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, 100 μm. (E&F) Representative images and quantification of liver sections stained with anti-cleaved caspase 3 antibody (red) and DAPI (blue). Scale bar, 100 μm. (G) Representative images of liver sections stained with anti-phospho-MLKL antibody (red) and DAPI (blue). Scale bar, 100 μm. (H) Fluorescence microscopy images of the liver stained with anti-Alb antibody (red), TUNEL (green), and DAPI (blue). Scale bar, 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 Ripk1Δhep mice. (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, 50 μm. (F) Representative flow-cytometry plots showing Ki67-positive cells in the liver tissue (n = 3 per group). (G) Relative increase of Ki67-positive cells in the liver tissue of Ripk1Δhep mice after 12 hours of fasting compared to that of Ripk1fl/fl mice (n = 3 per group). 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 Ripk1Δhep group. (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 liver tissue from Ripk1fl/fl and Ripk1Δhep mice. (A) t-SNE plots display color-coded cell subtypes of cells in the Ripk1fl/fl (left) and Ripk1Δhep (right) mice liver tissues. (B) Bar charts display the proportion of major cell subtypes within all different genotypes after fasting. (C) The altered signaling pathways associated with differential gene changes in macrophages were enriched by KEGG analysis. (D) t-SNE plots display color-coded cell subtypes of macrophages in the Ripk1fl/fl (left) and Ripk1Δhep (right) mice liver tissues. (E) Bar charts display the proportion of major cell subtypes within macrophages after fasting. (F) Representative flow cytometry plots of F4/80-positive and CD11b-positive cells in the liver tissue (n = 3 per group). (G) Relative increase of F4/80-positive and CD11b-positive cells in the liver tissue of Ripk1Δhep mice after 12 hours of fasting, compared to that in Ripk1fl/fl mice. (n = 3 per group; unpaired t test).(H) Bar charts display the number of interactions among cell types across the experiments. (I) Bar charts displaying the interaction strength among cell types across the experiments. (J) Circle plots displaying the brand link pairs between macrophages and other cell types, along with their corresponding event counts. (K) Heatmaps summarizing specific signals among interacting cell types in the liver tissue of Ripk1Δhep mice after 12 hours of fasting, compared to that in Ripk1fl/fl mice.. Interactions are categorized into outgoing and incoming events for specific cell types. The color gradient indicates the relative strength of these interactions.(NK&T : Natural Killer cells and T cells; ECs : Endothelial Cells; PCs : Plasma Cells; Mac : Macrophages; DCs : Dendritic Cells)

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 = 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, 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, 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.

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 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 = 6 per group). ns, no significant, * P < 0.05, *** P < 0.001, **** P < 0.0001.