Oxysterol sulfation by cytosolic sulfotransferase suppresses liver X receptor/sterol regulatory element binding protein-1c signaling pathway and reduces serum and hepatic lipids in mouse models of nonalcoholic fatty liver disease

Abstract

Cytosolic sulfotransferase (SULT2B1b) catalyzes oxysterol sulfation. 5-Cholesten-3β-25-diol-3-sulfate (25HC3S), one product of this reaction, decreases intracellular lipids in vitro by suppressing liver X receptor/sterol regulatory element binding protein (SREBP)-1c signaling, with regulatory properties opposite to those of its precursor 25-hydroxycholesterol. Upregulation of SULT2B1b may be an effective strategy to treat hyperlipidemia and hepatic steatosis. The objective of the study was to explore the effect and mechanism of oxysterol sulfation by SULT2B1b on lipid metabolism in vivo. C57BL/6 and LDLR(-/-) mice were fed with high-cholesterol diet or high-fat diet for 10 weeks and infected with adenovirus encoding SULT2B1b. SULT2B1b expressions in different tissues were determined by immunohistochemistry and Western blot. Sulfated oxysterols in liver were analyzed by high-pressure liquid chromatography. Serum and hepatic lipid levels were determined by kit reagents and hematoxylin and eosin staining. Gene expressions were determined by real-time reverse transcriptase polymerase chain reaction and Western Blot. Following infection, SULT2B1b was successfully overexpressed in the liver, aorta, and lung tissues, but not in the heart or kidney. SULT2B1b overexpression, combined with administration of 25-hydroxycholesterol, significantly increased the formation of 25HC3S in liver tissue and significantly decreased serum and hepatic lipid levels, including triglycerides, total cholesterol, free cholesterol, and free fatty acids, as compared with controls in both C57BL/6 and LDLR(-/-) mice. Gene expression analysis showed that increases in SULT2B1b expression were accompanied by reduction in key regulators and enzymes involved in lipid metabolism, including liver X receptor α, SREBP-1, SREBP-2, acetyl-CoA carboxylase-1, and fatty acid synthase. These findings support the hypothesis that 25HC3S is an important endogenous regulator of lipid biosynthesis.

Fig. 1

Determination of SULT2B1b expression in different tissues and SULT2B1b activities in liver tissue following infection with Ad-SULT2B1b. C57BL/6 mice, 8w, were fed with high cholesterol diet (HCD) for 10 weeks, and infected with Ad-control or Ad-SULT2B1b (1×10⁸ pfu) in the presence or absence of 25HC as indicated. SULT2B1b protein expressions in different tissues were analyzed by immunohistochemistry at day 6 following infection (A) and its protein levels were analyzed by Western blot (B and C). Total intracellular lipids were extracted with chloroform/methanol. Sulfated oxysterols in the liver infected with Ad-β-Gal (D and E) or Ad-SULT2B1b (F and G) were analyzed by HPLC. 24-hydrocholesterol (24HC), 25-hydrocholesterol (25HC), 27-hydrocholesterol (27HC), 24, 25-epoxycholesterol (24, 25EC), 7-ketocholesterol (7KC), 6β-hydrocholesterol (6βHC), 7α-hydrocholesterol (7αHC), and 7β-hydrocholesterol (7βHC) were used as standard controls. * P<0.05, ** P<0.01 vs. Control. N = 5 or 6.

Fig. 1

Determination of SULT2B1b expression in different tissues and SULT2B1b activities in liver tissue following infection with Ad-SULT2B1b. C57BL/6 mice, 8w, were fed with high cholesterol diet (HCD) for 10 weeks, and infected with Ad-control or Ad-SULT2B1b (1×10⁸ pfu) in the presence or absence of 25HC as indicated. SULT2B1b protein expressions in different tissues were analyzed by immunohistochemistry at day 6 following infection (A) and its protein levels were analyzed by Western blot (B and C). Total intracellular lipids were extracted with chloroform/methanol. Sulfated oxysterols in the liver infected with Ad-β-Gal (D and E) or Ad-SULT2B1b (F and G) were analyzed by HPLC. 24-hydrocholesterol (24HC), 25-hydrocholesterol (25HC), 27-hydrocholesterol (27HC), 24, 25-epoxycholesterol (24, 25EC), 7-ketocholesterol (7KC), 6β-hydrocholesterol (6βHC), 7α-hydrocholesterol (7αHC), and 7β-hydrocholesterol (7βHC) were used as standard controls. * P<0.05, ** P<0.01 vs. Control. N = 5 or 6.

Fig. 2

Effect of SULT2B1b overexpression on lipoprotein cholesterol in serum by HPLC. C57BL/6 mice and LDLR^−/− mice, 8w, were fed with high cholesterol diet (HCD) or high fat diet (HFD) for 10 weeks, and then infected with Ad-control or Ad-SULT2B1b (1×10⁸ pfu) in the presence or absence of 25HC as indicated. The lipoprotein (VLDL, LDL, and HDL) cholesterol levels in sera both in C57BL/6 mice and LDLR^−/− mice were analyzed by HPLC (A, B, C). The lipoprotein protein levels were determined by absorbance at 280 nm (D, E, F). The data represent one of three separate experiments.

Fig. 3

Effect of SULT2B1b overexpression on lipid levels in the liver tissues. The mice were fed and infected as stated in Fig. 2. Total intracellular lipids were extracted with chloroform/methanol. Triglycerides (TG), free fatty acids (FFA), total cholesterol (TC) and free cholesterol (FC) in liver both in C57BL/6 mice and LDLR^−/− mice (A–C) were analyzed as described in Methods. Liver morphology was examined by H&E staining (D). * P<0.05, ** P<0.01 vs. Control. N = 5 or 6.

Fig. 4

Effect of SULT2B1b overexpressoin on gene expressions involved in lipid metabolism at protein level. The mice were fed and infected as stated in Fig. 2. Nuclear proteins (LXRα, SREBP-1 mature, SREBP-2 mature) and cytosolic proteins (FAS, ACC1, SREBP-1 precursor, SULT2B1b) in liver tissue were analyzed by Western blot with specific antibodies (A). Western blot data were quantitatively analyzed (B). Cytoplamic proteins were normalized to β-actin; nuclear proteins, to Lamin B1. * P<0.05, ** P<0.01 vs. Control. N = 5 or 6.