Abstract
Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from beta-oxidation and toward glycerolipid synthesis. Adenoviral-mediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have >2-fold increased enzyme activity. Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.
Highlights
Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis
To verify that our construct was properly expressing mtGPAT, Chinese hamster ovary (CHO) cells were transiently transfected with mtGPAT in a pcDNA expression vector and compared with cells transfected with an empty vector
Total cellular proteins and mitochondrial proteins were prepared, and the mtGPAT protein level was assayed with Western blot using antibodies directed against either the FLAG epitope (Fig. 1A) or antibodies generated against aa 312-326 in mouse mtGPAT (Fig. 1B)
Summary
Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. The promotor of mtGPAT contains both sterol- and carbohydrate-responsive elements [10, 11], and mtGPAT mRNA level is upregulated by a high-carbohydrate, fat-free diet and by insulin administration to streptozotocin-diabetic mice [12] Taken together, these results show that mtGPAT activity is upregulated in tissues from obese animals or from animals fed high-carbohydrate diets and that increased activity may promote TG synthesis
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