Eri Mukai

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...

Exendin-4 Suppresses Src Activation and Reactive Oxygen Species Production in Diabetic Goto-Kakizaki Rat Islets in an Epac-Dependent Manner

OBJECTIVE Reactive oxygen species (ROS) is one of most important factors in impaired metabolism secretion coupling in pancreatic β-cells. We recently reported that elevated ROS production and impaired ATP production at high glucose in diabetic Goto-Kakizaki (GK) rat islets are effectively ameliorated by Src inhibition, suggesting that Src activity is upregulated. In the present study, we investigated whether the glucagon-like peptide-1 signal regulates Src activity and ameliorates endogenous ROS production and ATP production in GK islets using exendin-4. RESEARCH DESIGN AND METHODS Isolated islets from GK and control Wistar rats were used for immunoblotting analyses and measurements of ROS production and ATP content. Src activity was examined by immunoprecipitation of islet lysates followed by immunoblotting. ROS production was measured with a fluorescent probe using dispersed islet cells. RESULTS Exendin-4 significantly decreased phosphorylation of Src Tyr416, which indicates Src activation, in GK islets under 16.7 mmol/l glucose exposure. Glucose-induced ROS production (16.7 mmol/l) in GK islet cells was significantly decreased by coexposure of exendin-4 as well as PP2, a Src inhibitor. The Src kinase–negative mutant expression in GK islets significantly decreased ROS production induced by high glucose. Exendin-4, as well as PP2, significantly increased impaired ATP elevation by high glucose in GK islets. The decrease in ROS production by exendin-4 was not affected by H-89, a PKA inhibitor, and an Epac-specific cAMP analog (8CPT-2Me-cAMP) significantly decreased Src Tyr416 phosphorylation and ROS production. CONCLUSIONS Exendin-4 decreases endogenous ROS production and increases ATP production in diabetic GK rat islets through suppression of Src activation, dependently on Epac.

GLP-1 receptor antagonist as a potential probe for pancreatic β-cell imaging

We examined exendin(9-39), an antagonist of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R), as a potential probe for imaging of pancreatic beta-cells. To evaluate in vitro receptor specificity, binding assay was performed using dispersed mouse islet cells. Binding assay showed competitive inhibition of [(125)I]BH-exendin(9-39) binding by non-radioactive exendin(9-39). To assess in vivo selectivity, the biodistribution was evaluated by intravenous administration of [(125)I]BH-exendin(9-39) to mice. Radioactivity of harvested pancreas reached highest levels at 60 and 120min among organs examined except lung. Pre-administration of excess non-radioactive exendin(9-39) remarkably and specifically blocked the radioactivity of pancreas. After [(125)I]BH-exendin(9-39) injection into transgenic mice with pancreatic beta-cells expressing GFP, fluorescent and radioactive signals of sections of pancreas were evaluated with an image analyzer. Imaging analysis showed that the fluorescent GFP signals and the radioactive signals were correspondingly located. Thus, the GLP-1R antagonist exendin(9-39) may serve as a useful probe for pancreatic beta-cell imaging.

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.

Metabolic inhibition impairs ATP-sensitive K+ channel block by sulfonylurea in pancreatic β-cells

The effect of metabolic inhibition on the blocking of β-cell ATP-sensitive K+ channels (KATP channels) by glibenclamide was investigated using a patch-clamp technique. Inhibition of KATP channels by glibenclamide was attenuated in the cell-attached mode under metabolic inhibition induced by 2,4-dinitrophenol. Under a low concentration (0.1 μM) of ATP applied in the inside-out mode, KATP channel activity was not fully abolished, even when a high dose of glibenclamide was applied, in contrast to the dose-dependent and complete KATP channel inhibition under 10 μM ATP. On the other hand, cibenzoline, a class Ia antiarrhythmic agent, inhibits KATP channel activity in a dose-dependent manner and completely blocks it, even under metabolic inhibition. In sulfonylurea receptor (SUR1)- and inward rectifier K+ channel (Kir6.2)-expressed proteins, cibenzoline binds directly to Kir6.2, unlike glibenclamide. Thus, KATPchannel inhibition by glibenclamide is impaired under the condition of decreased intracellular ATP in pancreatic β-cells, probably because of a defect in signal transmission between SUR1 and Kir6.2 downstream of the site of sulfonylurea binding to SUR1.

Rapamycin impairs metabolism-secretion coupling in rat pancreatic islets by suppressing carbohydrate metabolism

Rapamycin, an immunosuppressant used in human transplantation, impairs beta-cell function, but the mechanism is unclear. Chronic (24 h) exposure to rapamycin concentration dependently suppressed 16.7 mM glucose-induced insulin release from islets (1.65+/-0.06, 30 nM rapamycin versus 2.35+/-0.11 ng/islet per 30 min, control, n=30, P<0.01) without affecting insulin and DNA contents. Rapamycin also decreased alpha-ketoisocaproate-induced insulin release, suggesting reduced mitochondrial carbohydrate metabolism. ATP content in the presence of 16.7 mM glucose was significantly reduced in rapamycin-treated islets (13.42+/-0.47, rapamycin versus 16.04+/-0.46 pmol/islet, control, n=30, P<0.01). Glucose oxidation, which indicates the velocity of metabolism in the Krebs cycle, was decreased by rapamycin in the presence of 16.7 mM glucose (30.1+/-2.7, rapamycin versus 42.2+/-3.3 pmol/islet per 90 min, control, n=9, P<0.01). Immunoblotting revealed that the expression of complex I, III, IV, and V was not affected by rapamycin. Mitochondrial ATP production indicated that the respiratory chain downstream of complex II was not affected, but that carbohydrate metabolism in the Krebs cycle was reduced by rapamycin. Analysis of enzymes in the Krebs cycle revealed that activity of alpha-ketoglutarate dehydrogenase (KGDH), which catalyzes one of the slowest reactions in the Krebs cycle, was reduced by rapamycin (10.08+/-0.82, rapamycin versus 13.82+/-0.84 nmol/mg mitochondrial protein per min, control, n=5, P<0.01). Considered together, these findings indicate that rapamycin suppresses high glucose-induced insulin secretion from pancreatic islets by reducing mitochondrial ATP production through suppression of carbohydrate metabolism in the Krebs cycle, together with reduced KGDH activity.

Metformin suppresses hepatic gluconeogenesis and lowers fasting blood glucose levels through reactive nitrogen species in mice

Aims/hypothesisMetformin, the major target of which is liver, is commonly used to treat type 2 diabetes. Although metformin activates AMP-activated protein kinase (AMPK) in hepatocytes, the mechanism of activation is still not well known. To investigate AMPK activation by metformin in liver, we examined the role of reactive nitrogen species (RNS) in suppression of hepatic gluconeogenesis.MethodsTo determine RNS, we performed fluorescence examination and immunocytochemical staining in mouse hepatocytes. Since metformin is a mild mitochondrial complex I inhibitor, we compared its effects on suppression of gluconeogenesis, AMPK activation and generation of the RNS peroxynitrite (ONOO−) with those of rotenone, a representative complex I inhibitor. To determine whether endogenous nitric oxide production is required for ONOO− generation and metformin action, we used mice lacking endothelial nitric oxide synthase (eNOS).ResultsMetformin and rotenone significantly decreased gluconeogenesis and increased phosphorylation of AMPK in wild-type mouse hepatocytes. However, unlike rotenone, metformin did not increase the AMP/ATP ratio. It did, however, increase ONOO− generation, whereas rotenone did not. Exposure of eNOS-deficient hepatocytes to metformin did not suppress gluconeogenesis, activate AMPK or increase ONOO− generation. Furthermore, metformin lowered fasting blood glucose levels in wild-type diabetic mice, but not in eNOS-deficient diabetic mice.Conclusions/interpretationActivation of AMPK by metformin is dependent on ONOO−. For metformin action in liver, intra-hepatocellular eNOS is required.

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

Aims/hypothesisNa+/K+-ATPase inhibition by ouabain suppresses ATP production by generating reactive oxygen species (ROS) and impairs glucose-induced insulin secretion from pancreatic islets. To clarify the signal-transducing function of Na+/K+-ATPase in decreasing ATP production by the generation of ROS in pancreatic islets, the involvement of Src was examined. In addition, the significance of Src activation in diabetic islets was examined.MethodsIsolated islets from Wistar rats and diabetic Goto–Kakizaki (GK) rats (a model for diabetes) were used. ROS was measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein fluorescence using dispersed islet cells. After lysates were immunoprecipitated by anti-Src antibody, immunoblotting was performed.ResultsOuabain caused a rapid Tyr418 phosphorylation, indicating activation of Src in the presence of high glucose. The specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) restored the ouabain-induced decrease in ATP content and the increase in ROS production. Both PP2 and ROS scavenger restored the impaired insulin release and impaired ATP elevation in GK islets, but had no such effect in control islets. PP2 reduced the high glucose-induced increase in ROS generation in GK islet cells but had no effect on that in control islet cells. Moreover, ouabain had no effect on ATP content and ROS production in the presence of high glucose in GK islets.Conclusions/interpretationThese results indicate that Src plays a role in the signal-transducing function of Na+/K+-ATPase, in which ROS generation decreases ATP production in control islets. Moreover, ROS generated by Src activation plays an important role in impaired glucose-induced insulin secretion in GK islets, in which Src is endogenously activated independently of ouabain.

Role of mitochondrial phosphate carrier in metabolism-secretion coupling in rat insulinoma cell line INS-1

In pancreatic β-cells, glucose-induced mitochondrial ATP production plays an important role in insulin secretion. The mitochondrial phosphate carrier PiC is a member of the SLC25 (solute carrier family 25) family and transports Pi from the cytosol into the mitochondrial matrix. Since intramitochondrial Pi is an essential substrate for mitochondrial ATP production by complex V (ATP synthase) and affects the activity of the respiratory chain, Pi transport via PiC may be a rate-limiting step for ATP production. We evaluated the role of PiC in metabolism-secretion coupling in pancreatic β-cells using INS-1 cells manipulated to reduce PiC expression by siRNA (small interfering RNA). Consequent reduction of the PiC protein level decreased glucose (10 mM)-stimulated insulin secretion, the ATP:ADP ratio in the presence of 10 mM glucose and elevation of intracellular calcium concentration in response to 10 mM glucose without affecting the mitochondrial membrane potential (Δψm) in INS-1 cells. In experiments using the mitochondrial fraction of INS-1 cells in the presence of 1 mM succinate, PiC down-regulation decreased ATP production at various Pi concentrations ranging from 0.001 to 10 mM, but did not affect Δψm at 3 mM Pi. In conclusion, the Pi supply to mitochondria via PiC plays a critical role in ATP production and metabolism-secretion coupling in INS-1 cells.

Lectin-like oxidized LDL receptor-1 (LOX-1) acts as a receptor for remnant-like lipoprotein particles (RLPs) and mediates RLP-induced migration of vascular smooth muscle cells

OBJECTIVE Remnant-like lipoprotein particles (RLPs) have been implicated in atherogenesis especially by diabetic dyslipidemia; however, their receptor(s) and effects on vascular smooth muscle cells (VSMCs) remain unclear. In this study, we examined if lectin-like oxidized LDL receptor-1 (LOX-1) acts as a receptor for RLPs and its biological effects in VSMCs. METHODS AND RESULTS RLPs were isolated from human plasma by immunoaffinity gel containing anti-apolipoprotein A-I and anti-apolipoprotein B-100 monoclonal antibodies. DiI-labeled RLPs were taken up by CHO-K1 cells stably expressing LOX-1 but not by wild-type CHO-K1 cells. RLPs induced LOX-1 expression and cell migration in bovine VSMCs (BVSMCs), which were significantly suppressed by transfection with LOX-1 specific siRNAs. Inhibitors of metalloproteinases, epidermal growth factor (EGF) receptor tyrosine kinase, heparin-binding EGF-like growth factor (HB-EGF), p38 mitogen-activated protein kinase (p38 MAPK), MAPK kinase (MEK1) and phosphoinositide 3-kinase (PI3K) significantly blocked RLP-induced LOX-1 expression and cell migration of BVSMCs. CONCLUSIONS The present study provides direct evidence that LOX-1 is a novel receptor for RLPs in VSMCs. LOX-1-mediated uptake of RLPs may thus play important roles in atherogenesis by inducing LOX-1 expression and VSMC migration especially in the settings of postprandial hyperlipidemia, diabetes and metabolic syndrome.