Imeglimin attenuates liver fibrosis by inhibiting vesicular ATP release from hepatic stellate cells

Abstract

The protective effects of imeglimin, a recently approved antidiabetic agent, against liver fibrosis have not been previously evaluated. In this study, we demonstrated that 8-week administration of imeglimin attenuated immune cell infiltration and reduced collagen deposition, improving fibrosis stage in a thioacetamide-induced murine model. Further analyses focusing on hepatic stellate cells (HSCs), the primary effector cells in fibrogenesis, revealed decreased expression of α-smooth muscle actin and desmin, markers of HSC activation. Mechanistically, a clinically relevant low concentration (10 μm) of imeglimin reduced intracellular vesicular ATP accumulation and subsequently suppressed ATP release from HSCs in vitro. These findings suggest that imeglimin may exert anti-inflammatory and antifibrotic effects by inhibiting vesicular ATP release and ATP-mediated purinergic signaling. Impact statement At clinically relevant doses, imeglimin inhibits vesicular ATP release from hepatic stellate cells, reducing inflammatory infiltration and fibrotic collagen accumulation. These findings support its evaluation as a combined metabolic and antifibrotic therapy for MASLD and other chronic liver conditions.

Keywords: ATP; VNUT; hepatic stellate cells; imeglimin; liver fibrosis; purinergic signaling; vesicular ATP release.

Fig. 1

Imeglimin ameliorates thioacetamide‐induced body weight loss and liver inflammation in C57BL/6J mice. (A) Schematic representation of the experimental protocol illustrating the treatment regimen for different groups of C57BL/6J mice subjected to saline or thioacetamide followed by oral administration of saline or imeglimin. (B) Time‐course analysis of body weight changes in mice post‐thioacetamide treatment. (C) Measurement of white adipose tissue (WAT) and liver weights at the end of the intervention period, presented separately for mice sacrificed under fasting (n = 4 for Sal; n = 3 for TAA; n = 3 for IMG) or ad libitum conditions (n = 2 for Sal; n = 4 for TAA; n = 4 for IMG). (D) Intraperitoneal glucose tolerance test (ipGTT) conducted at 12 weeks of age. The area under the curve (AUC) is depicted in the right panel (n = 6 for Sal; n = 7 for TAA; n = 7 for IMG). (E) Representative micrographs of hematoxylin and eosin (HE) staining and F4/80 immunostaining of liver sections postintervention (n = 6 for Sal; n = 7 for TAA; n = 7 for IMG). Enlarged views (middle panel) highlight yellow dashed rectangle areas. Inflammatory cell clusters are denoted by green circles, while large necroptotic hepatocytes surrounded by inflammatory cells are indicated by green arrows. Yellow arrows point to blank areas surrounded by inflammatory cells. Red arrows indicate F4/80‐positive macrophages. (F) Quantitative analysis of inflammatory cell infiltration area and F4/80‐positive cell numbers. Inflammatory cell areas were quantified as the mean of five high‐powered fields per animal (n = 5 per group). F4/80 positive cells were quantified as the mean of five high‐powered fields per animal (n = 3 for Sal; n = 4 for TAA; n = 3 for IMG). Scale bar = 200 μm. Data are expressed as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test and indicated as follows: *P < 0.05, **P < 0.01. CV, central vein; IMG, imeglimin; i.p., intraperitoneal injection; p.o., per os (oral administration); Sal, saline; TAA, thioacetamide; WAT, white adipose tissue.

Fig. 2

Effect of Imeglimin on serum and hepatic inflammatory mediators in C57BL/6J mice exposed to thioacetamide (TAA). (A) Serum activities of AST, ALT, and ALP measured using the Vetscan VS2 chemistry analyzer and blood platelet counts measured using the Vetscan HM5 hematology analyzer (n = 4). (B) Serum concentrations of IL‐1β, TNF, IL‐6, and MCP1 assessed using BD CBA (n = 6 for Sal; n = 7 for the intervention groups). (C) Hepatic mRNA expression of Il1b, Tnfa, Il6, and Mcp1 quantified by quantitative real‐time PCR (qRT‐PCR) (n = 4 for Sal; n = 7 for the intervention groups). (D) Tissue levels of IL‐1β, TNF, IL‐6, and MCP‐1 in liver homogenates assessed by BD CBA (n = 6 for Sal; n = 7 for the intervention groups). Data presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test and indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ALT, alanine aminotransferase; ALP, alkaline phosphatase; AST, aspartate aminotransferase; BD CBA, BD® Cytometric Bead Array; IL, interleukin; IMG, imeglimin; MCP1, monocyte chemoattractant protein‐1; Sal, saline; TAA, thioacetamide; TNF, tumor necrosis factor.

Fig. 3

Imeglimin ameliorates thioacetamide‐induced liver fibrosis in C57BL/6J mice. (A, B) Representative micrographs depicting liver sections stained with Picro‐Sirius Red (PSR) and α‐Smooth Muscle Actin (α‐SMA) following intervention and quantitative analysis of METAVIR fibrosis scores (n = 6 for Sal; n = 7 for the intervention groups) and α‐SMA‐positive area (n = 3 for each group) as the mean of five high‐powered fields per animal. Scale bar = 200 μm. (C, D) Fluorescence micrographs displaying α‐SMA staining of liver sections postintervention and quantitative analysis of α‐SMA‐positive area (n = 4 for Sal; n = 4 for TAA; n = 3 for IMG). Nuclei stained with DAPI (blue). Scale bar = 50 μm. (E, F) Fluorescence micrographs of Desmin staining. Red arrows indicate the stellate shape of HSCs. (F) Quantitative analysis of Desmin positive area. (n = 6 for Sal; n = 7 for TAA; n = 6 for IMG). (G) Real‐time PCR (RT‐PCR) analysis of fibrosis marker genes Timp1, Sma, and Col1a1 in liver tissue. (n = 4 for Sal; n = 7 for the intervention groups). (H, I) Western blot analysis and densitometric quantification of α‐SMA, COL1A1, TGF‐β1, and MMP1 in mouse livers. β‐Actin used as an internal control. Biological replicate samples labeled as #1–#6. Data presented as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test and indicated as follows: *P < 0.05, **P < 0.01, ****P < 0.0001. Col1a1, collagen type I alpha 1; DAPI, 4′,6‐diamidino‐2‐phenylindole; IMG, imeglimin; MMP1, matrix metalloproteinase 1; Sal, saline; TAA, thioacetamide; TGF‐β1, transforming growth factor beta 1; Timp1, tissue inhibitor of metalloproteinases 1.

Fig. 4

Impact of imeglimin on ATP accumulation and ATP secretion in LX‐2 cells. (A, B) Live‐cell imaging illustrating MANT‐ATP fluorescence within ATP‐containing secretory vesicles. Clodronate served as a positive control, known to hinder ATP accumulation. Scale bar = 20 μm. (B) Quantification of vesicular MANT‐ATP accumulation as total intracellular corrected vesicular fluorescence (CVF) (n = 8 from two independent experiments). (C, D) Live‐cell imaging illustrating MANT‐ATP fluorescence within ATP‐containing secretory vesicles. Scale bar = 20 μm. (D) Quantification of vesicular MANT‐ATP accumulation as total intracellular CVF (n = 38 for NC, n = 43 from two independent experiments). (E, F) Assessment of ATP secretion in LX‐2 cells upon exposure to the calcium ionophore ionomycin at a concentration of 5 μm after 18 h of drug pre‐incubation. (n = 6) f. with 12 h of TGF‐β1 stimulation at a concentration of 5 ng·mL⁻¹ (n = 6 for NC, Clo; n = 4 for NC with Ionomycin and IMG). Data shown are from a single representative experiment, which was repeated three times with comparable outcomes. Data shown as mean ± SEM. Statistical significance was determined by one‐way ANOVA followed by Tukey's post hoc test (B, E, F) and by unpaired two‐tailed Student's t‐test and indicated as follows: denoted as **P < 0.01, ***P < 0.001, ****P < 0.0001. Clo, clodronate; DIC, differential interference contrast; IMG, imeglimin; NC, negative control.