Fig 2: Proposed mechanism for metformin-alleviated steatohepatitis. A SIRT1-dependent cholesterol catabolism and M1 macrophages polarization are involved in metformin-induced alleviation of steatohepatitis in DIO mice. Metformin upregulates CH25H and may result in increased level of 25-HC, which has been reported to inhibit IL-1ß-driven inflammation, thus ameliorates hepatic inflammation. On the other hand, metformin also downregulates the M1 macrophage markers, which suggests that metformin may inhibit the M1 macrophages polarization to alleviate steatohepatitis. Importantly, SIRT1 deficiency impairs the beneficial effects of metformin on steatohepatitis.

Fig 3: SIRT1 deficiency impairs the effect of metformin on regulating cholesterol catabolism. Metformin reduced (A) hepatic cholesterol content in DIO mice (p < 0.05). (B) The relative mRNA level of CYP7B1 in the liver of DIO mice was reduced by metformin (p < 0.05). (C,D) The relative protein expression of CH25H was significantly upregulated (p < 0.05) but that of CYP7B1 was unaffected (p > 0.05) by metformin in the liver of DIO mice. Although (E) metformin also reduced hepatic cholesterol content (p < 0.05) and (F) downregulated the mRNA expression of CYP7B1 in Sirt1 +/- mice (p < 0.05), (G,H) the protein levels of CH25H were unaffected by metformin in the liver of Sirt11 +/- mice (p > 0.05). In C57BL/6J mice, *p < 0.05, HFD group vs Chow diet group, # p < 0.05, HFD + Metformin vs. HFD group. In Sirt1 +/- mice, *p < 0.05, WT + HFD + Metformin or Sirt1 +/- + HFD vs. WT + HFD group, # p < 0.05, Sirt1 +/- + HFD + Metformin vs. Sirt1 +/- + HFD group.

Fig 4: Ch25h overexpression alleviates HFD-induced fatty liver. (A) Representative liver images of C57BL/6J mice administered AAV8-control or AAV8-Ch25h, then fed an HFD for 12 weeks (n = 9 mice/group). (B) RT-qPCR analysis of hepatic Ch25h mRNA level. (C) Hematoxylin and eosin (H&E) and Oil-red O staining. Scale bars, 50 µm. (D) Steatosis score. (E) Liver weight and liver weight/body weight ratio. (F) Hepatic TC and TG levels. (G) Serum TC, TG, and LDL-C levels. (H) Hepatic expression of mRNAs encoded by inflammatory-related genes (IL-6, IL-1ß, TNFa, and MCP-1) from AAV8-control or AAV8-Ch25h-injected mice fed an HFD for 12 weeks (n = 9 mice/group). (I-K) Hepatic expression of mRNAs encoded by lipogenesis-related genes (SREBP1c, FASN, Cd36, Fabp1, Fabp3, Fabp4, SCD1, and Elovl6) (I), fatty acid ß-oxidation related genes (Acox1, Acox2, Cpt1, Cpt2, Ascl1, Acadl, Acaa1a, and Ehhadh) (J), and VLDL secretion-related genes (ApoB and Mttp) (K) from AAV8-control and AAV8-Ch25h injected mice fed an HFD for 8 weeks (n = 8–9 mice/group). Data are mean ± standard error of the mean. For data in (D) and left panels in (E, F), significance was determined by the Mann-Whitney U test. For right panels in (E, F), and data in (G–K), significance was determined by the Student 2-tailed t test. *P < .05, **P < .01; ***P < .001. i.v., Intravenous.

Fig 5: Ch25h activation of CYP7A1 via LXRa. RT-qPCR analysis of mRNA levels of CYP7A1 (A) and CYP27A1 (B) in cultured primary hepatocytes from WT and Ch25h-/- mice. (C) Western blot analysis of protein levels of CYP7A1 and CYP27A1. (C) Western blot analysis was performed to show the protein level of CYP7A1 and CYP27A1 in cultured primary hepatocytes isolated from AAV8-control or AAV8-Ch25h-injected mice (n = 3 mice/group), along with treated with or without LXRa antagonist (GSK2033) for 24 hours. Data are presented as mean ± standard error of the mean. For data in (A) and (C), significance was determined by the Student 2-tailed t test. For data in (B), significance was determined by the Mann-Whitney U test. For data in (D), significance was determined by the 1-way analysis of variance test between multiple groups. *P < .05; **P < .01.