Inhibition of hepatocyte apoB secretion by naringenin: enhanced rapid intracellular degradation independent of reduced microsomal cholesteryl esters

The grapefruit flavonoid, naringenin, is hypocholesterolemic in vivo, and inhibits basal apolipoprotein B (apoB) secretion and the expression and activities of both ACAT and microsomal triglyceride transfer protein (MTP) in human hepatoma cells (HepG2). In this report, we examined the effects of naringenin on apoB kinetics in oleate-stimulated HepG2 cells and determined the contribution of microsomal lumen cholesteryl ester (CE) availability to apoB secretion. Pulse-chase studies of apoB secretion and intracellular degradation were analyzed by multicompartmental modeling. The model for apoB metabolism in HepG2 cells includes an intracellular compartment from which apoB can be either secreted or degraded by both rapid and slow pathways. In the presence of 0.1 mM oleic acid, naringenin (200 micro M) reduced the secretion of newly synthesized apoB by 52%, due to a 56% reduction in the rate constant for secretion. Intracellular degradation was significantly increased due to a selective increase in rapid degradation, while slow degradation was unaffected. Incubation with either N-acetyl-leucinyl-leucinyl-norleucinal (ALLN) or lactacystin showed that degradation via the rapid pathway was largely proteasomal. Although these changes in apoB metabolism were accompanied by significant reductions in CE synthesis and mass, subcellular fractionation experiments comparing naringenin to specific ACAT and HMG-CoA reductase inhibitors revealed that reduced accumulation of newly synthesized CE in the microsomal lumen is not consistently associated with reduced apoB secretion. However, naringenin, unlike the ACAT and HMG-CoA reductase inhibitors, significantly reduced lumenal TG accumulation. We conclude that naringenin inhibits apoB secretion in oleate-stimulated HepG2 cells and selectively increases intracellular degradation via a largely proteasomal, rapid kinetic pathway. Although naringenin inhibits ACAT, CE availability in the endoplasmic reticulum (ER) lumen does not appear to regulate apoB secretion in HepG2 cells. Rather, inhibition of TG accumulation in the ER lumen via inhibition of MTP is the primary mechanism blocking apoB secretion.     

Differential regulation of apolipoprotein B secretion from HepG2 cells by two HMG-CoA reductase inhibitors, atorvastatin and simvastatin.

The concept that hepatic cholesterol synthesis regulates hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to examine the regulation of apoB secretion by the HMG-CoA reductase inhibitor atorvastatin. ApoB accumulation in the media was decreased by 24% and 36% at 10 microm (P < 0.02) and 20 microm (P < 0.01) of atorvastatin, respectively. Atorvastatin inhibited HepG2 cell cholesterol synthesis by up to 96% (P < 0.001) and cellular cholesteryl ester (CE) mass by 54% (P < 0.001). Another HMG-CoA reductase inhibitor, simvastatin, decreased cellular cholesterol synthesis and CE mass by up to 96% (P < 0.001) and 52% (P < 0.001), respectively. However, in contrast to atorvastatin, simvastatin had no effect on apoB secretion. To characterize the reduction in apoB secretion by atorvastatin (10 microm), pulse-chase experiments were performed and the kinetic data were analyzed by multicompartmental modeling using SAAM II. Atorvastatin had no affect on the synthesis of apoB, however, apoB secretion into the media was decreased by 44% (P = 0.048). Intracellular apoB degradation increased proportionately (P = 0.048). Simvastatin (10 microm) treatment did not significantly alter either the secretion or intracellular degradation of apoB, relative to control. The kinetics of apoB degradation were best described by a rapidly and a slowly turning over degradation compartment. The effect of atorvastatin on apoB degradation was largely confined to the rapid compartment. Neither inhibitor affected apoB mRNA concentrations, however, both significantly increased LDL receptor and HMG-CoA reductase mRNA levels. Atorvastatin treatment also decreased the mRNA for the microsomal triglyceride transfer protein (MTP) by 22% (P < 0.02). These results show that atorvastatin decreases apoB secretion, by a mechanism that results in an enhanced intracellular degradation in apoB.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

Studies of the sites of intracellular degradation of apolipoprotein B in Hep G2 cells.

We previously reported that treatment of Hep G2 cells with oleate significantly increased apolipoprotein B (apoB) secretion by reducing early intracellular degradation of nascent apoB. In the current study, inhibitors of secretory protein transport (brefeldin A and monensin), cell fractionation studies, and protease protection assays were utilized to determine the location of apoB degradation and to better define the mechanism whereby oleate treatment reduces nascent apoB intracellular degradation. When cells were treated with brefeldin A, which blocks endoplasmic reticulum (ER) to Golgi protein transport, apoB degradation continued in control cells, suggesting that apoB is degraded in the ER. When oleate-treated cells were blocked with brefeldin A, oleate failed to protect apoB from intracellular degradation. The effects of brefeldin A were not due to effects on lipid synthesis as brefeldin A did not inhibit the synthesis of triglyceride, phospholipid, free cholesterol, or cholesteryl ester in control cells and did not prevent the increases in triglyceride (14-fold) and phospholipid (1.4-fold) synthesis seen in oleate-treated cells. Simultaneous treatment of cells with brefeldin A and nocodazole, which inhibits retrograde transport of proteins from Golgi to ER, added to the evidence for the ER as the site of apoB degradation. This conclusion received further support from experiments in which cells were treated with monensin, a Na+ ionophore which halts protein secretion at the level of the trans-Golgi network. Early degradation of nascent apoB (between 10 and 20 min of chase) was observed in monensin-treated cells, but then cellular apoB degradation ceased and apoB was stable during the remaining chase period. More apoB accumulated in the Golgi of cells that had been treated with oleate and monensin. These results suggest that ER degradation occurs in monensin-treated cells, but then stops as apoB is transferred to the Golgi. The results obtained in whole cells were confirmed in studies using isolated ER and Golgi, which indicated that ER contains a proteolytic activity which degrades apoB, in vitro, whereas Golgi does not. ApoB degradation in isolated ER was not reduced by pretreatment with oleate. Finally, protease protection assays carried out with isolated microsomes indicated that a majority of the apoB in both control or oleate-treated HepG2 cells was located on the cytosolic side of the membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of translocation of nascent apolipoprotein B across the endoplasmic reticulum membrane is associated with selective inhibition of the synthesis of apolipoprotein B

In HepG2 cells, inhibition of apolipoprotein B100 (apoB) translocation across the endoplasmic reticulum by an microsomal triglyceride transfer protein (MTP) inhibitor (CP-10447) in the presence of N-acetyl-leucinyl-norleucinal, a proteasomal inhibitor, results in accumulation of newly synthesized apoB in the translocation channel. Here we demonstrated that such accumulation led to a specific reduction of apoB synthesis. ApoB mRNA levels remained unchanged, but we observed reduced rates of elongation of nascent apoB in puromycin-synchronized cells pretreated with MTP inhibitor. This observation was consistent with a longer half-ribosome transit time for the synthesis of apoB in MTP-inhibited cells. Initiation of translation of apoB mRNA was not impaired by MTP inhibition. Overall, these findings suggest that translocation arrest of apoB in the endoplasmic reticulum channel can exert a selective and negative effect on the synthesis of apoB at the stage of elongation.     

Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP

The citrus flavonoids, naringenin and hesperetin, lower plasma cholesterol in vivo. However, the underlying mechanisms are not fully understood. The ability of these flavonoids to modulate apolipoprotein B (apoB) secretion and cellular cholesterol homeostasis was determined in the human hepatoma cell line, HepG2. apoB accumulation in the media decreased in a dose-dependent manner following 24-h incubations with naringenin (up to 82%, P < 0.00001) or hesperetin (up to 74%, P < 0.002). Decreased apoB secretion was associated with reduced cellular cholesteryl ester mass. Cholesterol esterification was decreased, dose-dependently, up to 84% (P < 0.0001) at flavonoid concentrations of 200 microM. Neither flavonoid demonstrated selective inhibition of either form of acyl CoA:cholesterol acyltransferase (ACAT) as determined using CHO cells stably transfected with either ACAT1 or ACAT2. However, in HepG2 cells, ACAT2 mRNA was selectively decreased (- 50%, P < 0.001) by both flavonoids, whereas ACAT1 mRNA was unaffected. In addition, naringenin and hesperetin decreased both the activity (- 20% to - 40%, P < 0.00004) and expression (- 30% to - 40%, P < 0.02) of microsomal triglyceride transfer protein (MTP). Both flavonoids caused a 5- to 7-fold increase (P < 0.02) in low density lipoprotein (LDL) receptor mRNA, which resulted in a 1.5- to 2-fold increase in uptake and degradation of (125)I-LDL. We conclude that both naringenin and hesperetin decrease the availability of lipids for assembly of apoB-containing lipoproteins, an effect mediated by 1) reduced activities of ACAT1 and ACAT2, 2) a selective decrease in ACAT2 expression, and 3) reduced MTP activity. Together with an enhanced expression of the LDL receptor, these mechanisms may explain the hypocholesterolemic properties of the citrus flavonoids.     

Translocational status of ApoB in the presence of an inhibitor of microsomal triglyceride transfer protein

Despite numerous studies demonstrating that microsomal triglyceride transfer protein (MTP) activity is critical to apoB secretion, there is still controversy as to whether MTP directly facilitates the translocation of apoB across the membrane of the endoplasmic reticulum (ER) through either the recruitment of lipids and/or chaperone activity. In the present study, a specific inhibitor of MTP (BMS 197636) was utilized in HepG2 cells to investigate whether a direct relationship exists between the translocation of apoB across the ER membrane and the lipid-transferring activity of MTP. Inhibition of MTP (with 10 and 50 nmol/L of the inhibitor) did not significantly affect the translocation of newly synthesized apoB (P = 0.77) or the translocational efficiency of the steady-state apoB mass (P = 0.45), despite a 49% decrease in apoB secretion and increased proteosomal degradation. These results compared well with subcellular fractionation experiments which showed no significant change in the fraction of apoB accumulated in the lumen of isolated microsomes in MTP-treated cells (P = 0.35). In summary, MTP lipid transfer activity does not appear to influence translocational status of apoB, but its inhibition is associated with an increased susceptibility to proteasome-mediated degradation and reduced assembly and secretion of apoB lipoprotein particles.     

Microsomal triglyceride transfer protein enhances cellular cholesteryl esterification by relieving product inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.     

Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.     

Secretion of lipids, apolipoproteins, and lipoproteins by human hepatoma cell line, HepG2: effects of oleic acid and insulin

The aim of this study was to determine the effect of oleic acid and insulin on the secretion of lipoproteins by HepG2 cells grown in minimum essential medium. Triglycerides were the major neutral lipid (57% of total) and apoB was the predominant apolipoprotein (56% of total) secreted by these cells. The addition of oleate resulted in a two-fold increase in the concentration of neutral lipids but only a slight to moderate increase in the apolipoprotein (A-I, A-II, B, and E) levels. The secretion of very low density lipoproteins (VLDL) was stimulated by 425%, low density lipoproteins (LDL) by 77%, and high density lipoproteins (HDL) by 68%. Whereas neutral lipid composition of LDL was unchanged, the VLDL particles contained a significantly higher percentage of triglyceride and lower percentages of cholesterol and cholesteryl esters compared with VLDL secreted in the absence of oleate. Oleate had no significant effect on the composition of apolipoproteins in VLDL, LDL and HDL. In basal medium, insulin caused a significant decrease in the secretion of neutral lipids and apolipoproteins, particularly triglycerides and apoB. In addition to a 60-68% reduction in the total concentration of VLDL and LDL, insulin altered their composition by producing particles that had a significantly lower content of triglycerides, contained less apoB, and were deficient in apoE. There were no major changes in the concentration or composition of HDL particles. Insulin had a similar but less pronounced effect on the concentration and composition of lipoproteins secreted in the presence of oleate. The increased accumulation of triglycerides in the HepG2 cells concomitant with their reduced levels in the medium suggests that insulin may affect the secretion rather than synthesis of triglyceride-rich lipoproteins.     

ACAT2 stimulates cholesteryl ester secretion in apoB-containing lipoproteins

Previous studies in nonhuman primates revealed a striking positive correlation between liver cholesteryl ester (CE) secretion rate and the development of coronary artery atherosclerosis. CE incorporated into hepatic VLDL is necessarily synthesized by ACAT2, the cholesterol-esterifying enzyme in hepatocytes. We tested the hypothesis that the level of ACAT2 expression, in concert with cellular cholesterol availability, affects the CE content of apolipoprotein B (apoB)-containing lipoproteins. In a model system of lipoprotein secretion using COS cells cotransfected with microsomal triglyceride transfer protein and truncated forms of apoB, ACAT2 expression resulted in a 3-fold increase in microsomal ACAT activity and a 4-fold increase in the radiolabeled CE content of apoB-lipoproteins. After cholesterol-cyclodextrin (Chol-CD) treatment, CE secretion was increased by 27-fold in ACAT2-transfected cells but by only 7-fold in control cells. Chol-CD treatment also caused the percentage of CE in the apoB-lipoproteins to increase from 3% to 33% in control cells and from 16% to 54% in ACAT2-transfected cells. In addition, ACAT2-transfected cells secreted 3-fold more apoB than control cells. These results indicate that under all conditions of cellular cholesterol availability tested, the relative level of ACAT2 expression affects the CE content and, hence, the potential atherogenicity, of nascent apoB-containing lipoproteins.     

Glucosamine-induced endoplasmic reticulum stress attenuates apolipoprotein B100 synthesis via PERK signaling

Glucosamine impairs hepatic apolipoprotein B100 (apoB100) production by inducing endoplasmic reticulum (ER) stress and enhancing cotranslational and posttranslational apoB100 degradation (Qiu, W., R. K. Avramoglu, A. C. Rutledge, J. Tsai, and K. Adeli. Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. J. Lipid Res. 2006. 47: 1749–1761). Here, we report that glucosamine also regulates apoB100 protein synthesis via ER-stress-induced PERK activation. Short-term (4 h) glucosamine treatment of HepG2 cells reduced both cellular (by 62%) and secreted apoB100 (by 43%) without altering apoB100 mRNA. Treatment with proteasomal inhibitors only partially prevented the suppressive effects of glucosamine, suggesting that mechanisms other than proteasomal degradation may also be involved. Glucosamine-induced ER stress was associated with a significantly reduced apoB100 synthesis with no significant change in posttranslational decay rates, suggesting that glucosamine exerted its effect early during apoB biosynthesis. The role of PERK and its substrate, α-subunit of eukaryotic initiation factor 2 (eIF2α), in the suppressive effects of glucosamine on apoB synthesis was then investigated. Coexpression of apoB15 (normally resistant to intracellular degradation) with wild-type double stranded (ds) RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) in COS-7 cells resulted in a dramatic reduction in the levels of newly synthesized apoB15. Interestingly, cotransfection with apoB15 and a kinase inactive PERK mutant (K618A) increased apoB15 expression. In addition, short-term glucosamine treatment stimulated an increase in phosphorylation of PERK and eIF2α. Taken together, these data suggest that in addition to the induction of ER-associated degradation and other degradative pathways, ER stress is associated with suppression of apoB synthesis via a PERK-dependent mechanism.     

Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.     

Co-translational interactions of apoprotein B with the ribosome and translocon during lipoprotein assembly or targeting to the proteasome

Hepatic lipoprotein assembly and secretion can be regulated by proteasomal degradation of newly synthesized apoB, especially if lipid synthesis or lipid transfer is low. Our previous studies in HepG2 cells showed that, under these conditions, newly synthesized apoB remains stably associated with the endoplasmic reticulum (ER) membrane (Mitchell, D. M., Zhou, M., Pariyarath, R., Wang, H., Aitchison, J. D., Ginsberg, H. N., and Fisher, E. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14733-14738). We now show that independent of lipid synthesis, apoB chains that appear full-length are, in fact, incompletely translated polypeptides still engaged by the ribosome and associated with the ER translocon. In the presence of active lipid synthesis and transfer, translation and lipoprotein assembly are completed, and the complexes exit the ER. Upon omitting fatty acids from, or adding a microsomal triglyceride transfer protein inhibitor to, culture media to reduce lipid synthesis or transfer, respectively, apoB was degraded while it remained associated with the ER and complexed with cytosolic hsp70 and proteasomes. Thus, unlike other ER substrates of the proteasome, such as major histocompatibility complex class I molecules, apoB does not fully retrotranslocate to the cytosol before entering the ubiquitin-proteasome pathway. Although, upon immunofluorescence, apoB in proteasome-inhibited cells accumulated in punctate structures similar in appearance to aggresomes (cytosolic structures containing molecules irreversibly lost from the secretory pathway), these apoB molecules could be secreted when lipid synthesis was stimulated. The results suggest a model in which 1) apoB translation does not complete until lipoprotein assembly terminates, and 2) assembly with lipids or entry into the ubiquitin-proteasome pathway occurs while apoB polypeptides remain associated with the translocon and attached to the ribosome.     

Efficient glycosylation site utilization by intracellular apolipoprotein B. Implications for proteasomal degradation

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) may control the secretion of apoB-containing lipoproteins. In one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation behavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-terminal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide cleavage, all of the sites were glycosylated irrespective of cell type, labeling time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn residues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-linked glycosylation occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2. 5-fold increase in intracellular apoB but failed to cause accumulation of an unglycosylated form. These results indicate that 1) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2) despite its large size and affinity for lipid, delivery of misassembled apoB to the proteasome requires retrograde translocation from the ER lumen to cytosol.     

Apple procyanidins decrease cholesterol esterification and lipoprotein secretion in Caco-2/TC7 enterocytes

Decrease of plasma lipid levels by polyphenols was linked to impairment of hepatic lipoprotein secretion. However, the intestine is the first epithelium that faces dietary compounds, and it contributes to lipid homeostasis by secreting triglyceride-rich lipoproteins during the postprandial state. The purpose of this study was to examine the effect of apple and wine polyphenol extracts on lipoprotein synthesis and secretion in human Caco-2/TC7 enterocytes apically supplied with complex lipid micelles. Our results clearly demonstrate that apple, but not wine, polyphenol extract dose-dependently decreases the esterification of cholesterol and the enterocyte secretion of lipoproteins. Apple polyphenols decrease apolipoprotein B (apoB) secretion by inhibiting apoB synthesis without increasing the degradation of the newly synthesized protein. Under our conditions, cholesterol uptake, apoB mRNA, and microsomal triglyceride protein activity were not modified by apple polyphenols. The main monomers present in our mixture did not interfere with the intestinal lipid metabolism. By contrast, apple procyanidins reproduced the inhibition of both cholesteryl ester synthesis and lipoprotein secretion. Overall, our results are compatible with a mechanism of action of polyphenols resulting in impaired lipid availability that could induce the inhibition of intestinal lipoprotein secretion and contribute to the hypolipidemic effect of these compounds in vivo.     

Hepatocellular triglyceride synthesis and transfer to lipid droplets and nascent very low density lipoproteins

The transfer of triglyceride from sites of synthesis in the endoplasmic reticulum to cytoplasmic lipid droplets and nascent VLDL (very low density lipoproteins) in rat liver in vivo has been examined with [3H]glycerol, cell fractionation, and electron microscopy. Rates of mass transfer of newly synthesized triglyceride were estimated from the specific radioactivity of triglyceride present in microsomal membranes and the radioactivity observed in recipient triglyceride pools. Fasting decreased the transfer of triglyceride to nascent VLDL without affecting transfer to lipid droplets. Stimulation of triglyceride synthesis with 2-tetradecylglycidic acid (TDGA) increased transfer of triglyceride to nascent VLDL 5-fold, and to lipid droplets 14-fold, 1 hr after TDGA administration. Triglyceride transfer to nascent VLDL was increased 6-fold, and to lipid droplets 37-fold, above control rates 6 hr following TDGA treatment, indicative of saturation of triglyceride assembly into nascent VLDL and storage of excess triglyceride in lipid droplet reservoirs. These liver triglyceride pools were concurrently expanded and electron microscopy demonstrated more abundant VLDL particles in the endoplasmic reticulum together with a proliferation of lipid droplets in hepatocytes. TDGA progressively decreased hepatic sn-glycerol-3-phosphate in fasting rats while triglyceride synthesis increased, indicating that sn-glycerol-3-phosphate does not limit the rate of triglyceride synthesis in this metabolic state. Results implicate triglyceride transfer from endoplasmic reticulum membranes to nascent VLDL as a regulated determinant of hepatic VLDL assembly and VLDL triglyceride secretion in vivo.     

Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipid-binding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270-570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins.