Metformin and berberine synergistically improve NAFLD via the AMPK-SREBP1-FASN signaling pathway

Abstract

Nonalcoholic fatty liver disease (NAFLD) is a prevalent metabolic condition linked to dyslipidemia, insulin resistance, and persistent inflammation. Due to its complex pathogenesis, no approved pharmacological treatments currently exist. The research sought to explore the combined impact of metformin (Met) and berberine (BBR) on NAFLD, focusing on the AMPK-SREBP1-FASN pathway implicated in liver lipid regulation. The study design incorporated in both living organisms and laboratory conditions to examine how these interventions influenced NAFLD-associated metabolic abnormalities. The HFD-fed mice provided insight into systemic effects, while the OA/PA-stimulated HepG2 cells offered a controlled environment to investigate cellular mechanisms. By employing this dual approach, the researchers could thoroughly characterize the efficacy of Met, BBR, and their combination in mitigating metabolic disturbances. An Adenosine 5'-monophosphate (AMP)-activated protein kinase(AMPK) inhibitor was used in cellular experiments to verify the AMPK-dependent mechanism. Our findings highlight that compared to monotherapies, combination treatment significantly enhanced AMPK activation and inhibited sterol regulatory element-binding protein 1 (SREBP1) expression and that of its downstream target fatty acid synthase (FASN). In HepG2 cells, these effects were partially reversed by the AMPK inhibitor, confirming AMPK dependence. In vivo, the combined therapy effectively inhibited body weight gain, reduced visceral fat accumulation, improved insulin sensitivity, and attenuated hepatic steatosis and inflammation. The combination of metformin and berberine exerts synergistic effects in ameliorating NAFLD by activating AMPK, downregulating SREBP1 and FASN, and improving lipid metabolism. These findings provide evidence supporting a potentially effective multi-modal treatment approach for NAFLD.

Keywords: AMPK signaling; Berberine; Metformin; NAFLD; Synergistic therapy.

Fig. 1

Metformin combined with berberine intervention ameliorates high-fat diet (HFD)-induced obesity phenotype and insulin resistance in mice. (a) Schematic diagram illustrating the experimental procedure and grouping scheme; (b) Weekly food intake curves of mice in each group; (c) Longitudinal changes in body weight over 12 weeks of treatment; (d) Area under the curve (AUC) of body weight for each group; (e) Liver weight in each group; (f) Liver-to-body weight ratio (liver weight/body weight); (g) Visceral fat weight; (h) GLU; (i) INS; (j) HOMA-IR. Data are presented as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2

Combined intervention with metformin and berberine effectively alleviates hepatic lipid accumulation and dyslipidemia induced by high-fat diet in mice. (a) Representative gross images of liver tissues from each group; (b) Oil Red O staining of liver sections (magnification = 20×; scale bar = 50 μm) (c) Quantitative analysis of Oil Red O-positive areas; (d–e) Hepatic TG and TC levels; (f–i) Serum levels of HDL-C, LDL-C, TG, and TC. Data are presented as mean ± SEM (n = 6). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3

Metformin combined with berberine alleviates HFD-induced hepatic pathological injury and inflammation in mice, and inhibits key lipogenic protein expression via activation of the hepatic AMPK signaling pathway. (a) Representative H&E staining of liver tissues (magnification = 20×; scale bar = 50 μm). Yellow arrows indicate fused lipid droplets; red arrows, Kupffer cells; black arrows, inflammatory cell infiltration; (b) Stacked bar chart of NAFLD activity scores including steatosis, inflammation, and ballooning components; (c) Serum ALT and AST levels; (d-f) Hepatic expression levels of TNF-α, interleukin-1 beta (IL-1β), and IL-6; (g) Western blot bands of AMPK and phosphorylated AMPK (p-AMPK); (h) Western blot images of SREBP1 and FASN proteins; (i–k) Quantitative analyses of relative expression levels of p-AMPK/AMPK, SREBP1 and FASN. Data in panels a–g are presented as mean ± SEM (n = 6 per group, animal experiments); data in panels h–l are presented as mean ± SEM (n = 3 per group, representative of three independent experiments). To highlight the target bands, the images were cropped; the full-length gel/blot images are provided in the Supplementary Material. **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant.

Fig. 4

Synergistic reduction of OA/PA-induced lipid accumulation by metformin combined with berberine in HepG2 cells. (a) Oil Red O staining images of lipid deposition induced by OA/PA at various concentrations (magnification = 40×; scale bar = 50 μm); (b-c) Quantitative analyses of lipid area and TG content; (d) Cell viability assay; (e) IC₅₀ determination of drug treatment; (f–h) Oil Red O staining, TG content measurement, and image quantification across different treatment groups (magnification = 40×; scale bar = 50 μm). Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5

Metformin combined with berberine synergistically activates AMPK and inhibits SREBP1 and FASN expression in HepG2 cells. (a–b) Western blot images of p-AMPK/AMPK, SREBP1 and FASN; (c–e) Densitometric analyses of protein expression levels. To highlight the target bands, the images were cropped; the full-length gel/blot images are provided in the Supplementary Material. Data are presented as mean ± SEM (n = 3). ***p < 0.001, ****p < 0.0001.

Fig. 6

Metformin combined with berberine inhibits lipid synthesis through the AMPK–SREBP1–FASN signaling pathway. (a) Oil Red O staining images after AMPK inhibitor treatment (magnification = 40×; scale bar = 50 μm); (b–c) Quantitative analysis of intracellular Oil Red O staining and triglyceride (TG) content. (d–e) Western blot showing expression changes of p-AMPK, SREBP1 and FASN proteins; (f–h) Quantitative analyses of protein grayscale values. To highlight the target bands, the images were cropped; the full-length gel/blot images are provided in the Supplementary Material. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001, ns: not significant.