Glycerol-3-phosphate Acyltransferase-1 Research Articles

Causes of Sex Differences in Serum Cholesterol and Triglyceride Levels in Meishan Pigs.

To clarify the causes of sex differences (male < female) in the serum total cholesterol (TCHO) and triglyceride (TG) levels in Meishan pigs, we examined the sex differences in mRNA levels of key hepatic enzymes involved in the biosynthesis/metabolism of cholesterol and TG using real-time RT-PCR. There were no sex differences in mRNA levels of 3-hydroxy-3-methylglutaryl-CoA reductase and CYP51A1 for cholesterol biosynthesis, or of the rate-limiting enzyme CYP7A1 for bile acid synthesis from cholesterol. By contrast, sex differences (male < female) were observed in mRNA levels of glycerol-3-phosphate acyltransferase 1 (GPAT1), a rate-limiting enzyme for TG biosynthesis. However, the sex differences in mRNA levels of carnitine palmitoyltransferase 1A (CPT1A) and acyl-CoA dehydrogenase long chain (ACADL), key enzymes for the oxidation of the fatty acids that are structural components of TG, were the opposite (male > female). Castration of male pigs led to an increase in the mRNA level of GPAT1 and decreases in those of CPT1A and ACADL. Furthermore, testosterone propionate (TP)-treatment of castrated males and intact females restored and changed, respectively, these mRNA levels to those of intact males. Notably, castration and TP-treatment increased and decreased, respectively, serum and hepatic TG levels. These findings suggest that sex differences in the serum and hepatic TG levels in Meishan pigs are closely correlated with differences in testosterone-associated mRNA expression levels of the key enzymes (GPAT1, CPT1A, and ACADL) involved in the TG biosynthesis process, although no causes of sex differences in serum and hepatic TCHO levels could be found.

GPAT1 Deficiency in Mice Modulates NASH Progression in a Model-Dependent Manner

Nonalcoholic fatty liver disease (NAFLD), and its more severe form, nonalcoholic steatohepatitis (NASH), is the leading cause for liver failure and liver cancer. Although the etiology is likely multifactorial, genes involved in regulating lipid metabolism are enriched in human NAFLD genome-wide association studies (GWAS), pointing to dysregulated lipid metabolism as a major pathogenic factor. Glycerol-3-phosphate acyltransferase 1 (GPAT1), encoded by GPAM, converts acyl-CoAs and glycerol-3-phosphate into lysophosphatidic acid and has been shown to regulate lipid accumulation in the liver. However, its role in mediating the progression from NAFLD to NASH has not been explored. GPAT1-deficient mice were generated and challenged with diets inducing hepatic steatosis and NASH. Effects of GPAT1 deficiency on lipid and systemic metabolic end points were evaluated. Ablating GPAT1 globally or specifically in mouse hepatocytes reduced hepatic steatosis in the context of diet-induced or genetic obesity. Interestingly, blunting of progression from NAFLD to NASH in global GPAT1 knockout (KO) mice was model dependent. GPAT1 KO mice were protected from choline deficient, amino acid defined high-fat diet-induced NASH development, but not from the high fat, high carbohydrate, and high cholesterol diet-induced NASH. Our preclinical data support the notion that lipid metabolism pathways regulated by GPAT1 in hepatocytes play an essential role in NASH progression, albeit in a model-dependent manner.

6-Gingerol Ameliorates Hepatic Steatosis via HNF4α/miR-467b-3p/GPAT1 Cascade.

Background & AimsThe development of nonalcoholic fatty liver disease (NAFLD) can be modulated by microRNAs (miRNA). Dietary polyphenols modulate the expression of miRNA such as miR-467b-3p in the liver. In addition, 6-gingerol (6-G), the functional polyphenol of ginger, has been reported to ameliorate hepatic steatosis; however, the exact mechanism involved and the role of miRNA remain elusive. In this study, we assessed the role of miR-467b-3p in the pathogenesis of hepatic steatosis and the regulation of miR-467b-3p by 6-G through the hepatocyte nuclear factor 4α (HNF4α).MethodsmiR-467b-3p expression was measured in free fatty acid (FFA)-treated hepatocytes or liver from high-fat diet (HFD)-fed mice. Gain- or loss-of-function of miR-467b-3p was induced using miR-467b-3p–specific miRNA mimic or miRNA inhibitor, respectively. 6-G was exposed to FFA-treated cells and HFD-fed mice. The HNF4α/miR-467b-3p/GPAT1 axis was measured in mouse and human fatty liver tissues.ResultsWe found that miR-467b-3p was down-regulated in liver tissues from HFD-fed mice and in FFA-treated Hepa1-6 cells. Overexpression of miR-467b-3p decreased intracellular lipid accumulation in FFA-treated hepatocytes and mitigated hepatic steatosis in HFD-fed mice via negative regulation of glycerol-3-phosphate acyltransferase-1 (GPAT1). In addition, miR-467b-3p up-regulation by 6-G was observed. 6-G inhibited FFA-induced lipid accumulation and mitigated hepatic steatosis. Moreover, it increased the transcriptional activity of HNF4α, resulting in the increase of miR-467b-3p and subsequent decrease of GPAT1. HNF4α/miR-467b-3p/GPAT1 signaling also was observed in human samples with hepatic steatosis.ConclusionsOur findings establish a novel mechanism by which 6-G improves NAFLD. This suggests that targeting of the HNF4α/miR-467b-3p/GPAT1 cascade may be used as a potential therapeutic strategy to control NAFLD.

Growth performance, meat quality and lipid metabolism in finishing lambs fed diets containing rumen-unprotected and rumen-protected betaine

This study investigated the influence of dietary supplementation of rumen-unprotected betaine (RUPB) and rumen-protected betaine (RPB) on growth performance, meat quality and fatty acid profile in intramuscular and subcutaneous fat of growing lambs. A total of 24 Hu male lambs were equally divided into three groups and fed a basal diet or basal diet supplemented with 1.6 g/kg of RUPB or 2.9 g/kg of RPB for 68 days. The water loss, redness of longissimus dorsi (LD) muscle and abdominal fat percentage were reduced in RUPB and RPB groups (p < .05). RPB supplementation decreased the shear force of LD muscle (p < .05). Polyunsaturated fatty acid (PUFA) content in LD muscle was decreased in RPB group, whereas n-3PUFA content was increased in RUPB group (p < .05). RUPB and RPB supplementation reduced the content of saturated fatty acid (SFA), and increased the contents of PUFA, monounsaturated fatty acid (MUFA), and n-6PUFA in subcutaneous fat (p < .05). Furthermore, dietary RUPB and RPB supplementation increased the mRNA expression of acetyl-CoA carboxylase (ACC) and glycerol-3-phosphate acyltransferase 1 (GPAT1) in LD muscle (p<.05); it increased the mRNA expression of fatty acid synthetase (FANS) and lipoprotein lipase (LPL) but reduced the mRNA expression of GPAT1 in subcutaneous fat (p < .05). In conclusion, either RUPB or RPB supplementation may improve the meat quality and slightly change the fat deposition and fatty acid composition of intramuscular and subcutaneous fat, and RUPB is recommended as a supplement for growing male lambs based on its lower cost. Highlights Dietary supplementation of both rumen-unprotected betaine and (RUPB) and rumen-protected betaine (RPB) improved meat quality and altered the fat deposition of growing male lambs. Both RUPB and RPB supplementation changed fatty acid composition in the longissimus dorsi muscle and subcutaneous adipose tissue. RUPB is recommended as a supplement for growing lambs based on its lower cost.

Glycerol-3-phosphate Acyltransferase1 Is a Model-Agnostic Node in Nonalcoholic Fatty Liver Disease: Implications for Drug Development and Precision Medicine

Left untreated nonalcoholic fatty liver disease (NAFLD) can progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The observed failure of clinical trials in NASH may suggest that current model systems do not fully recapitulate human disease, and/or hallmark pathological features of NASH may not be driven by the same pathway in every animal model let alone in each patient. Identification of a model-agnostic disease-associated node can spur the development of effective drugs for the treatment of liver disease. Glycerol-3-phosphate acyltransferase1 (GPAT1) plays a pivotal role in lipid accumulation by shunting fats away from oxidation. In the present study, hepatic GPAT1 expression was evaluated in three etiologically different models of NAFLD. Compared to the sham cohort, hepatic GPAT1 mRNA levels were elevated by ∼5-fold in steatosis and NASH with fibrosis with immunofluorescent staining revealing increased GPAT1 in the fatty liver. A significant and direct correlation (r = 0.88) was observed between hepatic GPAT1 mRNA expression and severity of the liver disease. Picrosirius red staining revealed a logarithmic relation between hepatic GPAT1 mRNA expression and scar. These data suggest that hepatic GPAT1 is an early disease-associated model-agnostic node in NAFLD and form the basis for the development of a potentially successful therapeutic against NASH.

Thioesterase superfamily member 2 promotes hepatic insulin resistance in the setting of glycerol-3-phosphate acyltransferase 1–induced steatosis

Hepatic insulin resistance in the setting of steatosis is attributable at least in part to the accumulation of bioactive lipids that suppress insulin signaling. The mitochondria-associated glycerol-3-phosphate acyltransferase 1 (GPAT1) catalyzes the first committed step in glycerolipid synthesis, and its activity diverts fatty acids from mitochondrial β-oxidation. GPAT1 overexpression in mouse liver leads to hepatic steatosis even in the absence of overnutrition. The mice develop insulin resistance owing to the generation of saturated diacylglycerol and phosphatidic acid molecular species that reduce insulin signaling by activating PKCϵ and by suppressing mTORC2, respectively. Them2, a mitochondria-associated acyl-CoA thioesterase, also participates in the trafficking of fatty acids into oxidative versus glycerolipid biosynthetic pathways. Them2-/- mice are protected against diet-induced hepatic steatosis and insulin resistance. To determine whether Them2 contributes to hepatic insulin resistance due to hepatic overexpression of GPAT1, recombinant adenovirus was used to overexpress GPAT1 in livers of chow-fed Them2+/+ and Them2-/- mice. Hepatic GPAT1 overexpression led to steatosis in both genotypes. In the setting of GPAT1 overexpression, glucose tolerance was reduced in Them2+/+ but not Them2-/- mice, without influencing whole-body insulin sensitivity or basal hepatic glucose production. Improved glucose tolerance in Them2-/- mice was associated with reduced PKCϵ translocation. Preserved insulin receptor activity was supported by Thr-308 phosphorylation of Akt following GPAT1 overexpression in Them2-/- hepatocytes. These findings suggest a pathogenic role of Them2 in the biosynthesis of glycerolipid metabolites that promote hepatic insulin resistance.

Epigenetic Switching and Neonatal Nutritional Environment.

The hepatic metabolic function changes sequentially during early life in mammals to adapt to the drastic changes in the nutritional environment. Accordingly, hepatic fatty acid β-oxidation is activated after birth to produce energy from breast milk lipids. De novo lipogenesis is induced upon the onset of oral intake, when the major nutritional source switches to carbohydrate. However, how a particular metabolic pathway is activated during the liver maturation is poorly understood. We found that the expression of glycerol-3-phosphate acyltransferase 1 (GPAT1), a rate-limiting enzyme of de novo hepatic lipogenesis, is epigenetically regulated in the mouse liver by DNA methylation. In the neonatal liver, DNA methylation of the GPAT1 gene (Gpam) promoter, which is likely to be induced by DNA methyltransferase (Dnmt) 3b, inhibited the recruitment of sterol regulatory element-binding protein-1c (SREBP-1c), whereas in the adult, decreased DNA methylation resulted in active chromatin conformation, allowing the recruitment of SREBP-1c. Maternal nutritional environment affects the DNA methylation status in the Gpam promoter, GPAT1 expression, and triglyceride content in the liver of the offspring. We also found DNA demethylation and increased mRNA expression of the fatty acid β-oxidation genes in the postnatal mouse liver. The DNA demethylation is specifically induced in the lactation period. Analysis of mice deficient in the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and maternal administration of a PPARα ligand during the gestation and lactation periods reveals that the DNA demethylation is PPARα-dependent. These findings indicate the gene- and lifestage-specific DNA demethylation of a particular metabolic pathway in the neonatal liver to adapt the marked changes in nutritional environment in early life.

Integrated Regulation of Hepatic Lipid and Glucose Metabolism by Adipose Triacylglycerol Lipase and FoxO Proteins.

Metabolism is a highly integrated process that is coordinately regulated between tissues and within individual cells. FoxO proteins are major targets of insulin action and contribute to the regulation of gluconeogenesis, glycolysis, and lipogenesis in the liver. However, the mechanisms by which FoxO proteins exert these diverse effects in an integrated fashion remain poorly understood. We report that FoxO proteins also exert important effects on intrahepatic lipolysis and fatty acid oxidation via the regulation of adipose triacylglycerol lipase (ATGL), which mediates the first step in lipolysis, and its inhibitor, the G0/S1 switch 2 gene (G0S2). We also find that ATGL-dependent lipolysis plays a critical role in mediating diverse effects of FoxO proteins in the liver, including effects on gluconeogenic, glycolytic, and lipogenic gene expression and metabolism. These results indicate that intrahepatic lipolysis plays a critical role in mediating and integrating the regulation of glucose and lipid metabolism downstream of FoxO proteins.

Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.

Thioesterase Superfamily Member 2 (Them2) Channels Fatty Acids to Glycerol‐3‐Phosphate Acyltransferase‐1 (GPAT1): Role in Hepatic Insulin Resistance

Them2 is a mitochondria‐associated acyl‐CoA thioesterase, which promotes fatty acid oxidation within hepatocytes apparently by generating free fatty acids that are re‐esterified to CoA by long chain acyl‐CoA synthetase 1 (ACSL1) for uptake by carnitine palmitoyl transferase 1. ACSL1‐generated fatty acyl‐CoAs may also become substrates for GPAT1, which initiates the synthesis of glycerolipids. Diacylglycerols and phosphatidic acid suppress insulin signaling by activating PKCε or inactivating mTORC2, respectively, and GPAT1 overexpression in liver promotes hepatic insulin resistance. Because livers of Them2‐/‐ mice exhibit increased sensitivity to insulin, we examined whether Them2 promotes GPAT1‐mediated suppression of insulin signaling. Using recombinant adenovirus, GPAT1 or GFP was overexpressed in mouse livers. GPAT1 but not GFP overexpression promoted hepatic synthesis of glycerolipids in both Them2+/+ and Them2‐/‐ mice. As evidenced by increased ratios of membrane/cytosolic PKCε, GPAT1 overexpression activated PKCε in livers of Them2+/+ mice, an effect that was attenuated in the absence of Them2 expression. Consistent with the contribution of Them2 to GPAT1‐induced hepatic insulin resistance, when primary mouse hepatocytes were infected with GPAT1 adenovirus, insulin‐mediated Akt phosphorylation was increased at S473 and T308 in the absence of Them2 expression. These findings implicate Them2 in channeling fatty acids to GPAT1 and in the pathogenesis of hepatic insulin resistance.Support: NIH DK48873, DK56626 and DK56598; Sumitomo Life Welfare and Culture Foundation

Up-Regulation Mttp and Apob Gene Expression in Rat Liver is Related to Post-Lipectomy Hypertriglyceridemia

Background/Aims: The aim of this study was to explain the molecular basis for elevated concentrations of circulating triglycerides (TAGs) after partial surgical removal of adipose tissue (lipectomy) in rats. Methods: The levels of mRNA and protein: a) involved in synthesis of fatty acids and TAGs; b) participating in TAG-rich lipoproteins assembly and secretion; and c) transcription factors essential for maintaining TAG homeostasis were determined by RT-PCR and Western Blot in the livers of control and lipectomized rats. Results: Partial lipectomy was associated with increase: a) in serum and liver concentration of TAGs, and b) in the liver levels of mRNA of microsomal TAG transfer protein (MTP) and apolipoprotein B-100 (ApoB-100). These changes were tightly associated with up-regulation of Hnf1a and Hnf4a gene expression in the liver. Lipectomy was also reflected by a significant increase in the expression of genes encoding: a) fatty acid synthase (FASN), b) glycerol 3-phosphate acyltransferase 1 (GPAT1), diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2), c) spot 14 protein (S14) and SREBP-1 in the liver. Conclusion: Coordinated up-regulation of Mttp, Apob, Hnf1a, Hnf4a, Fasn, Gpam and Dgat (1 and 2) gene expressions may contribute to the increase in circulating and liver concentrations of TAGs after lipectomy in an experimental rat model.

Commentary on “Age-related alterations in T-lymphocytes modulate key pathways in prostate tumorigenesis.” De Angulo A, Faris R, Cavazos D, Jolly C, Daniel B, DeGraffenried L, Department of Nutritional Sciences, Dell Pediatric Research Institute, University of Texas, Austin, TX, USA.: Prostate 2013;73(8):855–64 [Epub 2013 Mar 26

BackgroundThe primary risk for prostate cancer is aging, often associated with inflammation. Evidence implicates progressive age-related immune dysfunction with increased prostate cancer incidence and mortality. The aged T-cell response is characterized by increased production of pro-inflammatory cytokines, which could significantly contribute to prostate tumorigenesis through induction of key inflammation-mediated pro-survival factors. MethodsT-cell function of the young (<6 month-old) glycerol-3-phosphate acyltransferase-1 (GPAT-1) knock-out mouse mimics many of the hallmarks observed in an aged (>22-month-old) mouse. Serum and splenic T-lymphocytes from young GPAT-1(−/−) (6 months) and aged wild type (22 months) mice were collected for in vitro studies, including a cytokine immunoarray for serum cytokine levels, luciferase assays for NF-κB activation and Western blot analyses for protein expression. ResultsThe T-cell cytokine profile of the GPAT-1(−/−) mice mirrored that observed in aged wild type mice, including higher expression levels of IL-17. Serum- and T-cell-derived factors induced NF-κB activity in normal, non-transformed and prostate cancer epithelial cells, correlating with re-localization of NF-κB and increased protein expression of downstream targets of NF-κB. ConclusionThe aging and aging-mimic mice produced circulating factors that induce pro-inflammatory pathways in prostate cells, most notably NF-κB. These findings provide evidence that an aged T-cell may directly contribute to the increased risk for prostate cancer in the elderly and establish that the GPAT-1(−/−) model, which mimics many of the characteristics of an aged immune system, is a viable tool for investigating this novel area of cancer risk.

Age‐related alterations in T‐lymphocytes modulate key pathways in prostate tumorigenesis

The primary risk for prostate cancer is aging, often associated with inflammation. Evidence implicates progressive age-related immune dysfunction with increased prostate cancer incidence and mortality. The aged T-cell response is characterized by increased production of pro-inflammatory cytokines, which could significantly contribute to prostate tumorigenesis through induction of key inflammation-mediated pro-survival factors. T-cell function of the young (<6 month-old) glycerol-3-phosphate acyltransferase-1 (GPAT-1) knock-out mouse mimics many of the hallmarks observed in an aged (>22-month-old) mouse. Serum and splenic T-lymphocytes from young GPAT-1(-/-) (6 months) and aged wild type (22 months) mice were collected for in vitro studies, including a cytokine immunoarray for serum cytokine levels, luciferase assays for NF-κB activation and Western blot analyses for protein expression. The T-cell cytokine profile of the GPAT-1(-/-) mice mirrored that observed in aged wild type mice, including higher expression levels of IL-17. Serum- and T-cell-derived factors induced NF-κB activity in normal, non-transformed and prostate cancer epithelial cells, correlating with re-localization of NF-κB and increased protein expression of downstream targets of NF-κB. The aging and aging-mimic mice produced circulating factors that induce pro-inflammatory pathways in prostate cells, most notably NF-κB. These findings provide evidence that an aged T-cell may directly contribute to the increased risk for prostate cancer in the elderly and establish that the GPAT-1(-/-) model, which mimics many of the characteristics of an aged immune system, is a viable tool for investigating this novel area of cancer risk.

Glycerol‐3‐Phosphate Acyltransferase‐1 Gene Ablation Results in Altered Thymocyte Lipid Content and Reduces Thymic T Cell Production in Mice

Glycerol 3-phosphate acyltransferase-1 (GPAT-1) catalyzes the initial and rate-limiting step in de novo glycerophospholipid and triacylglycerol (TAG) biosynthesis. We have previously shown that peripheral T cell proliferation and cytokine production is altered in GPAT-1 gene-ablated (KO) mice. This finding is important in light of the reduction in GPAT-1 activity associated with aged T cells. To determine if the mechanism for altered peripheral T cell function is linked to altered T cell development, we assessed thymic function in 3, 6 and 16-week old GPAT-1 KO compared to wild type (WT) mice. At 16 weeks of age, there was a significant reduction in thymic T cell production in KO compared to WT mice but not at 6 weeks of age. The reduced thymic T cell production was associated with altered thymic development as confirmed by increased numbers of double-negative (DN) thymocytes and a significant reduction in the double positive (DP) thymocytes suggesting a developmental block at the DN stage. This change was accompanied by an increase in the single positive CD4 subset. These changes were associated with reduced glycerophospholipid mass while thymic cortex and medulla architecture was not altered by GPAT-1 KO. Taken together, these data suggest that GPAT-1 deletion is capable of reducing the number of new T cells produced via alterations in membrane receptor function rather than by causing deleterious changes within the thymic microenvironment explaining in part the observed alterations in peripheral T cell function.

Identification and functional characterization of human glycerol-3-phosphate acyltransferase 1 gene promoters

Glycerol-3-phosphate acyltransferase 1 (GPAT1) acts as a rate limiting enzyme in triacylglycerol and phospholipid synthesis in mammals. GPAT1 regulates hepatic lipid accumulation associated with metabolic disorders. Here we have identified two transcriptional initiation sites and two promoters (promoter I and II) required for expression of the human GPAT1 (hGPAT1) gene. Promoter I regulates transcription of three alternative hGPAT1 mRNA variants, hGPAT1-V1, V2, and V3, while promoter II induces expression of a fourth variant, hGPAT1-V4. RT-PCR analysis and luciferase reporter assays revealed that promoter II acts in lipogenic tissues like the liver (and liver-derived HepG2 cells), whereas promoter I is differentially regulated and also acts in non-liver HeLa cells. Among liver-enriched transcription factors, HNF4α and C/EBPα slightly activated hGPAT1 promoter I, while factors including HNF1α altered promoter II activity. The lipogenic transcription factor SREBP1c greatly increased promoter II activity in HepG2 cells. The use of various truncated or mutated fragments of promoter II revealed that one sterol regulatory element-like motif and one inverted CCAAT box on promoter II contributed to the SREBP1c response. These cis-acting elements and trans-acting factors can be potential targets for manipulation of hepatic GPAT1 levels in humans.

PAS Kinase Regulates Hepatic Lipid Metabolism through Activating SREBP‐1

PAS kinase (PASK) is a serine/threonine protein kinase conserved from yeast to human. PASK activity has been shown to be controlled by nutrient status, but its roles in regulating metabolism remain largely unclear. In order to study the function of PASK in mammals, we used PASK deficient (PASK−/−) mice as a model system. PASK−/− mice showed decreased liver accumulation of triglyceride induced by a high‐fat diet. This was accompanied by lower expression levels of glycerol‐3‐phosphate acyltransferase 1 (GPAT1), stearoyl‐CoA desaturase 1 (SCD1) and fatty acid elongase (FAE), three important enzymes responsible for biosynthesis of fatty acids and triglyceride. We also observed the down‐regulation of these genes upon PASK knockdown in cultured HepG2 (Hepatocellular carcinoma, human) cells. The expression of GPAT1, SCD1 and FAE are regulated by a family of transcription factors called sterol regulatory element binding proteins (SREBPs). Our data suggests that PASK promotes the activity of SREBP‐1, probably by enhancing the processing of the SREBP‐1 precursor to form the active nuclear form. We are now trying to determine the detailed mechanisms of how PASK regulates the processing step. These studies will further our understanding of the physiological roles of PASK, and hopefully provide a potential therapeutic target for metabolic diseases.

Expression of fat deposition and fat removal genes is associated with intramuscular fat content in longissimus dorsi muscle of Korean cattle steers1

Intramuscular fat (IMF) in cattle is an important component of traits that influence meat quality. We measured carcass characteristics and gene expression in Korean steers to clarify the molecular mechanism(s) underlying IMF deposition in LM tissue by determining the correlation between IMF content and gene expression abundance and by developing models to predict IMF content using gene expression abundance. The deposition of IMF is determined by a balance between fat deposition and fat removal in the LM. We measured mRNA abundance of lipid metabolic genes including lipogenesis [acetyl CoA carboxylase (ACC), fatty acid synthase (FASN)], fatty lipid uptake [lipoprotein lipase (LPL), fatty acid translocase (CD36), fatty acid transport protein 1 (FATP1)], fatty acid esterification [glycerol-3-phosphate acyltransferase 1 (GPAT1), acylglycerol phosphate acyltransferase 1 (AGPAT1), diacylglycerol acyltransferase 1 (DGAT1), DGAT2], lipolysis [adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), monoglyceride lipase (MGL)], and fatty acid oxidation [carnitine palmitoyl transferase 1B, very long-chain acyl-CoA dehydrogenase (VLCAD), medium-chain acyl-CoA dehydrogenase (MCAD)] in the LM. The mRNA abundance of the GPAT1 gene showed the greatest correlation (r = 0.74; P < 0.001) with IMF content among 9 fat deposition genes. The gene expression abundance of other fat deposition genes including ACC, FASN, LPL, CD36, FATP1, AGPAT1, DGAT1, and DGAT2 also exhibited significant positive correlations (P < 0.05) with IMF content in the LM. Conversely, ATGL mRNA abundance showed the greatest negative correlation (r = -0.68; P < 0.001) with IMF content in the LM among 6 fat removal genes. The expression of other fat removal genes including MGL, VLCAD, and MCAD showed significant negative correlations (P < 0.05) with IMF content. Our findings show that the combined effects of increases in lipogenesis, fatty acid uptake, fatty acid esterification, and of decreases in lipolysis and fatty acid oxidation contribute to increasing IMF deposition in Korean steers. The multiple regression analysis revealed that the mRNA abundance of the GPAT1 gene in the LM was the first major variable predicting IMF content (54%) among 15 lipid metabolic genes. The second was mRNA abundance of ATGL (11%). In conclusion, these results suggest that GPAT1 and ATGL genes could be used as genetic markers to predict IMF deposition in the LM.

Glycerolipid signals alter mTOR complex 2 (mTORC2) to diminish insulin signaling

Increased flux through the glycerolipid synthesis pathway impairs the ability of insulin to inhibit hepatic gluconeogenesis, but the exact mechanism remains unknown. To determine the mechanism by which glycerolipids impair insulin signaling, we overexpressed glycerol-3-phosphate acyltransferase-1 (GPAT1) in primary mouse hepatocytes. GPAT1 overexpression impaired insulin-stimulated phosphorylation of Akt-S473 and -T308, diminished insulin-suppression of glucose production, significantly inhibited mTOR complex 2 (mTORC2) activity and decreased the association of mTOR and rictor. Conversely, in hepatocytes from Gpat1(-/-) mice, mTOR-rictor association and mTORC2 activity were enhanced. However, this increase in mTORC2 activity in Gpat1(-/-) hepatocytes was ablated when rictor was knocked down. To determine which lipid intermediate was responsible for inactivating mTORC2, we overexpressed GPAT1, AGPAT, or lipin to increase the cellular content of lysophosphatidic acid (LPA), phosphatidic acid (PA), or diacylglycerol (DAG), respectively. The inhibition of mTOR/rictor binding and mTORC2 activity coincided with the levels of PA and DAG species that contained 16:0, the preferred substrate of GPAT1. Furthermore, di-16:0-PA strongly inhibited mTORC2 activity and disassociated mTOR/rictor in vitro. Taken together, these data reveal a signaling pathway by which phosphatidic acid synthesized via the glycerol-3-phosphate pathway inhibits mTORC2 activity by decreasing the association of rictor and mTOR, thereby down-regulating insulin action. These data demonstrate a critical link between nutrient excess, TAG synthesis, and hepatic insulin resistance.

Human 1-Acylglycerol-3-phosphate O-Acyltransferase Isoforms 1 and 2: BIOCHEMICAL CHARACTERIZATION AND INABILITY TO RESCUE HEPATIC STEATOSIS IN Agpat2−/− GENE LIPODYSTROPHIC MICE

Loss-of-function mutations in 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) 2 in humans and mice result in loss of both the white and brown adipose tissues from birth. AGPAT2 generates precursors for the synthesis of glycerophospholipids and triacylglycerols. Loss of adipose tissue, or lipodystrophy, results in hyperinsulinemia, diabetes mellitus, and severe hepatic steatosis. Here, we analyzed biochemical properties of human AGPAT2 and its close homolog, AGPAT1, and we studied their role in liver by transducing their expression via recombinant adenoviruses in Agpat2(-/-) mice. The in vitro substrate specificities of AGPAT1 and AGPAT2 are quite similar for lysophosphatidic acid and acyl-CoA. Protein homology modeling of both the AGPATs with glycerol-3-phosphate acyltransferase 1 (GPAT1) revealed that they have similar tertiary protein structure, which is consistent with their similar substrate specificities. When co-expressed, both isoforms co-localize to the endoplasmic reticulum. Despite such similarities, restoring AGPAT activity in liver by overexpression of either AGPAT1 or AGPAT2 in Agpat2(-/-) mice failed to ameliorate the hepatic steatosis. From these studies, we suggest that the role of AGPAT1 or AGPAT2 in liver lipogenesis is minimal and that accumulation of liver fat is primarily a consequence of insulin resistance and loss of adipose tissue in Agpat2(-/-) mice.

Lysophosphatidic Acid Activates Peroxisome Proliferator Activated Receptor-γ in CHO Cells That Over-Express Glycerol 3-Phosphate Acyltransferase-1

Lysophosphatidic acid (LPA) is an agonist for peroxisome proliferator activated receptor-γ (PPARγ). Although glycerol-3-phosphate acyltransferase-1 (GPAT1) esterifies glycerol-3-phosphate to form LPA, an intermediate in the de novo synthesis of glycerolipids, it has been assumed that LPA synthesized by this route does not have a signaling role. The availability of Chinese Hamster Ovary (CHO) cells that stably overexpress GPAT1, allowed us to analyze PPARγ activation in the presence of LPA produced as an intracellular intermediate. LPA levels in CHO-GPAT1 cells were 6-fold higher than in wild-type CHO cells, and the mRNA abundance of CD36, a PPARγ target, was 2-fold higher. Transactivation assays showed that PPARγ activity was higher in the cells that overexpressed GPAT1. PPARγ activity was enhanced further in CHO-GPAT1 cells treated with the PPARγ ligand troglitazone. Extracellular LPA, phosphatidic acid (PA) or a membrane-permeable diacylglycerol had no effect, showing that PPARγ had been activated by LPA generated intracellularly. Transient transfection of a vector expressing 1-acylglycerol-3-phosphate acyltransferase-2, which converts endogenous LPA to PA, markedly reduced PPARγ activity, as did over-expressing diacylglycerol kinase, which converts DAG to PA, indicating that PA could be a potent inhibitor of PPARγ. These data suggest that LPA synthesized via the glycerol-3-phosphate pathway can activate PPARγ and that intermediates of de novo glycerolipid synthesis regulate gene expression.