Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy.

Dysregulated sphingolipid metabolism causes neuronal cell death and is associated with insulin resistance and diseases. Thus, we hypothesized that diabetes-induced changes in retinal sphingolipid metabolism may contribute to neuronal pathologies in diabetic retinopathy. ESI-MS/MS was used to measure ceramide content and ceramide metabolites in whole retinas after 2, 4, and 8 weeks of streptozotocin-induced diabetes. After 4 and 8 weeks of diabetes, a approximately 30% decrease in total ceramide content was observed, concomitant with a significant approximately 30% increase in glucosylceramide levels in fed diabetic rats compared with their age-matched controls. Acute insulin therapy as well as a short-term lowering of glucose via fasting did not affect the increase in glucosylceramide composition. To assess the putative biological consequences of the increase in glucosylceramide composition, R28 retinal neurons were treated with glucosylceramide synthase inhibitors. Inhibiting glycosphingolipid metabolism increased insulin sensitivity in retinal neurons. Glycosphingolipid inhibitors augmented insulin-stimulated p70 S6kinase activity in the presence of inhibitory concentrations of high glucose or glucosamine. Inhibition of glycosphingolipid synthesis also suppressed glucosamine- and interleukin-1beta-induced death. Consistent with these inhibitor studies, pharmacological accumulation of glycosphingolipids increased activation of the endoplasmic reticulum stress response, a putative modulator of insulin resistance and neuronal apoptosis. It is speculated that an increase in glucosylceramide, and possibly higher-order glycosphingolipids, could contribute to the pathogenesis of diabetic retinopathy by contributing to local insulin resistance, resulting in neuronal cell death. Thus, dysfunctional glycosphingolipid metabolism may contribute to metabolic stress in diabetes, and therapeutic strategies to restore normal sphingolipid metabolism may be a viable approach for treatment of diabetic retinopathy.

Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.

PURPOSE: In recent years, detergent-resistant membranes (DRMs) have been isolated in in vitro models of lipid rafts, from photoreceptor outer segments (ROS), and the localization of a specific complement of photoreceptor proteins has been demonstrated. However, surprisingly little is known about the lipid composition of these important membrane domains. The present study provides the first characterization of phospholipids and fatty acids from ROS-derived DRMs. METHODS: Bovine ROS membranes were incubated with 1% Triton X-100 at 4 degrees C and subjected to density gradient centrifugation to isolate DRMs from the parent membranes. Lipids of ROS and DRMs were separated by two-dimensional, thin-layer chromatography and converted to methyl esters, and fatty acids were analyzed by gas chromatography. Proteins of ROS and DRMs were analyzed by SDS-PAGE and Western blot analysis. RESULTS: The DRMs represented 8% and 3%, respectively, of total ROS lipid and protein. In general, DRMs were enriched in saturated fatty acids when compared with ROS membranes. Relative to ROS, DRMs were enriched in free fatty acids (FFAs) and a specific phosphatidylcholine (PC) fraction that was almost devoid of polyunsaturated fatty acids (PUFAs). DRMs contained less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Ceramide (CM) from ROS contained PUFAs but no saturated fatty acids; the converse was true of CM from DRMs. Docosahexaenoic acid was diminished in DRM PS and was not detected in the FFAs, but was equally abundant in ROS and DRM PE. ROS-derived DRMs were dramatically enriched in caveolin-1, contained significant amounts of transducin-alpha and c-Src, and were relatively devoid of arrestin. CONCLUSIONS: The relatively saturated lipid environment observed in DRMs is likely to promote the localization of signaling proteins modified with saturated fatty acyl chains. Based on the lipid composition of DRMs, the authors conclude that they would not efficiently support phototransduction.

Lipid differences in rod outer segment membranes of rats with P23H and S334ter opsin mutations.

PURPOSE: Retinal degenerations and diets low in n-3 fatty acids are associated with decreased docosahexaenoic acid (22:6n-3) in retina and plasma and with sterol abnormalities in retina and sperm. Using wild type (WT) and transgenic rats with P23H and S334ter opsin mutations, we evaluated retinal cholesterol levels, cholesterol synthesis, and fatty acid compositions of phospholipid classes in animals fed diets enriched in n-3 or n-6 polyunsaturated fatty acids. METHODS: Pregnant WT and heterozygous P23H and S334ter transgenic (TG) rats were fed safflower (safflower oil [SO], high n-6, trace n-3 fatty acids) or flaxseed oil (flaxseed oil [FO], high n-3, moderate n-6 fatty acids) diets beginning at E15, and pups were continued on the diets after weaning. Rod outer segment (ROS) membranes were prepared from 55-day-old rats, and the ratios of total fatty acid to cholesterol and the fatty acid compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in ROS were determined. Intravitreal injections of [3H]acetate were given to 35-day-old WT and TG rats fed standard chow-diets. Endogenous cholesterol mass and de novo [3H]cholesterol synthesis were measured and normalized to total ROS fatty acid content. Multivariate analysis of variance (ANOVA) with post hoc Newman-Keuls tests were used to determine statistical differences. RESULTS: The relative levels of PC, PE, and PS were similar in all three rat strains independent of diet. Total lipids, PC, PS, and PE of ROS FO fed rats had higher levels of 22:6n-3 and lower levels of 22:5n-6 than those fed SO. Rats fed SO had higher levels of 22:5n-6 than those fed FO. Significant increases in 18:1n-9 were seen in PC and PS of P23H and S334ter rats; arachidonate (20:4n-6) increased only in PE. These changes were independent of diet. ROS membranes of transgenic rats were cholesterol enriched, relative to WT ROS, yet retinal cholesterol synthesis was not altered. Plasma cholesterol levels of transgenic rats were not different from those of WT rats. CONCLUSIONS: Endogenous levels of cholesterol, 18:1n-9, 20:4n-6, 22:5n-6, and 22:6n-3 were altered in ROS membranes of P23H and S334ter compared to WT rats. There appear to be two pools of 22:6n-3 in rat ROS, one that is sensitive to retinal degenerations and one that is not. The stress induced reduction in 22:6n-3 was not specific to any phospholipid class and was not caused by alteration of relative amounts of PC, PS, or PE in the membrane. Elevated retinal cholesterol may be a result of either an increased half life or an increased uptake of cholesterol from the blood.

Bright cyclic rearing protects albino mouse retina against acute light-induced apoptosis.

PURPOSE: Previous studies have shown that albino rats born and raised in bright cyclic light are protected from light-induced apoptosis. The present study was designed to determine if bright cyclic rearing provides protection against retinal degeneration caused by acute light exposure in albino mice. METHODS: BALB/c mice were born in dim cyclic light (5 lux, 12 h ON/OFF). At 1 week of age, half of the litters were moved into 400 lux cyclic light. At 5 weeks of age, mice raised in the dim or bright cyclic conditions were divided into two groups. One group was placed in constant light (3,000 lux for 72 h) and the other was maintained in its original cyclic light environment. Control and constant light-stressed mice were dark-adapted for 24 and 48 h, respectively, after which their eyes were removed immediately for morphologic evaluation or preparation of rod outer segment (ROS) membranes. ROS lipids were extracted and fatty acid methyl esters were analyzed by gas-liquid chromatography. Eyes used for TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) and DNA fragmentation assays were enucleated immediately after the 72 h light exposure. RESULTS: Measurement of outer nuclear layer (ONL) thickness indicated there was no difference in the number of viable photoreceptor cells in the dim-reared controls compared to bright-reared controls. Constant light exposure significantly reduced the ONL thickness in dim- and bright-reared groups, with the largest change occurring in the dim-reared mice. TUNEL assay showed no apoptotic photoreceptor cells in either control group; however, apoptotic nuclei could be detected in both exposed groups, with the largest number found in the dim-reared mice. After light exposure, DNA fragmentation was prominent in dim-reared mice, but was not present in bright-reared animals. There was no significant difference in the fatty acid composition of ROS membranes in the dim- and bright-reared control mice. However, constant light exposure resulted in a greater loss of docosahexaenoic acid (22:6n-3) in the ROS of dim-reared animals. CONCLUSIONS: Mice raised in a bright cyclic light environment are protected against light-induced apoptosis. We suggest that the protection is due to the up-regulation of cell survival pathways or the down-regulation of pathways that are vulnerable to acute cell stress.

P23H and S334ter opsin mutations: Increasing photoreceptor outer segment n-3 fatty acid content does not affect the course of retinal degeneration.

PURPOSE: The n-3 polyunsaturated fatty acids (PUFA) facilitate retinal development and function. Rats carrying transgenes with P23H and S334ter rhodopsin mutations lose their photoreceptors and have lower levels of 22:6n-3 in rod photoreceptor outer segments (ROS) than wild type (WT) animals. We tested the hypothesis that the rate of retinal degeneration in these mutant animals could be sensitive to the n-3 fatty acid content of retina. METHODS: Beginning embryonic day 15, WT and heterozygous transgenic rats with P23H and S344ter rhodopsin mutations were fed semi-synthetic diets enriched in n-6 (safflower oil, SO) or n-3 (flaxseed oil, FO) PUFA. At 35 and 55 days of age, electroretinographic (ERG) response, outer nuclear layer (ONL) thickness, and fatty acid composition of plasma and ROS were determined. Student's t-tests and multivariate analysis of variance with post hoc tests determined statistical differences. RESULTS: Rats fed FO or SO diets had different n-6/n-3 PUFA ratios in plasma (1.3 and 62) and ROS (0.2 and 1.1, respectively). Although there were profound effects of the diets on the plasma fatty acid composition, there were only minor differences between WT and transgenic animals within each dietary regime. The ROS of FO fed rats had 70% more 22:6n-3 than those fed SO, and the WT had higher concentrations of 22:6n-3 than the transgenic animals (WT>P23H>S334ter). In contrast, there was no difference in 22:6n-3 levels in ROS of WT and transgenic rats fed the SO diet. At P55, both transgenic lines had diminished ERGs and ONL thickness relative to the WT. There was no detectable effect of ROS fatty acid enrichment on the rate of retinal degeneration in the transgenic animals. However, the FO-diet provided a modest protection of function (b-wave) in S334ter animals. CONCLUSIONS: Feeding n-3 fatty acids to rats with mutant rhodopsin transgenes significantly increased the levels of 22:6n-3 in ROS membranes, but had no effect on the rate of retinal degeneration. Therefore, the degeneration is not the result of low (or high) 22:6n-3 in ROS and supplementation with 18:3n-3 will not rescue dying photoreceptor cells in these animal models of inherited retinal degenerations.

Dietary essential fatty acids and gender-specific behavioral responses in cranially irradiated rats.

Specific memory deficits, reduced intellectual processing speed, and a variety of social and behavioral problems have been implicated as long-term effects of cranial radiation therapy (CRT). These deficits are thought to be related to changes in brain cytology and structure associated with microvascular aberrations. N-3 fatty acids may serve as protectants in pediatric patients who receive CRT for brain tumors. Timed-pregnant rat dams were fed one of four diets that were identical in all respects, except for their essential fatty acid content. The dams were placed on these diets at the beginning of the third trimester of gestation and their pups remained on them throughout the study. The rats' behavioral response as judged by acoustic startle response (ASR) and neurocognitive response (performance in a radial maze, RM) were evaluated in relation to diet, gender, and CRT. The following hypotheses were tested: (1) female rats will show greater CRT-induced neurocognitive and behavioral deficits; (2) dietary n-3 fatty acids will diminish CRT-induced neurocognitive and behavioral deficits; (3) gender-specific differences would be dampened by n-3 fatty acids in the diet. All three hypotheses were partially supported. These findings are discussed in light of the potential neuroprotective effects of n-3 fatty acids.