Chromatin remodeling in spermatids: a sensitive step for the genetic integrity of the male gamete.

Several causes of male infertility remain idiopathic. Recently, the condensed state of the sperm head has been demonstrated as a discriminating parameter for the assessment of male infertility. Altered DNA condensation is associated with an increase in DNA strand breakage so the genetic integrity of the male gamete is threatened. The origin of the DNA strand breaks in unknown. However, transient DNA strand breaks appear in the whole population of elongating spermatids during mid-spermiogenesis steps. Most likely, these transient breaks are required to support the change in DNA topology associated with chromatin remodeling at these steps. Histones hyperacetylation is also coincident with the DNA strand breakage steps. Hyperacetylation of histones may represent a necessary condition for strand breakages to form allowing access to the yet unknown enzymatic activity involved in the removal of DNA supercoils. A better characterization of this enzyme activity at these steps is necessary as this may represent a very sensitive process where altercations in the genetic integrity of the male gamete may arise and persist up to the mature spermatozoa. During the chromatin remodeling in spermatids, the combined DNA-condensing activities provides by the basic transition proteins and protamines may optimize the strand repair process emphasizing the link between altered sperm DNA condensation and DNA fragmentation. The mutagenic potential of these events may have been overlooked as it may result in fertility and/or developmental problems.

Evidence of apoptosis in the castration-induced atrophy of the rat levator ani muscle.

Apoptosis or programmed cell death has been previously reported in androgen-responsive tissue such as the prostate. We tested the hypothesis that apoptosis may also represent a component of the castration-induced atrophy of the sexually dimorphic levator ani (LA) muscle of the male rat. Gonadectomy (GDX) induced a severe decrease in the LA muscle wet weight accompanied by important modifications in cytoarchitecture including an increase in myofibrillar interspace and condensed mitochondria. These alterations were almost completely reversed after 7 days of testosterone propionate replacement therapy (GDX + TP). In GDX rats, internucleosomal DNA fragmentation was confirmed by both agarose gel electrophoresis and electron microscopy. DNA fragmentation was no longer detected in GDX + TP rats. In GDX rats, overexpression of the trpm-2/clusterin gene, used as an early marker of apoptosis, further confirmed that an apoptotic response, typical of androgen-responsive tissues, is taking place following androgen withdrawal.

Stimulation of DNA repair by the spermatidal TP1 protein.

The chromatin remodeling process that takes place during spermiogenesis in mammals is characterized by a transient increase in DNA single-strand breaks (SSB). The mammalian transition proteins (TPs) are expressed at a high level at mid-spermiogenesis steps coincident with chromatin remodeling and could be involved in the repair of these lesions since SSB are no longer detected in terminally differentiated spermatids. We report that TP1 can stimulate the repair of SSB in vitro and demonstrate that in vivo repair of UV-induced DNA lesions is enhanced in mammalian cells stably expressing TP1. These results suggest that, aside from its role in DNA compaction, this major transition protein may contribute to the yet unidentified enzymatic activity responsible for the repair of SSB at mid-spermiogenesis steps. These results also suggest that the TP1 proteins have the potential to participate in the repair process following genotoxic insults and therefore may play an active role in the maintenance of the integrity of the male haploid genome during spermiogenesis.

High levels of dietary carnosine are associated with increased concentrations of carnosine and histidine in rat soleus muscle.

The aims of this investigation were to: 1) determine the effect of a moderately high dose of carnosine on muscle concentrations of carnosine, histidine and vitamin E at deficient, minimally adequate and sufficient levels of dietary vitamin E and 2) compare the effects of moderately high and pharmacological doses of carnosine on muscle concentrations of carnosine, histidine and vitamin E when dietary vitamin E is minimally adequate. Muscle concentrations of carnosine, histidine and vitamin E were measured in the lateral gastrocnemius and red and white vastus lateralis; carnosine and histidine concentrations were also measured in soleus muscle. Male Sprague-Dawley rats (n = 12/group) were fed a basal vitamin E-deficient diet supplemented with either 0, 0.001 or 0.01% vitamin E and 0, 0.1 or 1.8% carnosine. After 8 wk, 1.8% carnosine resulted in significant fivefold increases in carnosine and twofold increases in histidine in the soleus muscle (P < or = 0.05). Muscle vitamin E concentrations were not significantly affected by dietary carnosine. Thus, very high levels of dietary carnosine are associated with increases in carnosine and histidine concentrations in rat soleus muscle.

Comparative study of the coupling between topoisomerase I activity and high-mobility group proteins in E. coli and mammalian cells.

It is now well established that the HMG box DNA-binding motif can alter the topology of double-stranded DNA in several ways. Using the spermatid-specific tsHMG as a model protein of the HMG-1/-2 family, we have demonstrated that its expression in E. coli produces an increase in plasmid supercoiling density that is likely a consequence of its ability to constrain free supercoils in vivo. As demonstrated in vitro, stabilization of free DNA supercoils by tsHMG prevents topoisomerase I from gaining access to the template and could represent a mechanism for the apparent inhibition of topoisomerase I in bacteria. A similar modulation of eukaryotic topoisomerase I activity was not detected after expression of the tsHMG in mammalian cells. This differential response is discussed in terms of the marked difference in DNA packaging and accessibility of free supercoils in prokaryotic vs. eukaryotic cells.

Influence of dietary protein and lipid on weight loss in obese ovariohysterectomized cats.

OBJECTIVE: To determine effects of dietary lipid and protein on development of hepatic lipidosis (HL) and on physical and biochemical indices following rapid weight loss in cats. ANIMALS: 24 ovariohysterectomized cats. PROCEDURE: Cats were fed a high energy diet until they gained 30% of their ideal body weight and then randomly assigned to receive 1 of 4 weight-reduction diets (6 cats/diet) at 25% of maintenance energy requirements per day. Diets contained a low or high quality protein source and a lipid source deficient or sufficient in long chain essential fatty acids (LCEFA). Serum and plasma samples and liver biopsy specimens were obtained for biochemical analyses and determination of hepatic lipid content before and after weight gain and during and after weight loss. RESULTS: Irrespective of weight-reduction diet fed, all cats lost weight at a comparable rate (4.51 to 5.00 g/d/kg of obese body weight). Three cats developed hepatic lipidosis. Significant changes in plasma insulin, cholesterol, triglyceride, and serum glucose concentrations were detected after weight gain and weight loss in all diet groups, but values for these variables did not differ among groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cats can lose 25 to 30% of their obese body weight over 7 to 9 weeks without developing overt clinical signs of HL, provided that weight-reduction diets are highly palatable, contain a high quality protein, have a source of LCEFA, and are fortified with vitamins and microminerals. However, rapid weight loss may increase risk factors associated with development of diabetes mellitus.

Architectural DNA-binding properties of the spermatidal transition proteins 1 and 2.

Mammalian spermiogenesis is characterized by replacement of somatic histones by a set of basic nuclear transition proteins thought to be actively involved in the chromatin remodeling process. The two major transition proteins of the elongating spermatids, namely TP1 and TP2, were expressed and purified using a bacterial expression system. Both topoisomerase and ligase-mediated supercoiling assays demonstrated that TP1, as well as TP2, did not produce detectable changes in the twist and/or writhe of DNA molecules upon binding. Ligase-mediated circularization assay further demonstrated that neither of the transition proteins under study produced bends in linear DNA but that they both have the capacity to stimulate oligomerization of linear DNA fragments. We further established that the transition proteins are in vitro substrates for the Ca+2-phospholipid-dependent protein kinase (PKC) as well as the cAMP-dependent protein kinase (PKA). PKC phosphorylation was found to strongly weaken the DNA-condensing ability of TP2. These results suggest that the major transition proteins represent architectural factors able to stabilize DNA in a nonsupercoiled state, thereby promoting DNA condensation.

Comparison of two ammoniation procedures to reduce the toxicity of endophyte-infected tall fescue seed fed to rats.

To determine the effect of extending the duration of ammonia (2% dry matter basis) treatment from 1 to 5 wk on the toxicity of endophyte-infected tall fescue seed, 60 male Harlan Sprague-Dawley rats were randomly assigned to the following six treatments during a 28-d trial: endophyte-free (E-), endophyte-infected (E+), 1 wk ammoniated endophyte-free (1AE-), 1 wk ammoniated endophyte-infected (1AE+), 5 wk ammoniated endophyte-free (5AE-), and 5 wk ammoniated endophyte-infected (5AE+) tall fescue seed. The concentration of total pyrrolizidine alkaloids (N-acetyl and N-formyl loline) of E+ fescue was reduced from 4203 micrograms/g to 3009 and 2533 micrograms/g by the 1AE+ and 5AE+ treatments, respectively. Ergovaline was lowered from 3.77 to 1.57 micrograms/g by 1AE+ and eliminated by 5AE+. Endophyte-infected treatment groups had depressed (P < 0.0001) daily feed intakes (DFI), daily weight gains (DWG), feed efficiencies (G/F), primary antibody responses, and T cell and B cell mitogenic responses than endophyte-free treatment groups. Ammoniation of endophyte-infected fescue seed improved DFI and DWG (P < 0.0001) and G/F (P < 0.05); however, there was no difference in performance criteria between the 1-wk and 5-wk ammoniation treatments. Endophyte-induced depressions in immune function were not alleviated by ammoniation.

Comparison of two ammoniation procedures to reduce the toxicity of endophyte-infected tall fescue seed fed to rats.

To determine the effect of extending the duration of ammonia (2% dry matter basis) treatment ti'om 1 to 5 wk on the toxicity of endophyte-infected tall fescue seed, 60 male Harlan Sprague-Dawley rats were randomly assigned to the following six treatments during a 28-d trial: endophyte-free (E-), endophyte-infected (E+), 1 wk ammoniated endophyte-fee (1AE-), 1 wk ammoniated endophyte-infected (1AE+), 5 wk ammoniated endophyte-free (5AE-), and 5 wk ammoniated endophyte-infected (5AE+) tall fescue seed. The concentration of total pyrrolizidine alkaloids (N-acetyl and N-formyl loline) or E+ rescue was reduced from 4203 12 g/g to 3009 and 2533 I-tg/g by the 1AE+ and 5AE+ treatments, respectively. Ergovaline was lowered from 3.77 to 1.57 12 g/g by 1AE+ and eliminated by 5AE+. Endophyte-infected treatment groups had depressed (P < 0.0001) dally feed intakes (DFI), daily weight gains (DWG), feed efficiencies (G/F), primary antibody responses, and T cell and B cell mitogenic responses than endophyte-free treatment groups. Ammoniation of endophyte-infected rescue seed improved DFI and DWG (P < 0.0001) and G/F (P < 0.05); however, there was no difference in performance criteria between the 1-wk and 5-wk ammoniation treatments. Endophyte-induced depressions in immune function were not alleviated by ammoniation.

The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids.

Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines. The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA. A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process. We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids. Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro. The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner. We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG. Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I. This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation.

Susceptibility to hepatic oxidative stress in rabbits fed different animal and plant fats.

OBJECTIVE: This study was designed to determine the effect of diets enriched with plant and animal fats on oxidative stress and glutathione metabolism in rabbit liver tissues. This study was conducted to investigate whether the type of dietary fat will impact fatty acid composition and oxidant/antioxidant status in tissues. METHODS: Rabbits were fed diets containing 2 g corn oil/100 g diet (low fat diet, LF) and LF supplemented with 16 g/100 g diet of either corn oil (CO), CO with added cholesterol (CO + C), milk fat (MF), chicken fat (CF), beef tallow (BT), or lard (L) for 30 days. After the feeding period, livers were analyzed for total fatty acid composition, thiobarbituric acid reactive substances (TBARS), conjugated dienes, and reduced glutathione (GSH), as well as for activities of glutathione peroxidase (GP) and glutathione reductase (GR). Moreover, to fully determine the oxidative stability and free radical trapping capacity, TBARS levels were measured after additional exposure of liver homogenates to 10 mM 2,2(1)-azo-bis-amidinopropane- hydrochloride (AAPH) for up to 21 hours. RESULTS: CO and CF, but not saturated fats such as MF, increased liver conjugated diene and TBARS levels and decreased liver GSH levels and GP activity. In tissues additionally exposed to AAPH, the maximum oxidation, measured as TBARS, was reached between 6 and 7 hours of treatment, independent of dietary fat. In addition, there was a marked effect of AAPH on the maximum rate of TBARS formation with the following descending order: CO > CF > CO + C > L > MF > BT > LF. This high susceptibility to oxidative stress in liver tissues of rabbits fed the CO diet may be explained in part by the significant elevation in linoleic acid (18:2n-6). DISCUSSION: There appears to be an inverse correlation between dietary fat-mediated oxidative stress and antioxidant enzyme activities. The present data suggest that high levels of dietary unsaturated fat should be avoided if oxidative stress is a critical issue in nutrition-related diseases. In addition, these data support our hypothesis that diets rich in MF provide a lipid environment with low susceptibility to oxidative stress.

Oxidized lipid-mediated alterations in proteoglycan metabolism in cultured pulmonary endothelial cells.

Compared to cholesterol or linoleic acid (18:2), oxidized lipids such as cholestan-3 beta, 5 alpha, 6 beta-triol (triol) and hydroperoxy linoleic acid (HPODE) markedly impair endothelial barrier function in culture [Hennig and Boissonneault, 1987; Hennig et al. 1986]. Because proteoglycans contribute to vascular permeability properties, the effects of cholesterol and 18:2 and their oxidation products, triol and HPODE, on endothelial proteoglycan metabolism were determined. While cholesterol was without effect, a concentration-dependent decrease in cellular proteoglycans (measured by 35S incorporation) was observed after exposure to triol. Compared to control cultures, cholesterol reduced mRNA levels for the proteoglycans, perlecan and biglycan. Triol had a similar effect on biglycan but not an perlecan mRNA levels. Compared to 18:2, 1,3 and 5 microM HPODE depressed cellular proteoglycans. Perlecan mRNA levels were reduced more by HPODE when compared to 18:2. Biglycan mRNA levels were reduced by 3 microM, but not by 5 microM HPODE. These data demonstrate that oxidized lipids such as triol and HPODE can decrease cellular proteoglycan metabolism in endothelial monolayers and alter mRNA levels of major specific proteoglycans in a concentration-dependent manner. This may have implications in lipid-mediated disruption of endothelial barrier function and atherosclerosis.

Role of fatty acids and eicosanoids in modulating proteoglycan metabolism in endothelial cells.

Endothelial cell dysfunction is considered to be a critical event in the etiology of atherosclerosis. Thus, the preservation of endothelial structure and function are a prerequisite for normal control of vascular permeability properties, mediation of both inflammatory and immunologic responses and the general 'communication' between blood-borne cells and abluminal tissues. Many of these properties can be influenced by proteoglycans present in vascular tissues. There is evidence that selected lipids can be atherogenic by altering endothelial proteoglycan metabolism. Little is known about the role of fatty acids in modulating proteoglycan composition in endothelial cells. Data suggest, however, that linoleic acid in particular can adversely alter proteoglycan metabolism, which may be related to an imbalance in eicosanoid synthesis patterns. These events could be sufficient to disrupt normal endothelial barrier function, initiate smooth muscle migration and proliferation, and result in other metabolic dysfunctions associated with the etiology of vascular diseases such as atherosclerosis. Thus, the focus of this review is on fatty acids and eicosanoids as they may alter proteoglycan metabolism of vascular tissues and in particular of the endothelium.

Animal and plant fats selectively modulate oxidizability of rabbit LDL and LDL-mediated disruption of endothelial barrier function.

Enrichment of lipoproteins with fatty acids derived from animal and/or plant fats may modify the oxidizability of lipoproteins and their effects on endothelial barrier function. To test this hypothesis, rabbits were fed for 30 days diets containing 2 g corn oil/100 g diet (low fat diet) or low fat supplemented with 16 g/100 g diet of corn oil, corn oil with added cholesterol, milk fat, chicken fat, beef tallow or lard. Compared with those fed the low fat, serum and LDL cholesterol concentrations were significantly lower in rabbits fed corn oil and greater in animals fed corn oil with added cholesterol or chicken fat. In contrast to the cholesterol data, lipid hydroperoxide levels were highest in oxidized LDL derived from rabbits fed corn oil or lard. LDL vitamin E levels were highest in rabbits fed corn oil with added cholesterol. The significant elevations in linoleic acid [18:2(n-6)] in serum and LDL may partially explain the high oxidizability of LDL in rabbits fed corn oil. LDL isolated from animals fed corn oil, lard or milk fat had significantly greater albumin transfer across cultured endothelial monolayers compared with those of the low fat diet group. Their oxidative modification further contributed to endothelial barrier dysfunction. Dietary cholesterol supplementation to the corn oil diet decreased oxidizability of LDL and partially protected the oxidized LDL-mediated endothelial cell dysfunction as compared with the corn oil diet group. These data suggest that beef tallow and chicken fat are the least atherogenic fats if oxidative modification of LDL is a critical issue in atherosclerosis.

Oxidized low-density lipoproteins delay endothelial wound healing: lack of effect of vitamin E.

The purpose of this study was to examine the influence of oxidized low-density lipoprotein (oxLDL) on endothelial regrowth in an in vitro wounding model and the possible protection afforded by vitamin E (E). Endothelial cells grown on micropore filters were wounded by scraping and allowed to reestablish growth on denuded areas in the presence of LDL or oxLDL (25-200 micrograms/ml), linoleic acid (FA, 90 microM) or linoleic acid hydroperoxide (OFA, 15 microM) for 24 h. Some monolayers were pretreated with 25 microM E for 24 h. Transendothelial albumin movement was used as a measure of endothelial barrier function and as an indicator of endothelial monolayer regrowth. Exposure to levels of oxLDL as low as 25 micrograms/ml for 24 h resulted in depressed endothelial monolayer regrowth, whereas native LDL was without effect and pre-enrichment with 25 microM E offered no protection. In comparison, E pre-enrichment improved endothelial regrowth to control levels in FA- and OFA-treated cultures, unlike oxLDL-treated cultures. It is concluded that circulating oxLDL may reduce regrowth of wounded endothelium and supplemental E may not offer protection. Moreover, fatty acids or their hydroperoxides are unlikely to be involved in this effect.

Obesity minimizes the immunopotentiation of food restriction in ob/ob mice.

The objective of this study was to investigate food restriction-related changes in several indices of immune competence in young (11 wk old) and adult (33 wk old) female lean (+/?) and obese (ob/ob) C57BL/6J mice. Body weight accumulation, tail length accretion and organ weights were more severely curtailed by food restriction in obese mice than in lean mice. Tail collagen denaturation time increased with age, although the magnitude was greater in obese mice, and this change was minimized by food restriction. Splenocyte mitogen responses were generally not altered with age in lean or obese mice, whereas food restriction augmented these responses in lean mice while having no effect or reducing them in obese mice. The concanavalin A and phytohemagglutinin responses of splenocytes from young and adult obese mice were greater than those for lean mice, whereas the bacterial lipopolysaccharide response was elevated only in adult obese vs. lean mice. Flow cytometric analysis of splenocytes revealed an increase in Thy-1+ cells with food restriction vs. freely fed obese and lean mice, with a proportional decrease in Ig+ cells. Percentages of CD4+ and CD8+ cells increased with food restriction in both lean and obese mice. These results suggest that genetic obesity largely eliminates the immunopotentiating effects of food restriction, although the rate of "aging" may be reduced by food restriction.

Effect of dietary carnosine on plasma and tissue antioxidant concentrations and on lipid oxidation in rat skeletal muscle.

The effect of dietary carnosine supplementation on plasma and tissue carnosine and alpha-tocopherol concentrations and on the formation of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle homogenates was evaluated. Plasma, heart, liver and hind leg muscle was obtained from rats fed basal semipurified diets or basal diets containing carnosine (0.0875%), alpha-tocopheryl acetate (50 ppm), or carnosine (0.0875%) plus alpha-tocopheryl acetate (50 ppm). Dietary carnosine supplementation did not increase carnosine concentrations in heart, liver and skeletal muscle. Dietary supplementation with both carnosine and alpha-tocopherol increased carnosine concentrations in liver 1.56, 1.51- and 1.51-fold as compared with diets lacking carnosine, alpha-tocopherol or both carnosine and alpha-tocopherol, respectively. Dietary supplementation with both carnosine and alpha-tocopherol also increased alpha-tocopherol concentrations in heart and liver 1-38-fold and 1.68-fold, respectively, as compared to supplementation with alpha-tocopherol alone. Dietary supplementation with carnosine, alpha-tocopherol or both carnosine and alpha-tocopherol was effective in decreasing the formation of TBARS in rat skeletal muscle homogenate, with dietary alpha-tocopherol and alpha-tocopherol plus carnosine being more effective than dietary carnosine alone. The data suggest that dietary supplementation with carnosine and alpha-tocopherol modulates some tissue carnosine and alpha-tocopherol concentrations and the formation of TBARS in rat skeletal muscle homogenates.

Immunomodulatory effect of fu-fang-tai-pan-pian, a traditional Chinese tonic medicine.

The purpose of this study was to evaluate the effect of the traditional Chinese medicine Fu-Fang-Tai-Pan-Pian on responsiveness of mouse spleen leukocytes to the mitogens concanavalin A (con A), phytohemagglutinin (PHA), and bacterial endotoxin (LPS). Aqueous and chloroform/methanol extracts of the drug were prepared and added to mitogen-stimulated cultures at doses ranging from 0.625% to 20% by volume. The aqueous extract depressed responsiveness to all mitogens at all doses tested, and was significantly more potent in this regard than the organic extract. The organic extract depressed responsiveness at low dilutions; however it significantly stimulated responsiveness to PHA and LPS, but not to con A, at dilutions of 2.5% or less. The relative ability of compounds partitioning into aqueous and organic extracts of the medicinal mixture to both stimulate and depress the ability of lymphocytes to proliferate may provide insight into the mechanism of action of this and related medicines.

Proteoglycans and endothelial barrier function: effect of linoleic acid exposure to porcine pulmonary artery endothelial cells.

Certain fatty acids induce changes in endothelial barrier function which may be mediated by alterations in normal proteoglycan synthesis/metabolism. To test this hypothesis, pulmonary artery derived endothelial cells were treated with media supplemented with linoleic acid (18:2), and/or a known proteoglycan synthesis inhibitor, beta-D-xyloside. Independent exposure to 1 mM beta-D-xyloside or 90 microM 18:2 increased albumin transfer, i.e., decreased barrier function, when compared with control cultures. 18:2 and beta-D-xyloside increased albumin transfer additively, suggesting that the mechanisms by which 18:2 and beta-D-xyloside alter the proteoglycan metabolism are different. Compared with the control group, treatment with 18:2 inhibited proteoglycan synthesis, decreased anionic properties of heparan sulfate proteoglycans in the cell monolayers and caused the release of a unique chondroitin sulfate proteoglycan into the culture media. Treatment with beta-D-xyloside caused an increased incorporation of radioactive sulfate into glycosaminoglycans but inhibited proteoglycan synthesis. These results suggest that the fatty acid- and beta-D-xyloside-induced impairment in endothelial barrier function may involve changes in the synthesis, release and physicochemical properties of proteoglycans.

Selective disruption of endothelial barrier function in culture by pure fatty acids and fatty acids derived from animal and plant fats.

Endothelial cell integrity has been suggested to play a role in the development of atherosclerosis. The effects of fatty acids on endothelial barrier function were tested by measuring albumin transport across endothelial monolayers cultured on polycarbonate filters. Compared with control cultures, a 24-h exposure to 90 mumol/L lauric (12:0) and linoleic acid (18:2) but not to butyric (4:0), hexanoic (6:0), octanoic (8:0), decanoic (10:0), myristic (14:0), palmitic (16:0) or stearic acid (18:0) caused an increase in albumin transfer across endothelial monolayers. Selective enrichment of a "physiological" serum fatty acid mixture (FA-Mix; 90 mumol/L) with 90 mumol/L of 12:0 or 18:2 significantly increased albumin transfer, whereas enrichment with 90 mumol/L of 4:0, 16:0 or 18:0 significantly decreased albumin transfer relative to 180 mumol/L FA-Mix. Only 12:0- or 18:2-treated cultures showed increased Ca(++)-ATPase activity and the presence of lipid droplets. Fatty acids (60 mumol/L) extracted from butter fat and beef tallow had no effect on albumin transfer, whereas fatty acids extracted from chicken fat and corn oil consistently disrupted endothelial barrier function. This fat-induced disruption of endothelial barrier function seems to be related to the amount of 18:2 present in each fat source. These data indicate that unsaturated fats cause cellular perturbations that result in a decrease in endothelial barrier function in this model system, and that high dietary levels of unsaturated fats may be detrimental to cell integrity.