Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells

Cholesteryl ester transfer protein (CETP) promotes reverse cholesterol transport via exchange of cholesteryl ester and triglyceride among lipoproteins. Here, we focused on HDL metabolism during inhibition of CETP expression by using CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells. CETP secretion was decreased by 70% in mRNA levels and by 52% in mass 20 h after ODNs against CETP were delivered to HepG2 cells. Furthermore, as a consequence of the downregulation of CETP, the expression of scavenger receptor class B type I (SR-BI), an HDL receptor, was also reduced by approximately 50% in mRNA and protein levels, whereas the apolipoprotein A-I (apoA-I) expression and secretion were increased by 30 and 92%, respectively. In a functional study, the selective uptake of (125)I-[(14)C]cholesteryl oleate-labeled HDL(3) was decreased. Cholesterol efflux to apoA-I and HDL(3) was significantly increased by 88 and 37%, respectively. Moreover, the CE levels in cells after antisense treatment were elevated by 20%, which was related to the about twofold increase of cholesterol esterification and increased acyl-CoA:cholesterol acyltransferase 1 mRNA levels. Taken together, these findings suggest that although acute suppression of CETP expression leads to an elevation in cellular cholesterol stores, apoA-I secretion, and cellular cholesterol efflux to apoA-I, the return of HDL-CE to hepatocytes via an SR-BI pathway was inhibited in vitro. Thus antisense inhibition of hepatic CETP expression manifests dual effects: namely, increased formation of HDL and suppression of catabolism of HDL-CE, probably via the SR-BI pathway.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

The amphipathic alpha-helical repeats of apolipoprotein A-I are responsible for binding of high density lipoproteins to HepG2 cells.

Nine monoclonal antibodies (mAbs) against apoA-I reacting with distinct but overlapping epitopes covering more than 90% of the sequence have been used to block the interaction of 125I-labeled high density lipoprotein (125I-HDL) with HepG2 cells in order to delineate the cell binding domain of apolipoprotein A-I (apoA-I). While 2 mAbs reacting with epitopes exclusively localized in the N-terminal region (residues 1 to 86) enhanced slightly association of 125I-HDL, all other mAbs, which react with epitopes localized in the regions of amphipathic alpha-helical repeats, inhibited that association by 9 to 15%. Although this inhibition is not significant compared to the effect of an irrelevant mAb, combination of these mAbs could significantly inhibit the association of 125I-HDL (32 to 43%) as could polyclonal antibodies (up to 95%). These results are compatible with the concept of HDL binding to these cells via the nonexclusive interaction of each of the amphipathic alpha-helical repeats of apoA-I. When the same approach was applied to block the association of 3H-cholesteryl ether (CE)-labeled HDL to HepG2 cells, each anti-apoA-I could inhibit by 15 to 25% the cellular association of cholesteryl ether while mAbs in combination or polyclonal antibodies could inhibit this association up to 45% or 60%, respectively. The cholesteryl ether radioactivity that remained associated with the cells (40%) in the presence of polyclonal antibodies could be effectively blocked by addition of an antibody against the receptor binding domain of apoE (1D7). Therefore, the differential cellular association of cholesteryl ether compared to apolipoprotein can be explained by the presence of apoE secreted by HepG2 and apoE or apoB/E receptors. Thus, we conclude that the optimum uptake of both cholesteryl ether and apoA-I of HDL by cells requires the accessibility of the entire apoA-I and the cooperative binding of the amphipathic alpha-helical repeats to HepG2 cell membranes. This type of interaction would explain the competitive binding observed for apoA-I, -A-II, and -A-IV by others.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Identification of an ApoA-I ligand domain that interacts with high-affinity binding sites on HepG2 cells

We have previously described the presence of two (high- and low-affinity) HDL binding sites on the hepatoma cell line (HepG2) (R. Barbaras, X. Collet, H. Chap, and B. Perret (1994) Biochemistry 33, 2335-2340]. Moreover, apoA-I, the major HDL apolipoprotein, interacts with these two binding sites, while lipid-free apoA-I binds only to the high-affinity sites. Using tryptic HDL fragments and HepG2 cell monolayers as an "affinity matrix," we identified an apoA-I peptide of 16 amino acids, spanning between residues 62 and 77, as a ligand domain. The corresponding synthetic peptide displays high-affinity (K(d) approximately 10(-7) M) and low-capacity (B(max) 8 pmol/mg of cell protein) binding components. Competition experiments with this peptide, using (125)I-labeled free apoA-I as a ligand, show that this binding corresponds to the high-affinity binding sites already described. In conclusion, we identified the apoA-I 62-77 region as a specific high-affinity ligand domain of HDL on HepG2 cells.     

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.     

Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake

Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that (125)I-labeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30-50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.     

Comparative acyl specificities for transfer and selective uptake of high density lipoprotein cholesteryl esters

This study compares the specificities of selective uptake and transfer mediated by plasma cholesteryl ester transfer protein (CETP) for various species of cholesteryl esters in high density lipoproteins (HDL). [3H]Cholesterol was esterified with a series of variable chain length saturated acids and a series of variably unsaturated 18-carbon acids. These were incorporated into synthetic HDL particles along with 125I-labeled apoA-I as a tracer of HDL particles and [14C]cholesteryl oleate as an internal standard for normalization between preparations. Selective uptake by Y1-BS1 mouse adrenal cortical tumor cells was most extensively studied, but uptake by human HepG2 hepatoma cells and fibroblasts of human, rat, and rabbit origin were also examined. Acyl chain specificities for selective uptake and for CETP-mediated transfer were conversely related; selective uptake by all cell types decreased with increasing acyl chain length and increased with the extent of unsaturation of C18 chains. In contrast, CETP-mediated transfer increased with acyl chain length, and decreased with unsaturation of C18 chains. The specificities of human and rabbit CETP were also compared, and were found to differ little. Associated experiments showed that HDL-associated triglycerides, traced by [3H]glyceryl trioleyl ether, were selectively taken up but at a lesser rate than cholesteryl esters. The mechanism of this uptake appears to be the same as for selective uptake of cholesteryl esters.     

Proteolytic degradation of HDL1 on the fat cell surface is nutritionally regulated

The importance of plasma HDL apolipoprotein concentration as a predictor of atherosclerotic risk is well recognized, yet the processes of HDL modification and degradation in various cells are not clearly understood. We examined the characteristics of HDL1 apolipoprotein degradation and cellular uptake by rat adipocytes and determined the effects of fasting on these processes. Epididymal and perirenal adipocytes were isolated from male Wistar rats (310 +/- 4 g) fed ad libidum and incubated with 5 micrograms of rat 125I-labeled HDL1 (d: 1.07-1.10 g/mL) mL-1 for 2 h at 37 degrees C. Cellular uptake of HDL1 was calculated as the trichloroacetic acid precipitable radioactivity associated with adipocytes following incubation. Intracellular and medium degradation of HDL1 were determined as trichloroacetic acid soluble 125I counts associated with cells and measured in the postincubation medium, respectively. Fifty to sixty percent of cellular uptake and degradation of HDL1 was inhibited by the addition of 25-fold excess unlabeled HDL. HDL1 degradation measured in the medium was 10- to 12-fold greater than cellular uptake of HDL1 apolipoproteins. Intracellular degradation of HDL1 was negligible. The presence of EDTA in the incubation medium reduced HDL1 degradation measured in the medium, but enhanced HDL1 cellular uptake. Conditioned medium separated from cells after 2 h of incubation at 37 degrees C in the absence of HDL and subsequently incubated with 125I-labeled HDL1 for an additional 2 h at 37 degrees C, degraded less than 5% of HDL compared with degradation in the presence of cells. These results suggest that rat adipocytes degrade, or modify, HDL1 particles, possibly by interactions with cell surface proteases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Niacin noncompetitively inhibits DGAT2 but not DGAT1 activity in HepG2 cells

Niacin is a widely used lipid-regulating agent in dyslipidemic patients. Previously, we have shown that niacin inhibits triacylglycerol synthesis. In this report, using HepG2 cells, we have examined the effect of niacin on the mRNA expression and microsomal activity of diacylglycerol acyltransferase 1 and 2 (DGAT1 and DGAT2), the last committed but distinctly different enzymes for triglyceride synthesis. Addition of niacin to the DGAT assay reaction mixture dose-dependently (0-3 mM) inhibited DGAT activity by 35-50%, and the IC(50) was found to be 0.1 mM. Enzyme kinetic studies showed apparent K(m) values of 8.3 microM and 100 microM using [(14)C]oleoyl-CoA and sn-1,2-dioleoylglycerol as substrates, respectively. A decrease in apparent V(max) was observed with niacin, whereas the apparent K(m) remained constant. A Lineweaver-Burk plot of DGAT inhibition by niacin showed a noncompetitive type of inhibition. Niacin selectively inhibited DGAT2 but not DGAT1 activity. Niacin inhibited overt DGAT activity. Niacin had no effect on the expression of DGAT1 and DGAT2 mRNA. These data suggest that niacin directly and noncompetitively inhibits DGAT2 but not DGAT1, resulting in decreased triglyceride synthesis and hepatic atherogenic lipoprotein secretion, thus indicating a major target site for its mechanism of action.     

The ��-subunit of ATP synthase is involved in cellular uptake and resecretion of apoA-I but does not control apoA-I-induced lipid efflux in adipocytes

Cellular uptake and resecretion of apoA-I (apoA-I recycling) could be an important factor in determining the circulating plasma levels of apoA-I and/or HDL. Using a novel method to study protein recycling, we have recently demonstrated recycling of apoA-I by adipocytes and suggested that this is a receptor mediated process independent of ABCA1 function. In the present study, it is shown that apoA-I recycling by adipocytes can be blocked by a monoclonal antibody against the β-subunit of ATP synthase, a protein that had been previously identified as an apoA-I receptor. Investigation of the cellular recycling of two other proteins, an apolipoprotein and a small globular protein, showed that recycling of apoA-I is a selective process. The present study also shows that blocking apoA-I recycling has no effect on the rate of apoA-I-induced cholesterol or phospholipid efflux. It is concluded that cellular recycling of apoA-I is a selective process that involves the ectopically expressed β-subunit of ATP synthase. The physiological function of apoA-I recycling remains to be elucidated. However, this study shows that the process of apoA-I uptake and resecretion is not required for apoA-I lipidation.     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.     

Niacin and cholesterol: role in cardiovascular disease (review)

Niacin has been widely used as a pharmacologic agent to regulate abnormalities in plasma lipid and lipoprotein metabolism and in the treatment of atherosclerotic cardiovascular disease. Although the use of niacin in the treatment of dyslipidemia has been reported as early as 1955, only recent studies have yielded an understanding about the cellular and molecular mechanism of action of niacin on lipid and lipoprotein metabolism. In brief, the beneficial effect of niacin to reduce triglycerides and apolipoprotein-B containing lipoproteins (e.g., VLDL and LDL) are mainly through: a) decreasing fatty acid mobilization from adipose tissue triglyceride stores, and b) inhibiting hepatocyte diacylglycerol acyltransferase and triglyceride synthesis leading to increased intracellular apo B degradation and subsequent decreased secretion of VLDL and LDL particles. The mechanism of action of niacin to raise HDL is by decreasing the fractional catabolic rate of HDL-apo AI without affecting the synthetic rates. Additionally, niacin selectively increases the plasma levels of Lp-AI (HDL subfraction without apo AII), a cardioprotective subfraction of HDL in patients with low HDL. Using human hepatocytes (Hep G2 cells) as an in vitro model system, recent studies indicate that niacin selectively inhibits the uptake/removal of HDL-apo AI (but not HDL-cholesterol ester) by hepatocytes, thereby increasing the capacity of retained HDL-apo AI to augment cholesterol efflux through reverse cholesterol transport pathway. The studies discussed in this review provide evidence to extend the role of niacin as a lipid-lowering drug beyond its role as a vitamin.     

Role of the kidney in regulating the metabolism of HDL in rabbits: evidence that iodination alters the catabolism of apolipoprotein A-I by the kidney

To evaluate the factors that regulate HDL catabolism in vivo, we have measured the clearance of human apoA-I from rabbit plasma by following the isotopic decay of (125)I-apoA-I and the clearance of unlabeled apoA-I using a radioimmunometric assay (RIA). We show that the clearance of unlabeled apoA-I is 3-fold slower than that of (125)I-apoA-I. The mass clearance of iodinated apoA-I, as determined by RIA, is superimposable with the isotopic clearance of (125)I-apoA-I. The data demonstrate that iodination of tyrosine residues alters the apoA-I molecule in a manner that promotes an accelerated catabolism. The clearance from rabbit plasma of unmodified apoA-I on HDL(3) and a reconstituted HDL particle (LpA-I) were very similar and about 3-4-fold slower than that for (125)I-apoA-I on the lipoproteins. Therefore, HDL turnover in the rabbit is much slower than that estimated from tracer kinetic studies. To determine the role of the kidney in HDL metabolism, the kinetics of unmodified apoA-I and LpA-I were reevaluated in animals after a unilateral nephrectomy. Removal of one kidney was associated with a 40-50% reduction in creatinine clearance rates and a 34% decrease in the clearance rate of unlabeled apoA-I and LpA-I particles. In contrast, the clearance of (125)I-labeled molecules was much less affected by the removal of a kidney; FCR for (125)I-LpA-I was reduced by <10%. The data show that the kidneys are responsible for most (70%) of the catabolism of apoA-I and HDL in vivo, while (125)I-labeled apoA-I and HDL are rapidly catabolized by different tissues. Thus, the kidney is the major site for HDL catabolism in vivo. Modification of tyrosine residues on apoA-I may increase its plasma clearance rate by enhancing extra-renal degradation pathways.     

HDL modification by secretory phospholipase A(2) promotes scavenger receptor class B type I interaction and accelerates HDL catabolism

During inflammatory states plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I) are reduced. Secretory group IIa phospholipase A(2) (sPLA(2)) is a cytokine-induced acute-phase enzyme associated with HDL. Transgenic mice overexpressing sPLA(2) have reduced HDL levels. Studies were performed to define the mechanism for the HDL reduction in these mice. HDL isolated from sPLA(2) transgenic mice have a significantly lower phospholipid content and greater triglyceride content. In autologous clearance studies, (125)I-labeled HDL from sPLA(2) transgenic mice was catabolized significantly faster than HDL from control mice (4.24 +/- 1.16 vs. 2.84 +/- 0.1 pools per day, P < 0.008). In both sPLA(2) transgenic and control mice, the cholesteryl ester component of HDL was more rapidly catabolized than the protein component, indicating a selective uptake mechanism. In vitro studies using CHO cells transfected with scavenger receptor class B type I (SR-BI) showed that sPLA(2)-modified HDL was nearly twice as efficient as a substrate for cholesteryl ester transfer. These data were confirmed in in vivo selective uptake experiments using adenoviral vector overexpression of SR-BI. In these studies, increased hepatic selective uptake was associated with increased (125)I-labeled apolipoprotein uptake in the kidney.We conclude that during inflammation sPLA(2) hydrolysis of HDL phospholipids alters the lipid composition of the particle, allowing for more efficient SR-BI-mediated selective cholesteryl ester uptake. This enhanced SR-BI activity generates HDL remnants that are preferentially catabolized in the kidney.     

Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts

Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated (125)I-HDL(3) secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4-7% of the total cell-associated HDL) that is almost complete within 30 min (half-life approximately 10 min). In COS-7 cells, the calculated rate of HDL secretion ( approximately 0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake ( approximately 17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface ( approximately 19 ng HDL/mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.     

Testosterone up-regulates scavenger receptor BI and stimulates cholesterol efflux from macrophages

By lowering high density lipoprotein (HDL) cholesterol, testosterone contributes to the gender difference in HDL cholesterol and has been accused to be pro-atherogenic. The mechanism by which testosterone influences HDL cholesterol is little understood. We therefore investigated the effect of testosterone on the gene expression of apolipoprotein A-I (apoA-I), hepatic lipase (HL), scavenger receptor B1 (SR-BI), and the ATP binding cassette transporter A1 (ABCA1), all of which are important regulators of HDL metabolism. In both cultivated HepG2 hepatocytes and primary human monocyte-derived macrophages, testosterone led to a dose-dependent up-regulation of SR-BI, which was assessed on both the mRNA and the protein levels. As a functional consequence, we observed an increased HDL(3)-induced cholesterol efflux from macrophages. At supraphysiological dosages, testosterone also increased the expression of HL in HepG2 cells. Testosterone had no effect on the expression of apoA-I in HepG2 cells and ABCA1 in either HepG2 cells or macrophages. These data suggest that testosterone, despite lowering HDL cholesterol, intensifies reverse cholesterol transport and thereby exerts an anti-atherogenic rather than a pro-atherogenic effect.     

Apolipoprotein A-I assayed in human serum by isotope dilution as a potential standard for immunoassay

We measured the amount of apoA-I in serum by isotope dilution, finding 1.33 mg/ml (standard deviation 0.177) in six normolipidemic, healthy subjects. We developed this method by adapting published techniques to purify apoA-I from 3 ml of serum in two steps: density gradient ultracentrifugation and high performance liquid chromatography gel filtration. The 125I-labeled apoA-I tracer was first screened, by incubation with serum, to select labeled apoA-I which retained the ability to exchange with native apoA-I and bind to HDL. A known amount of 125I-labeled apoA-I-labeled HDL was added to unknown serum samples; apoA-I was reisolated from the serum and its specific radioactivity was used to calculate the dilution of the added, labeled apoA-I by the unlabeled apoA-I in the unknown serum. By not relying on immunochemical techniques, the isotope dilution assay provided results that are independent of the expression of individual apoA-I antigenic sites. Therefore, sera that have been assayed by isotope dilution can serve as standards to evaluate the accuracy of immunoassays for serum apoA-I and provide primary standards for such immunoassays.