Regulation Of Lipid Synthesis Research Articles

A comparative metabolomic study of NHR-49 in Caenorhabditis elegans and PPAR-α in the mouse

Proton Nuclear Magnetic Resonance spectroscopy and Gas Chromatography Mass Spectrometry based metabolomics has been used in conjunction with multivariate statistics to examine the metabolic changes in Caenorhabditis elegans following the deletion of nuclear hormone receptor-49 ( nhr-49). Deletion of the receptor produced profound changes in fatty acid metabolism, in particular an increase in the ratio of unsaturated to saturated fatty acids, a decrease in the concentration of glucose and increases in lactate and alanine. Given the proposed functional similarity between nhr-49 and the mammalian peroxisome proliferator-activated receptors (PPARs) these changes were compared with the metabolome of the PPAR-α null mouse. The metabolomic approach demonstrated a number of similarities including the regulation of lipid synthesis, β-oxidation of fatty acids and changes in glycolysis/gluconeogenesis.

Molecular Mechanism of Age‐Specific Hepatic Lipid Accumulation in PPARα (+/−):LDLR (+/−) Mice, an Obese Mouse Model

This study aimed to clarify the molecular mechanisms of age-specific hepatic lipid accumulation accompanying hyperinsulinemia in a peroxisome proliferator-activated receptor alpha (PPARalpha) (+/-):low-density lipoprotein receptor (LDLR) (+/-) mouse line. The hepatic fat content, protein amounts, and mRNA levels of genes involved in hepatic lipid metabolism were analyzed in 25-, 50-, 75- and 100-week-old mice. Severe fatty liver was confirmed only in 50- and 75-week-old mice. The hepatic expression of proteins that function in lipid transport and catabolism did not differ among the groups. In contrast, the mRNA levels and protein amounts of lipogenic enzymes, including acetyl-coenzyme A carboxylase-1, fatty acid synthase, and glycerol-3-phosphate acyltransferase, enhanced in the mice with fatty liver. Elevated mRNA and protein levels of lipoprotein lipase and fatty acid translocase, which are involved in hepatic lipid uptake, were also detected in mice with fatty liver. Moreover, both protein and mRNA levels of sterol regulatory element-binding protein-1 (SREBP-1), a transcription factor regulating lipid synthesis, had age-specific patterns similar to those of the proteins described above. Therefore, the age-specific fatty liver found in the PPARalpha (+/-):LDLR (+/-) mouse line is probably caused by age-specific expression of SREBP-1 and its downstream lipogenic genes, coordinated by the increased uptake of lipids. All of these factors might be affected by age-specific changes in serum insulin concentration.

ChREBP, but not LXRs, is required for the induction of glucose-regulated genes in mouse liver

The transcription factor carbohydrate-responsive element-binding protein (ChREBP) has emerged as a central regulator of lipid synthesis in liver because it is required for glucose-induced expression of the glycolytic enzyme liver-pyruvate kinase (L-PK) and acts in synergy with SREBP to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Liver X receptors (LXRs) are also important regulators of the lipogenic pathway, and the recent finding that ChREBP is a direct target of LXRs and that glucose itself can bind and activate LXRs prompted us to study the role of LXRs in the induction of glucose-regulated genes in liver. Using an LXR agonist in wild-type mice, we found that LXR stimulation did not promote ChREBP phosphorylation or nuclear localization in the absence of an increased intrahepatic glucose flux. Furthermore, the induction of ChREBP, L-PK, and ACC by glucose or high-carbohydrate diet was similar in LXRalpha/beta knockout compared with wild-type mice, suggesting that the activation of these genes by glucose occurs by an LXR-independent mechanism. We used fluorescence resonance energy transfer analysis to demonstrate that glucose failed to promote the interaction of LXRalpha/beta with specific cofactors. Finally, siRNA silencing of ChREBP in LXRalpha/beta knockout hepatocytes abrogated glucose-induced expression of L-PK and ACC, further demonstrating the central role of ChREBP in glucose signaling. Taken together, our results demonstrate that glucose is required for ChREBP functional activity and that LXRs are not necessary for the induction of glucose-regulated genes in liver.

The UPS in diabetes and obesity

Type 2 diabetes is caused by defects in both insulin signaling and insulin secretion. Though the role of the ubiquitin proteasome system (UPS) in the pathogenesis of type 2 diabetes remains largely unexplored, the few examples present in the literature are interesting and suggest targets for drug development. Studies indicate that insulin resistance can be induced by stimulating the degradation of important molecules in the insulin signaling pathway, in particular the insulin receptor substrate proteins IRS1, IRS2 and the kinase AKT1 (Akt). In addition, a defect in insulin secretion could occur due to UPS-mediated degradation of IRS2 in the β-cells of the pancreas. The UPS also appears to be involved in regulating lipid synthesis in adipocytes and lipid production by the liver and could influence the development of obesity. Other possible mechanisms for inducing defects in insulin signaling and secretion remain to be explored, including the role of ubiquitylation in insulin receptor internalization and trafficking.Republished from Current BioData's Targeted Proteins database (TPdb; ).

Plasma cell differentiation and lipid metabolism (83.18)

Abstract Plasma cells are terminally differentiated B cells that produce and secrete profuse amounts of antibodies. Plasma cell differentiation involves an increase in exocytic pathway components and expansion of the endoplasmic reticulum (ER) in order to accommodate high-rate immunoglobulin synthesis. ER expansion requires membrane biogenesis, a process that requires phospholipids. Increased phospholipid synthesis is dependent upon a sufficient supply of fatty acids. We have found that de novo fatty acid biosynthesis increases in CH12 B cells differentiating in response to lipopolysaccharide (LPS). Increased fatty acid biosynthesis correlates with upregulation of acetyl CoA carboxylase (ACC) and fatty acid synthase (FAS), enzymes that catalyze the formation of fatty acids. Further investigation will shed light into mechanisms that regulate lipid synthesis and ER expansion in differentiating plasma cells. Supported by NIH-GM61970 and NIH-T32 A1007508

Sphingolipids: major regulators of lipid metabolism

Sphingolipids and their metabolites regulate a great variety of cellular processes. Recent findings implicate sphingolipids in the regulation of lipid synthesis, lipoprotein metabolism and the development of atherosclerosis. Sphingolipid synthesis correlates with the regulation of the sterol-regulatory element-binding proteins - key transcription factors of genes of lipid metabolism. Inhibition of sphingolipid synthesis decreases synthesis of genes regulated by sterol regulatory element-binding protein, such as the rate-limiting enzymes of fatty acid and cholesterol synthesis as well as fatty-acyl-CoA synthases, important in the synthesis of phospholipids. In animal models, inhibition of sphingolipid synthesis correlates with decreased atherosclerotic lesions and a decreased susceptibility of lipoproteins to aggregate--a key mechanism in the development of the atherosclerotic lesion. The demonstration that ceramide and glucosylceramide (metabolites of sphingolipid synthesis) affect cholesterol efflux and mechanisms that regulate plasma high-density lipoprotein concentrations is further evidence for a role of sphingolipids in the regulation of lipid homeostasis. Direct mechanisms of how sphingolipid synthesis regulates lipid synthesis are currently unknown. The recent identification of key proteins of synthesis and specific transport proteins that regulate sphingolipid synthesis, however, is expected to contribute to the understanding about the interdependent regulation of sphingolipid and lipid metabolism. Emerging data strongly suggest a role of sphingolipid synthesis in the regulation of transcription factors and regulatory proteins that control cellular lipid homeostasis.

Gene networks regulating lipid synthesis in bovine mammary gland during the lactation cycle

Regulation of lipid synthesis in the bovine mammary gland is a complex dynamic process likely controlled by interacting gene networks. Little is known about these sensing and signal transduction circuits. Mammary biopsies from 6 Holstein cows were obtained at -15, 1, 15, 30, 60, 120, and 240 days relative to parturition (DIM), and RNA used for qPCR of ABCA1, ABCG2, ACACA, ADFP, BTN1A1, CD36, DGAT1, ESR1, FABP3, FADS1, FASN, GPAM, INSIG1, INSIG2, LPIN1, LPL, PPARG, PPARGC1A, PPARGC1B, RETN, SCAP, SCD, SREBF1, SREBF2, and THRSP. These genes are relatively well-studied in non-ruminant cells and tissues due to their key roles in aspects of carbohydrate and lipid metabolism. Except for PPARGC1B, lactation increased (P < 0.01) mRNA expression of other genes by 1.5 (ABCA1) to 80-fold (FABP3, LPL) relative to day -15. Peak expression patterns were observed at 60 DIM for all genes except PPARG, PPARGC1A, SCAP, and THRSP, which peaked (3-, 10-, 1.7-, and 3-fold, respectively) at 120 DIM. Network analysis using Ingenuity Pathway Analysis revealed regulatory loops consisting of SREBF2, SREBF1, PPARG, PPARGC1A, and PPARGC1B along with their down-stream targets (e.g. SCD, ACACA, INSIG1, FABP3). LPIN1, INSIG2, and SCAP appeared to independently regulate expression of several lipogenic genes. LPIN1 (25-fold higher on day 60) and AGPAT6 (16-fold higher on day 60) seem to play a role in bovine milk fat synthesis regulation. Results suggest that mammary lipid synthesis is regulated by complex gene networks including transcription factors and novel genes.

Good soldier falls: the liver in sleep apnea

severe obstructive sleep apnea fires a complex array of physiological stressors rapidly and relentlessly across each period of sleep. With each occlusion of the upper airway, particularly in obese individuals, arterial oxygen content and delivery fall precipitously, carbon dioxide rises, acidosis

Metabolic regulation in the lactating mammary gland: a lipid synthesizing machine

The mammary gland of the lactating mouse synthesizes and secretes milk lipid equivalent to its entire body weight in a single 20-day lactation cycle, making it one of the most active lipid synthetic organs known. We test the hypothesis that multiple control points and potential regulatory mechanisms regulate milk lipid synthesis at the level of gene expression. The mammary transcriptome of 130 genes involved in glucose metabolism was examined at late pregnancy and early lactation, utilizing data obtained from microarray analysis of mammary glands from quadruplicate FVB mice at pregnancy day 17 and lactation day 2. To correlate changes with physiological parameters, the metabolome obtained from magnetic resonance spectroscopy of flash-frozen glands at day 17 of pregnancy was compared with that at day 2 of lactation. A significant increase in carbohydrates (glucose, lactose, sialic acid) and amino acids (alanine, aspartate, arginine, glutamate) with a moderate increase in important osmolytes (myo-inositol, betaine, choline derivatives) were observed in the lactating gland. In addition, diets containing 8% or 40% lipid were fed from lactation days 5-10 and mammary glands and livers of triplicate FVB mice prepared for microarray analysis. The results show that substantial regulation of lipid synthesis occurs at the level of mRNA expression and that some of the regulation points differ substantially from the liver. They also implicate the transcription factor SREBP-1c in regulation of part of the pathway.

The endocrine regulation of milk lipid synthesis and secretion in tammar wallaby ( Macropus eugenii)

Lipids in tammar milk are predominantly triacylglycerols, and the fatty acid composition varies during the lactation cycle. Little is known about the regulation of their synthesis. This study investigates the endocrine regulation of lipid synthesis in mammary explants from pregnant tammars. Treatment of mammary explants with insulin resulted in a high level of lipid synthesis, but the lipids accumulated in the cytosol. Culture with prolactin resulted in a small increase in lipid synthesis, but electron microscopy showed lipid globules were synthesized in the mammary epithelial cells and secreted into the lumen. Culture with both insulin and prolactin demonstrated elevated levels of synthesis and secretion of lipid. Analysis of the type of fatty acids synthesized in these mammary explants showed that the initiation of synthesis of C 16:0, which also occurs in the first week of lactation, could be reproduced in the pregnant explants cultured with prolactin alone. However, treatment of mammary explants with hydrocortisone did not show a significant effect on lipid synthesis, secretion or the fatty acid synthesized. These results provide new information identifying the role of insulin and prolactin in regulating milk lipid synthesis and secretion in the tammar.

Novel Regulation of Lipid Synthesis: The Spot 14 Protein Controls Fatty Acid Synthase Activity in Vivo and in Vitro

Novel Regulation of Lipid Synthesis: The Spot 14 Protein Controls Fatty Acid Synthase Activity in Vivo and in Vitro

Identification of a functional DNA binding site for the SREBP-1c transcription factor in the first intron of the human caspase-2 gene

Sterol Regulatory Element Binding Proteins (SREBP) are transcription factors that regulate lipid synthesis. We have shown recently that the human CASP-2 gene, encoding procaspase-2, was under the positive control of SREBP-2 that transactivates several responsive elements in the promoter region. We describe here the function of an additional SREBP-responsive element located in the first intron. Remarkably, this site is uniquely responsive to SREBP-1c that is mainly involved in fatty acids synthesis. This observation, together with our recent findings, strengthens the notion that the CASP-2 gene belongs to the SREBP-responsive gene battery in human cells.

The Inhibitory Effect of trans-10, cis-12 CLA on Lipid Synthesis in Bovine Mammary Epithelial Cells Involves Reduced Proteolytic Activation of the Transcription Factor SREBP-1

The trans-10, cis-12 CLA isomer has been causally related to milk fat depression in dairy cows, although no molecular mechanism has been established. Sterol response element-binding protein (SREBP)-1 is a transcription factor synthesized and retained as a membrane-bound precursor in the endoplasmic reticulum and proteolytically cleaved to release an active fragment that migrates to the nucleus to stimulate lipogenic gene transcription. Certain lipid molecules (i.e., PUFA) were shown to inhibit the proteolytic activation of SREBP-1 in rodent liver models, although there has been no previous demonstration of its presence in bovine tissues or in mammary tissue of any species. We used a bovine mammary cell line (MAC-T) to assess the involvement of SREBP-1 in the regulation of lipid synthesis in bovine mammary cells by trans-10, cis-12 CLA. Treatment with 75 micromol/L trans-10, cis-12 CLA for 48 h resulted in an approximately 50% reduction of (14)C-acetate incorporation into total lipid and corresponding reductions in mRNA abundance for acetyl CoA carboxylase, fatty acid synthase, and stearoyl CoA desaturase, whereas cis-9, trans-11 CLA had no effect on these genes. There was no reduction in SREBP-1 mRNA or precursor protein, but the abundance of the activated nuclear fragment of the protein was significantly reduced by treatment with 75 micromol/L trans-10, cis-12 CLA. These results indicate that trans-10, cis-12 CLA reduces lipid synthesis in the bovine mammary gland through inhibition of the proteolytic activation of SREBP-1 and subsequent reduction in transcriptional activation of lipogenic genes.

Kupffer cell-derived prostaglandin E2 is involved in regulation of lipid synthesis in rat liver tissue.

Our recent studies suggest that Kupffer cells play a role in the physiological regulation of lipid metabolism of the adjacent hepatocytes. In the present study, we have tested the hypothesis that inhibition of Kupffer cells decreases prostaglandin E(2) (PGE(2)) release inside liver tissue, a phenomenon contributing to lipid accumulation in hepatocytes. PGE(2) secretion as well as lipid synthesis were assessed in precision-cut liver slices (PCLS) from rats previously treated with Kupffer cell inhibitors (GdCl(3) 10 mg kg(-1) body wt, i.v. injection and glycine 5% in diet). In addition, lipid synthesis was assessed in primary rat hepatocytes cultured in the absence or presence of PGE(2) (0.01, 1 and 10 microM). Inhibition of Kupffer cell activity by GdCl(3) decreases PGE(2) secretion by PCLS and resulted in a higher lipid synthesis. Since incubation with PGE(2) over 48 h decreases lipid synthesis from acetate in cultured hepatocytes, we propose that the lower PGE(2) secretion linked to Kupffer cell inhibition, partly explains a higher rate of synthesis of lipids with a subsequent accumulation in liver tissue, as previously shown in fasted rats.

SREBPs suppress IRS-2-mediated insulin signalling in the liver.

Insulin receptor substrate 2 (IRS-2) is the main mediator of insulin signalling in the liver, controlling insulin sensitivity. Sterol regulatory element binding proteins (SREBPs) have been established as transcriptional regulators of lipid synthesis. Here, we show that SREBPs directly repress transcription of IRS-2 and inhibit hepatic insulin signalling. The IRS-2 promoter is activated by forkhead proteins through an insulin response element (IRE). Nuclear SREBPs effectively replace and interfere in the binding of these transactivators, resulting in inhibition of the downstream PI(3)K/Akt pathway, followed by decreased glycogen synthesis. These data suggest a molecular mechanism for the physiological switching from glycogen synthesis to lipogenesis and hepatic insulin resistance that is associated with hepatosteatosis.

Membrane Topology of Human Insig-1, a Protein Regulator of Lipid Synthesis

Insig-1 is an intrinsic protein of the endoplasmic reticulum (ER) that regulates the proteolytic processing of membrane-bound sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in mammalian cells. When cellular levels of sterols rise, Insig-1 binds to the membranous sterol-sensing domain of SREBP cleavage-activating protein (SCAP), retaining the SCAP/SREBP complex in the ER and preventing it from moving to the Golgi for proteolytic processing. Under conditions of sterol excess, Insig-1 also binds to the ER enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, facilitating its ubiquitination and proteasomal degradation. Here, we use protease protection, glycosylation site mapping, and cysteine derivitization to define the topology of the 277-amino acid human Insig-1. The data indicate that short segments at the N and C termini of Insig-1 face the cytosol. Most of the protein is buried within the membrane, forming six transmembrane segments separated by five short luminal and cytosolic loops that range from approximately 5 to 16 amino acids. The membranous nature of Insig-1 is consistent with its sterol-dependent binding to hydrophobic sterol-sensing domains in SCAP and HMG CoA reductase.

Precision-cut liver slices in culture as a tool to assess the physiological involvement of Kupffer cells in hepatic metabolism

Hepatic macrophages have the capacity to secrete a tremendous array of molecules, which can be divided into 3 categories e cytokines (TNF-alpha), lipid mediators (prostaglandins PGE2) and reactive intermediates (NOe) e in response to stimulus, such as lipopolysaccharides (LPS) [1,2]. Such mediators are capable to modulate both the metabolism and the integrity of hepatocytes in vitro [2]. The physiological role of Kupffer cell in hepatic metabolism regulation has been approached in the present study by using the original in vitro model of precision-cut liver slices (PCLS) in culture; this model allows preserving the liver lobule architecture, by maintaining namely cell diversity in physiological proportion and cell-cell interactions [3]. First, we established whether non-parenchymal cells are still viable in rat PCLS and are able to respond to LPS in vitro; TNF-alpha, PGE2, NOx (reflecting NOe release) were measured in the incubation medium of PCLS from rats previously treated with GdCl3 e a specific inhibitor of Kupffer cell phagocytosis [4] e or NaCl as a control- in order to evaluate the contribution of Kupffer cell in mediator release. Moreover, by using the same model, we have investigated the role of Kupffer cell in the regulation of lipid synthesis in PCLS, in order to approach the biochemical mechanism explaining our last results, which indicate that the inhibition of Kupffer cell by GdCl3 leads to triglycerides accumulation in liver tissue [5].

Inhibition of hepatocyte lipogenesis by nitric oxide donor: could nitric oxide regulate lipid synthesis?

Tissue lipogenesis is variably controlled by substrate supply and hormones. The possibility that nitric oxide (NO) might regulate lipogenesis derives from the action of NO on coenzyme A (CoA) to produce metabolically inactive S-nitrosoCoA. The effect of the nitric oxide donor S-nitrosoglutathione (GSNO) on long chain fatty acid and cholesterol synthesis was measured in isolated cultured rat hepatocytes. [1-14C] Butyrate was used as substrate to measure 14C incorporation into lipids as butyrate is twice as effective as acetate in hepatic lipogenesis and is ketogenic via the Lynen cycle. NO very significantly (P < 0.01) impaired long chain fatty acid and cholesterol synthesis an observation dependent upon time of exposure (3 h pre-incubation or 6 h continuous exposure) and concentration of GSNO (500 microM to 2.0 mM). Decrease in hepatic lipogenesis was paralleled by decrease in ketogenesis. ATP levels remained unchanged following short-term exposure to GSNO. Exposure of hepatocytes to GSNO together with 2.0 mM glutathione significantly diminished the inhibition of lipogenesis induced by GSNO alone. Impairment of lipogenesis by GSNO appears not to be limited by energy supply and now adduced, but not proven, to be operative via the degree of inactivation of cytosolic CoA. NO control of lipogenesis could be clinically important where NO production is increased as in demyelinating diseases, chronic arthritis or colitis and in wasting diseases such as AIDS.

Subcellular localization of yeast Sec14 homologues and their involvement in regulation of phospholipid turnover.

Sec14p of the yeast Saccharomyces cerevisiae is involved in protein secretion and regulation of lipid synthesis and turnover in vivo, but acts as a phosphatidylinositol-phosphatidylcholine transfer protein in vitro. In this work, the five homologues of Sec14p, Sfh1p-Sfh5p, were subjected to biochemical and cell biological analysis to get a better view of their physiological role. We show that overexpression of SFH2 and SFH4 suppressed the sec14 growth defect in a more and SFH1 in a less efficient way, whereas overexpression of SFH3 and SFH5 did not complement sec14. Using C-terminal yEGFP fusions, Sfh2p, Sfh4p and Sfh5p are mainly localized to the cytosol and microsomes similar to Sec14p. Sfh1p was detected in the nucleus and Sfh3p in lipid particles and in microsomes. In contrast to Sec14p, which inhibits phospholipase D1 (Pld1p), overproduction of Sfh2p and Sfh4p resulted in the activation of Pld1p-mediated phosphatidylcholine turnover. Interestingly, Sec14p and the two homologues Sfh2p and Sfh4p downregulate phospholipase B1 (Plb1p)-mediated turnover of phosphatidylcholine in vivo. In summary, Sfh2p and Sfh4p are the Sec14p homologues with the most pronounced functional similarity to Sec14p, whereas the other Sfh proteins appear to be functionally less related to Sec14p.

Mutant Mammalian Cells as Tools to Delineate the Sterol Regulatory Element-Binding Protein Pathway for Feedback Regulation of Lipid Synthesis

The tools of somatic cell genetics have been instrumental in unraveling the pathway by which sterol regulatory element-binding proteins (SREBPs) control lipid metabolism in animal cells. SREBPs are membrane-bound transcription factors that enhance the synthesis and uptake of cholesterol and fatty acids. The activities of the SREBPs are controlled by the cholesterol content of cells through feedback inhibition of proteolytic processing. When cells are replete with sterols, SREBPs remain bound to membranes of the endoplasmic reticulum (ER) and are therefore inactive. When cells are depleted of sterols, the SREBPs move to the Golgi complex where two proteases release the active portions of the SREBPs, which then enter the nucleus and activate transcription of target genes. This processing requires three membrane proteins—a sterol-sensing escort protein (SCAP) that transports SREBPs from the ER to the Golgi and two Golgi-located proteases (S1P and S2P) that release SREBPs from membranes. The existence of all three proteins was revealed through analysis of mutant mammalian cells in tissue culture. Their cDNAs and genes were isolated by genetic complementation or by expression cloning. The somatic cell genetic approach described in this article should prove useful for unraveling other complex biochemical pathways in animal cells.