The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPAR␥ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPAR␥ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPAR␥ and cytosol IB and decreased nuclear NF-B protein levels. PPAR␥ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPAR␥ competitor assay showed that the Ki for 25HC3S was ϳ1 M, similar to those of other known natural ligands. NF-B-dependent promoter reporter gene assays showed that 25HC3S suppressed TNF␣-induced luciferase activities only when cotransfected with pcDNAI-PPAR␥ plasmid. In addition, 25HC3S decreased LPSinduced expression and release of IL-1␤. In the PPAR␥-specific siRNA transfected macrophages or in the presence of PPAR␥-specific antagonist, 25HC3S failed to increase IB and to suppress TNF␣ and IL-1␤ expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPAR␥ and cytosol IB and increased nuclear NF-B protein levels. We conclude that 25HC3S acts in macrophages as a PPAR␥ ligand and suppresses inflammatory responses via the PPAR␥/IB/NF-B signaling pathway.peroxisome proliferator-activated receptor-␥; oxysterols; oxysterol sulfation; cholesterol metabolites; inflammatory response; macrophages; nuclear factor-B signaling pathway MACROPHAGES ARE THE KEY CELLULAR PLAYERS in the pathogenesis of atherosclerosis. In the early stage of atherosclerosis, macrophages in arterial walls accumulate lipids. These lipid-loaded macrophages, termed foam cells, are characteristic of a reversible early cellular phase of atherosclerotic lesions. Progressive lipid accumulation leads to further escalation of inflammatory responses and infiltration of inflammatory cells (26). Through this process, early cellular lesions are transformed to late, fibrous, atherosclerotic plaques. Physiological or pharmacological maneuvers that reduce macrophage lipids and inflammatory responses may be effective in preventing or reversing atherosclerosis.Nuclear receptors are ligand-activated transcription factors that regulate the expression of target genes to affect processes as diverse as reproduction, inflammation, development, and metabolism (17). Nuclear receptor peroxisome proliferatoractivated receptors (PPARs) play major roles in the regulation of lipid metabolism, glucose homeostasis, and inflammatory processes and may be ideal targets for therapeutic management strategies for cardiovascular diseases (4,6,11,16,23,42). PPAR␥ appears particularly important in regulating genes involved in lipid metabolism and inflammation (1,8,20,21). The PPAR␥...

Sterol regulatory element-binding protein-1c (SREBP-1c) increases lipogenesis at the transcriptional level, and its expression is upregulated by liver X receptor a (LXRa). The LXRa/ 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-3b,25-diol 3-sulfate (25HC3S), inhibits the LXRa 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 a/b 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.

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3β, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.