Kazuo Tsuji

Characteristic features of insulin secretion in the streptozotocin-induced NIDDM rat model

Male Wistar neonatal rats at age 1.5 days (Streptozotocin [STZ] group 1) and 5 days (STZ group 2) received a subcutaneous injection of 90 mg/kg STZ. After 10 weeks, the rats were subjected to an oral glucose tolerance test (OGTT) (2 g/kg) in a conscious state. The pancreas perfusion experiments were conducted 2 weeks after the OGTT. There was no statistical difference in insulin response between the STZ group 1 and the control group. On the contrary, in the STZ group 2, the plasma glucose response to OGTT showed a typical diabetic pattern, and the plasma insulin response was markedly blunted. In the isolated perfused rat pancreas, the infusion of glucose evoked a biphasic insulin secretion, but the peak insulin levels induced by 16.7 mmol/L glucose in the STZ group 1 were significantly lower than in the controls. We further investigated characteristics of insulin secretion in response to different secretagogues in these animal models using isolated islets. The insulin content of the islets of the STZ group 1 were about one half that of the control group. Insulin secretion in the STZ group 1 was impaired in response to glucose stimulation, but remained normal in response to arginine and forskolin. These results suggest that insulin secretion of non-insulin-dependent diabetes mellitus (NIDDM) rat model is selectively impaired in response to glucose stimulation, possibly due to a disorder of signaling mechanism other than adenylate cyclase.

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.

Selective impairment of the cytoplasmic Ca2+ response to glucose in pancreatic β cells of streptozocin-induced non-insulin-dependent diabetic rats☆

Pancreatic islets from the streptozocin-induced non-insulin-dependent diabetes mellitus (NIDDM) rat model showed a diminished insulin response to 16.7 mmol/L glucose, but the insulin response to arginine remained intact. To evaluate the importance of intracellular calcium concentration ([Ca2+]i) in the diminished insulin response to glucose, the [Ca2+]i of pancreatic beta cells was investigated using fura-2. Glucose produced heterogeneous responses of [Ca2+]i, which were in beta-cell clusters of both the control and NIDDM groups. Many cells showed initial slight decreases of [Ca2+]i, which were followed by gradual and large increments of [Ca2+]i after glucose stimulation of beta cells in the control group. On the other hand, the increase of [Ca2+]i in response to glucose was markedly diminished in beta cells of the NIDDM group compared with controls. The average lag time to [Ca2+]i elevation of beta cells in the NIDDM group was significantly longer than that of the control group. Arginine produced marked increases of [Ca2+]i, in contrast to the effect of glucose stimulation in the NIDDM group. These results suggest that the diminished and delayed [Ca2+]i increases in beta cells of NIDDM rats in response to glucose stimulation are responsible for the selectively impaired insulin response to glucose in the rat model of NIDDM.

Increased calcium-channel currents of pancreatic β cells in neonatally streptozocin-induced diabetic rats

Using a whole-cell patch-clamp technique, voltage-dependent Ca(2+)-channel activities were found to be increased in cultured single beta cells isolated from neonatally streptozocin-induced diabetic rats (NSZ rats). The current-voltage relationship and inactivation time course of Ba2+ currents via L-type Ca2+ channels were indistinguishable between NSZ and control rats. However, the current density observed in NSZ rats was significantly greater than that in control rats. Ba2+ currents via T-type Ca2+ channels were also found to be enhanced in NSZ beta cells. The insulin-secretory capacity of cultured pancreatic islets in response to a depolarizing stimulus (20 mmol/L arginine or 30 mmol/L KCl) in the presence of 11.1 mmol/L glucose was augmented in NSZ rats, whereas that in response to 11.1 and 16.7 mmol/L glucose alone was significantly reduced. It is concluded that the impaired insulinotropic action of glucose in beta cells in NSZ rats is not due to reduced activity of voltage-dependent Ca2+ channels. The fact that insulin secretion induced by a depolarizing stimulus was enhanced in NSZ rats may be related to the augmented activity of the voltage-dependent calcium current found in NSZ beta cells.

Effects of naloxone on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin from the isolated perfused rat stomach

The effects of naloxone, an opiate antagonist, on basal and vagus nerve-induced secretions of GRP, gastrin, and somatostatin were examined using the isolated perfused rat stomach prepared with vagal innervation. Naloxone (10(-6) M) significantly inhibited basal somatostatin secretion in the presence and absence of atropine and of hexamethonium, whereas basal GRP and gastrin secretion was not affected by naloxone. Electrical stimulation (10 Hz, lms duration, 10V) of the distal end of the subdiaphragmatic vagal trunks elicited a significant increase in both GRP and gastrin but a decrease in somatostatin. Naloxone (10(-6) M) failed to affect these responses in the presence or absence of atropine. On the other hand, when hexamethonium was infused, naloxone significantly inhibited both the GRP and gastrin responses to electrical vagal stimulation. Somatostatin secretion was unchanged by vagal stimulation during the infusion of hexamethonium with or without naloxone. These findings suggest that basal somatostatin secretion is under the control of an opiate neuron and that opioid peptides might be involved in vagal regulation of GRP and gastrin secretion.

Effects of oral glucose administration on preproinsulin mRNA in rats in vivo.

An experiment was performed to examine whether the acute increase in insulin release induced by oral glucose ingestion is associated with alterations in pancreatic preproinsulin mRNA (PImRNA) levels. Rats either fed ad libitum or after overnight fast were used. Fasted rats were administered glucose orally in a conscious, unrestrained state. Pancreatic RNA was extracted and the relative level of PImRNA was determined by dot blot analysis with a cloned rat preproinsulin I cDNA probe. In fasted animals PImRNA levels decreased compared with those of fed animals and showed no significant changes after acute glucose administration, while blood glucose and plasma insulin increased rapidly to maximum values at 15 min after glucose administration. Although the transcription rate was not directly measured in this experiment, it is possible that the rapid increase in insulin release from the beta cell under physiological conditions is controlled by post-transcriptional regulations.

Effect of vitamin D on gastrin and gastric somatostatin secretion from the isolated perfused rat stomach

We have studied the role of vitamin D in the regulation of gastrin and gastric somatostatin secretion from the isolated perfused rat stomach. In Ca-deficient vitamin D-deficient rats (Ca(-)D(-) group), the basal and bombesin-stimulated gastrin and gastric somatostatin release (basal IRGa, basal IRS, sigma delta IRGa, and sigma delta IRS) all were significantly lower than in Ca-replete vitamin D-replete rats (Ca(+)D(+) group), and also lower than in Ca-replete vitamin D-deficient rats (Ca(+)D(-) group) except for the basal IRGa. In the Ca(+)D(-) group, the basal IRGa and IRS, and sigma delta IRS were not significantly lower than in the Ca(+)D(+) group. Although there was no significant impairment in basal IRGa, sigma delta IRGa in the Ca(+)D(-) group was significantly lower than in the Ca(+)D(+) control group. Thus, the gastrin and gastric somatostatin secretion from the Ca-deficient vitamin D-deficient rats were impaired. In addition, the impaired gastrin and gastric somatostatin secretions seem to be caused not only by a decrease in serum Ca but also by the reduced effect of the vitamin D on the G and gastric D cells.