Figure 3. LCA induces expression of SULT via the Vitamin D receptor (VDR), resulting in production of CA7S and GLP-1 secretion.
(A) qRT-PCR quantification of hSULT2A expression level in HepG2 cells treated with indicated concentrations of CA, TDCA, CDCA, or LCA normalized to human GAPDH. LCA increased hSULT2A expression in a dose-dependent manner relative to DMSO control. (≥3 biological replicates per condition, data marked with asterisk(s) are only for induction of hSULT2A. CA, 0.1 μM **p=8.90x10−3, 1 μM **p=5.40x10−3, 10 μM **p=5.30x10−3, 100 μM **p=8.40x10−3; TDCA, 0.1 μM ****p=1.00x10−4, 1 μM ****p=1.00x10−4, 10 μM ****p=1.00x10−4, 100 μM ****p=1.00x10−4; CDCA, 0.1 μM **p=1.80x10−3, 1 μM **p=2.50x10−3, 10 μM **p=9.30x10−3, 100 μM p=0.21; LCA, 0.1 μM p=0.92, 1 μM p=0.95, 10 μM **p=5.86x10−3, 100 μM *p=0.04, one-way ANOVA followed by Dunnett’s multiple comparisons test).
(B) siRNA-mediated knockdown of VDR significantly reduced LCA-mediated induction of hSULT2A in HepG2 cells compared to negative control siRNA (≥3 biological replicates per condition, *p=0.01, Welch’s t test).
(C) Vdr expression levels in mouse livers were increased in SG compared to sham mice as determined by qRT-PCR. Expression was normalized to mouse ribosomal 18S (n=11 in each group; *p=0.02, Welch’s t test).
(D) Hepatic expression of Vdr was increased in DIO mice compared to DIO + Abx. mice and DIO; GF mice. Expression levels were normalized to mouse ribosomal 18S (DIO, n=9, DIO + Abx., n=10, DIO;GF, n=8; DIO vs. DIO + Abx. **p=3.50x10−3, DIO vs. DIO;GF **p=3.90x10−3, one-way ANOVA followed by Dunnett’s multiple comparisons test).
(E) CA7S levels were reduced in feces of Vdr-knockout (KO) mice compared to wild-type (WT) mice (female WT, n=4, Vdr-KO, n=7, *p=0.01, male WT, n=7, Vdr-KO, n=9, *p=0.01, Welch’s t test).
(F) Schematic of portal vein injection with LCA (GB=gallbladder).
(G,H) Quantitative real time PCR quantification of mSult2A1 (G) and Vdr (H) expression levels in mouse livers injected with LCA or PBS normalized to mouse ribosomal 18S (n=3; for (G) *p=0.02, for (H) *p=0.03, Welch’s t test).
(I) CA7S levels in the gallbladder of mice injected with LCA or PBS normalized to mouse ribosomal 18S (n=3; *p=0.02, Welch’s t test).
(J) Synthesis of CA7S in HepG2 cells requires the cofactor PAPS, is induced upon incubation with LCA, and is dependent on VDR. Vdr siRNA was used to knockdown Vdr expression. 48 hours post-knockdown, substrate CA and ligand LCA was added as indicated. CA7S production was quantified by UPLC-MS after 16 hours. (≥3 biological replicates per condition, data marked with asterisk(s) are only for production of CA7S; CA+LCA+PAPS vs. CA+PAPS **p=2.70x10−3, CA+LCA+PAPS vs. CA+LCA+PAPS+Vdr siRNA **p=1.60x10−3, one-way ANOVA followed by Dunnett’s multiple comparisons test).
(K) Schematic of co-culture study. Liver HepG2 cells were cultured in the basolateral chamber, while NCI-H716 enteroendocrine cells were cultured separately in transwell inserts, and combined to measure GLP-1 secretion for indicated treatments.
(L) Secretion of GLP-1 by NCI-H716 cells co-cultured with HepG2 cells was induced by addition of LCA, CA, and PAPS to liver cells and was reduced by siRNA-mediated knockdown of VDR in liver cells. (≥3 biological replicates per condition, data not marked by asterisk(s) are not significant, DMSO vs. CA p=0.96; DMSO vs. CA+PAPS p=0.26; DMSO vs. CA+LCA p=0.12; DMSO vs. CA+LCA+PAPS ****p<1.00x10−4; DMSO vs. CA+LCA+PAPS+VDR siRNA p=0.49; CA vs. CA+LCA+PAPS ***p=3.00x10−3; CA+LCA vs. CA+LCA+PAPS *p=0.04; CA+PAPS vs. CA+LCA+PAPS *p=0.01; CA+LCA+PAPS vs. CA+LCA+PAPS+VDR siRNA *p=0.02, one-way ANOVA followed by Dunnett’s multiple comparisons test).
All data are presented as mean ± SEM.