The adiponectin-derived peptide ALY688 protects against the development of metabolic dysfunction-associated steatohepatitis

Abstract

Metabolic dysfunction-associated steatohepatitis (MASH) is the severe form of non-alcoholic fatty liver disease which has a high potential to progress to cirrhosis and hepatocellular carcinoma, yet adequate effective therapies are lacking. Hypoadiponectinemia is causally involved in the pathogenesis of MASH. This study investigated the pharmacological effects of adiponectin replacement therapy with the adiponectin-derived peptide ALY688 (ALY688-SR) in a mouse model of MASH. Human induced pluripotent stem (iPS) cell-derived hepatocytes were used to test cytotoxicity and signaling of unmodified ALY688 in vitro. High-fat diet with low methionine and no added choline (CDAHF) was used to induce MASH and test the effects of ALY688-SR in vivo. Histological MASH activity score (NAS) and fibrosis score were determined to assess the effect of ALY688-SR. Transcriptional characterization of mice through RNA sequencing was performed to indicate potential molecular mechanisms involved. In cultured hepatocytes, ALY688 efficiently induced adiponectin-like signaling, including the AMP-activated protein kinase and p38 mitogen-activated protein kinase pathways, and did not elicit cytotoxicity. Administration of ALY688-SR in mice did not influence body weight but significantly ameliorated CDAHF-induced hepatic steatosis, inflammation, and fibrosis, therefore effectively preventing the development and progression of MASH. Mechanistically, ALY688-SR treatment markedly induced hepatic expression of genes involved in fatty acid oxidation, whereas it significantly suppressed the expression of pro-inflammatory and pro-fibrotic genes as demonstrated by transcriptomic analysis. ALY688-SR may represent an effective approach in MASH treatment. Its mode of action involves inhibition of hepatic steatosis, inflammation, and fibrosis, possibly via canonical adiponectin-mediated signaling.

FIGURE 1

ALY688 ameliorates the development of metabolic dysfunction‐associated steatohepatitis (MASH) in streptozotocin and high‐fat diet (STAM) mice. (a) Schematic diagram for induction of MASH in mice and the treatment regime. Male C57BL/6N mice at 2 days after birth received a single subcutaneous injection of 200 μg streptozotocin (STZ) solution; these mice were fed with a high‐fat diet (HFD) from 4 to 9 weeks of age to induce MASH. From 6 to 9 weeks of age, the mice were administered with vehicle (veh) or two doses of ALY688 (low: 0.5 mg/kg/day, high: 5 mg/kg/day) via daily subcutaneous injection (SC) or continuous subcutaneous infusion via osmotic pumps (OP). (b) Body weight of the mice treated with vehicle or ALY688 during the treatment. Serum alanine transaminase (ALT) (c), serum aspartate transaminase (AST) (d), and liver weight (e) of the mice at the end of treatment. (f) Representative images of liver sections stained with hematoxylin and eosin (H&E) (scale bar, 100 μm). Lower panel, quantitative analyses of the grades of steatosis, inflammation, ballooning, and the total MASH activity score (NAS) for the H&E staining. Data represent mean ± standard error of the mean; n = 6 per group; *p < 0.05, **p < 0.01, ***p < 0.001 versus veh‐STC (standard chow), ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus veh‐CDAHFD (choline‐deficient with low methionine high‐fat diet).

FIGURE 2

Sustained release form of ALY688 (ALY688‐SR) protects against diet‐induced metabolic dysfunction‐associated steatohepatitis (MASH) in mice. (a) Schematic diagram for establishment of diet‐induced mouse model of MASH and the treatment regime. Male C57BL/6N mice at 8 weeks of age were fed with choline‐deficient with low methionine high‐fat diet (CDAHFD) for 12 weeks to induce MASH. Three weeks after CDAHFD feeding, the mice were treated with vehicle or ALY688‐SR (low: 3 mg/kg/day, high: 15 mg/kg/day) by daily subcutaneous injection for 9 weeks. Obeticholic acid (OCA) was used as a positive control, standard chow (STC)‐fed mice treated with vehicle were considered as the negative control group. (b) Body weight of the mice receiving vehicle (veh) or ALY688‐SR during the treatment. Serum alanine transaminase (ALT) (c), aspartate transaminase (AST) (d), liver weight (e), and hepatic triglyceride (TG) concentration (f) of the mice at the end of treatment. Panel (g), representative gross photos of livers and histological images of liver sections stained with hematoxylin and eosin (H&E) (scale bar, 100 μm). Lower panel, quantitative analyses of the grades of steatosis, inflammation, and the total MASH activity score for the H&E staining. Data represent mean ± standard error of the mean; n = 10 per group; *p < 0.05, **p < 0.01, ***p < 0.001 versus veh‐STC, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus veh‐CDAHFD (see also Figure S3).

FIGURE 3

ALY688‐SR protects against metabolic dysfunction‐associated steatohepatitis (MASH)‐induced hepatic fibrosis in mice. (a) Representative images of liver sections from standard chow (STC) or choline‐deficient with low methionine high‐fat diet (CDAHFD)‐fed mice treated with vehicle (veh) or ALY688‐SR stained with Sirius red or Masson's trichrome (scale bar, 100 μm). (b) Quantitative analysis for Sirius red staining. (c) Quantitative analysis for Masson's trichrome staining. (d) Hepatic hydroxyproline (OH‐proline) content of the mice. (e) Western blot analysis for 𝛂‐SMA protein expression in the liver. The bottom panel is the densitometric analysis for the relative abundance of 𝛂‐SMA normalized with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). (f) Quantitative analysis for 𝛂‐SMA. Data represent mean ± standard error of the mean; n = 10 per group; *p < 0.05, **p < 0.01, ***p < 0.001 versus veh‐STC, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus veh‐CDAHFD.

FIGURE 4

ALY688‐SR improves lipid metabolism, reduces inflammation and fibrosis in the liver in mice with metabolic dysfunction‐associated steatohepatitis (MASH). (a–d) After 9 weeks of treatment, liver samples from vehicle (veh) and high‐dose ALY688‐SR‐treated mice fed with choline‐deficient with low methionine high‐fat diet (CDAHFD) were harvested for transcriptomic analysis. (a) Heat map illustrates all differentially expressed transcripts (adjusted p‐value <0.05) in the liver. Colors refer to gene expression z‐score. (b) Venn diagram identifies the genes induced by MASH and reversed by ALY688‐SR treatment or suppressed by MASH and reversed by ALY688‐SR treatment. The publicly available dataset (MASH vs. Control: GSE162249) was used for the analysis. Gene ontology (GO) enrichment analysis of the genes downregulated by MASH and upregulated by ALY688‐SR (c) and upregulated by MASH and downregulated by ALY688‐SR (d). The top 20 regulated pathways are shown. n = 6 per group. Real‐time polymerase chain reaction was performed to determine the mRNA expression of genes related to lipid metabolism (e), fibrosis (f), and inflammation (g) in the livers of the indicated groups. Data represent mean ± standard error of the mean; n = 10 per group; *p < 0.05, **p < 0.01, ***p < 0.001 versus veh‐STC, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus veh‐CDAHFD. (h, i) TRRUST database was used to create a transcriptional regulatory model using the 917 differentially expressed genes (DEGs) downregulated by MASH, upregulated by ALY688‐SR (h) and the 605 DEGs upregulated by MASH, downregulated by ALY688‐SR (adjusted p < 0.01).