ANGPTL8 deficiency attenuates lipopolysaccharide-induced liver injury by improving lipid metabolic dysregulation

Figures

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  Fig. 1. Increased lipid deposition and lipid peroxidation in the liver after LPS stimulation. A–C: Serum IL-6 (A), IL-1β (B) and TNF-α (C) levels after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h (n = 3∼5 per group) were detected using ELISA kits. D: Serum FFAs levels after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h (n = 5 per group) were detected using NEFA assay kits. E: H&E staining (A) of liver tissue after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h (n = 5 per group). F and G: Serum alanine transaminase (ALT) (F) and aspartate transaminase (AST) (G) levels after LPS stimulation for 0 h, 24 h, 48 h, or 72 h (n = 4 per group) were measured via ALT and AST assay kits. H: Oil Red O staining of liver tissue after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h (n = 5 per group). I and J: TG levels (I) and MDA levels (J) in liver homogenates from LPS-induced septic mice were detected using TG and MDA detection kits (n = 3∼5 per group). The data are shown as the mean ± SEM and were analyzed using an unpaired two-tailed Student’s test (C–F); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns > 0.05.

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  Fig. 2. LPS upregulates the expression of ANGPTL8 in vivo and in vitro. A: The transcription level of ANGPTL8 in mice with sepsis was analyzed via the GEO database (GSE184167). B and C: the expression of ANGPTL8 in mouse liver tissue after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h was detected via qRT‒PCR and Western blot (n = 3∼5 per group). D: serum ANGPTL8 levels after LPS stimulation for 48 h were measured using an ANGPTL8 ELISA kit (n = 3∼4 per group). E: qRT‒PCR analysis of the mRNA expression of ANGPTL8 in HepG2 cells after LPS (4 μg/ml) stimulation for 0 h, 12 h, 24 h, 48 h, or 72 h (n = 3 per group). F: the protein expression of ANGPTL8 in HepG2 cells and mouse primary hepatocytes after LPS treatment for 24 h or 4 h was determined by immunofluorescence (n = 3 per group). The data are shown as the mean ± SEM and were analyzed using an unpaired two-tailed Student’s test (B, D, E); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns > 0.05.

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  Fig. 3. Knockout of Angptl8 improves LPS-induced lipid deposition and peroxidation in the liver. A: The survival of Angptl8 KO and WT septic mice induced by LPS (n = 10 per group). B: The serum ALT and AST levels after LPS stimulation for 0 h, 24 h and 48 h were measured via ALT and AST assay kits (n = 3∼4 per group). C: H&E staining was performed on the liver tissue of Angptl8 KO and WT mice after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h (n = 3∼4 per group). D and E: IL-1β (D) and TNF-α (E) expression in the livers of Angptl8 KO and WT mice after LPS stimulation for 0 h, 24 h, and 48 h (n = 3∼4 per group) was detected and analyzed by qRT‒PCR. F: Oil Red O staining was performed on the liver tissue of Angptl8 KO and WT mice after LPS (10 mg/kg) stimulation for 0 h, 24 h, 48 h, or 72 h (n = 3∼4 per group). G and H: TG levels (G) and MDA levels (H) in the liver homogenates of Angptl8 KO and WT mice after LPS treatment were detected using TG and MDA detection kits (n = 3∼4 per group). The data are shown as the mean ± SEM and were analyzed using an unpaired two-tailed Student’s test (A, B, D, E, G, H); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns > 0.05.

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  Fig. 4. ANGPTL8 deficiency potentiates liver PPARα expression. A: The above results demonstrated that the lipid deposition and peroxidation indices in the livers of Angptl8 KO mice and control mice were significantly different after LPS stimulation for 48 h. Volcano plot analysis of the RNA sequences from the livers of Angptl8 KO and WT mice after LPS stimulation for 48 h is shown (n = 3 per group). B: KEGG enrichment analysis of the Angptl8 KO and WT groups to predict the functions of ANGPTL8 during LPS-induced liver injury (n = 3 per group). C and D: The transcriptional levels of PPARα (C) and PPARγ (D) in the liver tissue of Angptl8 KO and WT mice after LPS (10 mg/kg) stimulation for 48 h (n = 3∼4 per group). E: The protein levels of PPARα and PPARγ in the liver tissue of Angptl8 KO and WT mice after LPS (10 mg/kg) stimulation for 48 h (n = 3 per group). F and G: qRT‒PCR analysis of the mRNA expression of PPARα (G) and PPARγ (H) in control HepG2 cells and ANGPTL8 KD HepG2 cells after LPS (4 μg/ml) stimulation for 24 h. H: Western blot analysis of the protein levels of PPARα and PPARγ in ANGPTL8 KD HepG2 cells and control HepG2 cells after LPS (4 μg/ml) stimulation for 24 h. I: ANGPTL8…

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  Fig. 5. The LPS-induced upregulation of PPARα depends on PGC1α. A and B: qRT‒PCR and Western blot analysis of the mRNA (A) and protein (B) expression of PGC1α in the liver tissue of Angptl8 KO and WT mice after LPS (10 mg/kg) stimulation for 48 h (n = 3∼5 per group). C and D: qRT‒PCR and Western blot analysis of the mRNA (C) and protein (D) expression of PGC1α in ANGPTL8 KD HepG2 cells and control HepG2 cells after LPS (4 μg/ml) stimulation for 24 h. E: Oil Red O staining analysis of lipid deposition in ANGPTL8 KD HepG2 cells and control HepG2 cells after stimulation with the PGC1α inhibitor SR-18292 (20 μM) and LPS for 24 h. F and G: qRT‒PCR and Western blot analysis of the mRNA (F) and protein (G) expression of PPARα in ANGPTL8 KD HepG2 cells after stimulation with SR-18292 and LPS for 24 h. H: Western blot analysis of the protein level of PGC1α in the cytoplasm and nucleus of ANGPTL8 KD HepG2 cells and control HepG2 cells after LPS (4 μg/ml) stimulation for 24 h; β-actin and lamin B1 were used as reference for the cytoplasm and nucleus, respectively. The data are shown as the mean ± SEM and were analysed using an unpaired two-tailed Student’s test (A, C, F), ∗P < 0.05, ∗∗P < 0.01,…

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  Fig. 6. Inhibition of TNF-α can ameliorate LPS-induced lipid deposition in vivo and in vitro. A: qRT‒PCR detection of TNF-α mRNA expression at different time points after LPS (4 μg/ml) stimulation. B and C: The mRNA (B) and protein (C) expression of ANGPTL8 in HepG2 cells after LPS or adalimumab (5 μg/ml) treatment for 24 h was measured by qRT‒PCR and Western blot, respectively. D: H&E staining and Oil Red O staining analysis of pathology and lipid deposition in the livers of septic mice treated with the TNF-α receptor inhibitor R-7050 (6 mg/kg) for 24 h and 48 h, respectively (n = 3∼4 per group). E and F: TG levels (D) and MDA levels (E) in liver homogenates from septic mice after R-7050 treatment for 24 h or 48 h were measured (n = 3∼4 per group). G and H: The serum ALT (G) and AST (H) levels of septic mice after R-7050 treatment for 24 h or 48 h were detected (n = 3∼4 per group). I–K: The expression of PGC1α and PPARα in the livers of septic mice after R-7050 treatment for 24 h or 48 h was measured by qRT‒PCR (I and J) and Western blot (K) (n = 3 per group). L: The survival of septic mice after R-7050 treatment was observed and statistically analyzed. The data are shown as the mean ±…

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  Fig. 7. Working model of the ANGTPL8-mediated regulation of PGC1α/PPARα signaling. Infection upregulates liver TNF-α expression, which further promotes ANGPTL8 expression. ANGPTL8 can inhibit the expression and activation of PGC1α. The expression of the downstream gene of PPARα, was also downregulated, which blocks fatty acid β-oxidation and leads to lipid deposition and liver damage.

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  Supplemental Figure S1. ANGPTL8 deficiency protects against LPS-induced apoptosis in liver tissue. A: The first exon of the ANGPTL8 gene in Angptl8 KO mice is missing 8 bases, and the Angptl8 KO mice were identified by DNA sequencing. B: TUNEL staining was used to analyse the number of apoptotic cells in liver tissue from Angptl8 KO and WT mice after LPS (10 mg/kg) stimulation for 48 h (n = 3 per group). The data are shown as the mean ± SEM and were analysed using an unpaired two-tailed Student’s test ∗∗∗∗p < 0.0001, ns > 0.05.

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  Supplemental Figure S2. LPS promotes lipid deposition in HepG2 cells. A: Oil Red O staining analysis of lipid deposition in HepG2 cells after LPS (2 μg/ml, 4 μg/ml or 8 μg/ml) stimulation for 24 h. B: Lipid deposition in HepG2 cells was detected by Oil Red O staining after LPS (4 μg/ml) stimulation for 24 h or 48 h. C: Lipid deposition in ANGPTL8 KD HepG2 cells and control HepG2 cells after LPS (4 μg/ml) stimulation for 24 h was detected by Oil Red O staining.

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  Supplemental Figure S3. The inhibitory effects of LPS on PPARα were dependent on PGC1α. A: The optimal treatment duration and concentration of the PGC1α inhibitor SR-18292 were determined via a CCK8 assay. B: Oil Red O staining analysis of lipid deposition in HepG2 cells after SR-18292 (20 μM) treatment for 24 h. C and D: The expression of PPARα in HepG2 cells after SR-18292 treatment was analysed through qRT‒PCR (C) and Western blot (D). The data are shown as the mean ± SEM and were analysed using an unpaired two-tailed Student’s test; ∗p < 0.05, ∗∗p < 0.01, ns > 0.05.

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  Supplemental Figure S4. Regulation of lipid metabolism by ANGPTL8 during sepsis depends on TNF-α. A and B: HepG2 cells were stimulated with TNF-α at 50, 100 and 200 ng/ml, respectively, and ANGPTL8 expression was measured by qRT‒PCR (A) and Western blot (B). C: ELISA was used to measure the serum TNF-α concentration after LPS stimulation for 24 h, 48 h, or 72 h (n = 4 per group). D: The mRNA expression of TNF-α in liver tissue from LPS challenged mice was detected through qRT‒PCR (n ≥ 3 per group). E: Oil Red O staining analysis of lipid deposition in ANGPTL8 KD HepG2 cells and control HepG2 cells after treatment with LPS (4 μg/ml) or the TNF-α monoclonal antibody adalimumab (5 μg/ml) for 24 h. The data are shown as the mean ± SEM and were analysed using an unpaired two-tailed Student’s test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.