Identification of a novel oncogenic mutation of FGFR4 in gastric cancer.

Gastric cancer remains one of the leading causes of cancer death worldwide. Despite intensive investigations of treatments over the past three decades, the poor prognosis of patients with unresectable advanced or recurrent gastric cancer has not significantly changed, and improved therapies are required. Here, we report the identification of an oncogenic mutation in FGFR4 in a human gastric tumour that leads to constitutive activation of its product, FGFR4. The G636C-FGFR4 tyrosine kinase domain mutation was found in 1 of 83 primary human gastric tumours. The G636C mutation increased FGFR4 autophosphorylation, and activated FGFR4 downstream signalling molecules and enhanced anchorage-independent cell growth when expressed in NIH/3T3 cells. 3D-structural analysis and modelling of FGFR4 suggest that G636C destabilizes an auto-inhibitory conformation and stabilizes an active conformation, leading to increased kinase activation. Ba/F3 cell lines expressing the G636C-FGFR4 mutant were significantly more sensitive to ASP5878, a selective FGFR inhibitor, than the control. Oral administration of ASP5878 significantly inhibited the growth of tumours in mice engrafted with G636C-FGFR4/3T3 cells. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncoprotein. These findings support the therapeutic targeting of FGFR4 in gastric cancer.

ASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4, Inhibits the Growth of FGF19-Expressing Hepatocellular Carcinoma.

Hepatocellular carcinoma is an aggressive cancer with poor prognosis. Fibroblast growth factor 19, a member of the fibroblast growth factor family, is a ligand for fibroblast growth factor receptor 4. Moreover, it plays a crucial role in the progression of hepatocellular carcinoma. ASP5878 is a novel inhibitor of fibroblast growth factor receptors 1, 2, 3, and 4 that is under development. It inhibits fibroblast growth factor receptor 4 kinase activity with an IC50 of 3.5 nmol/L. ASP5878 potently suppressed the growth of the fibroblast growth factor 19-expressing hepatocellular carcinoma cell lines Hep3B2.1-7, HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878 inhibited the phosphorylation of fibroblast growth factor receptor 4 and its downstream signaling molecules as well as induced apoptosis. Oral administration of ASP5878 at 3 mg/kg induced sustained tumor regression in a subcutaneous xenograft mouse model using Hep3B2.1-7. In HuH-7, an orthotopic xenograft mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival of the mice. These results suggest that ASP5878 is a potentially effective therapeutic agent for hepatocellular carcinoma patients with tumors expressing fibroblast growth factor 19. Mol Cancer Ther; 16(1); 68-75. ©2016 AACR.

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

OBJECTIVE: 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. METHODS: Type 1 diabetic rats were prepared by intravenous administration of streptozotocin (STZ). Ipragliflozin was acutely or chronically administered, and therapeutic effects were investigated. KEY FINDINGS: Single administration of ipragliflozin significantly increased urinary glucose excretion, and its effect lasted over 12 h. In addition, ipragliflozin improved glucose tolerance and sustainably reduced hyperglycaemia. Repeated administration of ipragliflozin to diabetic rats for 4 weeks significantly improved not only hyperglycaemia, but also hyperlipidaemia and hepatic steatosis with concomitant increases in urinary glucose excretion. In addition, ipragliflozin ameliorates renal glomerular hyperfiltration and albuminuria. Further, ipragliflozin reduced liver levels of oxidative stress biomarkers and plasma levels of inflammatory markers, and improved liver injury as assessed by plasma levels of aminotransferases. CONCLUSION: These results suggest that SGLT2 selective inhibitor ipragliflozin exerts a beneficial effect on glycaemic control and ameliorates diabetes-associated metabolic abnormalities and complications in STZ-induced diabetic rats, and would be a potential agent for the treatment of type 1 diabetes.

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.

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.

Pharmacological Characterization of YM543, a Newly Synthesized, Orally Active SGLT2 Selective Inhibitor.

BACKGROUND AND AIM: Sodium-glucose cotransporter (SGLT) 2 is a specifically expressed transporter in the kidney that plays an important role in renal glucose reabsorption, and its inhibition may present a novel therapeutic strategy for treating diabetes. Here, we pharmacologically characterized YM543, a newly synthesized SGLT2 selective inhibitor to test this theory. RESULTS: In vitro studies revealed that YM543 potently and selectively inhibited mouse and human SGLT2 activities at nanomolar ranges. In vivo single oral administration of YM543 dose-dependently and significantly reduced blood glucose levels and improved glucose tolerance with a concomitant increase in urinary glucose excretion in KK/Ay type 2 diabetic mice, effects that were sustained even after 12 h. Repeated once-daily oral administration of YM543 for 5 weeks significantly reduced hyperglycemia in type 2 diabetic mice. In addition, combination treatment of YM543 with rosiglitazone or metformin additively improved diabetic symptoms. In contrast, YM543 did not affect normoglycemia at pharmacological doses in normal mice. CONCLUSIONS: Results from the present study suggest that YM543 is an orally active SGLT2 selective inhibitor which reduces hyperglycemia with a concomitant increase in urinary glucose excretion, indicating its promise as an effective treatment against type 2 diabetes.

Antidiabetic effects of SGLT2-selective inhibitor ipragliflozin in streptozotocin-nicotinamide-induced mildly diabetic mice.

Sodium-glucose cotransporter (SGLT) 2 plays an important role in renal glucose reabsorption, and inhibition of renal SGLT2 activity represents an innovative strategy for the treatment of hyperglycemia in diabetic patients. The present study investigated the antidiabetic effects of ipragliflozin, a SGLT2-selective inhibitor, in streptozotocin-nicotinamide-induced mildly diabetic mice, which exhibited a mild decline in glucose tolerance associated with the loss of early-phase insulin secretion. Oral administration of ipragliflozin increased urinary glucose excretion in a dose-dependent manner, an effect which was significant at doses of 0.3 mg/kg or higher and lasted over 12 h. In addition, ipragliflozin dose-dependently improved hyperglycemia and glucose intolerance with concomitant decreases in plasma insulin levels without causing hypoglycemia. Once-daily dosing of ipragliflozin (0.1 - 3 mg/kg) for 4 weeks attenuated hyperglycemia, glucose intolerance, and impaired insulin secretion. These results suggest that the SGLT2-selective inhibitor ipragliflozin increases urinary glucose excretion by inhibiting renal glucose reabsorption, improves hyperglycemia in streptozotocin-nicotinamide-induced mildly diabetic mice, and may be useful for treating type 2 diabetes.

Pharmacological profile of ipragliflozin (ASP1941), a novel selective SGLT2 inhibitor, in vitro and in vivo.

The pharmacological profile of ipragliflozin (ASP1941; (1S)-1,5-anhydro-1-C-{3-[(1-benzothiophen-2-yl)methyl]-4-fluorophenyl}-D: -glucitol compound with L: -proline (1:1)), a novel SGLT2 selective inhibitor, was investigated. In vitro, the potency of ipragliflozin to inhibit SGLT2 and SGLT1 and stability were assessed. In vivo, the pharmacokinetic and pharmacologic profiles of ipragliflozin were investigated in normal mice, streptozotocin-induced type 1 diabetic rats, and KK-A(y) type 2 diabetic mice. Ipragliflozin potently and selectively inhibited human, rat, and mouse SGLT2 at nanomolar ranges and exhibited stability against intestinal glucosidases. Ipragliflozin showed good pharmacokinetic properties following oral dosing, and dose-dependently increased urinary glucose excretion, which lasted for over 12 h in normal mice. Single administration of ipragliflozin resulted in dose-dependent and sustained antihyperglycemic effects in both diabetic models. In addition, once-daily ipragliflozin treatment over 4 weeks improved hyperglycemia with a concomitant increase in urinary glucose excretion in both diabetic models. In contrast, ipragliflozin at pharmacological doses did not affect normoglycemia, as was the case with glibenclamide, and did not influence intestinal glucose absorption and electrolyte balance. These results suggest that ipragliflozin is an orally active SGLT2 selective inhibitor that induces sustained increases in urinary glucose excretion by inhibiting renal glucose reabsorption, with subsequent antihyperglycemic effect and a low risk of hypoglycemia. Ipragliflozin has, therefore, the therapeutic potential to treat hyperglycemia in diabetes by increasing glucose excretion into urine.

Identification of ISG12b as a putative interferon-inducible adipocytokine which is highly expressed in white adipose tissue.

AIM: A number of adipocytokines have been suggested to be involved in the disruption of glucose metabolism, and also in the development of various diabetic complications. We attempted to identify and analyze additional adipocytokines, to better understanding the roles of adipocytes and adipocytokines. METHODS: An oligo-capping signal sequence trap, developed in our laboratory for screening the cDNAs of secretory proteins, was used to sreen cDNAs expressed in mouse white adipose tissue. Profiles of the genes identified in mice and cultured cells were further investigated by northern blotting and luciferase assay. RESULTS: A cDNA fragment of interferon-stimulated gene 12b (ISG12b) was obtained in the search. A northern blot analysis revealed ISG12b to be highly expressed in white adipose tissue. Interferon alpha (IFNalpha) was shown to induce ISG12b expression in the adipose tissue of BL6 mice in vivo, and also in a 3T3-L1 preadipocyte cell line in vitro. The level of ISG12b was higher in mature adipocytes than in preadipocytes. A promoter analysis demonstrated that the 369bp upstream from the transcription initiation site of ISG12b mRNA contain strong promoter activity, and the interferon-stimulated response elements (ISREs) were not present within the 5593bp upstream region. CONCLUSION: ISG12b is an additional candidate for a adipocytokine induced to express in adipose tissue by interferon.

Effects of atorvastatin on glucose metabolism and insulin resistance in KK/Ay mice.

Insulin resistance plays an important role not only in the development and progression of diabetes mellitus but also in the establishment of metabolic syndrome. Improvement of insulin resistance is thus of great importance both in improving glucose metabolism and preventing atherosclerosis. Although HMG-CoA reductase inhibitors appear to favorably affect glucose metabolism, as indicated by the results of a subanalysis in the West of Scotland Coronary Prevention Study (WOSCOPS), their effects on glucose metabolism and insulin resistance have not been thoroughly investigated in animal models. In this study, the effects of atorvastatin on the glucose metabolism and insulin resistance of KK/Ay mice, an animal model of type II diabetes, were investigated. Atorvastatin significantly decreased the non-HDL-cholesterol level in the oral glucose tolerance test, inhibited increase in the 30-min glucose level, decreased plasma insulin levels before and 30 and 60 minutes after glucose loading, and decreased the insulin resistance index, compared with corresponding values in controls, indicating that atorvastatin appeared to improve glucose metabolism by improving insulin resistance. Northern blot analysis revealed decreases in levels of mRNA of sterol regulatory element binding protein-1 (SREBP-1) and glucose-6-phosphatase (G6Pase), and it may play a role in the improvement of glucose metabolism and insulin resistance.

WGEF is a novel RhoGEF expressed in intestine, liver, heart, and kidney.

Rho family GTPases regulate multiple cellular processes through their downstream effectors, where their activities are stimulated by the guanine nucleotide exchange factors. Here, we report a new member of RhoGEF, WGEF, which has the classical structure of DH-PH domain and a C-terminal SH3 domain. WGEF was shown to activate RhoA, Cdc42, and Rac1 by pulldown assay, and forced expression of WGEF resulted in marked rearrangement of the actin cytoskeleton, which is typically seen by the activation of RhoA, Cdc42, and Rac1. WGEF was highly expressed in intestine and also in liver, heart and kidney, which may suggest the involvement of WGEF in the development and functions of these organs. The expression pattern may also suggest the possible importance of WGEF in the understanding of diseases based on metabolic disorder.