Masao Nawano

Insulin Prevents Cardiomyocytes From Oxidative Stress–Induced Apoptosis Through Activation of PI3 Kinase/Akt

BackgroundLoss of cardiomyocytes by apoptosis is proposed to cause heart failure. Reactive oxygen species induce apoptosis in many types of cells including cardiomyocytes. Because insulin has been reported to have protective effects, we examined whether insulin prevents cardiomyocytes from oxidative stress–induced apoptotic death. Methods and ResultsCultured cardiomyocytes of neonatal rats were stimulated by hydrogen peroxide (H2O2). Apoptosis was evaluated by means of the TUNEL method and DNA laddering. Incubation with 100 &mgr;mol/L H2O2 for 24 hours increased the number of TUNEL-positive cardiac myocytes (control, ≈4% versus H2O2, ≈23%). Pretreatment with 10−6 mol/L insulin significantly decreased the number of H2O2-induced TUNEL-positive cardiac myocytes (≈12%) and DNA fragmentation induced by H2O2. Pretreatment with a specific phosphatidylinositol 3 kinase (PI3K) inhibitor, wortmannin, and overexpression of dominant negative mutant of PI3K abolished the cytoprotective effect of insulin. Insulin strongly activated both PI3K and the putative downstream effector Akt. Moreover, a proapoptotic protein, Bad, was significantly phosphorylated and inactivated by insulin through PI3K. ConclusionsThese results suggest that insulin protects cardiomyocytes from oxidative stress–induced apoptosis through the PI3K pathway.

Improved diabetic syndrome in C57BL/KsJ-db/db mice by oral administration of the Na+-glucose cotransporter inhibitor T-1095

The therapeutic effects of an orally active inhibitor of Na+‐glucose cotransporter (SGLT), T‐1095 (a derivative of phlorizin; 3‐(benzo[b]furan‐5‐yl)‐2′,6′‐dihydroxy‐4′‐methylpropiophenone 2′‐O‐(6‐O‐methoxycarbonyl‐β‐D‐glycopyranoside)) were examined in C57BL/KsJ‐db/db (db/db) mice, a genetic animal model of obese type 2 diabetes. The higher renal SGLT activity in db/db mice than normoglycaemic C57BL/KsJ‐db/+m (db/+m) mice may support the rationale for using an SGLT inhibitor in the treatment regimen for type 2 diabetes. Both T‐1095 and its metabolite, T‐1095A, which had approximately 10 times more potency, effectively inhibited renal SGLT activity of these mice in vitro. Single oral administration of T‐1095 (10, 30, 100 mg kg−1, p.o.) to db/db mice caused a dose‐dependent reduction in blood glucose levels and a concomitant increase in glucose excretion into urine. In contrast, T‐1095 only slightly affected blood glucose levels in db/+m mice. Chronic administration of T‐1095 (0.1% w w−1 pellet chow, for 12 weeks) decreased blood glucose and haemoglobin A1C levels, and improved glucose intolerance in db/db mice. The age‐related decrease in plasma insulin levels was markedly inhibited and there was a 2.5 fold increase of insulin content in the pancreas of T‐1095‐treated db/db mice. Food consumption was not changed, while impaired body weight gain was ameliorated by T‐1095 treatment. Both the development of albuminuria and the expansion of glomerular mesangial area in db/db mice were significantly suppressed by chronic T‐1095 treatment, indicating the prevention of the progression of diabetic nephropathy. These results demonstrate that the SGLT inhibitor T‐1095 is able to improve the metabolic abnormalities and inhibit the development of diabetic complications in db/db mice. Thus, T‐1095 can be used for therapy of type 2 diabetic patients.

Enhanced insulin-stimulated activation of phosphatidylinositol 3-kinase in the liver of high-fat-fed rats.

Insulin receptor substrate (IRS)-1 and IRS-2, which mediate phosphatidylinositol (PI) 3-kinase activation, play essential roles in insulin-induced translocation of GLUT4 and in glycogen synthesis. In this study, we investigated the process of PI 3-kinase activation via binding with IRS-1 and -2 in liver, muscle, and fat of high-fat-fed rats, a model of insulin-resistant diabetes. In the liver of high-fat-fed rats, insulin increased the PI 3-kinase regulatory subunit p85alpha and the PI 3-kinase activities associated with IRS-1 3.6- and 2.4-fold, and with IRS-2, 4.7- and 3.0-fold, respectively, compared with those in control rats. The tyrosine phosphorylation levels of IRS-1 and IRS-2 were not significantly altered, however. In contrast with the liver, tyrosine phosphorylation levels and associated PI 3-kinase proteins and activities were decreased in the muscle and adipose tissue of high-fat-fed rats. Thus, high-fat feeding appears to cause insulin resistance in the liver by a mechanism different from the impaired PI 3-kinase activation observed in muscle and adipose tissue. Taking into consideration that hepatic PI 3-kinase activation is severely impaired in obese diabetic models such as Zucker fatty rats, it is possible that the mechanism by which a high-fat diet causes insulin resistance is quite different from that associated with obesity and overeating due to abnormality in the leptin system. This is the first report to show increased PI 3-kinase activation by insulin in an insulin-resistant diabetic animal model. These findings may be important for understanding the mechanism of insulin resistance in human NIDDM, since a high-fat diet is considered to be one of the major factors exacerbating insulin insensitivity in humans.

Imidapril, an angiotensin-converting enzyme inhibitor, improves insulin sensitivity by enhancing signal transduction via insulin receptor substrate proteins and improving vascular resistance in the Zucker fatty rat☆

Angiotensin-converting enzyme (ACE) inhibitors are antihypertensive agents, that inhibit the conversion of angiotensin I to angiotensin II, resulting in smooth-muscle relaxation and a reduction of vascular resistance. Recently, it has been suggested that ACE inhibitors improve insulin resistance in diabetic patients. To investigate the effect of an ACE inhibitor on insulin sensitivity, insulin signaling, and circulation, imidapril was administered orally or intraduodenally to Zucker fatty rats. Oral administration of imidapril improved insulin sensitivity based on the results of an oral glucose tolerance test (OGTT) and a decrease in urinary glucose secretion. Phosphatidylinositol 3-kinase (PI 3-kinase) activity associated with hepatic insulin receptor substrate-1 (IRS-1) in the insulin-stimulated condition was significantly enhanced 110% without a significant alteration in tyrosine phosphorylation of IRS-1 in the imidapril-treated group. In muscle, IRS-1 tyrosine phosphorylation and PI 3-kinase activity associated with IRS-1 in the insulin-stimulated condition were enhanced 70% and 20%, respectively, in the imidapril-treated group. In contrast, an alteration of the IRS-2 pathway was observed only in liver; a significant insulin-induced increase in the IRS-2-associated PI 3-kinase over the basal level was observed in the imidapril-treated group but not in the control. In addition, treatment with imidapril was shown to significantly reduce blood pressure and increase blood flow in the liver and muscle. These results suggest that the ACE inhibitor imidapril may improve insulin sensitivity not only by acting directly on the insulin signaling pathway but also by increasing blood flow in tissues via normalization of vascular resistance, a major cause of hypertension.

SAR, Pharmacokinetics, Safety, and Efficacy of Glucokinase Activating 2-(4-Sulfonylphenyl)-N-thiazol-2-ylacetamides : Discovery of PSN-GK1

Allosteric activators of the glucose-sensing enzyme glucokinase (GK) are currently attracting much interest as potential antidiabetic therapies because they can achieve powerful blood glucose lowering through actions in multiple organs. Here, the optimization of a weakly active high-throughput screening hit to (2 R)-2-(4-cyclopropanesulfonylphenyl)- N-(5-fluorothiazol-2-yl)-3-(tetrahydropyran-4-yl)propionamide (PSN-GK1), a potent GK activator with an improved pharmacokinetic and safety profile, is described. Following oral administration, this compound elicited robust glucose lowering in rats.

p85/p110-type Phosphatidylinositol Kinase Phosphorylates Not Only the D-3, but Also the D-4 Position of the Inositol Ring

Activation of p85/p110-type phosphatidylinositol (PI) kinase has been implicated in various cellular activities. This PI kinase phosphorylates the D-4 position with a similar or higher efficiency than the D-3 position when trichloroacetic acid-treated cell membrane is used as a substrate, although it phosphorylates almost exclusively the D-3 position of the inositol ring in phosphoinositides when purified PI is used as a substrate. Furthermore, the lipid kinase activities of p110 for both the D-3 and D-4 positions were completely abolished by introducing kinase-dead point mutations in their lipid kinase domains (ΔKinα and ΔKinβ, respectively). In addition, both PI 3- and PI 4-kinase activities of p110α and p110β immunoprecipitates were similarly inhibited by either wortmannin or LY294002, specific inhibitors of p110. Insulin induced phosphorylation of not only the D-3 position, but also the D-4 position. Indeed, overexpression of p110 in Sf9 or 3T3-L1 cells induced marked phosphorylation of the D-4 position to a level comparable to or much greater than that of D-3, whereas inhibition of endogenous p85/p110-type PI kinase via overexpression of dominant-negative p85α (Δp85α) in 3T3-L1 adipocytes abolished insulin-induced synthesis of both. Thus, p85/p110-type PI kinase phosphorylates the D-4 position of phosphoinositides more efficiently than the D-3 positionin vivo, and each of the D-3- or D-4-phosphorylated phosphoinositides may transmit signals downstream.

Localization of the histamine H2 receptor, a target for antiulcer drugs, in gastric parietal cells

Background/Aims: Histamine H₂ receptor antagonists are widely used for the treatment of peptic ulcer disorders. However, whether the H₂ receptor is present in parietal or immune cells in the lamina propria remains controversial. This study is designed to determine the H₂ receptor localization immunohistochemically using an antibody against the newly cloned mouse histamine H₂ receptor. Methods: We cloned the mouse histamine H₂ receptor gene and generated a specific antipeptide antibody against the C terminus. Immunohistochemical studies were performed with this antibody and with a monoclonal antibody against H⁺/K⁺ adenosine triphosphatase (ATPase). Results: Histamine H₂ receptors were localized on the plasma membrane and on the cytoplasm just beneath the plasma membrane on the basolateral sides of gastric cells. Confocal microscopy of double-stained sections using the monoclonal antibody against H⁺/K⁺ ATPase, a specific parietal cell marker, showed that histamine H₂ receptors colocalized with H⁺/K⁺ ATPase. No specific histamine H₂ receptor immunoreactivities were observed in the submucosal regions. Conclusion: The H₂ receptor is localized in the gastric parietal cell.