Fig 2: Crtc1 deficiency in mice induces spontaneous hepatic steatosis.(a–l) Blood (a), livers (b–e,g,i,j,m), WAT (f), isolated hepatocytes (h), MEFs (k, l) from Crtc1 KO mice and their wild-type littermates were compared. Unless otherwise specified, results for male mice were shown. (a) Oral glucose and intraperitoneal insulin tolerance tests (GTT and ITT, respectively) (n > 8). (b,c) Lipids extracted from livers of 11 weeks old mice; total lipid assayed by thin layer chromatography (TLC) (b) and biochemical quantification of triglyceride (TG) (c). (d) Akt expression and phosphorylation in livers harvested 10 min after intraperitoneal injection of insulin (3 U/kg). Each panel has been run separately under the same experimental condition. (e) Hematoxylin and eosin (H&E) stain of livers of 25 weeks old mice. (f) H&E stain of WAT (25 weeks old), and macrophage infiltration shown by immunohistochemical stain for F4/80. (g–j,m) Liver (g,i,j,m) and isolated hepatocytes (h) from overnight fasted 11 weeks old mice were compared. Gene expression was measured using RT-PCR. (g) Expression of lipogenic genes. (h) Rate of de novo lipogenesis. (i) Expression of genes involved in lipid uptake. (j) Expression of genes regulating hepatic fatty acid oxidation (left) and ex vivo palmitate β-oxidation rate (right). (g,i,j) Results by Crtc1 KO liver are presented relative to results by wild-type liver (n > 8). All: p < 0.05 by paired sample t-test. (k) PGC1α expression and mitochondrial DNA content of MEFs, presented as in (g) (all: p < 0.05). (l) Mitochondrial function in MEFs shown by stain with MitoTracker Green and Red. (m) Expression of genes involved in lipid release, presented as in (g) (*p < 0.05).

Fig 3: CRTC1 negatively regulates lipogenic gene expression.(a,b) Effect of the CRTC family members on FASN expression in 293ETN cells. (a) Luciferase activity of cells transfected with a human FASN reporter (FASN-Luc) or 3x ChREBP response elements (ChRE-Luc), with or without priming with LXRα or SREBP1c (left) or ChREBP:MLX (1:1) (right). (b) Effect of forskolin (1 μM) treatment on the FASN expression induced by LXRα. (c) Effect of CRTC1 on lipogenic gene expression (RT-PCR analysis). HepG2 cells were incubated with vehicle or forskolin (5 μM) for 1 h, and then treated with 1 μM of a LXRα agonist T0901317 (T09) for 12 h. Expression levels are presented relative to expression by vehicle treated, mock-transfected control (*p < 0.05). (d–f) Immunoprecipitation (IP) analyses in 293ETN cells. Each panel has been run separately under the same experimental condition. Middle and bottom, immunoblot analysis of 3% of input lysate. Un-cropped blots are available upon request. (d) Interaction of Flag-CRTC1 with HA-tagged lipogenic transcription factors. (e) Interaction of CRTC1 with the NCoR corepressor complex (NCoR1 and HDAC3). (f) Effect of CRTC1 expression on interaction of the NCoR complex with LXRα. (g,h) Recruitment of CRTC1 and the NCoR complex to the promoters of lipogenic genes (chromatin immunoprecipitation (ChIP)). (g) Effect of forskolin treatment (10 μM for 2 h) in HepG2 cells. (h) Effect of in vivo rolipram (5 mg/kg) administration or fasting (12 h) in the liver of wild-type mice. Results are presented as enrichment of specific promoter immunoprecipitated with specific antibody relative to that with immunoglobulin G (n = 4) (*p < 0.05). Data are represented as mean ± SD.

Fig 4: Crtc1 deficiency deteriorates lipid, cholesterol and bile acid homeostasis.(a–j) livers (a–e,h), serum (f,g), ileum (i), and feces (j) from 17 weeks old Crtc1-null (KO) and their wild-type (WT) littermates were compared. Unless otherwise specified, results for male mice were shown. (a–c) RNA-Seq analyses. M: male and F: female. Hepatic RNAs were pooled from 3 mice per sample. (a) Gene Ontology (GO) enrichment analysis of differentially expressed genes between Crtc1 KO and WT livers. (b) Heatmap of differentially expressed genes between KO and WT. Only those identified by the GO analysis (a) were shown. (c) Principal component analysis (PCA) and gene contribution plot. Principal component one (PC1) and two (PC2) primarily capture sex and genotype differences, respectively. Red: each sample in the PC space. Blue: contribution of each gene to the PCs. Downward (or upward) arrow indicates increased (or decreased) gene expression in Crtc1 KO mice. (d) Hepatic expression of genes involved in cholesterol homeostasis (RT-PCR). Expressions by Crtc1 KO liver are presented relative to expressions by wild-type liver (n > 7). All: p < 0.05 by paired sample t-test. (e) Total hepatic cholesterol. (f) Serum HDL and LDL cholesterol. (g) Serum total bile acid. (h) Hepatic expression of genes involved in bile acid homeostasis, presented as in d (all: p < 0.05). (i) Ileal expression of genes involved in bile acid homeostasis, presented as in d (all: p < 0.05). (j) Daily fecal bile acid excretion rate.

Fig 5: Effects of intrathecal administration of Ad-CRTC1, Ad-EGFP, and vehicle on spinal p-CREB, t-CREB, CRTC1, NR2B, and BDNF protein expression and miRNA-212/132 expression. Mice were treated with once-daily injections of Adenoviruses or vehicle on days 14–16 after inoculation. The spinal cord was removed on day 17 after inoculation. Treatment with Ad-CRTC1 downregulated spinal p-CREB, CRTC1, NR2B, BDNF, and miRNA-212/132 expression in tumor-bearing mice. (a) Western blot for NR2B, CRTC1, β-actin, t-CREB, p-CREB, and BDNF resulted in products of 180, 78, 45, 37–40, 37, and 28 kDa, as expected (markers show predicted band sizes). (b–f) Densitometric quantification of β-actin, p-CREB, t-CREB, CRTC1, NR2B, and BDNF immunoreactivity on Western blots. β-actin was used as a loading control. (g and h) Quantitative real-time PCR analysis of changes of spinal miRNA-212 and miRNA-132 after intrathecal administration. U6 was used as a reference gene. Each group consisted of five mice. The data were expressed as the mean ± SD. *p < 0.05 versus Sham + vehicle mice, #p < 0.01 versus Tumor + vehicle mice. BDNF: brain-derived neurotrophic factor; CREB: cAMP response element binding protein; CRTC: CREB-regulated transcription coactivator; EGFP: enhanced green fluorescent protein.