Mechanisms involved in cellular ceramide homeostasis

ABSTRACT: Sphingolipids are ubiquitous and critical components of biological membranes. Their biosynthesis starts with soluble precursors in the endoplasmic reticulum and culminates in the Golgi complex and plasma membrane. Ceramides are important intermediates in the biosynthesis of sphingolipids, such as sphingomyelin, and their overload in the membranes is injurious to cells. The major product of ceramide metabolism is sphingomyelin. We observed that sphingomyelin synthase (SMS) 1 or SMS2 deficiencies significantly decreased plasma and liver sphingomyelin levels. However, SMS2 but not SMS1 deficiency increased plasma ceramides. Surprisingly, SMS1 deficiency significantly increased glucosylceramide and ganglioside GM3, but SMS2 deficiency did not. To explain these unexpected findings about modest to no significant changes in ceramides and increases in other sphingolipids after the ablation of SMS1, we hypothesize that cells have evolved several organelle specific mechanisms to maintain ceramide homeostasis. First, ceramides in the endoplasmic reticulum membranes are controlled by its export to Golgi by protein mediated transfer. Second, in the Golgi, ceramide levels are modulated by their enzymatic conversion to different sphingolipids such as sphingomyelin, and glucosylceramides. Additionally, these sphingolipids can become part of triglyceride-rich apolipoprotein B-containing lipoproteins and be secreted. Third, in the plasma membrane ceramide levels are maintained by ceramide/sphingomyelin cycle, delivery to lysosomes, and efflux to extracellular plasma acceptors. All these pathways might have evolved to ensure steady cellular ceramide levels.     

Analysis of ceramide metabolites in differentiating epidermal keratinocytes treated with calcium or vitamin C

Ceramides (Cer) comprise the major constituent of sphingolipids in the epidermis and are known to play diverse roles in the outermost layers of the skin including water retention and provision of a physical barrier. In addition, they can be hydrolyzed into free sphingoid bases such as C₁₈ sphingosine (SO) and C₁₈ sphinganine (SA) or can be further metabolized to C₁₈ So-1-phosphate (S1P) and C₁₈ Sa-1-phosphate (Sa1P) in keratinocytes. The significance of ceramide metabolites emerged from studies reporting altered levels of SO and SA in skin disorders and the role of S1P and Sa1P as signaling lipids. However, the overall metabolism of sphingoid bases and their phosphates during keratinocyte differentiation remains not fully understood. Therefore, in this study, we analyzed these Cer metabolites in the process of keratinocyte differentiation. Three distinct keratinocyte differentiation stages were prepared using 0.07 mM calcium (Ca²⁺) (proliferation stage), 1.2 mM Ca²⁺ (early differentiation stage) in serum-free medium, or serum-containing medium with vitamin C (50 µL/mL) (late differentiation stage). Serum-containing medium was also used to determine whether vitamin C increases the concentrations of sphingoid bases and their phosphates. The production of sphingoid bases and their phosphates after hydrolysis by alkaline phosphatase was determined using high-performance liquid chromatography. Compared to cells treated with 0.07 mM Ca²⁺, levels of SO, SA, S1P, and SA1P were not altered after treatment with 1.2 mM Ca²⁺. However, in keratinocytes cultured in serum-containing medium with vitamin C, levels of SO, SA, S1P, and SA1P were dramatically higher than those in 0.07- and 1.2-mM Ca²⁺-treated cells; however, compared to serum-containing medium alone, vitamin C did not significantly enhance their production. Taken together, we demonstrate that late differentiation induced by vitamin C and serum was accompanied by dramatic increases in the concentration of sphingoid bases and their phosphates, although vitamin C alone had no effect on their production.     

Ceramide composition of whole brain synaptosomal gangliosides from mice genetically bred for divergent ethanol sensitivities.

A comparison of the two major ceramide molecular species (d18:1-C18:0 and d20:1-C18:0) of synaptosomal gangliosides GM1, GD1a+GT1a, GD1b, GT1b, revealed a difference between the ceramide composition of ethanol-sensitive LS and ethanol-insensitive SS whole brain synaptosomal gangliosides. In all comparisons, the ratio of the two major molecular species, (d18:1-C18:0/d20:1-C18:0) was less for LS than for SS mice.     

茶多酚对皮肤角朊细胞生长与凋亡的影响

为探讨茶叶中的重要活性成分茶多酚对皮肤角朊细胞的生长周期动力学和对细胞凋亡的影响。在原代培养大鼠皮肤角朊细胞基础上，根据培养基中乳酸脱氢酶释放情况确定的茶多酚浓度范围，采用细胞计数法和流式细胞仪研究角朊细胞的生长，周期动力学和凋亡发生率。结果发现，在一定浓度范围内（０．１－１．５ｇ／Ｌ）茶多酚可以促进角朊细胞生长。在细胞周期动力学上，表现为促进细胞从Ｇ１期和静止期（Ｇ０）转向降到１７．９７％。表明     

Comparison of cytokine modulation by natural peroxisome proliferator-activated receptor gamma ligands with synthetic ligands in intestinal-like Caco-2 cells and human dendritic cells--potential for dietary modulation of peroxisome proliferator-activated receptor gamma in intestinal inflammation

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a role in the regulation of intestinal inflammation and is activated by both natural (polyunsaturated fatty acid; PUFAs) and synthetic (troglitazone) ligands. The fatty acid content of defined formula diets may play a role in mediating the antiinflammatory effect, but the mechanism is unclear. OBJECTIVE: We evaluated to what extent the effect of PUFAs on intestinal inflammation is mediated via PPARgamma. DESIGN: The human enterocyte-like cell line Caco-2 and human dendritic cells were stimulated by interleukin (IL) 1beta and lipoprotein polysaccharide, respectively, in the presence of PPARgamma agonists (troglitazone or PUFAs) or antagonist (GW9662). Five PUFAs were tested: alpha-linolenic acid (ALA), conjugated linoleic acid (CLA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and gamma-linolenic acid (GLA). Cytokine production was measured by enzyme-linked immunosorbent assay and PPARgamma, I-kappaB, and inducible nitric oxide synthase (iNOS) expression by Western blot. RESULTS: In Caco-2 cells, IL-6 secretion was significantly decreased by troglitazone, DHA, EPA, and GLA. IL-8 production was significantly decreased by troglitazone, ALA, DHA, EPA, and GLA. PPARgamma expression was significantly increased by troglitazone, DHA, and EPA. iNOS expression was significantly decreased by troglitazone, DHA, and EPA. Troglitazone and PUFAs at 0.1 mumol/L tended to increase the expression of I-kappaB. Addition of GW9662 reversed the effect of troglitazone and PUFAs at 0.1 mumol/L on IL-8 production and decreased the expression of PPARgamma. EPA and DHA also modulated the dendritic cell response to lipoprotein polysaccharide. CONCLUSIONS: The tested PUFAs exerted an antiinflammatory effect in vitro in both models. This effect of PUFAs in Caco-2 cells is similar to that of troglitazone on intestinal inflammation mediated by PPARgamma, and the potency of the antiinflammatory effect is linked to the number of double bonds.     

Peripartal feeding strategy with different n-6:n-3 ratios in sows: effect on gene expression in backfat white adipose tissue postpartum

The aim of this study was to describe the effects of two diets differing in n-6:n-3 ratio and prepartal feeding regime on gene expression of PPARgamma1a/1b, PPARgamma1c/1d, PPARgamma2, PPARgamma coactivator 1A (PPARGC1A), GLUT4, TNFalpha, adiponectin, leptin, leptin receptor (LEPR), fatty acid binding protein 4 (FABP4), lipoprotein lipase (LPL) in sows' white adipose tissue on the first day of lactation. The relationship between mRNA expression of these genes and circulating insulin, leptin and thyroid hormones was also considered. Diets contained a low (supplemented with fish oil; f group) or a high (supplemented with sunflower oil; s group) n-6:n-3 ratio and were provided from 8 (f8, s8) or 3d (f3, s3) before parturition (onset day 8 or 3). A low n-6:n-3 ratio reduced the 1d postpartum expression of PPARgamma2 and PPARGC1A but only when applied from 3 d before parturition. Circulating leptin was negatively correlated with mRNA expression of adiponectin, LEPR and LPL, whereas thyroxine was positively correlated with levels of PPARGC1A. In conclusion, the effect of dietary treatments, e.g. altering the n-6:n-3 ratio, around parturition on the expression of crucial genes in nutrient metabolism can be modulated by the duration of application before parturition.     

Resveratrol triggers apoptosis through regulating ceramide metabolizing genes in human K562 chronic myeloid leukemia cells

Resveratrol, an important phytoalexin in many plants, has been reported to have cytotoxic effects on various types of cancer. Ceramide is a bioactive sphingolipid that regulates many signaling pathways, including cell growth and proliferation, senescence and quiescence, apoptosis, and cell cycle. Ceramides are generated by longevity assurance genes (LASS). Glucosylceramide synthase (GCS) and sphingosine kinase-1 (SK-1) enzymes can convert ceramides to antiapoptotic molecules, glucosylceramide, and sphingosine-1-phosphate, respectively. C8:ceramide, an important cell-permeable analogue of natural ceramides, increases intracellular ceramide levels significantly, while 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and SK-1 inhibitor increase accumulation of ceramides by inhibiting GCS and SK-1, respectively. Chronic myelogenous leukemia (CML) is a hematological disorder resulting from generation of BCR/ABL oncogene. In this study, we examined the roles of ceramide metabolizing genes in resveratrol-induced apoptosis in K562 CML cells. There were synergistic cytotoxic and apoptotic effects of resveratrol with coadministration of C8:ceramide, PDMP, and SK-1 inhibitor. Interestingly, there were also significant increases in expression levels of LASS genes and decreases in expression levels of GCS and SK-1 in K562 cells in response to resveratrol. Our data, in total, showed for the first time that resveratrol might kill CML cells through increasing intracellular generation and accumulation of apoptotic ceramides.     

Trans-10 octadecenoic acid does not reduce milk fat synthesis in dairy cows

Diet-induced milk fat depression (MFD) involves the interrelation between rumen fermentation and mammary synthesis of milk fat, and the reduction in milk fat coincides with a marked increase in the trans-10 18:1 content of milk fat. Our objective was to directly examine the effect of trans-10 18:1 on milk fat synthesis in dairy cows. Three mid-lactation cows were used in a 3 x 3 Latin square design; treatments were abomasal infusion of: 1) ethanol (control); 2) trans-10 18:1 (t10); and 3) trans-10, cis-12 conjugated linoleic acid (CLA; positive control). The t10 and CLA supplements (>90% purity) were infused for 4 d and provided 42.6 and 4.3 g/d of trans-10 18:1 and trans-10, cis-12 CLA, respectively. Milk yield, feed intake, milk protein, and milk lactose were unaffected by treatment. Compared with the control, the t10 treatment had no effect on milk fat synthesis, whereas the CLA treatment resulted in a 27 and 24% reduction in milk fat content and yield, respectively. The transfer efficiency of the abomasally infused trans-10 18:1 and trans-10, cis-12 CLA into milk fat was 15 +/- 1 and 23 +/- 5% (means +/- SD), respectively. Overall, trans-10 18:1 had no effect on milk fat synthesis when abomasally infused at approximately 43 g/d, although it was taken up by the mammary glands and incorporated into milk fat. Therefore, our results offer no support for the concept that changes in rumen production of trans-10 18:1 within the physiological range play a role in the regulation of fatty acid synthesis during diet-induced MFD.     

Effects of dietary cis 9, trans 11-18:2, trans 10, cis 12-18:2, or vaccenic acid (trans 11-18:1) during lactation on body composition, tissue fatty acid profiles, and litter growth in mice

Cis 9, trans 11 (c 9, t11)-18:2 and trans 10, cis 12 (t10, c12)-18:2 are the major conjugated linoleic acid (CLA) isomers in dietary supplements which reduce milk fat content in nursing women. The present study evaluated the effects of each CLA isomer or vaccenic acid on body composition and tissue fatty acids during lactation in mice. Dams were fed 30 g rapeseed oil (control)/kg diet or 20 g control plus 10 g 18:0, trans 11-18:1 (t11-18:1), c 9, t11-18:2, or t10, c12-18:2. Dietary t10, c12-18:2 reduced food intake by 18 % and carcass fat weight of the dams by 49 % compared with the other treatments. Milk fat percentage ranked by treatment was 18:0>t11-18:1=c 9, t11-18:2>t10, c12-18:2. The sum of saturated 12:0 to 16:0 in milk fat was lower when c 9, t11-18:2 was fed compared with the control, 18:0, or t11-18:1 treatments. Dietary t10, c12-18:2 caused further reductions in milk fat 12:0 to 16:0. The proportion of CLA isomers was 3-fold greater in milk fat than in the carcasses of the dams. The pups nursing from the dams fed t10, c12-18:2 had the lowest body weights and carcass fat, protein, and ash contents. Nursing from the dams fed c 9, t11-18:2 also resulted in lower carcass fat compared with the 18:0 or t11-18:1 treatments. The ratios of cis 9-16:1:16:0 or cis 9-18:1:18:0, proxies for Delta(9)-desaturase activity, were markedly lower in the carcasses of the dams and pups fed t10, c12-18:2. The ratio of 20:4n-6:18 : 2n-6, a proxy for Delta(6)- and Delta(5)-desaturase and elongase activity, in the liver of the dams and pups fed t10, c12-18:2 also was lower. Dietary t11-18:1 enhanced the content of c 9, t11-18:2 in milk fat and carcasses. As in previous studies, the reduction in food intake by t10, c12-18:2 could not entirely account for the marked decrease in carcass fat content and milk fat concentration. T10, c12-18:2 probably had a negative effect on Delta(9)-desaturase and mammary de novo fatty acid synthesis. Although these effects need to be confirmed in lactating women, the results suggest that the consumption of supplements containing t10, c12-18:2 should be avoided during the nursing period.     

Fatty acid-mediated inhibition of IL-12 production by murine macrophages is independent of PPARgamma

Our laboratory has reported that n-3 PUFA can reduce host resistance to Listeria infection, in part, by impairing in vivo IL-12 biosynthesis. Recently, PUFA were shown to be ligands for PPAR, a novel family of nuclear receptors with three isoforms: PPARalpha, PPARdelta/beta and PPARgamma. PPARgamma is expressed in immune cells, such as T cells and macrophages. Two PPARgamma agonists, 15-deoxy-delta(12,14)-prostaglandin (PG) J2 and rosiglitazone, have been shown to have immunomodulatory activity in vitro, including inhibiting IL-12 biosynthesis. We hypothesized that n-3 PUFA inhibit IL-12 production through activating PPARgamma. We used thioglycolate-elicited mouse peritoneal macrophages to study the effect of various fatty acids and their oxidized metabolites on in vitro IL-12 production. Our present results demonstrate that both n-3 and n-6 PUFA can reduce in vitro IL-12 biosynthesis, though less potently than 15-deoxy-PGJ2 and rosiglitazone. GW9662, a PPARgamma antagonist, reversed the inhibitory effect of rosiglitazone, but not that of PUFA. Our present findings suggest that fatty acid-mediated inhibition of IL-12 production is independent of PPARgamma.     

Trans-10, cis-12-conjugated linoleic acid increases phagocytosis of porcine peripheral blood polymorphonuclear cells in vitro

Trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) has been shown to alter immune function. PPARgamma has been shown to potentially play an important role in regulating inflammatory and immune responses by modulating the activity of monocytes and macrophages. Previous studies have indicated that the phagocytic capacity of porcine peripheral blood polymorphonuclear cells (PMN) was enhanced by the culture supernatant fraction from t10c12-CL-stimulated porcine peripheral blood mononuclear cells (PBMC) but not by t10c12-CLA itself. In the present study, we examined the effects of t10c12-CLA on PPARgamma and TNF-alpha expression of porcine PBMC and the phagocytic capacity of PMN. t10c12-CLA increased TNF-alpha mRNA expression and production by PBMC. The phagocytic capacity of porcine PMN was enhanced by either culture supernatant fraction from PBMC treated with t10c12-CLA or recombinant porcine (rp) TNF-alpha. Anti-rpTNF-alpha polyclonal antibody inhibited the enhancement of PMN phagocytic capacity. t10c12-CLA also up regulated PPARgamma mRNA expression in porcine PBMC. Bisphenol A diglycidyl ether, a PPARgamma antagonist, not only completely negated the t10c12-CLA-stimulating effects on TNF-alpha expression and production by porcine PBMC, but also decreased the enhancement of PMN phagocytic capacity by the t10c12-CLA-stimulated porcine PBMC culture supernatant fraction. These results suggest that t10c12-CLA has an immunostimulating effect on porcine PMN phagocytic capacity, which is mediated by TNF-alpha from PBMC via a PPARgamma-dependent pathway.     

Conjugated linoleic acid attenuates the production and gene expression of proinflammatory cytokines in weaned pigs challenged with lipopolysaccharide

We investigated the anti-inflammatory role of conjugated linoleic acid (CLA) in inflammation-challenged weaned pigs and in in vitro cultured peripheral blood mononuclear cells (PBMCs). To test the hypothesis that inflammation responses can be attenuated by dietary CLA supplementation, we used an acute inflammation model in which pigs were injected with lipopolysaccharide (LPS). After 14 d of dietary supplementation with either 2% soybean oil or 2% CLA, half of the pigs in each diet group were challenged with LPS. Dietary CLA alleviated growth depression and prevented the elevations in production and mRNA expression of proinflammatory cytokines [i.e., interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha] induced by the LPS challenge. CLA enhanced the expression of interleukin-10 (IL-10) and peroxisome proliferator-activated receptor-gamma (PPARgamma) in spleen and thymus. To further elucidate the inhibitory effects and the mechanism of action of CLA on cytokine profiles (i.e., IL-1beta, IL-6, and TNF-alpha), PBMCs were isolated from weaned pigs and cultured in media containing cis-9, trans-11 (9c,11t) CLA and trans-10, cis-12 (10t,12c) CLA. Each CLA isomer suppressed the production and expression of IL-1beta, IL-6, and TNF-alpha, and enhanced PPARgamma activation and gene expression in cultured PBMCs. At the molecular level, the inhibitory actions of CLA on IL-1beta, IL-6, and TNF-alpha are attributable mainly to 10t,12c-CLA and the anti-inflammatory properties of CLA are mediated, at least in part, through a PPARgamma-dependent mechanism.     

Cis-9,trans-11-conjugated linoleic acid inhibits allergic sensitization and airway inflammation via a PPARgamma-related mechanism in mice

Milk consumption from early childhood on has been found to be inversely correlated with allergic sensitization and the onset of bronchial asthma. We tested whether cis-9,trans-11-conjugated linoleic acid (c9,t11-CLA), naturally occurring in milk fat, may prevent allergic sensitization and inhibit airway inflammation in a murine asthma model. BALB/c mice were fed a diet enriched in 1 wt% of c9,t11-CLA or a control diet 7 d prior to and for 32 d during sensitization [d 1 and 14, 100 mg/L ovalbumin (OVA) in adjuvant vs. PBS] and airway challenges (d 28-30, 1% OVA in PBS vs. PBS). Subgroups of mice were coadministered 20 micromol/L of the selective PPARgamma antagonist GW9662 during each OVA challenge. C9,t11-CLA feeding resulted in significantly reduced IgE production and allergen-induced in vivo airway hyperresponsiveness. Further, less mucous plugging of segmental bronchi and significantly reduced interleukin-5 and eosinophils were determined in bronchoalveolar lavage fluids of c9,t11-CLA-fed mice. C9,t11-CLA feeding prevented the downregulation of PPARgamma mRNA in the lung tissues observed after allergen sensitization and airway challenges in control mice. The inhibitory effects of c9,t11-CLA on airway inflammation were partially prevented by coadministration of GW9962. Further, c9,t11-CLA feeding resulted in a significantly lower concentration of the eicosanoid precursor, arachidonic acid, in tissue lipids. These findings demonstrate that dietary c9,t11-CLA can reduce allergic airway inflammation, most likely via a PPARgamma-related mechanism and by reducing eicosanoid precursors. They give new insights into the fatty acid-mediated mechanism of immunomodulation and may represent a step toward an attractive novel strategy in the dietary prevention and treatment of allergic asthma.     

De novo lipogenesis during controlled overfeeding with sucrose or glucose in lean and obese women.

BACKGROUND: The results of previous studies suggest that de novo lipogenesis may play an important role in the etiology of obesity, particularly during overconsumption of different carbohydrates. OBJECTIVE: We hypothesized that de novo lipogenesis would increase during overfeeding, would vary depending on the type of carbohydrate consumed, and would be greater in obese than in lean women. DESIGN: De novo lipogenesis was measured during 96 h of overfeeding by 50% with either sucrose or glucose and during an energy balance treatment (control) in 8 lean and 5 obese women. De novo lipogenesis was determined by measuring the amount of deuterium incorporation into plasma triacylglycerols. Fat and carbohydrate balance were measured simultaneously by continuous whole-body calorimetry. RESULTS: De novo lipogenesis did not differ significantly between lean and obese subjects, except with the control treatment, for which de novo lipogenesis was greater in the obese subjects. De novo lipogenesis was 2- to 3-fold higher after overfeeding by 50% than after the control treatment in all subjects. The type of carbohydrate overfeeding (sucrose or glucose) had no significant effect on de novo lipogenesis in either subject group. Estimated amounts of absolute VLDL production ranged from a minimum of 2 g/d (control) to a maximum of 10 g/d after overfeeding. This compares with a mean fat balance of approximately 275 g after 96 h of overfeeding. Individual subjects showed characteristic amounts of de novo lipogenesis, suggesting constitutive (possibly genetic) differences. CONCLUSION: De novo lipogenesis increases after overfeeding with glucose and sucrose to the same extent in lean and obese women but does not contribute greatly to total fat balance.     

Acute-on-chronic effects of fatty acids on intestinal triacylglycerol-rich lipoprotein metabolism

Postprandial triacylglycerol (TAG) metabolism is an important metabolic state that has been associated with cardiovascular disease. The magnitude of the postprandial TAG response is determined by dietary fat composition, which alters intestinal and hepatic TAG-rich lipoprotein (TRL) metabolism. Caco-2 cell monolayers are morphologically and physiologically similar to the human intestinal enterocytes, hence they are a good model to study intestinal lipoprotein metabolism. To date only the acute effect of fatty acid composition on intestinal TRL metabolism in Caco-2 cells has been investigated. Little is known of the effect of habitual, or chronic, dietary fat composition on intestinal TRL metabolism. Using the Caco-2 cell model, the present study investigated the acute-on-chronic effect of fatty acid composition on TRL metabolism. Caco-2 cells were grown in the presence of 0.05 mm-palmitic acid (PA; 16 : 0), -oleic acid (OA; 18 : 1n-9),-eicosapentaenoic acid (EPA; 20 : 5n-3) or no fatty acid (control) for 19 d, then one of four acute treatments (control (bovine serum albumin (BSA; 5 g/l)) or BSA (5 g/l) plus 0.5 mm-PA, -OA or -EPA) were administered for 22 h. Significant acutexchronic interactions for the effect of fatty acid composition on cellular TAG:secreted de novo TAG, and cellular de novo TAG:de novo phospholipid were observed. Thus the effect of a fatty acid was determined by the duration of exposure to the fatty acid intervention. Acute PA treatment increased de novo TAG synthesis, but chronic PA supplementation did not. Acute and chronic OA treatments increased de novo TAG secretion. For EPA, chronic supplementation had the greatest effect on TAG synthesis and secretion. The acute-on-chronic effects of fatty acids on apolipoprotein B metabolism were relatively minor compared with the changes noted for TRL lipid composition. The present study shows that the Caco-2 cell model is valuable for studying intestinal TRL metabolism and that fatty acids modulate this process, the nature of which can be determined by the length of exposure of the cell to the fatty acid.     

Involvement of the sphingolipid ceramide in heat-shock-induced apoptosis of bovine oocytes

Programmed cell death via the sphingomyelin pathway has been suggested to underlie heat-shock disturbance of oocyte developmental competence. A series of experiments were performed to characterise the role of the sphingolipid ceramide in heat-shock-induced apoptosis, and to determine whether ceramide formation can be regulated. Bovine cumulus-oocyte complexes (COCs) were aspirated from ovaries collected in the cold season (November-April), in vitro-matured, fertilised and cultured for 8 days. Exposure of COCs to heat shock (41°C) during maturation reduced cleavage rate and blastocyst formation relative to the control group (38.5°C). Annexin-V binding (V-FITC assay), which is associated with the early apoptotic event of membrane phosphatidylserine turnover, was higher in oocytes exposed to short-term versus long-term heat shock, suggesting that heat-shock-induced apoptosis involves membrane alterations. Similar to heat exposure, oocyte maturation with C(2)-ceramide had a dose-dependent deleterious effect on the first cleavages and subsequent embryonic development in association with increased annexin-V binding. Blocking endogenous ceramide generation with fumonisin B1, a specific inhibitor of dihydroceramide synthase (i.e. de novo formation), moderated, to some extent, the effects of heat shock on oocyte developmental competence, suggesting that ceramide plays an important role in heat-shock-induced apoptosis.