Sterol regulatory element binding protein 1a regulates hepatic fatty acid partitioning by activating acetyl coenzyme A carboxylase 2.

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

Mice deficient in mitochondrial glycerol-3-phosphate acyltransferase-1 have diminished myocardial triacylglycerol accumulation during lipogenic diet and altered phospholipid fatty acid composition.

Glycerol-3-phosphate acyltransferase-1 (GPAT1), which is located on the outer mitochondrial membrane comprises up to 30% of total GPAT activity in the heart. It is one of at least four mammalian GPAT isoforms known to catalyze the initial, committed, and rate-limiting step of glycerolipid synthesis. Because excess triacylglycerol (TAG) accumulates in cardiomyocytes in obesity and type 2 diabetes, we determined whether lack of GPAT1 would alter the synthesis of heart TAG and phospholipids after a 2-week high-sucrose diet or a 3-month high-fat diet. Even in the absence of hypertriglyceridemia, TAG increased 2-fold with both diets in hearts from wildtype mice. In contrast, hearts from Gpat1(-/-) mice contained 20-80% less TAG than the wildtype controls. In addition, hearts from Gpat1(-/-) mice fed the high-sucrose diet incorporate 60% less [(14)C]palmitate into heart TAG as compared to wildtype mice. Because GPAT1 prefers 16:0-CoA to other long-chain acyl-CoA substrates, we determined the fatty acid composition of heart phospholipids. Compared to wildtype littermate controls, hearts from Gpat1(-/-)(-/-) mice contained a lower amount of 16:0 in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine/phosphatidylinositol and significantly more C20:4n6. Phosphatidylcholine and phosphatidylethanolamine from Gpat1(-/-)(-/-) hearts also contained higher amounts of 18:0 and 18:1. Although at least three other GPAT isoforms are expressed in the heart, our data suggest that GPAT1 contributes significantly to cardiomyocyte TAG synthesis during lipogenic or high-fat diets and influences the incorporation of 20:4n6 into heart phospholipids.

Increased oxidative stress is associated with balanced increases in hepatocyte apoptosis and proliferation in glycerol-3-phosphate acyltransferase-1 deficient mice.

The absence of mouse mitochondrial glycerol-3-phosphate acyltransferase-1 (Gpat1-/-) increases the amount of arachidonate in liver phospholipids and increases beta-hydroxybutyrate and acyl-carnitines, suggesting an elevated rate of liver fatty acid oxidation. We asked whether these alterations might increase reactive oxygen species (ROS), apoptosis, or hepatocyte proliferation. Compared to wildtype controls, liver mitochondria from Gpat1-/- mice showed a 20% increase in the rate of ROS production and a markedly increased sensitivity to the induction of the mitochondrial permeability transition. Mitochondrial phosphatidylethanolamine and phosphatidylcholine from Gpat1-/- liver contained 21% and 67% more arachidonate, respectively, than wildtype controls, and higher amounts of 4-hydroxynonenal, a product of arachidonate peroxidation. Oxidative stress was associated with an increase in apoptosis, and with 3-fold and 15-fold higher TUNEL positive cells in liver from young and old Gpat1-/- mice, respectively, compared to age-matched controls. Compared to controls, bromodeoxyuridine labeling was 50% and 7-fold higher in livers from young and old Gpat1-/- mice, respectively, but fewer glutathione-S-transferase positive cells were present. Thus, Gpat1-/- liver exhibits increased oxidative stress and sensitivity of the mitochondrial permeability transition pore, and a balanced increase in apoptosis and proliferation.

Prevention of hepatic steatosis and hepatic insulin resistance in mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 knockout mice.

In order to investigate the role of mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 (mtGPAT1) in the pathogenesis of hepatic steatosis and hepatic insulin resistance, we examined whole-body insulin action in awake mtGPAT1 knockout (mtGPAT1(-/-)) and wild-type (wt) mice after regular control diet or three weeks of high-fat feeding. In contrast to high-fat-fed wt mice, mtGPAT1(-/-) mice displayed markedly lower hepatic triacylglycerol and diacylglycerol concentrations and were protected from hepatic insulin resistance possibly due to a lower diacylglycerol-mediated PKC activation. Hepatic acyl-CoA has previously been implicated in the pathogenesis of insulin resistance. Surprisingly, compared to wt mice, mtGPAT1(-/-) mice exhibited increased hepatic insulin sensitivity despite an almost 2-fold elevation in hepatic acyl-CoA content. These data suggest that mtGPAT1 might serve as a novel target for treatment of hepatic steatosis and hepatic insulin resistance and that long chain acyl-CoA's do not mediate fat-induced hepatic insulin resistance in this model.

Mitochondrial glycerol-3-phosphate acyltransferase-1 is essential in liver for the metabolism of excess acyl-CoAs.

In vitro studies suggest that the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform catalyzes the initial and rate-controlling step in glycerolipid synthesis and aids in partitioning acyl-CoAs toward triacylglycerol synthesis and away from degradative pathways. To determine whether the absence of mtGPAT1 would increase oxidation of acyl-CoAs and restrict the development of hepatic steatosis, we fed wild type and mtGPAT1-/- mice a diet high in fat and sucrose (HH) for 4 months to induce the development of obesity and a fatty liver. Control mice were fed a diet low in fat and sucrose (LL). With the HH diet, absence of mtGPAT1 resulted in increased partitioning of acyl-CoAs toward oxidative pathways, demonstrated by 60% lower hepatic triacylglycerol content and 2-fold increases in plasma beta-hydroxybutyrate, acylcarnitines, and hepatic mRNA expression of mitochondrial HMG-CoA synthase. Despite the increase in fatty acid oxidation, liver acyl-CoA levels were 3-fold higher in the mtGPAT1-/- mice fed both diets. A lack of difference in CPT1 and FAS mRNA expression between genotypes suggested that the increased acyl-CoA content was not because of increased de novo synthesis, but instead, to an impaired ability to use long-chain acyl-CoAs derived from the diet, even when the dietary fat content was low. Hyperinsulinemia and reduced glucose tolerance on the HH diet was greater in the mtGPAT1-/- mice, which did not suppress the expression of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This study demonstrates that mtGPAT1 is essential for normal acyl-CoA metabolism, and that the absence of hepatic mtGPAT1 results in the partitioning of fatty acids away from triacylglycerol synthesis and toward oxidation and ketogenesis.

Mitochondrial glycerol-3-phosphate acyltransferase-deficient mice have reduced weight and liver triacylglycerol content and altered glycerolipid fatty acid composition.

Microsomal and mitochondrial isoforms of glycerol-3-phosphate acyltransferase (GPAT; E.C. 2.3.1.15) catalyze the committed step in glycerolipid synthesis. The mitochondrial isoform, mtGPAT, was believed to control the positioning of saturated fatty acids at the sn-1 position of phospholipids, and nutritional, hormonal, and overexpression studies suggested that mtGPAT activity is important for the synthesis of triacylglycerol. To determine whether these purported functions were true, we constructed mice deficient in mtGPAT. mtGPAT(-/-) mice weighed less than controls and had reduced gonadal fat pad weights and lower hepatic triacylglycerol content, plasma triacylglycerol, and very low density lipoprotein triacylglycerol secretion. As predicted, in mtGPAT(-/-) liver, the palmitate content was lower in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Positional analysis revealed that mtGPAT(-/-) liver phosphatidylethanolamine and phosphatidylcholine had about 21% less palmitate in the sn-1 position and 36 and 40%, respectively, more arachidonate in the sn-2 position. These data confirm the important role of mtGPAT in the synthesis of triacylglycerol, in the fatty acid content of triacylglycerol and cholesterol esters, and in the positioning of specific fatty acids, particularly palmitate and arachidonate, in phospholipids. The increase in arachidonate may be functionally significant in terms of eicosanoid production.