High Density Lipoprotein Cholesteryl Research Articles

Total Outflow of High-Density Lipoprotein–Cholesteryl Esters from Plasma Is Decreased in a Model of 3/4 Renal Mass Reduction

(1) Background: Previous studies have enriched high-density lipoproteins (HDL) using cholesteryl esters in rabbits with a three-quarter reduction in functional renal mass, suggesting that the kidneys participate in the cholesterol homeostasis of these lipoproteins. However, the possible role of the kidneys in lipoprotein metabolism is still controversial. To understand the role of the kidneys in regulating the HDL lipid content, we determined the turnover of HDL-cholesteryl esters in rabbits with a three-quarter renal mass reduction. (2) Methods: HDL subclass characterization was conducted, and the kinetics of plasma HDL-cholesteryl esters, labeled with tritium, were studied in rabbits with a 75% reduction in functional renal mass (Ntx). (3) Results: The reduced renal mass triggered the enrichment of cholesterol, specifically cholesteryl esters, in HDL subclasses. The exchange of cholesteryl esters between HDL and apo B-containing lipoproteins (VLDL/LDL) was not significantly modified in Ntx rabbits. Moreover, the cholesteryl esters of HDL and VLDL/LDL fluxes from the plasmatic compartment tended to decrease, but they only reached statistical significance when both fluxes were added to the Nxt group. Accordingly, the fractional catabolic rate (FCR) of the HDL-cholesteryl esters was lower in Ntx rabbits, concomitantly with its accumulation in HDL subclasses, probably because of the reduced mass of renal cells requiring this lipid from lipoproteins.

Identification of Novel Compounds Enhancing SR-BI mRNA Stability through High-Throughput Screening.

Atherosclerosis is the pathological basis of most cardiovascular diseases. Reverse cholesterol transport (RCT) is a main mechanism of cholesterol homeostasis and involves the direct transport of high-density lipoprotein (HDL) cholesteryl ester by selective cholesterol uptake. Hepatic scavenger receptor class B member 1 (SR-BI) overexpression can effectively promote RCT and reduce atherosclerosis. SR-BI may be an important target for prevention or treatment of atherosclerotic disease. In our study, we inserted human SR-BI mRNA 3' untranslated region (3'UTR) downstream of the luciferase reporter gene, to establish a high-throughput screening model based on stably transfected HepG2 cells and to screen small-molecule compounds that can significantly enhance the mRNA stability of the SR-BI gene. Through multiple screenings of 25 755 compounds, the top five active compounds that have similar structures were obtained, with a positive rate of 0.19%. The five positive compounds could enhance the SR-BI expression and uptake of DiI-HDL in the hepatocyte HepG2. E238B-63 could also effectively extend the half-life of SR-BI mRNA and enhance the SR-BI mRNA and protein level and the uptake of DiI-HDL in hepatocytes in a time-dependent and dose-dependent manner. The structure-activity relationship analysis showed that the structure N-(3-hydroxy-2-pyridyl) carboxamide is possibly the key pharmacophore of the active compound, providing reference for acquiring candidate compounds with better activity. The positive small molecular compounds obtained in this study might become new drug candidates or lead compounds for the treatment of cardiovascular diseases and contribute to the further study of the posttranscriptional regulation mechanism of the SR-BI gene.

Incorporation of whole oat, especially bran, into a high-fat diet, improves cardio-metabolic risk factors in type 2 diabetic rats

PurposeThe purpose of this study was to determine the effects of whole oat, oat bran and refined oat incorporation in a high-fat diet (HFD) on cardio-metabolic risk biomarkers in rats with type 2 diabetes mellitus (T2DM).Design/methodology/approachT2DM was induced by feeding male rats with an HFD for 10 weeks, followed by a low dose of streptozotocin. T2DM rats were then divided into four homogeneous groups. Three groups consumed an HFD containing 45 per cent (g/100 g diet) whole oat, oat bran or refined oat. The fourth untreated group (control) received the HFD.FindingsThe results showed that whole oat and oat bran, compared with refined oat and control, effectively reduced food intake (p < 0.007), arterial blood pressure (p = 0.0001), glycemia (p < 0.001), insulinemia (p < 0.01), glycosylated haemoglobin (p < 0.001) as well as homeostasis insulin resistance (HOMA-IR) (p < 0.001). They also improved blood lipid levels and reverse cholesterol transport by reducing serum total cholesterol (p = 0.0001), triacylglycerols (p < 0.05), very-low- (p = 0.0001) and low-density lipoproteins cholesterol contents (p < 0.02) increasing lipids (p < 0.002) and cholesterol excretion (p = 0.0001), and high-density lipoprotein cholesteryl esters (HDL2-CE) concentrations (p = 0.0001) and stimulating lecithin: cholesterol acyltransferase (LCAT) activity (p = 0.0001). Moreover, they attenuated lipid peroxidation by increasing paraoxonase-1 (PON-1) atheroprotective activity (p < 0.05).Originality/valueIn T2DM rats, whole oat and particularly, its bran incorporated into an HFD improves arterial blood pressure, glycemic balance and lipid metabolic pathway by reducing hypertriglyceridemia and hypercholesterolemia and increasing atheroprotective activities of LCAT and PON-1. In contrast, refined oat accentuates the risk factors associated with diabetes.

Blood Metabolomic Measures Associate With Present and Future Glycemic Control in Type 2 Diabetes.

We studied whether blood metabolomic measures in people with type 2 diabetes (T2D) are associated with insufficient glycemic control and whether this association is influenced differentially by various diabetes drugs. We then tested whether the same metabolomic profiles were associated with the initiation of insulin therapy. A total of 162 metabolomic measures were analyzed using a nuclear magnetic resonance-based method in people with T2D from four cohort studies (n = 2641) and one replication cohort (n = 395). Linear and logistic regression analyses with adjustment for potential confounders, followed by meta-analyses, were performed to analyze associations with hemoglobin A1c levels, six glucose-lowering drug categories, and insulin initiation during a 7-year follow-up period (n = 698). After Bonferroni correction, 26 measures were associated with insufficient glycemic control (HbA1c >53 mmol/mol). The strongest association was with glutamine (OR, 0.66; 95% CI, 0.61 to 0.73; P = 7.6 × 10-19). In addition, compared with treatment-naive patients, 31 metabolomic measures were associated with glucose-lowering drug use (representing various metabolite categories; P ≤ 3.1 × 10-4 for all). In drug-stratified analyses, associations with insufficient glycemic control were only mildly affected by different glucose-lowering drugs. Five of the 26 metabolomic measures (apolipoprotein A1 and medium high-density lipoprotein subclasses) were also associated with insulin initiation during follow-up in both discovery and replication. The strongest association was observed for medium high-density lipoprotein cholesteryl ester (OR, 0.54; 95% CI, 0.42 to 0.71; P = 4.5 × 10-6). Blood metabolomic measures were associated with present and future glycemic control and might thus provide relevant cues to identify those at increased risk of treatment failure.

Obesity-induced overexpression of miRNA-24 regulates cholesterol uptake and lipid metabolism by targeting SR-B1

Scavenger Receptor B1 (SR-B1) is an 82 kDa integral membrane glycoprotein that mediates selective uptake of high-density lipoprotein cholesteryl ester (CE) in vitro and in vivo. Previously, we defined several kinds of regulatory mechanisms of SR-B1 expression and function. Here, we have dissected the function of a novel miR-24 on SR-B1 expression, HDL uptake and lipid metabolism. We showed that miR-24 was upregulated in HepG2 cells cultured in the mimicked hyperlipidemic condition and in the livers of dietary induced and genetic obesity mice. Overexpression of miR-24 inhibited SR-B1 expression by directly targeting SR-B1 3′ UTR and repressed HDL uptake and steroidogenesis in steroidogenic cells. HepG2 cells with miR-24 showed attenuation of TG levels and lipid accumulation. Moreover, we validated that overexpression of miR-24 downregulated the expression of certain genes involved in lipogenesis, FASN, ACLY and SCD1, and increased the expression of genes of cholesterol synthesis, HMGCR, DHCR24 and SREBP2. Taken together, we demonstrated that obesity induced miR-24 repressed HDL uptake, steroid hormone synthesis and lipid metabolism by targeting SR-B1.

Nicotinic Acid Accelerates HDL Cholesteryl Ester Turnover in Obese Insulin-Resistant Dogs.

AimNicotinic acid (NA) treatment decreases plasma triglycerides and increases HDL cholesterol, but the mechanisms involved in these change are not fully understood. A reduction in cholesteryl ester transfer protein (CETP) activity has been advanced to explain most lipid-modulating effects of NA. However, due to the central role of CETP in reverse cholesterol transport in humans, other effects of NA may have been hidden. As dogs have no CETP activity, we conducted this study to examine the specific effects of extended-release niacin (NA) on lipids and high-density lipoprotein (HDL) cholesteryl ester (CE) turnover in obese Insulin-Resistant dogs with increase plasma triglycerides.MethodsHDL kinetics were assessed in fasting dogs before and four weeks after NA treatment through endogenous labeling of cholesterol and apolipoprotein AI by simultaneous infusion of [1,2 13C2] acetate and [5,5,5 2H3] leucine for 8 h. Kinetic data were analyzed by compartmental modeling. In vitro cell cholesterol efflux of serum from NA-treated dogs was also measured.ResultsNA reduced plasma total cholesterol, low-density lipoprotein cholesterol, HDL cholesterol, triglycerides (TG), and very-low-density lipoprotein TG concentrations (p < 0.05). The kinetic study also showed a higher cholesterol esterification rate (p < 0.05). HDL-CE turnover was accelerated (p < 0.05) via HDL removal through endocytosis and selective CE uptake (p < 0.05). We measured an elevated in vitro cell cholesterol efflux (p < 0.05) with NA treatment in accordance with a higher cholesterol esterification.ConclusionNA decreased HDL cholesterol but promoted cholesterol efflux and esterification, leading to improved reverse cholesterol transport. These results highlight the CETP-independent effects of NA in changes of plasma lipid profile.

Cooperation between hepatic cholesteryl ester hydrolase and scavenger receptor BI for hydrolysis of HDL-CE

Liver is the sole organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or bile acids. High density lipoprotein (HDL)-derived cholesterol is the major source of biliary sterols and represents a mechanism for the removal of cholesterol from peripheral tissues including artery wall-associated macrophage foam cells. Via selective uptake through scavenger receptor BI (SR-BI), HDL-cholesterol is thought to be directly secreted into bile, and HDL cholesteryl esters (HDL-CEs) enter the hepatic metabolic pool and need to be hydrolyzed prior to conversion to bile acids. However, the identity of hepatic CE hydrolase (CEH) as well as the role of SR-BI in bile acid synthesis remains elusive. In this study we examined the role of human hepatic CEH (CES1) in facilitating hydrolysis of SR-BI-delivered HDL-CEs. Over-expression of CEH led to increased hydrolysis of HDL-[³H]CE in primary hepatocytes and SR-BI expression was required for this process. Intracellular CEH associated with BODIPY-CE delivered by selective uptake via SR-BI. CEH and SR-BI expression enhanced the movement of [³H]label from HDL-[³H]CE to bile acids in vitro and in vivo. Taken together, these studies demonstrate that SR-BI-delivered HDL-CEs are hydrolyzed by hepatic CEH and utilized for bile acid synthesis.

MicroRNAs 125a and 455 repress lipoprotein-supported steroidogenesis by targeting scavenger receptor class B type I in steroidogenic cells.

We sought to identify and characterize microRNA (miRNAs) that posttranscriptionally regulate the expression of scavenger receptor class B type I (SR-BI) and SR-BI-linked selective high-density lipoprotein (HDL) cholesteryl ester (CE) transport and steroidogenesis. Four miRNAs (miRNA-125a, miRNA-125b, miRNA-145, and miRNA-455) with a potential to regulate SR-BI were identified in silico and validated by quantitative real-time PCR (qRT-PCR), Western blot analysis, and SR-BI 3' untranslated region (UTR) reporter assays. In vitro treatment of primary rat granulosa cells and MLTC-1 cells with cyclic AMP (cAMP) or in vivo treatment of rat adrenals with adrenocorticotropic hormone (ACTH) decreased the expression of miRNA-125a, miRNA-125b, and miRNA-455 and reciprocally increased SR-BI expression. Using luciferase constructs containing the 3' untranslated region of SR-BI combined with miRNA overexpression and mutagenesis, we have provided evidence that steroidogenic SR-BI is a direct target of miRNA-125a and miRNA-455. Moreover, the transfection of Leydig tumor cells with precursor miRNA 125a (pre-miRNA-125a) or pre-miRNA-455 resulted in the suppression of SR-BI at both the transcript and protein levels and reduced selective HDL CE uptake and HDL-stimulated progesterone production. Transfection of liver Hepa 1-6 cells with pre-miRNA-125a significantly reduced SR-BI expression and its selective transport function. In contrast, overexpression of miRNA-145 did not affect SR-BI expression or selective HDL CE uptake mediated by SR-BI in steroidogenic cell lines. These data suggest that a trophic hormone and cAMP inversely regulate the expression of SR-BI and miRNA-125a and miRNA-455 in steroidogenic tissues/cells and that both miRNA-125a and miRNA-455, by targeting steroidogenic SR-BI, negatively regulate selective HDL CE uptake and HDL CE-supported steroid hormone production.

High-capacity selective uptake of cholesteryl ester from native LDL during macrophage foam cell formation

Macrophage foam cells are a defining pathologic feature of atherosclerotic lesions. Recent studies have demonstrated that at high concentrations associated with hypercholesterolemia, native LDL induces macrophage lipid accumulation. LDL particles are taken up by macrophages as part of bulk fluid pinocytosis. However, the uptake and metabolism of cholesterol from native LDL during foam cell formation has not been clearly defined. Previous reports have suggested that selective cholesteryl ester (CE) uptake might contribute to cholesterol uptake from LDL independently of particle endocytosis. In this study we demonstrate that the majority of macrophage LDL-derived cholesterol is acquired by selective CE uptake in excess of LDL pinocytosis and degradation. Macrophage selective CE uptake does not saturate at high LDL concentrations and is not down-regulated during cholesterol accumulation. In contrast to CE uptake, macrophages exhibit little selective uptake of free cholesterol (FC) from LDL. Following selective uptake from LDL, CE is rapidly hydrolyzed by a novel chloroquine-sensitive pathway. FC released from LDL-derived CE hydrolysis is largely effluxed from cells but also is subject to ACAT-mediated reesterification. These results indicate that selective CE uptake plays a major role in macrophage metabolism of LDL.

Reverse cholesterol transport is elevated in carboxyl ester lipase‐knockout mice

Mechanisms to increase reverse cholesterol transport (RCT) and biliary sterol disposal are currently sought to prevent atherosclerosis. Previous work with HepG2 cells and primary hepatocytes showed that carboxyl ester lipase (CEL), a broad-spectrum lipase secreted by pancreas and liver, plays an important role in hydrolysis of high-density lipoprotein (HDL) cholesteryl esters (CEs) after selective uptake by hepatocytes. The effect of CEL on RCT of HDL cholesterol was assessed by measuring biliary and fecal disposal of radiolabeled HDL-CE in control and Cel(-/-) mice. Radiolabeled CE was increased 3-fold in hepatic bile of Cel(-/-) mice, and the mass of CE in gall bladder bile was elevated. Total radiolabeled transport from plasma to hepatic bile was more rapid in Cel(-/-) mice. Fecal disposal of radiolabel from HDL-CE, as well as total sterol mass, was markedly elevated for Cel(-/-) mice, primarily due to more CE. RCT of macrophage CE was also increased in Cel(-/-) mice, as measured by excretion of radiolabel from injected J774 cells. Increased sterol loss was compensated by increased cholesterol synthesis in Cel(-/-) mice. Together, the data demonstrate significantly increased RCT in the absence of CEL and suggest a novel mechanism by which to manipulate plasma cholesterol flux.

Expression of the Scavenger Receptor Class B type I (SR-BI) family in Drosophila melanogaster

In mammals, cholesterol is transformed into steroid hormones in the adrenal gland, the ovaries or the testes. The Scavenger Receptors Class B Type I (SR-BI) are membrane proteins that belong to the CD36 family and participate in the selective uptake of high density lipoprotein cholesteryl ester in the mammalian steroidogenic tissues. Fourteen members of the CD36 family have been identified in Diptera, although their expression patterns remain uncharacterized. Using in situ hybridization we have characterized the expression patterns of the fourteen SR-BIs in Drosophila melanogaster. We analyzed three different developmental larval stages prior to and during the peak of the insect steroid hormone ecdysone, which triggers the larval to pupal transition. We focused on the steroidogenic tissues, such as the prothoracic gland, the ovaries and the testes, and extended our analysis to non-steroidogenic tissues, such as the fat body, salivary glands, the gut, the gastric caeca or the central nervous system. Our results show highly regulated expression patterns, with three genes crq, pes and Snmp being upregulated in steroidogenic tissues at the onset of pupariation when steroidogenesis is crucial. This study underlines the importance of the transport of cholesterol and steroids in the process of ecdysone synthesis.

Paraoxonase 1 deficiency in mice is associated with reduced steroid biosynthesis: Effects on HDL binding, cholesteryl ester accumulation and scavenger receptor type BI expression

ObjectiveSelective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) is considered as the major source of cholesterol for production of steroids in the adrenal gland in rodents. As paraoxonase 1 (PON1) is an HDL-associated lipo-lactonase that has been shown to increase binding of HDL to macrophages, we used PON1 knock-out (PON1KO) mice to test the possible role of PON1 in corticosterone (CS) biosynthesis. Methods and resultsPON1 deficiency was associated with reduced serum CS concentration. Adrenal glands obtained from PON1KO mice had significantly lower CE content compared to adrenals from C57Bl6 control mice. Binding of HDL obtained from PON1KO mice to human adrenocortical carcinoma cell line was found to be significantly lower than that of control HDL, and was associated with decreased CS biosynthesis. Addition of purified PON1 to HDL from PON1KO mice increased HDL binding and CS synthesis. Furthermore, the expression of the HDL receptor, SR-BI, protein and mRNA, was reduced in adrenals from PON1KO mice compared to control mice. When challenged with low salt diet, PON1KO mice demonstrated an increase in adrenal SR-BI gene expression and in serum corticosterone which reached levels similar to those obtained in control mice. ConclusionPON1 regulates adrenal CS biosynthesis at two levels: (a) via an accessory role in HDL binding properties, and (b) a supportive role in SR-BI expression and CE supply to the cells.

Hepatic SR-BI, not endothelial lipase, expression determines biliary cholesterol secretion in mice

High density lipoprotein cholesterol is thought to represent a preferred source of sterols secreted into bile following hepatic uptake by scavenger receptor class B type I (SR-BI). The present study aimed to determine the metabolic effects of an endothelial lipase (EL)-mediated stimulation of HDL cholesterol uptake on liver lipid metabolism and biliary cholesterol secretion in wild-type, SR-BI knockout, and SR-BI overexpressing mice. In each model, injection of an EL expressing adenovirus decreased plasma HDL cholesterol (P < 0.001) whereas hepatic cholesterol content increased (P < 0.05), translating into decreased expression of sterol-regulatory element binding protein 2 (SREBP2) and its target genes HMG-CoA reductase and LDL receptor (each P < 0.01). Biliary cholesterol secretion was dependent on hepatic SR-BI expression, being decreased in SR-BI knockouts (P < 0.001) and increased following hepatic SR-BI overexpression (P < 0.001). However, in each model, biliary secretion of cholesterol, bile acids, and phospholipids as well as fecal bile acid and neutral sterol content, remained unchanged in response to EL overexpression. Importantly, hepatic ABCG5/G8 expression did not correlate with biliary cholesterol secretion rates under these conditions. These results demonstrate that an acute decrease of plasma HDL cholesterol levels by overexpressing EL increases hepatic cholesterol content but leaves biliary sterol secretion unaltered. Instead, biliary cholesterol secretion rates are related to the hepatic expression level of SR-BI. These data stress the importance of SR-BI for biliary cholesterol secretion and might have relevance for concepts of reverse cholesterol transport.

Antiproteinuric therapy decreases LDL-cholesterol as well as HDL-cholesterol in non-diabetic proteinuric patients: relationships with cholesteryl ester transfer protein mass and adiponectin

Objective: Dyslipidemia contributes to increased cardiovascular risk in nephrotic syndrome. We questioned whether reduction in proteinuria not only lowers low-density lipoprotein cholesterol (LDL-C), but also high-density lipoprotein cholesterol (HDL-C) and cholesteryl ester transfer protein (CETP) mass and whether changes in HDL-C were related to changes in plasma adiponectin. Methods: Thirty-two non-diabetic proteinuric patients (12 on statin therapy), were followed during two double blind 6-week periods of placebo and treatment (low sodium + 100mg losartan + 25 mg hydrochlorothiazide). Results: With placebo HDL-C was lower but LDL-C and CETP were not different in proteinuric patients compared with matched controls. LDL-C, HDL-C and CETP decreased upon proteinuria reduction. The decrease in LDL-C correlated with the drop in CETP and the degree of proteinuria reduction. HDL-C also decreased in proportion to proteinuria lowering. Individual changes in HDL-C were correlated with changes in adiponectin. Conclusion: LDL-C lowering upon robust reduction of proteinuria may be affected by changes in plasma CETP mass, but this treatment also decreases HDL-C in relation to the degree of proteinuria reduction. This adverse effect on HDL-C may in part be attributable to changes in adiponectin.

The Transcriptional Factor Prolactin Regulatory Element-Binding Protein Mediates the Gene Transcription of Adrenal Scavenger Receptor Class B Type I via 3′,5′-Cyclic Adenosine 5′-Monophosphate

Prolactin regulatory element-binding (PREB) protein is a transcription factor that regulates prolactin promoter activity in the rat anterior pituitary. The PREB protein is not only expressed in the anterior pituitary but also in the adrenal gland. However, the role of PREB in the adrenal gland is not clearly understood. Scavenger receptor class B type I (SR-BI) is a receptor for high-density lipoprotein that mediates the cellular uptake of high-density lipoprotein-cholesteryl ester and is a major route for cholesterol delivery to the steroidogenic pathway in the adrenal gland. In the present study, we have examined the role of PREB in regulating SR-BI. SR-BI expression was found to be regulated by cAMP, which stimulated the expression of PREB in a dose-dependent manner. Conversely, overexpression of PREB using a PREB-expressing adenovirus increased the expression of the SR-BI protein in the adrenocortical cell line Y-1. In addition, PREB induced the expression of the luciferase reporter protein that was under the control of the SR-BI promoter. EMSA showed that PREB mediates its transcriptional effect by binding to the PREB-responsive cis-element of the SR-BI promoter. Finally, we used small interfering RNA to inhibit PREB expression in the Y-1 cells and demonstrated that the knockdown of PREB expression attenuated the effects of cAMP on SR-BI expression. In summary, our data showed that in the adrenal gland, PREB regulates the transcription of the SR-BI gene via cAMP.

Where are we with high-density lipoprotein raising and inhibition of cholesteryl ester transfer for heart disease risk reduction?

To review recent research in the area of high-density lipoprotein raising and coronary heart disease risk reduction. A decreased high-density lipoprotein-cholesterol is an important coronary heart disease risk factor and raising high-density lipoprotein-cholesterol has been associated with coronary heart disease risk reduction. A relative new strategy for raising high-density lipoprotein-cholesterol, i.e. inhibition of cholesteryl ester transfer protein, is markedly effective. Cholesteryl ester transfer protein inhibitors prevent the transfer of cholesteryl ester from high-density lipoprotein to triglyceride-rich lipoproteins in exchange for triglyceride. One inhibitor, torcetrapib, binds to cholesteryl ester transfer protein on high-density lipoprotein, markedly raises high-density lipoprotein-cholesteryl ester and has no effect on fecal cholesterol excretion, but can raise blood pressure. A large clinical trial in coronary heart disease patients on atorvastatin was recently stopped prematurely because of excess mortality in those receiving torcetrapib vs. placebo and two other trials reported no benefit of torcetrapib on coronary atherosclerosis or carotid artery intimal medial thickness as compared with subjects on atorvastatin alone. The adverse effects of torcetrapib may be compound-specific and, since the crystal structure of cholesteryl ester transfer protein is now known, it should be possible to develop more optimal cholesteryl ester transfer protein inhibitors that do not form a nonproductive complex with cholesteryl ester transfer protein on the high-density lipoprotein particle, as has been reported for torcetrapib. The alternative for high-density lipoprotein raising is to develop more effective and better tolerated niacin preparations.

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.

Hepatic ATP-Binding Cassette Transporter A1 Is a Key Molecule in High-Density Lipoprotein Cholesteryl Ester Metabolism in Mice

Mutations in ATP-binding cassette transporter A1 (ABCA1), the cellular lipid transport molecule mutated in Tangier disease, result in the rapid turnover of high-density lipoprotein (HDL)-associated apolipoproteins that presumably are cleared by the kidneys. However, the role of ABCA1 in the liver for HDL apolipoprotein and cholesteryl ester (CE) catabolism in vivo is unknown. Murine HDL was radiolabeled with 125I in its apolipoprotein and with [3H]cholesteryl oleyl ether in its CE moiety. HDL protein and lipid metabolism in plasma and HDL uptake by tissues were investigated in ABCA1-overexpressing bacterial artificial chromosome (BAC)-transgenic (BAC+) mice and in mice harboring deletions of total (ABCA1-/-) and liver-specific ABCA1 (ABCA1(-L/-L)). In BAC+ mice with elevated ABCA1 expression, fractional catabolic rates (FCRs) of both the protein and the lipid tracer were significantly decreased in plasma and in the liver, yielding a diminished hepatic selective CE uptake from HDL. In contrast, ABCA1-/- or ABCA1(-L/-L) mice had significantly increased plasma and liver FCRs for both HDL tracers. An ABCA1 deficiency was associated with increased selective HDL CE uptake by the liver under all experimental conditions. Hepatic ABCA1 has a critical role for HDL catabolism in plasma and for HDL selective CE uptake by the liver.

Metabolism of high density lipoprotein apolipoprotein A‐I and cholesteryl ester in insulin resistant dog: a stable isotope study

In reverse cholesterol transport (RCT), hepatic Scavenger Receptor class B type I (SR-BI) plays an important role by mediating the selective uptake of high-density lipoprotein cholesteryl ester (HDL-CE). However, little is known about this antiatherogenic mechanism in insulin resistance. HDL-CE selective uptake represents the main process for HDL-CE turnover in dog, a species lacking cholesteryl ester transfer protein activity. We therefore investigate the effects of diet induced insulin resistance on RCT. Five beagle dogs, in healthy and insulin resistant states, underwent a primed constant infusion of [1,2(13)C(2)]acetate and [5,5,5-(2)H(3)]leucine, as labelled precursors of CE and apolipoprotein (apo) A-I, respectively. Data were analysed using modelling methods. HDL-apo A-I concentration did not change in insulin resistant state but apo A-I absolute production rate (APR) and fractional catabolic rate (FCR) were both higher (2.2- and 2.4-fold, respectively, p < 0.05). HDL-CE levels were lower (1.2-fold, p < 0.05). HDL-CE APR and FCR were both lower (2.3- and 2-fold, respectively, p < 0.05), as well as selective uptake (2.6-fold, p < 0.05). Lower HDL-CE selective uptake suggests that RCT is impaired in obese insulin resistant dog.

Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy

Scavenger receptor class B type I (SR-BI) plays a critical role in the delivery of HDL cholesterol and cholesteryl esters (CEs) to liver and steroidogenic tissues by a selective process that does not result in significant degradation of HDL protein. Recently, SR-BI-mediated endocytosis and recycling of HDL have been demonstrated. However, it remains unclear whether efficient SR-BI-mediated selective uptake occurs strictly at the plasma membrane or at additional sites along its endocytic itinerary. To examine the requirement for SR-BI endocytosis in HDL selective uptake, we determined the effects of energy depletion on the levels of cell-associated HDL protein and CE in primary mouse hepatocytes. Compared with CHO cells, we observed a much larger energy-dependent effect on CE uptake in primary mouse hepatocytes. Although varying the levels of caveolin-1 and carboxyl ester lipase altered the efficiency of selective uptake, neither was able to account for the energy-dependent component of HDL-CE uptake. Finally, we demonstrate that the hepatocyte-specific, energy-dependent effects on HDL-apolipoprotein A-I and -CE uptake are independent of SR-BI and are not required to achieve efficient SR-BI-mediated selective uptake of CE. Together, these data support the conclusion that neither the intracellular trafficking of HDL nor any energy-dependent cellular process affects the ability of the cell to maximally acquire CE through SR-BI-mediated selective uptake from HDL.