Preventive effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on diethylnitrosamine-induced liver tumorigenesis in obese and diabetic mice

Abstract

Sodium glucose cotransporter 2 inhibitors are expected to ameliorate the abnormalities associated with metabolic syndrome including non-alcoholic fatty liver disease. In this study, we investigated the effects of the sodium glucose cotransporter 2 inhibitor tofogliflozin on the development of non-alcoholic fatty liver disease-related liver tumorigenesis in C57BL/KsJ-+Lepr ^db /+Lepr ^db obese and diabetic mice. The direct effects of tofogliflozin on human liver cancer cell proliferation were also evaluated. Mice were administered diethylnitrosamine-containing water for 2 weeks and were treated with tofogliflozin throughout the experiment. In mice treated with tofogliflozin, the development of hepatic preneoplastic lesions was markedly suppressed, and hepatic steatosis and inflammation significantly reduced, as evaluated using the non-alcoholic fatty liver disease activity score, in comparison with the control mice. Serum levels of glucose and free fatty acid and mRNA expression levels of pro-inflammatory markers in the liver were reduced by tofogliflozin treatment. Conversely, the proliferation of sodium glucose cotransporter 2 protein-expressing liver cancer cells was not inhibited by this agent. These findings suggest that tofogliflozin suppressed the early phase of obesity- and non-alcoholic fatty liver disease-related hepatocarcinogenesis by attenuating chronic inflammation and hepatic steatosis. Therefore, sodium glucose cotransporter 2 inhibitors may have a chemopreventive effect on obesity-related hepatocellular carcinoma.

Keywords: diabetes mellitus; hepatocellular carcinoma (HCC); non-alcoholic fatty liver disease (NAFLD); obesity; sodium glucose cotransporter 2 (SGLT2).

Figure 1. Effects of tofogliflozin on the development of pre-neoplastic lesions and histopathology in the liver of the experimental mice

(A) Representative photomicrographs of hepatic pre-neoplastic lesions, foci of cellular alteration (FCA). (B) Representative photomicrographs of H&E staining at high-power field (upper panels; bars, 200 μm) and low-power field (lower panels; bars, 50 μm) of liver sections from the DEN alone-treated control mice (Ctrl), low-dose tofogliflozin-treated mice (Low), and high-dose tofogliflozin-treated mice (High) at the end of experiment. Ballooned hepatocytes are indicated by the black arrows. (C) Presence of NAFLD activity score (NAS; steatosis, inflammation, and ballooning) was determined using histopathological analysis. Values are expressed as mean ± SD. *P < 0.05.

Figure 2. Effects of tofogliflozin on the expression levels of mRNA involved in inflammation in the liver of the experimental mice

Total RNA was isolated from the livers of the experimental mice, and expression levels of mRNA associated with inflammation (CCL2, F4/80, IL1-β, IL-6, and TNF-α) were determined using quantitative real-time RT-PCR with specific primers. Values are expressed as mean ± SD. *P < 0.05.

Figure 3. Protein expression levels of SGLT2 in human hepatocyte and hepatoma cell lines and effects of tofogliflozin on the proliferation of the HCC cells

(A) Total protein was extracted from cultured cells and equivalent amounts of protein (10 μg/lane) were examined by western blot analysis. Primary antibodies for SGLT2 and GAPDH were used. GAPDH served as a loading control. (B) Cell proliferation assay of Huh7 and JHH7 cells treated with different concentrations of tofogliflozin in vitro. (C) Cell proliferation assay of Huh7 and JHH7 cells treated under normal glucose, high-glucose, high-glucose and high-insulin, and high-glucose and high-insulin plus tofogliflozin conditions. Values are expressed as mean ± SD.