signaling cascade previously characterized in adipocytes. These data suggest that high glucose specifically inhibits insulin-stimulated NO synthesis and down-regulates some aspects of insulin signaling, including the CAP-Cbl signaling pathway, yet this is not a result of reduced PKB-mediated eNOS phosphorylation at Ser1177. Therefore, we propose that phosphorylation of eNOS at Ser1177 is not sufficient to stimulate NO production in cells cultured at 25 mM glucose.     

DPP-4 inhibition improves glucose tolerance and increases insulin and GLP-1 responses to gastric glucose in association with normalized islet topography in mice with beta-cell-specific overexpression of human islet amyloid polypeptide

Inhibition of dipeptidyl peptidase-4 (DPP-4) is currently explored as a novel therapy of type 2 diabetes. The strategy has been shown to improve glycemia in most, but not all, rodent forms of glucose intolerance. In this study, we explored the effects of DPP-4 inhibition in mice with beta-cell overexpression of human islet amyloid polypeptide (IAPP). We therefore administered the orally active and highly selective DPP-4 inhibitor, vildagliptin (3 micromol/mouse daily) to female mice with beta-cell overexpression of human IAPP. Controls were given plain water, and a series of untreated wildtype mice was also included. After five weeks, an intravenous glucose tolerance test showed improved glucose disposal and a markedly enhanced insulin response in mice treated with vildagliptin. After eight weeks, a gastric tolerance test showed that vildagliptin improved glucose tolerance and markedly (approximately ten-fold) augmented the insulin response in association with augmented (approximately five-fold) levels of intact glucagon-like peptide-1 (GLP-1). Furthermore, after nine weeks, islets were isolated. Islets from vildagliptin-treated mice showed augmented glucose-stimulated insulin response and a normalization of the islet insulin content, which was reduced by approximately 50% in transgenic controls versus wildtype animals. Double immunostaining of pancreatic islets for insulin and glucagon revealed that transgenic islets displayed severely disturbed intra-islet topography with frequently observed centrally located alpha-cells. Treatment with vildagliptin restored the islet topography. We therefore conclude that DPP-4 inhibition improves islet function and islet topography in mice with beta-cell specific transgenic overexpression of human IAPP.     

Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes

Blood glucose concentrations are maintained by insulin secreted from beta-cells located in the islets of Langerhans. There are approximately 2000 beta-cells per islet, and approximately one million islets of Langerhans scattered throughout the pancreas. The islet in type 2 diabetes mellitus (T2D) has deficient beta-cell mass due to increased beta-cell apoptosis and islet amyloid derived from islet amyloid polypeptide (IAPP). Accumulating evidence implicates toxic IAPP oligomers in the mediation of beta-cell apoptosis in T2D. Humans, monkeys, and cats express an amyloidogenic toxic form of IAPP and spontaneously develop diabetes characterized by islet amyloid deposits. However, longitudinal studies of islet pathology in humans are impossible, and studies in nonhuman primates and cats are costly and impractical. Rodent IAPP is not amyloidogenic, thus commonly used rodent models of diabetes do not recapitulate islet pathology in humans. To investigate the diabetogenic role of human IAPP (h-IAPP), several mouse models and, more recently, a rat model transgenic for h-IAPP have been developed. Studies in these models have revealed that the toxic effect of h-IAPP on beta-cell apoptosis demonstrates a threshold-dependent effect. Specifically, increasing h-IAPP transgene expression by breeding or induction of insulin resistance leads to increased beta-cell apoptosis and diabetes. These transgenic rodent models for h-IAPP provide an opportunity to elucidate the mechanisms responsible for h-IAPP-induced beta-cell apoptosis further and to test novel approaches to the prevention and treatment of T2D.     

Tissue-specificity of insulin action and resistance

Insulin resistance is the most important pathophysiological feature in many pre-diabetic states. Type 2 diabetes mellitus is a complex metabolic disease and its pathogenesis involves abnormalities in both peripheral insulin action and insulin secretion by pancreatic beta cells. The creation of monogenic or polygenic genetically manipulated mice models in a tissue-specific manner was of great help to elucidate the tissue-specificity of insulin action and its contribution to the overall insulin resistance. However, complete understanding of the molecular bases of the insulin action and resistance requires the identification of the intracellular pathways that regulate insulin-stimulated proliferation, differentiation and metabolism. Accordingly, cell lines derived from insulin target tissues such as brown adipose tissue, liver and beta islets lacking insulin receptors or sensitive candidate genes such as IRS-1, IRS-2, IRS-3, IR and PTP1B were developed. Indeed, these cell lines have been also very useful to understand the tissue-specificity of insulin action and inaction.     

Nicotinamide nucleotide transhydrogenase: a key role in insulin secretion

The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. QTL mapping identified Nicotinamide Nucleotide Transhydrogenase (Nnt), a nuclear-encoded mitochondrial protein thought to be involved in free radical detoxification, as a candidate gene. To investigate its functional role, we used siRNA to knock down Nnt in insulin-secreting MIN6 cells. This produced a dramatic reduction in insulin secretion and the rise in [Ca2+]i evoked by glucose, but not tolbutamide. We identified two ENU-induced point mutations in Nnt (N68K, G745D). Nnt mutant mice were glucose intolerant and secreted less insulin during a glucose tolerance test. Isolated islets showed impaired insulin secretion in response to glucose, but not to tolbutamide, and glucose failed to enhance ATP levels. Glucose utilization and production of reactive oxygen species were increased in Nnt beta cells. We hypothesize that Nnt mutations/deletion uncouple beta cell mitochondrial metabolism leading to less ATP production, enhanced KATP channel activity, and consequently impaired insulin secretion.     

Abnormal glucose homeostasis and pancreatic islet function in mice with inactivation of the Fem1b gene

Type 2 diabetes mellitus is a disorder of glucose homeostasis involving complex gene and environmental interactions that are incompletely understood. Mammalian homologs of nematode sex determination genes have recently been implicated in glucose homeostasis and type 2 diabetes mellitus. These are the Hedgehog receptor Patched and Calpain-10, which have homology to the nematode tra-2 and tra-3 sex determination genes, respectively. Here, we have developed Fem1b knockout (Fem1b-KO) mice, with targeted inactivation of Fem1b, a homolog of the nematode fem-1 sex determination gene. We show that the Fem1b-KO mice display abnormal glucose tolerance and that this is due predominantly to defective glucose-stimulated insulin secretion. Arginine-stimulated insulin secretion is also affected. The Fem1b gene is expressed in pancreatic islets, within both beta cells and non-beta cells, and is highly expressed in INS-1E cells, a pancreatic beta-cell line. In conclusion, these data implicate Fem1b in pancreatic islet function and insulin secretion, strengthening evidence that a genetic pathway homologous to nematode sex determination may be involved in glucose homeostasis and suggesting novel genes and processes as potential candidates in the pathogenesis of diabetes mellitus.     

Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia

Autophagy is a cellular degradation-recycling system for aggregated proteins and damaged organelles. Although dysregulated autophagy is implicated in various diseases including neurodegeneration, its role in pancreatic beta cells and glucose homeostasis has not been described. We produced mice with beta cell-specific deletion of Atg7 (autophagy-related 7). Atg7 mutant mice showed impaired glucose tolerance and decreased serum insulin level. beta cell mass and pancreatic insulin content were reduced because of increased apoptosis and decreased proliferation of beta cells. Physiological studies showed reduced basal and glucose-stimulated insulin secretion and impaired glucose-induced cytosolic Ca2+ transients in autophagy-deficient beta cells. Morphologic analysis revealed accumulation of ubiquitinated protein aggregates colocalized with p62, which was accompanied by mitochondrial swelling, endoplasmic reticulum distension, and vacuolar changes in beta cells. These results suggest that autophagy is necessary to maintain structure, mass and function of pancreatic beta cells, and its impairment causes insulin deficiency and hyperglycemia because of abnormal turnover and function of cellular organelles.     

Development of Type 2 Diabetes Mellitus in the Obese Adolescent:A Growing Challenge

ObjectiveTo describe the metabolic phenotype of type 2 diabetes mellitus in youth and possible metabolic defects leading to its development with particular emphasis on fatty liver.MethodsWe present data gathered from studies performed in obese adolescents across the spectrum of glucose tolerance to assess both alterations in insulin sensitivity and secretion. Discussion regarding treatment options is presented using the data from the Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) study.ResultsAs the number of children with obesity continues to grow, the health implications of the condition are becoming increasingly evident. An unprecedented phenomenon rarely seen before has emerged: type 2 diabetes mellitus. At the time of diabetes diagnosis, cardiovascular disease may already be present, even in young adults. The progression from normal glucose tolerance to type 2 diabetes in adults occurs through an intermediate phase of altered glucose metabolism known as impaired glucose tolerance or prediabetes. Previous studies from our group and others reported a high prevalence of impaired glucose tolerance among children and adolescents with marked obesity. Cross-sectional studies demonstrate that impaired glucose tolerance in obese youth is associated with severe insulin resistance, β-cell dysfunction, and altered abdominal and muscle fat partitioning. We end briefly by discussing the current data available on treatment of this condition from the TODAY study, the largest clinical trial ever performed in youth with type 2 diabetes.ConclusionThe observed rapid progression of the glucose homeostasis alterations in adolescents underlines the importance of focusing attention on the earliest stages of the disease before the onset of any alterations in glucose tolerance. (Endocr Pract. 2012;18:791-795)     

Insulin signaling and glucose transport in insulin resistant human skeletal muscle

Insulin increases glucose uptake and metabolism in skeletal muscle by signal transduction via protein phosphorylation cascades. Insulin action on signal transduction is impaired in skeletal muscle from Type 2 diabetic subjects, underscoring the contribution of molecular defects to the insulin resistant phenotype. This review summarizes recent work to identify downstream intermediates in the insulin signaling pathways governing glucose homeostasis, in an attempt to characterize the molecular mechanism accounting for skeletal muscle insulin resistance in Type 2 diabetes. Furthermore, the effects of pharmaceutical treatment of Type 2 diabetic patients on insulin signaling and glucose uptake are discussed. The identification and characterization of pathways governing insulin action on glucose metabolism will facilitate the development of strategies to improve insulin sensitivity in an effort to prevent and treat Type 2 diabetes mellitus.     

Islet amyloid polypeptide (IAPP) does not inhibit glucose-stimulated insulin secretion from isolated perfused rat pancreas

Islet amyloid polypeptide (IAPP) is a recently discovered pancreatic islet hormone which is stored with insulin in the secretory vesicles of beta cells. Several lines of evidence suggested that IAPP might affect glucose-stimulated insulin secretion and, therefore, might play a role in the development of impaired insulin secretion which is typical of type 2 diabetes. In this study, the effects of human IAPP (amide) on glucose-stimulated insulin secretion was evaluated in the isolated perfused rat pancreas. IAPP in concentrations from 5 x 10(-12) to 10(-7) M had no significant effects on insulin secretion. IAPP, therefore, does not appear to be a significant modulator of glucose-stimulated insulin secretion at concentrations that are physiologically relevant.     

The role of glycogen synthase kinase 3beta in insulin-stimulated glucose metabolism.

To characterize the contribution of glycogen synthase kinase 3beta (GSK3beta) inactivation to insulin-stimulated glucose metabolism, wild-type (WT-GSK), catalytically inactive (KM-GSK), and uninhibitable (S9A-GSK) forms of GSK3beta were expressed in insulin-responsive 3T3-L1 adipocytes using adenovirus technology. WT-GSK, but not KM-GSK, reduced basal and insulin-stimulated glycogen synthase activity without affecting the -fold stimulation of the enzyme by insulin. S9A-GSK similarly decreased cellular glycogen synthase activity, but also partially blocked insulin stimulation of the enzyme. S9A-GSK expression also markedly inhibited insulin stimulation of IRS-1-associated phosphatidylinositol 3-kinase activity, but only weakly inhibited insulin-stimulated Akt/PKB phosphorylation and glucose uptake, with no effect on GLUT4 translocation. To further evaluate the role of GSK3beta in insulin signaling, the GSK3beta inhibitor lithium was used to mimic the consequences of insulin-stimulated GSK3beta inactivation. Although lithium stimulated the incorporation of glucose into glycogen and glycogen synthase enzyme activity, the inhibitor was without effect on GLUT4 translocation and pp70 S6 kinase. Lithium stimulation of glycogen synthesis was insensitive to wortmannin, which is consistent with its acting directly on GSK3beta downstream of phosphatidylinositol 3-kinase. These data support the hypothesis that GSK3beta contributes to insulin regulation of glycogen synthesis, but is not responsible for the increase in glucose transport.     

Effects of chronic N(omega)-nitro-L-arginine methyl ester administration on glucose tolerance and skeletal muscle glucose transport in the rat.

It has been suggested that nitric oxide (NO) is a key regulator of carbohydrate metabolism in skeletal muscle. The present study was undertaken to examine the effects of chronic in vivo competitive antagonism of NO synthase (NOS) by the administration of N(omega)-nitro-L-arginine methyl ester (L-NAME) in the drinking water (1 mg/ml) for 14 days on glucose tolerance and skeletal muscle glucose transport in rats. Oral glucose tolerance tests (OGTT) revealed an impaired glucose tolerance in the L-NAME-treated rats as reflected by the area under the glucose curve (4675 +/- 514 mg% x 120 min (control) vs 6653 +/- 571 mg% x 120 min (L-NAME treated); P < 0.03). While a large rise in plasma insulin concentration was present in the control rats (0.87 +/- 0.34 ng/ml, P < 0.001) during the first 15 min of the OGTT, rises in plasma insulin concentration were absent in the L-NAME-treated rats (0.18 +/- 0.13 ng/ml, P = NS). Intravenous glucose tolerance tests confirmed an impaired insulin secretion in the L-NAME-treated rats. In contrast, insulin-stimulated 2-deoxyglucose transport was enhanced (P < 0.03) by chronic NOS inhibition (5.29 +/- 0.83 nmol/g/min) compared to control rats (2.21 +/- 0.90 nmol/g/min). Plasma sodium concentrations were lower and plasma potassium concentrations were higher in the L-NAME-treated group, indicating an impaired electrolyte status. We conclude that chronic in vivo administration of a NOS inhibitor, while not impairing basal parameters of carbohydrate metabolism, may manifest different responses than acute exposure to the same agent in vitro.     

Time-dependent effects of fatty acids on skeletal muscle metabolism

Increased plasma levels of free fatty acids (FFA) occur in states of insulin resistance such as type 2 diabetes mellitus, obesity, and metabolic syndrome. These high levels of plasma FFA seem to play an important role for the development of insulin resistance but the mechanisms involved are not known. We demonstrated that acute exposure to FFA (1 h) in rat incubated skeletal muscle leads to an increase in the insulin-stimulated glycogen synthesis and glucose oxidation. In conditions of prolonged exposure to FFA, however, the insulin-stimulated glucose uptake and metabolism is impaired in skeletal muscle. In this review, we discuss the differences between the effects of acute and prolonged exposure to FFA on skeletal muscle glucose metabolism and the possible mechanisms involved in the FFA-induced insulin resistance.     

Glucosamine-induced insulin resistance in 3T3-L1 adipocytes

To study molecular mechanisms for glucosamine-induced insulin resistance, we induced complete and reversible insulin resistance in 3T3-L1 adipocytes with glucosamine in a dose- and time-dependent manner (maximal effects at 50 mM glucosamine after 6 h). In these cells, glucosamine impaired insulin-stimulated GLUT-4 translocation. Glucosamine (6 h) did not affect insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 and -2 and weakly, if at all, impaired insulin stimulation of phosphatidylinositol 3-kinase. Glucosamine, however, severely impaired insulin stimulation of Akt. Inhibition of insulin-stimulated glucose transport was correlated with that of Akt activity. In these cells, glucosamine also inhibited insulin stimulation of p70 S6 kinase. Glucosamine did not alter basal glucose transport and insulin stimulation of GLUT-1 translocation and mitogen-activated protein kinase. In summary, glucosamine induced complete and reversible insulin resistance in 3T3-L1 adipocytes. This insulin resistance was accompanied by impaired insulin stimulation of GLUT-4 translocation and Akt activity, without significant impairment of upstream molecules in insulin-signaling pathway.