Myeloid AMPK signaling restricts fibrosis but is not required for metformin improvements during CDAHFD-induced NASH in mice

Abstract

Metabolic programming underpins inflammation and liver macrophage activation in the setting of chronic liver disease. Here, we sought to identify the role of an important metabolic regulator, AMP-activated protein kinase (AMPK), specifically within myeloid cells during the progression of non-alcoholic steatohepatitis (NASH) and whether treatment with metformin, a firstline therapy for diabetes and activator of AMPK could stem disease progression. Male and female Prkaa1^fl/fl/Prkaa2^fl/fl (Flox) control and Flox-LysM-Cre⁺ (MacKO) mice were fed a low-fat control or a choline-deficient, amino acid defined 45% Kcal high-fat diet (CDAHFD) for 8 weeks, where metformin was introduced in the drinking water (50 or 250 mg/kg/day) for the last 4 weeks. Hepatic steatosis and fibrosis were dramatically increased in response to CDAHFD-feeding compared to low-fat control. While myeloid AMPK signaling had no effect on markers of hepatic steatosis or circulating markers, fibrosis as measured by total liver collagen was significantly elevated in livers from MacKO mice, independent of sex. Although treatment with 50 mg/kg/day metformin had no effect on any parameter, intervention with 250 mg/kg/day metformin completely ameliorated hepatic steatosis and fibrosis in both male and female mice. While the protective effect of metformin was associated with lower final body weight, and decreased expression of lipogenic and Col1a1 transcripts, it was independent of myeloid AMPK signaling. These results suggest that endogenous AMPK signaling in myeloid cells, both liver-resident and infiltrating, acts to restrict fibrogenesis during CDAHFD-induced NASH progression but is not the mechanism by which metformin improves markers of NASH.

Keywords: AMPK; NASH; fibrosis; immunometabolism; liver; macrophage; metformin; non-alcoholic steatohepatitis.

Graphical abstract

Fig. 1

Metformin intervention improves hepatic steatosis in a myeloid AMPK-independent manner. A and G: Western blots of whole liver lysate from male and female control Prkaa1/2^fl/fl (Flox) and Prkaa1/2^fl/fl/LysM-Cre⁺ (MacKO) mice fed a LFD, CDAHFD or CDAHFD + 250 mg/kg/d metformin. B and H: final body weights (g) C and I) liver weight (g) D and J) representative H&E-stained liver sections (20x magnification, 100 μm scale bar). E and K: hepatic cholesterol and triglyceride normalized to total protein. F and L: serum total cholesterol, total triglycerides, LDL-cholesterol and HDL-cholesterol. Data were analyzed by Two-way ANOVA with Tukey’s test for multiple comparisons where ∗, ∗∗, ∗∗∗ and ∗∗∗∗ represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. The geometric mean of LFD Flox controls is represented by the hashed lines.

Fig. 2

Myeloid AMPK restricts fibrosis but is not critical for metformin-associated improvements. A and G: representative Picrosirius Red-stained liver sections (20x magnification, 100 μm scale bar) of male and female control Prkaa1/2^fl/fl (Flox) and Prkaa1/2^fl/fl/LysM-Cre⁺ (MacKO) mice fed a CDAHFD or CDAHFD + 250 mg/kg/d metformin. B and H: total collagen quantification. C–E and I–K: hepatic mRNA transcript levels of markers of extracellular matrix-regulating transcripts (Col3a1, Col1a1, Acta2). F and L: serum levels of liver injury markers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL)). Transcript expression was normalized to the average expression of Actb and Hprt and expressed relative to LFD-fed Flox mice. Data were analyzed by Two-way ANOVA with Tukey’s test for multiple comparisons where ∗, ∗∗, ∗∗∗, and ∗∗∗∗ represent P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively. The geometric mean of LFD Flox controls is represented by a hashed line.

Fig. 3

Metformin intervention reduces some markers of hepatic inflammation in a myeloid AMPK-independent manner. A and D: Hepatic mRNA transcript levels of macrophages markers (Emr1, Trem2) from male and female control Prkaa1/2^fl/fl (Flox) and Prkaa1/2^fl/fl/LysM-Cre⁺ (MacKO) mice fed a CDAHFD or CDAHFD + 250 mg/kg/d metformin. B and E: the marker of monocyte recruitment (Ccl2) and (C and F) inflammatory cytokines (Tnf, Il1b). Transcript expression was normalized to the average expression of Actb and Hprt and shown relative to LFD-fed Flox mice. Data were analyzed by Two-way ANOVA with Tukey’s test for multiple comparisons where ∗ and ∗∗∗∗ represent P < 0.05 and P < 0.0001, respectively. The geometric mean of LFD Flox controls is represented by a hashed line.