Takeshi Kurose

Impaired β-cell function and insulin sensitivity in Japanese subjects with normal glucose tolerance

The development of type 2 diabetes mellitus is characterized by both impaired beta-cell function and increasing insulin resistance. To clarify the roles of them in developing type 2 diabetes, we evaluated insulin resistance by HOMA-IR and insulin secretion by HOMA beta-cell in 453 Japanese subjects whose fasting plasma glucose (FPG) and HbA(1c) levels were within normal range. HOMA beta-cell was found to decrease in the over 30 years groups, while HOMA-IR increased with body mass index (BMI). To analyze the reserve capacity of insulin secretion and insulin sensitivity, the 67 of them, who underwent a standard oral glucose tolerance test and were diagnosed with normal glucose tolerance (NGT), were divided into four degrees of BMI age-adjusted to 50 years. They were compared for insulinogenic index and ISI composite proposed by Matsuda and DeFronzo across the range of BMI. ISI composite was significantly less in the highest BMI group, while insulin secretion did not increase in the higher BMI groups. The subjects with higher BMI had remarkably lower insulinogenic indices than those with lower BMI. These data suggest that insulin secretory reserve is insufficient to compensate for increased insulin resistance in Japanese people with NGT at about 50 years of age.

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.

Little enhancement of meal-induced glucagon-like peptide 1 secretion in Japanese: Comparison of type 2 diabetes patients and healthy controls

Although glucose‐dependent insulinotropic polypeptide (GIP) levels have been characterized previously, GLP‐1 levels in Asians remain unclear. Here, we investigate total and intact levels of GLP‐1, as well as GIP during oral glucose and meal tolerance tests (OGTT and MTT) in Japanese patients with or without type 2 diabetes (T2DM). Seventeen Japanese healthy controls and 18 age‐matched and untreated patients with T2DM of short duration participated in the present study. Fasting levels of total GPL‐1 were similar between the two groups (approximately 15 pM), and intact GLP‐1 levels were considerably low in both groups (less than 1 pM). In both groups, total GLP‐1 reached a peak 30 min after glucose ingestion (30–40 pM), whereas intact GLP‐1 levels remained low with no significant peak. In MTT, total and intact GLP‐1 showed no obvious peak. The current data indicate that intact GLP‐1 levels are considerably low in the Japanese and that meal‐induced enhancement of GLP‐1 secretion is negligible in the Japanese. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00010.x, 2010)

Comparison of incretin immunoassays with or without plasma extraction: Incretin secretion in Japanese patients with type 2 diabetes

Aims/Introduction:  The effectiveness of incretin‐based therapies in Asian type 2 diabetes requires investigation of the secretion and metabolism of glucose‐dependent insulinotropic polypepide (GIP) and glucagon‐like peptide 1 (GLP‐1). Plasma extractions have been suggested to reduce variability in intact GLP‐1 levels among individuals by removing interference that affects immunoassays, although no direct demonstration of this method has been reported. We have evaluated the effects of ethanol and solid‐phase extractions on incretin immunoassays. We determined incretin levels during meal tolerance tests in Japanese patients with type 2 diabetes and characterized predictors for incretin secretion.

Localization and Functional Role of Synaptotagmin III in Insulin Secretory Vesicles in Pancreatic β-Cells

Pancreatic β-cells secrete insulin by Ca2+-triggered exocytosis of insulin-containing large dense-core vesicles. Synaptotagmin is a Ca2+/phospholipid-binding protein and is a good candidate for the Ca2+ sensor for exocytosis of synaptic vesicles in neurons. In the present study, we generated a polyclonal antibody against synaptotagmin III, and found that synaptotagmin III immunoreactivity was present at high levels in insulincontaining pancreatic islet cells and insulin-secreting clonal MIN6 cells. In subcellular fractionations of MIN6 cells, synaptotagmin III was recovered in the vesicular fractions containing both insulin and vesicleassociated membrane protein-2 (VAMP-2), but not in synaptophysin-positive fractions. The secretory vesicles immunoprecipitated by anti-VAMP-2 antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MIN6 cells with anti-synaptotagmin III antibody significantly inhibited Ca2+-triggered insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic β-cells, and further strongly suggest that synaptotagmin III is the Ca2+ sensor in the exocytosis of insulin secretory vesicles.

High Prevalence of Autoantibodies Against Carbonic Anhydrase II and Lactoferrin in Type 1 Diabetes : Concept of Autoimmune Exocrinopathy and Endocrinopathy of the Pancreas

Introduction Dysfunction of the exocrine as well as the endocrine pancreas has been reported in type 1 diabetes. Lymphocytic infiltration of the exocrine pancreas is observed in approximately half of Japanese type 1 diabetic patients. Aims To investigate the involvement of autoimmunity against the exocrine pancreas in type 1 diabetes. Methodology We examined autoantibodies against human carbonic anhydrase II (ACA) and lactoferrin (ALF), antigens in the pancreatic duct cells and the pancreatic acinus, respectively, in 43 type 1 diabetic patients and 20 type 2 diabetic patients using the enzyme-linked immunosorbent assay method. Results Of 43 type 1 diabetic patients, ACA was detected in 28 patients (65%) and ALF was detected in 29 patients (67%). One or both of the antibodies were detected in 33 type 1 diabetic patients (77%). In contrast, neither ACA nor ALF were detected in type 2 diabetic patients. Conclusions The high prevalence of both ACA and ALF strongly suggests the involvement of autoimmunity against the exocrine pancreas as well as the endocrine pancreas in some type 1 diabetic patients. We propose that these conditions be referred to as autoimmune exocrinopathy and endocrinopathy of the pancreas.

Early events involved in the development of insulin resistance in Zucker fatty rat.

AIM: To clarify the mechanism by which insulin resistance develops in obesity, Zucker fatty rats (ZFR) and lean litter mates (ZLR) were temporally subjected to oral glucose tolerance tests (OGTT) at 6 and 15 weeks of age.METHOD: As candidates for causative factors of insulin resistance, plasma leptin, free fatty acids (FFA) and tumor necrosis factor (TNF)-α levels were evaluated.RESULTS: There was no difference in the body weight between the two groups at 6 weeks of age, but ZFR were significantly heavier than ZLR at 15 weeks of age. At 6 weeks of age, blood glucose levels and area under the curve of glucose (AUCg) during OGTT were not significantly different between the two groups, while plasma insulin levels and area under the curve of insulin (AUCi) in the ZFR group were significantly higher than those in the ZLR group. At 15 weeks of age, the blood glucose levels and AUCg as well as plasma insulin levels and AUCi in the ZFR group during OGTT were significantly higher than those in the ZLR group. The ratio of fasting insulin to glucose in the ZFR group was significantly higher than that in the ZLR group at 6 and 15 weeks of age. Peripheral and portal plasma leptin and FFA levels were significantly higher in ZFR than ZLR both at 6 weeks and 15 weeks of age. Meanwhile, at 6 weeks, plasma TNF-α levels and expression of TNF-α protein in subcutaneous and visceral fat tissues were similar in both groups; however at 15 weeks, these were significantly higher in the ZFR group than the ZLR group.CONCLUSION: These results suggest that FFA rather than TNF-α may play an important role in early events involved in the development of insulin resistance and TNF-α accelerates insulin resistance together with FFA in the later stage.

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.

Suppressive effect of GABA on insulin secretion from the pancreatic beta-cells in the rat.

In order to investigate a possible role of GABA in the regulation of insulin secretion, we have studied the effect of GABA on insulin secretion from the isolated perfused rat pancreas in vitro and on the changes in the cytoplasmic Ca2+ of Beta-cells from the isolated rat islets. When glucose is present, GABA caused a dose dependent inhibition of the first phase of arginine-induced insulin secretion during the range of 10-1000 microM, but GABA did not affect arginine-induced insulin secretion in the absence of glucose. GABA inhibited not only the first phase but also the second phase of glucose-induced insulin secretion. A GABAB-receptor agonist, baclofen, also inhibited both phases of insulin secretion induced by 16.7 mM glucose. Furthermore, GABA inhibited the rise in cytoplasmic Ca2+ of Beta-cells in response to 16.7 mM glucose. These studies indicate that GABA decreases Beta cell secretory activity mainly in response to glucose. These inhibitory effects of GABA on insulin secretion may be mediated through GABAB-receptor and the inhibition of the rise in cytoplasmic Ca2+.

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.