Reversal of High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Metformin Combined with PGG, an Inducer of Glycine N-Methyltransferase

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidities and mortality, and no effective drug treatment currently exists. We aimed to develop a novel treatment strategy to induce the expression of glycine N-methyltransferase (GNMT), which is an important enzyme regulating S-adenosylmethionine metabolism whose expression is downregulated in patients with NAFLD. Because 1,2,3,4,6-pentagalloyl glucose (PGG) is a GNMT inducer, and metformin was shown to upregulate liver mitochondrial GNMT protein expression, the effect of PGG and metformin was evaluated. Biochemical analysis, histopathological examination, immunohistochemical staining, reverse transcription-quantitative PCR (RT-qPCR), Western blotting (WB), proteomic analysis and Seahorse XF Cell Mito Stress Test were performed. The high-fat diet (HFD)-induced NAFLD mice were treated with PGG and metformin. Combination of PGG and metformin nearly completely reversed weight gain, elevation of serum aminotransferases, and hepatic steatosis and steatohepatitis. In addition, the downregulated GNMT expression in liver tissues of HFD-induced NAFLD mice was restored. The GNMT expression was further confirmed by RT-qPCR and WB analysis using both in vitro and in vivo systems. In addition, PGG treatment was shown to increase oxygen consumption rate (OCR) maximum capacity in a dose-dependent manner, and was capable of rescuing the suppression of mitochondrial OCR induced by metformin. Proteomic analysis identified increased expression of glutathione S-transferase mu 4 (GSTM4), heat shock protein 72 (HSP72), pyruvate carboxylase (PYC) and 40S ribosomal protein S28 (RS28) in the metformin plus PGG treatment group. Our findings show that GNMT expression plays an important role in the pathogenesis of NAFLD, and combination of an inducer of GNMT and metformin can be of therapeutic potential for patients with NAFLD.