Evidence for triacylglycerol synthesis in the lumen of microsomes via a lipolysis-esterification pathway involving carnitine acyltransferases

In this study a pathway for the synthesis of triacylglycerol (TAG) within the lumen of the endoplasmic reticulum has been identified, using microsomes that had been preconditioned by depleting their endogenous substrates and then fusing them with biotinylated phosphatidylserine liposomes containing CoASH and Mg2+ Incubating these fused microsomes with tri[H-3] oleoylglycerol and [C-14]oleoyl-CoA yielded microsome-associated triacylglycerol, which resisted extensive washing and had a [3H]:[14C] ratio close to 2:1, The data suggest that the precursor tri[H-3]oleoylglycerol was hydrolyzed by microsomal lipase to membrane-bound di[3H]oleoylglycerol and subsequently re-esterified with luminal [C-14]oleoyl-CoA. The accumulation of TAG within the microsomes, even when overt diacylglycerol acyltransferase (DGAT I) was inactive, is consistent with the existence of a latent diacylglycerol acyltransferase (DGAT II) within the microsomal lumen. Moreover, because luminal synthesis of TAG was carnitine-dependent and markedly reduced by glybenclamide, a potent carnitine acyltransferase inhibitor, microsomal carnitine acyltransferase appears to be essential for trafficking the [C-14]oleoyl CoA into the microsomal lumen for subsequent incorporation into newly synthesized TAG. This study thus provides the first direct demonstration of an enzymatic process leading to the synthesis of luminal triacylglycerol, which is a major component of very low density lipoproteins.