Expression Of Diacylglycerol Acyltransferase Research Articles

Engineering E. coli for triglyceride accumulation through native and heterologous metabolic reactions

Triglycerides, traditionally sourced from plant oils, are heavily used in both industrial and healthcare applications. Commercially significant products produced from triglycerides include biodiesel, lubricants, moisturizers, and oils for cooking and dietary supplements. The need to rely upon plant-based production, however, raises concerns of increasing demand and sustainability. The reliance on crop yields and a strong demand for triglycerides provides motivation to engineer production from a robust microbial platform. In this study, Escherichia coli was engineered to synthesize and accumulate triglycerides. Triglycerides were produced from cell wall phospholipid precursors through engineered expression of two enzymes, phosphatidic acid phosphatase (PAP) and diacylglycerol acyltransferase (DGAT). A liquid chromatography-mass spectrometry (LC-MS) method was developed to analyze the production of triglycerides by the engineered E. coli strains. This proof-of-concept study demonstrated a yield of 1.1 mg/L triglycerides (2 g/L dry cell weight) in lysogeny broth medium containing 5 g/L glucose at 8 h following induction of PAP and DGAT expression. LC-MS results also demonstrated that the intracellular triglyceride composition of E. coli was highly conserved. Triglycerides containing the fatty acid distributions 16:0/16:0/16:1, 16:0/16:0/18:1, and 18:1/16:0/16:1 were found in highest concentrations and represent ∼70 % of triglycerides observed.

Suppression of Sebum Production and Accumulation by <i>β</i>-Cryptoxanthin Due to the Inhibition of the Expression of Diacylglycerol Acyltransferase-1 and Perilipin in Hamster Sebocytes

Background: Acne vulgaris is characterized by the enhancement of sebaceous lipogenesis and sebum secretion, and apart from retinoids and some natural products there are few effective antiacne agents that directly suppress sebum production and accumulation in sebaceous glands. Objective: We examined the effects of β-cryptoxanthin (β-CRX), which is a carotenoid pigment most abundant in Citrus unshiu Marcovich (Satsuma mandarin orange) and plays a role as a vitamin A precursor on sebum production and accumulation in hamster sebaceous gland cells (sebocytes). Materials and methods: The regulation of sebum production was examined by the measurement of triacylglycerols (TGs), the major sebum component, and oil red O staining in insulindifferentiated hamster sebocytes. The expression of diacylglycerol acyltransferase-1 (DGAT-1), a rate-limiting enzyme of TG biosynthesis, and perilipin 1 (PLIN1), a lipid storage droplet protein, was analyzed using real-time PCR and Western blotting. Results: Hamster sebocytes constitutively produced TGs during cultivation and the production of TGs was enhanced by insulin treatment. Both constitutive and insulin-enhanced TG productions were dose- and time-dependently inhibited by β-CRX as well as 13-cis retinoic acid. In addition, the gene expression of DGAT-1 was suppressed by β-CRX in the sebocytes. Furthermore, the insulin-en- hanced sebum accumulation as lipid droplets was reduced in the β-CRX-treated cells. Moreover, β-CRX was found to suppress the gene expression and production of PLIN1 in insulin-differentiated hamster sebocytes. Conclusions: These results provide novel evidence that β-CRX is an effective candidate for acne therapy by its ability to exert dual inhibitory actions against DGAT-1-dependent TG production and PLIN1-mediated lipiddroplet formation in hamster sebocytes.

Aqueous Extract from Pepino (Solanum muricatumAit.) Attenuated Hyperlipidemia and Cardiac Oxidative Stress in Diabetic Mice

This study examined the lipid-lowering and cardiac protective effects of aqueous extract of pepino (Solanum muricatum Ait.) in type 2 diabetic mice. Pepino at 1, 2, or 5% was supplied for 8 weeks. Results showed that pepino significantly decreased water intake and epididymal fat pad weight in diabetic mice (P < 0.05). Pepino treatments also significantly reduced plasma glucose and insulin levels, HOMA-IR index, and improved oral glucose tolerance (P < 0.05). Plasma and hepatic levels of triglyceride and total cholesterol (TC) were higher in diabetic groups when compared with normal group (P < 0.05), pepino treatments at 2 and 5% decreased triglyceride and TC levels in both plasma and liver (P < 0.05). Diabetes enhanced mRNA expression of resistin and diacylglycerol acyltransferase1 (DGAT1) in epididymal fat pad (P < 0.05); however, pepino intake significantly suppressed mRNA expression of resistin and DGAT1 in epididymal fat pad (P < 0.05). Pepino intake significantly reduced reactive oxygen species level, increased glutathione level, and retained glutathione peroxidase and catalase activities in cardiac tissues (P < 0.05). These findings suggest that pepino could be considered as a functional food for the alleviation of type 2 diabetes.

Lipid droplet formation on opposing sides of the endoplasmic reticulum

In animal cells, the primary repositories of esterified fatty acids and alcohols (neutral lipids) are lipid droplets that form on the lumenal and/or cytoplasmic side of the endoplasmic reticulum (ER) membrane. A monolayer of amphipathic lipids, intermeshed with key proteins, serves to solubilize neutral lipids as they are synthesized and desorbed. In specialized cells, mobilization of the lipid cargo for delivery to other tissues occurs by secretion of lipoproteins into the plasma compartment. Serum lipoprotein assembly requires an obligate structural protein anchor (apolipoprotein B) and a dedicated chaperone, microsomal triglyceride transfer protein. By contrast, lipid droplets that form on the cytoplasmic face of the ER lack an obligate protein scaffold or any required chaperone/lipid transfer protein. Mobilization of neutral lipids from the cytosol requires regulated hydrolysis followed by transfer of the products to different organelles or export from cells. Several proteins play a key role in controlling droplet number, stability, and catabolism; however, it is our premise that their formation initiates spontaneously, solely as a consequence of neutral lipid synthesis. This default pathway directs droplets into the cytoplasm where they accumulate in many lipid disorders.

Diacylglycerol acyl transferase 1 overexpression detoxifies cardiac lipids in PPARγ transgenic mice

Accumulation of excess lipids is associated with heart failure. The effects of transgenic expression of diacylglycerol acyl transferase 1 (DGAT1) in cardiomyocytes is controversial. We explored whether mice expressing DGAT1 via the myosin heavy chain (MHC) promoter develop heart dysfunction with aging or after crossing with mice over expressing peroxisome proliferator-activated receptor γ (PPARγ) in the heart. MHC-DGAT1 transgenic mice had increased heart triglyceride but no evidence of heart dysfunction, even up to age 12 months. The MHC-DGAT1 transgene improved heart dysfunction and survival of MHC-PPARγ-expressing transgenic mice. Both diacylglycerol and ceramide levels in the heart were reduced by this cross, as were the levels of several mRNAs of genes involved in lipid metabolism. There were fewer large lipid droplets in MHC-DGAT1×MHC-PPARγ mice compared with MHC-PPARγ, but total lipid content was not changed. Therefore, overexpression of DGAT1 is not toxic to the heart but reduces levels of toxic lipids and improves lipotoxic cardiomyopathy. Moreover, the beneficial effects of DGAT1 illustrate the interrelationship of several lipid metabolic pathways and the difficulty of assigning benefit to an isolated change in one potentially toxic lipid species.

A Novel Soluble Beta-Glucan Salecan Protects against Acute Alcohol-Induced Hepatotoxicity in Mice

This investigation was designed to determine the effect of a novel soluble beta-glucan salecan on acute alcohol-induced hepatic injury in mice. Mice were given salecan (15 or 30 mg/kg) or PBS for 4 d. Ethanol (6 g/kg) was administered orally 1 h after the last injection. The animals were sacrificed at 10 h after alcohol administration. Pretreatment with salecan significantly ameliorated the hepatic damage induced by ethanol, as evidenced by markedly reduced serum aminotransferase activities and hepatocyte steatosis. Salecan administration remarkably alleviated the formation of thiobarbituric acid-reactive substances and counteracted glutathione depletion. The mRNA level of peroxisome proliferator activated receptor alpha, a major gene responsible for fatty acid oxidation, was significantly increased after salecan pretreatment. The expression of diacylglycerol acyltransferase 1, an important gene responsible for triacylglycerol synthesis, was markedly decreased after salecan was administrated. These findings suggest that salecan might represent a novel protective strategy against alcoholic liver injury.

ABI4 Activates DGAT1 Expression in Arabidopsis Seedlings during Nitrogen Deficiency

Triacylglycerol (TAG) is the major seed storage lipid and is important for biofuel and other renewable chemical uses. Acyl-coenzyme A:diacylglycerol acyltransferase1 (DGAT1) is the rate-limiting enzyme in the TAG biosynthesis pathway, but the mechanism of its regulation is unknown. Here, we show that TAG accumulation in Arabidopsis (Arabidopsis thaliana) seedlings increased significantly during nitrogen deprivation (0.1 mm nitrogen) with concomitant induction of genes involved in TAG biosynthesis and accumulation, such as DGAT1 and OLEOSIN1. Nitrogen-deficient seedlings were used to determine the key factors contributing to ectopic TAG accumulation in vegetative tissues. Under low-nitrogen conditions, the phytohormone abscisic acid plays a crucial role in promoting TAG accumulation in Arabidopsis seedlings. Yeast one-hybrid and electrophoretic mobility shift assays demonstrated that ABSCISIC ACID INSENSITIVE4 (ABI4), an important transcriptional factor in the abscisic acid signaling pathway, bound directly to the CE1-like elements (CACCG) present in DGAT1 promoters. Genetic studies also revealed that TAG accumulation and DGAT1 expression were reduced in the abi4 mutant. Taken together, our results indicate that abscisic acid signaling is part of the regulatory machinery governing TAG ectopic accumulation and that ABI4 is essential for the activation of DGAT1 in Arabidopsis seedlings during nitrogen deficiency.

Role of Cardiac Steatosis and Lipotoxicity in Obesity Cardiomyopathy

See related article, pp 216–222 The pandemic of obesity is a devastating health problem and contributes to premature morbidity and mortality. Results from clinical and experimental studies have identified a variety of unfavorable consequences of obesity including cardiovascular diseases, pulmonary diseases, cancer, and sleep disorders. An obesity-triggered parallel increase in the prevalence of type 2 diabetes mellitus is also expected to add to the overall cardiovascular burden of obesity. Components of metabolic syndrome such as dyslipidemia, hyperglycemia, insulin resistance, and hypertension are thought to play pivotal roles in obesity-associated sequelae responsible for atherosclerosis, cardiac hypertrophy, and ventricular dysfunction. The presence of 1 or more of these metabolic syndrome components can adversely affect multiple metabolic pathways resulting in alterations in glucose and lipid metabolism, fatty acid (FA) transport/storage/oxidation, oxygen consumption, redox status, and high-energy phosphate metabolism. Although the precise mechanism(s) of action responsible for metabolic derangement-induced cardiac abnormalities in obesity remains poorly understood, 1 theory that has received increasing attention focuses on lipid transport and storage, excessive FA oxidation (FAO), and lipotoxic injury to the heart.1,2 When energy intake exceeds expenditure, fat is stored as triacylglycerol (TG) in adipose tissue. In turn, once fat levels exceed the storage capacity of adipocytes, a variety of neutral lipids are released and accumulated in other cells and tissues including the heart. The presence of lipid inclusions within cardiomyocytes, a condition referred to as cardiac steatosis, has been confirmed in obesity and diabetes.3,4 Although recent evidence indicates that cardiac steatosis, increased availability of FA and excess FAO contribute to cardiac anomalies associated with obesity and type 2 diabetes, it has also been suggested that cardiac steatosis may be a compensatory mechanism used to neutralize FAs and their metabolites through esterification to neutral lipids. Generation of ATP for normal cardiac …

Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the ∆snf2 disruptant produces a significant increase in its enzyme activity

We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the Deltasnf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the Deltasnf2 disruptant. To analyze the expression of Dga1p in wild type and the Deltasnf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the Deltasnf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the Deltasnf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the Deltasnf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1DeltaN2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1DeltaN2p is activated Dga1p. Dga1DeltaN2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.

DGAT1-dependent triacylglycerol storage by macrophages protects mice from diet-induced insulin resistance and inflammation

Diet-induced obesity (DIO) leads to inflammatory activation of macrophages in white adipose tissue (WAT) and subsequently to insulin resistance. PPARgamma agonists are antidiabetic agents known to suppress inflammatory macrophage activation and to induce expression of the triacylglycerol (TG) synthesis enzyme acyl CoA: diacylglycerol acyltransferase 1 (DGAT1) in WAT and in adipocytes. Here, we investigated in mice the relationship between macrophage lipid storage capacity and DIO-associated inflammatory macrophage activation. Mice overexpressing DGAT1 in both macrophages and adipocytes (referred to herein as aP2-Dgat1 mice) were more prone to DIO but were protected against inflammatory macrophage activation, macrophage accumulation in WAT, systemic inflammation, and insulin resistance. To assess the contribution of macrophage DGAT1 expression to this phenotype, we transplanted wild-type mice with aP2-Dgat1 BM. These mice developed DIO similar to that of control mice but retained the protection from WAT inflammation and insulin resistance seen in aP2-Dgat1 mice. In isolated macrophages, Dgat1 mRNA levels correlated directly with TG storage capacity and inversely with inflammatory activation by saturated fatty acids (FAs). Moreover, PPARgamma agonists increased macrophage Dgat1 mRNA levels, and the protective effects of these agonists against FA-induced inflammatory macrophage activation were absent in macrophages isolated from Dgat1-null mice. Thus, increasing DGAT1 expression in murine macrophages increases their capacity for TG storage, protects against FA-induced inflammatory activation, and is sufficient to reduce the inflammatory and metabolic consequences of DIO.

Adipose triglyceride lipase in human skeletal muscle is upregulated by exercise training

Mobilization of fatty acids from stored triacylglycerol (TG) in adipose tissue and skeletal muscle [intramyocellular triacylglycerol (IMTG)] requires activity of lipases. Although exercise training increases the lipolytic capacity of skeletal muscle, the expression of hormone-sensitive lipase (HSL) is not changed. Recently, adipose triglyceride lipase (ATGL) was identified as a TG-specific lipase in various rodent tissues. To investigate whether human skeletal muscle ATGL protein is regulated by endurance exercise training, 10 healthy young men completed 8 wk of supervised endurance exercise training. Western blotting analysis on lysates of skeletal muscle biopsy samples revealed that exercise training induced a twofold increase in skeletal muscle ATGL protein content. In contrast to ATGL, expression of comparative gene identification 58 (CGI-58), the activating protein of ATGL, and HSL protein was not significantly changed after the training period. The IMTG concentration was significantly decreased by 28% at termination of the training program compared with before. HSL-phoshorylation at Ser(660) was increased, HSL-Ser(659) phosporylation was unchanged, and HSL-phoshorylation at Ser(565) was decreased altogether, indicating an enhanced basal activity of this lipase. No change was found in the expression of diacylglycerol acyl transferase 1 (DGAT1) after training. Inhibition of HSL with a monospecific, small molecule inhibitor (76-0079) and stimulation of ATGL with CGI-58 revealed that significant ATGL activity is present in human skeletal muscle. These results suggest that ATGL in addition to HSL may be important for human skeletal muscle lipolysis.

Abstract 5383: The Circadian Clock within the Cardiomyocyte is a Novel Mechanism Regulating Myocardial Triglyceride Metabolism

Virtually every mammalian cell, including the cardiomyocyte, has an intrinsic molecular circadian clock. The cardiomyocyte circadian clock has recently been shown to directly mediate diurnal variations in myocardial gene expression, metabolism, and contractile function. This includes modulation of the heart’s acute transcriptional response to increased circulating fatty acids, in a time-of-day-dependent manner. We therefore hypothesized that disruption of the cardiomyocyte circadian clock would impair metabolic adaptation of the heart to high fat feeding. Wild type (WT) and cardiomyocyte-specific circadian clock mutant (CCM) mice were fed either a control or high fat diet (10% and 45% calories from fat, respectively) for 16 weeks, after which myocardial adaptation was assessed at transcriptional, metabolic, and functional levels. Ex vivo mouse heart perfusions in the working mode revealed that high fat feeding induced alterations in myocardial metabolism ( e.g. reduced carbohydrate oxidation; p&lt;0.01) and contractile function ( e.g. depressed cardiac power; p&lt;0.01) in WT, but not CCM, hearts. Furthermore, the ability to decrease reliance on endogenous substrate utilization during an acute increase in fatty acid availability ex vivo was abolished in hearts from CCM mice fed the high fat diet (p&lt;0.05), suggesting potential alterations in triglyceride metabolism. Consistent with the latter, microarray analysis revealed that the cardiomyocyte circadian clock regulates expression of several genes involved in triglyceride turnover. For example, diurnal variations in expression of diacylglycerol acyltransferase 2, which promotes lipogenesis, and rates of myocardial triglyceride synthesis both peak in the middle of the active phase (2- and 3- fold, trough-to-peak, respectively; p&lt;0.05), which are abolished in CCM hearts. These data provide direct evidence that the cardiomyocyte circadian clock regulates myocardial triglyceride metabolism at multiple levels. We postulate that disruption of this intramyocellular mechanism may contribute to altered myocardial metabolism and contractile dysfunction observed during cardiac hypertrophy, diabetes mellitus, and/or shift work.

Effect of CLA and Other C18 Unsaturated Fatty Acids on DGAT in Bovine Milk Fat Biosynthetic Systems

Production of dairy products with increased amounts of nutraceutic FA such as conjugated linoleic acid (CLA) represents a recent approach for dairy producers and processors to increase the value of their products. The effect of CLA and other FA on the expression of diacylglycerol acyltransferase-1 (DGAT-1) and DGAT-2, and DGAT activity were investigated in bovine mammary gland epithelial (MAC-T) cells. DGAT gene expression analyses were also conducted using bovine mammary gland tissue from dairy cows. In the studies with MAC-T cells, there were no significant effects of CLA isomers or other FA on DGAT1 expression, whereas all FA tested showed enhanced DGAT2 expression (P < 0.05 to P < 0.001), with alpha-linolenic acid (alpha-18:3) having the greatest effect. Additionally, DGAT2 expression was co-ordinated with expression of lysophosphatidic acid acyltransferase (LPAAT), an observation that was also apparent in mammary gland from lactating dairy cows. In contrast, treatment of MAC-T cells with trans-10, cis-12 18:2 or alpha-18:3 resulted in a significant (P < 0.05) decrease in overall DGAT enzyme activity, although the mechanisms resulting in these effects are unclear. Competition assays using microsomes from bovine mammary gland tissue and 1-[(14)C]oleoyl-CoA suggested that DGAT activity was more selective for oleoyl (cis-9 18:1)-CoA than cis-9, trans-11 18:2-, trans-10, cis-12 18:2- or cis-9, cis-12 18:2-CoA. Collectively, the results suggest the relationship between trans-10, cis-12 18:2 and reduced TAG production in bovine milk is not linked to the production of DGAT1 or DGAT2 transcripts, but probably involves effects of this CLA isomer at events beyond transcription, such as post-translational and/or enzyme activity effects.

Nonalcoholic fatty liver disease: emerging mechanisms and consequences

One of the critical complications of obesity and diabetes is nonalcoholic fatty liver disease, a disorder of triacylglycerol accumulation in the liver that has potential to develop into end stage liver failure. In this review, the recent progress in understanding the role of hepatic triacylglycerol synthesis in the development of nonalcoholic fatty liver disease is discussed. It has become apparent that the development of hepatic steatosis is a complex, multifactorial process. Although the molecular pathways underlying its development have been described, there are no established therapies for nonalcoholic fatty liver disease. Recently, however, DGAT1 and DGAT2, the enzymes responsible for the final step in triacylglycerol synthesis, have been characterized as playing a vital role in hepatic triacylglycerol metabolism. Cellular and murine models in which diacylglycerol acyltransferase expression is altered suggest that these enzymes may play a role in the development hepatic steatosis, are feasible targets in the treatment of nonalcoholic fatty liver disease, but also function as lipotoxic buffers. Hepatic steatosis remains the watershed event in the progression of nonalcoholic fatty liver disease. The diacylglycerol acyltransferases are emerging as important mediators of hepatic triacylglycerol accumulation. Therefore, these enzymes are attractive targets in the development of therapies to prevent liver triacylglycerol accumulation and the consequences of nonalcoholic fatty liver disease.

Hepatic free fatty acids accumulate in experimental steatohepatitis: Role of adaptive pathways

We determined the effects of dietary lipid composition on steatohepatitis development with particular attention to the nature of lipid molecules that accumulate in the liver and pathways of hepatic triglyceride synthesis. Mice were fed methionine and choline deficient (MCD) diets supplemented with 20% fat as lard (saturated) or olive oil (monounsaturated), for 3 weeks. Irrespective of dietary lipid composition, MCD-fed mice developed steatosis, ballooning degeneration and lobular inflammation. MCD-feeding increased hepatic free fatty acid (FFA) levels 2-3-fold, as well as total triglyceride levels. Hepatic FFA composition was characterized by increased ratio of monounsaturated: saturated FFA. There were reduced nuclear levels of the lipogenic transcription factor sterol regulatory element binding protein-1 in MCD-fed mice, but no consistent reduction in fatty acid synthesis genes (acetyl-CoA carboxylase and fatty acid synthase). Consistent with pathways of hepatic triglyceride synthesis, expression of diacylglycerol acyltransferase-1 and -2 was increased, as were delta-5- and delta-6- fatty acid desaturase mRNA levels. In this nutritional model of steatohepatitis, accumulation of FFA occurs despite substantial suppression of lipogenesis and induction of triglyceride synthesis genes. Accumulation of FFA supports a lipotoxicity mechanism for liver injury in this form of fatty liver disease.

Acylation-stimulating protein deficiency and altered adipose tissue in alternative complement pathway knockout mice

Acylation-stimulating protein (C3adesArg/ASP) is an adipokine that acts on its receptor C5L2 to stimulate triglyceride (TG) synthesis in adipose tissue. The present study investigated ASP levels in mouse models of obesity and leanness and the effect of ASP deficiency in C3 knockout (C3KO) mice on adipose tissue morphology. Plasma ASP levels in wild-type (WT) mice correlated positively with plasma nonesterified fatty acids (NEFA) (R = 0.664, P < 0.001) and total cholesterol (R = 0.515, P < 0.001). Plasma ASP was increased by 85% in obese ob/ob leptin-deficient mice and decreased in lean diacylglycerol acyltransferase 1 (DGAT1) KO mice (-54%) and C/EBPalpha(beta/beta) transgenic mice (-70%) compared with WT. Mice lacking alternative complement factor B or adipsin (FBKO or ADKO), required for ASP production, were also ASP deficient. Both FBKO and C3KO mice had delayed postprandial TG and NEFA clearance on low-fat (LF) and high-fat (HF) diets, suggesting that lack of ASP, not C3, drives the metabolic phenotype. Adipocyte size distribution in C3KO mice was polarized (increased number of both small and large cells), with decreased adipsin expression (-33% gonadal HF), DGAT1 expression (-31% to -50%) and DGAT activity (-41%). Overall, a reduction/deficiency in ASP is associated with an antiadipogenic state and ASP may provide a target for controlling fat storage.

Identification of Mouse Prp19p as a Lipid Droplet-associated Protein and Its Possible Involvement in the Biogenesis of Lipid Droplets

Prp19p is an integral component of the heteromeric protein complex (the NineTeen complex) in the nucleus, and it is essential for the structural integrity of NineTeen complex and its subsequent activation of the spliceosome. We identified Prp19p, which has never been reported in relation to any function outside of the nucleus, as a member of proteins associated with lipid droplets. Down-regulation of Prp19p expression with RNA interference in 3T3-L1 cells repressed lipid droplet formation with the reduction in the level of expression of perilipin and S3-12. The levels of expression of SCD1 (stearoyl-CoA desaturase-1), DGAT-1 (acyl-CoA diacylglycerol acyltransferase-1), and glycerol-3-phosphate acyltransferase were also reduced in Prp19p down-regulated cells, and a significant decrease in triglycerides was observed. Unlike perilipin, which is one of the most extensively studied lipid droplet-associated proteins, Prp19p is not essential for cAMP- and hormone-sensitive lipase-dependent lipolysis pathways, even though Prp19p is a component of the lipid droplet phospholipid monolayer, and down-regulation of Prp19p represses fat accretion significantly. These results suggest that Prp19p or Prp19-interacting proteins during lipid droplet biogenesis in adipocytes may be considered as another class of potential targets for attacking obesity and obesity-related problems.

Up-regulation of hepatic Acyl CoA: Diacylglycerol acyltransferase-1 (DGAT-1) expression in nephrotic syndrome

Nephrotic syndrome is associated with hypercholesterolemia, hypertriglyceridemia, and marked elevations of plasma low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL). Hypertriglyceridemia in nephrotic syndrome is accompanied by increased hepatic fatty acid synthesis, elevated triglyceride secretion, as well as lipoprotein lipase, VLDL-receptor, and hepatic triglyceride lipase deficiencies, which lead to impaired clearance of triglyceride-rich lipoproteins. Acyl CoA: diacylglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl CoA to 1, 2-diacylglycerol to form triglyceride. Two distinct DGATs (DGAT-1 and DGAT2) have recently been identified in the liver and other tissues. The present study tested the hypothesis that the reported increase in hepatic triglyceride secretion in nephrotic syndrome may be caused by up-regulation of DGAT. Male Sprague-Dawley rats were rendered nephrotic by two sequential injections of puromycin aminonucleoside (130 mg/kg on day 1 and 60 mg/kg on day 14) and studied on day 30. Placebo-treated rats served as controls. Hepatic DGAT-1 and DGAT-2 mRNA abundance and enzymatic activity were measured. The nephrotic group exhibited heavy proteinuria, hypoalbuminemia, hypercholesterolemia, hypertriglyceridemia, and marked elevation of VLDL concentration. Hepatic DGAT-1 mRNA, DGAT-1, and total DGAT activity were significantly increased, whereas DGAT-2 mRNA abundance and activity were unchanged in the nephrotic rats compared to the control animals. The functional significance of elevation of DGAT activity was illustrated by the reduction in microsomal free fatty acid concentration in the liver of nephrotic animals. Nephrotic syndrome results in up-regulation of hepatic DGAT-1 expression and activity, which can potentially contribute to the associated hypertriglyceridemia by enhancing triglyceride synthesis. Thus, it appears that both depressed catabolism and increased synthetic capacity contribute to hypertriglyceridemia of nephrotic syndrome.

Posttranscriptional Control of the Expression and Function of Diacylglycerol Acyltransferase-1 in Mouse Adipocytes

Acyl-CoA:diacylglycerol acyltransferase-1 (DGAT1) catalyzes the final step of triglyceride synthesis in mammalian cells. Data obtained from DGAT1-knockout mice have indicated that this enzyme plays an important role in energy homeostasis. We investigated the regulation of the expression and function of DGAT1 in mouse 3T3-L1 cell as a model for mammalian adipocytes. We demonstrated that the DGAT1 protein level increased by approximately 90-fold following differentiation of 3T3-L1 into mature adipocytes, a change that was accompanied by approximately 7-fold increase in DGAT1 mRNA. On the other hand, forced overexpression of DGAT1 mRNA by >20-fold via a recombinant adenovirus only resulted in approximately 2-fold increase in DGAT1 protein in mature adipocytes and little increase in preadipocytes. These results indicated that gene expression of DGAT1 in adipocytes is subjected to rigorous posttranscriptional regulation, which is modulated significantly by the differentiation status of 3T3-L1 cells. Protein stability is not a significant factor in the control of DGAT1 expression. The steady-state levels of DGAT1 were unaffected by blockage of proteolytic pathways by ALLN. However, translational control was suggested by sequence analysis of the 5'-untranslated region of human DGAT1 (hDGAT1) mRNA. We found that the level of DGAT1 activity was predominantly a function of the steady-state level of DGAT1 protein. No significant functional changes were observed when the conserved tyrosine phosphorylation site in hDGAT1 was mutated by a single base pair substitution. Despite only a approximately 2-fold increase in DGAT1 protein caused by recombinant viral transduction, a proportionate increase in cellular triglyceride synthesis resulted without affecting the triglyceride lipolysis rate, leading to >2-fold increase in intracellular triglyceride accumulation. No change in adipocyte morphology or in the expression levels of lipoprotein lipase, proxisomal proliferation-activating receptor-gamma, and aP2 was evident secondary to DGAT1 overexpression at different stages in 3T3-L1 differentiation. These data suggest that dysregulation of DGAT1 may play a role in the development of obesity, and manipulation of the steady-state level of DGAT1 protein may offer a potential means to treat or prevent obesity.

Dissociation of obesity and impaired glucose disposal in mice overexpressing acyl coenzyme a:diacylglycerol acyltransferase 1 in white adipose tissue.

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two DGAT enzymes known to catalyze the final step in mammalian triglyceride synthesis. Mice deficient in DGAT1 are resistant to obesity and have enhanced insulin sensitivity. To understand better the relationship between triglyceride synthesis and energy and glucose metabolism, we generated transgenic (aP2-Dgat1) mice in which expression of murine DGAT1 in the white adipose tissue (WAT) was twofold higher than normal. aP2-Dgat1 mice that were fed a regular diet had larger adipocytes and greater total fat pad weight than wild-type (WT) mice. In response to a high-fat diet, aP2-Dgat1 mice became more obese ( approximately 20% greater body weight after 15 weeks) than WT mice. However, the increase in adiposity in aP2-Dgat1 mice was not associated with impaired glucose disposal, as demonstrated by glucose and insulin tolerance tests. Correlating with this finding, triglyceride deposition in the liver and skeletal muscle, two major target tissues of insulin, was similar in aP2-Dgat1 and WT mice. Thus, DGAT1 overexpression in murine WAT provides a model in which obesity does not impair glucose disposal. Our findings support the lipotoxicity hypothesis that the deposition of triglycerides in insulin-sensitive tissues other than adipocytes causes insulin resistance.