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.

Hepatic triacylglycerol accumulation and insulin resistance.

The association of hepatic steatosis with hepatic insulin resistance and type 2 diabetes has prompted investigators to elucidate the underlying mechanism. In this review we focus on pathways of lipid metabolism, and we review recent data, primarily from mouse models, that link lipid intermediates with insulin resistance. Most of the studies that implicate acyl-CoA, lysophosphatidic acid, phosphatidic acid, diacylglycerol, or ceramide rely on indirect associations. Convincing data to support the hypothesis that specific lipid intermediates initiate pathways that alter insulin signaling will require studies in which the concentration of each purported signaling molecule can be manipulated independently.

Identification of a novel sn-glycerol-3-phosphate acyltransferase isoform, GPAT4, as the enzyme deficient in Agpat6-/- mice.

Elucidation of the metabolic pathways of triacylglycerol (TAG) synthesis is critical to the understanding of chronic metabolic disorders such as obesity, cardiovascular disease, and diabetes. sn-Glycerol-3-phosphate acyltransferase (GPAT) and sn-1-acylglycerol-3-phosphate acyltransferase (AGPAT) catalyze the first and second steps in de novo TAG synthesis. AGPAT6 is one of eight AGPAT isoforms identified through sequence homology, but the enzyme activity for AGPAT6 has not been confirmed. We found that in liver and brown adipose tissue from Agpat6-deficient (Agpat6(-/-)) mice, N-ethylmaleimide (NEM)-sensitive GPAT specific activity was 65% lower than in tissues from wild-type mice, but AGPAT specific activity was similar. Overexpression of Agpat6 in Cos-7 cells increased an NEM-sensitive GPAT specific activity, but AGPAT specific activity was not increased. Agpat6 and Gpat1 overexpression in Cos-7 cells increased the incorporation of [(14)C]oleate into diacylglycerol (DAG) or into DAG and TAG, respectively, suggesting that the lysophosphatidic acid, phosphatidic acid, and DAG intermediates initiated by each of these isoforms lie in different cellular pools. Together, these data show that "Agpat6(-/-) mice" are actually deficient in a novel NEM-sensitive GPAT, GPAT4, and indicate that the alterations in lipid metabolism in adipose tissue, liver, and mammary epithelium of these mice are attributable to the absence of GPAT4.

Hepatic overexpression of glycerol-sn-3-phosphate acyltransferase 1 in rats causes insulin resistance.

Fatty liver is commonly associated with insulin resistance and type 2 diabetes, but it is unclear whether triacylglycerol accumulation or an excess flux of lipid intermediates in the pathway of triacyglycerol synthesis are sufficient to cause insulin resistance in the absence of genetic or diet-induced obesity. To determine whether increased glycerolipid flux can, by itself, cause hepatic insulin resistance, we used an adenoviral construct to overexpress glycerol-sn-3-phosphate acyltransferase-1 (Ad-GPAT1), the committed step in de novo triacylglycerol synthesis. After 5-7 days, food intake, body weight, and fat pad weight did not differ between Ad-GPAT1 and Ad-enhanced green fluorescent protein control rats, but the chow-fed Ad-GPAT1 rats developed fatty liver, hyperlipidemia, and insulin resistance. Liver was the predominant site of insulin resistance; Ad-GPAT1 rats had 2.5-fold higher hepatic glucose output than controls during a hyperinsulinemic-euglycemic clamp. Hepatic diacylglycerol and lysophosphatidate were elevated in Ad-GPAT1 rats, suggesting a role for these lipid metabolites in the development of hepatic insulin resistance, and hepatic protein kinase Cepsilon was activated, providing a potential mechanism for insulin resistance. Ad-GPAT1-treated rats had 50% lower hepatic NF-kappaB activity and no difference in expression of tumor necrosis factor-alpha and interleukin-beta, consistent with hepatic insulin resistance in the absence of increased hepatic inflammation. Glycogen synthesis and uptake of 2-deoxyglucose were reduced in skeletal muscle, suggesting mild peripheral insulin resistance associated with a higher content of skeletal muscle triacylglycerol. These results indicate that increased flux through the pathway of hepatic de novo triacylglycerol synthesis can cause hepatic and systemic insulin resistance in the absence of obesity or a lipogenic diet.

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes.

Because excess triacylglycerol (TAG) in nonadipose tissues is closely associated with the development of insulin resistance, interest has increased in the metabolism of long-chain acyl-CoAs toward beta-oxidation or the synthesis and storage of TAG. To learn whether a mitochondrial isoform of glycerol-3-phosphate acyltransferase (mtGPAT1) competes with carnitine palmitoyltransferase I (CPT I) for acyl-CoAs and whether it contributes to the formation of TAG, we overexpressed rat mtGPAT1 13-fold in primary hepatocytes obtained from fasted rats. When 100, 250, or 750 microM oleate was present, both TAG mass and the incorporation of [14C]oleate into TAG increased more than twofold in hepatocytes overexpressing mtGPAT1 compared with vector controls. Although the incorporation of [14C]oleate into CO2 and acid-soluble metabolites increased with increasing amounts of oleate in the media, these metabolites were approximately 40% lower in the Ad-mtGPAT1 infected cells, consistent with competition for acyl-CoAs between CPT I and mtGPAT1. A 50-60% decrease was also observed in [14C]oleate incorporation into cholesteryl ester. With increasing amounts of exogenous oleate, [14C]TAG secretion increased appropriately in vector control-infected hepatocytes, suggesting that the machinery for VLDL-TAG biogenesis and secretion was unaffected. Despite the marked increases in TAG synthesis and storage in the Ad-mtGPAT1 cells, however, the Ad-mtGPAT1 cells secreted the same amount of [14C]TAG as the vector control cells. Thus, in isolated hepatocytes, mtGPAT1 may synthesize a cytosolic pool of TAG that cannot be secreted.