Yoshimasa Okamoto

An Insulinotropic Effect of Vitamin D Analog with Increasing Intracellular Ca2+ Concentration in Pancreatic β-Cells through Nongenomic Signal Transduction1

The effect of 1α,25-dihydroxylumisterol3 (1α,25(OH)2lumisterol3) on insulin release from rat pancreatic β-cells was measured to investigate the nongenomic action of vitamin D via the putative membrane vitamin D receptor (mVDR). 1α,25(OH)2lumisterol3, a specific agonist of mVDR, dose-dependently augmented 16.7 mm glucose-induced insulin release from rat pancreatic islets and increased the intracellular Ca2+ concentration ([Ca2+]i), though not increasing Ca2+ efficacy in the exocytotic system. These effects were completely abolished by an antagonist of mVDR, 1β,25-dihydroxyvitamin D3 (1β,25(OH)2D3), or by a blocker of voltage-dependent Ca2+ channels, nitrendipine. Moreover, both [Ca2+]i elevation, caused by membrane depolarization, and sufficient intracellular glucose metabolism are required for the expression of these effects. 1α,25(OH)2lumisterol3, therefore, has a rapid insulinotropic effect, through nongenomic signal transduction via mVDR, that would be dependent on the augmentation of Ca2+ influx throu...

Glucose Sensitivity of ATP-Sensitive K+ Channels Is Impaired in β-Cells of the GK Rat: A New Genetic Model of NIDDM

In the Goto-Kakizaki rat, a new genetic model of NIDDM, insulin response to glucose is selectively impaired. To elucidate the mechanism of this abnormality, we studied the properties of ATP-sensitive K+ channels, the inhibition of which is a key step of insulin secretion induced by fuel substrates, using the patch-clamp technique. The glucose-sensitivity of KATP channels was considerably reduced in GK rats. However, the inhibitory effects of ATP on channel activity and unitary conductance were not significantly different between control and GK rats. Thus, it appears that the impaired insulinotropic action of glucose in β-cells of GK rats is attributable to insufficient closure of the KATP channels, probably because of deficient ATP production by impaired glucose metabolism. KATP-channel activities in both control and diabetic β-cells were found to be equally suppressed by glyceraldehyde and 2-ketoisocaproate. These results strongly suggest that the step responsible for the metabolic dysfunction of diabetic β-cells is located within the glycolytic pathway before glyceraldehyde-3-phosphate or in the glycerol phosphate shuttle.

Effects of Troglitazone (CS-045) on insulin secretion in isolated rat pancreatic islets and HIT cells: an insulinotropic mechanism distinct from glibenclamide

SummaryIn order to elucidate the direct effects of (±)-5-[4-(6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-yl-methoxy) benzyl]-2,4-thiazolidinedione (Troglitazone), a newly-developed oral hypoglycaemic agent, on pancreatic beta-cell function, in vitro investigation of isolated rat pancreatic islets and a hamster beta-cell line (HIT cell) were performed. Troglitazone stimulates both glucose, and glibenclamide-induced insulin release at a concentration of 10−6 mol/l in these cells but, conversely, inhibits insulin secretion at 10−4 mol/l. Glucose uptake in HIT cells is similarly enhanced by 10−6 mol/l Troglitazone, but is reduced in the presence of 10−4 mol/l Troglitazone. However, a quantitative immunoblot analysis with a specific antibody for GLUT 2 glucose transporter revealed no significant change in GLUT 2 protein in HIT cells with 10−6 mol/l Troglitazone. Specific binding of [3H]-glibenclamide to beta-cell membranes is replaced by Troglitazone in a non-competitive manner, but 10−6 mol/l Troglitazone failed to eliminate ATP-sensitive K++ channel activity. These results suggest that Troglitazone has a putative non-competitive binding site at, or in the vicinity of, the sulphonylurea receptor in rat pancreatic islets and HIT cells and that the dual effect of Troglitazone on insulin secretory capacity is mediated through the modulation of glucose transport activity, possibly due to the modification of intrinsic activity in glucose transporter in pancreatic beta cells by this novel agent. [Diabetologia (1995) 38: 24–30]

The Novel Insulinotropic Mechanism of Pimobendan: Direct Enhancement of the Exocytotic Process of Insulin Secretory Granules by Increased Ca2+ Sensitivity inβ -Cells1

Pimobendan is a new class of inotropic drug that augments Ca2+ sensitivity and inhibits phosphodiesterase (PDE) activity in cardiomyocytes. To examine the insulinotropic effect of pimobendan in pancreatic beta-cells, which have an intracellular signaling mechanism similar to that of cardiomyocytes, we measured insulin release from rat isolated islets of Langerhans. Pimobendan augmented glucose-induced insulin release in a dose-dependent manner, but did not increase cAMP content in pancreatic islets, indicating that the PDE inhibitory effects may not be important in beta-cells. This agent increased the intracellular Ca2+ concentration ([Ca2+]i) in the presence of 30 mM K+, 16.7 mM glucose, and 200 microM diazoxide, but failed to enhance the 30 mM K+-evoked [Ca2+]i rise in the presence of 3.3 mM glucose. Insulin release evoked by 30 mM K+ in 3.3 mM glucose was augmented. Then, the direct effects of pimobendan on the Ca2+-sensitive exocytotic apparatus were examined using electrically permeabilized islets in which [Ca2+]i can be manipulated. Pimobendan (50 microM) significantly augmented insulin release at 0.32 microM Ca2+, and a lower threshold for Ca2+-induced insulin release was apparent in pimobendan-treated islets. Moreover, 1 microM KN93 (Ca2+/calmodulin-dependent protein kinase II inhibitor) significantly suppressed this augmentation. Pimobendan, therefore, enhances insulin release by directly sensitizing the intracellular Ca2+-sensitive exocytotic mechanism distal to the [Ca2+]i rise. In addition, Ca2+/calmodulin-dependent protein kinase II activation may at least in part be involved in this Ca2+ sensitization for exocytosis of insulin secretory granules.

Role of Cytosolic Ca2+ in Impaired Sensitivity to Glucose of Rat Pancreatic Islets Exposed to High Glucose In Vitro

Sustained exposure to high concentrations of glucose selectively impairs the ability of pancreatic islets to secrete insulin in acute glucose stimulation. In order to evaluate the interrelationship between impaired insulin secretion and the dynamics of the cytosolic free Ca2+ level ([Ca2+]i), we have investigated the effect of high glucose exposure on both [Ca2+]i dynamics in single rat β-cells and insulin release from rat pancreatic islets. Islets cultured at a high glucose concentration (16.7 mM) for 24 h showed significant reductions of the 16.7 mM GSIR compared with islets cultured at a normal glucose concentration (5.5 mM) (3.38 ± 0.24 vs. 4.26 ± 0.34%, respectively, P < 0.05). The capacity of glucose to raise the [Ca2+]i level also was significantly reduced in the β-cells maintained for 24 h at 16.7 mM glucose (P < 0.001). An additional culture in the medium with 5.5 mM glucose for 16 h restored both the GSIR and the [Ca2+]i response of islets cultured at high glucose. On the other hand, insulin release and [Ca2+]i rise in response to 20 mM L-Arg were well preserved. These observations confirm that exposure of pancreatic β-cells to high glucose concentrations induces a selective reduction of the GSIR and, further, shows that this impaired response is reversibly restored by an additional culture with normal glucose. We also suggest that the inability of glucose to provoke a [Ca2+]i rise, which is observed in the β-cells exposed to high glucose, may be responsible for the selective impairment of the GSIR.

Expression of GLUT1 and GLUT2 Glucose Transporter Isoforms in Rat Islets of Langerhans and Their Regulation by Glucose

Previous studies revealed that rat islets express the GLUT2-liver facilitative glucose transporter isoform, a glucose carrier with a low affinity for glucose but a high capacity for glucose transport. These studies indicated the presence of a second glucose transporter in rat islets; however, they did not indicate to which of the five known facilitative glucose transporters it corresponded. In this study, we isolated RNA from rat islets of Langerhans and confirmed the presence of GLUT2 mRNA. In addition, we present dataindicating that the second isoform expressed in islets is the GLUT1-erythrocyte isoform.The effect of culturing islets in 5.5, 8.3, or 11.1 mM glucose on the levels of GLUT1 and GLUT2 mRNA also was examined. The levels of GLUT1 and GLUT2 mRNA were two- and threefold higher, respectively, in islets cultured for 24 h in 11.1 mM glucose compared with those incubated in the presence of 5.5 mM glucose. Therefore, the previously observed increase in GLUT2 mRNA levels in the islets of rats made hyperglycemic by chronic infusion of glucose can be mimicked in vitro, implying that glucose regulates GLUT2 mRNA expression.

Glucagon, insulin and somatostatin secretion in response to sympathetic neural activation in streptozotocin-induced diabetic rats. A study with the isolated perfused rat pancreas in vitro

SummaryChanges in glucagon, insulin and somatostatin secretion induced by electrical splanchnic nerve stimulation were examined in rats treated with streptozotocin as neonates and as adults. In order to study the direct neural effects we used the isolated perfused rat pancreas with intact left splanchnic nerve in vitro. In normal rats splanchnic nerve stimulation causes significant decreases in insulin (30–40%) and somatostatin (30–50%) secretion at both 16.7 mmol/l and 1 mmol/l glucose concentrations. In the neonatal streptozotocin-diabetic rats splanchnic nerve stimulation at 16.7 mmol/l glucose decreased insulin secretion (14%) further than in the control rats (30%), however, somatostatin secretion did not decrease to the same extent. Similar results were also observed at the low (1 mmol/l) glucose concentration. On the other hand, percent decreases of insulin and somatostatin secretion induced by splanchnic nerve stimulation in the streptozotocin-diabetic rats were similar to the values observed in the normal control rats. The glucagon secretion in response to splanchnic nerve stimulation at 16.7 mmol/l glucose from pancreatic Alpha cells in both types of induced diabetes is exaggerated, and the degree of exaggeration seems to parallel the severity of the hyperglycaemia. However, the splanchnic nerve stimulation-induced glucagon secretion at 1 mmol/l glucose was impaired in the streptozotocin-diabetic rats, but not in the neonatal streptozotocin-diabetic rats. These data suggest that the sensitivity of diabetic Alpha and Delta cells to sympathetic neural activation are blunted, whereas the sensitivity of Beta cells is enhanced in the diabetic animal model.

Impaired Glucose Sensitivity of ATP-Sensitive K + Channels in Pancreatic β-cells in Streptozotocin-Induced NIDDM Rats

ATP-sensitive K+ channels (KATP channels) are known to play a key role in the cellular mechanism of insulin secretion from pancreatic β cells. In order to examine the possible impairment of KATP channel function in non-insulin-dependent diabetes mellitus (NIDDM), we have studied the properties of the KATP channels in single β cells of neonatally streptozotocin-induced diabetic rats (NSZ rats) using the patch-clamp technique. The unitary conductance of the channel in diabetic β-cells was virtually identical to that in control beta cells and there was no difference in the sensitivity to ATP and glibenclamide of KATP channels between the NIDDM and control groups. In response to glucose, the activity of the KATP channels was diminished in a dose-dependent manner in both control and diabetic cells. However, the inhibition of the KATP channels in β-cells of NSZ rats was significantly < that in control cells. Even in the presence of 11.1 mM glucose, the openings of a few single KATP channels were consistently observed in cell-attached patch membranes of diabetic, but not control, β-cells. Thus, it appears that the impaired insulinotropic action of glucose in β-cells in NSZ rats is associated with a reduced sensitivity of the KATP channel to glucose, but not to ATP, presumably due to a deficiency in glucose metabolism.

Beneficial effect of T-1095, a selective inhibitor of renal Na+-glucose cotransporters, on metabolic index and insulin secretion in spontaneously diabetic GK rats

1. To investigate the pharmacological effects of T‐1095, this novel derivative of phlorizin was administered to GK rats for 8 weeks. T‐1095 treatment significantly lowered plasma glucose and glycosylated haemoglobin (HbA1c) levels, but did not significantly affect bodyweight.

Hyperresponse in calcium-induced insulin release from electrically permeabilized pancreatic islets of diabetic GK rats and its defective augmentation by glucose

SummaryIn spontaneously diabetic GK rats, insulin secretion from pancreatic beta cells in response to glucose is selectively impaired, probably due to deficient intracellular metabolism of glucose and impaired closure of KATP channels during glucose stimulation. By using electrically permeabilized islets of GK rats, we explored the functional modulations in exocytotic steps distal to the rise in [Ca2+]i in the diabetic condition. At 30 nmol/l Ca2+ (basal conditions) insulin release was similar between GK and non-diabetic control Wistar rats. In response to 3.0 μmol/l Ca2+(maximum stimulatory conditions), insulin release was significantly augmented in permeabilized GK islets (p < 0.01). Raising glucose concentrations from 2.8 to 16.7 mmol/l further augmented insulin release induced by 3.0 μmol/l Ca2+ from permeabilized control islets (p < 0.001), but had no effect on that from permeabilized GK islets. The stimulatory effect of glucose on insulin release from permeabilized control islets was partly inhibited by 2,4-dinitrophenol, an inhibitor of mitochondrial oxidative phosphorylation (p < 0.01). The hyperresponse to Ca2+ in GK islets may play a physiologically compensatory role on the putative functional impairment both in [Ca2+]i rise and energy state in response to glucose in diabetic β cells, and may explain the relative preservation of insulin release induced by non-glucose depolarizing stimuli, such as arginine, from pancreatic islets in non-insulin-dependent diabetes mellitus.