Long noncoding RNA licensing of obesity-linked hepatic lipogenesis and NAFLD pathogenesis

Fig. 1. Hepatic Blnc1 is linked to obesity and lipogenic activation. a qPCR analysis of Blnc1 and Srebp1c expression in the livers from lean (open) and obese (filled) mice. For diet-induced obesity (DIO), WT mice were fed chow (n = 4) or HFD (n = 12) for 10 weeks. For genetic obesity, WT (n = 5), ob/ob (n = 3), or db/db (n = 6) mice were fed standard chow. Data represent mean ± SEM. **p < 0.01, ***p < 0.001, obese vs. lean, two-tailed unpaired Student’s t-test. b Correlation between liver Blnc1 RNA levels and body weight (left) or liver fat content (right) in HFD-fed mice. c qPCR analysis of gene expression in hepatocytes transduced with GFP (open) or Blnc1 (filled) adenovirus followed by treatment with vehicle (Veh) or 5 μM T0901317 (T1317) for 24 h. d Incorporation of 14C-acetate into lipids in transduced hepatocytes treated with Veh or T1317 for 24 h. Data in (c) and (d) represent mean ± SD (n = 3). **p < 0.01, ***p < 0.001, GFP vs. Blnc1, two-way ANOVA. e Plasma and liver TAG and cholesterol in mice transduced with AAV vectors expressing GFP (n = 5) or Blnc1 (n = 7). f Immunoblots of total liver lysates and liver nuclear extracts (NE) from transduced mice. g qPCR analysis of hepatic…

Fig. 2. Blnc1 is required for LXR-mediated lipogenic activation. a Plasma and liver TAG content from WT or Blnc1 KO mice receiving oral gavage of vehicle (Veh, open, WT, n = 4; KO, n = 3) or T0901317 (T1317, filled, WT, n = 3; KO, n = 4) for 4 days. b H&E staining of liver sections. Scale bar = 100 μm. c qPCR analysis of hepatic gene expression. Data in (a) and (c) represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, WT vs. KO, two-way ANOVA. d Immunoblots of total liver lysates and liver nuclear extracts. e qPCR analysis of gene expression in WT and Blnc1 KO hepatocytes treated with vehicle (Veh) or 5 μM T1317 for 24 h. f Incorporation of 14C-acetate into lipids in hepatocytes treated with Veh or T1317. Data in (e, f) represent mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, WT vs. KO, two-way ANOVA. g qPCR analysis of gene expression in hepatocytes transduced with GFP or Blnc1 adenovirus treated with Veh or 10 μM SR9238 for 24 h. h Incorporation of 14C-acetate into lipids in treated hepatocytes. Data in (g) and (h) represent mean ± SD (n = 3). **p < 0.01, ***p < 0.001, GFP vs. Blnc1, #p < 0.001, Veh vs. SR9238, two-way ANOVA

Fig. 3. Liver-specific inactivation of Blnc1 alleviates diet-induced obesity. a A schematic of the strategy for the generation of Blnc1 LKO mice using CRISPR/Cas9. Adenoviral expression of sgRNAs in Cas9 transgenic mice results in liver-specific deletion of Blnc1. b Body weight of control (ctrl, filled, n = 8) and LKO (open, n = 11) mice fed HFD for 10 weeks. Data represent mean ± SEM. *p < 0.01, ctrl vs. LKO, two-way ANOVA with multiple comparisons. c Blood glucose (left) and plasma insulin (right) concentrations in ctrl (n = 9) and LKO (n = 9) mice after HFD feeding for 17 weeks (fast) and 19 weeks (fed). d, e GTT (d) and ITT (e) in ctrl (filled, n = 9) and LKO (open, n = 9) mice fed HFD for 8 and 9 weeks, respectively. Data represent mean ± SEM. **p < 0.01, ***p < 0.001; ctrl vs. LKO, two-way ANOVA with multiple comparisons. f Plasma parameters in mice fed HFD for 19 weeks. Data in (c) and (f) represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ctrl vs. LKO, two-tailed unpaired Student’s t-test

Fig. 4. Liver-specific inactivation of Blnc1 attenuates hepatic lipogenesis and preserves metabolic health. a Liver weight, TAG, and cholesterol content in ctrl (filled, n = 9) and LKO (open, n = 9) mice fed HFD for 19 weeks. b H&E staining of liver sections. Scale bar = 100 μm. c Immunoblots of total liver lysates and liver nuclear extracts. d qPCR analysis of hepatic gene expression. Data in (a) and (d) represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ctrl vs. LKO, two-tailed unpaired Student’s t-test

Fig. 5. Hepatic ablation of Blnc1 stimulates thermogenesis and energy expenditure. a, b VO2 and energy expenditure (a), food intake and total activity (b) in ctrl (filled, n = 8) and LKO (open, n = 8) mice fed HFD for 15 weeks. Data in (a) and (b) represent mean ± SEM. *p < 0.05; ctrl vs. LKO, two-tailed unpaired Student’s t-test. c H&E staining of BAT section from HFD-fed mice. Scale bar = 100 μm. d qPCR analysis of brown fat gene expression in HFD-fed ctrl (filled, n = 9) and LKO (open, n = 9) mice. Data represent mean ± SEM. *p < 0.05, **p < 0.01; ctrl vs. LKO, two-tailed unpaired Student’s t-test

Fig. 6. Blnc1 inactivation ameliorates diet-induced NASH. a Blnc1 expression in mice fed chow (n = 3) or NASH (n = 3) diet for 6 months. Data represent mean ± SEM. ***p < 0.001, chow vs. NASH, two-tailed unpaired Student’s t-test. (b–d) Plasma lipids (b), liver weight, TAG and cholesterol content (c) and plasma ALT and AST levels (d) in ctrl (filled, n = 6) and LKO (open, n = 6) mice fed NASH diet for 24 weeks. e H&E and Sirius red staining of liver sections. Scare bar = 100 μm. f Liver hydroxyproline content and quantitation of Sirius red-positive area in e. g TUNEL staining and quantitation (scale bar = 100 μm). Data in (b–d), (f) and (g) represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Ctrl vs. LKO, two-tailed unpaired Student’s t-test

Fig. 7. Blnc1 inactivation suppresses the molecular signature of diet-induced NASH. a Heat map representation of upregulated and downregulated genes identified in RNAseq analysis of the livers from NASH-fed mice. b Enrichment score for downregulated (blue) and upregulated (yellow) genes. c qPCR analysis of hepatic genes involved in lipid metabolism from ctrl (filled, n = 6) and LKO (open, n = 6) mice fed NASH diet for 24 weeks. d Immunoblots of total liver lysates and liver nuclear extracts from NASH-fed mice. e qPCR analysis of hepatic genes involved in inflammation and fibrosis. Data in (c) and (e) represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.01; ctrl vs. LKO, two-tailed unpaired Student’s t-test

Fig. 8. Proteomic analysis of the liver Blnc1 ribonucleoprotein complex. a Silver-stained gel of affinity-purified Blnc1 complexes. b Immunoblots of precipitated proteins on streptavidin (SA) beads or total liver lysates. c IP/qPCR analyses of endogenous Blnc1 on the immunocomplexes as indicated. Immunoblots of precipitated proteins are shown on the right. Data represent mean ± SD (n = 3). ***p < 0.001, two-tailed unpaired Student’s t-test. d IP/qPCR analyses of Blnc1 in EDF1 (d) or LXRα (e) immunocomplexes (top) or input (bottom) from transiently transfected HEK293T cells. Data in (d) and (e) represent mean ± SD (n = 3). ***p < 0.001, two-way ANOVA. f Immunoblots of HA-EDF1 (top) or HA-LXRα (bottom) from total lysates and precipitated proteins on SA beads from transfected HEK293T cells

Fig. 9. Physical and functional interaction between Blnc1, LXRα, and EDF1. a Diagram of Blnc1 RNA domains (RD, left) and truncation mutants (right). b, c Immunoblots of total lysates and streptavidin (SA) beads precipitated proteins from transiently transfected HEK293T cells. d IP/qPCR analyses of Blnc1 in α-HA immunocomplexes (right) and input (left) from transiently transfected HEK293T cells. e Immunoblots of total lysates and α-Flag immunocomplexes from transiently transfected HEK293T cells. f Immunoblots of total lysates and α-LXRα immunocomplexes from WT and Blnc1 KO hepatocyte transduced with HA-LXRα and HA-EDF1 adenoviruses. g Srebp1c-luciferase reporter assay. Data in (d) and (g) represent mean ± SD (n = 3). ***p < 0.001, vs. vector, one-way ANOVA. h ChIP assay of LXRα occupancy in the livers from WT (open) and KO (filled) mice gavaged with T1317 for 4 days. Data represent mean ± SD (n = 3). **p < 0.01, two-tailed unpaired Student’s t-test

Fig. 10. Role of EDF1 in lipogenic gene regulation. a qPCR gene expression analyses in hepatocytes transduced with recombinant adenoviruses as indicated. Data represent mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, GFP vs. Blnc1, two-way ANOVA. b Gene expression analysis in transduced WT or Blnc1 KO hepatocytes. Data represent mean ± SD (n = 3). *p < 0.05, ***p < 0.001, WT vs. KO, two-way ANOVA. c qPCR analysis of gene expression in transduced hepatocytes receiving control siRNA (V) or siRNAs targeting EDF1 (#1 and #2) followed by treatments with vehicle or 5 μM T1317 for 24 h. Data represent mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, vector vs. #1 or #2, two-way ANOVA. d A model depicting licensing of hepatic lipogenesis and NAFLD progression by the LXR/EDF1/Blnc1 ribonucleoprotein transcriptional complex