Missense mutation in APOC3 within the C-terminal lipid binding domain of human ApoC-III results in impaired assembly and secretion of triacylglycerol-rich very low density lipoproteins: evidence that ApoC-III plays a major role in the formation of lipid precursors within the microsomal lumen.

Hepatic assembly of triacylglycerol (TAG)-rich very low density lipoproteins (VLDL) is achieved through recruitment of bulk TAG (presumably in the form of lipid droplets within the microsomal lumen) into VLDL precursor containing apolipoprotein (apo) B-100. We determined protein/lipid components of lumenal lipid droplets (LLD) in cells expressing recombinant human apoC-III (C3wt) or a mutant form (K58E, C3KE) initially identified in humans that displayed hypotriglyceridemia. Although expression of C3wt markedly stimulated secretion of TAG and apoB-100 as VLDL(1), the K58E mutation (located at the C-terminal lipid binding domain) abolished the effect in transfected McA-RH7777 cells and in apoc3-null mice. Metabolic labeling studies revealed that accumulation of TAG in LLD was decreased (by 50%) in cells expressing C3KE. A Fat Western lipid protein overlay assay showed drastically reduced lipid binding of the mutant protein. Substituting Lys(58) with Arg demonstrated that the positive charge at position 58 is crucial for apoC-III binding to lipid and for promoting TAG secretion. On the other hand, substituting both Lys(58) and Lys(60) with Glu resulted in almost entire elimination of lipid binding and loss of function in promoting TAG secretion. Thus, the lipid binding domain of apoC-III plays a key role in the formation of LLD for hepatic VLDL assembly and secretion.

Secretion of triacylglycerol-poor VLDL particles from McA-RH7777 cells expressing human hepatic lipase.

Hepatic lipase (HL) plays a role in the catabolism of apolipoprotein (apo)B-containing lipoproteins through its lipolytic and ligand-binding properties. We describe a potential intracellular role of HL in the assembly and secretion of VLDL. Transient or stable expression of HL in McA-RH7777 cells resulted in decreased (by 40%) incorporation of [(3)H]glycerol into cell-associated and secreted triacylglycerol (TAG) relative to control cells. However, incorporation of [(35)S]methionine/cysteine into cell and medium apoB-100 was not decreased by HL expression. The decreased (3)H-TAG synthesis/secretion in HL expressing cells was not attributable to decreased expression of genes involved in lipogenesis. Fractionation of medium revealed that the decreased [(3)H]TAG from HL expressing cells was mainly attributable to decreased VLDL. Expression of catalytically-inactive HL (HL(SG)) (Ser-145 at the catalytic site was substituted with Gly) in the cells also resulted in decreased secretion of VLDL-[(3)H]TAG. Examination of lumenal contents of microsomes showed a 40% decrease in [(3)H]TAG associated with lumenal lipid droplets in HL or HL(SG) expressing cells as compared with control. The microsomal membrane-associated [(3)H]TAG was decreased by 50% in HL expressing cells but not in HL(SG) expressing cells. Thus, expression of HL, irrespective of its lipolytic function, impairs formation of VLDL precursor [(3)H]TAG in the form of lumenal lipid droplets. These results suggest that HL expression in McA-RH7777 cells result in secretion of [(3)H]TAG-poor VLDL.

Nonsynonymous mutations within APOB in human familial hypobetalipoproteinemia: evidence for feedback inhibition of lipogenesis and postendoplasmic reticulum degradation of apolipoprotein B.

Five nontruncating missense APOB mutations, namely A31P, G275S, L324M, G912D, and G945S, were identified in heterozygous carriers of familial hypobetalipoproteinemia (FHBL) in the Italian population. To test that the FHBL phenotype was a result of impaired hepatic secretion of mutant apoB proteins, we performed transfection studies using McA-RH7777 cells stably expressing wild type or mutant forms of human apolipoprotein B-48 (apoB-48). All mutant proteins displayed varied impairment in secretion, with G912D the least affected and A31P barely secreted. Although some A31P was degraded by proteasomes, a significant proportion of it (although inappropriately glycosylated) escaped endoplasmic reticulum (ER) quality control and presented in the Golgi compartment. Degradation of the post-ER A31P was achieved by autophagy. Expression of A31P also decreased secretion of endogenous apoB and triglycerides, yet the impaired lipoprotein secretion did not lead to lipid accumulation in the cells or ER stress. Rather, expression of genes involved in lipogenesis was down-regulated, including liver X receptor alpha, sterol regulator element-binding protein 1c, fatty acid synthase, acetyl-CoA carboxylase 1, stearoyl-CoA desaturase 1, and lipin-1. These results suggest that feedback inhibition of hepatic lipogenesis in conjunction with post-ER degradation of misfolded apoB proteins can contribute to reduce fat accumulation in the FHBL liver.