Presence Of HDL Research Articles

Simultaneous expression of apolipoprotein B mRNA editing enzyme and scavenger receptor BI mediated by a therapeutic gene expression system

Cardiovascular diseases are often accompanied by elevated LDL particles and endothelial dysfunction. We have examined the possibility of concurrently reducing LDL levels and modulating endothelial function using a single helper-dependent adenovirus vector system to simultaneously express the apolipoprotein B mRNA editing enzyme (Apobec1) and the scavenger receptor, class B, type I (SR-BI) genes under the control of separate promoters (designated HD-C2). Apobec1 edits apoB mRNA at nucleotide C-6666 to produce truncated apoB48 and is normally expressed in small intestine only. SR-BI is a receptor for multiple ligands with distinct tissue-specific functions. Expression of Apobec1 in HepG2 cells resulted in apoB mRNA editing, leading to decreased apoB100 abundance (to 6% of control) and the appearance of apoB48. Editing of apoB mRNA in HepG2 cells resulted in decline in apoB mRNA levels of 50%. This was probably the result of nonsense-mediated decay of edited message, since over-expression of Apobec1 increased neither Apobec1 complementary factor (ACF) mRNA nor protein abundance. Over-expression of SR-BI in human endothelial cells activated endothelial nitric oxide synthase (eNOS) activity by phosphorylation of eNOS at residue Ser-1177 in the presence of HDL, leading to increased production of the anti-atherogenic molecule nitric oxide (NO). Taken together, this study demonstrates that using one vector delivery system to express two genes in two different cell types results in the cell-specific beneficial effects of decreasing apoB100 production and increasing eNOS activities. This combined gene expression approach may provide an improved therapeutic strategy by targeting multiple sites in the mechanism of cardiovascular injury.

Expression of cholesterol sulfotransferase (SULT2B1b) in human platelets.

Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation. We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4 degrees C but substantially diminished over a period of 4 hours at 37 degrees C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37 degrees C is subject to regulation by the apoA-I component of HDL.

Degradation of Phospholipid Transfer Protein (PLTP) and PLTP-generated Pre-β-high Density Lipoprotein by Mast Cell Chymase Impairs High Affinity Efflux of Cholesterol from Macrophage Foam Cells

Human atherosclerotic lesions contain mast cells filled with the neutral protease chymase. Here we studied the effect of human chymase on (i) phospholipid transfer protein (PLTP)-mediated phospholipid (PL) transfer activity, and (ii) the ability of PLTP to generate pre-beta-high density lipoprotein (HDL). Immunoblot analysis of PLTP after incubation with chymase for 6 h revealed, in addition to the original 80-kDa band, four specific proteolytic fragments of PLTP with approximate molecular masses of 70, 52, 48, and 31 kDa. This specific pattern of PLTP degradation remained stable for at least 24 h of incubation with chymase. Such proteolyzed PLTP had reduced ability (i) to transfer PL from liposome donor particles to acceptor HDL(3) particles, and (ii) to facilitate the formation of pre-beta-HDL. However, when PLTP was incubated with chymase in the presence of HDL(3), only one major cleavage product of PLTP (48 kDa) was generated, and PL transfer activity was almost fully preserved. Moreover, chymase effectively depleted the pre-beta-HDL particles generated from HDL(3) by PLTP and significantly inhibited the high affinity component of cholesterol efflux from macrophage foam cells. These results suggest that the mast cells in human atherosclerotic lesions, by secreting chymase, may prevent PLTP-dependent formation of pre-beta-HDL particles from HDL(3) and so impair the anti-atherogenic function of PLTP.

Caveolin-1 and Caveolin-2 Expression in Mouse Macrophages: HIGH DENSITY LIPOPROTEIN 3-STIMULATED SECRETION AND A LACK OF SIGNIFICANT SUBCELLULAR CO-LOCALIZATION

Evidence for caveolin expression in macrophages is scarce and conflicting. We therefore examined caveolin-1 and caveolin-2 expression in resident and thioglycollate-elicited mouse peritoneal macrophages (tg-MPM) and in the J774 mouse macrophage cell line by RT-PCR, ribonuclease protection assay, immunoblotting, and immunofluorescence. We found that relative to 3T3 cells, resident MPM and tg-MPM express low amounts of caveolin-1 (45 and 15% of those in 3T3 fibroblasts, respectively), while J774.A1 cells do not express any. Caveolin-2, on the other hand, is expressed in all cells examined, with highest expression in tg-MPM and the lowest in J774 cells. The relative levels of caveolin expression in the various cells correspond well with their respective mRNA levels, as measured by ribonuclease protection assay. Caveolin-1, present primarily on the cell surface, does not co-localize significantly with caveolin-2, which is present primarily in the Golgi compartment in all macrophages studied. Loading of tg-MPM with cholesterol or variations in unesterified cholesterol content appear to have little effect on the level of caveolin-1 or -2 expression or their distribution. Stimulation of cholesterol efflux by HDL(3) leads to caveolin-1 and caveolin-2 secretion to the cell culture medium, a process not detected in the absence of HDL(3). The lack of significant co-localization of the two caveolin isoforms in primary macrophages and their secretion in the presence of HDL(3) provides an interesting and physiologically relevant model system to study additional aspects of caveolin function.

Serum paraoxonase is reduced in type 1 diabetic patients compared to non-diabetic, first degree relatives; influence on the ability of HDL to protect LDL from oxidation

Paraoxonase is a serum enzyme with an anti-oxidant function, protecting low density lipoproteins (LDL) from oxidative modifications. Diabetic patients are suggested to be at greater risk of oxidative stress, which may contribute to the significantly higher incidence of vascular disease in this population. Less efficient protection mechanisms may be one feature of the greater susceptibility to oxidation in diabetes. In this context, the present study examined the hypothesis that serum paraoxonase is reduced in type 1 (insulin-dependent) diabetic patients and that the reduction can affect the anti-oxidant capacity of HDL. Serum paraoxonase concentrations and activities were compared in type 1 patients and first degree, non-diabetic relatives with particular attention paid to the confounding effects of paraoxonase gene polymorphisms. In addition, the ability of HDL-paraoxonase to protect low density lipoproteins from oxidation was analysed in an in vitro system. Serum concentrations and enzyme activities of paraoxonase were significantly lower in type 1 patients compared to non-diabetic, first degree relatives. The differences were independent of promoter and coding region polymorphisms, which influence serum concentrations and activities of the enzyme. Overall, paraoxonase concentrations were a mean 13.3±4.5% lower ( P<0.02) in type 1 patients. Specific activities did not differ between diabetic and non-diabetic groups. The concentration ratios of LDL cholesterol:paraoxonase (1.37±0.51 vs. 1.18±0.37, P=0.003) and apolipoprotein B:paraoxonase (0.84±0.33 vs. 0.71±0.40; P=0.012) were significantly higher in diabetic patients, consistent with a reduced capacity to protect LDL from oxidation. In vitro oxidation studies showed that a significantly higher level of lipid hydroperoxides was generated in LDL in the presence of HDL, containing paraoxonase levels equivalent to those of type 1 patients, compared to HDL containing paraoxonase levels equivalent to those of control subjects (mean difference 8.1%, P<0.05). The study demonstrates that serum concentrations of the antioxidant enzyme paraoxonase are significantly lower in type 1 (insulin-dependent) diabetic patients compared to non-diabetic, first-degree relatives, independently of known gene polymorphisms. Concentrations are reduced to an extent that can affect its anti-oxidant capacity. The results are consistent with the contention that modifications to serum paraoxonase in type 1 patients can increase risk of lipoprotein oxidation and, consequently, risk of vascular disease.

The human breast carcinoma cell line HBL-100 acquires exogenous cholesterol from high-density lipoprotein via CLA-1 (CD-36 and LIMPII analogous 1)-mediated selective cholesteryl ester uptake.

Aberrant cell proliferation is one of the hallmarks of carcinogenesis, and cholesterol is thought to play an important role during cell proliferation and cancer progression. In the present study we examined the pathways that could contribute to enhanced proliferation rates of HBL-100 cells in the presence of apolipoprotein E-depleted high-density lipoprotein subclass 3 (HDL(3)). When HBL-100 cells were cultivated in the presence of HDL(3) (up to 200 microg/ml HDL(3) protein), the growth rates and cellular cholesterol content were directly related to the concentrations of HDL(3) in the culture medium. In principle, two pathways can contribute to cholesterol/cholesteryl ester (CE) uptake from HDL(3), (i) holoparticle- and (ii) scavenger-receptor BI (SR-BI)-mediated selective uptake of HDL(3)-associated CEs. Northern- and Western-blot analyses revealed the expression of CLA-1 (CD-36 and LIMPII analogous 1), the human homologue of the rodent HDL receptor SR-BI. In line with CLA-1 expression, selective uptake of HDL(3)-CEs exceeded HDL(3)-holoparticle uptake between 12- and 58-fold. Competition experiments demonstrated that CLA-1 ligands (oxidized HDL, oxidized and acetylated low-density lipoprotein and phosphatidylserine) inhibited selective HDL(3)-CE uptake. In line with the ligand-binding specificity of CLA-1, phosphatidylcholine did not compete for selective HDL(3)-CE uptake. Selective uptake was regulated by the availability of exogenous cholesterol and PMA, but not by adrenocorticotropic hormone. HPLC analysis revealed that a substantial part of HDL(3)-CE, which was taken up selectively, was subjected to intracellular hydrolysis. A potential candidate facilitating extralysosomal hydrolysis of HDL(3)-CE is hormone-sensitive lipase, an enzyme which was identified in HBL-100 cells by Western blots. Our findings demonstrate that HBL-100 cells are able to acquire HDL-CEs via selective uptake. Subsequent partial hydrolysis by hormone-sensitive lipase could provide 'free' cholesterol that is available for the synthesis of cellular membranes during proliferation of cancer cells.

Effects of chronic alcohol treatment on the synthesis, sialylation, and disposition of nascent apolipoprotein E by peritoneal macrophages of rats

Plasma apolipoprotein (apo) E, a sialoprotein, plays an important role in reverse cholesterol transport. Previously, we showed that chronic alcohol consumption impairs glycosylation of apo E in rat liver. Peritoneal macrophages are another significant apo E synthesis site. The main purpose of this study was to determine the effects of chronic alcohol feeding of rats on the synthesis, sialylation, and sialic acid content of macrophage apo E and its ability to bind to the HDL(3) molecule in vitro. Rats were fed an alcoholic diet or an isoenergetic control diet for 8 wk, after which peritoneal macrophages isolated from them were cultured and analyzed for apo E metabolism. Macrophages from alcohol-fed rats accumulated 33.3% more (P < 0.05) cholesterol than did those from control rats when incubated with acetylated LDL. These macrophages showed a 51-57% lower relative sialylation rate of apo E (P < 0.001) but no significant difference in relative protein synthetic rate. The sialic acid content of the intracellular and secreted forms of apo E was reduced by 41.8% (P < 0.001) and 50.3% (P < 0.001), respectively, with chronic alcohol treatment. Secretion of newly synthesized apo E was impaired by 53.7% (P < 0.001) and 26. 1% (P < 0.001) in the absence and presence of HDL in the medium, respectively. Macrophages of alcohol-treated rats secreted apo E with 47.6-67.2% lower (P < 0.001) HDL(3) binding ability; binding ability was restored completely by resialylation of the desialylated apo E. In rats, an alcohol-mediated decrease in sialylation rate resulting in loss of sialic acid residues in apo E impairs the ability of apo E to bind to HDL and consequently in defective reverse cholesterol transport.

Effects of low density and high density lipoproteins isolated from non-insulin dependent diabetic patients on prostaglandin secretion by mouse macrophage cell line P388D1

We have previously shown that low-density (LDL) and high-density (HDL) lipoprotein from healthy subjects can promote in vitro prostaglandin (PG) release by murine macrophages. In this pilot study, we have measured PG production induced by lipoproteins of six diabetic patients with poor metabolic control, compared to five healthy controls. Plasma lipoprotein levels were similar in both groups. Lipoprotein fractions were purified by sequential ultracentrifugation. After lipoprotein incubation with cells, supernatants were extracted and PG quantified by HPLC. In presence of LDL, in control subjects, there was an increase in total PG production, mainly due to thromboxane B2 (TxB2). In diabetic patients, the secretion pattern was similar. In presence of HDL, in control subjects, total PG secretion was also increased, but it was balanced between TxB2 and prostacyclin. In diabetic patients, at low HDL concentration (10 mg/l) the secretion was mainly due to TxB2, while at higher HDL concentrations (100 mg/l), the secretion was balanced between TxB2 and prostacyclin. Comparison of means of areas under curve for the two groups studied showed that LDL increased all PG secretion in diabetic patients compared to controls ( P<0.05 for PGF2 α), while HDL increased all PG secretion in controls compared to diabetic patients, except PGF2 α. Our work suggests a key role of LDL in TxB2 secretion in diabetic patients, which is a major proaggregant and vasoconstrictive agent. There was also an increased secretion of all PG in diabetic patients.

Mechanism of high-density lipoprotein subfractions inhibiting copper-catalyzed oxidation of low-density lipoprotein

Objective: To investigate the role of HDL subfractions, HDL 2 and HDL 3, on the oxidation of LDL catalyzed by 5 μM Cu 2+ ion, and to illustrate the mechanism of the generation of conjugated diene and thiobarbituric acid reactive substances (TBARS) during LDL oxidation. Methods: LDL was incubated for 8 h with 5 μM Cu 2+ ion in phosphate-buffered saline (PBS) alone, or in the presence of HDL 2, HDL 3, HSA, BSA, or transferrin. Meantime, LDL was incubated for 24 h with 10 μM Ni 2+ ions in PBS. The amount of conjugated diene and TBARS in each sample of LDL were measured. Results: (a) HDL 2 and HDL 3 could inhibit the generation of conjugated diene, but could not inhibit the generation of TBARS; (b) the transferrin containing HDL 3 shows the ability of inhibiting the generation of both conjugated diene and TBARS; (c) the transferrin presented in blood exhibits the inhibitory effect on the generation of conjugated diene and TBARS, however, when the transferrin is saturated with Fe 3+ ion, it could not inhibit the generation of TBARS; (d) HSA and BSA could prevent the generation of conjugated diene and TBARS; (e) Ni 2+ ion could induce the generation of conjugated diene, but the amount of TBARS was much smaller than that induced by Cu 2+ ion. Conclusion: HDL 2 and HDL 3 play important role in the copper-catalyzed oxidation of LDL; it is absolutely necessary to require chelation of Cu 2+ ion for inhibiting generation of TBARS; whereas, inhibition of conjugated diene can be fulfilled either by chelating Cu 2+ ion, or the free radicals scavenger.

Subendothelial retention of lipoprotein (a). Evidence that reduced heparan sulfate promotes lipoprotein binding to subendothelial matrix.

Vessel wall subendothelial extracellular matrix, a dense mesh formed of collagens, fibronectin, laminin, and proteoglycans, has important roles in lipid and lipoprotein retention and cell adhesion. In atherosclerosis, vessel wall heparan sulfate proteoglycans (HSPG) are decreased and we therefore tested whether selective loss of HSPG affects lipoprotein retention. A matrix synthesized by aortic endothelial cells and a commercially available matrix (Matrigel; , Rutherford, NJ) were used. Treatment of matrix with heparinase/heparitinase (1 U/ml each) increased LDL binding by approximately 1.5-fold. Binding of lipoprotein (a) [Lp(a)] to both subendothelial matrix and Matrigel(R) increased 2-10-fold when the HSPG were removed by heparinase treatment. Incubation of endothelial cells with oxidized LDL (OxLDL) or lysolecithin resulted in decreased matrix proteoglycans and increased Lp(a) retention by matrix. The effect of OxLDL or lysolecithin on endothelial PG was abolished in the presence of HDL. The decrease in matrix HSPG was associated with production of a heparanase-like activity by OxLDL-stimulated endothelial cells. To test whether removal of HSPG exposes fibronectin, a candidate Lp(a) binding protein in the matrix, antifibronectin antibodies were used. The increased Lp(a) binding after HSPG removal was inhibited 60% by antifibronectin antibodies. Similarly, the increased Lp(a) binding to matrix from OxLDL-treated endothelial cells was inhibited by antifibronectin antibodies. We hypothesize that atherogenic lipoproteins stimulate endothelial cell production of heparanase. This enzyme reduces HSPG which in turn promotes Lp(a) retention.

HDL 3 exerts more powerful anti-oxidative, protective effects against copper-catalyzed LDL oxidation than HDL 2

Objective: To evaluate which HDL subfraction, HDL 2 or HDL 3, exerts the greater preventive effect on the Cu 2+-induced LDL oxidation. Methods: LDL was incubated for 6 h with 2.5 μM Cu 2+ in phosphate-buffered saline alone, or in the presence of HDL 2 or HDL 3 at various protein concentrations. Each sample was subjected to agarose gel electrophoresis, and the amount of lipid hydroperoxide in each sample of LDL was measured. Results: There was no significant difference in the levels of LPO between the LDL and LDL + HDL 2 cases, whereas a significant reduction was apparent with LDL + HDL 3. Both HDL 2 and HDL 3 significantly inhibited oxidative modification of LDL, as assessed by electrophoretic mobility, in a concentration dependent manner, but this effect was much more pronounced with HDL 3. Conclusion: HDL 3 may play an important role in the prevention of atherosclerosis in vivo, more effectively inhibiting oxidation of LDL than HDL 2.

Lipoprotein receptor 'CK'--dependent signalling in human platelets.

The study addressed to understand whether or not lipoproteins at low concentrations could modulate Receptor-'C' dependent platelet signalling revealed that LDL, like exogenous cholesterol, had the capacity to initiate PLD-dependent platelet signalling in a dose dependent fashion and this effect was inhibited in presence of HDL; cAMP; DTT; Zn++ and butanol whereas cGMP had no effect upon this PLD-dependent signalling. Further Receptor 'C' from platelet in the purified-form displayed LDL-or cholesterol-dependent autophosphorylation at the tyrosine residues and this Receptor-'C' tyrosine kinase (Receptor-Ck) activity contributed to the observed LDL-or cholesterol -dependent PLD activity in human platelets. Based upon these results coupled with earlier results, an attempt was made to define the lipoprotein-dependent platelet signalling pathway.

Sodium oleate-facilitated reassembly of apolipoprotein A-I with phosphatidylcholine

The influence of sodium oleate (oleate) on complexing of apolipoprotein A-I (apo A-I) with egg yolk phosphatidylcholine (EYPC) was evaluated. Without the use of additional detergent such as sodium cholate, oleate facilitates formation of a single complex of unique stoichiometry, approx. 76:2:20, molar ratio EYPC/apo A-I/oleate, and mean size 7.4 nm with round to ellipsoidal morphology. Near complete reassembly of apo A-I into the complex occurs when the stoichiometry of the mixture approximates that of the complex itself. With increasing content of EYPC in the mixture, the same complex is formed but in decreasing yield; larger complexes are not formed. The rate of complex formation decreases with increase of EYPC in the mixture. Reduction of pH in the reassembly mixture from 8.0 to 5.4 results in a marked reduction in complex formation indicating that ionized oleate facilitates lipidation. Removal of oleate by interaction of the complex with fatty acid-free human serum albumin does not degrade the complex. Incorporation of increasing amounts of unesterified cholesterol into the EYPC-sonicate progressively inhibits oleate-facilitated complex formation. This study shows that oleate, a physiologically relevant lipolysis-derived product, facilitates reassembly of apo A-I with EYPC and promotes formation of a small lipid-poor particle similar to that observed in nascent HDL and during in vivo or in vitro lipolysis of triacylglycerol-rich lipoproteins in the presence of HDL.

Modification of amphotericin B's therapeutic index by increasing its association with serum high-density lipoproteins.

AmpB remains one of the drugs of choice in the treatment of systemic fungal infection; however, its therapy is limited by the development of renal toxicity. When AmpB was incorporated into negatively charged liposomes composed of DMPC and DMPG (L-AmpB), it was less toxic but as effective as free AmpB. However, the mechanism of L-AmpB's enhanced therapeutic index remains unknown. We have demonstrated that AmpB predominantly associates with HDL when incorporated into positively and negatively charged liposomes. To further understand the therapeutic importance of AmpB predominantly associating with HDL, we next examined the influence of lipoproteins on the antifungal activity and renal cytotoxicity of AmpB. The antifungal activity of AmpB and L-AmpB was not altered in the presence of HDL or LDL. The reduced nephrotoxicity associated with the use of L-AmpB, however, was related to a decreased uptake of AmpB by renal cells when AmpB was associated with HDL, and it may be a result of the low expression of HDL receptors in the LLC PK1 renal cells.

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.

Effects of sphingomyelin degradation on cholesterol mobilization and efflux to high-density lipoproteins in cultured fibroblasts

The hydrolysis of sphingomyelin from cellular plasma membranes imposes many consequences on cellular cholesterol homeostasis by causing a rapid and dramatic redistribution of plasma membrane cholesterol within the cells (Slotte, J.P. and Bierman, E.L. (1988) Biochem. J. 250, 653–658). The objective of this study was to examine the effects of an extracellular cholesterol acceptor on the directions of the sphingomyelinase-induced cholesterol flow in cultured fibroblasts. We have used HDL 3 as a physiological acceptor for cholesterol, and measured the effects of sphingomyelin hydrolysis on efflux and endogenous esterification of cellular [ 3H]cholesterol. Treatment of cells with sphingomyelinase did induce a dramatically increased esterification of plasma-membrane-derived [ 3H]cholesterol. The presence of HDL 3 in the medium (100 μg/ml) did not prevent or reduce the extent of the sphingomyelinase-induced cellular esterification of [ 3H]cholesterol. Degradation of cellular sphingomyelin (75% hydrolysis) also did not enhance the rate of [ 3H]cholesterol efflux from the plasma membranes to HDL 3. In addition, we also observed that the degradation of sphingomyelin in the HDL 3 particles (complete degradation) did not change the apparent rate of [ 3H]cholesterol transfer from HDL 3 to the cells. These findings together indicate that hydrolysis of sphingomyelin did not markedly affect the rates of cholesterol surface transfer between HDL 3 and cells. By whatever mechanism cholesterol is forced to be translocated from the plasma membranes subsequent to the degradation of sphingomyelin, it appears that the sterol flow is specifically directed towards the interior of the cells.

Effets des sels minéraux sur l'activité de la lipoprotéine lipase du lait de vache in vitro

Summary - Effects of mineraI salts on the actlvlty of the bovine mllk IIpoproteln lipase ln vItro. The effects of some milk constituents such as mineraI elements (Na+, W, Ca++ and Mg>+) have been studied in vitro on the mLPL (skim milk) activity and that of the purified LPL, in presence and in absence of serum lipoprotein (HDL), with a view to highlighting the incidence of these factors on the lipolysis intensity and the regulation of the enzyme lipase through these factors. The results have shown a clear similarity of the LPL behaviour of skim milk or in a purified form. The presence of HDL in the incubation medium increases seven times the mLPL activity and five times the purified LPL activity, Amongst ail mineraI constituents involved in this study, divalent ions undoubtedly hold the highest inhibitory power of the lipoprotein lipase activity. This inhibitory effect, which has been modulated by the presence of HDL, depends mainly upon the concentration and the nature of ions added to the medium, but by no means upon the ionic strengh. With 1 = 0.24, the inhibition of the LPL by CaCI2 is 75% in presence of HDL and 90% without HDL in the incubation medium. Contrastingly, with a similar ionic strengh and in presence of NaCI, this activity is subject to any modification neither in presence nor in absence of HDL in the incubation medium.

Modification of LDL by platelet secretory products induces enhanced uptake and cholesterol accumulation in macrophages

LDL modified by incubation with platelet secretory products caused cholesterol accumulation and stimulation of cholesterol esterification in mouse peritoneal macrophages. Its uptake by the macrophages was a receptor-mediated process, not susceptible to competition by acetyl-LDL or polyanions suggesting independence of the scavenger receptor. Stimulation of the esterification process in macrophages by this modified LDL was inhibited by the lysosomal inhibitor chloroquine, indicating requirement for cellular uptake and lysosomal hydrolysis of the lipoprotein. Within the cell, the modified LDL inhibited cellular biosynthesis of triglycerides in a manner similar to the action of acetyl-LDL but different to the effect of native LDL. In the presence of HDL, acting in the medium as an acceptor for cholesterol, a low rate of cholesterol efflux from cells incubated with this modified LDL as well as with acetyl-LDL was demonstrated. A small reduction in cholesteryl ester synthesis was found in these cells, compared to a 60% reduction in cells incubated with native LDL. Thus it was demonstrated that LDL modified by platelet secretory products could induce macrophage cholesterol accumulation even though it was recognized and taken up via the regulatory LDL receptor.

The effect of inhibition of cholesterol esterification on the fate of cholesterol derived from HDL in rat hepatocyte monolayers

Rat HDL 2 is known to stimulate bile acid synthesis in rat hepatocyte monolayers. The intracellular fate of the cholesterol derived from the HDL 2 was studied using the inhibitor of cholesterol esterification, Sandoz compound 58-035. Rat HDL 2 added to rat hepatocyte monolayers caused a stimulation of cholesterol esterification of 32%. This stimulation could be inhibited by 58-035. A small significant increase in bile acid synthesis was also observed in cells in the presence of HDL 2, confirming our earlier observations. 58-035 prevented this increase. These observations imply that cholesterol entering the cell from HDL 2 is first esterified and can only enter the substrate pool for bile acid synthesis after subsequent intracellular hydrolysis.

The catabolism of very low density lipoproteins.

The lipolysis of very low density lipoproteins (VLDL) in vitro is a useful model for the study of the process of conversion of this triacylglycerol rich lipoprotein into low (LDL) and high (HDL) density lipoproteins. Data is reviewed to show that a portion of surface cholesterol and phospholipid which becomes redundant during lipolysis is lost from the lipoprotein. In the absence of HDL, the material forms lipoprotein-X (LpX) like vesicles which are not readily disrupted by HDL once they are formed. In the presence of HDL during lipolysis, the material is largely incorporated into HDL. The data is used to suggest a mechanism of formation of LpX-like vesicles in conditions where the production of surface remnants exceeds the capacity of HDL to disrupt them. Evidence is also provided to show that apolipoproteins (apo) C-II, C-III, and E are lost from VLDL and that this loss is primarily in association with a neutral core particle of HDL size which can subsequently exchange lipids and apolipoproteins with plasma HDL. Such a mechanism could account for the removal of apo E and excess cholesteryl ester which is necessary for conversion of VLDL to LDL. The role of hepatic lipase in this process remains speculative. Recent evidence is reviewed and used to propose that the enzyme may serve to rearrange the neutral core and surface composition of LDL and HDL subfractions to allow for the packaging of cholesteryl esters and the cycling of apolipoproteins.