Triacylglycerol synthesis in cultured rat hepatocytes

Abstract

The limiting role of diacylglycerol acyltransferase with respect to triacylglycerol synthesis in cultured rat hepatocytes was evaluated by following the inhibition of the overall synthetic flux by 2-bromooctanoate acting as an inhibitor of the diacylglycerol acyltransferase step. The flux-control coefficient of diacylglycerol acyltransferase in intact cultured hepatocytes amounted to 0.76 in the presence of saturating glycerol and either palmitate or oleate as the fatty acyl substrates. The flux-control coefficient of diacylglycerol acyltransferase in lysolecithin-permeabilized cultured hepatocytes amounted to 0.80 and 0.99 in the presence of saturating glycerol 3-phosphate and either palmitate or oleate as the fatty acyl substrate, respectively. Hence, triacylglycerol synthesis in liver cells under the experimental conditions employed is rate-limited by the diacylglycerol acyltransferase.The rate of esterification of long-chain fatty acids into neutral glycerides in liver cells is dependent on the availability of the respective substrates and the in situ activity of the participating enzymes. The availability of the fatty acid substrate is dependent on its endogenous de novo synthesis and its uptake from the medium while the glycerol 3-phosphate precursor may be produced from glucose, gluconeogenic precursors or by glycerol phosphorylation. Under conditions of unlimited availability of the respective substrates, the flux of neutral lipid synthesis will depend on the activities of the enzymes involved and the distribution of their control strengths [l, 21.The rate-limiting step of neutral lipid synthesis was inferred only indirectly by either studying the relative capacities of enzymes of the pathway or searching for those enzymatic activities which could be modulated in vivo or in culture in correlation with modulation of the overall synthetic flux. These studies have repeatedly implicated Mg2 +-dependent phosphatidate phosphohydrolase (PPH) as the rate-limiting step of hepatic neutral lipid synthesis [3] induced by long-chain fatty acids and their CoA thioesters offered an apparent linkage between the availability of the fatty acid substrate and modulation of the proposed ratelimiting step. Thus, cytosolic PPH in liver was suggested to represent an inactive enzyme reservoir which, when translocated to the endoplasmic reticulum, could become active in microsomal lipid synthesis. The rate-limiting role of microsomal PPH in liver lipid synthesis was however challenged by other studies where the content of liver microsomal PPH was found to be uncorrelated with the overall flux of neutral lipid synthesis [17, 181. The limiting role proposed for PPH was similarly questioned recently in suspended rat adipocytes where particulate PPH and the rate of triacylglycerol synthesis were found to be only partially correlated [19, 201. These studies were complemented by findings pointing to the induction of diacylglycerol acyltransferase (DGAT) under conditions which favored the synthesis of liver neutral lipid as well as its ...

Defective Uptake and Utilization of Long Chain Fatty Acids in Muscle and Adipose Tissues of CD36 Knockout Mice

“…The lower affinity of DGAT for acyl-CoA as compared with other enzymes in the FA esterification pathway (Fig. 5) has been suggested from results of enzymatic assays in intact and permeabilized hepatocytes (42,43). However, the findings presented here mark the first demonstration of a key regulatory role for DGAT in vivo and point to the pivotal role of membrane FA transport in maintaining acyl-CoA at the levels required for optimal DGAT activity.…”

Niacin noncompetitively inhibits DGAT2 but not DGAT1 activity in HepG2 cells

“…DGAT is the terminal and only committed enzyme that catalyzes the acylation of DAG in the synthesis of TGs (25)(26)(27)(28). However, DAG used in the DGAT reaction can be derived from multiple pathways, including 1) hydrolysis of phosphatidic acid produced by the de novo synthesis pathway from glycerol-3-phosphate catalyzed by glycerol-3-phosphate acyltransferase; 2) esterification of monoacylglycerol catalyzed by MGAT; and 3) hydrolysis of TG or phospholipids.…”

Seed-Specific Over-Expression of an Arabidopsis cDNA Encoding a Diacylglycerol Acyltransferase Enhances Seed Oil Content and Seed Weight

“…A number of studies with both mammalian (Mayorek et al, 1989;Tijburg et al, 1989) and plant (Ichihara et al, 1988;Harwood, 1993a, 1993b;Settlage et al, 1995;Perry et al, 1999) systems have suggested that DGAT may catalyze a rate-limiting reaction in TAG bioassembly. For example, developing seeds of B. napus L., cv Shiralee, have been shown to produce significant levels of DAG during the active phase of oil accumulation Harwood, 1993a, 1993b).…”