Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements.

Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) suppress sterol regulatory element-binding protein 1c (SREBP-1c) expression and, thus, lipogenesis. In the current study, the molecular mechanism for this suppressive effect was investigated with luciferase reporter gene assays using the SREBP-1c promoter in HEK293 cells. Consistent with previous data, the addition of PUFAs to the medium in the assays robustly inhibited the SREBP-1c promoter activity. Deletion and mutation of the two liver X receptor (LXR)-responsive elements (LXREs) in the SREBP-1c promoter region eliminated this suppressive effect, indicating that both LXREs are important PUFA-suppressive elements. The luciferase activities of both SREBP-1c promoter and LXRE enhancer constructs induced by co-expression of LXRalpha or -beta were strongly suppressed by the addition of various PUFAs (arachidonic acid > eicosapentaenoic acid > docosahexaenoic acid > linoleic acid), whereas saturated or mono-unsaturated fatty acids had minimal effects. Gel shift mobility and ligand binding domain activation assays demonstrated that PUFA suppression of SREBP-1c expression is mediated through its competition with LXR ligand in the activation of the ligand binding domain of LXR, thereby inhibiting binding of LXR/retinoid X receptor heterodimer to the LXREs in the SREBP-1c promoter. These data suggest that PUFAs could be deeply involved in nutritional regulation of cellular fatty acid levels by inhibiting an LXR-SREBP-1c system crucial for lipogenesis.     

Effects of gender and GH secretory pattern on sterol regulatory element-binding protein-1c and its target genes in rat liver

We investigated whether the sexually dimorphic secretory pattern of growth hormone (GH) in the rat regulates hepatic gene expression of sterol regulatory element-binding protein-1c (SREBP-1c) and its target genes. SREBP-1c, fatty acid synthase (FAS), and glycerol-3-phosphate acyltransferase (GPAT) mRNA were more abundant in female than in male livers, whereas acetyl-CoA carboxylase-1 (ACC1) and stearoyl-CoA desaturase-1 (SCD-1) were similarly expressed in both sexes. Hypophysectomized female rats were given GH as a continuous infusion or as two daily injections for 7 days to mimic the female- and male-specific GH secretory patterns, respectively. The female pattern of GH administration increased the expression of SREBP-1c, ACC1, FAS, SCD-1, and GPAT mRNA, whereas the male pattern of GH administration increased only SCD-1 mRNA. FAS and SCD-1 protein levels were regulated in a similar manner by GH. Incubation of primary rat hepatocytes with GH increased SCD-1 mRNA levels and decreased FAS and GPAT mRNA levels but had no effect on SREBP-1c mRNA. GH decreased hepatic liver X receptor-alpha (LXRalpha) mRNA levels both in vivo and in vitro. Feminization of the GH plasma pattern in male rats by administration of GH as a continuous infusion decreased insulin sensitivity and increased expression of FAS and GPAT mRNA but had no effect on SREBP-1c, ACC1, SCD-1, or LXRalpha mRNA. In conclusion, FAS and GPAT are specifically upregulated by the female secretory pattern of GH. This regulation is not a direct effect of GH on hepatocytes and does not involve changed expression of SREBP-1c or LXRalpha mRNA but is associated with decreased insulin sensitivity.     

Increased expression of the sterol regulatory element-binding protein-1 gene in insulin receptor substrate-2(-/-) mouse liver

Insulin receptor substrate (IRS)-2(-/-) mice develop diabetes because of insulin resistance in the liver and failure to undergo beta-cell hyperplasia. Here we show by DNA chip microarray analysis that expression of the sterol regulatory element-binding protein (SREBP)-1 gene, a downstream target of insulin, was paradoxically increased in 16-week-old IRS-2(-/-) mouse liver, where insulin-mediated intracellular signaling events were substantially attenuated. The expression of SREBP-1 downstream genes, such as the spot 14, ATP citrate-lyase, and fatty acid synthase genes, was also increased. Increased liver triglyceride content in IRS-2(-/-) mice assures the physiological importance of SREBP-1 gene induction. IRS-2(-/-) mice showed leptin resistance; low dose leptin administration, enough to reduce food intake and body weight in wild-type mice, failed to do so in IRS-2(-/-) mice. Interestingly, high dose leptin administration reduced SREBP-1 expression in IRS-2(-/-) mouse liver. Thus, IRS-2 gene disruption results in leptin resistance, causing an SREBP-1 gene induction, obesity, fatty liver, and diabetes.     

TNF-alpha induces hepatic steatosis in mice by enhancing gene expression of sterol regulatory element binding protein-1c (SREBP-1c)

We investigated the effect of tumor necrosis factor-alpha (TNF-alpha), a member of the proinflammatory cytokine family, on steatosis of the mouse liver by analyzing morphological changes and hepatic triglyceride content in response to TNF-alpha. We also examined expression of the sterol regulatory element binding protein-1c gene. Intraperitoneal injection of TNF-alpha acutely and dramatically accelerated the accumulation of fat in the liver, as evidenced by histological analysis and hepatic triglyceride content. This treatment increased liver weight, increased serum levels of free fatty acids, and increased fatty acid synthase and sterol regulatory element binding protein-1c mRNA expression. Furthermore, intraperitoneal injection of lipopolysaccaride (LPS) to induce TNF-alpha expression also accelerated hepatic fat accumulation. Pretreatment with anti-TNF-alpha antibody attenuated the development of LPS-induced fatty change in the liver. Antibody pretreatment not only decreased sterol regulatory element binding protein-1c expression in LPS-treated mice but also attenuated the expression of suppressors of cytokine signaling-3 mRNA. This study suggests that TNF-alpha, acting downstream of LPS, increases intrahepatic fat deposition by affecting hepatic lipogenetic metabolism involving sterol regulatory element binding protein-1c.     

A novel pathway to enhance adipocyte differentiation of 3T3-L1 cells by up-regulation of lipocalin-type prostaglandin D synthase mediated by liver X receptor-activated sterol regulatory element-binding protein-1c

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is expressed in adipocytes and is proposed to be involved in the regulation of glucose tolerance and atherosclerosis in type 2 diabetes, because L-PGDS gene knock-out mice show abnormalities in these functions. However, the role of L-PGDS and the regulation mechanism governing its gene expression in adipocytes remain unclear. Here, we applied small interference RNA of L-PGDS to mouse 3T3-L1 cells and found that it suppressed differentiation of these cells into adipocytes. Reporter analysis of the mouse L-PGDS promoter demonstrated that a responsive element for liver receptor homolog-1 (LRH-1) at -233 plays a critical role in preadipocytic 3T3-L1 cells. Moreover, we identified two sterol regulatory elements (SREs) at -194 to be cis-elements for activation of L-PGDS gene expression in adipocytic 3T3-L1 cells. L-PGDS mRNA was induced in response to synthetic liver X receptor agonist, T0901317, through activation of the expression of SRE-binding protein-1c (SREBP-1c) in the adipocytic 3T3-L1 cells. The results of electrophoretic mobility shift assay and chromatin immunoprecipitation assay revealed that LRH-1 and SREBP-1c bound to their respective binding elements in the promoter of L-PGDS gene. Small interference RNA-mediated suppression of LRH-1 or SREBP-1c decreased L-PGDS gene expression in preadipocytic or adipocytic 3T3-L1 cells, respectively. These results indicate that L-PGDS gene expression is activated by LRH-1 in preadipocytes and by SREBP-1c in adipocytes. Liver X receptor-mediated up-regulation of L-PGDS through activation of SREBP-1c is a novel path-way to enhance adipocyte differentiation.     

Lxralpha deficiency hampers the hepatic adaptive response to fasting in mice

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing.     

High density lipoprotein inhibits the activation of sterol regulatory element-binding protein-1 in cultured cells

A link between cellular uptake of high density lipoprotein (HDL) and regulation of sterol regulatory element-binding protein-1 (SREBP-1) was investigated in vitro. HDL decreased nuclear SREBP-1 levels as well as SREBP-1 target gene expression in HepG2 and HEK293 cells. However, HDL did not repress an exogenously expressed, constitutively active form of SREBP-1. HDL increased cellular cholesterol levels, and cellular cholesterol depletion by methyl-β-cyclodextrin abolished the effects of HDL. These results suggest that HDL inhibits the activation of SREBP-1 through a cholesterol-dependent mechanism, which may play an important role in regulating lipid synthetic pathways mediated by SREBP-1.