RESUMEN
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-3ß, 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.
Asunto(s)
Ésteres del Colesterol/análisis , Colesterol/metabolismo , Hidroxicolesteroles/análisis , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Adenoviridae/metabolismo , Animales , Células Cultivadas , Colesterol/análisis , Colesterol/biosíntesis , Ésteres del Colesterol/síntesis química , Ésteres del Colesterol/farmacología , Modelos Animales de Enfermedad , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Femenino , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Hidroxicolesteroles/síntesis química , Hidroxicolesteroles/farmacología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Ratas , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
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-3ß,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/ß 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.
Asunto(s)
Ésteres del Colesterol/farmacología , Dieta Alta en Grasa/efectos adversos , Hígado Graso/tratamiento farmacológico , Hidroxicolesteroles/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/sangre , Hígado/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Ácidos Grasos/metabolismo , Hígado Graso/sangre , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/metabolismo , Femenino , Expresión Génica/genética , Prueba de Tolerancia a la Glucosa/métodos , Glicerol-3-Fosfato O-Aciltransferasa/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Inflamación/metabolismo , Insulina/genética , Insulina/metabolismo , Resistencia a la Insulina/genética , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Metabolismo de los Lípidos/genética , Hígado/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Receptor fas/genética , Receptor fas/metabolismoRESUMEN
The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ 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 PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was â¼1 µM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1ß. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1ß expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.
