RIPK1 deficiency in hepatocytes sensitizes the liver to short-term fasting-induced liver injury and hepatocyte apoptosis. (A) Serum alanine amino-transferase (ALT) levels. (B) Serum aspartate amino-transferase (AST) levels. (C&D) Representative images and quantification of TUNEL staining. Scale bar, 50 or 100 μm. (E&F) Representative images and quantification of liver sections stained with anti-cleaved caspase 3 antibody (red) and DAPI (blue). Scale bar, 50 or 100 μm. (G) Representative images of liver sections stained with anti-phospho-MLKL antibody (red) and DAPI (blue). Scale bar, 50 or 100 μm. (H) Fluorescence microscopy images of the liver stained with anti-Alb antibody (red), TUNEL (green), and DAPI (blue). Scale bar, 50 or 100 μm. (I) Blood glucose levels. (J) Serum triglycerides (TG) levels. (K) Serum total cholesterol (TC) levels. (L) Serum free fatty acids (FFA) levels. (M) Serum β-hydroxybutyrate (BHB) levels. (N) Hepatic triglyceride (TG) levels (mg/g tissue). (O) Liver tissue was stained by Oil Red O. Scale bar, 100 μm. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=6 per group). Asterisks denote statistical significance. ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Short-term fasting induced hepatic inflammation and compensatory proliferation in Ripk1Δhepmice. (A) Hepatic mRNA expression of the inflammatory molecules. (B&C) Representative images and quantification of the liver stained with anti-TNFα antibody (red) and DAPI (blue). Scale bar, 100 μm. (D) Transcriptional expression of Afp and Ki67 in liver tissue. (E) Representative images of the liver stained with anti-Ki67 antibody (brown) and hematoxylin (blue). Scale bar, 100 μm. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=6 per group). ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Transcriptome sequencing of the liver tissue from Ripk1fl/fl and Ripk1Δhep mice. (A) The volcano plot of differentially expressed genes was illustrated. The blue spots represent the down-regulated genes in Ripk1Δhep group compared with control (Ripk1fl/fl) group, and the red spots represent the up-regulated genes in Ripk1Δhepgroup. (B) The altered signaling pathways were enriched by KEGG analysis. (C) The genes which expression were significantly altered in Ripk1Δhep group were depicted in the heat map. (D&E) Representative fluorescence microscopy images and quantification of the liver stained with anti-F4/80 antibody (red), anti-CD11b antibody (red), anti-CCR2 antibody (red), anti-CX3CR1 antibody (red) and DAPI (blue), respectively. Scale bar, 100 μm. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=6 per group). ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Single-cell RNA sequencing of the liver tissue from Ripk1fl/fl and Ripk1Δhep mice. (A) t-SNE visualization of liver cell clusters based on 22274 single-cell transcriptomes. (B) Heatmap of cluster marker genes. (C) t-SNE plots display color-coded cell clusters of cells in the Ripk1fl/fl (left) and Ripk1Δhep (right) mice liver tissues. (D) Bar charts show the proportion of major cell clusters among all cells at different genotypes after fasting by scRNA-seq. (E) t-SNE plots display color-coded cell subtypes of cells in the Ripk1fl/fl (left) and Ripk1Δhep (right) mice liver tissues. (F) Bar charts display the proportion of major cell subtypes within all different genotypes after fasting. (G) The altered signaling pathways associated with differential gene changes in macrophages were enriched by KEGG analysis. (H) t-SNE plots display color-coded cell subtypes of macrophages in the Ripk1fl/fl (left) and Ripk1Δhep (right) mice liver tissues. (I) Bar charts display the proportion of major cell subtypes within macrophages after fasting.

Inhibitor of ER stress 4-PBA effectively rescued the fasting-induced liver injury and inflammation in Ripk1Δhep mice. (A) Western blot analysis of p-IRE1α, IRE1α, GRP78, CHOP, RIPK1 and GAPDH in liver tissue. (B) Experiment schema. (C) Transcriptional expression of ER stress genes in mouse liver. (D) Serum alanine amino-transferase (ALT) and aspartate amino-transferase (AST) levels. (E&F) Fluorescence microscopy image and quantification of TUNEL staining. Scale bar, 100 μm. (G) Expression (qPCR) of inflammatory genes in the livers. (H) Expression (qPCR) of Afp in the livers. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=4-6 per group). ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

AAV-TBG-Cre-mediated liver-specific RIPK1 knockout confirms fasting-induced acute liver injury in mice (A) Schema of AAV8-TBG-Cre administration. (B) Western blot analysis of p-IRE1α, IRE1α, GRP78, CHOP, TNFα, RIPK1 and GAPDH in liver tissue. (C) Serum alanine amino-transferase (ALT) levels. (D) Serum aspartate amino-transferase (AST) levels. (E&F) Fluorescence microscopy image and quantification of TUNEL staining. Scale bar, 50 or 100 μm. (G) Serum triglycerides (TG) levels. (H) Serum total cholesterol (TC) levels. (I) Hepatic triglyceride (TG) levels (mg/g tissue). (J) Expression (qPCR) of ER stress genes in the livers. (K) Expression (qPCR) of inflammatory genes in the livers. (L&M) Fluorescence microscopy images and quantification of liver of Ripk1Δhep and control mice stained with anti-cleaved caspase 3 antibody (red) and DAPI (blue). Scale bar, 50 or 100 μm. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=4-6 per group). ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Short-term high fat diet feeding induced liver injury, hepatic apoptosis, inflammation and endoplasmic reticulum stress in Ripk1Δhep mice. (A) Schema of HFD administration. (B) Liver tissue was stained by Oil Red O. Scale bar, 100 μm. (C) Hepatic triglyceride (TG) levels (mg/g tissue). (D) Serum total cholesterol (TC) levels. (E) Serum alanine amino- transferase (ALT) levels. (F) Serum aspartate amino-transferase (AST) levels. (G&H) Fluorescence microscopy image and quantification of TUNEL staining. Scale bar, 50 or 100 μm. (I) Expression (qPCR) of inflammatory genes in the livers. (J) Expression (qPCR) of Afp in the livers. (K) Expression (qPCR) of ER stress markers in the livers. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=4-6 per group). ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

RIPK1 deficiency in hepatocytes sensitizes the liver to short-term fasting-induced liver injury and hepatocyte apoptosis in female mice. (A) Western blot analysis of RIPK1 and GAPDH in liver tissue. (B) Serum alanine amino-transferase (ALT) levels. (C) Serum aspartate amino-transferase (AST) levels. (D) Representative fluorescence microscopy images of TUNEL staining. Scale bar, 50 μm. The data was analyzed via two-way ANOVA or one-way ANOVA. Data are expressed as mean ± SEM (n=4-6 per group). Asterisks denote statistical significance. ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.

Transcriptome sequencing of the liver tissue from Ripk1fl/fl and Ripk1Δhepmice. (A) The volcano plot of differentially expressed genes was illustrated. The blue spots represent the down-regulated genes in fasted group compared with control (fed) group and the red spots represent the up-regulated genes in Ripk1fl/fl mice. (B) The altered signaling pathways were enriched by KEGG analysis. (C) The genes which expression were significantly altered in fasted group were depicted in the heat map. (D) The volcano plot of differentially expressed genes was illustrated. The blue spots represent the down-regulated genes in Ripk1Δhep group compared with control (Ripk1fl/fl) group and the red spots represent the up-regulated genes in Ripk1Δhep group. (E) The altered signaling pathways were enriched by KEGG analysis.