Hepatic Zbtb18 (Zinc Finger and BTB Domain Containing 18) alleviates hepatic steatohepatitis via FXR (Farnesoid X Receptor)

Abstract

A lasting imbalance between fatty acid synthesis and consumption leads to non-alcoholic fatty liver disease (NAFLD), coupled with hepatitis and insulin resistance. Yet the details of the underlying mechanisms are not fully understood. Here, we unraveled that the expression of the transcription factor Zbtb18 is markedly decreased in the livers of both patients and murine models of NAFLD. Hepatic Zbtb18 knockout promoted NAFLD features like impaired energy expenditure and fatty acid oxidation (FAO), and induced insulin resistance. Conversely, hepatic Zbtb18 overexpression alleviated hepato-steatosis, insulin resistance, and hyperglycemia in mice fed on a high-fat diet (HFD) or in diabetic mice. Notably, in vitro and in vivo mechanistic studies revealed that Zbtb18 transcriptional activation of Farnesoid X receptor (FXR) mediated FAO and Clathrin Heavy Chain (CLTC) protein hinders NLRP3 inflammasome activity. This key mechanism by which hepatocyte's Zbtb18 expression alleviates NAFLD and consequent liver fibrosis was further verified by FXR's deletion and forced expression in mice and cultured mouse primary hepatocytes (MPHs). Moreover, CLTC deletion significantly abrogated the hepatic Zbtb18 overexpression-driven inhibition of NLRP3 inflammasome activity in macrophages. Altogether, Zbtb18 transcriptionally activates the FXR-mediated FAO and CLTC expression, which inhibits NLRP3 inflammasome's activity alleviating inflammatory stress and insulin resistance, representing an attractive remedy for hepatic steatosis and fibrosis.

Fig. 1

Hepatic Zbtb18 mRNA and protein down-regulation is closely related to the development of NAFLD. a Venn diagram representing common significantly changed transcripts in liver from mice with NAFLD (GSE35961, P < 0.01), NAFLD patients (GSE213621, P < 0.05) and our clinic transcript data (P < 0.05). b–e Representative quantitative PCR and Western blot analysis of hepatic Zbtb18 in clinical samples (b, normal controls = 7, NAFLD patients = 8); diabetic mice (c, n = 6); obese mice (d, n = 6); HFD-fed mice (e, n = 6). f, g Representative quantitative PCR, Western blot and immunofluorescence (IF) analysis of Zbtb18 in MPHs treated with OA&PA; n = 4. h Representative quantitative PCR and Western blot data show an effective overexpression of Zbtb18 in Ad-Zbtb18 infected MPHs; n ≥ 5. i Representative quantitative PCR data show that Zbtb18 overexpression significantly upregulates the genes involved in FAO and OXPHOS; n ≥ 5. j Zbtb18 overexpression elevates the FAO rates in MPHs; n = 4. k, l Zbtb18 overexpression decreases the lipid accumulation (k) and TGs contents (l) in MPHs; n = 6. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001

Fig. 2

Hepatic ablation of Zbtb18 aggravates steatohepatitis in mice fed on HFD. a, b Hepatic Zbtb18 deletion leads to increases in body weight (a) and fat mass percentage (b) in mice fed on HFD; n ≥ 5. c Adipocyte hypertrophy of epididymal WAT, inguinal WAT and interscapular BAT of hepatic Zbtb18 deleted mice fed on HFD. d, e Hepatic Zbtb18 deletion decreases thermogenic genes (d) and energy expenditure (e) in BAT of mice fed on HFD; n ≥ 5. f, g Hepatic Zbtb18 deletion increases the fasting blood glucose (f) and impairs glucose tolerance, and insulin sensitivity (g) in mice fed on HFD; n = 6. h Hepatic Zbtb18 deficiency decreases the phosphorylation of AKT and GSK-3β in the livers of mice fed on HFD. i, j Hepatic Zbtb18 deficiency alters the function of genes related to glucose and lipid metabolism (i) and leads to severe fatty liver phenotype (j) in mice fed on HFD, n = 6. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 3

Hepatic Zbtb18 overexpression defends against HFD-induced hepatic steatosis. a, b Hepatic Zbtb18 overexpression decreases body weight (a) and fat mass (b) of mice fed on HFD; n ≥ 4. c, d Hepatic Zbtb18 overexpression increases energy expenditure (c) and Ucp1 expression in the BAT (d) of mice fed on HFD; n ≥ 5. e, f Hepatic Zbtb18 overexpression improves the hepatic steatosis (e), increases serum ketone body levels (f) and decreases TGs contents in serum and livers of mice fed on HFD; n = 6. g, h Hepatic Zbtb18 overexpression decreased serum and hepatic TG contents (g) and altered hepatic genes related to glucose and lipid metabolism (h) in mice fed on HFD; n ≥ 5. i Hepatic Zbtb18 overexpression decreases F4/80⁺ cells in the livers of mice fed on HFD. j, k Hepatic Zbtb18 overexpression decreases the mRNA levels of inflammatory genes (j) and serum proinflammatory cytokines levels (k) of mice fed on HFD; n = 6. l Hepatic Zbtb18 overexpression decreases serum ALT and AST levels in mice fed on HFD; n = 6. m–o Hepatic Zbtb18 overexpression decreases fasting blood glucose (m), and insulin levels (n) and improves glucose intolerance and insulin resistance (o) in mice fed on HFD; n = 6. p Hepatic Zbtb18 overexpression enhances the phosphorylation of AKT and GSK-3β in the livers of mice fed on HFD. Data are shown as means ± SEM. *P < 0.05; **P < 0.01

Fig. 4

Rescued hepatic Zbtb18 expression alleviates hepato-steatosis in diabetic mice. a–c Rescued hepatic Zbtb18 expression decreases the body weight (a), the ratio values of liver weight to body weight (b) and TGs contents in the serum and livers (c) of db/db mice; n = 6. d AAV-Zbtb18 infected db/db mice show an improved liver steatosis phenotype. e, f Hepatic Zbtb18 overexpression significantly reduces fasting blood glucose and insulin levels (e), and improves glucose tolerance and insulin sensitivity (f) of db/db mice; n = 6. g, h Hepatic Zbtb18 overexpression increases serum ketone body levels (g) and alters the expression of genes related to glucose and lipid metabolism (h) in db/db mice; n = 6. i Rescued Zbtb18 expression in livers increases the phosphorylation of AKT and GSK-3β in db/db mice. j Hepatic Zbtb18 overexpression decreases F4/80⁺ and Cd11b⁺ cells in the livers of db/db mice. k, l Hepatic Zbtb18 overexpression reduces serum proinflammatory cytokines levels (k) and ALT, and AST levels (l) in db/db mice, n = 6. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001

Fig. 5

Zbtb18 transcriptional activation of FXR accelerates lipid catabolism via FAO. a, b RNA-seq data of Zbtb18-overexpressing and control MPHs show that Zbtb18 alters the expression of genes related to FAO and the FXR signaling pathway, as shown by Scatter plot (a) and KEGG analysis (b). c Venn diagram showing the overlapping significantly differentially expressed transcripts identified in MPHs overexpressing Zbtb18 (P < 0.01) and the liver form the NAFLD patients (P < 0.01). d Heatmap of transcriptome data showing the mRNA levels of genes involved in the FAO and FXR signaling pathway. e GSEA analysis of cellular components of DEGs identified in Zbtb18-overexpressing primary hepatocytes and the patients with NAFLD. f, g Representative quantitative PCR and Western blot analysis of FXR and of its target genes in hepatic Zbtb18 overexpressing mice and control mice, n ≥ 5. h Immunofluorescence analysis reveals that Zbtb18 overexpression increases FXR and SHP levels in the cytoplasm and nucleus of cultured MPHs. i Map of the FXR locus revealing Zbtb18 binding in MPHs; aligned reads were visualized by Integrated Genomics Viewer 2. The signal of the IgG or Anti-Zbtb18 is represented with gray or red peaks, respectively. j The predicted Zbtb18-binding motif. k, l Luciferase assays indicate that Zbtb18 protein binds to a unique site (−993 bp to −984 bp) to transcriptionally activate FXR expression, n ≥ 5. m ChIP-qPCR analyses of the Zbtb18 protein occupancy on the FXR promoter; n = 3. n FXR deletion diminished the Zbtb18 protein-driven protective effects on lipid accumulation. o, p FXR deletion reduces the stimulatory effects on FAO induced by Zbtb18 protein overexpression; n = 4 (o) and TGs contents (p) in MPHs; n ≥ 3. q Forced expression of FXR counteracts the Zbtb18-deficiency-induced elevation of TGs contents in MPHs; n ≥ 4. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001

Fig. 6

Hepatic Zbtb18 protein inhibits NLRP3 inflammasome’s activation in macrophage via an FXR-mediated CLTC protein expression. a Hepatic Zbtb18 deletion increases HFD-induced inflammasome-related proteins NLRP3, ASC, Caspase-1, and NF-кB phosphorylation in livers. b Hepatic Zbtb18 overexpression decreases HFD-induced inflammasome-related proteins NLRP3, ASC, Caspase-1, and NF-кB phosphorylation in livers. c Hepatic Zbtb18 overexpression decreases inflammasome-related proteins NLRP3, ASC, Caspase-1, and NF-кB phosphorylation in the livers of db/db mice. d Representative Western blot analyses indicated that the treatment with the conditioned medium of Ad-Zbtb18 infected MPHs cultures suppressed the OA&PA&LPS-induced expression of inflammasome related proteins, NLRP3, ASC, Caspase-1, and NF-κb phosphorylation in BMDM macrophages. e FXR deletion diminishes the hepatic Zbtb18 overexpression-induced protective effects on inflammasome-related proteins NLRP3, ASC, Caspase-1, and NF-кB phosphorylation in livers. f Hepatic FXR forced expression rescued the Zbtb18 protein deficiency-induced expression of inflammasome-related proteins NLRP3, ASC, Caspase-1, and NF-кB phosphorylation in livers. g, h Representative Western blot and Immunofluorescence data show that the treatment with a conditioned medium of Ad-Zbtb18-infected CLTC knockout MPHs cultures fails to alter the OA&PA&LPS-induced expression of inflammasome related proteins NLRP3, ASC, Caspase-1, and NF-кB phosphorylation in BMDM cells

Fig. 7

FXR ablation diminished hepatic Zbtb18-induced protective effects against steatohepatitis. a, b Hepatic Zbtb18 overexpression fails to reduce the body weight (a) and fat mass (b) of FXR knockout mice; n = 6. c, d FXR deficiency abrogates the Zbtb18 protein-stimulated elevation of serum ketone body (c) and expression of hepatic genes related to FAO (d); n = 6. e, f Hepatic Zbtb18 overexpression fails to alter the TGs contents in the serum and liver (e), and the fatty liver phenotype (f) due to FXR deletion; n = 6. g Hepatic Zbtb18 overexpression fails to change the fasting blood glucose and insulin levels in FXR knockout mice; n = 6. h The Zbtb18-driven improvement of glucose and insulin resistance was diminished in FXR knockout mice; n = 6. i–j Hepatic Zbtb18 overexpression fails to change the phosphorylation of AKT and GSK-3β (i) and of glucogenic genes (j) in FXR knockout mice, n = 6. k, l Hepatic Zbtb18 overexpression does not change serum proinflammatory cytokines (k) and ALT and AST levels (l) following FXR ablation; n = 6. Data are shown as means ± SEM. ns=no significant, *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001

Fig. 8

Hepatic FXR forced expression alleviates NAFLD phenotype in hepatic Zbtb18 deleted mice. a, b Hepatic FXR overexpression (a) decreases the fat mass (b) of Zbtb18^LKO mice; n = 6. c Hepatic FXR overexpression rescues Zbtb18 deficiency-induced impairment of energy expenditure; n ≥ 4. d Hepatic FXR overexpression decreases Zbtb18 deficiency-induced liver weight gain and TGs accumulation in liver and serum, while recovering normal levels of serum ketone body; n = 6. e, f Hepatic FXR overexpression reduces Zbtb18 deficiency-induced fatty liver phenotype (e), and the dysregulation of genes related to lipid metabolism (f); n = 6. g Hepatic FXR overexpression decreases the fasting blood glucose and insulin levels in Zbtb18^LKO mice; n = 6. h Hepatic FXR overexpression improves glucose and insulin resistance in Zbtb18^LKOmice; n = 6. i Hepatic FXR overexpression decreases Zbtb18 deficiency-induced glucogenic genes in mice; n = 6. j Hepatic FXR overexpression alleviates the impaired phosphorylation of AKT and GSK-3β in the livers of Zbtb18^LKO mice. k Hepatic FXR overexpression reduces the Zbtb18 deficiency-induced gathering of F4/80⁺ and Cd11b⁺ cells in livers. l, m Hepatic FXR overexpression decreases the hepatic mRNA expression of inflammatory genes (l) and serum proinflammatory cytokines levels (m) in Zbtb18^LKO mice, n = 6. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001

Fig. 9

Hepatic Zbtb18 deletion aggravates MCD-induced progression of liver fibrosis. a, b Serum ALT, and AST levels (a) and TGs contents in serum and livers (b) of hepatic Zbtb18 deleted mice and control mice fed on MCD diet or on normal diet, n = 6. c Hepatic Zbtb18 deletion aggravates MCD-induced liver injury and lipid deposition in livers. d Sirius and α-SMA staining of liver sections from hepatic Zbtb18 deleted mice and control mice fed on MCD diet or a normal diet. e Hepatic Zbtb18 deletion alters the expression of genes related to liver fibrosis and to an inflammatory response in the liver of mice fed on MCD diet or on a normal diet; n = 6. f Hepatic Zbtb18 deletion aggravates the MCD-induced suppression of FXR and its downstream target genes in the liver. g, h Hepatic Zbtb18 deletion aggravates the MCD-induced elevation of serum proinflammatory cytokines (g) and the hepatic gathering of F4/80⁺ and CD11b⁺cells (h); n = 6. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 10

Hepatic Zbtb18 overexpression protects mice against MCD-induced liver fibrosis. a, b Serum ALT, and AST levels (a) and TGs contents in serum and liver (b) samples from hepatic Zbtb18 overexpressing mice and control mice fed on MCD diet or normal diet; n = 6. c Hepatic Zbtb18 overexpression attenuates MCD-induced liver injury and hepato-steatosis. d Sirius and α-SMA staining of liver sections from hepatic Zbtb18 transgenic mice and control mice fed on MCD diet or normal diet. e Hepatic Zbtb18 overexpression alters the expression of genes related to liver inflammatory response and fibrosis in mice fed on MCD diet or normal diet; n = 6. f, g Hepatic Zbtb18 overexpression reduces the MCD-induced elevation of serum proinflammatory cytokines (f) and the hepatic accumulation of F4/80⁺ and CD11b⁺ cells (g); n = 6. Data are shown as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005

Fig. 11

Zbtb18 transcriptionally activates the FXR-mediated hepatic lipid metabolism, which inhibits NLRP3 inflammasome’s activity alleviating inflammatory stress and insulin resistance