Regulatory Element Binding Protein-1a Research Articles

Beneficial effects of IH-901 on glucose and lipid metabolisms via activating adenosine monophosphate–activated protein kinase and phosphatidylinositol-3 kinase pathways

IH-901 is an intestinal metabolite of ginsenosides found in Panax ginseng. In the present study, effects of IH-901 on glucose and lipid metabolisms were examined using C2C12 myotubes and C57BL/ksJ db/db mice. A significant increase in phosphorylated adenosine monophosphate–activated protein kinase was observed when differentiated C2C12 myotubes were treated with IH-901. Glucose transporter 4 protein expressions were also up-regulated when muscle cells were treated with of IH-901 up to 60 minutes, resulting in stimulation of glucose uptake by 25% as compared with untreated cells. In addition, phosphatidylinositol-3 kinase and Akt protein expressions were increased when C2C12 myotubes were exposed to IH-901 for up to 3 hours; and these effects including glucose uptake were attenuated by pretreatment with LY294002, a selective phosphatidylinositol-3 kinase inhibitor. In animal study, IH-901 at 25 mg/kg lowered the plasma glucose, triglyceride, cholesterol, and nonesterified fatty acid levels by 20.7%, 41.6%, 20.2%, and 24.6%, respectively, compared with control mice. In the meantime, plasma insulin levels were significantly increased by 2.2 and 3.4 times in 10 and 25 mg/kg–treated mice, respectively, compared with control mice, in parallel with the histologic observation showing a preserved architecture of the pancreatic islet. Protein and gene expression patterns for adenosine monophosphate–activated protein kinase, sterol regulatory element binding protein–1a, and glucose transporter 4 in the liver and skeletal muscles were similar to those in cell studies. In summary, IH-901 might be a promising therapeutic agent improving altered glucose and lipid metabolisms revealed in type 2 diabetes mellitus patients.

Eugenosedin-A prevents hyperglycaemia, hyperlipidaemia and lipid peroxidation in C57BL/6J mice fed a high-fat diet

Abstract Objectives Eugenosedin-A is a serotonin (5-hydroxytryptamine; 5-HT) 5-HT1b/2a and α1/α2/β1-adrenoceptor blocker with anti-oxidative, anti-inflammatory and free-radical scavenging activities. Previous reports demonstrated that 5-HT2a blockers could diminish hyperlipidaemia. This study therefore aimed to investigate the possible uses and mechanisms of eugenosedin-A and other agents in treating hyperlipidaemia. Methods C57BL/6J mice were randomly divided into seven groups, fed a regular diet or a high-fat diet alone or supplemented with one of five agents: eugenosedin-A, ketanserin, prazosin, propranolol or atorvastatin (5 mg/kg p.o.) for 8 weeks. Key findings Compared with the regular diet, the mice fed the high-fat diet had significantly higher body weight and glucose, insulin and lipid levels. Brain malondialdehyde concentration was increased and liver glutathione peroxidase activity decreased. Addition of eugenosedin-A to the high-fat diet resulted in less weight gain and reduced hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. Lipid and glucose homeostasis were related to decreased hepatic lipogenesis mRNAs and proteins (sterol regulatory element binding protein 1a, fatty acid synthase, sterol-CoA desaturase) and restored adipose peroxisome proliferator-activated receptor γ expression. Eugenosedin-A also enhanced low-density lipoprotein receptor mRNA expression. Conclusions Eugenosedin-A may improve plasma lipid metabolism by increasing low-density lipoprotein receptor and peroxisome proliferator-activated receptor γ expression and diminishing sterol regulatory element binding protein 1a, fatty acid synthase and sterol-CoA desaturase. Reduction of plasma glucose and lipid levels may, in turn, reduce insulin concentration, which would explain the marked improvement in obesity-related hyperglycaemia and hyperlipidaemia. Furthermore, eugenosedin-A affected malondialdehyde concentration and glutathione peroxidase activity, suggesting it may have anti-peroxidation effects in mice fed a high-fat diet.

The unique role of proprotein convertase subtilisin/kexin 9 in cholesterol homeostasis

The LDL receptor (LDLR) plays an essential role in the regulation of plasma (LDL) cholesterol concentrations by virtue of its ability to clear plasma LDL. Down-regulation of the LDLR by proprotein convertase subtilisin/kexin 9 (PCSK9) has recently emerged as a regulatory mechanism that controls plasma LDL cholesterol concentrations. Studies in which PCSK9 is over-expressed in mice, have demonstrated that PCSK9, by enhancing hepatic LDLR degradation, decreases the availability of the LDLR for LDL uptake, resulting in increased plasma LDL cholesterol levels. However, PCSK9 has also recently been shown to mediate down-regulation of surface receptors other than the LDLR, suggesting that it may have much broader roles than initially thought.

Sterol Regulatory Element Binding Protein 1a Regulates Hepatic Fatty Acid Partitioning by Activating Acetyl Coenzyme A Carboxylase 2

Sterol Regulatory Element Binding Protein 1a Regulates Hepatic Fatty Acid Partitioning by Activating Acetyl Coenzyme A Carboxylase 2

Sterol Regulatory Element Binding Protein 1a Regulates Hepatic Fatty Acid Partitioning by Activating Acetyl Coenzyme A Carboxylase 2

We generated a line of mice in which sterol regulatory element binding protein 1a (SREBP-1a) was specifically inactivated by insertional mutagenesis. Homozygous mutant mice were completely viable despite expressing SREBP-1a mRNA below 5% of normal, and there were minimal effects on expression of either SREBP-1c or -2. Microarray expression studies in liver, where SREBP-1a mRNA is 1/10 the level of the highly similar SREBP-1c, demonstrated that only a few genes were affected. The only downregulated genes directly linked to lipid metabolism were Srebf1 (which encodes SREBP-1) and Acacb (which encodes acetyl coenzyme A [acetyl-CoA] carboxylase 2 [ACC2], a critical regulator of fatty acyl-CoA partitioning between cytosol and mitochondria). ACC2 regulation is particularly important during food restriction. Similar to Acacb knockout mice, SREBP-1a-deficient mice have lower hepatic triglycerides and higher serum ketones during fasting than wild-type mice. SREBP-1a and -1c have identical DNA binding and dimerization domains; thus, the failure of the more abundant SREBP-1c to substitute for activating hepatic ACC2 must relate to more efficient recruitment of transcriptional coactivators to the more potent SREBP-1a activation domain. Our chromatin immunoprecipitation results support this hypothesis.

Fibrillar Collagen Inhibits Cholesterol Biosynthesis in Human Aortic Smooth Muscle Cells

Integrin-mediated cell adhesion to type I fibrillar collagen regulates gene and protein expression, whereas little is known of its effect on lipid metabolism. In the present study, we examined the effect of type I fibrillar collagen on cholesterol biosynthesis in human aortic smooth muscle cells (SMCs). SMCs were cultured on either fibrillar or monomer collagen for 48 hours and [(14)C]-acetate incorporation into cholesterol was evaluated. Fibrillar collagen reduced by 72.9+/-2.6% cholesterol biosynthesis without affecting cellular cholesterol levels. Fibrillar collagen also reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) promoter activity (-72.6+/-7.3%), mRNA (-58.7+/-6.4%), protein levels (-35.5+/-8.5%), and enzyme activity (-37.7+/-2.2%). Intracellular levels of the active form of sterol regulatory element binding proteins (SREBP) 1a was decreased by 60.7+/-21.7% in SMCs cultured on fibrillar collagen, whereas SREBP2 was not significantly affected (+12.1+/-7.1%). The overexpression of the active form of SREBP1a rescued the downregulation of fibrillar collagen on HMG-CoA reductase levels. Blocking antibody to alpha2 integrin partially reversed the downregulation of HMG-CoA reductase mRNA expression. Finally, fibrillar collagen led to an intracellular accumulation of unprenylated Ras. Our study demonstrated that alpha2 beta 1 integrin interaction with fibrillar collagen affected the expression of HMG-CoA reductase, which led to the inhibition of cholesterol biosynthesis in human SMCs.

Eugenosedin-A prevents hyperglycaemia, hyperlipidaemia and lipid peroxidation in C57BL/6J mice fed a high-fat diet

Eugenosedin-A is a serotonin (5-hydroxytryptamine; 5-HT) 5-HT(1B/2A) and alpha(1)/alpha(2)/beta(1)-adrenoceptor blocker with anti-oxidative, anti-inflammatory and free-radical scavenging activities. Previous reports demonstrated that 5-HT(2A) blockers could diminish hyperlipidaemia. This study therefore aimed to investigate the possible uses and mechanisms of eugenosedin-A and other agents in treating hyperlipidaemia. C57BL/6J mice were randomly divided into seven groups, fed a regular diet or a high-fat diet alone or supplemented with one of five agents: eugenosedin-A, ketanserin, prazosin, propranolol or atorvastatin (5 mg/kg p.o.) for 8 weeks. Compared with the regular diet, the mice fed the high-fat diet had significantly higher body weight and glucose, insulin and lipid levels. Brain malondialdehyde concentration was increased and liver glutathione peroxidase activity decreased. Addition of eugenosedin-A to the high-fat diet resulted in less weight gain and reduced hyperglycaemia, hyperinsulinaemia and hyperlipidaemia. Lipid and glucose homeostasis were related to decreased hepatic lipogenesis mRNAs and proteins (sterol regulatory element binding protein 1a, fatty acid synthase, sterol-CoA desaturase) and restored adipose peroxisome proliferator-activated receptor gamma expression. Eugenosedin-A also enhanced low-density lipoprotein receptor mRNA expression. Eugenosedin-A may improve plasma lipid metabolism by increasing low-density lipoprotein receptor and peroxisome proliferator-activated receptor gamma expression and diminishing sterol regulatory element binding protein 1a, fatty acid synthase and sterol-CoA desaturase. Reduction of plasma glucose and lipid levels may, in turn, reduce insulin concentration, which would explain the marked improvement in obesity-related hyperglycaemia and hyperlipidaemia. Furthermore, eugenosedin-A affected malondialdehyde concentration and glutathione peroxidase activity, suggesting it may have anti-peroxidation effects in mice fed a high-fat diet.

Nuclear SREBP-1a causes loss of pancreatic β-cells and impaired insulin secretion

Transgenic mice expressing nuclear sterol regulatory element-binding protein-1a under the control of the insulin promoter were generated to determine the role of SREBP-1a in pancreatic β-cells. Only low expressors could be established, which exhibited mild hyperglycemia, impaired glucose tolerance, and reduced plasma insulin levels compared to C57BL/6 controls. The islets isolated from the transgenic mice were fewer and smaller, and had decreased insulin content and unaltered glucagon staining. Both glucose- and potassium-stimulated insulin secretions were decreased. The transgenic islets consistently expressed genes for fatty acids and cholesterol synthesis, resulting in accumulation of triglycerides but not cholesterol. PDX-1, ΒΕΤΑ2, MafA, and IRS-2 were suppressed, partially explaining the loss and dysfunction of β-cell mass. The transgenic mice on a high fat/high sucrose diet still exhibited impaired insulin secretion and continuous β-cell growth defect. Therefore, nuclear SREBP-1a, even at a low level, strongly disrupts β-cell mass and function.

188: Fibrillar collagen inhibits cholesterol biosynthesis in human aortic smooth muscle cells

Objective— Integrin-mediated cell adhesion to type I fibrillar collagen regulates gene and protein expression, whereas little is known of its effect on lipid metabolism. In the present study, we examined the effect of type I fibrillar collagen on cholesterol biosynthesis in human aortic smooth muscle cells (SMCs). Methods and Results— SMCs were cultured on either fibrillar or monomer collagen for 48 hours and [14C]-acetate incorporation into cholesterol was evaluated. Fibrillar collagen reduced by 72.9±2.6% cholesterol biosynthesis without affecting cellular cholesterol levels. Fibrillar collagen also reduced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) promoter activity (−72.6±7.3%), mRNA (−58.7±6.4%), protein levels (−35.5±8.5%), and enzyme activity (−37.7±2.2%). Intracellular levels of the active form of sterol regulatory element binding proteins (SREBP) 1a was decreased by 60.7±21.7% in SMCs cultured on fibrillar collagen, whereas SREBP2 was not significantly affected (+12.1±7.1%). The ov...

Inhibition of stearoyl CoA desaturase activity induces hypercholesterolemia in the cholesterol-fed hamster

Reduction of stearoyl CoA desaturase (SCD) activity has been shown to induce resistance to diet-induced obesity in mice. In the present study, SCD was inhibited by feeding sterculic oil (SO) to male Golden Syrian Hamsters fed high-fat diets with or without added dietary cholesterol. In the absence of cholesterol, SO had little impact on adipose tissue mass or plasma lipoprotein concentrations. When cholesterol was included in the diet, inhibition of SCD resulted in reduced body weight, adipose tissue mass, and feed efficiency. These animals also exhibited a marked hypercholesterolemia, with an accumulation of free-cholesterol-rich particles within the LDL density range, and reduced hepatic cholesterol esterification. This was accompanied by a 20-fold increase in plasma alanine aminotransferase, which was suggestive of significant hepatic damage. Hepatic acetyl CoA carboxylase and fatty acid synthase mRNA concentrations were reduced by feeding cholesterol and SO, whereas lipoprotein lipase and SCD mRNA were increased. These changes were associated with decreased hepatic sterol regulatory element binding protein 1a and 1c mRNA concentrations. Thus, inhibition of SCD activity in the cholesterol-fed hamster results in a reduction in overall body weight and adipose tissue deposition. However, this also causes marked hypercholesterolemia and potential liver damage.

Genetic Deletion of Low Density Lipoprotein Receptor Impairs Sterol-induced Mouse Macrophage ABCA1 Expression: A NEW SREBP1-DEPENDENT MECHANISM

Low density lipoprotein receptor (LDLR) mutations cause familial hypercholesterolemia and early atherosclerosis. ABCA1 facilitates free cholesterol efflux from peripheral tissues. We investigated the effects of LDLR deletion (LDLR(-/-)) on ABCA1 expression. LDLR(-/-) macrophages had reduced basal levels of ABCA1, ABCG1, and cholesterol efflux. A high fat diet increased cholesterol in LDLR(-/-) macrophages but not wild type cells. A liver X receptor (LXR) agonist induced expression of ABCA1, ABCG1, and cholesterol efflux in both LDLR(-/-) and wild type macrophages, whereas expression of LXRalpha or LXRbeta was similar. Interestingly, oxidized LDL induced more ABCA1 in wild type macrophages than LDLR(-/-) cells. LDL induced ABCA1 expression in wild type cells but inhibited it in LDLR(-/-) macrophages in a concentration-dependent manner. However, lipoproteins regulated ABCG1 expression similarly in LDLR(-/-) and wild type macrophages. Cholesterol or oxysterols induced ABCA1 expression in wild type macrophages but had little or inhibitory effects on ABCA1 expression in LDLR(-/-) macrophages. Active sterol regulatory element-binding protein 1a (SREBP1a) inhibited ABCA1 promoter activity in an LXRE-dependent manner and decreased both macrophage ABCA1 expression and cholesterol efflux. Expression of ABCA1 in animal tissues was inversely correlated to active SREBP1. Oxysterols inactivated SREBP1 in wild type macrophages but not in LDLR(-/-) cells. Oxysterol synergized with nonsteroid LXR ligand induced ABCA1 expression in wild type macrophages but blocked induction in LDLR(-/-) cells. Taken together, our studies suggest that LDLR is critical in the regulation of cholesterol efflux and ABCA1 expression in macrophage. Lack of the LDLR impairs sterol-induced macrophage ABCA1 expression by a sterol regulatory element-binding protein 1-dependent mechanism that can result in reduced cholesterol efflux and lipid accumulation in macrophages under hypercholesterolemic conditions.

Role of Sp1 and SREBP-1a in the insulin-mediated regulation of glucokinase transcription in the liver of gilthead sea bream ( Sparus aurata)

Insulin induction of glucokinase (GCK) transcription in the liver is essential for maintaining glucose homeostasis. To study the molecular mechanism underlying the regulation of hepatic GCK expression in the carnivorous fish gilthead sea bream ( Sparus aurata), we analysed the role of sterol regulatory element binding protein-1a (SREBP-1a) and specificity protein (Sp) 1 in insulin-dependent GCK transcription. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays allowed us to identify a cis-element in the proximal region of GCK promoter implicated in transactivation by SREBP-1a. Consistently, mutations in the SRE binding site completely abolished the enhancing effect of SREBP-1a. These results and previous findings suggest that SREBP-1a plays a role in the transcriptional regulation of key enzymes in glycolysis–gluconeogenesis. Since SREBP-1a and Sp1 may mediate insulin action on S. aurata GCK transcription, we analysed the effect of insulin on HepG2 cells transfected with GCK promoter reporter constructs carrying intact or mutated SRE or Sp boxes. Insulin transactivated GCK irrespective of the presence of an intact or mutated SRE box. However, insulin failed to induce GCK transcription when using reporter constructs that had either a mutated Sp site or no Sp site. Our findings indicate that Sp1, rather than SREBP-1a, mediates the insulin-dependent induction of S. aurata GCK.

Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene

The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers.

Genetics and molecular biology: macrophage ACAT depletion – mechanisms of atherogenesis

Genetics and molecular biology: macrophage ACAT depletion – mechanisms of atherogenesis

Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins

In the process of membrane biogenesis several dozen proteins must operate in precise concert to generate approximately 100 lipids at appropriate concentrations. To study the regulation of bilayer assembly in a cell cycle-independent manner, we have exploited the fact that phagocytes replenish membranes expended during particle engulfment in a rapid phase of lipid synthesis. In response to phagocytosis of latex beads, human embryonic kidney 293 cells synthesized cholesterol and phospholipids at amounts equivalent to the surface area of the internalized particles. Lipid synthesis was accompanied by increased transcription of several lipogenic proteins, including the low-density lipoprotein receptor, enzymes required for cholesterol synthesis (3-hydroxy-3-methylglutaryl CoA synthase, 3-hydroxy-3-methylglutaryl CoA reductase), and fatty acid synthase. Phagocytosis triggered the proteolytic activation of two lipogenic transcription factors, sterol regulatory element binding protein-1a (SREBP-1a) and SREBP-2. Proteolysis of SREBPs coincided with the appearance of their transcriptionally active N termini in the nucleus and 3-fold activation of an SREBP-specific reporter gene. In previous studies with cultured cells, proteolytic activation of SREBP-1a and SREBP-2 has been observed in response to selective starvation of cells for cholesterol and unsaturated fatty acids. However, under the current conditions, SREBP-1a and SREBP-2 are induced without lipid deprivation. SREBP activation is inhibited by high levels of the SREBP-interacting proteins Insig1 or the cytosolic domain of SREBP cleavage-activating protein. Upon overexpression of these proteins, phagocytosis-induced transcription and lipid synthesis were blocked. These results identify SREBPs as essential regulators of membrane biogenesis and provide a useful system for further studies on membrane homeostasis.

Spatial Distribution and Function of Sterol Regulatory Element-Binding Protein 1a and 2 Homo- and Heterodimers by In Vivo Two-Photon Imaging and Spectroscopy Fluorescence Resonance Energy Transfer

Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

Insulin and Human Chorionic Gonadotropin Cause a Shift in the Balance of Sterol Regulatory Element-Binding Protein (SREBP) Isoforms Toward the SREBP-1c Isoform in Cultures of Human Granulosa Cells

The isoforms of sterol regulatory element-binding proteins (SREBP) (1a, 1c, and 2) are key transcriptional regulators of lipid biosynthesis. We examined their regulation by gonadotropin and insulin in human granulosa cells. After removal of leukocytes, granulosa cells were exposed to hormonal additions for 16 h starting on d 2 of culture. Progesterone, lactate, and IGF binding protein-1 were measured in culture medium and cellular mRNA measured by competitive RT-PCR. Addition of human chorionic gonadotropin (hCG) (100 ng/ml) stimulated progesterone production (7.0-fold, P < 0.001 vs. control), whereas lactate was increased by hCG (1.6-fold, P < 0.001) and insulin (1.4-fold, P < 0.001; 1000 ng/ml). Insulin decreased IGF binding protein-1 production by 85% (P < 0.001). There were no significant effects on the expression of SREBP-1a but significant increases in mRNA for SREBP-1c with insulin (6.3-fold), hCG (10.4-fold) and in combination (15.2-fold; P < 0.01 for all comparisons). No consistent effects on SREBP-2 were observed. The expression of mRNA for fatty acid synthase, a target gene for SREBP-1c, was increased by hCG (24-fold, P = 0.006) and insulin (19-fold, P = 0.024), which also increased the level of cellular, total fatty acid (1.34-fold; P = 0.03). Thus, hCG and insulin cause a switch toward expression of the SREBP-1c isoform with consequent effects on fatty acid synthesis. We suggest that high circulating insulin, associated with clinically defined insulin resistance, may up-regulate SREBP-1c expression in the ovary.

Effect of Sterol Regulatory Element Binding Protein-1a on the Mitochondrial Protein Pattern in Human Liver Cells Detected by 2D-DIGE

Sterol regulatory element binding protein-1a (SREBP-1a) is a transcription factor that is a major player in lipid metabolism and insulin action. We have generated human liver cells (HepG2) overexpressing active SREBP-1a constitutively called SREBP-1a (+). These cells show massive intracellular lipid accumulation. To elucidate the effect of SREBP-1a on lipid metabolism at the level of the cellular protein network, we have analyzed the protein pattern of mitochondria using the novel technique two-dimensional difference gel electrophoresis (2D-DIGE). Mitochondria were enriched by subcellular fractionation using differential and isopyknic centrifugation. Proteins of isolated organelles were labeled with Cy dyes and separated on 2D gels. These gels revealed more than 100 protein spots, which were significantly different in their abundance between wild-type and SREBP-1a (+) cells. MALDI mass spectrometry showed that 68% of identified proteins belong to mitochondria. In SREBP-1a (+) cells, several enzymes involved in lipid metabolism were significantly altered, suggesting that cellular lipid metabolism is triggered by accumulation of fatty acids rather than by its degradation. To test the possible functional relevance of this finding, intracellular fatty acid (FA) patterns were analyzed by gas chromatography. The results showed a significant increase in total fatty acid content with a shift in composition to long-chain unsaturated FAs. Therefore, the detected protein differences might be an explanation for the observed intracellular lipid accumulation and might link SREBP-1a to features like steatosis hepatis.

A Novel in Vivo Lecithin-Cholesterol Acyltransferase (LCAT)-Deficient Mouse Expressing Predominantly LpX Is Associated with Spontaneous Glomerulopathy

Complete lecithin cholesterol acyltransferase (LCAT) deficiency is a rare genetic cause of extreme reduction in high density lipoproteins and there is a high prevalence of chronic renal dysfunction that may progress to renal failure. Previous in vitro studies suggest the vesicular lipoprotein X (LpX) particles commonly seen in LCAT-deficient plasmas may be causative. To test this hypothesis, we have generated a novel murine model that selectively accumulate LpX in the circulation by cross breeding the sterol regulatory element binding protein (SREBP) 1a transgenic mice (S+) with the LCAT knockout (lcat-/-) mice. Fast protein liquid chromatography fractionation of pooled plasma lipids revealed that virtually all cholesterol is concentrated in the very low density lipoprotein (VLDL)-sized fractions. These fractions are enriched in free cholesterol and phospholipid but extremely poor in triglyceride. Electron microscopy of the d <1.063 g/ml fraction of the S+lcat-/- mice revealed abnormal large vesicular particles, suggestive of LpX. The S+lcat-/- mice developed glomerular lesions spontaneously evident at 6 months with glomerular and tubulointerstitial lipid-deposits. Immunohistochemical staining with RhoA showed marked positive focal staining in glomeruli in the S+lcat-/- mice and undetectable in the S+/lcat+/+ control. By 10 months of age, the kidneys showed progressive glomerular injury including segmental foam cell infiltrates, mesangial expansion, and hyalinosis. Renal abnormalities are very similar to those seen in human LCAT deficiency. We conclude that the selective high-level accumulation of plasma LpX in the S+lcat-/- mice is strongly associated with a spontaneous glomerulopathy, providing in vivo evidence that LpX contributes to the LCAT deficiency-related nephropathy.

Valproic acid inhibits leptin secretion and reduces leptin messenger ribonucleic acid levels in adipocytes.

Treatment of epilepsy or bipolar disorder with valproic acid (VPA) induces weight gain and increased serum levels for the satiety hormone, leptin, through an unidentified mechanism. In this study we tested the effects of VPA, a short-chain branched fatty acid (C8:0), on leptin biology and fatty acid metabolism in 3T3-L1 adipocytes. VPA significantly reduced leptin secretion in a dose-dependent manner. Because fatty acid accumulation has been hypothesized to block leptin secretion, we tested the effect of VPA on fatty acid metabolism. Using 14C-radiolabeled VPA, we found that the 14C was mainly incorporated into triacylglycerol. VPA did not alter lipogenesis from acetate, nor did it change the amount of intracellular free fatty acids available for triacylglycerol synthesis. Decreased leptin secretion was accompanied by a reduction in leptin mRNA, even though VPA treatment did not alter the protein levels for known transcription factors affecting leptin transcription including: CCAAT/enhancer binding protein-alpha, peroxisome proliferator-activated receptor-gamma, or steroid regulatory element binding protein 1a. VPA altered levels of leptin mRNA independent of de novo protein synthesis without affecting leptin mRNA degradation. This report demonstrates that VPA decreases leptin secretion and mRNA levels in adipocytes in vitro, suggesting that VPA therapy may be associated with altered leptin homeostasis contributing to weight gain in vivo.