Postprandial lipemia enhances the capacity of large HDL2 particles to mediate free cholesterol efflux via SR-BI and ABCG1 pathways in type IIB hyperlipidemia

Lipid and cholesterol metabolism in the postprandial phase is associated with both quantitative and qualitative remodeling of HDL particle subspecies that may influence their anti-atherogenic functions in the reverse cholesterol transport pathway. We evaluated the capacity of whole plasma or isolated HDL particles to mediate cellular free cholesterol (FC) efflux, cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester (CE) transfer, and selective hepatic CE uptake during the postprandial phase in subjects displaying type IIB hyperlipidemia (n = 16). Postprandial, large HDL2 displayed an enhanced capacity to mediate FC efflux via both scavenger receptor class B type I (SR-BI)-dependent (+12%; P < 0.02) and ATP binding cassette transporter G1 (ABCG1)-dependent (+31%; P < 0.008) pathways in in vitro cell systems. In addition, the capacity of whole postprandial plasma (4 h and 8 h postprandially) to mediate cellular FC efflux via the ABCA1-dependent pathway was significantly increased (+19%; P < 0.0003). Concomitantly, postprandial lipemia was associated with elevated endogenous CE transfer rates from HDL2 to apoB lipoproteins and with attenuated capacity (−17%; P < 0.02) of total HDL to deliver CE to hepatic cells. Postprandial lipemia enhanced SR-BI and ABCG1-dependent efflux to large HDL2 particles. However, postprandial lipemia is equally associated with deleterious features by enhancing formation of CE-enriched, triglyceride-rich lipoprotein particles through the action of CETP and by reducing the direct return of HDL-CE to the liver.     

Cholesteryl ester is transported from caveolae to internal membranes as part of a caveolin-annexin II lipid-protein complex.

We previously demonstrated that in Chinese hamster ovary cells scavenger receptor, class B, type I-dependent selective cholesteryl ester uptake occurs in caveolae. In the present study we hypothesized that cholesteryl ester is transported from caveolae through the cytosol to an internal membrane by a caveolin chaperone complex similar to the one we originally described for the transport of newly synthesized cholesterol. To test this hypothesis we incubated Chinese hamster ovary cells expressing scavenger receptor, class B, type I with [(3)H]cholesteryl ester-labeled high density lipoprotein, subfractionated the cells and looked for a cytosolic pool of [(3)H]cholesteryl ester. The radiolabeled sterol initially appeared in the caveolae fraction, then in the cytosol, and finally in the internal membrane fraction. Caveolin IgG precipitated all of the [(3)H]cholesteryl ester associated with the cytosol. Co-immunoprecipitation studies demonstrated that in the presence of high density lipoprotein, but not low density lipoprotein or lipoprotein-deficient serum, caveolin IgG precipitated four proteins: annexin II, cyclophilin 40, caveolin, and cyclophilin A. Caveolin acylation-deficient mutants were used to demonstrate that acylation of cysteine 133 but not cysteine 143 or 156 is required for annexin II association with caveolin and the rapid transport of cholesteryl esters out of caveolae. We conclude that a caveolin-annexin II lipid-protein complex facilitates the rapid internalization of cholesteryl esters from caveolae.     

Alteration in lipoprotein lipase activity bound to triglyceride-rich lipoproteins in the postprandial state in type 2 diabetes

Postprandial lipid metabolism is largely dependent upon lipoprotein lipase (LPL), which hydrolyses triglycerides (TGs). The time course of LPL activity in the postprandial state following a single meal has never been studied, because its determination required heparin injection. Recently, we have shown that LPL activity could be accurately measured in nonheparinized VLDL using a new assay aiming to determine the LPL-dependent VLDL-TG hydrolysis. Based on the same principle, we used in this study TG-rich lipoprotein (TRL)-bound LPL-dependent TRL-TG hydrolysis (LTTH) to compare the time course of LPL activity of 10 type 2 diabetics to that of 10 controls, following the ingestion of a lipid-rich meal. The peak TG concentration, reached after 4 h, was 67% higher in diabetics than in controls (P < 0.005). Fasting LTTHs were 91.3 +/- 15.6 in controls versus 70.1 +/- 4.8 nmol NEFA/ml/h in diabetics (P < 0.001). LTTH was increased 2 h postprandially by 190% in controls and by only 89% in diabetics, resulting in a 35% lowering of the LTTH area under the curve in diabetics. Postprandial LTTH was inversely correlated with TG or remnant concentrations in controls but not in diabetics, and with insulin resistance in both groups. These data show that TRL-bound LPL activity increases in the postprandial state and is strongly reduced in type 2 diabetes, contributing to postprandial hypertriglyceridemia.     

Removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo is not enhanced by plasma cholesteryl ester transfer protein

The putative role of cholesteryl ester transfer protein (CETP) in the removal of cholesteryl ester from hepatic reticuloendothelial cells in vivo was studied in hamsters. The parameter tested was retention of [3H]cholesteryl linoleyl ether ([3H]CLE), a nonhydrolysable analog of cholesteryl ester, in the liver after injection of [3H]CLE labeled acetylated LDL, which is targetted to nonparenchymatous littoral cells. In hamsters fed laboratory chow, plasma cholesteryl ester transfer activity (CETA) was 10.6 +/- 0.9 units and the retention of [3H]CLE in the liver 28 days after injection was 86% of the 4 h value. It was about 55% in rats fed the same diet, in which CETA was not detectable. When the diet was supplemented with 2% cholesterol and 15% margarine, CETA activity in hamsters increased 2-fold, yet no change in retention of [3H]CLE in liver was seen after 28 days. In rats, the retention of [3H]CLE in the liver was also not changed by the dietary fat supplementation. These results do not support the role of CETP in vivo in removal of cholesteryl ester from intact reticuloendothelial cells.     

Glucoregulatory responses to intense exercise performed in the postprandial state

A seven- to eightfold increment in hepatic glucose production (endogenous R(a)) occurs in postabsorptive (PA) intense exercise (IE). A similar response is likely present in the postprandial (PP) state, when most such exercise is performed, because 1) little evidence for increased intestinal absorption of glucose during exercise exists, and 2) intravenous glucose does not prevent it. We investigated IE in 10 PA and 8 PP fit, lean, young males who had exercised for 15 min at >84% maximum O(2) uptake, starting 3 h after a 412-kcal mixed meal. The meal induced a small rise in glycemia with sustained insulin and glucagon increases. Preexercise glucose total R(a) and utilization (R(d)) were equal and approximately 130% of the PA level. Exercise hyperglycemia in PP was delayed and diminished and, in early recovery, was of shorter duration and lesser magnitude (P = 0.042). Peak catecholamine (12- to 16-fold increase) and R(a) (PP: 11.5 +/- 1.4, PA: 13.8 +/- 1.4 mg. kg(-1). min(-1)) responses did not differ, and their responses during exercise were significantly correlated. Exercise glucagon, insulin, and glucagon-to-insulin responses were small or not significant. R(d) reached the same peak (PP: 8.0 +/- 0.6, PA: 9.3 +/- 0.8 mg. kg(-1). min(-1)) but was greater at 20-120 min of recovery in PP (P = 0.001). Therefore, the total R(a) response to IE is preserved despite the possibility of prior PP suppression of endogenous R(a) and is consistent with catecholamine mediation. Post-IE hyperglycemia is reduced in the postprandial state.     

VLDL-bound lipoprotein lipase facilitates the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from HDL to VLDL

In recent years, it has been established that lipoprotein lipase (LPL) is partly associated with circulating lipoproteins. This report describes the effects of physiological amounts of very low density lipoprotein (VLDL)-bound LPL on the cholesteryl ester transfer protein (CETP)-mediated cholesteryl ester transfer (CET) from high density lipoprotein (HDL) to VLDL. Three patients with severe LPL deficiency exhibited a strong decrease in net mass CET that was more than 80% lower than that of common hypertriglyceridemic subjects. Recombination experiments showed that this was due to an abnormal behavior of the VLDL fraction. Replacement of the latter by normal VLDL totally normalized net mass CET. We therefore prepared VLDL containing controlled amounts of bound LPL that we used as CE acceptors in experiments involving unidirectional radioisotopic CET measurements. These were carried out either in the absence or in the presence of inhibitors of LPL lipolytic activity. When LPL-induced lipolysis was totally blocked, the stimulating effect of the enzyme on the CETP-dependent CET was only reduced by about 50%, showing that it did not entirely result from its lipolytic action. These data were dependent upon neither the type of LPL inhibitor (E600 or THL) nor the source of CETP (delipidated plasma or partially purified CETP). Thus, in addition to the well-known stimulating effect of LPL-dependent lipolysis on CET, our work demonstrates that physiological amounts of VLDL-bound LPL may facilitate CET through a mechanism partially independent of its lipolytic activity.     

HDL from apoA1 transgenic mice expressing the 4WF isoform is resistant to oxidative loss of function

HDL functions are impaired by myeloperoxidase (MPO), which selectively targets and oxidizes human apoA1. We previously found that the 4WF isoform of human apoA1, in which the four tryptophan residues are substituted with phenylalanine, is resistant to MPO-mediated loss of function. The purpose of this study was to generate 4WF apoA1 transgenic mice and compare functional properties of the 4WF and wild-type human apoA1 isoforms in vivo. Male mice had significantly higher plasma apoA1 levels than females for both isoforms of human apoA1, attributed to different production rates. With matched plasma apoA1 levels, 4WF transgenics had a trend for slightly less HDL-cholesterol versus human apoA1 transgenics. While 4WF transgenics had 31% less reverse cholesterol transport (RCT) to the plasma compartment, equivalent RCT to the liver and feces was observed. Plasma from both strains had similar ability to accept cholesterol and facilitate ex vivo cholesterol efflux from macrophages. Furthermore, we observed that 4WF transgenic HDL was partially (∼50%) protected from MPO-mediated loss of function while human apoA1 transgenic HDL lost all ABCA1-dependent cholesterol acceptor activity. In conclusion, the structure and function of HDL from 4WF transgenic mice was not different than HDL derived from human apoA1 transgenic mice.     

Co-expression of scavenger receptor-BI and caveolin-1 is associated with enhanced selective cholesteryl ester uptake in THP-1 macrophages.

Scavenger receptor (SR)-BI mediates the selective uptake of high density lipoprotein (HDL) cholesteryl esters and the efflux of free cholesterol. In Chinese hamster ovary (CHO) cells, SR-BI is predominantly associated with caveolae which we have recently demonstrated are the initial loci for membrane transfer of HDL cholesteryl esters. Because cholesterol accumulation in macrophages is a critical event in atherogenesis, we investigated the expression of SR-BI and caveolin-1 in several macrophage cell lines. Human THP-1 monocytes were examined before and after differentiation to macrophages by treatment with 200 nm phorbol ester for 72 h. Undifferentiated THP-1 cells expressed caveolin-1 weakly whereas differentiation up-regulated caveolin-1 expression greater than 50-fold. In contrast, both undifferentiated and differentiated THP-1 cells expressed similar levels of SR-BI. Differentiation of THP-1 cells increased the percent of membrane cholesterol associated with caveolae from 12% +/- 1.9% to 38% +/- 3.1%. The increase in caveolin-1 expression was associated with a 2- to 3-fold increase in selective cholesterol ether uptake from HDL. Two mouse macrophage cell lines, J774 and RAW, expressed levels of SR-BI similar to differentiated THP-1 cells but did not express detectable levels of caveolin-1. In comparison to differentiated THP-1 cells, RAW and J774 cells internalized 9- to 10-fold less cholesteryl ester. We conclude that differentiated THP-1 cells express both caveolin-1 and SR-BI and that their co-expression is associated with enhanced selective cholesteryl ester uptake.     

Catabolism of HDL1 cholesteryl ester in the rat. Effect of ethinyl estradiol treatment.

The present study was performed in control and ethinyl estradiol-treated rats in order to determine the mechanisms involved in the catabolism of HDL1 cholesteryl ester. Ligand blottings on liver membranes showed that purified HDL1, containing about 70% apolipoprotein E and 10% apolipoprotein AI, bind to the LDL receptor (130 kDa) and not to HB2 (100 kDa) or SR-BI (82 kDa), candidate HDL receptors. Immunoblots showed that the treatment increased the hepatic level of the LDL receptor five- to ten-fold, strongly decreased that of SRBI and did not change that of HB2. An in vivo kinetic study showed that the turnover of HDL1 cholesteryl ester is more rapid in treated than control rats. The liver participation (60%) in this clearance was not modified by the treatment. Therefore, it can be concluded that the catabolism of HDL1 cholesteryl ester, in control as in treated rats, is essentially ensured by the uptake of entire particles in the hepatocytes via LDL receptors.     

Identification of proteins whose synthesis is preferentially enhanced by polyamines at the level of translation in mammalian cells

In Escherichia coli, several proteins whose synthesis is enhanced by polyamines at the level of translation have been identified. We looked for proteins that are similarly regulated in eukaryotes using a mouse mammary carcinoma FM3A cell culture system. Polyamine deficiency was induced by adding an inhibitor of ornithine decarboxylase, α-difluoromethylornithine, to the medium. Proteins enhanced by polyamines were determined by comparison of protein levels in control and polyamine-deficient cells using two-dimensional gel electrophoresis, and were identified by Edman degradation and/or LC/MALDI-TOF/TOF tandem mass spectrometry. Polyamine stimulation of the synthesis of these proteins at the level of translation was confirmed by measuring levels of the corresponding mRNAs and proteins, and levels of the [³⁵S]methionine pulse-labeled proteins. The proteins identified in this way were T-complex protein 1, β subunit (Cct2); heterogenous nuclear ribonucleoprotein L (Hnrpl); and phosphoglycerate mutase 1 (Pgam1). Since Cct2 was most strongly enhanced by polyamines among three proteins, the mechanism of polyamine stimulation of Cct2 synthesis was studied using NIH3T3 cells transiently transfected with genes encoding Cct2-EGFP fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Cct2 mRNA. Polyamines most likely enhanced ribosome shunting on the 5′-UTR of Cct2 mRNA.     

Biogenesis of HDL by SAA is dependent on ABCA1 in the liver in vivo

Serum amyloid A (SAA) was markedly increased in the plasma and in the liver upon acute inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) in mice, and SAA in the plasma was exclusively associated with HDL. In contrast, no HDL was present in the plasma and only a small amount of SAA was found in the VLDL/LDL fraction (d < 1.063 g/ml) after the induction of inflammation in ABCA1-knockout (KO) mice, although SAA increased in the liver. Primary hepatocytes isolated from LPS-treated wild-type (WT) and ABCA1-KO mice both secreted SAA into the medium. SAA secreted from WT hepatocytes was associated with HDL, whereas SAA from ABCA1-KO hepatocytes was recovered in the fraction that was >1.21 g/ml. The behavior of apolipoprotein A-I (apoA-I) was the same as that of SAA in HDL biogenesis by WT and ABCA1-KO mouse hepatocytes. Lipid-free SAA and apoA-I both stabilized ABCA1 and caused cellular lipid release in WT mouse-derived fibroblasts, but not in ABCA1-KO mouse-derived fibroblasts, in vitro when added exogenously. We conclude that both SAA and apoA-I generate HDL largely in hepatocytes only in the presence of ABCA1, likely being secreted in a lipid-free form to interact with cellular ABCA1. In the absence of ABCA1, nonlipidated SAA is seemingly removed rapidly from the extracellular space.     

Cluap1 localizes preferentially to the base and tip of cilia and is required for ciliogenesis in the mouse embryo

Qilin is one of several genes in zebrafish whose mutation results in cystic kidney. We have now studied the role of its mouse ortholog, Cluap1, in embryonic development by generating Cluap1 knockout (Cluap1^−/−) mice. Cluap1^−/− embryos died mid-gestation manifesting impairment of ciliogenesis in various regions including the node and neural tube. The basal body was found to be properly docked to the apical membrane of cells in the mutant, but the axoneme failed to grow. Cluap1 is a ciliary protein and is preferentially localized at the base and tip of cilia. Hedgehog signaling, as revealed with a Pacthed1-lacZ reporter gene, was lost in Cluap1^−/− embryos at embryonic day (E) 8.5 but was ectopically expanded at E9.0. The Cluap1 knockout embryos also failed to manifest left–right asymmetric expression of Nodal in the lateral plate, most likely as a result of the loss of Hedgehog signaling in node crown cells that in turn leads to pronounced down-regulation of Gdf1 expression in these cells. Crown cell-specific restoration of Cluap1 expression rescued Gdf1 expression in crown cells and left-sided Nodal expression in the lateral plate of mutant embryos. Our results suggest that Cluap1 contributes to ciliogenesis by regulating the intraflagellar transport (IFT) cycle at the base and tip of the cilium.     

Serum IF1 concentration is independently associated to HDL levels and to coronary heart disease: the GENES study

HDL is strongly inversely related to cardiovascular risk. Hepatic HDL uptake is controlled by ecto-F₁-ATPase activity, and potentially inhibited by mitochondrial inhibitor factor 1 (IF1). We recently found that IF1 is present in serum and correlates with HDL-cholesterol (HDL-C). Here, we have evaluated the relationship between circulating IF1 and plasma lipoproteins, and we determined whether IF1 concentration is associated with the risk of coronary heart disease (CHD). Serum IF1 was measured in 648 coronary patients ages 45–74 and in 669 matched male controls, in the context of a cross-sectional study on CHD. Cardiovascular risk factors were documented for each participant, including life-style habits and biological and clinical markers. In controls, multivariate analysis demonstrated that IF1 was independently positively associated with HDL-C and apoA-I (r = 0.27 and 0.28, respectively, P < 0.001) and negatively with triglycerides (r = −0.23, P < 0.001). Mean IF1 concentration was lower in CHD patients than in controls (0.43 mg/l and 0.53 mg/l, respectively, P < 0.001). In multivariate analyses, following adjustments on cardiovascular risk factors or markers, IF1 was negatively related to CHD (P < 0.001). This relationship was maintained after adjustment for HDL-C or apoA-I. This study identifies IF1 as a new determinant of HDL-C that is inversely associated with CHD.     

In vivo conversion of human HDL3 to HDL2 and apoE-rich HDL1 in the rat: effects of lipid transfer protein

In this study we determined in vivo conversions of human 3H-labeled cholesteryl ester-labeled HDL3 [( 3H]CE-HDL3) in male rats and the effects of partially purified lipid transfer protein on the conversion processes. Zonal centrifugation techniques were used to prepare the [3H]CE-HDL3 and to follow the conversion processes. One hour after the injection, a complete conversion of HDL3 to the HDL2-density species was found. With time, [3H]CE separated with apoE-rich HDL1 and, by 18 hr, 35.9% of plasma radioactivity was associated with the apoE-rich HDL1 lipoprotein fraction. In vitro incubation of [3H]CE-HDL3 in rat plasma reproduced in part the HDL3----HDL2 conversion, but no movement of radioactivity to HDL1 was observed. Injection of the rats with partially purified lipid transfer proteins induced [3H]CE exchange between lipoproteins. The conversion of HDL3 to HDL2, however, was minimally affected. Formation of [3H]CE-HDL1, in contrast, was reduced to about one-half of that found in control animals. It is concluded that in vivo conditions are necessary for conversions of HDL3 (and HDL2) to HDL1, and that lipid transfer reactions delay this process.     

The slow S to M fluorescence rise in cyanobacteria is due to a state 2 to state 1 transition

In dark-adapted plants and algae, chlorophyll a fluorescence induction peaks within 1s after irradiation due to well documented photochemical and non-photochemical processes. Here we show that the much slower fluorescence rise in cyanobacteria (the so-called "S to M rise" in tens of seconds) is due to state 2 to state 1 transition. This has been demonstrated in particular for Synechocystis PCC6803, using its RpaC(-) mutant (locked in state 1) and its wild-type cells kept in hyperosmotic suspension (locked in state 2). In both cases, the inhibition of state changes correlates with the disappearance of the S to M fluorescence rise, confirming its assignment to the state 2 to state 1 transition. The general physiological relevance of the SM rise is supported by its occurrence in several cyanobacterial strains: Synechococcus (PCC 7942, WH 5701) and diazotrophic single cell cyanobacterium (Cyanothece sp. ATCC 51142). We also show here that the SM fluorescence rise, and also the state transition changes are less prominent in filamentous diazotrophic cyanobacterium Nostoc sp. (PCC 7120) and absent in phycobilisome-less cyanobacterium Prochlorococcus marinus PCC 9511. Surprisingly, it is also absent in the phycobiliprotein rod containing Acaryochloris marina (MBIC 11017). All these results show that the S to M fluorescence rise reflects state 2 to state 1 transition in cyanobacteria with phycobilisomes formed by rods and core parts. We show that the pronounced SM fluorescence rise may reflect a protective mechanism for excess energy dissipation in those cyanobacteria (e.g. in Synechococcus PCC 7942) that are less efficient in other protective mechanisms, such as blue light induced non-photochemical quenching. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Stable overexpression of human macrophage cholesteryl ester hydrolase results in enhanced free cholesterol efflux from human THP1 macrophages

Reduction of the lipid burden of atherosclerotic lesion-associated macrophage foam cells is a logical strategy to reduce the plaque volume. Since extracellular cholesterol acceptor-mediated cholesterol efflux is the only recognized mechanism of cholesterol removal from foam cells and this process is rate limited at the level of intracellular cholesterol ester hydrolysis, a reaction catalyzed by neutral cholesteryl ester hydrolase (CEH), we examined the hypothesis that CEH overexpression in the human macrophage monocyte/macrophage cell line THP1 results in increased cholesterol efflux, as well as decreased cellular cholesterol ester accumulation. We generated THP1-CEH cells with stable integration of human macrophage CEH cDNA driven by the cytomegalovirus promoter. Compared with wild-type THP1 cells (THP1-WT), THP1-CEH cells showed increased CEH mRNA expression and increased CEH activity. Efflux of free or unesterified cholesterol by acetylated LDL-loaded THP1-CEH cells to ApoA-I by an ABCA1-dependent pathway or to HDL by an ABCG1-dependent pathway was significantly higher than that in THP1-WT cells. In addition, THP1-CEH cells accumulated significantly lower amount of esterified cholesterol. CEH overexpression, therefore, not only enhances cholesterol efflux but also reduces cellular accumulation of cholesteryl esters. Taken together, these data provide evidence for evaluating CEH expression in human macrophages as a potential target for attenuation of foam cell formation and regression of atherosclerotic plaques. lipoproteins; lipid burden; foam cells