Postprandial variations in the cholesteryl ester transfer protein activity, phospholipid transfer protein activity and plasma cholesterol efflux capacity in normolipidemic men.

BACKGROUND AND AIM: Plasma cholesterol efflux capacity is stimulated during postprandial (PP) hypertriglycerdemia. Plasma cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) are the key proteins in lipoprotein metabolism and remodelling, but their role during the PP cholesterol efflux process remains indeterminate. The aim of this study was to determine the effect of a fatty meal intake on plasma CETP and PLTP activities, and the capacity of plasma to promote cholesterol efflux, as well as to evaluate the relationship between these three key mechanisms of the reverse cholesterol transport process. METHODS AND RESULTS: CETP and PLTP activities and the cholesterol efflux capacity of plasma were measured over eight hours following a fatty meal (1000 kcal, 62% fat) in 13 normolipidemic men. CETP activity and the cholesterol efflux capacity of plasma from Fu5AH cells increased after the meal, reaching a maximum after eight hours (respectively 32%, p = 0.06, and 6.5%, p = 0.045), whereas PLTP activity remained unchanged. CETP and PLTP activities did not correlate with plasma cholesterol efflux capacity in the fasting or PP state. Plasma CETP activity in the fasting state positively correlated with the plasma non-esterified fatty acid (NEFA) levels, but no correlation was found with any lipid or apolipoprotein postprandially. The cholesterol efflux capacity of plasma correlated positively with high-density lipoprotein (HDL) components, the best correlation being with the HDL phospholipid fraction in both the fasting and PP states. CONCLUSIONS: These findings suggest that plasma CETP and PLTP activities in healthy normolipidemic subjects are differently regulated in the PP state, and are not correlated with the increased cholesterol efflux capacity of PP plasma. HDL-phospholipid remains the key factor in the regulation of the capacity of plasma to promote Fu5AH cell cholesterol efflux.

Beneficial effects of a moderate consumption of red wine on cellular cholesterol efflux in young men.

BACKGROUND AND AIM: Protection against coronary artery disease (CAD) by moderate alcohol consumption is thought to be partly mediated through an increase in high density lipoprotein (HDL) levels. The protective effect of HDL can be related to its role in reverse cholesterol transport. Some studies have shown that wine intake is associated with a lower CAD risk compared to other alcoholic beverages. METHODS AND RESULTS: In order to separate the possible beneficial effects of the alcoholic and the non-alcoholic components of red wine, three beverages were compared in a group of 56 healthy young men: red wine (W) (30 g alcohol/day), a solution with the same degree of alcohol (A) and alcohol-free red wine (AFW). Beverages were consumed in random order over a period of 14 days. W significantly increased serum HDL-C, Apo A-I, HDL3-C, LpA-I and LpA-I:A-II particles. With A, only ApoA-I, HDL3-C, LpA-I:A-II were increased, though triglycerides were also increased. AFW had no effect apart from decreasing HDL-C. Plasma CETP was never altered. Serum-promoted cellular cholesterol efflux was measured on 3H labelled Fu5AH cells. Fractional cholesterol efflux was increased only after W intake, by 7%. Efflux variations correlated positively with HDL-C, HDL3-C and HDL-phospholipid variations. CONCLUSIONS: A modest, specific beneficial effect of moderate red wine consumption was demonstrated in comparison to an alcoholic solution. This was due to its effects on lipoproteins and its stimulation of serum ability to induce efflux of cellular cholesterol.

Oleic acid-rich fats increase the capacity of postprandial serum to promote cholesterol efflux from Fu5AH cells.

Cell cholesterol efflux to serum is stimulated after an oral fat load. The impact of meal fatty acid composition was explored by measure of serum promoted cholesterol efflux from Fu5AH cells after ingestion of 4 different fats: sunflower (Sf), oleic-sunflower (Ol), a mixed oil (Mx), and beef tallow (Bt). High density lipoprotein (HDL)2 and HDL3 were isolated and analyzed. Cholesterol efflux increased regularly after Ol (P<0.05 at 4 h and P<0.02 at 8 h), and 8 h after Mx (P<0.02) or Bt (P<0.05), but not after Sf. Percent HDL3 phospholipids increased after Ol (P<0.05 at 6 h and P<0.01 at 8 H) and 8 h after Mx (P<0.01). After Ol, variations in efflux and percent phospholipids in HDL3 (but not HDL2) were positively correlated (r=0.929; P=0.007 at 6 h). Using HDL3, efflux increased 6 h after Ol (P<0.05) but not after Sf, and efflux was correlated with HDL3 phospholipid concentration in medium (r=0.913; P=0.011). Thus postprandial increase in cholesterol efflux in influenced by ingested fats in relation to increased phospholipid availability on HDL3. The protective effect of monounsaturated fatty acids against atherogenesis might be partly mediated by an enhanced ability of postprandial serum to accept cell cholesterol.

Binding of HDL to basolateral membranes of the renal cortex. Evidence for two components in the HDL-membrane association.

The binding of porcine 125I-HDL to purified basolateral membrane fractions isolated from pig kidney cortex displays two categories of sites, one with high affinity ((Kd = (3.0 +/- 0.7) x 10(-9) M) and low capacity (Bmax = 52 +/- 32 ng/mg proteins) another with low affinity (Kd = (5.3 +/- 0.7) x 10(-8) M) but a higher capacity (Bmax = 795 +/- 115 ng/mg proteins). Binding was competitively inhibited to the same extent by unlabeled HDL from swine, human or rat, demonstrating an absence of species specificity. Porcine LDL partially competed for binding even in the presence of 30 mM EDTA which prevents apo B/E specific binding. Membrane proteins solubilized with CHAPS were analyzed by electrophoresis followed by ligand blotting using porcine 125I-HDL and 125I-apoAI-HDL to show that HDL bound to two proteins of respective molecular masses 120 +/- 2 and 95 +/- 9 kDa. 125I-apoAI associated mostly with the 95 kDa protein. A 100-fold excess of unlabeled HDL greatly decreased binding to the 95 kDa protein but less to the 120 kDa protein. We conclude that part of HDL binding occurs through the lipid moiety, while another is the result of a specific interaction between apoAI and a membrane protein of 95 kDa.

Effect of a moderate alcohol intake on the lipoproteins of normotriglyceridemic obese subjects compared with normoponderal controls.

Moderate alcohol intake is frequently associated with an elevated concentration of high-density lipoprotein (HDL), which is one of the potential causes for the relative decrease in cardiovascular risk reported in moderate drinkers. Conversely, low HDL concentrations, particularly HDL2, in obese subjects may be a risk factor. The effect of 30 g alcohol daily (wine) during 14 days following a period of abstinence was studied in seven normolipidemic obese subjects (body mass index [BMI], 30 +/- 1.7 kg/m2) compared with seven normoponderal controls (BMI, 22 +/- 1.2 kg/m2). Alcohol caused apolipoprotein (apo) AI and apo AII concentrations to increase in all controls by 12% and 16% (P less than .05), but not in obese subjects. Lipoprotein (Lp) AI HDL particles (without AII) were initially in the same proportions in the two groups. Their increase in controls only (P less than .03) was not matched by an increase in HDL2 in all subjects. In obese subjects, neither Lp AI nor HDL2 were increased by alcohol, but their HDL-triglyceride (TG) contents, initially elevated, were normalized. Cholesterol ester (CE) transfer activity was not different in controls and obese subjects during abstinence (105.7 +/- 40.8 v 104.8 +/- 34.5 mmol/mg protein/h). It was notably depressed by alcohol in controls (74.2 +/- 27.4, P less than .002), but not in obese subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sympathetic de-activation on thermogenic function and membrane lipid composition in mitochondria of brown adipose tissue.

Male Long-Evans rats (9 weeks of age) were exposed to cold (5 degrees C) for 10 days. Then, sympathetic de-activation of brown adipose tissue (BAT) was performed either by BAT surgical denervation (Sy) or by warm re-exposure at 28 degrees C (WE) for 4 days. The incidence of the two treatments on thermogenic activity of BAT mitochondrial membranes and their lipid composition was investigated. Sy and WE induced a large decrease in GDP binding on the uncoupling protein (UCP) (43% and 82%, respectively). Several parameters of mitochondrial energization were investigated. Sy and WE substantially decreased UCP-dependent proton conductance (CmH+) over the whole range of protonmotive force. CmH+ showed greater variation than GDP binding. The low basal UCP-independent CmH+ was the same in all groups. Comparison of GDP binding and CmH+ with UCP content which is not modified revealed a masking of both the nucleotide binding site and the proton channel. Sy and WE induced the same increase of phosphatidylcholine to phosphatidylethanolamine ratio (16%) but had opposite effects on fatty acid unsaturation. The results were discussed with reference to functional significance of these variations in BAT mitochondrial thermogenic activity and lipid composition.

Effect of apoprotein cross-linking on the metabolism of human HDL3 in rat.

Apo E-free human high-density lipoprotein (HDL3) was labeled with 125I in apoprotein and with 3H in cholesteryl linoleyl ether (a non-hydrolyzable analogue of cholesteryl ester). The labeled HDL3 was modified by cross-linking of apoproteins with dimethylsuberimidate (DMS) to inhibit binding to HDL specific receptors. The control and the DMS HDL3 were characterized with respect to their rate of clearance from rat blood, in vivo binding to major rat organs and in vitro binding to purified rat liver plasma membranes. Both 125I and 3H labels from control HDL3 were cleared from rat blood monoexponentially, but 3H at a faster rate than 125I (3H t1/2 = 3.0-4.1 h; 125I t1/2 = 7.0-7.7 h). This difference is consistent with reports of the nonendocytotic selective uptake of HDL-associated cholesteryl ester. DMS modification did not affect the rate of 3H clearance whereas it increased the rate of 125I clearance (HDL3 t1/2 = 7.7 h; DMS HDL3 t1/2 = 4.1 h). Both in vivo binding to rat organs and in vitro binding to rat liver membranes confirmed that DMS modification inhibited the specific binding of HDL, but also suggested that the modification produced saturable binding of HDL to a separate class of sites. Thus, the present data do not rule out the involvement of direct HDL-cell interaction in the selective uptake of HDL cholesteryl ester. However, results suggest that the binding of HDL to its specific cell surface sites is not necessary for this uptake.

Non-shivering thermogenesis and brown adipose tissue activity in essential fatty acid deficient rats.

The effects of essential fatty acid (EFA) deficiency on energetic metabolism and interscapular brown adipose tissue (BAT) activity were examined in the cold acclimated rat. Weanling male Long-Evans rats were fed on a low fat semipurified diet (control diet, 2% sunflower oil; EFA deficient diet, 2% hydrogenated coconut oil) for 9 weeks. They were exposed at 5 degrees C for the last 5 weeks. In EFA deficient rats, compared to controls, growth retardation reached 22% at sacrifice. Caloric intake being the same in the two groups, it follows that food efficiency was decreased by 40%. Resting metabolism in relation to body surface area was 25% increased. Calorigenic effect of norepinephrine (NE) in vivo (test of non-shivering thermogenesis) underwent a marked decrease of 34%. BAT weight was 21% decreased but total and mitochondrial protein content showed no variation. A 26% increase in purine nucleotide binding per BAT (taken as an index of thermogenic activity) was observed, suggesting that the enhancement in resting metabolism observed was mainly due to increased BAT thermogenesis. However, BAT mitochondria respiratory studies which are more direct functional tests showed a marked impairment of maximal O2 consumption of about 30% with palmitoyl-carnitine or acetyl-carnitine (both in presence of malate) or with alpha-glycerophosphate as substrate. It is likely that this impaired maximal BAT oxidative capacity may explain the impaired NE calorigenic effect in vivo. A possible increase in mitochondrial basal permeability is also discussed.

Relation between membrane phospholipid composition, fluidity and function in mitochondria of rat brown adipose tissue. Effect of thermal adaptation and essential fatty acid deficiency.

Male weanling rats were maintained either at 28 degrees C (thermoneutrality) or at 5 degrees C (cold adaptation). During 9 weeks they were fed either a 2% hydrogenated coconut oil diet deficient in essential fatty acids or a diet containing 2% sunflower oil. The respective incidences of cold adaptation and of EFA deficiency on lipid composition of mitochondrial membranes from brown adipose tissue (BAT) were investigated. Using 1,6 diphenylhexatriene (DPH) as a probe, the parameters of membrane fluidity were estimated by steady-state fluorescence polarization measurements (rs) and by time-resolved fluorescence anisotropy decay (order parameter S). Cold acclimation induced a decrease of phosphatidylcholine to phosphatidylethanolamine (PC/PE ratio), an increase of the total fatty acid unsaturation index (T.U.). EFA deficiency had the same effect as cold on the PC/PE ratio, but decreased T.U. Cold adaptation induced a larger decrease of S than of rs, whereas EFA deficiency only increased rs and did not modify S. In liposomes prepared from mitochondrial lipids, rs values were smaller than in whole mitochondria. Both in cold-adapted and in EFA-deficient rats the variations of rs were correlated with lipid unsaturation. Comparison between BAT thermogenic activity, assessed by GDP binding and proportions of PE and PC showed a high correlation suggesting a change in the membrane occurring with the increase of mitochondrial activity that could be related to phospholipid composition rather than to membrane fluidity.

The effects of essential fatty acid deficiency on brown adipose tissue activity in rats maintained at thermal neutrality.

1. The consequences of essential fatty acid (EFA) deficiency on the resting metabolism, food efficiency and brown adipose tissue (BAT) thermogenic activity were examined in rats maintained at thermal neutrality (28 C). 2. Weanling male Long-Evans rats were fed a hypolipidic semi-purified diet (control diet: 2% sunflower oil; EFA-deficient diet: 2% hydrogenated coconut oil) for 9 weeks. 3. They were kept at 28 C for the last 5 weeks. Compared to controls, in EFA-deficient rats the growth shortfall reached 21% at killing. 4. As food intake was the same in EFA-deficient and control rats, food efficiency was thus decreased by 40%. 5. Resting metabolism expressed per surface unit was 15% increased. 6. Non-renal water loss was increased by 88%. 7. BAT weight was 28% decreased but total and mitochondrial proteins were not modified. 8. Heat production capacity, tested by GDP binding per BAT was 69% increased in BAT of deficient rats. 9. The stimulation of BAT was established by two other tests: GDP inhibition of mitochondrial O2 consumption and swelling of mitochondria. 10. It is suggested that the observed enhancement of resting metabolism in EFA-deficient rats is, in part, due to an activation of heat production in BAT.

In vitro study of adrenergic stimulation of 32P incorporation into phospholipids of brown adipose tissue of control and cold-acclimated rats.

1. 32P-labelled inorganic phosphate incorporation into total and mitochondrial phospholipids was studied, in vitro, on brown adipose tissue (BAT) of control and cold-acclimated rats. 2. It was found that norepinephrine acts as in vivo, on BAT phospholipid metabolism via alpha 1 adrenergic receptors specifically increasing phosphatidic acid and phosphatidylinositol turnover with the same magnitude in both groups. 3. Cold-induced alpha 1 adrenergic desensitization is not as important as cold-induced beta adrenergic desensitization. 4. No specific effect of norepinephrine was seen in mitochondrial phospholipid turnover.

Characteristics of beta-adrenergic receptors in isolated cells and in crude membranes of brown adipose tissue.

In relation to decreased metabolic sensitivity to catecholamines observed, in vitro, in brown fat of cold-acclimated rats, beta-adrenergic receptors were studied in isolated cells and in a crude membrane preparation from rat interscapular brown adipose tissue. [3H] dihydroalprenolol binding had the same characteristics in both types of preparation; competition studies of [3H] dihydroalprenolol binding led to the characterization of beta 1 subtype adrenergic receptors with a lower affinity of beta-adrenergic agonists for [3H] dihydroalprenolol binding sites in membranes than that found in isolated cells. Cold acclimation produced, in isolated cells only, a decrease of 41% in the [3H] dihydroalprenolol binding sites and a beta-adrenergic agonist affinity increase. It is concluded that beta-adrenergic receptor decrease could be a factor, at the hormone receptor interaction level, in the regulation of the transmission of biological action responsible for the cold-induced decrease in catecholamine responsiveness in brown adipose tissue. For a study of the desensitization process in brown fat, isolated cells seem to offer certain advantages over a crude membrane preparation.

Beta-adrenergic receptors in white fat cells of cold-acclimated rats.

Beta-adrenergic receptors were studied in membranes prepared from isolated fat cells obtained from both control and cold-acclimated rats. Beta-adrenergic receptors were identified using beta-adrenergic antagonist, (-) - [3H] dihydroalprenolol. Scatchard plots of specific binding suggest the existence of two binding sites for (-) - [3H] dihydroalprenolol with different affinities. Beta-adrenergic agonists competed for specific binding sites with typical beta 1-adrenergic specificity. There appears to be approximately 5,000 sites of high affinity per fat cell. Cold-acclimation (4 weeks at 4 degrees C) reduced adipocyte size; it did not appear to alter either the maximal number of binding sites of high and low affinity, or their affinity for beta-antagonists or agonists; however the density of binding sites in the fat cell was increased in cold-acclimated rats. It is concluded that the cold-adaptative process which induces in vivo the enhancement of calorigenic effect of norepinephrine is not found at the level of beta-receptors in white fat cells. The increased lipolytic response to norepinephrine observed in vitro in epididymal fat may be explained by cold-induced modifications in cell size and number.

Cold-induced developmental changes in fat cell size and number in brown adipose tissue of the rat.

Seven-week-old Long-Evans rats were acclimated to a constant temperature of either 28 degrees C (control group) or 5 degrees C (cold-acclimated group). Cold acclimation induced a 70% increase in the interscapular brown adipose tissue (IBAT) relative mass, a 35% increase in DNA content, and a 44% decrease in triglyceride (TG) content, which resulted in a 51% decrease of the TG/DNA ratio. A procedure is described by which brown fat cells were isolated, with a yield of 21% from the IBAT of the control group and of 38% in the cold-acclimated group. In both groups, the brown fat cells accounted for 35-37% of the total cells in the tissue. Cold acclimation induced decreases in the mean fat cell diameter (about 20%), the mean fat cell TG content (50%), and the fat cell TG/DNA ratio (50%). The total number of IBAT fat cells was significantly increased in cold-acclimated rats. It is concluded that cold acclimation involves a hyperplasia of the IBAT, associated with a decrease of fat cell size without any alteration of the fat cell-to-nonfat cell ratio.

Cellularity and composition of epididymal adipose tissue from cold-acclimatized rats.

Cold acclimatization induces morphological and compositional modifications of rat epididymal adipose tissue: a decrease in fat cell size, an increase of fat cell number per g of tissue, but no significant increase in total fat cell number in the tissue; finally, an increase in protein content and a decrease in triglyceride content.

The lipid composition of plasma membrane and mitochondrial fractions from epididymal adipocytes of cold-acclimated rats.

1. The effects of cold acclimation (5 degrees C) on the lipid composition of plasma membrane and mitochondrial fractions from epididymal adipocytes of rats were studied. 2. The adipocyte plasma membrane fraction of the cold-acclimated rats had lower lipid, phospholipid and cholesterol to protein weight ratios, a lower cholesterol to sphingomyelin molar ratio, and a higher linoleic acid content in the phospholipids than controls. 3. The mitochondrial fraction of the cold-acclimated rat adipocyte had lower ratios of cholesterol to protein (weight), to phospholipid and to cardiolipin (molar), and less sphingomyelin content than did controls. 4. These data, discussed in terms of alterations in physical and biochemical properties, indicate cold-induced changes at the membrane level in rat epididymal adipocytes.

Prostaglandins E2 and F alpha levels in white and brown adipose tissues of cold acclimated rats as measured by a new micromethod.

A new micro-method was used to determine the effects of cold acclimation of rats on the levels of prostaglandin E2 and F alpha in both white and brown adipose tissues. Whereas PGF alpha levels were significantly higher than PGE2 levels in white fat, no difference between the amounts of the two prostaglandins was observed in brown fat. In both tissues, cold acclimation did not induce any change in prostaglandin levels.