Isoflavone-poor soy protein alters the lipid metabolism of rats by SREBP-mediated down-regulation of hepatic genes

Soy intake acts hypolipidemically. Besides isoflavones, soy protein itself is suggested to influence plasma lipid concentrations. We investigated the effects of an alcohol-washed isoflavone-poor soy protein isolate on plasma and liver lipids and the hepatic expression of genes encoding proteins involved in cholesterol and fatty acid metabolism. Therefore, rats were fed diets containing 200 g/kg of either ethanol-extracted soy protein isolate or casein over 22 days. Rats fed soy protein isolate had markedly lower concentrations of liver cholesterol and lower concentrations of triglycerides in the liver and in plasma than rats fed casein (P<.05). Rats fed soy protein isolate had lower relative mRNA concentrations of sterol-regulatory element-binding protein (SREBP)-2, 3-hydroxy-3-methylglutaryl coenzyme A reductase, low-density lipoprotein receptor, cholesterol 7alpha-hydroxylase, apolipoprotein B, Delta9-desaturase and glucose-6-phosphate dehydrogenase in the liver than rats fed casein (P<.05). Hepatic mRNA concentration of SREBP-1c tended to be lower in rats fed soy protein isolate (P<.10). Hepatic mRNA concentrations of insulin-induced gene (Insig) 1 and Insig-2 and of microsomal triglyceride transfer protein, as well as plasma concentrations of free fatty acids, insulin and glucagon, were not different between the two groups. In conclusion, this study suggests that isoflavone-poor soy protein isolate affects cellular lipid homeostasis by the down-regulation of SREBPs and its target genes in the liver, which are involved in the synthesis of cholesterol and triglycerides.     

FXRalpha down-regulates LXRalpha signaling at the CETP promoter via a common element

The cholesteryl ester transfer protein (CETP), a key player in cholesterol metabolism, has been shown to promote the transfer of triglycerides from very low density lipoprotein (VLDL) and low density lipoprotein (LDL) to high density lipoprotein (HDL) in exchange for cholesterol ester. Here we demonstrate that farnesoid X receptor alpha (FXRalpha; NR1H4) down-regulates CETP expression in HepG2 cells. A FXRalpha ligand, chenodeoxycholic acid (CDCA), suppressed basal mRNA levels of the CETP gene in HepG2 cells in a dose-dependent manner. Using gel shift and chromatin immunoprecipitation (ChIP) assays, we found that FXRalpha could bind to the liver X receptor alpha (LXRalpha; NR1H3) binding site (LXRE; DR4RE) located within the CETP 5' promoter region. FXRalpha suppressed LXRalpha-induced DR4RE-luciferase activity and this effect was mediated by a binding competition between FXRalpha and LXRalpha for DR4RE. Furthermore, the addition of CDCA together with a LXRalpha ligand, GW3965, to HepG2 cells was shown to substantially decrease mRNA levels of hepatic CETP gene, which is typically induced by GW3965. Together, our data demonstrate that FXRalpha down-regulates CETP gene expression via binding to the DR4RE sequence within the CETP 5' promoter and this FXRalpha binding is essential for FXRalpha inhibition of LXRalpha-induced CETP expression.     

Effects of peroxisome proliferator-activated receptor alpha activation on pathways contributing to cholesterol homeostasis in rat hepatocytes

Peroxisome proliferator-activated receptor alpha (PPARalpha) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [(14)C] acetate and [(3)H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARalpha-null mice, showing an essential role of PPARalpha. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARalpha ligands.     

Hormonal regulation of testicular steroid and cholesterol homeostasis

The male sex steroid, testosterone (T), is synthesized from cholesterol in the testicular Leydig cell under control of the pituitary gonadotropin LH. Unlike most cells that use cholesterol primarily for membrane synthesis, steroidogenic cells have additional requirements for cholesterol, because it is the essential precursor for all steroid hormones. Little is known about how Leydig cells satisfy their specialized cholesterol requirements for steroid synthesis. We show that in mice with a unique hypomorphic androgen mutation, which disrupts the feedback loop governing T synthesis, that genes involved in cholesterol biosynthesis/uptake and steroid biosynthesis are up-regulated. We identify LH as the central regulatory molecule that controls both steroidogenesis and Leydig cell cholesterol homeostasis in vivo. In addition to the primary defect caused by high levels of LH, absence of T signaling exacerbates the lipid homeostasis defect in Leydig cells by eliminating a short feedback loop. We show that T signaling can affect the synthesis of steroids and modulates the expression of genes involved in de novo cholesterol synthesis. Surprisingly, accumulation of active sterol response element-binding protein 2 is not required for up-regulation of genes involved in cholesterol biosynthesis and uptake in Leydig cells.     

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2^−/− mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2^−/− mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.     

Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.     

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein''s cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.     

Mig-6 plays a critical role in the regulation of cholesterol homeostasis and bile Acid synthesis

The disruption of cholesterol homeostasis leads to an increase in cholesterol levels which results in the development of cardiovascular disease. Mitogen Inducible Gene 6 (Mig-6) is an immediate early response gene that can be induced by various mitogens, stresses, and hormones. To identify the metabolic role of Mig-6 in the liver, we conditionally ablated Mig-6 in the liver using the Albumin-Cre mouse model (Alb(cre/+)Mig-6(f/f); Mig-6(d/d)). Mig-6(d/d) mice exhibit hepatomegaly and fatty liver. Serum levels of total, LDL, and HDL cholesterol and hepatic lipid were significantly increased in the Mig-6(d/d) mice. The daily excretion of fecal bile acids was significantly decreased in the Mig-6(d/d) mice. DNA microarray analysis of mRNA isolated from the livers of these mice showed alterations in genes that regulate lipid metabolism, bile acid, and cholesterol synthesis, while the expression of genes that regulate biliary excretion of bile acid and triglyceride synthesis showed no difference in the Mig-6(d/d) mice compared to Mig-6(f/f) controls. These results indicate that Mig-6 plays an important role in cholesterol homeostasis and bile acid synthesis. Mice with liver specific conditional ablation of Mig-6 develop hepatomegaly and increased intrahepatic lipid and provide a novel model system to investigate the genetic and molecular events involved in the regulation of cholesterol homeostasis and bile acid synthesis. Defining the molecular mechanisms by which Mig-6 regulates cholesterol homeostasis will provide new insights into the development of more effective ways for the treatment and prevention of cardiovascular disease.