Glutathione S-transferase Mu 2 inhibits hepatic steatosis via ASK1 suppression

ARTICLE https://doi.org/10.1038/s42003-022-03251-w OPEN Glutathione S-transferase Mu 2 inhibits hepatic steatosis via ASK1 suppression 1234567890():,; Yi Jin1,2, Yanjie Tan1, Pengxiang Zhao1, Yu Guo1, Shilin Chen1, Jian Wu1 ✉ & Zhuqing Ren 1,2 ✉ Hepatic steatosis is the main characteristic of some liver metabolism diseases. However, unclear molecular mechanism of hepatic steatosis impedes the therapy of this hepatic steatosis. Glutathione-S-transferase mu 2 (GSTM2), as a member of phase II drug metabolizing enzymes (DMEs), regulates cellular antioxidant and detoxificant. GSTM2 was highly upregulated in hepatic steatosis tissues and high-fat diet (HFD) fed mice. Loss-of-function GSTM2 mouse model demonstrated that GSTM2 protected mice from excess fat accumulation. Mechanistically, GSTM2 interacted with ASK1 and suppressed its phosphorylation and the activation of subsequent downstream p38-JNK signalling. Moreover, GSTM2 overexpression in the liver effectively ameliorated hepatic lipid accumulation. Therefore, we identified GSTM2 as an important negative regulator in progression of hepatic steatosis via both its detoxification/antioxidant and inhibition of ASK1-p38/JNK signalling. This study showed potential therapeutic function of the DME in progression of hepatic steatosis. 1 Key Laboratory of Agriculture Animal Genetics, Breeding and Reproduction of the Ministry of Education & Key Laboratory of Swine Genetics and Breeding of the Ministry of Agriculture, College of Animal Science, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China. 2 Hubei Hongshan Laboratory, Wuhan, Hubei 430070, PR China. ✉email: ; COMMUNICATIONS BIOLOGY | (2022)5:326 | https://doi.org/10.1038/s42003-022-03251-w | www.nature.com/commsbio 1 ARTICLE COMMUNICATIONS BIOLOGY | https://doi.org/10.1038/s42003-022-03251-w H epatic steatosis, characterized by excessive accumulation of triglycerides (TGs) in hepatocytes, is considered the cause of non‐alcoholic fatty liver disease (NAFLD)1–3. Disordered lipid metabolism leads to the hepatic steatosis causing lots of excess fat accumulation in hepatocytes. High hepatic fat content is the important inducement of the impairment of redox imbalance and insulin resistance. However, the molecular mechanisms of hepatic steatosis occurrence and progression are poorly understood. Drug-metabolising enzymes (DMEs) play an important role in scavenging the waste products of lipid metabolism and oxidative metabolism and maintaining homeostasis of liver4,5, consisted of groups of enzymes, such as glutathione S-transferases (GSTs). Several studies reported DMEs could respond to the hepatic steatosis by protecting hepatocytes from free radicals6,7, additionally, GSTs are always considered as the scavenger of reactive oxygen species. Among other GSTs, glutathione S-transferase M2 (GSTM2) seems to be different. Previous researches from Huenchuguala et al. reported that they found cells with GSTM2 knockdown accumulated more lipid droplets8–11. Moreover, GSTM2 was highly up-regulated in high-fat diet mouse liver6,7. These studies suggested GSTM2 regulated lipid metabolism specially beyond detoxification/antioxidant, the basic functions of GSTs. In the present study, the close association between GSTM2 knockout and hepatic steatosis was examined by using GSTM2null mice. We demonstrated that GSTM2 protects against hepatic steatosis by inhibiting excess fat accumulation by interacting with and suppressing activation of apoptosis signal-regulating kinase1 (ASK1) and subsequent p38-JNK signalling, besides its antioxidant capacity. Moreover, GSTM2 overexpression reversed methionine choline-deficient diet (MCDD)-induced steatosis. Thus, we identified GSTM2 as an important negative regulator in hepatic steatosis progress. Results GSTM2 was upregulated in hepatic tissues of mice fed HFD and MCDD. The high-fat diet and methionine choline deficient diet were used to make hepatic steatosis. We validated the expression pattern of GSTM2 in the HFD and MCDD mouse models. The level of GSTM2 mRNA and protein were detected. GSTM2 was significantly up-regulated in mice fed HFD (Fig. 1a–c) and MCDD (Fig. 1d–f). GSTM2 deletion strongly promotes hepatic fat accumulation. To investigate the function of GSTM2 in the progression of hepatic steatosis, we generated a GSTM2 knockout (GSTM2 KO) mouse (Supplementary Fig. 1a, b). Hepatic histomorphology and TG content detection showed that GSTM2 KO mice had higher hepatic fat content (Fig. 1g, h). Moreover, there was no significant difference in the ratio of liver weight to body weight between GSTM2 KO and WT mice (Supplementary Fig. 1c, d). We also investigated the fat content in white adipose and skeletal muscle tissues via Oil Red O staining. More intramuscular fat was observed in GSTM2 KO mice, whereas no significant difference was found in white adipose tissue between KO and WT mice (Supplementary Fig. 1e, f). To further investigate the function of GSTM2 in hepatic lipid metabolism, we challenged the mice with HFD. KO mice accumulated more fat at a faster rate than WT mice (Fig. 1i, j, Supplementary Fig. 1g). Moreover, the hepatic alanine aminotransferase (ALT) level was higher in KO mice, while the γ-glutamyltranspetidase (GGT) level was not changed (Supplementary Fig. 1h, i). We next challenged the mice with 0.2 mM oleic acid medium (dissolved by BSA in 0.9% NaCl solution) to investigate the effect of GSTM2 on the rapid 2 formation of LDs. Hepatic morphology analysis showed that GSTM2-KO-livers formed more LDs than WT livers (Supplementary Fig. 1j, k). These results suggest that GSTM2 plays an important role in excess hepatic fat accumulation. GSTM2 overexpression suppresses hepatic steatosis. Although GSTM2 was up-regulated in mice with hepatic steatosis, we also overexpressed this protein in mouse model fed with MCDD (Supplementary Fig. 2a). Western blot analysis showed that GSTM2 was highly expressed in liver tissues (Supplementary Fig. 2b). As the feeding time increased, control mice accumulated much more hepatic fat, while GSTM2 overexpression suppressed hepatic fat accumulation (Fig. 2a). Control mice showed significant hepatic steatosis after 1 week of MCDD feeding, whereas mice with GSTM2 overexpression showed this effect at 4 weeks. Additionally, GSTM2-overexpressing mice showed mild LD accumulation in the first 3 weeks (Fig. 2b). Because fibrosis is the key signal of steatosis aggravation, we further detected the fibrosis level in liver tissues. Masson staining analysis indicated that control mice had significant hepatic fibrosis compared to mice with GSTM2 overexpression (Supplementary Fig. 2c, d). A significant degree of fibrosis was observed in liver tissues of control mice at 3 weeks, whereas GSTM2-overexpressing mice showed mild fibrosis at 8 weeks (Supplementary Fig. 2c, d). Moreover, we detected the marker genes of fibrosis α-SMA and CoL1A1. The results showed the expression of α-SMA and CoL1A1 was upregulated at 8 weeks signific (...truncated)