Phosphatidate phosphohydrolase and the regulation of glycerolipid biosynthesis.

Phosphatidate is an intermediate in the biosynthesis of diacylglycerol, triacylglycerol, phosphatidylethanolamine, phosphatidylcholine and CDP-diacylglycerol. In addition, phosphatidate may be subject to degradation by particulate phospholipases. The factors regulating the disposition of phosphatidate have interested numerous investigators. We have studied the reactions of phosphatidate metabolism in several tissues and under various environmental conditions. Phosphatidate phosphohydrolase activity is found primarily in the microsomal and supernatant fractions of liver, adipose tissue and intestine. Presumably the physiologically important substrate for this reaction is the particulate-bound phosphatidate and methods for the preparation of this substrate have been developed (Fallon et al., 1975). Radioactively labelled phosphatidate bound to microsomal particles is prepared from sn-[1 ,3-14C]glycerol 3-phosphate. A wide range of microsomal phosphatidate concentrations may be prepared by this technique, although the usual microsomal concentration is approx. 5 nmol/mg of microsomal protein. This substrate has been used in most of our studies of phosphatidate phosphohydrolase, CDP-diacylglycerol formation and phospholipase A. There are several lines of evidence which support an important role for phosphatidate phosphohydrolase in the regulation of neutral-lipid formation by liver or adipose-tissue particulate preparations. (1) The reaction rate of phosphatidate phosphohydrolase under optimum conditions in vitro is the lowest in the overall synthetic pathway in liver microsomal fractions (Lamb & Fallon, 1974a). The rate of phosphohydrolase activity was determined as 0.6-0.8 nmol/min per mg of microsomal protein when radiolabelled particulate-bound phosphatidate was used as substrate. The overall rate of glycerolipid formation by these microsomal preparations was approx. l.Onmol/min per mg of microsomal protein. The optimum rate of microsomal sn-glycerol 3-phosphate acyltransferase was 5-7-fold higher. In the presence of added rat liver supernatant fraction the rate of microsomal phosphatidate phosphohydrolase is increased 5-20-fold. However, at ratios of microsomal protein to supernatant protein estimated for the intact cell, the increase in phosphohydrolase activity is much more modest. (2) Induction of increased liver triacylglycerol formation by administration of a high-fructose diet is accompanied by an equivalent rise in the activity of the phosphohydrolase, as shown in Table 1. These changes in lipid metabolism occur simultaneously over a 10-day period (Lamb & Fallon, 19746). Further, the phosphatidate content of liver microsomal preparation is diminished by approx. 15 %, and diacylglycerol increased by approx. 90 % under these dietary conditions. (3) The increase in liver triacylglycerol content after partial hepatectomy is accompanied by a corresponding increase in phosphatidate phosphohydrolase activity (Mangiapane et al., 1973). (4) Several drugs which cause substantial inhibition of phosphatidate phosphohydrolase activity in vitro also result in inhibition of neutral-lipid formation when administered to the intact animal (Brindley & Bowley, 1975; Lamb et al., 1977). A series of 1,3-bis(substituted phenoxy)-2-propanones inhibited microsomal phosphatidate phosphohydrolase activity as measured in vitro. The inhibition ranged from 8 to 92% total activity. In animals fed on these agents in laboratory chow the incorporation of [14C]glycerol into hepatic triacylglycerol was decreased by 3-75 %. Agents which caused a 50 % or greater decrease in phosphatidate phosphohydrolase activity in vitro also lowered