Identification of sterol acceptors that stimulate cholesterol efflux from human spermatozoa during in vitro capacitation

The nature of cholesterol-binding proteins acting upon human spermatozoa during in vitro capacitation was determined by measuring the efflux of [³H]cholesterol and of [³H]cholesteryl sulfate from labeled spermatozoa. Efflux of [³H]sterols was stimulated when the labeled gametes were incubated in Ham's F-10 medium supplemented with female serum or follicular fluid. Upon centrifugation of capacitated spermatozoa and application of the supernatant to density-gradient ultracentrifugation for lipoprotein analysis, both [³H]cholesterol and [³H]cholesteryl sulfate were found to be carried by very-low-density lipoproteins (VLDL), low-density lipoproteins (VLDL), high-density lipoproteins (HDL), as well as the albumin fraction (d > 1.21) in serum. When the capacitation medium was supplemented with follicular fluid, the [³H]sterols were bound to HDL's and to the albumin fraction; when the latter fraction was analysed by molecular sieve chromatography, 60–70% of the radioactivity eluted in fractions with a mean molecular weight corresponding to that of human serum albumin. Sperm cholesterol efflux was also stimulated when serum or follicular fluid was added to a simplified medium (50 mM Tris-HCl, 0.56% NaCl, pH 7.8); efflux of [³H]cholesterol from labeled gametes progressed in a time-dependent manner, but was low in the absence of serum components. The [³H]cholesterol/cholesterol ratios were higher in the albumin and HDL fractions, indicating some degree of specificity of these sterol acceptors. It was observed that follicular fluid albumin has a [³H]sterol binding capacity that is 2—3-fold higher than that of serum albumin. Commercial human serum albumin also promoted sperm cholesterol efflux. These results provide new information concerning those components of follicular fluid which may play a role in human sperm capacitation and provide further support for the concept that loss of cholesterol from the sperm plasma membrane is an important component of the capacitation process.     

Transport of poly-β-hydroxybutyrate in human plasma

Poly-β-hydroxybutyrate (PHB) is an amphiphilic lipid that has been found to be a ubiquitous component of the cellular membranes of bacteria, plants and animals. The distribution of PHB in human plasma was investigated using chemical and immunological methods. PHB concentrations proved highly variable; in a random group of 24 blood donors, total plasma PHB ranged from 0.60 to 18.2 mg/l, with a mean of 3.5 mg/l. In plasma separated by density gradient ultracentrifugation, lipoproteins carried 20–30% of total plasma PHB; 6–14% in the very low density lipoproteins (VLDL), 8–16% in the low density lipoproteins (LDL), and < 3% in the high density lipoproteins (HDL). The majority of plasma PHB (70–80%) was found in protein fractions of density > 1.22 g/ml. Western blot analysis of the high density fractions with anti-PHB F(ab')₂ identified albumin as the major PHB-binding protein. The affinity of albumin for PHB was confirmed by in vitro studies which demonstrated transfer of ¹⁴C-PHB from chloroform into aqueous solutions of human and bovine serum albumins. PHB was less tightly bound to LDL than to other plasma components; the polymer could be isolated from LDL by extraction with chloroform, or by digestion with alkaline hypochlorite, but it could not similarly be recovered from VLDL or albumin. PHB in the LDL correlated positively with total plasma cholesterol and LDL cholesterol, and negatively with HDL cholesterol. The wide concentration range of PHB in plasma, its presence in VLDL and LDL and absence in HDL, coupled with its physical properties, suggest it may have important physiological effects.     

Serum lipoproteins as cholesterol donors to mycoplasma membranes.

Mycoplasma hominis andAcholeplasma laidlawii were grown in media in which a fraction of human serum lipoproteins provided the sole source of cholesterol. Increasing levels of very low density lipoproteins had an inhibitory effect on the growth of the organisms. Low and high density lipoproteins in all concentrations proved to be excellent sources of cholesterol. Both organisms were able to limit the amount of cholesterol taken up and to preferentially incorporate free cholesterol despite an excess of esterified cholesterol in the medium. When similar levels of free cholesterol were provided by low density or high density lipoproteins, the organisms incorporated from 20–45% more cholesterol from the former. This preference for cholesterol from low density lipoproteins partially supports the theory that the low density lipoproteins act as a donor while the high density lipoproteins are a scavenger of cholesterol.     

Effect of dietary coconut oil on lipoprotein composition of young chick (Gallus domesticus)

• 1.1. The composition of HDL, the major lipoprotein fraction from chick serum, drastically changed after 2 weeks of coconut oil feeding. Total cholesterol and triacylglycerols significantly increased following dietary 10 or 20% coconut oil supplementation.
• 2.2. Changes in LDL composition were less profound, cholesterol being the only component that increased by coconut oil supplementation (10 or 20%).
• 3.3. IDL proteins were the only components that increased following the same dietary treatment (20%).
• 4.4. VLDL cholesterol and proteins also increased after 1–2 weeks of 20% coconut oil supplementation to the diet.
• 5.5. Of total lipoproteins, the cholesterol content strongly increased after dietary treatment, while triacylglycerols did not change significantly.

     

Serum lipoproteins and albumin in the lecithin: Cholesterol acyltransferase reaction

The unique features of pig ovarian follicular fluids, i.e., presence of high density lipoprotein (HDL) only and lecithin: cholesterol acyltransferase (EC 2.3.1.43; LCAT) activity, provides a good model to study the effect of serum lipoproteins and serum albumin on the LCAT reaction. $\text{In}\text{vitro}$ cholesterol esterification is enhanced when very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions are added, but is inhibited when one or the other of these lipoproteins is absent. High concentrations of HDL₂ result in decreased activation which can be compensated for by the addition of the VLDL-LDL mixture. These findings suggest that the rate of cholesterol esterification in ovarian follicular fluid may be enhanced by providing the exogenous VLDL and LDL as the recipients of HDL-cholesteryl ester. The inhibition of LCAT activity caused by free fatty acid and lysophosphatidylcholine can be partially reversed by the addition of serum albumin, suggesting that serum albumin may regulate the LCAT reaction.     

Activation of lipoprotein lipase by lipoprotein fractions of human serum

Triglycerides in fat emulsions are hydrolyzed by lipoprotein lipase only when they are "activated" by serum lipoproteins. The contribution of different lipoprotein fractions to hydrolysis of triglycerides in soybean oil emulsion was assessed by determining the quantity of lipoprotein fraction required to give half-maximal hydrolysis. Most of the activator property of whole serum from normolipidemic, postabsorptive subjects was in high density lipoproteins. Low density lipoproteins and serum from which all lipoprotein classes were removed had little or no activity. Also, little activator was present in guinea pig serum or in very low density poor serum from an individual with lecithin:cholesterol acyltransferase deficiency, both of which are deficient in high density lipoproteins. Human very low density lipoproteins are potent activators and are much more active than predicted from their content of high density lipoprotein-protein. Per unit weight of protein, very low density lipoproteins had 13 times the activity of high density lipoproteins. These observations suggest that one or more of the major apoproteins of very low density lipoproteins, present as a minor constituent of high density lipoproteins, may be required for the activation process.     

Factors affecting the esterification of lipoprotein cholesterol by lecithin: cholesterol acyl transferase.

Lecithin: Cholesterol Acyltransferase (LCAT) esterified relatively small amounts of cholesterol from very low density lipoproteins (VLDL), low density lipoproteins (LDL) or high density lipoproteins (HDL) in the presence of 5% human serum albumin (HSA). On the other hand, in the presence of very high density (>1.225 g/ml) plasma fraction (F-4), the enzyme esterified cholesterol from VLDL at considerably higher rates than from LDL or HDL. VLDL together with some component present in the very high density plasma fraction (F-4) may thus provide a highly efficient complex resulting in a favorable configuration of substrate lipids for the enzyme.     

A method of water-soluble solid fraction saturation concentration evaluation in dry thalli of Antarctic lichenized fungi,in vivo

BackgroundAt initial steps of rehydration from cryptobiosis of anhydrobiotic organisms or at rehydration of dry tissues the liquid ¹H NMR signal increased anomaly. The surplus in liquid signal may appear if some solid constituents dissolved, or if they were decomposed by enzymatic action.MethodsHydration kinetics, sorption isotherm, ¹H NMR spectra and high power relaxometry were applied to monitor gaseous phase rehydration of Antarctic lichen Cetraria aculeata. Tightly and loosely bound water signal were distinguished, and the upper hydration limit for dissolution of water soluble solid fraction was not observed. A simple theoretical model was proposed.ResultsThe hydration courses showed a very tightly bound water fraction, a tightly bound water, and a loosely bound water fraction. Sigmoidal in form sorption isotherm was fitted well by multilayer sorption model. ¹H NMR showed one Gaussian signal component from solid matrix of thallus and one or two Lorentzian line components from tightly bound, and from loosely bound water. The hydration dependency of liquid signal was fitted by rational function.ConclusionsAlthough in dehydrated C.aculeata the level of carbohydrates and polyols was low, the lichenase action during rehydration process increased it; the averaged saturation concentration c_s=(57.3±12.0)%, which resembled that for sucrose.General significanceThe proposed method of water soluble solid fraction saturation concentration, c_s, calculation from ¹H NMR data may be applied for other organisms experiencing extreme dehydration or for dry tissues. We recalculated the published elsewhere data for horse chestnut (Aesculus hippocastanum) bast [water-soluble solid fraction recognized as sucrose, c_s=(74.5±5.1)%]; and for Usnea antarctica, where c_s=0.81±0.04.     

Effect of fatty acids on the synthesis and secretion of apolipoprotein B by rat hepatocytes

The modulation of apolipoprotein B synthesis and secretion by fatty acids in rat hepatocytes was studied. Maximum apolipoprotein B production was obtained in the case of oleic acid followed by linoleic, stearic and palmitic/linolenic acid when compared to control which was not supplemented with any fatty acids. Oleic acid was found to exert a concentration dependent increase in the secretion of [³H] apolipoprotein B into the medium while that associated with the cell layer was not affected. Pulse chase experiments in the presence of oleic acid showed that it caused an increase in the secretion of apolipoprotein B into the medium.¹⁴C-acetate incorporation into cholesterol and cholesteryl ester associated with the cell layer and secreted very low density lipoproteins also showed an increase in the presence of oleic acid indicating an increase in cholesterogenesis. The effect of oleic acid on [³H] apolipoprotein B and very low density lipoproteins secretion appeared to be mediated through cholesterol as (i) ketoconazole, an inhibitor of cholesterol synthesis caused significant reduction in the stimulatory effect of oleic acid on apolipoprotein secretion and (ii) mevinolin, another inhibitor of cholesterol synthesis also reversed the stimulatory effect of oleic acid on apolipoprotein B secretion. These results indicated that oleic acid may influence apolipoprotein B synthesis and secretion in hepatocytes probably by affecting cholesterol/cholesteryl ester formation which may be a critical component in the secretion of apolipoprotein B as lipoproteins     

Detergent-mediated phospholipidation of plasma lipoproteins increases HDL cholesterophilicity and cholesterol efflux via SR-BI

Cellular cholesterol efflux is an early, obligatory step in reverse cholesterol transport, the putative antiatherogenic mechanism by which human plasma high-density lipoproteins (HDL) transport cholesterol from peripheral tissue to the liver for recycling or disposal. HDL-phospholipid content is the essential cholesterol-binding component of lipoproteins and therefore a major determinant of cholesterol efflux. Thus, increased phospholipidation of lipoproteins, particularly HDL, is one strategy for increasing cholesterol efflux. This study validates a simple, new detergent perturbation method for the phospholipidation of plasma lipoproteins; we have quantified the cholesterophilicity of human plasma lipoproteins and the effects of lipoprotein phospholipidation on cholesterophilicity and cellular cholesterol efflux mediated by the class B type I scavenger receptor (SR-BI). We determined that low-density lipoproteins (LDL) are more cholesterophilic than HDL and that LDL has a higher affinity for phospholipids than HDL whereas HDL has a higher phospholipid capacity than LDL. Phospholipidation of total human plasma lipoproteins enhances cholesterol efflux, an effect that occurs largely through the preferential phospholipidation of HDL. We conclude that increasing HDL phospholipid increases its cholesterophilicity, thereby making it a better acceptor of cellular cholesterol efflux. Phospholipidation of lipoproteins by detergent perturbation is a simple way to increase HDL cholesterophilicity and cholesterol efflux in a way that may be clinically useful.     

Enhanced Hepatic apoA-I Secretion and Peripheral Efflux of Cholesterol and Phospholipid in CD36 Null Mice

CD36 facilitates oxidized low density lipoprotein uptake and is implicated in development of atherosclerotic lesions. CD36 also binds unmodified high and very low density lipoproteins (HDL, VLDL) but its role in the metabolism of these particles is unclear. Several polymorphisms in the CD36 gene were recently shown to associate with serum HDL cholesterol. To gain insight into potential mechanisms for these associations we examined HDL metabolism in CD36 null (CD36^−/−) mice. Feeding CD36^−/− mice a high cholesterol diet significantly increased serum HDL, cholesterol and phospholipids, as compared to wild type mice. HDL apolipoproteins apoA-I and apoA-IV were increased and shifted to higher density HDL fractions suggesting altered particle maturation. Clearance of dual-labeled HDL was unchanged in CD36^−/− mice and cholesterol uptake from HDL or LDL by isolated CD36^−/− hepatocytes was unaltered. However, CD36^−/− hepatocytes had higher cholesterol and phospholipid efflux rates. In addition, expression and secretion of apoA-I and apoA-IV were increased reflecting enhanced PXR. Similar to hepatocytes, cholesterol and phospholipid efflux were enhanced in CD36^−/− macrophages without changes in protein levels of ABCA1, ABCG1 or SR-B1. However, biotinylation assays showed increased surface ABCA1 localization in CD36^−/− cells. In conclusion, CD36 influences reverse cholesterol transport and hepatic ApoA-I production. Both pathways are enhanced in CD36 deficiency, increasing HDL concentrations, which suggests the potential benefit of CD36 inhibition.     

Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats

The mechanism of hypercholesterolemia effect of Cu²⁺ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu²⁺ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu²⁺ deficient rats.     

A study on the exchange of tightly bound nucleotides on the membrane-associated chloroplast ATP synthase complex

Light-induced exchange of tightly bound ADP on the membrane-associated chloroplast coupling factor 1 (CF₁) was concluded to be a two-step mechanism involving a loose enzyme-ADP complex (Strotmann, H., Bickel-Sandkötter, S. and Shoshan, V. (1979) FEBS Lett. 101, 316–320). Rapid binding of [¹⁴C]ADP to the coupling factor after deenergization of thylakoids which were illuminated in the presence of [¹⁴C]ADP was suggested to reflect the conversion of loosely bound to tightly bound ADP. Experimental data of the present paper support the assumption of an intermediate enzyme form with loosely bound ADP: (a) the amplitude of the rapid binding phase is independent on the concentration of uncoupler added in the light; (b) the amplitude is virtually unaffected by dilution of the medium [¹⁴]CADP concentration; (c) high concentrations of unlabeled ADP are required to reduce the rapid binding phase while binding of medium [¹⁴C]ADP is inhibited by unlabeled ADP in the micromolar range. These results exclude the possibility that the rapid initial formation of tightly bound [¹⁴C]ADP on deenergization might be caused by an energized nucleotide-free enzyme form which is able to pick up [¹⁴C]ADP from the medium at a higher rate than the deenergized nucleotide-free form. At saturating [¹⁴C]ADP concentrations in the light, the amount of the loose enzyme-ADP complex is about 35%, while 65% of the coupling factors contain a tightly bound ADP. Dissociation of the loose complex is slow in the absence of medium nucleotides but accelerated if ADP is present, suggesting that ADP binding to another site of the enzyme promotes release of the former ADP molecule. The significance of the loosely bound nucleotide in the catalytic mechanism is discussed.     

A Murine Model of Obesity With Accelerated Atherosclerosis

The epidemic of obesity sweeping developed nations is accompanied by an increase in atherosclerotic cardiovascular diseases. Dyslipidemia, diabetes, hypertension, and obesity are risk factors for cardiovascular disease. However, delineating the mechanism of obesity‐accelerated atherosclerosis has been hampered by a paucity of animal models. Similar to humans, apolipoprotein E–deficient (apoE^?/?) mice spontaneously develop atherosclerosis over their lifetime. To determine whether apoE^?/? mice would develop obesity with accelerated atherosclerosis, we fed mice diets containing 10 (low fat (LF)) or 60 (high fat (HF)) kcal % from fat for 17 weeks. Mice fed the HF diet had a marked increase in body weight and atherosclerotic lesion formation compared to mice fed the LF diet. There were no significant differences between groups in serum total cholesterol, triglycerides, or leptin concentrations. Plasma concentrations of the acute‐phase reactant serum amyloid A (SAA) are elevated in both obesity and cardiovascular disease. Accordingly, plasma SAA concentrations were increased fourfold (P < 0.01) in mice fed the HF diet. SAA was associated with both pro‐ and antiatherogenic lipoproteins in mice fed the HF diet compared to those fed the LF diet, in which SAA was primarily associated with the antiatherogenic lipoprotein high‐density lipoprotein (HDL). Moreover, SAA was localized with apoB‐containing lipoproteins and biglycan in the vascular wall. Taken together, these data suggest male apoE‐deficient mice are a model of metabolic syndrome and that chronic low level inflammation associated with increased SAA concentrations may mediate atherosclerotic lesion formation.     

Lipid transfer between human serum high density lipoproteins and egg yolk lipoproteins in incubation mixtures

Ultracentrifugally isolated human serum high density lipoproteins of d 1.063-1.21 (HDL) were incubated with egg yolk lipoproteins of d < 1.006 for up to 24 hr at various concentrations. Transfer of HDL cholesterol esters to egg yolk lipoproteins occurred simultaneously with transfer of glycerides from egg yolk lipoproteins to HDL. These observations show that exchange of lipids can take place between lipoproteins in the absence of other serum proteins and enzymes. The mole ratios of HDL cholesterol esters to glycerides approached an integral value of 1 : 1 during the course of the incubation. These results suggest that lipid components form complexes within the HDL structure.     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

Acute inflammation and infection maintain circulating phospholipid levels and enhance lipopolysaccharide binding to plasma lipoproteins

Circulating lipoproteins are thought to play an important role in the detoxification of lipopolysaccharide (LPS) by binding the bioactive lipid A portion of LPS to the lipoprotein surface. It has been assumed that hypocholesterolemia contributes to inflammation during critical illness by impairing LPS neutralization. We tested whether critical illness impaired LPS binding to lipoproteins and found, to the contrary, that LPS binding was enhanced and that LPS binding to the lipoprotein classes correlated with their phospholipid content. Whereas low serum cholesterol was almost entirely due to the loss of esterified cholesterol (a lipoprotein core component), phospholipids (the major lipoprotein surface lipid) were maintained at near normal levels and were increased in a hypertriglyceridemic subset of septic patients. The levels of phospholipids found in the LDL and VLDL fractions varied inversely with those in the HDL fraction, and LPS bound predominantly to lipoproteins in the LDL and VLDL fractions when HDL levels were low. Lipoproteins isolated from the serum of septic patients neutralized the bioactivity of the LPS that had bound to them. Our results show that the host response to acute inflammation and infection tends to maintain lipoprotein phospholipid levels and that, despite hypocholesterolemia and reduced HDL levels, circulating lipoproteins maintain their ability to bind and neutralize an important bacterial agonist, LPS.     

Mechanism of regulation of steroidogenesis in the adrenals by blood serum lipoproteins

It has been shown that high density lipoproteins obtained by ultracentrifugation and loaded with cholesterol and pregnenolone exert a stimulant effect on in vitro production of glucocorticoids by rat adrenals. Low density native lipoproteins possess an inhibitory effect connected with the protein component. The action of various blood serum lipoproteins on steroidogenesis in rat adrenals has significantly different mechanisms.     

Cholesterol transport between red blood cells and lipoproteins contributes to cholesterol metabolism in blood

Lipoproteins play a key role in transport of cholesterol to and from tissues. Recent studies have also demonstrated that red blood cells (RBCs), which carry large quantities of free cholesterol in their membrane, play an important role in reverse cholesterol transport. However, the exact role of RBCs in systemic cholesterol metabolism is poorly understood. RBCs were incubated with autologous plasma or isolated lipoproteins resulting in a significant net amount of cholesterol moved from RBCs to HDL, while cholesterol from LDL moved in the opposite direction. Furthermore, the bi-directional cholesterol transport between RBCs and plasma lipoproteins was saturable and temperature-, energy-, and time-dependent, consistent with an active process. We did not find LDLR, ABCG1, or scavenger receptor class B type 1 in RBCs but found a substantial amount of ABCA1 mRNA and protein. However, specific cholesterol efflux from RBCs to isolated apoA-I was negligible, and ABCA1 silencing with siRNA or inhibition with vanadate and Probucol did not inhibit the efflux to apoA-I, HDL, or plasma. Cholesterol efflux from and cholesterol uptake by RBCs from Abca1^+/+ and Abca1^−/− mice were similar, arguing against the role of ABCA1 in cholesterol flux between RBCs and lipoproteins. Bioinformatics analysis identified ABCA7, ABCG5, lipoprotein lipase, and mitochondrial translocator protein as possible candidates that may mediate the cholesterol flux. Together, these results suggest that RBCs actively participate in cholesterol transport in the blood, but the role of cholesterol transporters in RBCs remains uncertain.     

Incorporation of cholesterol into serum high and low density lipoproteins

Excess cholesterol (CHL) was incorporated into serum lipoproteins by incubation with Celite^®-dispersed CHL or CHL crystals. The initial rates of uptake were 15 and 7 mol CHL/mol lipoprotein × h^? for low (LDL) and high (HDL) density lipoprotein, respectively. Saturation values were obtained after 48 h and were 90 and 65 mol CHL/mol LDL, and 42 and 32 mol/mol HDL using CHL on Celite and CHL cyrstals, respectively. Characterization of the lipo-proteins showed a small change in electrophoretic mobility and an increase in molecular masses, especially after incubation with CHL on Celite. Spectroscopic methods showed only minor effects on the protein moieties.