S100a16 Deficiency Prevents Alcohol-induced Fatty Liver Injury via Inducing MANF Expression in Mice

Abstract

Alcoholic liver disease (ALD) encompasses conditions ranging from simple steatosis to cirrhosis and even liver cancer. It has gained significant global attention in recent years. Despite this, effective pharmacological treatments for ALD remain elusive, and the core mechanisms underlying the disease are not yet fully comprehended. S100A16, a newly identified calcium-binding protein, is linked to lipid metabolism. Our research has discovered elevated levels of the S100A16 protein in both serum and liver tissue of ALD patients. A similar surge in hepatic S100A16 expression was noted in a Gao-binge alcohol feeding mouse model. S100a16 knockdown alleviated ethanol-induced liver injury, steatosis and inflammation. Conversely, S100a16 transgenic mice showed aggravating phenomenon. Mechanistically, we identify mesencephalic astrocyte-derived neurotrophic factor (MANF) as a regulated entity downstream of S100a16 deletion. MANF inhibited ER-stress signal transduction induced by alcohol stimulation. Meanwhile, MANF silencing suppressed the inhibition effect of S100a16 knockout on ethanol-induced lipid droplets accumulation in primary hepatocytes. Our data suggested that S100a16 deletion protects mice against alcoholic liver lipid accumulation and inflammation dependent on upregulating MANF and inhibiting ER stress. This offers a potential therapeutic avenue for ALD treatment.

Keywords: Alcoholic liver disease; ER stress.; MANF; S100A16; hepatic steatosis.

Figure 1

Alcohol consumption up-regulated S100A16 expression in human and mice. (A) Representative immunofluorescence staining of S100A16 in liver paraffin slices from healthy controls or patients with alcoholic-associated liver disease (ALD). Scale bar = 50 μm. (B) ELISA analysis of S100A16 expression in serum of ALD patients(n=19) and healthy controls(n=9). (C-E) Mice were pair-fed or alcohol-fed for 10 d and administered a single binge of ethanol (NIAAA model). (C) Western blot analysis of S100A16 expression in mice liver. Quantification of western blots on the right. (D) S100a16 mRNA levels in mice liver. (E) Immunofluorescence assessment of S100A16 expression in mice liver. (F-G) Protein and mRNA expression of S100A16 in primary hepatocytes treated with ethanol (50 mM) for 24 h. (H-I) Protein and mRNA expression of S100A16 in AML 12 cells treated with ethanol (50 mM) for 24 h. α-Tubulin or GAPDH served as the loading control. All data are represented as the mean ± SEM values. *: p < 0.05 vs. the control group, **: p < 0.01 vs. the control group. ALD: alcohol-associated liver disease. PF: pair-fed group. AF: alcohol-fed group.

Figure 2

S100a16 deletion protected mice against alcohol-induced fatty liver injury. WT or S100a16^KO+/- mice were fed separately with the corresponding liquid diet 10 days and administered a single binge of ethanol. (A) Pictures of mice liver from different groups. Protein (B) and mRNA (C) levels of S100A16 in the liver from WT and S100a16^KO+/- mice. Quantification of western blots on the right. (D) Liver weight/body weight (%). (E) Serum ALT, AST levels. (F) Serum TG, TC levels and liver TG, TC levels. (G) Representative H&E and Oil Red O staining of mice livers. Scale bars: 50 µm. (H) Relative mRNA levels of Srebf1, Acaca, Fasn, and Pparg. (I) Relative mRNA levels of Il6, Tnfa, and Il1b. (J) Primary hepatocytes from WT and S100a16^KO+/-mice were treated with ethanol (50 mM) for 24 h. Representative images of BODIPY 493/503 staining of lipid droplets. Scale bars: 50 µm. (K) AML 12 cells with knockdown of S100A16 by siRNA (S100A16 KD) were treated with ethanol (50 mM) for 24 h. Representative images of BODIPY 493/503 staining of lipid droplets. Scale bars: 50 µm. α-Tubulin served as the loading control. All data are represented as the mean ± SEM values. *: p < 0.05 vs. the control group, **: p < 0.01 vs. the control group. ALT, alanine aminotransferase; AST, aspartate aminotransferase; TG: triacylglycerol; T-CHO: total cholesterol; H&E, hematoxylin and eosin.

Figure 3

S100a16 overexpression aggravated alcohol-induced fatty liver injury in mice. WT or S100a16^TG mice were fed separately the corresponding liquid diet 10 days and administered a single binge of ethanol. (A) Pictures of mice liver from different groups. Protein (B) and mRNA (C) levels of S100A16 in the liver from WT and S100a16^TG mice. Quantification of western blots on the right. (D) Liver weight/body weight (%). (E) Serum ALT, AST levels. (F) Serum TG, TC levels and liver TG, TC levels. (G) Representative H&E and Oil Red O staining of mice livers. Scale bars: 50 µm. (H) Relative mRNA levels of Srebf1, Acaca, Fasn, and Pparg. (I) Relative mRNA levels of Il6, Tnfa, and Il1b. (J) Primary hepatocytes from WT and S100a16^TG mice were treated with ethanol (50 mM) for 24 h. Representative images of BODIPY 493/503 staining of lipid droplets. Scale bars: 50 µm. (K) AML 12 cells with overexpression of S100A16 by overexpression plasmid (S100A16+) were treated with ethanol (50 mM) for 24 h. Representative images of BODIPY 493/503 staining of lipid droplets. Scale bars: 50 µm. α-Tubulin served as the loading control. All data are represented as the mean ± SEM values. *: p < 0.05 vs. the control group, **: p < 0.01 vs. the control group.

Figure 4

S100a16 deficiency upregulated MANF expression in alcohol-fed mice. (A-C) Primary hepatocytes(n=3/group) were isolated from alcohol-fed WT and S100a16^KO+/- mice and genome-wide analysis of gene expression was performed by transcriptome sequencing. (A) The number of up-regulation (350) and down-regulation(200) of differentially expressed genes (p ≤ 0.05, fold change ≥ 2). (B) GO and KEGG pathways enrichment analyses. (C) Hub genes of differentially expressed genes. We constructed a protein-protein interaction network and identified hub genes related to alcoholic fatty liver using STRING and Cytoscape. (D) mRNA levels of S100a16 and Manf in primary hepatocytes from WT and S100a16^KO+/- mice after alcohol feeding. (E) protein and(F) mRNA levels of MANF in the alcohol-fed WT and S100a16^KO+/- mice livers. Quantification of western blots on the right. (G)Representative immunofluorescence images of MANF in the alcohol-fed WT and S100a16^KO+/- mice livers. Scale bar: 50 μm. (H) protein and(I) mRNA levels of MANF in the alcohol -fed WT or S100a16^TG mice livers. Quantification of western blots on the right. (J) Representative immunofluorescence images of MANF in the alcohol -fed WT and S100a16^TG mice livers. Scale bar:50 μm. α-Tubulin served as the loading control. All data are represented as the mean ± SEM values. *: p < 0.05 vs. the control group, **: p < 0.01 vs. the control group. MANF: mesencephalic astrocyte-derived neurotrophic factor.

Figure 5

S100A16 restrained MANF and activated ER stress in ALD. (A) Western blot analysis of the S100A16, MANF and the ER stress-related genes GRP78, p-IRE1α, IRE1α, ATF6, ATF4, p-EIF2α, EIF2α in the livers of alcohol-fed WT or S100a16^KO+/- mice. Quantification of western blots on the right. (B) Western blot analysis of the S100A16, MANF and the ER stress-related genes in the livers of alcohol-fed WT or S100a16^TG mice. Quantification of western blots on the right. α-Tubulin served as the loading control. All data are represented as the mean ± SEM values. *: p < 0.05 vs. the control group, **: p < 0.01 vs. the control group. ATF4: activating transcription factor-4; ATF6: activating transcription factor-6; EIF2: eukaryotic initiation factor 2; GRP78: glucose-regulated protein 78; IRE1: inositol-requiring protein-1.

Figure 6

S100A16 regulated alcohol-induced lipid accumulation dependent on altering MANF expression. (A) mRNA levels of S100a16 and Manf in ethanol-induced primary hepatocytes from S100a16^f/f or S100a16^LKO mice transfected with Manf siRNA. (B) Representative BODIPY staining images are shown on the right side, Scale bar: 50 μm. (C) mRNA levels of S100a16 and Manf in ethanol-induced AML 12 cells co-transfected with S100a16 and Manf siRNA. (D) Representative BODIPY staining images are shown on the right side, Scale bar: 50 μm. (E) mRNA levels of S100a16 and Manf in ethanol-induced primary hepatocytes from WT or S100a16^TG mice transfected with overexpression plasmid. (F) Representative BODIPY staining images are shown on the right side, Scale bar: 50 μm. (G) mRNA levels of S100a16 and Manf in ethanol-induced AML 12 cells co-transfected with S100A16 and MANF overexpression plasmid. (H) Representative BODIPY staining images are shown on the right side, Scale bar: 50 μm. All data are represented as the mean ± SEM values. *: p < 0.05 vs. the control group, **: p < 0.01 vs. the control group. S100a16^LKO: hepatocyte-specific S100a16 knockout.

Figure 7

Schematic representation of the role of S100A16 in the pathogenesis of ALD. Alcohol consumption induced S100A16 upregulation. S100A16 aggravated ethanol-induced liver injury, steatosis and inflammation through inhibiting MANF expression and promoting ER stress-related signaling pathways.