NAFLD is characterized by dysregulated lipid metabolism, which is frequently associated with obesity and metabolic syndrome. In this study, we present empirical evidence suggesting that metformin may confer protection to mice with NAFLD induced by a high-fat diet by targeting the AMPK/SERCA2b pathway via miR-200a-5p. Intriguingly, miR-200a-5p expression is elevated in NAFLD, whereas metformin treatment significantly reduces its levels. Therefore, our findings reveal a novel molecular mechanism by which metformin ameliorates NAFLD, and underscore the potential beneficial role of miRNAs in the treatment of this condition (Fig. 5).
Lipotoxicity is a key driver of NAFLD, making effective lipid management in the liver crucial for advancing NAFLD treatments. Triglycerides can be stored within hepatocytes or secreted into the bloodstream as very low-density lipoproteins. Disruptions in lipid metabolism lead to an increase in circulating free fatty acids. When the storage capacity of adipose tissue and the oxidative capabilities of tissues for free fatty acids are exceeded, an excess conversion of free fatty acids into triglycerides occurs, leading to their excessive deposition in non-adipose tissues and causing tissue damage. Studies have indicated that free cholesterol can compromise mitochondrial function, trigger hepatocyte death, and thus promote the progression of NAFLD25. In the case of simple steatosis, the accumulation of excessive lipids such as cholesterol and triglycerides disrupts the internal stability of liver cells, resulting in liver damage. Lipid disruption, mitochondrial dysfunction, ER stress, and reactive oxygen species have all been implicated in hepatocyte damage and the progression of simple steatosis to NASH with subsequent fibrosis26,27. Our study has shown that in both the PA and HFD groups, the expression of lipogenic genes (Fasn, Srebp1, Acc1, Scd1) and the fatty acid translocase Cd36 was elevated, alongside a significant increase in TG, TC, and LDL-C, and a decrease in HDL-C. Conversely, in the metformin group, the expression of lipogenic genes (Fasn, Scd1, Acc1) and the fatty acid translocase Cd36 was reduced, which correlated with decreased levels of TG, TC, LDL-C, and an increase in HDL-C. Consistent with previous findings28, our findings reaffirm that Metformin can alleviate hepatic lipid metabolism disorders.
MicroRNAs play a pivotal role in a myriad of biological functions, encompassing development, lifespan, cellular proliferation, differentiation, signaling pathways, apoptosis, and metabolism29. Previous research has demonstrated that in patients with type 1 diabetes, Metformin can downregulate MicroRNA-222, MicroRNA-195, and MicroRNA-21a, thereby safeguarding cardiac function and improving glycemic control30. Further studies have revealed that hepatocytes treated with the lipotoxic palmitic acid exhibit elevated levels of MicroRNA-122 and MicroRNA-192, which correlate with the progression of steatohepatitis to liver fibrosis through the upregulation of fibrosis-related genes, such as Smooth Muscle Actin31,32. A diet known to induce steatohepatitis, the choline-deficient L-amino acid-defined diet, has also been shown to increase MicroRNA-122 and MicroRNA-192 levels in murine studies33. Ezaz et al. recently reported significant elevations in MicroRNA-34a, MicroRNA-122, MicroRNA-192, and MicroRNA-200a expressions in a cohort of 132 patients with biopsy-confirmed NAFLD, establishing a close histological association of MicroRNA-200a with the severity of NAFLD23. Moreover, in rat livers with diet-induced NAFLD, there was a significant downregulation of MicroRNA-122, MicroRNA-451, and MicroRNA-27, while MicroRNA-200a, MicroRNA-200b, and MicroRNA-429 were markedly upregulated34. An increasing body of evidence suggests that circulating MicroRNAs are stable and the techniques to detect them are available, making MicroRNAs potential candidates for monitoring the severity of NAFLD and non-invasive diagnosis of the disease. In our study, we focused on the variations of miR-200a-5p across different groups. Notably, we observed a differential expression of miR-200a-5p between the HFD and Metformin-treated groups. To further corroborate its function, we initially validated the interaction between miR-200a-5p and AMPK using a luciferase reporter assay in cell models. Subsequent overexpression of miR-200a-5p resulted in the reduction of AMPK levels.
Metformin is also known to alleviate ER stress. SERCA2b plays a crucial role in maintaining ER homeostasis, and the reduction in ER calcium levels due to decreased expression or activity of SERCA2b is a potential mechanism for the activation of ER stress induced by obesity35. In our previous studies, we discovered that miR-30b activates ER stress by targeting SERCA2b, thereby inducing insulin resistance36. Additionally, research has shown that MAR1 improves hepatic steatosis by inhibiting ER stress mediated by the AMPK/SERCA2b pathway21. Our current findings suggest that in the treatment of NAFLD with Metformin, miR-200a-5p may modulate the expression of AMPK/SERCA2b, thereby ameliorating lipid deposition and ER stress.
Our findings suggest that miR-200a-5p may ameliorate lipid deposition and ER stress by modulating the expression of AMPK/SERCA2b. This offers new insights into the potential mechanisms through which Metformin could be effective in treating NAFLD. However, our study has its limitations, as we did not validate the relationship between miR-200a-5p and AMPK at the animal level. Our research merely provides initial clues, indicating that further investigation is necessary to fully understand these mechanisms.