Hepatocyte activity of the cholesterol sensor smoothened regulates cholesterol and bile acid homeostasis in mice

Abstract

Cellular cholesterol is regulated by at least two transcriptional mechanisms involving sterol-regulatory-element-binding proteins (SREBPs) and liver X receptors (LXRs). Although SREBP and LXR pathways are the predominant mechanisms that sense cholesterol in the endoplasmic reticulum and nucleus to alter sterol-regulated gene expression, evidence suggests cholesterol in plasma membrane can be sensed by proteins in the Hedgehog (Hh) pathway which regulate organ self-renewal and are a morphogenic driver during embryonic development. Cholesterol interacts with the G-protein-coupled receptor Smoothened (Smo), which impacts downstream Hh signaling. Although evidence suggests cholesterol influences Hh signaling, it is not known whether Smo-dependent sterol sensing impacts cholesterol homeostasis in vivo. We examined dietary-cholesterol-induced reorganization of whole-body sterol and bile acid (BA) homeostasis in adult mice with inducible hepatocyte-specific Smo deletion. These studies demonstrate Smo in hepatocytes plays a regulatory role in sensing and feedback regulation of cholesterol balance driven by excess dietary cholesterol.

Keywords: Lipid; Molecular biology; Molecular physiology.

Graphical abstract

Figure 1

Smoothened deletion in hepatocytes alters hepatic cholesterol homeostasis in chow-fed mice Male Smo (+) and Smo (−) mice were fed a low-cholesterol chow (0.02% w/w) diet for 7 days. (A) Total liver cholesterol. (B) Liver cholesterol esters. (C) Hepatocyte gene expression of SREBP2, HMGCR, LSS, AACS, INSIG1, and ACAT2. (D and E) Western blot and densitometric analysis of hepatocyte membrane and nuclear SREBP2 (precursor, P; active A), EGFR, and TAF15 protein levels. (F and G) Western blot and densitometric analysis of hepatocyte HMGCR, AACS, SRB1, and β-tubulin protein levels. (H) Hepatocyte gene expression of LXRα, ABCA1, ABCG5, ABCG8, and SRB1. Results reported as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 vs. Smo (+) chow. See also Figure S1.

Figure 2

Smoothened deletion in hepatocytes dysregulates hepatic responses to dietary cholesterol challenge Male Smo (+) and Smo (−) mice were fed a high-cholesterol (HC, 0.2% w/w) diet for 10 days. (A) Hepatic SMO gene expression. (B) Liver total cholesterol. (C) Liver cholesterol esters. (D and E) Western blot and densitometric analysis of hepatic membrane and nuclear SREBP2 (precursor, P; active, A), EGFR, and TAF15 protein levels. (F) Hepatic gene expression of SREBP2, HMGCR, AACS, and LSS. (G and H) Western blot and densitometric analysis of hepatic HMGCR, AACS, SRB1, and β-tubulin protein levels. (I and J) Western blot and densitometric analysis of liver microsome ACAT2 and β-actin protein levels. (K) Hepatic gene expression of LXRα, ABCA1, AGCG5, ABCG8, and SRB1. Results reported as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 vs. Smo (+) Chow; #p < 0.05, ##p < 0.01 vs. Smo (+) HC.

Figure 3

Smoothened deletion in hepatocytes increases plasma and fecal bile acids Male Smo (+) and Smo (−) mice were fed either a low-cholesterol chow (0.02% w/w) or high-cholesterol (HC, 0.2% w/w) diet for 10 days. (A) Fold change in primary bile acids over Smo (+) chow-fed mice. (B) Fold change in secondary bile acids over Smo (+) chow-fed mice. (C) Fold change in total bile acids in feces over Smo (+) chow-fed mice. (D) Fold change in fecal neutral sterol loss over Smo (+) chow-fed mice. Results reported as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 vs. Smo (+) Chow; #p < 0.05, ##p < 0.01 vs. Smo (+) HC. TCA, taurocholic acid; TαMCA, tauro-α-murocholic acid; TβMCA, tauro-β-murocholic acid; TωMCA, tauro-ω-murocholic acid; TCDCA, taurochenoxycholic acid; TDCA, taurodeocycholic acid; CA, cholic acid; ωMCA, ω-murocholic acid; TUDCA, tauroursodeoxycholic acid; DCA, deoxycholic acid.

Figure 4

Smoothened deletion in hepatocytes dysregulates pathways that critically control hepatic bile acid synthesis Male Smo (+) and Smo (−) mice were fed either a low-cholesterol chow (0.02% w/w) or high-cholesterol (HC, 0.2% w/w) diet for 10 days. (A) Hepatic gene expression of CYP7A1, CYP8B1, and SHP. (B and C) Western blot and densitometric analysis of hepatic CYP7A1, CYP8B1, SHP, and β-tubulin protein levels. (D) Gene expression of FGF15 in the small intestine and APOC2/APOC3 in the liver. (E and F) Western blot and densitometric analysis of hepatic FXR, APOC3, APOC2, FGF15, and β-tubulin protein levels. (G) Effect of FXR on FXR target gene expression in HC Smo (−) liver and small intestine. Results reported as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 vs. Smo (+) Chow; #p < 0.05, ##p < 0.01 vs. Smo (+) HC.

Figure 5

Smoothened deletion in hepatocytes disrupts mechanisms that constrain liver triglyceride accumulation during dietary cholesterol challenge Male Smo (+) and Smo (−) mice were fed either a low-cholesterol chow (0.02% w/w) or a high-cholesterol (HC, 0.2% w/w) diet for 10 days. (A) Hepatic triglyceride levels. (B) Oil Red O staining in the liver. Scale bar equals 100 μm. (C and D) Western blot and densitometric analysis of the hepatic membrane and nuclear SREBP1C (precursor, P; active, A), EGFR, and TAF15 protein levels. (E) Hepatic SCD1 and DGAT2 gene expression. (F) Hepatic gene expression of ESRRA, PGC1α, and PGC1β. Results reported as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 vs. Smo (+) Chow; #p < 0.05, ##p < 0.01 vs. Smo (+) HC. See also Figure S1.

Figure 6

Smoothened deletion in hepatocytes alters activities of kinases involved in Hedgehog signaling Male Smo (+) and Smo (−) mice were fed either a low-cholesterol chow (0.02% w/w) or high-cholesterol (HC, 0.2% w/w) diet for 10 days. (A) Hepatic gene expression of Patched, Shh, Gli1, Gli2, and Gli3. (B and C) Western blot analysis of hepatic cytosolic and nuclear Gli1, Gli2, Gli3, β-tubulin, and TAF15 protein levels. (D–G) Western blot and densitometric analysis of phospho-AMPK Threonine 172, total AMPK, phospho-Akt Serine 473, total Akt, phospho-GSK3β Serine 9, and β-tubulin protein levels. Results reported as MEAN ± SEM. ∗p < 0.05, ∗∗p < 0.01 vs. Smo (+) Chow; #p < 0.05, ##p < 0.01 vs. Smo (+) HC. A, activator; R, repressor.