Sodium-glucose co-transporter-2 (SGLT2) inhibitors are new oral antidiabetic drugs that reduce hyperglycemia by promoting urinary glucose excretion. Glycosuria produced by SGLT2 inhibitors is associated with weight loss, mainly due to reduced fat volume. We investigated the effects of empagliflozin (selective SGLT2 inhibitor) and linagliptin (DPP-4 inhibitor) on steatohepatitis and fibrosis in a mouse model of non-alcoholic steatohepatitis (NASH) with diabetes. A novel NASH model was generated by administration of streptozotocin to C57BL/6J mice at 2 days old, with a high-fat diet from 4 weeks. NASH mice aged 6 weeks were divided into four groups of 6 animals: vehicle, linagliptin (10 mg/kg), empagliflozin (10 mg/kg), and linagliptin + empagliflozin. The histological non-alcoholic fatty liver disease activity score was significantly lower in the empagliflozin and linagliptin + empagliflozin groups than in the vehicle or linagliptin groups. Hepatic expression of inflammatory genes (tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1) was decreased in the empagliflozin and linagliptin + empagliflozin groups compared with the vehicle group. The collagen deposition with Sirius red staining was significantly reduced in the linagliptin + empagliflozin group compared with the linagliptin or the empagliflozin group. Immunohistochemistry showed that expression of α-smooth muscle actin, a marker of myofibroblasts (fibrosis), was reduced in the linagliptin + empagliflozin group compared with the vehicle group, as was expression of type 1 and 3 collagen mRNA. Linagliptin + empagliflozin decreased expression of mRNAs for genes related to fatty acid synthesis, but did not increase mRNAs for β-oxidation-related genes. While empagliflozin alone attenuates development of NASH showing anti-steatotic and anti-inflammatory effects, combined administration of empagliflozin and linagliptin can synergistically ameliorates NASH with stronger anti-fibrotic effects.

The aim of the present study is to investigate the effects of canagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, on non-alcoholic steatohepatitis (NASH) and NASH-related hepatocellular carcinoma (HCC) in a mouse model of diabetes and NASH-HCC. First, mice aged five weeks were divided into two groups (vehicle group and canagliflozin group) and were treated for three weeks. Then, mice aged five weeks were divided into three groups of nine animals each: the vehicle group, early canagliflozin group (treated from five to nine weeks), and continuous canagliflozin group (treated from five to 16 weeks). Canagliflozin was administered at a dose of 30 mg/kg in these experiments. In addition, the in vitro effects of canagliflozin were investigated using HepG2 cells, a human HCC cell line. At the age of eight or 16 weeks, the histological non-alcoholic fatty liver disease activity score was lower in the canagliflozin-treated mice than in vehicle-treated mice. There were significantly fewer hepatic tumors in the continuous canagliflozin group than in the vehicle group. Immunohistochemistry showed significantly fewer glutamine synthetase-positive nodules in the continuous canagliflozin group than in the vehicle group. Expression of α-fetoprotein mRNA, a marker of HCC, was downregulated in the continuous canagliflozin group when compared with the vehicle group. At 16 weeks, there was diffuse SGLT1 expression in the hepatic lobules and strong expression by hepatocytes in the vehicle group, while SGLT2 expression was stronger in liver tumors than in the lobules. In the in vitro study, canagliflozin (10 μM) suppressed the proliferation of HepG2 cells. Flow cytometry showed that canagliflozin reduced the percentage of HepG2 cells in the G2/M phase due to arrest in the G1 phase along with decreased expression of cyclin D and Cdk4 proteins, while it increased the percentage of cells in the G0/1 phase. Canagliflozin also induced apoptosis of HepG2 cells via activation of caspase 3. In this mouse model of diabetes and NASH/HCC, canagliflozin showed anti-steatotic and anti-inflammatory effects that attenuated the development of NASH and prevented the progression of NASH to HCC, partly due to the induction of cell cycle arrest and/or apoptosis as well as the reduction of tumor growth through the direct inhibition of SGLT2 in tumor cells.