Hepatic Slug epigenetically promotes liver lipogenesis, fatty liver disease, and type 2 diabetes

Abstract

De novo lipogenesis is tightly regulated by insulin and nutritional signals to maintain metabolic homeostasis. Excessive lipogenesis induces lipotoxicity, leading to nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Genetic lipogenic programs have been extensively investigated, but epigenetic regulation of lipogenesis is poorly understood. Here, we identified Slug as an important epigenetic regulator of lipogenesis. Hepatic Slug levels were markedly upregulated in mice by either feeding or insulin treatment. In primary hepatocytes, insulin stimulation increased Slug expression, stability, and interactions with epigenetic enzyme lysine-specific demethylase-1 (Lsd1). Slug bound to the fatty acid synthase (Fasn) promoter where Slug-associated Lsd1 catalyzed H3K9 demethylation, thereby stimulating Fasn expression and lipogenesis. Ablation of Slug blunted insulin-stimulated lipogenesis. Conversely, overexpression of Slug, but not a Lsd1 binding-defective Slug mutant, stimulated Fasn expression and lipogenesis. Lsd1 inhibitor treatment also blocked Slug-stimulated lipogenesis. Remarkably, hepatocyte-specific deletion of Slug inhibited the hepatic lipogenic program and protected against obesity-associated NAFLD, insulin resistance, and glucose intolerance in mice. Conversely, liver-restricted overexpression of Slug, but not the Lsd1 binding-defective Slug mutant, had the opposite effects. These results unveil an insulin/Slug/Lsd1/H3K9 demethylation lipogenic pathway that promotes NAFLD and type 2 diabetes.

Keywords: Diabetes; Hepatology; Insulin; Insulin signaling; Metabolism.

Figure 1. Hepatic Slug is upregulated by insulin and is elevated in NAFLD.

(A) C57BL/6J males were overnight-fasted and then fed again for 3 hours. Liver nuclear extracts were immunoblotted with the indicated antibodies. (B) C57BL/6J males were fasted overnight and treated with insulin (1 U/kg body weight for 4 hours). Liver nuclear extracts were immunoblotted with indicated antibodies. Slug levels were normalized to lamin A/C levels (n = 3 per group). (C) Liver Slug mRNA abundance (normalized to 36B4 levels; n = 3 per group). (D) Primary hepatocytes were pretreated with wortmannin (100 nM) or MK2066 (100 nM) for 0.5 hours before insulin stimulation (100 nM for 2 hours). Nuclear extracts and cell extracts were immunoblotted with the indicated antibodies. (E and F) Primary hepatocytes were transduced with Slug adenoviral vectors, treated with insulin in the presence or absence of cycloheximide. Nuclear Slug levels were normalized to lamin A/C (n = 3 per group). (G) Primary hepatocytes were transduced with Slug adenoviral vectors for 12 hours, and then stimulated with insulin (100 nM for 1 hour) in the presence or absence of MG132 (5 μM). Cell extracts were immunoprecipitated with antibody against Slug and immunoblotted with the indicated antibodies. (H) Liver Slug mRNA levels (normalized to 36B4 levels). Chow: HFD (n = 5, for 10 weeks); ob/ob (n = 5, 14 weeks of age). (I) Liver nuclear extracts were prepared from WT and ob/ob mice at 14 weeks of age or from WT mice fed a chow diet or HFD for 10 weeks, and immunoblotted with antibodies against Slug and lamin A/C. (J) Liver SLUG mRNA levels in NASH patients (n = 11) and normal subjects (Con) (n = 10) (normalized to GAPDH). Proteins were resolved in parallel gels. Data are presented as mean ± SEM. *P < 0.05, 2-tailed Student’s t test (B, C, and J) or 1-way ANOVA/Sidak posttest (H).

Figure 2. Hepatocyte-specific deletion of Slug protects against liver steatosis in obesity.

(A–E) Slug^Δhep and Slug^fl/fl mice were fed a HFD for 11 weeks. (A) Growth curves (n = 11 per group). (B) Liver size and weight. Male: n = 9 per group; female: n = 10 per group. (C) Reprehensive liver sections (n = 3–9 mice per group). Scale bar: 100 μm. (D) Liver TAG levels (normalized to liver weight). Male: n = 9 per group; female: n = 6 per group. (E) Overnight fasting plasma TAG levels in male (n = 9 per group). (F–J) Slug^fl/fl ob/ob males were transduced with GFP or Cre adenoviral vectors for 3 weeks. (F) Liver extracts were immunoblotted with antibodies against Slug and α-tubulin. (G) Growth curves (n = 6 per group). (H) Representative liver sections (3 pairs). Scale bar: 100 μm. (I) Liver TAG levels (normalized to liver weight). GFP: n = 6; Cre: n = 5. Data are presented as mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Figure 3. Ablation of hepatic Slug ameliorates diet-induced insulin resistance and glucose intolerance.

(A–E) Slug^Δhep and Slug^fl/fl mice were fed a HFD for 8 to 11 weeks. (A) Overnight-fasted plasma insulin levels (male: n = 8 per group; female: n = 6 per group). (B–D) GTT, ITT and PTT. Male: n = 11 per group; female: n = 11 per group. AUC: area under curves. (E and F) Mice were fasted overnight and stimulated with insulin (1 U/kg body weight for 5 minutes). Liver extracts were immunoblotted with antibodies against phospho-Akt or Akt. Phospho-Akt levels were normalized to total Akt levels (n = 3). (G–I) Tam^fl/fl and Tam^Δhep males were fed a HFD for 10 weeks. (G) Overnight-fasted plasma insulin levels (n = 6 per group). (H and I) GTT and ITT. Tam^fl/fl: n = 12; Tam^Δhep: n = 9. Data are presented as mean ± SEM. *P < 0.05, 2-tailed Student’s t test (A–D, F–J: AUC) and 2-way ANOVA/Bonferroni’s posttest (B–D, H, I: curves).

Figure 4. Ablation of hepatic Slug suppresses the hepatic lipogenic program.

Slug^Δhep and Slug^fl/fl males were fed a HFD for 11 weeks. (A–C) Affymetrix analysis (n = 6 per group). (A) Gene ontology analysis. (B) Volcano plots. (C) Heatmaps. (D) Liver extracts were immunoblotted with the indicated antibodies. Arrow indicates the mature form of Srepb1c. (E) Liver mRNA abundance (normalized to 36B4 levels). Slug^fl/fl: n = 7; Slug^Δhep: n = 9. Data are presented as mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Figure 5. Liver-specific overexpression of Slug but not ΔN30 promotes liver steatosis and insulin resistance.

C57BL/6J males were transduced with AAV-CAG-GFP, AAV-CAG-Slug, or AAV-CAG-ΔN30 vectors, and fed a HFD for 11 weeks. (A) Liver Slug mRNA levels (normalized to 36B4 levels, n = 4–5 per group). (B) Growth curves (n = 10 per group). (C and D) Representative livers and liver sections (n = 3 mice per group). Scale bar: 100 μm. (E) Liver TAG levels (normalized to liver weight); n = 6 per group. (F) Liver extracts were immunoblotted with the indicated antibodies. Fasn and Acc1 levels were normalized to α-tubulin levels. (G) Overnight-fasted plasma insulin levels (n = 6 per group), GTT, and ITT (n = 10, per group) 8 to 9 weeks after AAV transduction. (H) Mice were fasted overnight and stimulated with insulin (1 U/kg body weight for 5 minutes). Liver extracts were immunoblotted with antibodies against phospho-Akt (pThr308, pSer473) and Akt. Data are presented as mean ± SEM. *P < 0.05, 1-way ANOVA/Sidak posttest.

Figure 6. Slug/Lsd1/H3K9 demethylation pathway stimulates lipogenesis.

(A) Coimmunoprecipitation of Slug with Lsd1 in HEK293 cells. (B) Primary hepatocytes were stimulated with insulin (100 nM for 1 hour). Cell extracts were immunoprecipitated with anti-Slug antibody and immunoblotted with antibodies against Lsd1 and Slug. (C–E) Primary hepatocytes were transduced with Slug or β-gal adenoviral vectors and treated with GSK2879552 (1 μM) or DMSO for 24 hours. (C) Lipogenesis rates (normalized to protein levels) (n = 3 per group). (D) Cell extracts were immunoblotted with the indicated antibodies. Fasn and Acc1 levels were normalized to α-tubulin levels (n = 3). (E) Fasn, Acc1, and Srebp1c mRNA abundance (normalized to 36B4 levels) (n = 3 per group). (F) HEK293 cells were transfected with AAV-CAG-Slug or AAV-CAG-ΔN30 vectors. Cell extracts were immunoblotted with antibodies against Slug and α-tubulin. Fasn luciferase reporter activity (normalized to β-gal internal control) in HepG2 cells (n = 3). (G) Slug^Δhep (n = 3) and Slug^fl/fl (n = 3) males were fed a HFD for 11 weeks. Fasn promoter H3K9 and H3K4 methylation levels were measured in the liver by ChIP-qPCR. (H) Liver Fasn promoter H3K9 and H3K4 methylation levels (n = 4 per group). C57BL/6J males were transduced with AAV-CAG-GFP, AAV-CAG-Slug, or AAV-CAG-ΔN30 vectors, and fed a HFD for 11 weeks. (I) C57BL/6J mice were transduced with GFP or Slug adenoviral vectors and treated with GSK2879552. Fasn promoter H3K9me1 levels were assessed in the liver using ChIP (exclusion criteria: greater than 3 times SD). Data are presented as mean ± SEM. *P < 0.05, 2-tailed Student’s t test (G) or 1-way ANOVA/Sidak posttest (C–F and I).

Figure 7. Insulin stimulated lipogenesis via Slug/Lsd1 epigenetic pathway.

(A and B) Primary hepatocytes were transduced with Slug or β-gal adenoviral vectors, and stimulated with insulin (100 nM for 2 hours). Slug occupancy on the Fasn promoter was assessed by ChIP-qPCR and normalized to inputs (n = 3 per group). (C–E) Primary hepatocytes were stimulated with insulin (50 nM) for 3 hours (C) or 12 hours (D and E). (C) Fasn mRNA abundance (normalized to 36B4 levels) (n = 3 per group). (D) Cell extracts were immunoblotted with the indicated antibodies. Fasn levels were normalized to α-tubulin levels. (E) Lipogenesis rates (n = 3 per group). (F) Lipogenesis (normalized to protein levels, n = 3 per group). Primary hepatocytes were transduced with Slug or β-gal adenoviral vectors and stimulated with insulin (50 nM for 12 hours). (G) Lipogenesis (n = 3 per group). Primary hepatocytes were pretreated with GSK2879552 (4 μM) or DMSO and stimulated with insulin (50 nM for 5 hours). (H) Insulin stimulates Slug expression, Slug-Lsd1 interactions, and recruitment of Slug/Lsd1 to the Fasn promoter where Lsd1 demethylates H3K9, thereby activating Fasn expression and de novo lipogenesis. Cytokines and metabolic stressors similarly activate the Slug/Lsd1 lipogenic pathway. Data are presented as mean ± SEM. *P < 0.05, 1-way ANOVA/Sidak posttest.