5-cholesten-3β,25-diol 3-sulfate decreases lipid accumulation in diet-induced nonalcoholic fatty liver disease mouse model

Abstract

Sterol regulatory element-binding protein-1c (SREBP-1c) increases lipogenesis at the transcriptional level, and its expression is upregulated by liver X receptor α (LXRα). The LXRα/SREBP-1c signaling may play a crucial role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). We previously reported that a cholesterol metabolite, 5-cholesten-3β,25-diol 3-sulfate (25HC3S), inhibits the LXRα signaling and reduces lipogenesis by decreasing SREBP-1c expression in primary hepatocytes. The present study aims to investigate the effects of 25HC3S on lipid homeostasis in diet-induced NAFLD mouse models. NAFLD was induced by feeding a high-fat diet (HFD) in C57BL/6J mice. The effects of 25HC3S on lipid homeostasis, inflammatory responses, and insulin sensitivity were evaluated after acute treatments or long-term treatments. Acute treatments with 25HC3S decreased serum lipid levels, and long-term treatments decreased hepatic lipid accumulation in the NAFLD mice. Gene expression analysis showed that 25HC3S significantly suppressed the SREBP-1c signaling pathway that was associated with the suppression of the key enzymes involved in lipogenesis: fatty acid synthase, acetyl-CoA carboxylase 1, and glycerol-3-phosphate acyltransferase. In addition, 25HC3S significantly reduced HFD-induced hepatic inflammation as evidenced by decreasing tumor necrosis factor and interleukin 1 α/β mRNA levels. A glucose tolerance test and insulin tolerance test showed that 25HC3S administration improved HFD-induced insulin resistance. The present results indicate that 25HC3S as a potent endogenous regulator decreases lipogenesis, and oxysterol sulfation can be a key protective regulatory pathway against lipid accumulation and lipid-induced inflammation in vivo.

Fig. 1.

Effects of 25HC3S on serum lipid profiles in HFD-fed mice. Eight-week-old C57BL/6J female mice were fed an HFD or chow diet (Chow) for 10 weeks, treated with either 25HC3S or vehicle twice, and fasted overnight. Plasma triglyceride (A), total cholesterol (B), and high-density lipoprotein cholesterol (C) were determined. All values are expressed as the mean ± S.E.M. **P < 0.01 versus chow diet–fed vehicle-treated mouse liver; †P < 0.05 and ††P < 0.01 versus HFD-fed vehicle-treated mouse liver (n = 15–17). Serum lipoproteins were separated by HPLC with a Superose 6 column (D), and each fraction was collected for the measurement of concentration of triglycerides (E) and cholesterol (F). The data represent one of three separate experiments. Cont, vehicle control; O.D., optical density (absorbence).

Fig. 2.

HPLC analysis of 25HC3S and 25HC levels in treated mouse liver tissues. Animals were treated as described in Fig. 1. Total neutral lipids were extracted with chloroform/methanol mixture and analyzed by HPLC. 24HC, 25HC, 27HC, and 7α-hydroxycholesterol (7α-HC) were used as standard controls; testosterone in the chloroform phase was used as an internal control. Oxysterols in the chloroform phase from vehicle- or 25HC3S-treated mouse liver were determined (left panel). Chemical synthesis of 25HC3S was used as a standard control in the water/methanol phase. 25HC3S levels in the water/methanol phase from vehicle- and 25HC3S-treated mouse liver were determined (right panel). The data represent one of three separate experiments.

Fig. 3.

The effects of 25HC3S on protein expressions in lipid metabolism in liver tissues. Animals were treated as described in Fig. 1. Specific protein levels in cytoplasmic and nuclear extracts were determined by Western blot analysis. Cytoplasmic ACC1 and FAS protein levels are shown in (A), normalized by β-actin, and nuclear SREBP1 and SREBP2 are shown in (B), normalized by lamin B1. All values are expressed as the mean ± S.E.M. *P < 0.05 versus HFD-fed vehicle-treated mouse liver (n = 6–8). CONT, vehicle control.

Fig. 4.

The effects of long term-treatment with 25HC3S on mouse body mass and food intake. C57BL/6J female mice were fed an HFD, separated into two groups, and treated with either 25HC3S or vehicle once every 3 days for 6 weeks. During these 6 weeks, total food intake (A) and body weight (B) were monitored. After 5 hours of fasting, liver weight was determined (C), as were plasma alkaline phosphatase (ALK) (D), ALT (E), and AST (F). All values are expressed as the mean ± S.E.M. (n = 10). *P < 0.05 and **P < 0.01 versus HFD-fed vehicle-treated mouse liver. CONT, vehicle control; CT, control vehicle treatment.

Fig. 5.

Long-term treatment with 25HC3S decreases lipid accumulation in liver tissue in mouse NAFLD models. Animals were treated as described in Fig. 4. Hepatic triglyceride (A), free fatty acid (B), total cholesterol (C), free cholesterol (D), and cholesterol ester (E) are shown. Each individual level was normalized by liver weight. All values are expressed as the mean ± S.D. #P < 0.001 versus chow-fed vehicle-treated mice; **P < 0.01 versus HFD-fed vehicle-treated mouse liver. In a morphology study, liver sections from chow diet–fed (chow), HFD-fed, and HFD-fed and 25HC3S-treated (HFD-25HC3S) mice were stained by H&E staining (F). Arrows indicate unstained lipid inclusions. CT, control vehicle treatment.

Fig. 6.

Long-term treatment with 25HC3S improved insulin sensitivity and glucose homeostasis in HFD-fed mice. Animals were treated as described in Fig. 4. GTT (A) and ITT (B) were performed. All values are expressed as the mean ± S.E.M. (n = 9–10). *P < 0.05 and **P < 0.01 versus HFD-fed vehicle-treated mice. Cont, control vehicle; GTT, glucose tolerance test; ITT, insulin tolerance test.