Influence of subcutaneous injection of essential fatty acids on the stress-induced modifications of rat platelet aggregation and membrane lipid composition

Membrane lipids play an important role in the function of blood platelets but the mechanisms by which the lipid composition of the platelet membrane is adjusted remain unclear. It has been shown that stress and poly-unsaturated fatty acids modified the lipid composition of blood plasma and platelet lipids, but very little is known about the effect of stress and fatty acids on membrane platelet lipid composition. The purpose of the present investigation was to study the influence of the essential fatty acids: linoleic, linolenic and arachidonic acids on the composition of the platelet membrane lipids of rats assigned to heat and restraint stress. It was shown that injections of polyunsaturated fatty acids decrease or suppress the stress-induced increase in platelet aggregation, suppress the stress-induced modification of the composition of the platelet membrane lipids and modify the fatty acid composition of the platelet membrane phospholipids.     

Évolution de la composante lipidique de la membrane plasmique des spermatozoïdes durant la maturation épididymaire

One aspect of mammalian post-testicular sperm maturation is the progressive change in their plasma membrane lipid composition. These modifications in lipids allow sperm cells to fuse with oocytes during fertilization. A significant share of these sperm lipid changes occurs during their descent through the epididymal tubule. It then continues within the female genital tract during the capacitation process, an essential prerequisite for acrosomic reaction and hence fertilization. This review presents what is known concerning the sperm plasma membrane lipid changes during epididymal maturation in various mammalian models. In the first section, after a brief presentation of the classic eukaryotic cell plasma membrane lipid organization, the emphasis is on the particularities of sperm plasma membrane lipids. The second section presents the different changes occurring in the three major classes of lipids (i.e. phospholipids, sterols and fatty acids) during the sperm’s epididymal descent. The final section briefly describes the mechanisms by which these lipid changes might happen in the epididymal lumen environment. The role played by lipid-rich vesicles secreted by the epididymal epithelium via apocrine secretory processes is highlighted.     

Effect of ingestion of saline, glucose, and ethanol on mobilization and hepatic incorporation of epididymal pad palmitate-1-14C in rats

The effect of ingestion of saline, glucose, and ethanol (isocaloric with the glucose) on the mobilization of radiopalmitate from epididymal fat prelabeled in vivo and the incorporation of the mobilized label into liver lipids was investigated in rats. The mobilization of radiopalmitate from epididymal fat and the incorporation of the mobilized label into liver triglyceride were most markedly elevated by ingestion of ethanol. Increased mobilization and diversion of epididymal adipose tissue fatty acids to liver lipids of ethanol-treated rats were shown also by the close resemblance of the fatty acids of liver triglyceride to the fatty acids of epididymal fat. The amount of radiopalmitate mobilized by the saline-treated rats, comprising approximately a third of that mobilized by the ethanol-treated animals, was larger than the amount mobilized by the rats treated with glucose; most of it was oxidized rather than incorporated into the liver fats. In glucose-treated rats a larger fraction of radiopalmitate mobilized from one prelabeled epididymal pad was diverted to and incorporated into the lipids of the contralateral pad of the same animal. The specific activity of hepatic triglyceride of ethanol- and saline-treated rats was similar and significantly higher than that of animals treated with glucose. These data indicate that the ethanol-induced fatty liver can be attributed to an increased mobilization and incorporation of adipose tissue fatty acids into liver lipid and to an altered hepatic metabolism of fatty acids and triglyceride.     

Opposing actions of dehydroepiandrosterone and corticosterone in rats.

The purpose of this study was to determine the impact of dehydroepiandrosterone (DHEA) and corticosterone (CORT) treatment, using implants as a route of administration, on specific hormones, metabolites, and enzymes involved in energy metabolism. Sixty male Sprague-Dawley rats, 325 g initial weight, were implanted subcutaneously for 3 weeks with time-release pellets containing either DHEA or CORT at doses of 0, 10, 25, 50, or 100 mg in this 2 x 5 factorial experiment. In general, body weights and food intakes decreased as the level of steroid hormones increased. In contrast to DHEA treatment, rats receiving the 50- and 100-mg doses of CORT had lighter thymus glands and spleens and heavier epididymal and retroperitoneal fat pads than their controls. Rats treated with 100 mg of DHEA had lowered serum levels of triglycerides and lipid hydroperoxides whereas rats treated with 100 mg of CORT had higher levels of these blood lipids compared to their respective controls. In contrast to DHEA treatment, there was a dose-dependent increase in liver lipid content and the specific activities of the hepatic lipogenic enzymes glucose-6-phosphate dehydrogenase, malic enzyme, and fatty acid synthase in response to CORT treatment. Rats treated with 100 mg of DHEA had higher serum levels of IGF-1 than control rats. Conversely, rats treated with 100 mg of CORT had lower serum levels of IGF-1 and higher serum levels of testosterone, progesterone, and insulin than their controls. These data demonstrate the lipogenic actions of corticosterone in rats. Conversely, DHEA treatment reduced serum and hepatic lipids. Furthermore, these data suggest that using implants instead of bolus injections of steroids may be a more physiological approach for studying the influence of these steroids on lipid metabolism.     

Age-related characteristics of lipid changes in the lungs, heart and brain of albino rats in relation to dietary factors

It has been studied how long-term periodical calorie-insufficient and growth-restraining nutrition of the Wistar albino rats influences the lipid metabolism. It is shown that diet has a marked effect on the levels of phospholipids, cholesterol, neutral lipids, fatty acids in different tissues. The lipid composition in aged rats with prolonged life is closer to that of young animals of the control group.     

Age-related characteristics of the effect of periodic low-calorie nutrition on lipid composition of the liver cells, small intestine, adipose tissue and blood serum in albino rats

It is studied how long-term periodical calorie-insufficient and growth-restraining nutrition promoting appreciable life prolongation in the Wistar-line albino rats influences the lipid metabolism. It is shown that diet has an essential influence on the levels of phospholipids, cholesterol, neutral lipids, fatty acids in different tissue cells. The age specificity is observed in all the studied tissue cells. The lipid composition of cells in aged rats with prolonged life is similar to that of young animals of control group.     

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.     

Membrane lipid fluidity and its effect on the activation energy of membrane-associated enzymes.

1. The fatty acid composition of mitochondrial membranes from sheep and rats was altered by feeding these animals diets which were rich in unsaturated fatty acids. Changes in membrane lipid fluidity resulting from the altered membrane lipid composition were assessed by determining the upper temperature limit of the disorder-order transition (Tf) and the Arrhenius activation energy (Ea) of succinate oxidase. 2. After feeding the unsaturated fatty acid-rich diet to sheep the Ea, in the temperature range above Tf, increased from 8 to 63 kJ . mol-1 while Tf decreased from 32 to 15 degrees C. Rats fed an unsaturated fatty acid-rich diet exhibited an increase in Ea from 17 to 63 kJ . mol-1 and a decrease in Tf from 23 to 4 degrees C. 3. This decrease in Tf was related to an increase in the ratio of linoleic acid to stearic acid in the membrane lipid. Tf was not related to the proportion of unsaturated fatty acids in the membrane lipids, although an increase in unsaturation usually led to a decrease in Tf. 4. The results show that membrane lipid fluidity has a direct influence on the conformation of the active site of some membrane-associated enzymes, with the result that such enzymes display a higher Ea when the membrane lipids are comparatively more fluid. The increase in Ea of membrane-associated enzymes which accompanies changes in the physical state of membrane suggests that some proteins may phase separate with the more fluid lipids at low temperatures.     

Influence of the bovine seminal plasma protein PDC-109 on the physical state of membranes.

PDC-109 is the main component of bovine seminal plasma and has been suggested to play an important role in the genesis of bovine sperm cells. Here, the effect of binding of PDC-109 to membranes on the structure and physical properties of the lipid phase was investigated. For that, ESR measurements were undertaken on model membranes (lipid vesicles) and on biological membranes (epididymal spermatozoa) by employing various spin-labeled phospholipids. We found that PDC-109 alters the membrane structure of lipid vesicles as well as of bovine epididymal spermatozoa in that the mobility of spin-labeled phospholipids was reduced in the presence of the protein. This immobilizing effect of the protein was not restricted to analogues of phosphatidylcholine but was also detected with spin-labeled phosphatidylethanolamine. However, the extent of immobilization was lower for phosphatidylethanolamine compared with phosphatidylcholine, supporting the lipid headgroup specificity of the protein. Besides phospholipid headgroups, the physical state of membrane lipids is also important for the interaction of PDC-109 with membranes, in that, e.g., the immobilizing effect of the protein on labeled lipids was larger in membranes above the phase transition temperature compared with the effect below this temperature. The results are of relevance for understanding the physiological role of PDC-109 in the genesis of sperm cells.     

Lipid diffusion in the plasma membrane of ram and boar spermatozoa during maturation in the epididymis measured by fluorescence recovery after photobleaching

Maturation of spermatozoa in the epididymis involves remodelling of many protein and lipid components of the plasma membrane. In this investigation we have examined whether (a) diffusion of lipid molecules in the surface membrane changes during epididymal maturation; (b) diffusion is spatially restricted; and (c) differences in lipid diffusion can be related to known changes in membrane composition. For this purpose we have used the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusion of the lipid reporter probe ODAF (5‐(octa‐decanoyl)aminofluorescein) in spermatozoa from two species: ram, where substantial changes in membrane lipids occur during passage through the epididymis, and boar, where there are relatively few changes. Results on ram spermatozoa show that between the testis and cauda epididymidis, diffusion coefficients values (D) for ODAF increase significantly in all the surface domains. Percentage recovery values (%R) remain constant irrespective of maturational status. In boar spermatozoa, however, D and %R values do not change significantly between epididymal regions. Cholesterol, which has widespread effects on the behaviour of lipid molecules in cell membranes, was visualized by binding of filipin. In both species filipin was concentrated over the acrosomal domain and cytoplasmic droplet of testicular spermatozoa, but in the epididymis it had a heterogenous distribution over the whole head and tail. These results are discussed in relation to the establishment and maintenance of lipid domains in spermatozoa and their influence on development of fertilizing capacity. Mol. Reprod. Dev. 52:207–215, 1999. © 1999 Wiley‐Liss, Inc.     

The effects of alpha-chlorohydrin on the composition of rat and rabbit epididymal plasma: a possible explanation of species difference.

The relationship between the antifertility effect of alpha-chlorohydrin and changes in composition of luminal plasma from the cauda epididymidis of rats and rabbits has been investigated. At each dose regimen studied, the fertilizing capacity of rats treated with alpha-chlorohydrin was reduced to zero. The levels of sodium, potassium, glycerylphosphorylcholine (GPC), acid phosphatase and alkaline phosphatase in epididymal plasma were not markedly affected by drug treatment. The most noticeable change was a considerable increase in the concentration of lactic dehydrogenase (LDH) at all dose levels and of glutamic-oxaloacetic transaminase (GOT) after 7 days of treatment with 8 and 16 mg/kg. The effect of cold shock on the composition of epididymal plasma showed that LDH and GOT are, at least in part, derived from spermatozoa. In contrast, alpha-chlorohydrin did not have an antifertility action in the rabbit, and the only notable change in the compositon of epididymal plasma was an increase in the level of GPC. These results provide evidence that, in the rat, alpha-chlorohydrin or a metabolite primarily exerts its antifertility effect by a direct action on the spermatozoa, whilst in the rabbit a barrier may exist to the entrance of the drug into the lumen of the epididymal duct.     

Essential fatty acids in tissue phospholipids and triglycerides of the zinc-deficient rat

This study addressed the possibility that zinc deficiency has different effects on the fatty acid composition of triglyceride compared to total phospholipid. Male weanling Sprague-Dawley rats were maintained for 6 weeks on a semisynthetic diet deficient in zinc (3 mg/kg zinc). Control rats (40 mg/kg zinc) were pair-fed. Lipid fractionation and fatty acid analysis were by thin-layer and gas chromatography, respectively. In zinc-deficient rats, the percentage of linoleic acid was increased or that of arachidonic acid was decreased in total phospholipids of plasma, liver, and testis, and in skin total lipids. Saturated and monounsaturated fatty acids were increased in the triglyceride of liver but decreased in the triglyceride of epididymal fat of zinc deficient rats. Essential fatty acids, as a proportion of total fatty acids, were decreased in triglyceride of liver but increased in triglyceride of epididymal fat of zinc-deficient rats. Our fatty acid data from tissue total phospholipids therefore support the concept that linoleic acid desaturation is impaired in zinc deficiency.     

Role of thyroid on testicular lipids in prepubertal, pubertal and adult rats. I. Hyperthyroidism

The influence of thyroxine-induced hyperthyroidism on testicular neutral and phospholipids was studied in prepubertal, pubertal and adult rats. Thyroxine treatment (25 micrograms/100 g body weight) for 1 month decreased testicular total lipids, total glyceride glycerols, total cholesterol and total phospholipids. Different classes of glyceride glycerol, cholesterol and phospholipid were also diminished due to thyroxine treatment. All classes of lipids returned to the euthyroid level after the withdrawal of thyroxine treatment. The data obtained in the present study suggest that thyroid hormones have a definite influence on testicular lipid metabolism in rats.     

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca²⁺-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca²⁺-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca²⁺-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.     

The evaluation of the age-related characteristics of the structural status of the plasma membrane lipid phase in rat fatty tissue by using the method of inductive-resonance energy transfer

Using the method of inductance-resonance energy transfer from tryptophanyl residues to fluorescent pyrene probe the structural state of plasmatic membranes from adipose tissue of different age rats has been studied. The structural heterogeneity of membrane lipid phase has been revealed. The differences in physical properties of annular and bilayer lipids don't depend on age. During aging the membrane lipid viscosity including lipids of near protein area decreases, the conformation of membrane protein components alters during aging as well. The data on various effectiveness of energy transfer from tryptophanyls to pyrene probe in young and aged animals with stable polypeptide composition of membrane proteins indicates that. The structure of membrane lipid phase is suggested to be the main factor affecting the conformational state and functional activity of membrane-bound proteins during aging.     

Milk Fat Content and DGAT1 Genotype Determine Lipid Composition of the Milk Fat Globule Membrane

During secretion of milk fat globules, triacylglycerol (TAG) droplets are enveloped by a phospholipid (PL) trilayer. Globule size has been found to be related to polar lipid composition and fat content, and milk fat content and fatty acid composition have been associated with the diacylglycerol acyltransferase 1 (DGAT1) K232A polymorphism; however, the association between the DGAT1 polymorphism and fat globule size and polar lipid composition has not been studied. The ratio between polar and neutral lipids as well as the composition of the polar lipids in milk has industrial as well as nutritional and health implications. Understanding phenotypic and genotypic factors influencing these parameters could contribute to improving milk lipid composition for dairy products. The focus of the present study was to determine the effect of both fat content and DGAT1 polymorphism on PL/TAG ratio, as a marker for milk fat globule size, and detailed PL composition. Milk samples were selected from 200 cows such that there were equal numbers of samples for the different fat contents as well as per DGAT1 genotype. Samples were analyzed for neutral and polar lipid concentration and composition. PL/TAG ratio was significantly associated with both fat content and DGAT1 genotype. Phosphatidylinositol and phosphatidylserine concentrations were associated with fat content*DGAT1 genotype with a stronger association for the AA than the KK genotype. Sphingomyelin concentration tended to interact with fat content*DGAT1 genotype. Phosphatidylethanolamine (PE) concentration showed a biphasic response to fat content, suggesting that multiple biological processes influence its concentration. These results provide a new direction for controlling polar lipid concentration and composition in milk through selective breeding of cows.     

Do Lipids Show State-dependent Affinity to the Voltage-gated Potassium Channel KvAP?

As all integral membrane proteins, voltage-gated ion channels are embedded in a lipid matrix that regulates their channel behavior either by physicochemical properties or by direct binding. Because manipulation of the lipid composition in cells is difficult, we investigated the influence of different lipids on purified KvAP channels reconstituted in planar lipid bilayers of known composition. Lipids developed two distinct and independent effects on the KvAP channels; lipids interacting with the pore lowered the energy barriers for the final transitions, whereas voltage sensor-bound lipids shifted the midpoint of activation dependent on their electrostatic charge. Above all, the midpoint of activation was determined only by those lipids the channels came in contact with first after purification and can seemingly only be exchanged if the channel resides in the open state. The high affinity of the bound lipids to the binding site has implications not only on our understanding of the gating mechanism but also on the general experimental design of any lipid dependence study.     

Effect of insulin on glucose transport and metabolism in isolated fat-cells of gonadal adipose tissue from mature age-matched male and female rats.

Insulin action on glucose transport and metabolism was studied in paraovarian adipocytes from 3-month-old female rats and compared with insulin action in epididymal adipocytes from closely age-matched males. At maximal insulin concentrations the stimulations of 2-deoxyglucose uptake (4-fold the basal value) and of [U-14C]glucose incorporation into CO2 and total lipids (3- and 2-fold the basal values respectively) were similar in adipocytes from rats of both sexes. At submaximal insulin concentrations (less than 0.2 nM) the ability of paraovarian adipocytes to transport and to metabolize glucose was higher than that of epididymal adipocytes; accordingly an increase in insulin binding was observed in paraovarian adipocytes as compared with epididymal adipocytes. These results show that paraovarian adipocytes from mature female rats were highly responsive to insulin, and exhibited a higher sensitivity to the hormone than did epididymal adipocytes from male rats of the same age.     

The role of adipose tissue in glucose utilization in irradiated rats

The authors studied the conversion of U-14C-glucose to total lipids, fatty acids and glyceride glycerol in the epididymal adipose tissue of rats X-irradiated with a single whole body dose of 14.4 Gy X-rays. Analyses were carried out 1, 24, 48 and 72 h after irradiation. In the adipose tissue of irradiated rats, the incorporation of 14C-glucose into all the lipid fractions was raised throughout the whole time of observation (300-600% of the control value). Most of the 14C-glucose was incorporated into the glyceride glycerol fraction.     

The influence of membrane lipid structure on plasma membrane Ca2+ -ATPase activity

Lipid composition and Ca(2+)-ATPase activity both change with age and disease in many tissues. We explored relationships between lipid composition/structure and plasma membrane Ca(2+)-ATPase (PMCA) activity. PMCA was purified from human erythrocytes and was reconstituted into liposomes prepared from human ocular lens membrane lipids and synthetic lipids. Lens lipids were used in this study as a model for naturally ordered lipids, but the influence of lens lipids on PMCA function is especially relevant to the lens since calcium homeostasis is vital to lens clarity. Compared to fiber cell lipids, epithelial lipids exhibited an ordered to disordered phase transition temperature that was 12 degrees C lower. Reconstitution of PMCA into lipids was essential for maximal activity. PMCA activity was two to three times higher when the surrounding phosphatidylcholine molecules contained acyl chains that were ordered (stiff) compared to disordered (fluid) acyl chains. In a completely ordered lipid hydrocarbon chain environment, PMCA associates more strongly with the acidic lipid phosphatidylserine in comparison to phosphatidylcholine. PMCA associates much more strongly with phosphatidylcholine containing disordered hydrocarbon chains than ordered hydrocarbon chains. PMCA activity is influenced by membrane lipid composition and structure. The naturally high degree of lipid order in plasma membranes such as those found in the human lens may serve to support PMCA activity. The absence of PMCA activity in the cortical region of human lenses is apparently not due to a different lipid environment. Changes in lipid composition such as those observed with age or disease could potentially influence PMCA function.