Glucose-sensitive Insulin Secretion Research Articles

Glucose metabolites inhibit protein phosphatases and directly promote insulin exocytosis in pancreatic beta-cells.

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion is an early pathogenetic event. Glucose is the cardinal physiological stimulator of insulin secretion from the pancreatic beta-cell, but the mechanisms involved in glucose sensing are not fully understood. Specific ser/thr protein phosphatase (PPase) inactivation by okadaic acid promotes Ca(2+) entry and insulin exocytosis in the beta-cell. We now show that glycolytic and Krebs cycle intermediates, whose concentrations increase upon glucose stimulation, not only dose dependently inhibit ser/thr PPase enzymatic activities, but also directly promote insulin exocytosis from permeabilized beta-cells. Thus, fructose-1,6-bisphosphate, phosphoenolpyruvate, 3-phosphoglycerate, citrate, and oxaloacetate inhibit PPases and significantly enhance insulin exocytosis, nonadditive to that of okadaic acid, at micromolar Ca2+ concentrations. In contrast, the effect of GTP is potentiated by okadaic acid, suggesting that the action of GTP does not require PPase inactivation. We conclude that specific glucose metabolites and GTP inhibit beta-cell PPase activities and directly stimulate Ca2+-independent insulin exocytosis. The glucose metabolites, but not GTP, seem to require PPase inactivation for their stimulatory effect on exocytosis. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by metabolic intermediates, may be a novel regulatory mechanism linking glucose sensing to insulin exocytosis in the beta-cell.

Impaired cyclic AMP response to stimuli in glucose-desensitized rat pancreatic islets

Isolated islets were either studied immediately after isolation (fresh: F), or were cultured for 6 days at 11 mM glucose (desensitized; D), or were incubated for 2 h at 5.5 mM glucose following D (recovered; R). Glucose-stimulated insulin secretion in D islets was reduced compared with F and R islets. In the presence of 3-isobutyl-1-methylxanthine, glucose also increased cyclic adenosine monophosphate (cAMP) levels in F islets, but failed to affect cAMP generation in R or D islets. Glucagon alone or in the presence of glucose stimulated insulin release in F and R islets, but the response was blunted in D islets. Glucagon-like peptide 1 (GLP) potentiated insulin secretion in R islets, but not in D islets. Glucagon (0.01–0.1 μM) did not increase cAMP levels in D islets, whereas GLP (0.1 μM) increased cAMP as much as 4.5-fold. R islets recovered adenylyl cyclase responsivity to glucagon, and GLP increased cAMP levels as much as 9-fold. In F islets preincubated with 11 mM glucose for only 2 h, the cAMP response to GLP was inhibited. In F islets pretreated with forskolin for 2 h, the cAMP responses to glucose and GLP were inhibited. The cAMP response to forskolin stimulation was similarly inhibited in D islets and in islets pretreated for 2 h with forskolin. Forskolin pretreatment significantly attenuated the islet insulin release response to glucose, although the combined stimulus of glucose and GLP restored insulin release to control values. Insulin secretion in response to glucose and the cAMP analogue (Sp)5,6-dichloro-1-β-D-ribofuranosylbenzimidazole-3′-5′-cyclic monophosphorothioate was lower than that observed in F islets. In conclusion, β-cell cAMP accumulation in response to several stimuli acting through different mechanisms is impaired following continuous glucose stimulation. However, cAMP levels are not the definitive second messenger in the recovery of glucose-sensitive insulin secretion in glucose desensitized islets.

High Km of GLUT-2 glucose transporter does not explain its role in insulin secretion

Evidence indicates that the high-Km GLUT-2 function of the islet cells is essential for insulin secretion in response to glucose. To examine possible significance of the high-Km transport function of GLUT-2 in this secretory response, we have studied by computer simulation the effects of high- and low-Km glucose uptake on the steady-state intracellular glucose concentration and glucose phosphorylation in beta-cells. Our computations reveal that both the intracellular glucose concentration and the glucose phosphorylation catalyzed by glucokinase increase significantly as the extracellular glucose concentration increases from 5 to 20 mM, even with a transport Km as low as 1.5 mM, the lowest value known for GLUT-1. Our results indicate that the apparent requirement of GLUT-2 for glucose-sensitive insulin secretion cannot be explained simply by its high-Km transport function alone and suggest that an isoform-specific, direct coupling of GLUT-2 with a certain glycolytic enzyme, such as glucokinase, is essential for the secretory response.

In vitro screening of putative compounds inducing fetal porcine pancreatic beta-cell differentiation: implications for cell transplantation in insulin-dependent diabetes mellitus.

Successful transplantation of fetal pancreatic beta-cells to diabetic recipients requires that differentiation of the immature beta-cells is achieved. Animal experiments have shown that this can occur in vivo, but it would be desirable to induce beta-cell maturation in vitro prior to transplantation. For that purpose the effect of several putative inducers of beta-cell differentiation and/or replication in explant cultures of fetal porcine pancreatic islet-like cell clusters (ICC) were investigated. Initial screening experiments indicated that dexamethasone (DEX; 200 ng/ml) and sodium butyrate (BUT; 2 mM) might promote beta-cell differentiation as evidenced by increased insulin/DNA contents in the ICC. In subsequent experiments these two substances, and also nicotinamide (NIC; 10 mM) which previously has been found to promote fetal beta-cell differentiation, were added alone or in combinations to the basal control medium consisting of RPMI 1640 + 1% human serum. All three test agents alone or in combinations increased the insulin content/DNA of the ICC compared with that of the control group. The combination of NIC+DEX increased the insulin mRNA levels of the ICC. No significant stimulation of insulin release was observed in any test group after short-term incubation with high glucose alone. Addition of 5 mM theophylline to high glucose stimulation, however, increased the insulin secretion in most groups of ICC. Finally, ICC in groups of about 600, which had developed in the presence of NIC or NIC+DEX, were transplanted under the kidney capsule of alloxan-diabetic nude mice. However, neither the time for reversal of diabetes (4 weeks) nor the amount of insulin secretion during perfusion from the grafted ICC were further affected by adding DEX to the NIC supplemented medium. The marked increase of the insulin content of the ICC cultured with DEX supplementation, appeared transient and was not manifested after transplantation. In conclusion, the present study demonstrated that some compounds can stimulate porcine fetal beta-cells in an in vitro system, but in order to attain terminal differentiation of the beta-cells including glucose-sensitive insulin secretion, longer observation periods might be required than used herein. Alternatively, an in vivo environment like that after transplantation is mandatory for this process.

Cellular and molecular biology of the beta cell.

Considerable progress has been made in our understanding of islet-cell function and its relationship to regulation of whole body glucose metabolism. At the genetic level, the regulatory regions in islet-specific genes are being characterised. Transcription factors that interact with these regions have been cloned and these will be instructive in elucidating how islet-specific genes are regulated during development and regeneration. Identification of the enzymes responsible for proteolytic conversion of proinsulin to insulin represents a major advance in understanding prohormone processing. Cleavage of proinsulin is mediated by at least two prohormone convertases (PC3/PC1 and PC2). Their activity is regulated by an acidic gradient between the Golgi and secretory granules and by calcium ions. It is not yet clear how insulin or the PC's are specifically diverted into the regulated secretory pathway. Regulation at this step may be defective in some diabetic patients resulting in relatively elevated circulating proinsulin levels. Specific features of GLUT 2 and glucokinase (GK), proteins that regulate Beta-cell glucose transport and phosphorylation, indicate that these may be key components of the glucose sensor. GLUT 2 is necessary to reconstitute glucose-sensitive insulin secretion in pituitary tumour cells expressing a proinsulin cDNA. Furthermore, the expression of GLUT 2 in Beta cells, but not in hepatocytes, is decreased in diabetes mellitus. However, under normal circumstances GK is probably rate limiting for Beta-cell glucose utilisation. Thus, it is likely that both GLUT 2 and GK determine the set point for glucose-stimulated insulin secretion. Elucidation of distal effectors that regulate insulin secretion is also crucial to our understanding of Beta-cell function.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of insulin secretion by KN-62, a specific inhibitor of the multifunctional Ca 2+/calmodulin-dependent protein kinase II

The effects of KN-62, a specific inhibitor of Ca 2+/calmodulin-dependent protein kinase II (CamPKII), on insulin secretion and protein phosphorylation were studied in rat pancreatic islets and RINm5F cells. KN-62 was found to dose-dependently inhibit autophosphorylation of CamPKII in subcellular preparations of RINm5F cells (K 0.5 = 3.1 ± 0.3 μM), but had no effect on protein kinase C or myosin light chain kinase activity. KN-62, but not the inactive analogue KN-04, dose-dependently inhibited glucose-induced insulin release (K 0.5 = 1.5 ± 0.5 μM) in a manner similar to the inhibition of CamPKII autophosphorylation. KN-62 (10 μM) inhibited carbachol (in the presence of 8 mM glucose) and potassium-stimulated insulin secretion from islets by 53% and 59%, respectively. These results support a role of CamPKII in glucose-sensitive insulin secretion.

Glucose transport and NIDDM.

Three major metabolic abnormalities contribute to hyperglycemia in non-insulin-dependent diabetes mellitus (NIDDM) including defective glucose-induced insulin secretion, elevated rates of hepatic glucose output, and insulin's impaired ability to stimulate glucose uptake in peripheral target tissues (insulin resistance). These functions involve cellular glucose transport in beta-cells, liver, adipose tissue, and skeletal muscle; and, in some instances, abnormalities in glucose transporter isoforms (GLUT) specifically expressed in these tissues may constitute key biochemical lesions underlying defective glucose homeostasis. In animal models of NIDDM, suppression of GLUT2 in beta-cells is correlated with loss of high-Km glucose transport and glucose-sensitive insulin secretion. Although there are no data on humans with NIDDM, GLUT2 loss would constitute an attractive mechanism for defective glucose sensing in beta-cells if it can be shown that transport then becomes rate limiting for glucose metabolism. In the liver, however, hepatocyte glucose transport via GLUT2 probably plays only a permissive role in sustaining increased glucose efflux. Peripheral insulin resistance is associated with decreased glucose transport activity, the likely rate-limiting step for glucose uptake in fat and muscle. Accordingly, the insulin-responsive GLUT4 isoform expressed exclusively in insulin target tissues has been studied intensively in NIDDM. In these studies, pretranslational suppression of GLUT4 appears to be the key mechanism of insulin resistance in adipocytes. However, levels of GLUT4 protein and mRNA are normal in vastus lateralis and rectus abdominis, inferring that defects in GLUT4 functional activity or insulin-mediated translocation cause insulin resistance in muscle. Thus, the intensified study of glucose transport has provided important new insights into NIDDM pathogenesis over the past 5 yr and has presented investigators with additional intriguing hypotheses.

Parallel effects of arachidonic acid on insulin secretion, calmodulin-dependent protein kinase activity and protein kinase C activity in pancreatic islets

A potential role of arachidonic acid in the modulation of insulin secretion was investigated by measuring its effects on calmodulin-dependent protein kinase and protein kinase C in islet subcellular fractions. The results were interpreted in the light of arachidonic acid effects on insulin secretion from intact islets. Arachidonic acid could replace phosphatidylserine in activation of cytosolic protein kinase C (K 0.5 of 10 μM) and maximum activation was observed at 50 μM arachidonate. Arachidonic acid did not affect the Ca 2+ requirement of the phosphatidylserine-stimulated activity. Arachidonic acid (200 μM) inhibited (> 90%) calmodulin-dependent protein kinase activity (K 0.5 = 50–100 μM) but modestly increased basal phosphorylation activity (no added calcium or calmodulin). Arachidonic acid inhibited glucose-sensitive insulin secretion from islets (K 0.5 = 24 μM) measured in static secretion assays. Maximum inhibition (approximately 70%) was achieved at 50–100 μM arachidonic acid. Basal insulin secretion (3 mM glucose) was modestly stimulated by 100 μM arachidonic acid but in a non-saturable manner. In perifusion secretion studies, arachidonic acid (20 μM) had no effect on the first phase of glucose-induced secretion but nearly completely suppressed second phase secretion. At basal glucose (4 mM), arachidonic acid induced a modest but reproducible biphasic insulin secretion response which mimicked glucose-sensitive secretion. However, phosphorylation of an 80 kD protein substrate of protein kinase C was not increased when intact islets were incubated with arachidonic acid, suggesting that the small increases in insulin secretion seen with arachidonic acid were not mediated by protein kinase C. These data suggest that arachidonic acid generated by exposure of islets to glucose may influence insulin secretion by inhibiting the activity of calmodulin-dependent protein kinase but probably has little effect on protein kinase C activity.