Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle.

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/- mice (cCx43-/- mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2',7'-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43-/- mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43-/- mice. In cCx43-/- mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43-/- mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43-/- mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

Effect of transient elevation of glucose on contractile properties in non-diabetic rat cardiac muscle.

In non-diabetic patients with severe disease, such as acute myocardial infarction or acute heart failure, admission blood glucose level is associated with their short-term and long-term mortality. We examined whether transient elevation of glucose affects contractile properties in non-diabetic hearts. Force, intracellular Ca2+ ([Ca2+]i), and sarcomere length were measured in trabeculae from rat hearts. To assess contractile properties, maximum velocity of contraction (Max dF/dt) and minimum velocity of relaxation (Min dF/dt) were calculated. The ratio of phosphorylated troponin I (P-TnI) to troponin I (TnI) was measured. One hour after elevation of glucose from 150 to 400 mg/dL, developed force, Max dF/dt, and Min dF/dt were reduced without changes in [Ca2+]i transients at 2.5 Hz stimulation and 2.0 mM [Ca2+]o, while developed force and [Ca2+]i transients showed no changes at 0.5 Hz stimulation and 0.7 mM [Ca2+]o. In the presence of 1 µM KN-93, a Ca2+/calmodulin-dependent protein kinaseII (CaMKII) inhibitor, or 50 µM diazo-5-oxonorleucine, a L-glutamine-D-fructose-6-phosphate amidotransferase inhibitor, the reduction of contractile properties after elevation of glucose was suppressed. Furthermore, 1 h after elevation of glucose to 400 mg/dL at 2.0 mM [Ca2+]o, the ratio of P-TnI to TnI was increased. These results suggest that in non-diabetic hearts under higher Ca2+-load, transient elevation of glucose for 1 h reduces contractile properties probably by activating CaMKII through O-GlcNAcylation. Thus, in the patients with severe disease, transient elevation of blood glucose, such as due to stress, may worsen cardiac function and thereby affect their mortality without known diabetes.

Chronic inhibition of dipeptidyl peptidase-IV with ASP8497 improved the HbA(1c) level, glucose intolerance, and lipid parameter level in streptozotocin-nicotinamide-induced diabetic mice.

Dipeptidyl peptidase-IV (DPP-IV) is the primary inactivator of glucoregulatory incretin hormones, and DPP-IV inhibitors are expected to become a useful new class of anti-diabetic agent. The aim of the present study is to characterize the chronic in vivo profile of the DPP-IV inhibitor ASP8497. In streptozotocin-nicotinamide-induced diabetic mice, ASP8497 was administered orally for 3 weeks at 1, 3, or 10 mg/kg once daily, which improved the hemoglobin A(1c), non-fasting plasma insulin, fasting blood glucose levels, glucose intolerance, and lipid profiles (plasma triglyceride, non-esterified fatty acid and total cholesterol) with neutral effect on body weight. The pancreatic insulin content and hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity recovered dose-dependently in ASP8497-treated groups. These results revealed that ASP8497 was successful in improving glycemic control and metabolic parameters in streptozotocin-nicotinamide-induced diabetic mice. It is therefore suggested that ASP8497 may be a potential agent for the treatment of type 2 diabetes.

Evaluation of the antidiabetic effects of dipeptidyl peptidase-IV inhibitor ASP8497 in streptozotocin-nicotinamide-induced mildly diabetic mice.

Gastrointestinal hormone glucagon-like peptide-1 (GLP-1) has a potent glucose-dependent insulinotropic effect and is rapidly degraded by dipeptidyl peptidase (DPP)-IV. Therefore, the use of DPP-IV inhibitors is being actively explored as a novel approach to the treatment of type 2 diabetes. The present study investigated the antidiabetic effects of the DPP-IV inhibitor ASP8497 in streptozotocin-nicotinamide-induced mildly diabetic mice which possess aggravation of glucose tolerance due to loss of early-phase insulin secretion. ASP8497 exhibited good oral bioavailability with potent inhibition of plasma DPP-IV activity. This inhibitory activity lasted up to 24 h when administered at 5 mg/kg twice a day or 10 mg/kg once a day. A single oral administration of ASP8497 (0.3-3 mg/kg) significantly improved glucose tolerance by increasing plasma insulin and GLP-1 levels during the oral glucose or liquid meal tolerance tests. These effects were seen not only immediately, but also 8 h after administration. In contrast, ASP8497 (0.3-10 mg/kg) had no significant effect on blood glucose and plasma insulin levels under fasting conditions. Furthermore, repeated administration of ASP8497 (5 mg/kg twice a day or 10 mg/kg once a day) for 25 days significantly decreased nonfasting blood glucose and HbA(1c) levels. These results suggest that ASP8497 is a potent and long-acting DPP-IV inhibitor that improves glucose tolerance through glucose-dependent insulinotropic action via the elevation of the GLP-1 level in streptozotocin-nicotinamide-induced mildly diabetic mice. It is expected to be useful as a therapeutic agent for impaired glucose tolerance and type 2 diabetes.

Pharmacological profile of ASP8497, a novel, selective, and competitive dipeptidyl peptidase-IV inhibitor, in vitro and in vivo.

Dipeptidyl peptidase (DPP)-IV is involved in the inactivation of glucagon-like peptide-1 (GLP-1), a potent insulinotropic peptide. Thus, DPP-IV inhibitors are expected to become a useful new class of antidiabetic agent. This report describes the pharmacological profile of the novel DPP-IV inhibitor, ASP8497 [(2S,4S)-4-fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate], both in vitro and in vivo. ASP8497 inhibited DPP-IV in plasma from mice, dogs, and humans with median inhibition concentration (IC(50)) values of 2.6 nM, 7.3 nM, and 6.2 nM, respectively. In contrast, ASP8497 did not potently inhibit human DPP8 or DPP9 activity (IC(50)=1,700 nM and 100 nM, respectively) and exhibited selectivity against several proteases, including proline-specific proteases (IC(50)>10 microM). Kinetic analysis indicated that ASP8497 is a competitive DPP-IV inhibitor. In normal mice, ASP8497 inhibited plasma DPP-IV activity even 12 h after administration. ASP8497 significantly inhibited increases in the blood glucose level during the oral glucose tolerance test (OGTT) conducted 0.5 h after administration. This was accompanied by increases in the plasma active GLP-1 and insulin levels. In addition, ASP8497 significantly inhibited increases in the blood glucose level during the OGTT conducted 8 h after administration. Furthermore, in Zucker fatty rats, ASP8497 dose dependently improved glucose tolerance with significance at doses of 1 mg/kg or higher. In contrast, ASP8497 did not cause hypoglycemia in fasted normal mice. These results indicate that ASP8497 is a potent, competitive, and selective DPP-IV inhibitor with antihyperglycemic activity.

Ipragliflozin, an SGLT2 inhibitor, exhibits a prophylactic effect on hepatic steatosis and fibrosis induced by choline-deficient l-amino acid-defined diet in rats.

Ipragliflozin is a selective sodium glucose cotransporter 2 (SGLT2) inhibitor that increases urinary glucose excretion by inhibiting renal glucose reabsorption and thereby causes a subsequent antihyperglycemic effect. As nonalcoholic fatty liver disease (NAFLD), including nonalcoholic steatohepatitis (NASH), is closely linked to metabolic diseases such as obesity and diabetes, we investigated the effect of ipragliflozin on NAFLD in rats fed a choline-deficient l-amino acid-defined (CDAA) diet. Five weeks after starting the CDAA diet, rats exhibited hepatic triglyceride (TG) accumulation, fibrosis, and mild inflammation. Repeated oral administration of ipragliflozin (3mg/g, once daily for 5 weeks) prevented both hepatic TG accumulation (188 vs.290 mg/g tissue vehicle-treated group; P<0.001) and large lipid droplet formation. Further, ipragliflozin exerted a prophylactic effect on liver fibrosis, as indicated by a marked decrease in hydroxyproline content and fibrosis score. Pioglitazone, which is known to be effective on hepatic fibrosis in CDAA diet-fed rats as well as NASH patients with type 2 diabetes mellitus (T2DM), also exerted a mild prophylactic effect on fibrosis, but not on hepatic TG accumulation or inflammation. In conclusion, ipragliflozin prevented hepatic TG accumulation and fibrosis in CDAA-diet rats. These findings suggest the therapeutic potential of ipragliflozin for patients with NAFLD.

Antihyperglycemic effects of ASP8497 in streptozotocin-nicotinamide induced diabetic rats: comparison with other dipeptidyl peptidase-IV inhibitors.

(2S,4S)-4-Fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate (ASP8497) is a novel dipeptidyl peptidase (DPP)-IV inhibitor. In this study, we investigated the antidiabetic potency, mechanism, and duration of action of ASP8497 both in vitro and in vivo, and compared it with the DPP-IV inhibitors vildagliptin, sitagliptin, and saxagliptin. ASP8497 inhibited rat plasma DPP-IV activity in vitro with an IC(50) value of 2.96 nmol/l, while those for vildagliptin, sitagliptin, and saxagliptin were 2.12, 8.98, and 2.00 nmol/l, respectively. In rats that had streptozotocin-nicotinamide-induced, mildly diabetes, oral administration of ASP8497 dose-dependently and sustainably inhibited plasma DPP-IV activity. In addition, ASP8497 dose-dependently and significantly improved glucose tolerance with a concomitant increase in plasma glucagon-like peptide 1 (GLP-1) and insulin levels at both 0.5 h and 8 h after dosing. The order of both potency and duration of action for plasma DPP-IV inhibition and glucose tolerance improvement was as follows: saxagliptin > ASP8497 = vildagliptin = sitagliptin. These results suggest that ASP8497 exerts a potent and long-acting DPP-IV inhibitory effect and improves glucose tolerance through glucose-dependent insulinotropic action via elevation of the GLP-1 level in streptozotocin-nicotinamide-induced mildly diabetic rats. This compound is expected to be useful as a therapeutic agent for impaired glucose tolerance and type 2 diabetes.

Effects of the combination of dipeptidyl peptidase-IV inhibitor ASP8497 and antidiabetic drugs in streptozotocin-nicotinamide-induced mildly diabetic mice.

Several combination therapies have been investigated in an effort to achieve and maintain glycemic control in patients with type 2 diabetes. In this study, we combined the novel dipeptidyl peptidase (DPP)-IV inhibitor ASP8497 with the antidiabetic drugs metformin, glibenclamide, voglibose, rosiglitazone, and insulin to examine the effects of each combination on glucose tolerance in streptozotocin-nicotinamide-induced mildly diabetic mice. Single treatments with ASP8497 (1 mg/kg), metformin (300 mg/kg), glibenclamide (3 mg/kg), voglibose (0.3 mg/kg), rosiglitazone (10 mg/kg), or insulin (0.2 IU/kg) significantly improved glucose tolerance during the liquid meal tolerance test. In addition, combination treatment with ASP8497 and each antidiabetic drug additively improved glucose tolerance. Among these, the combination of ASP8497 and glibenclamide or insulin additively ameliorated glucose tolerance with an additive increase in the plasma insulin level; however, it did not affect the fasting blood glucose lowering effects of glibenclamide or insulin. These profiles indicate that the combination of ASP8497 with existing antidiabetic drugs could be useful for correcting the postprandial hyperglycemia seen with type 2 diabetes.

Antidiabetic effects of dipeptidyl peptidase-IV inhibitors and sulfonylureas in streptozotocin-nicotinamide-induced mildly diabetic mice.

The present study investigated the antidiabetic effects of the dipeptidyl peptidase (DPP)-IV inhibitors ASP8497 and vildagliptin, and the sulfonylureas glibenclamide and gliclazide in streptozotocin-nicotinamide-induced mildly diabetic mice. A single administration of ASP8497 and vildagliptin significantly improved glucose tolerance by increasing plasma insulin and glucagon-like peptide-1 levels. In addition, a single administration of glibenclamide and gliclazide also caused significant improvement in glucose tolerance with an accompanying increase in the plasma insulin level. Subsequently, the effects of a 1-week chronic daily dosing of DPP-IV inhibitors and sulfonylureas were investigated. All drugs significantly improved glucose tolerance on day 1 of chronic daily dosing. After 1 week of chronic daily dosing, the DPP-IV inhibitors caused a significant improvement in glucose tolerance similar to those observed on day 1 by increasing the plasma insulin and glucagon-like peptide-1 levels. In contrast, the sulfonylureas had no significant improving or insulinotropic effect. Furthermore, ASP8497 also had an antihyperglycemic effect and improved pancreatic histopathologic lesions in a 4-week chronic daily dosing study. These results suggest that chronic daily dosing of sulfonylureas had virtually no antidiabetic effects because of marked attenuation of the insulinotropic action in streptozotocin-nicotinamide-induced mildly diabetic mice. In contrast, the antidiabetic efficacy of DPP-IV inhibitors, including ASP8497, did not change even after chronic daily dosing; therefore, DPP-IV inhibitors are useful as a therapeutic agent for impaired glucose tolerance and type 2 diabetes mellitus.

Effects of the dipeptidyl peptidase-IV inhibitor ASP8497 on glucose tolerance in animal models of secondary failure.

UNLABELLED: Sulfonylureas promote insulin secretion and potently lower blood glucose levels, however, they induce hypoglycemia and undergo a reduction in efficacy when administered long-term (secondary failure). The dipeptidyl peptidase (DPP)-IV inhibitor ASP8497, (2S,4S)-4-fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate, inhibits the degradation of glucagon-like peptide-1 (GLP-1), an incretin hormone, and promotes insulin secretion in a glucose-dependent manner. ASP8497 is therefore less likely to induce hypoglycemia and less likely to show reduced efficacy even after repeated administration. Here, to determine whether or not ASP8497 improves glucose tolerance in Zucker fatty rats, we examined the effects of ASP8497 and gliclazide, a sulfonylurea, on glucose tolerance after repeated administration. We also developed an animal model of secondary failure using streptozotocin-nicotinamide-induced diabetic mice. RESULTS: ASP8497 (3mg/kg) improved glucose intolerance in Zucker fatty rat without any attenuation (blood glucose AUC: P=0.034 vs. vehicle) while gliclazide (10mg/kg) did not (P=0.916 vs. vehicle). Furthermore, ASP8497 (3, 10mg/kg) was found to effect glucose tolerance dose-dependently (3mg/kg: P=0.230, 10mg/kg: P=0.003 vs. glibenclamide (10mg/kg)) by enhancing insulin secretion in mice inadequately controlled with glibenclamide. Our results suggest that ASP8497 may be effective even in patients with secondary failure who are unable to maintain satisfactory glycemic control using sulfonylureas.

Hypoglycaemic effects of antidiabetic drugs in streptozotocin-nicotinamide-induced mildly diabetic and streptozotocin-induced severely diabetic rats.

In this study, streptozotocin-induced severely diabetic rats and streptozotocin-nicotinamide-induced mildly diabetic rats were established to compare their characteristics and to investigate the hypoglycaemic effects of antidiabetic drugs. Results show that in streptozotocin-induced severely diabetic rats, the pancreatic insulin content decreased to approximately 10% of that in normal rats. These severely diabetic rats also exhibited marked hyperglycaemia and impaired glucose tolerance due to insulin secretory deficiency. In contrast, the pancreatic insulin content was approximately 50% of normal levels in streptozotocin-nicotinamide-induced mildly diabetic rats. These mildly diabetic rats exhibited moderate hyperglycaemia and impaired glucose tolerance due to loss of early-phase insulin secretion. Voglibose (alpha-glucosidase inhibitor), metformin (biguanide), glibenclamide (sulfonylurea), sitagliptin (dipeptidyl peptidase-4 inhibitor) and insulin significantly improved glucose tolerance in streptozotocin-nicotinamide-induced mildly diabetic rats. In contrast, in streptozotocin-induced severely diabetic rats, voglibose, metformin and insulin significantly improved glucose tolerance, but no significant effect was observed for glibenclamide and sitagliptin due to a decreased insulinotropic effect. These results suggest that streptozotocin-nicotinamide-induced mildly diabetic rats, which exhibit a mild decline in glucose tolerance due to loss of early-phase insulin secretion, are sensitive to the hypoglycaemic effects of insulinotropic agents and have many pathological features resembling type 2 diabetes, which may be useful in the pharmacological investigation of numerous antidiabetic drugs.

ASP8497 is a novel selective and competitive dipeptidyl peptidase-IV inhibitor with antihyperglycemic activity.

Dipeptidyl peptidase (DPP)-IV inhibitors are expected to become a useful new class of antidiabetic agent. The aim of the present study is to characterize the in vitro and in vivo profile of ASP8497, (2S,4S)-4-fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate, which is a novel DPP-IV inhibitor. ASP8497 inhibited DPP-IV in plasma from mice, rats, dogs and humans with IC(50) values of 3.86, 2.36, 5.53 and 5.30 nM, respectively. In contrast, ASP8497 did not potently inhibit DPP8 or DPP9 activity (IC(50)>200 nM). Kinetic analysis indicated that ASP8497 inhibits DPP-IV activity in a competitive manner. In streptozotocin-nicotinamide-induced diabetic mice, ASP8497 (3 mg/kg) significantly reduced glucose excursion during the oral glucose tolerance test conducted 0.5 and 8.5 h after administration, with increases in plasma insulin and active glucagon-like peptide-1 (GLP-1) levels. In contrast, ASP8497 (3 and 30 mg/kg) did not cause hypoglycemia in fasted normal mice. Furthermore, administration of exogenous GLP-1 induced significant inhibition of gastric emptying and small intestinal transit rates, but ASP8497 (30 mg/kg) had no significant effects in normal mice. These present preclinical studies indicate that ASP8497 is a novel selective DPP-IV inhibitor with long-acting antidiabetic effect that might be a potential agent for type 2 diabetes.