Fig 2: 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 3: mCherry-Cre viral transfections of neurons in fCaNB1 mouse slices prevents the nuclear translocation of CRTC1. DAPI is used to stain nuclei while mCherry (shown here in green) reports neuronal transfections. A1–A8, Under control (CON) conditions in both wild-type (WT) and fCaNB1 slices, CRTC1 is cytoplasmic and excluded from nuclei. A9–A12, In slices from wild-type mice, bicuculline (BIC) treatment (100 µM for 4 h) resulted in CRTC1 nuclear translocation whether the neuron was transfected or not. To illustrate, five cells are circled in A9. Two of which were transfected (A10) and CRTC1 was translocated to the nuclei of all five cells (A11, A12). A13–A16, In contrast are results from fCaNB1 mice, where bicuculline treatment did not induce a CRTC1 nuclear translocation in transfected cells but did so in untransfected cells. Five representative cells are circled in A13. Three of these were transfected (A14). Bicuculline treatment resulted in the nuclear translocation of CRTC1 in the two untransfected cells but not in the three transfected neurons (A15 and A16). B, The computed ratio of the intensity of nuclear to cytoplasmic CRTC1 immunostaining was significantly increased in transfected neurons from wild-type mice but was not altered in transfected cells from fCaNB1 mice. The bar graphs summarize results from three separate experiments, nine slices total for each of the four conditions. Scale bar, 50 µm; *α = 0.05; ***α = 0.001.

Fig 4: (A) Genotyping result (Crtc1loxP/loxP, Crtc2loxP/loxP, and Sim1-Cre) of Sim1-CRTCDKO mice. (B) Crtc1, Crtc2, and Crtc3 mRNA expression in PVH in control and Sim1-CRTCDKO mice (n = 6–7). Values are presented as mean ± SEM. **: P < 0.01, *: P < 0.05. (C) Immunohistochemical analysis of CRTC1 and CRTC2 expression in the PVH of Sim1-CRTCDKO mice. Brain slices were stained with anti-CRTC1 antibody (red) or anti-CRTC2 antibody (red) and DAPI (blue).

Fig 5: A proposed antimelanogenic mechanism of α-viniferin. α-MSH increases cAMP levels via melanocortin 1 receptor (MC1R)-mediated activation of adenylate cyclase in melanocytes. cAMP binds to the regulatory (R)-subunit within PKA holoenzyme (R2C2), concomitantly unleashing the catalytic (C)-subunit for activation. On the other hand, the C-subunit of PKA reassociates with cAMP-free R-subunit, becoming an inactive complex, in the signal termination. α-Viniferin, an active constituent of C. sinica, primarily accelerates PKA inactivation via reassociation between C-subunit and R-subunit. This mechanism resultantly inhibits not only CREB phosphorylation, CRTC1 dephosphorylation but also nuclear translocation of CRTC1, thus down-regulating expression of MITF-M gene, a master transcription factor in cAMP-induced facultative melanogenesis.