Yoshiyuki Tsuura

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]

Alterations in basal and glucose-stimulated voltage-dependent Ca2+ channel activities in pancreatic beta cells of non-insulin-dependent diabetes mellitus GK rats.

In genetically occurring non-insulin-dependent diabetes mellitus (NIDDM) model rats (GK rats), the activities of L- and T-type Ca2+ channels in pancreatic beta cells are found to be augmented, by measuring the Ba2+ currents via these channels using whole-cell patch-clamp technique, while the patterns of the current-voltage curves are indistinguishable. The hyper-responsiveness of insulin secretion to nonglucose depolarizing stimuli observed in NIDDM beta cells could be the result, therefore, of increased voltage-dependent Ca2+ channel activity. Perforated patch-clamp recordings reveal that the augmentation of L-type Ca2+ channel activity by glucose is markedly less pronounced in GK beta cells than in control beta cells, while glucose-induced augmentation of T-type Ca2+ channel activity is observed neither in the control nor in the GK beta cells. This lack of glucose-induced augmentation of L-type Ca2+ channel activity in GK beta cells might be causatively related to the selective impairment of glucose-induced insulin secretion in NIDDM beta cells, in conjunction with an insufficient plasma membrane depolarization due to impaired closure of the ATP-sensitive K+ channels caused by the disturbed intracellular glucose metabolism in NIDDM beta cells.

Overexpression of Inducible Cyclic AMP Early Repressor Inhibits Transactivation of Genes and Cell Proliferation in Pancreatic β Cells

ABSTRACT Transcriptional control mediated by the cyclic AMP-responsive element (CRE) represents an important mechanism of gene regulation. To test our hypothesis that increased inducible cyclic AMP early repressor (ICER) Iγ inhibits function of CRE-binding proteins and thus disrupts CRE-mediated transcription in pancreatic β cells, we generated transgenic mice with β-cell-directed expression of ICER Iγ, a powerful repressor that is greatly increased in diabetes. Three transgenic lines clearly show that increased ICER Iγ expression in β cells results in early severe diabetes. From birth islets were severely disorganized with a significantly increased proportion of α cells throughout the islet. Diabetes results from the combined effects of impaired insulin expression and a decreased number of β cells. The decrease in β cells appears to result from impaired proliferation rather than from increased apoptosis after birth. Cyclin A gene expression is impaired by the strong inhibition of ICER; the suppression of cyclin A results in a substantially decreased proliferation of β cells in the postnatal period. These results suggest that CRE and CRE-binding factors have an important role in pancreatic β-cell physiology not only directly by regulation of gene trans-activation but also indirectly by regulation of β-cell mass.

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.

Genomic variation in pancreatic ion channel genes in Japanese type 2 diabetic patients.

Many genetic diseases are caused by mutations in ion channel genes. Because type 2 diabetes is characterized by pancreatic β‐cell insensitivity to glucose, the genes responsible for glucose metabolism and calcium signaling in pancreatic β‐cells are candidate type 2 diabetes susceptibility genes.

Block of pancreatic ATP-sensitive K+ channels and insulinotrophic action by the antiarrhythmic agent, cibenzoline

1 We investigated the effect of cibenzoline (a class Ia antiarrhythmic drug) on basal insulin secretory activity of rat pancreatic islets and ATP‐sensitive K+ channels (KATP) in single pancreatic β cells of the same species, using radioimmunoassay and patch clamp techniques. 2 Micromolar cibenzoline had a dose‐dependent insulinotrophic action with an EC50 of 94.2±46.4 μm;. The compound inhibited the activity of the KATP channel recorded from a single β‐cell in a concentration‐dependent manner. The IC50 was 0.4 μm in the inside‐out mode and 5.2 μm in the cell‐ attached mode, at pH 7.4. 3 In the cell‐attached mode, alkalinization of extracellular solution increased the inhibitory action of cibenzoline and the IC50 was reduced from 26.8 μm at pH 6.2 to 0.9 μm at pH 8.4. On the other hand, the action of cibenzoline in the excised inside‐out mode was acute in onset with a small IC50, indicating that the drug attains its binding site from the cytoplasmic side of the cell membrane. 4 In the inside‐out mode, micromolar ADP reactivated the cibenzoline‐blocked KATP channels in a manner similar to that by which ADP restored ATP‐dependent block of the channel. 5 The binding of [3H]‐glibenclamide to pancreatic islets was inhibited by glibenclamide but not by cibenzoline. In contrast, the [3H]‐cibenzoline binding was displaced by unlabelled cibenzoline but not by glibenclamide. It is concluded that cibenzoline blocks pancreatic KATP channels via a binding site distinct from the sulphonylurea receptor.

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.