mRNA Levels Of SREBP-1 Research Articles

Follicle-stimulating hormone promotes the transformation of cholesterol to estrogen in mouse adipose tissue

Follicle-stimulating hormone (FSH) levels increase estrogen biosynthesis in obese menopausal women. Ovariectomized mice and 3T3-L1 cells were used to explore estrogen biosynthesis in the decline of ovarian function. After ovariectomy, lipid deposition, and FSH and estrogen levels changed, and feed intake increased significantly. In mouse adipose tissue, FSH was found to have a role in accelerating lipid deposition via the peroxisome proliferators-activated receptor pathway, and in inducing estrogen biosynthesis via the steroid hormone metabolism pathway. Furthermore, FSH bound to the FSH receptor promoted CREB phosphorylation, which was activated by cAMP-PKA. Moreover, pCREB could up-regulate PPARγ and SREBP2 mRNA levels, resulting in an increased transformation of cholesterol to estrogen. Overall, this study shows that FSH induces fat deposition and promotes the transformation of cholesterol to estrogen through CREB activation by cAMP-PKA in mouse adipose tissue. Our findings provide a new understanding of menopause treatment.

Chinese Olive (Canarium album L.) Fruit Extract Attenuates Metabolic Dysfunction in Diabetic Rats.

Hyperglycemia and dysregulation of lipid metabolism play a crucial role in metabolic dysfunction. The aims of present study were to evaluate the ameliorative effect of the ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc) on high-fat diet (HFD) and streptozotocin (STZ)-induced diabetic rats. CO-EtOAc, rich in gallic acid and ellagic acid, could markedly decreased the body weight and epididymal adipose mass. In addition, CO-EtOAc increased serum HDL-C levels, hepatic GSH levels, and antioxidant enzyme activities; lowered blood glucose, serum levels of total cholesterol (TC), triglycerides (TG), bile acid, and tumor necrosis factor alpha (TNFα); and reduced TC and TG in liver. We further demonstrated that CO-EtOAc mildly suppressed hepatic levels of phosphorylated IRS-1, TNF-α, and IL-6, but enhanced Akt phosphorylation. The possible mechanisms of cholesterol metabolism were assessed by determining the expression of genes involved in cholesterol transportation, biosynthesis, and degradation. It was found that CO-EtOAc not only inhibited mRNA levels of SREBP-2, HMG-CoAR, SR-B1, and CYP7A1 but also increased the expression of genes, such as ABCA1 and LDLR that governed cholesterol efflux and cholesterol uptake. Moreover, the protein expressions of ABCA1 and LDLR were also significantly increased in the liver of rats supplemented with CO-EtOAc. We suggest that Chinese olive fruit may ameliorate metabolic dysfunction in diabetic rats under HFD challenge.

POSSIBLE REGULATION OF LDL-RECEPTOR BY NARINGENIN IN HEPG2 HEPATOMA CELL LINE.

Background:High plasma concentration of low-density lipoprotein cholesterol (LDL-c) plays a significant role in the incidence of atherosclerosis and coronary heart diseases (CHD).Materials and Methods:The purpose of this study was to investigate the mechanism by which citrus flavonoids, naringenin regulate the LDL receptor (LDLr) gene in human liver using the human hepatoma cell line, HepG2 as a model.Results:Time-course transient transfection of HepG2 cells with luciferase reporter-gene constructs incorporating the promoters of SREBP-1a,-1c, -2 and LDLr, revealed that in lipoprotein-deficient medium (LPDM), only SREBP-1a promoter activity was increased significantly after 4h exposure to 200μM naringenin respectively. However, after 24h incubation with 200μM naringenin the gene expression activities of all the SREBP-1a, -1c, -2 and LDLr promoter-constructs were increased significantly. The effects of both 200μM naringenin on elevating LDLr mRNA are possibly due to regulation of gene transcription by SREBP-la and SREBP-2. However, the suppression effect of 200μM naringenin on hepatic SREBP-1c mRNA expression is likely associated with the reduction in mRNA expression of both acetyl-CoA carboxylase and fatty acid synthase in human hepatoma HepG2 cells. It was found that, 200μM naringenin was likely to stimulate LDLr gene expression via increase phosphorylation of PI3K and ERK1/2 which enhance the transcription factors SREBP-1a and SREBP-2 mRNA levels and increased their protein maturation in human hepatoma HepG2 cell.Conclusion:Diets supplemented with naringenin could effectively reduce mortality and morbidity from coronary heart diseases and as cardio-protective effects in humans.

Role of hesperetin in LDL-receptor expression in hepatoma HepG2 cells.

BackgroundHigh plasma concentration of low-density lipoprotein cholesterol (LDL-c) plays a significant role in the incidence of atherosclerosis and coronary heart diseases. The aim of this study was to investigate the mechanism by which the citrus flavonoid, hesperetin, regulates the LDL receptor (LDLr) gene in the human liver using the human hepatoma cell line, HepG2.MethodsLuciferase reporter gene assays were performed (in the absence of lipoprotein) to measure the activity of the LDLr promoter and the promoters of the sterol regulatory element binding protein (SREBP) transcription factors that control the LDLr promoter.ResultsOnly SREBP-1 promoter activity was significantly increased 4 h after exposure to 200 μM hesperetin. However, after 24 h incubation with 200 μM hesperetin, the activities of all the promoter-constructs, SREBP-1a, -1c, -2 and LDLr, were significantly increased. The effects of 200 μM hesperetin on elevating LDLr mRNA levels were possibly due to regulation of LDLr gene transcription by SREBP-la and SREBP-2.ConclusionsWe conclude that 200 μM hesperetin was likely to have stimulated LDLr gene expression in human hepatoma HepG2 cells via increased phosphorylation of PI3K andERK1/2, which increased SREBP-1a and SREBP-2 mRNA levels and enhanced the maturation of the encoded proteins. This may lead to lower plasma LDL cholesterol; therefore, diets supplemented with hesperidin might provide cardio-protective effects and reduce mortality and morbidity from coronary heart diseases.

Fe reduced hepatic lipid deposition in Synechogobius hasta exposed to waterborne Cu

Recent evidences suggested that Fe influenced Cu metabolism in vertebrates. The present study was conducted to test the hypothesis that Fe could alleviate Cu-induced change of lipid deposition in the fish species. Synechogobius hasta were exposed to 0, 0.606 and 1.212μM Cu, in combination with 0 and 1.128μM Fe, respectively. Sampling occurred on day 28 and day 56, respectively. Growth performance, hepatic lipid deposition, Fe and Cu level, and activities and mRNA expression of enzymes and genes involved in lipid metabolism were analyzed. Fe addition in water improved survival in S. hasta exposed to the highest waterborne Cu concentration on day 56. Fe addition also increased hepatic Fe content both at day 28 and day 56, and reduced hepatic Cu content. Fe exposure tended to reduce the activities and mRNA expressions of lipogenic enzymes and genes (G6PD and FAS), and up-regulated the mRNA expression of ATGL. With the same Cu concentration, Fe addition tended to down-regulate mRNA levels of SREBP-1 and PPARγ, and up-regulate PPARα mRNA level on day 28. However, on day 56, the mRNA levels of SREBP-1, PPARγ and PPARα are very variable and not related with waterborne Fe addition. Some correlative relationship was observed between the mRNA of transcriptional factors, and the activities of enzymes and the mRNA expression of genes encoding them, implying their transcription regulation of these enzymatic genes by transcriptional factors after Fe addition. Overall, Fe addition mitigated Cu-induced changes of lipid deposition in fish by down-regulation of lipogenesis and up-regulation of lipolysis. Different response patterns of these enzyme activities and gene expressions in the liver of S. hasta following waterborne Fe exposure indicated that Fe effects on Cu-induced change of lipid metabolism are time-dependent.

Emodin improves lipid and glucose metabolism in high fat diet-induced obese mice through regulating SREBP pathway

Currently, obesity has become a worldwide epidemic associated with Type 2 diabetes, dyslipidemia, cardiovascular disease and chronic metabolic diseases. Emodin is one of the active anthraquinone derivatives from Rheum palmatum and some other Chinese herbs with anti-inflammatory, anticancer and hepatoprotective properties. In the present study, we investigated the anti-obesity effects of emodin in obese mice and explore its potential pharmacological mechanisms. Male C57BL/6 mice were fed with high-fat diet for 12 weeks to induce obesity. Then the obese mice were divided into four groups randomly, HFD or emodin (40mg/kg/day and 80mg/kg/day) or lovastatin (30mg/kg/ day) for another 6 weeks. Body weight and food intake were recorded every week. At the end of the treatment, the fasting blood glucose, glucose and insulin tolerance test, serum and hepatic lipid levels were assayed. The gene expressions of liver and adipose tissues were analyzed with a quantitative PCR assay. Here, we found that emodin inhibited sterol regulatory element-binding proteins (SREBPs) transactivity in huh7 cell line. Furthermore, emodin (80mg/kg/day) treatment blocked body weight gain, decreased blood lipids, hepatic cholesterol and triglyceride content, ameliorated insulin sensitivity, and reduced the size of white and brown adipocytes. Consistently, SREBP-1 and SREBP-2 mRNA levels were significantly reduced in the liver and adipose tissue after emodin treatment. These data demonstrated that emodin could improve high-fat diet-induced obesity and associated metabolic disturbances. The underlying mechanism is probably associated with regulating SREBP pathway.

Antidiabetic and Antihyperlipidemic Properties of a Triterpenoid Compound, Dehydroeburicoic Acid, from Antrodia camphorata in Vitro and in Streptozotocin-Induced Mice.

The aim of this study was to examine the effects of dehydroeburicoic acid (TT) on type 1 diabetes mellitus and dyslipidemia in streptozotocin (STZ)-induced diabetic mice. STZ-induced diabetic mice were randomly divided into six groups and given orally by gavage TT (at three dosages), metformin (Metf), fenfibrate (Feno), or vehicle for 4 weeks. STZ-induced diabetic mice showed elevations in blood glucose levels (P < 0.001). TT treatment markedly decreased blood glucose levels by 42.6-46.5%. Moreover, STZ-induced diabetic mice displayed an increase in circulating triglyceride (TG) and total cholesterol (TC) levels (P < 0.001 and P < 0.01, respectively) but a decrease in blood insulin and adiponectin levels (P < 0.01 and P < 0.05, respectively). These substances are also reversed by TT treatment, indicating TT ameliorated diabetes and dyslipidemia. Membrane skeletal muscular expression levels of glucose transporter 4 (GLUT4) and expression levels of AMPK phosphorylation (phospho-AMPK) in both liver and skeletal muscle were reduced in STZ-induced diabetic mice, which normalized upon TT treatment and correction of hyperglycemia accompanied with a decrease in mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6 Pase), which was related to the inhibition of hepatic glucose production and attenuating diabetic state. In addition, TT also showed hypolipidemic effect by increasing hepatic expression levels of peroxisome proliferator-activated receptor α (PPARα) and mRNA levels of carnitine palmitoyl transferase Ia (CPT-1a) but decreasing expression levels of fatty acid synthase (FAS), which further contributed to a decrease in circulating TG levels. TT-treated mice displayed decreased SREBP2 mRNA levels and reduced blood TC levels. These findings strongly support that TT prevents diabetic and dyslipidemic states in STZ-induced diabetic mice evidenced by regulation of GLUT4, PPARα, FAS, and phosphorylation of AMPK.

Abstract 636: The Nuclear Receptor FXR Uncouples the Actions of Srebp2 and MiR-33

The role of microRNAs in fine-tuning gene expression has now been well established, but the mechanisms for microRNA regulation at the transcriptional level remain understudied. The physiologic context in which microRNAs are acting is likely linked to the function of these same microRNAs and how they are regulated. Here we identify miR-33 as a direct target gene of the bile acid nuclear receptor FXR. We show that pharmacologic FXR activation induces miR-33 in wild-type, but not FXR knockout mice. MiR-33 is located in the Srebp2 locus, and we show that FXR activation also induces the expression of Srebp2 mRNA. Using ChIP-Seq, we identify at least one functional FXRE in an intronic region of Srebp2, demonstrating that miR-33 and Srebp2 are both direct FXR targets. Although FXR activation increases Srebp2 mRNA levels, we also demonstrate that FXR in fact decreases Srebp2 proteolytic processing, which determines its transcriptional activity. This decreased activity is a result of the FXR dependent induction of the integral ER membrane protein Insig2, which is itself also a direct FXR target. Together these results identify a molecular mechanism for the transcriptional regulation of Srebp2 and miR-33, as well as the mechanism for the decreased activity of Srebp2 processing in the ER by FXR. The best characterized target of miR-33 is ABCA1. We have previously demonstrated that FXR activation decreases ABCA1 protein levels via the induction of miR-144, a microRNA that also targets ABCA1. Our new findings suggest that FXR induces two independent microRNAs that function together to repress ABCA1 and reduce HDL levels.

Lipid Regulators during Atherogenesis: Expression of LXR, PPAR, and SREBP mRNA in the Human Aorta

Transcription factors LXRs, PPARs, and SREBPs have been implicated in a multitude of physiological and pathological processes including atherogenesis. However, little is known about the regulation of these transcription factors at different stages of atherosclerosis progression. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to compare the contents of mRNAs in pairs intact-injured aorta fragments taken from the same donors. Only minor changes in LXRα, LXRβ, PPARα, PPARγ, SREBP1, and SREBP2 mRNA levels were found in initial lesions as compared with intact non-diseased tissue. The contents of all mRNAs but SREBP2 mRNA were found to be progressively up-regulated in fatty streaks and fibrous lipoid plaques. These changes were only partially reproduced in cultured macrophages upon lipid loading. Wave-shaped changes in abundance of correlations between given group of mRNAs and 28 atherosclerosis-related mRNA species in the course of atherogenesis were observed. The impact of specific mRNA correlations on the total correlations also significantly varied between different lesion types. The study suggests that the extent and forms of LXR/PPAR/SREBP participation in intima functions vary nonlinear in individual fashion in atherogenesis. We speculate that the observed changes in mRNAs expression and coupling reflect shifts in lipid ligands availability and cellular composition in the course of atherosclerosis progression.

Cholesterol-lowering effect of dietary Lupinus angustifolius proteins in adult rats through regulation of genes involved in cholesterol homeostasis

In the absence of a clear indication from previous studies, a rat study was designed to evaluate a possible hypolipidaemic effect of Lupinus angustifolius (blue lupin) proteins. Rats were fed for 28days Nath’s hypercholesterolaemic diets containing 20% casein or blue lupin proteins. After 14 and 28days of dietary treatment, blue-lupin-fed rats had markedly lower plasma total cholesterol levels than rats fed casein (−53.0% and −55.3%, respectively, p<0.0005). No significant differences were instead observed for triglyceride and HDL-cholesterol levels between the two groups. Lupin-protein-fed rats displayed higher hepatic mRNA levels of SREBP-2, a major transcriptional regulator of intracellular cholesterol levels, and CYP7A1, the rate-limiting enzyme in bile acid biosynthesis (p<0.05). In conclusion, the present study demonstrates a marked cholesterol-lowering activity of proteins from L. angustifolius in rats. Moreover, blue lupin proteins appear to affect cellular lipid homeostasis by up-regulating SREBP-2 and CYP7A1 genes.

24 S-hydroxycholesterol effects on lipid metabolism genes are modeled in traumatic brain injury

Membrane damage during traumatic brain injury (TBI) alters the brain homeostasis of cholesterol and other lipids. Cholesterol 24 S-hydroxylase (Cyp46) is a cholesterol metabolic enzyme that is increased after TBI. Here, we systematically examined the effects of the enzymatic product of Cyp46, 24 S-hydroxycholesterol, on the cholesterol regulatory genes, SREBP-1 and 2, their posttranslational regulation, and their effects on gene transcription. 24 S-hydroxycholesterol increased levels of SREBP-1 mRNA and full-length protein but did not change levels of cleaved SREBP-1, consistent with the role of 24-hydroxycholesterol as an LXR agonist. In contrast, 24 S-hydroxycholesterol decreased levels of LXR-independent SREBP-2 mRNA, full-length protein, and SREBP-2 active cleavage product. We examined the downstream effects of changes to these lipid regulatory factors by studying cholesterol and fatty acid synthesis genes. In neuroblastoma cells, 24 S-hydroxycholesterol decreased mRNA levels of the cholesterol synthesis genes HMG CoA reductase, squalene synthase, and FPP synthase but did not alter levels of the mRNA of fatty acid synthesis genes acetyl CoA carboxylase or fatty acid synthase. After TBI, as after 24 S-hydroxycholesterol treatment in vitro, SREBP-1 mRNA levels were increased while SREBP-2 mRNA levels were decreased. Also similar to the in vitro results with 24 S-hydroxycholesterol, HMG CoA reductase and squalene synthase mRNA levels were significantly decreased. Fatty acid synthase mRNA levels were not altered but acetyl CoA carboxylase mRNA levels were significantly decreased. Thus, changes to transcription of cholesterol synthesis genes after TBI were consistent with increases in Cyp46 activity, but changes to fatty acid synthesis genes must be regulated by other mechanisms.

Activation of liver X receptor regulates fatty acid synthase expression in diabetic liver

To investigate the effects of liver X receptor (LXR) on the expression of fatty acid synthase (FAS) in diabetic liver. Sixteen-week-old male db/db mice with C57BL/6 background were administered via gavaging of T0901317 (TO), a LXR synthetic agonist, at the dose of 3 mg x kg(-1) x d(-1) or dimethyl sulfide (DMSO), a vehicle alone for 7 days. Then the mice were killed with their livers taken out to undergo immunohistochemistry to observe the distribution of FAS protein. Human hepatocellular liver carcinoma cell of the line HepG2 were cultured with TO (10 micromol/L) or DMSO for 24 hours. Another HepG2 cells were transfected with mouse FAS promoter-luciferase reporter recombinants with or without pcDNA3.1, LXR expression vector, or an active sterol regulatory element binding protein-1c (SREBP-1c) expression vector for 12 hours. Real-time PCR and Western blotting were used to detect the levels of mRNA and protein of FAS and SREBP-1c respectively. Luciferase reporter assay was utilized to examine the activity of mouse FAS promoter. FAS was abundantly expressed in the mouse livers, especially in the cytoplasm of liver cells. The FAS mRNA levels of the livers of the db/db mice was about 5.5 times as high as that of the db/m mice (P < 0.01). The FAS protein levels in the livers of db/db and db/m mice treated with TO were 1.7 and 3.5 times higher than those of the control mice (both P < 0.05). The SREBP-1 mRNA levels in the liver of the db/m and db/db mice treated with TO were 2.4 and 2.1 times higher compared with the control mice (P < 0.05, P < 0.01). Luciferase test showed that the FAS promoter activity of the HepG2 cells treated with TO was 1.5 times that of the control cells (P < 0.01). The FAS promoter activities of the HepG2 cells transfected with LXR and SREBP-1c were 1.9 and 1.6 times those of the control cells (botn P < 0.01). LXRE directly or indirect (via SREBP-lc) upregulates the expression of FAS gene in the diabetic liver. LXR may mediate the lipid accumulation in liver of diabetes.

Effect of deficiency in SREBP cleavage-activating protein on lipid metabolism during intermittent hypoxia

Obstructive sleep apnea (OSA), a condition leading to intermittent hypoxia (IH) during sleep, has been associated with dyslipidemia, atherosclerosis, and increased cardiovascular mortality. We previously showed in C57BL/6J mice that IH causes hypercholesterolemia and upregulation of sterol regulatory element binding protein (SREBP)-1, a transcription factor of lipid biosynthesis in the liver. The goal of the present study was to provide mechanistic evidence that IH causes hypercholesterolemia via the SREBP-1 pathway. We utilized mice with a conditional knockout of SREBP cleavage-activating protein (SCAP) in the liver (L-Scap- mice), which exhibit low levels of an active nuclear isoform of SREBP-1 (nSREBP-1). We exposed L-Scap- mice and wild-type (WT) littermates to IH or intermittent air control for 5 days. IH was induced during the 12-h light phase by decreasing Fi(O(2)) from 20.9% to 5% for a period of 30 s with rapid reoxygenation to 20.9% through the subsequent 30 s. In WT mice, IH increased fasting levels of serum total and HDL cholesterol, serum triglycerides, serum and liver phospholipids, mRNA levels of SREBP-1 and mitochondrial glycerol-3-phosphate acyltransferase (mtGPAT), and protein levels of SCAP, nSREBP-1, and mtGPAT in the liver. In L-Scap- mice, IH did not have any effect on serum and liver lipids, and expression of lipid metabolic genes was not altered. We conclude that hyperlipidemia in response to IH is mediated via the SREBP-1 pathway. Our data suggest that the SREBP-1 pathway could be used as a therapeutic target in patients with both OSA and hyperlipidemia.

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes

Recently, a novel oxysterol, 5-cholesten-3β, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 μM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.

GH and insulin affect fatty acid synthase activity in isolated porcine adipocytes in culture without any modifications of sterol regulatory element binding protein-1 expression

The ability of GH to decrease fatness and insulin-regulated events such as lipogenic enzyme activities is well known in pigs. Nevertheless, the precise mechanism underlying these actions has not been elucidated yet. Expression of the transcription factor sterol regulatory element binding protein (SREBP)-1 has been reported as a key mediator of insulin action in rat hepatocytes and adipose cell lines. The present study aimed to determine whether the regulation of lipogenesis by GH and/or insulin in porcine adipocytes also involved SREBP-1. Isolated adipocytes, obtained from perirenal or s.c. adipose tissue samples of female pigs (51+/-0.4 kg; n=17), were cultured in serum-free medium in the absence or presence of these hormones for up to 4 days. Glucose incorporation and fatty acid synthase activity were increased by insulin in a dose-dependent manner in adipocytes of both sites. The increase was maximal at 1.7 and 17 nM in s.c. and perirenal adipocytes respectively, suggesting inter-depot differences in the regulation of lipogenesis by insulin. These insulin-stimulated events were decreased by GH (1 nM). No change in SREBP-1 mRNA levels was observed in response to GH and/or insulin. Taken together, these data indicate that the regulation of lipogenesis by insulin and GH appears to not involve changes in SREBP-1 mRNA levels in porcine adipocytes.

Impaired regulation of sterol regulatory element binding protein 2 in cholesterol gallstone-susceptible mice.

Lipid synthesis is under tight transcriptional control involving sterol regulatory element binding proteins (SREBP1, SREBP2). Rising cellular cholesterol levels prevent SREBP2 from entering the nucleus to directly activate the expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr), the key enzyme of cholesterol synthesis. The failure to down-regulate cholesterol synthesis in gallstone-susceptible C57L/J but not AKR/J mice prompted us to study the processing of SREBP2 in these mice. Male mice of each strain received a control or lithogenic diet for 28 days. Membrane and nuclear extracts of pooled livers were prepared for immunoblot analysis of SREBP. Steady-state mRNA levels of Hmgcr, Srebp1, Srebp2 and the SREBP cleavage activating protein (Scap) were measured from individual livers. There was no marked difference between the two strains with regard to processing of SREBP1 as well as steady-state mRNA levels of Srebp1, Srebp2 and Scap. However, a near-complete suppression of nuclear SREBP2 related to low Hmgcr mRNA levels was noticed only for gallstone-resistant AKR mice. Abnormal regulation of SREBP2 appears to be responsible for the failure to suppress cholesterol synthesis in genetically cholesterol gallstone-susceptible mice. This defect may contribute to cholesterol hypersecretion and gallstone formation.

Soybean beta-conglycinin diet suppresses serum triglyceride levels in normal and genetically obese mice by induction of beta-oxidation, downregulation of fatty acid synthase, and inhibition of triglyceride absorption.

The purpose of this study was to discover the effects of soybean beta-conglycinin (7S-globulin) and glycinin (11S-globulin) on serum lipid levels and metabolism in the livers of normal and genetically obese mice. Male normal (ICR) and obese (KK-Ay) mice were fed ad libitum high fat diets for two weeks, followed by a 2-week restriction of diet (2 g diet/mouse/day) containing 20% casein, soybean beta-conglycinin, or soybean glycinin, and then sacrificed immediately. Serum triglyceride (TG), glucose, and insulin levels of beta-conglycinin-fed mice were lower than in casein- and glycinin-fed mice of both strains. In order to analyze the related events to these effects, enzyme activities and relative mRNA levels of lipid metabolism-related proteins were measured. The activities of two enzymes related to fatty acid beta-oxidation were higher while that of fatty acid synthase was lower in livers of beta-conglycinin-fed mice than of casein-fed both mice. Messenger RNA levels of acyl-CoA oxidase (fatty acid beta-oxidation related enzyme) were significantly higher in livers of beta-conglycinin-fed mice than of both casein-fed mice. On the contrary, mRNA levels of SREBP-1 and 2 tended to be lowered in livers of soy protein-fed mice than of both casein-fed mice. Fecal excretion of TG was higher in beta-conglycinin-fed mice than in casein-fed mice. Our results demonstrated that the soy beta-conglycinin diet reduced serum TG levels by acceleration of beta-oxidation, suppression of fatty acid synthase and/or increased TG fecal excretion, and also diminished serum glucose and insulin levels. Some of these events might be caused at the transcriptional levels, judged from the result that relative messenger RNA levels of lipid metabolism-related proteins were altered. These results suggest that soy beta-conglycinin could be a potentially useful dietary protein source for the prevention of hypertriglyceridemia, hyperinsulinemia, and hyperglycemia, which are recognized as risk factors for atherosclerosis.

Modulation of ERG25 expression by LDL in vascular cells.

Plasma low density lipoproteins (LDL) play a key role in the pathogenesis of atherosclerosis. LDL modify gene expression in vascular cells leading to disturbances in the functional state of the vessel wall. Expression levels of C-4 sterol methyl oxidase gene (ERG25), sterol regulatory element binding protein (SREBP)-1 and -2 were evaluated in porcine aortic endothelial cells (PAEC), porcine and human smooth muscle cells (SMC) and in the vascular wall from normolipemic and hyperlipemic pigs by RT-PCR. SREBP-1 protein levels were assessed by Western blot and SREBP-SRE binding by EMSA. SREBP-2 was overexpressed by transient transfection with lipofectin. We have identified expression of the ERG25 in vascular cells and analyzed its regulation by LDL. ERG25, an enzyme involved in cholesterol biosynthesis, is expressed in vascular endothelial and SMC from porcine and human origin and is downregulated by LDL in a time- and dose-dependent manner. Downregulation of ERG25 by LDL was abolished by an inhibitor of neutral cysteine proteases (N-acetyl-leucyl-leucyl-norleucinal) that abrogates SREBP catabolism. LDL downregulated SREBP-2 mRNA levels but not SREBP-1 expression in these cells and both ERG25 and SREBP-2 gene expression was significantly decreased in the vascular wall of diet-induced hypercholesterolemic swine. Finally, in cell transfection experiments SREBP-2 overexpression blocks ERG25 downregulation caused by LDL. Our results indicate that LDL modulate ERG25 expression in the vascular wall and suggest the involvement of SREBP-2 in this mechanism.

Regulation of sterol regulatory element-binding proteins by cholesterol flux in CaCo-2 cells

The regulation of sterol regulatory element-binding proteins (SREBP) by cholesterol flux was studied in the intestinal cell line CaCo-2. CaCo-2 cells were incubated for 18 h with micelles containing 5 mM taurocholate and 500 μM oleic acid or micelles containing either 200 μM cholesterol or 150 μM lysophosphatidylcholine. In some incubations, an ACAT inhibitor was added or 25-hydroxycholesterol was substituted for cholesterol. The SREBP-1a transcript was 2-fold more abundant than the SREBP-1c transcript. In cells incubated with micelles containing cholesterol, rates of cholesterol synthesis were decreased and rates of esterification were increased. Cholesterol synthesis was decreased further by ACAT inhibition. Cholesterol influx decreased mRNA levels of SREBP-2, HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. ACAT inhibition modestly suppressed gene expression further. Neither SREBP-1a nor SREBP-1c mRNA levels were altered by cholesterol. Despite decreases in gene expression of the sterol-responsive genes by cholesterol, the amounts of precursor and mature forms of SREBP-1 and SREBP-2 were not altered. In contrast, if 25-hydroxycholesterol was substituted for cholesterol, both the precursor and mature forms of SREBP-2 were decreased. The polar sterol decreased the mature form of SREBP-1 but the amount of the precursor form was unchanged. In cells incubated with micelles containing lysophosphatidylcholine, which causes cholesterol to efflux from cells, sterol-responsive gene expression was increased. The amounts of precursor and mature forms of SREBP-1 and SREBP-2, however, were not altered. In contrast, if the cells were depleted of cholesterol by incubating them with lovastatin and cyclodextrin, the mature forms of SREBP-1 and SREBP-2 were increased, as were mRNA levels for the sterol-responsive genes.The data would suggest that cholesterol influx/efflux regulates mRNA levels of sterol-responsive genes independently of changes in the amount of mature SREBP. In contrast, 25-hydroxycholesterol influx or cholesterol depletion alters the amount of mature SREBP, leading to the regulation of sterol-responsive gene expression.

Gene expression of sterol regulatory element-binding proteins in hamster small intestine

Gene expression of sterol regulatory element-binding proteins 1a, 1c, and 2 (SREBP-1a, -1c, and -2) and of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and the low density lipoprotein (LDL) receptor was examined in hamster small intestine. SREBP-1c transcript predominated over SREBP-1a. mRNA levels for SREBP-1a, -1c, and -2, LDL receptor, and HMG-CoA synthase were highest in jejunum and ileum. Expression of SREBP-1a and SREBP-1c was highest in cells of the upper villus and decreased in cells of the lower villus. Gene expression of SREBP-2 was lowest in cells of the upper villus and increased in cells of the lower villus. Ileal SREBP-2 gene expression was highest in cells of the midvillus. mRNA levels for HMG-CoA synthase and the LDL receptor followed a pattern similar to that of SREBP-2. A positive correlation existed between SREBP-2 gene expression and rates of cholesterol synthesis. Fatty acid synthesis was highest in jejunum and ileum, correlating positively with the expression of SREBP-1c. Sterol influx into intestinal cells decreased mRNA levels of SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and LDL receptor. In ileum, sterol influx decreased gene expression of SREBP-1a and increased expression of SREBP-1c. The results suggest that SREBP-2 regulates cholesterol synthesis in the small intestine. SREBP-1a is a minor transcript and its expression does not correlate with cholesterol-synthesizing activity. SREBP-1c is a major transcript in small intestine and its expression along the length of the gut correlates with fatty acid synthesis. Sterols regulate gene expression of sterol-responsive genes, including SREBP-2, in small intestine.—Field, F. J., E. Born, S. Murthy, and S. N. Mathur. Gene expression of sterol regulatory element-binding proteins in hamster small intestine. J. Lipid Res. 2001. 42: 1–8.