Atsuo Tahara

Effects of SGLT2 selective inhibitor ipragliflozin on hyperglycemia, hyperlipidemia, hepatic steatosis, oxidative stress, inflammation, and obesity in type 2 diabetic mice

The sodium-glucose cotransporter 2 (SGLT2) is responsible for most glucose reabsorption in the kidney and has been proposed as a novel therapeutic target for the treatment of type 2 diabetes. In the present study, the therapeutic effects of SGLT2 selective inhibitor ipragliflozin were examined in high-fat diet and streptozotocin-nicotinamide-induced type 2 diabetic mice which exhibit impaired insulin secretion, insulin resistance, hyperlipidemia, hepatic steatosis, and obesity. Single administration of ipragliflozin dose-dependently increased urinary glucose excretion, reduced blood glucose and plasma insulin levels, and improved glucose intolerance. Four-week repeated administration of ipragliflozin improved not only glucose tolerance, hyperglycemia, and hyperinsulinemia but also impaired insulin secretion, hyperlipidemia, hepatic steatosis, and obesity with a concomitant increase in urinary glucose excretion. In addition, ipragliflozin reduced plasma and liver levels of oxidative stress biomarkers (thiobarbituric acid reactive substances and protein carbonyl) and inflammatory markers (interleukin 6, tumor necrosis factor α, monocyte chemotactic protein-1, and c-reactive protein), and improved liver injury as assessed by plasma levels of aminotransferases. These results demonstrate that SGLT2 selective inhibitor ipragliflozin improves not only hyperglycemia but also diabetes/obesity-associated metabolic abnormalities in type 2 diabetic mice and suggest that ipragliflozin may be useful in treating type 2 diabetes with metabolic syndrome.

SGLT2 selective inhibitor ipragliflozin reduces body fat mass by increasing fatty acid oxidation in high-fat diet-induced obese rats

Ipragliflozin is a novel and selective sodium-glucose cotransporter 2 (SGLT2) inhibitor that induces sustained increases in urinary glucose excretion by inhibiting renal glucose reabsorption and thereby exerting a subsequent antihyperglycemic effect. Here, we examined the effect of ipragliflozin on body weight in high-fat diet-induced (HFD) obese rats. Treatment of ipragliflozin (10mg/kg once daily) reduced body weight despite a slight increase in food intake. Dual-energy X-ray absorptiometry and computed tomography demonstrated that the reduction in body weight was accompanied by reduced visceral and subcutaneous fat masses but not lean mass or bone mineral content. Analysis of plasma and urinary parameters suggested the possibility that ipragliflozin enhanced lipolysis and fatty acid oxidation, and indirect calorimetry showed that ipragliflozin decreased the heat production rate from glucose but increased the rate from fat and lowered the respiratory exchange ratio. In conclusion, these data demonstrate that ipragliflozin-induced urinary glucose excretion specifically reduces fat mass with steady calorie loss by promoting the use of fatty acids instead of glucose as an energy source in HFD rats. By improving hyperglycemia and promoting weight reduction, ipragliflozin may prove useful in treating type 2 diabetes in obese individuals.

Effect of YM087, a potent nonpeptide vasopressin antagonist, on vasopressin-induced protein synthesis in neonatal rat cardiomyocyte

OBJECTIVE Hypertrophy of cardiomyocytes may play an important role in the pathogenesis of cardiac hypertrophy associated with various cardiovascular diseases such as congestive heart failure. The aim of this study was to investigate whether vasopressin (AVP) induces protein synthesis in cultured neonatal rat cardiomyocytes through its specific receptor and whether YM087, a newly synthesized nonpeptide AVP receptor antagonist, inhibits AVP-induced protein synthesis in vitro. METHODS AVP receptors on cardiomyocytes were characterized using the radioligand [3H] AVP. The effects of AVP and YM087 on intracellular free calcium concentration ([Ca2+]i), mitogen-activated protein (MAP) kinase and [3H]-leucine incorporation were investigated in cultured neonatal rat cardiomyocytes. RESULTS In cardiomyocytes, Scatchard analysis showed a single population of high-affinity binding sites with the expected AVP V1A receptor subtype profile. YM087 showed high affinity for cardiomyocyte V1A receptors with a Ki value of 0.63 nM. In these same cells, YM087 potently inhibited AVP-induced increases in [CA2+]I and activation of MAP kinase in a concentration-dependent manner. In addition, AVP concentration-dependently stimulated the synthesis of protein without changing the rate of DNA synthesis, and YM087 prevented AVP-induced protein synthesis in a concentration-dependent manner. CONCLUSIONS These results suggest that AVP directly causes protein synthesis and YM087 is a potent inhibitor of AVP-induced protein synthesis of cardiomyocytes and thus may have beneficial effects in the development and regression of cardiomyocytic hypertrophy.

1-desamino-8-D-arginine vasopressin (DDAVP) as an agonist on V1b vasopressin receptor.

1-desamino-8-D-arginine vasopressin (DDAVP) is considered a standard vasopressin V2 receptor-selective agonist with a potent antidiuretic effect through V2 receptor without the induction of vasoconstriction through V1a receptor. Furthermore, DDAVP was reported to act as an agonist on non-V1a, non-V2 receptor to cause the accumulation of intracellular Ca2+ in several tissues. However, the agonistic activity of DDAVP against the other vasopressin receptor, V1b (or V3), which can accumulate intracellular Ca2+ and which we recently cloned, has not been clarified. Hence, we compared the characteristics of DDAVP on V1b receptor with those on the other vasopressin receptors. In binding experiments, DDAVP more strongly inhibited [3H]arginine vasopressin binding to V1b than to V2 receptor (Ki: 5.84 nM vs 65.9 nM). In addition, DDAVP dose-dependently stimulated inositol turnover in human V1b receptor-expressing COS-1 cells. DDAVP acted as a full agonist on human V1b receptor (EC50: 11.4 nM) as well as on human V2 receptor (EC50: 23.9 nM). However, DDAVP behaved as a partial agonist toward rat V1b receptor (intrinsic activity: 0.7, EC50: 43.5 nM), while there was no significant difference in the agonistic properties of arginine vasopressin on human and rat V1b receptor. In conclusion, DDAVP acts as an agonist on V1b receptor, as it does on V2 receptor. These findings will allow us to better understand the physiological role of V1b receptor in pancreatic beta cells and in the renal inner medullary collecting duct, and help us to identify as yet unknown vasopressin receptors through which DDAVP cause the accumulation of intracellular Ca2+ in other tissues.

Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells

1 Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]‐AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2 Scatchard analysis of saturation isotherms for the specific binding of [3H]‐AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nm and a maximum receptor density (Bmax) of 1580 fmol mg−1 protein, 5230 fmol mg−1 protein and 7020 fmol mg−1 protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non‐interacting receptor populations. 3 Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC‐21268, OPC‐31260, SR 49059 and SR 121463A, to inhibit binding of [3H]‐AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC‐31260>SR 121463A>>OPC‐21268 and at hV2 receptors, YM087=SR 121463A>OPC‐31260>SR 49059>>OPC‐21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059>>SR 121463A=YM087=OPC‐31260=OPC‐21268. 4 In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nm and 0.90 nm, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nm. The potency order of antagonists in inhibiting AVP‐induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays. 5 In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.

Discovery and functional characterization of a novel small molecule inhibitor of the intracellular phosphatase, SHIP2

Background and purpose:  The lipid phosphatase known as SH2 domain‐containing inositol 5′‐phosphatase 2 (SHIP2) plays an important role in the regulation of the intracellular insulin signalling pathway. Recent studies have suggested that inhibition of SHIP2 could produce significant benefits in treatment of type 2 diabetes. However, there were no small molecule SHIP2 inhibitors and we, therefore, aimed to identify this type of compound.

Effects of antidiabetic drugs in high-fat diet and streptozotocin–nicotinamide-induced type 2 diabetic mice

Based on previously established methods, we developed an easily available type 2 diabetic mouse model that exhibits obesity and insulin resistance. We investigated the effects of several antidiabetic drugs on this new model, which was induced by a high-fat diet in combination with streptozotocin and nicotinamide injection. Male ICR mice were fed a high-fat diet (45% of calories as fat) for 3weeks and then intraperitoneally administered with nicotinamide (1000mg/kg) and streptozotocin (150mg/kg). These diabetic mice exhibited hyperglycemia and glucose intolerance as a result of the loss of early-phase insulin secretion. The mice also developed significant insulin resistance, hyperlipidemia and obesity. A single dose of mitiglinide, glibenclamide, sitagliptin, insulin, metformin and voglibose significantly improved glucose tolerance during a liquid meal tolerance test. Repeated administration of sitagliptin and rosiglitazone also improved hyperglycemia and insulin resistance. These results demonstrate that a high-fat diet combined with nicotinamide and streptozotocin injection induces a diabetic mouse model that replicates the metabolic characteristics of human type 2 diabetes. This diabetic model, which exhibits impaired insulin secretion, glucose intolerance, insulin resistance, and obesity, may be suitable to evaluate antidiabetic agents for the treatment of type 2 diabetes.

Effects of sodium-glucose cotransporter 2 selective inhibitor ipragliflozin on hyperglycaemia, oxidative stress, inflammation and liver injury in streptozotocin-induced type 1 diabetic rats.

Sodium‐glucose cotransporter (SGLT) 2 plays an important role in renal glucose reabsorption and has been highlighted as a therapeutic target for the treatment of diabetes. Here, we investigated the therapeutic effects of SGLT2 selective inhibitor ipragliflozin in type 1 diabetic rats.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

[3H]‐oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [3H]‐oxytocin to USMC plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high‐affinity binding sites with an apparent equilibrium dissociation constant (Kd) of 0.76 nM and a maximum receptor density (Bmax) of 153 fmol mg−1 protein. The Hill coefficient (nH) did not differ significantly from unity, suggesting binding to homogenous, non‐interacting receptor populations. Competitive inhibition of [3H]‐oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [3H]‐oxytocin in a concentration‐dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu1,6]‐oxytocin>AVP= atosiban>d(CH2)5Tyr(Me)AVP>[Thr4,Gly7]‐oxytocin>dDAVP, and for nonpeptide antagonists was: L‐371257>YM087>SR 49059>OPC‐21268>SR 121463A>OPC‐31260. Oxytocin significantly induced concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L‐371257, potently and concentration‐dependently inhibited oxytocin‐induced [Ca2+]i increase and hyperplasia. In contrast, the V1A receptor selective antagonist, SR 49059, and the V2 receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin‐induced [Ca2+]i increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin‐induced [Ca2+]i increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high‐affinity [3H]‐oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca2+]i increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus.

Pharmacological characterization of YM087, a potent, nonpeptide human vasopressin V1A and V2 receptor antagonist

The effects of YM087 (4’-[(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzazepin-6-yl)-carbonyl]-2-phenylbenzanilide monohydrochloride), a novel nonpeptide vasopressin (AVP) receptor antagonist, on [3H]AVP binding to human AVP receptors (V1A, V1B and V2) cloned and transiently expressed in COS-1 cells generated from monkey renal tissue were studied. Scatchard analysis of saturation isotherms for the specific binding of [3H]AVP to membranes, prepared from COS-1 cells transfected with human V1A, V1B and V2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.67nM, 0.28nM and 2.14nM and a maximum receptor density (Bmax) of 2180fmol/mg protein, 369fmol/mg protein and 2660fmol/mg protein, respectively. YM087 showed high affinity for AVP V1A and V2 receptors with Ki values of 6.3 and 1.1nM, respectively, but had no effect on [3H]AVP binding to AVP V1B receptors.In COS-1 cells expressing either AVP V1A or V1B receptors, AVP caused a concentration-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). YM087 inhibited the AVP-induced increase in [Ca2+]i in COS-1 cells expressing AVP V1A receptors in a concentration-dependent manner with an IC50 value of 14.3nM, but did not influence this increase in AVP V1B-receptor expressing cells. In contrast, stimulation of COS-1 cells expressing AVP V2 receptors resulted in an accumulation of cAMP. YM087 inhibited AVP-induced cAMP production in COS-1 cells expressing AVP V2 receptors in a concentration-dependent manner with an IC50 value of 1.95nM. In all assays used, YM087 was devoid of any agonistic activity.These results suggest that YM087 is a potent nonpeptide dual human AVP V1A and V2 receptor antagonist, and that YM087 will be a powerful tool in investigation of the physiological and pathophysiological roles of AVP.