Shumei Zhong

Expression of apolipoprotein C-III in McA-RH7777 cells enhances VLDL assembly and secretion under lipid-rich conditions

Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion efficiency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL1. Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL1 secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identified in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions.

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 VLDL1, 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 Lys58 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 Lys58 and Lys60 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.

Missense Mutations in APOB within the βα1 Domain of Human APOB-100 Result in Impaired Secretion of ApoB and ApoB-containing Lipoproteins in Familial Hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the α-helical domain within the N terminus of apoB. Thus, proper folding of the α-helical domain of apoB-100 is essential for efficient secretion.

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 α, 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.

Functional analysis of the missense APOC3 mutation Ala23Thr associated with human hypotriglyceridemia

We have shown that expression of apolipoprotein (apo) C-III promotes VLDL secretion from transfected McA-RH7777 cells under lipid-rich conditions. To determine structural elements within apoC-III that confer to this function, we contrasted wild-type apoC-III with a mutant Ala23Thr originally identified in hypotriglyceridemia subjects. Although synthesis of [3H]glycerol-labeled TAG was comparable between cells expressing wild-type apoC-III (C3wt cells) or Ala23Thr mutant (C3AT cells), secretion of [3H]TAG from C3AT cells was markedly decreased. The lowered [3H]TAG secretion was associated with an inability of C3AT cells to assemble VLDL1. Moreover, [3H]TAG within the microsomal lumen in C3AT cells was 60% higher than that in C3wt cells, yet the activity of microsomal triglyceride-transfer protein in C3AT cells was not elevated. The accumulated [3H]TAG in C3AT microsomal lumen was mainly associated with lumenal IDL/LDL-like lipoproteins. Phenotypically, this [3H]TAG fractionation profiling resembled what was observed in cells treated with brefeldin A, which at low dose specifically blocked the second-step VLDL1 maturation. Furthermore, lumenal [35S]Ala23Thr protein accumulated in IDL/LDL fractions and was absent in VLDL fractions in C3AT cells. These results suggest that the presence of Ala23Thr protein in lumenal IDL/LDL particles might prevent effective fusion between lipid droplets and VLDL precursors. Thus, the current study reveals an important structural element residing within the N-terminal region of apoC-III that governs the second step VLDL1 maturation.

Improved recovery and identification of membrane proteins from rat hepatic cells using a centrifugal proteomic reactor

Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism.

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 [3H]glycerol into cell-associated and secreted triacylglycerol (TAG) relative to control cells. However, incorporation of [35S]methionine/cysteine into cell and medium apoB-100 was not decreased by HL expression. The decreased 3H-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 [3H]TAG from HL expressing cells was mainly attributable to decreased VLDL. Expression of catalytically-inactive HL (HLSG) (Ser-145 at the catalytic site was substituted with Gly) in the cells also resulted in decreased secretion of VLDL-[3H]TAG. Examination of lumenal contents of microsomes showed a 40% decrease in [3H]TAG associated with lumenal lipid droplets in HL or HLSG expressing cells as compared with control. The microsomal membrane-associated [3H]TAG was decreased by 50% in HL expressing cells but not in HLSG expressing cells. Thus, expression of HL, irrespective of its lipolytic function, impairs formation of VLDL precursor [3H]TAG in the form of lumenal lipid droplets. These results suggest that HL expression in McA-RH7777 cells result in secretion of [3H]TAG-poor VLDL.

Global Inactivation of the Pla2g6 Gene in Mice Does Not Cause Dyslipidemia under Chow or High-fat Diet Conditions.

Background: Genome-wide association studies suggest that plasma triacylglyceride (TAG) in humans was associated with variation in the PLA2G6 locus, a gene that encodes calcium-independent phospholipase A2 (iPLA2β). The objective of the present study is to understand the impact of genetic inactivation of iPLA2β on hepatic TAG metabolism in C57BL/6 mice. Methods: Male iPLA2β+/− mice were backcrossed with female iPLA2β−/− mice for up to 10 generations prior to experiments. Lipid and lipoprotein metabolism from plasma, hepatocytes, thigh subcutaneous adipose and thigh skeletal muscle tissues of the mice were determined under various experimental conditions. Results: The iPLA2β−/− mice, either male or female as compared with iPLA2β+/+ littermates, showed no change in fasted or postprandial plasma TAG or total cholesterol at young (12–15 weeks) or old (40–44 weeks) ages under chow diet or high-fat diet (HFD) conditions. However, fractionation of plasma lipoproteins showed that under HFD conditions, there was a significant increase (by 40%) in apoB-100 association with VLDL1 fractions in iPLA2β−/− mice as compared with iPLA2β+/+ littermates. There was no significant difference in triglyceride or cholesterol contents in the liver, muscle, or adipose tissue between iPLA2β−/− and iPLA2β+/+ littermates. Metabolic labeling experiments with cultured primary hepatocytes isolated from iPLA2β−/− mice also showed 2-fold increase in the secretion of [35S]methionine-labeled apoB-100 in VLDL1 fractions as compared with that from iPLA2β+/+ hepatocytes. Likewise, secretion of [3H]palmitate-labeled TAG from the iPLA2β−/− hepatocytes was increased by 2-fold. Conclusions: Although iPLA2β may play a role in TAG-rich VLDL1 production from cultured hepatocytes, there is no evidence that inactivation of iPLA2β would lead to dyslipidemia in mice in vivo.