Changes in the daily rhythm of lipid metabolism in the diabetic retina.

Disruption of circadian regulation was recently shown to cause diabetes and metabolic disease. We have previously demonstrated that retinal lipid metabolism contributed to the development of diabetic retinopathy. The goal of this study was to determine the effect of diabetes on circadian regulation of clock genes and lipid metabolism genes in the retina and retinal endothelial cells (REC). Diabetes had a pronounced inhibitory effect on the negative clock arm with lower amplitude of the period (per) 1 in the retina; lower amplitude and a phase shift of per2 in the liver; and a loss of cryptochrome (cry) 2 rhythmic pattern in suprachiasmatic nucleus (SCN). The positive clock arm was increased by diabetes with higher amplitude of circadian locomotor output cycles kaput (CLOCK) and brain and muscle aryl-hydrocarbon receptor nuclear translocator-like 1 (bmal1) and phase shift in bmal1 rhythmic oscillations in the retina; and higher bmal1 amplitude in the SCN. Peroxisome proliferator-activated receptor (PPAR) α exhibited rhythmic oscillation in retina and liver; PPARγ had lower amplitude in diabetic liver; sterol regulatory element-binding protein (srebp) 1c had higher amplitude in the retina but lower in the liver in STZ- induced diabetic animals. Both of Elongase (Elovl) 2 and Elovl4 had a rhythmic oscillation pattern in the control retina. Diabetic retinas lost Elovl4 rhythmic oscillation and had lower amplitude of Elovl2 oscillations. In line with the in vivo data, circadian expression levels of CLOCK, bmal1 and srebp1c had higher amplitude in rat REC (rREC) isolated from diabetic rats compared with control rats, while PPARγ and Elovl2 had lower amplitude in diabetic rREC. In conclusion, diabetes causes dysregulation of circadian expression of clock genes and the genes controlling lipid metabolism in the retina with potential implications for the development of diabetic retinopathy.

N-3 polyunsaturated Fatty acids prevent diabetic retinopathy by inhibition of retinal vascular damage and enhanced endothelial progenitor cell reparative function.

OBJECTIVE: The vasodegenerative phase of diabetic retinopathy is characterized by not only retinal vascular degeneration but also inadequate vascular repair due to compromised bone marrow derived endothelial progenitor cells (EPCs). We propose that n-3 polyunsaturated fatty acid (PUFA) deficiency in diabetes results in activation of the central enzyme of sphingolipid metabolism, acid sphingomyelinase (ASM) and that ASM represents a molecular metabolic link connecting the initial damage in the retina and the dysfunction of EPCs. RESEARCH DESIGN AND METHODS: Type 2 diabetic rats on control or docosahexaenoic acid (DHA)-rich diet were studied. The number of acellular capillaries in the retinas was assessed by trypsin digest. mRNA levels of interleukin (IL)-1ß, IL-6, intracellular adhesion molecule (ICAM)-1 in the retinas from diabetic animals were compared to controls and ASM protein was assessed by western analysis. EPCs were isolated from blood and bone marrow and their numbers and ability to form colonies in vitro, ASM activity and lipid profiles were determined. RESULTS: DHA-rich diet prevented diabetes-induced increase in the number of retinal acellular capillaries and significantly enhanced the life span of type 2 diabetic animals. DHA-rich diet blocked upregulation of ASM and other inflammatory markers in diabetic retina and prevented the increase in ASM activity in EPCs, normalized the numbers of circulating EPCs and improved EPC colony formation. CONCLUSIONS: In a type 2 diabetes animal model, DHA-rich diet fully prevented retinal vascular pathology through inhibition of ASM in both retina and EPCs, leading to a concomitant suppression of retinal inflammation and correction of EPC number and function.

The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models.

OBJECTIVE: Acid sphingomyelinase (ASM) is an important early responder in inflammatory cytokine signaling. The role of ASM in retinal vascular inflammation and vessel loss associated with diabetic retinopathy is not known and represents the goal of this study. RESEARCH DESIGN AND METHODS: Protein and gene expression profiles were determined by quantitative RT-PCR and Western blot. ASM activity was determined using Amplex Red sphingomyelinase assay. Caveolar lipid composition was analyzed by nano-electrospray ionization tandem mass spectrometry. Streptozotocin-induced diabetes and retinal ischemia-reperfusion models were used in in vivo studies. RESULTS: We identify endothelial caveolae-associated ASM as an essential component in mediating inflammation and vascular pathology in in vivo and in vitro models of diabetic retinopathy. Human retinal endothelial cells (HREC), in contrast with glial and epithelial cells, express the plasma membrane form of ASM that overlaps with caveolin-1. Treatment of HREC with docosahexaenoic acid (DHA) specifically reduces expression of the caveolae-associated ASM, prevents a tumor necrosis factor-α-induced increase in the ceramide-to-sphingomyelin ratio in the caveolae, and inhibits cytokine-induced inflammatory signaling. ASM is expressed in both vascular and neuroretina; however, only vascular ASM is specifically increased in the retinas of animal models at the vasodegenerative phase of diabetic retinopathy. The absence of ASM in ASM(-/-) mice or inhibition of ASM activity by DHA prevents acellular capillary formation. CONCLUSIONS: This is the first study demonstrating activation of ASM in the retinal vasculature of diabetic retinopathy animal models. Inhibition of ASM could be further explored as a potential therapeutic strategy in treating diabetic retinopathy.

Inhibition of cytokine signaling in human retinal endothelial cells through downregulation of sphingomyelinases by docosahexaenoic acid.

PURPOSE: The authors have previously demonstrated that DHA inhibits cytokine-induced inflammation in human retinal endothelial cells (HRECs), the resident vasculature affected by diabetic retinopathy. However, the anti-inflammatory mechanism of docosahexaenoic acid (DHA) is still not well understood. Sphingolipids represent a major component of membrane microdomains, and ceramide-enriched microdomains appear to be a prerequisite for inflammatory cytokine signaling. Acid sphingomyelinase (ASMase) and neutral sphingomyelinase (NSMase) are key regulatory enzymes of sphingolipid metabolism, promoting sphingomyelin hydrolysis to proinflammatory ceramide. The authors address the hypothesis that DHA inhibits cytokine-induced inflammatory signaling in HRECs by downregulating sphingomyelinases. METHODS: ASMase and NSMase activity was determined by sphingomyelinase assay in primary cultures of HRECs. The expression of ASMase, NSMase, ICAM-1, and VCAM-1 was assessed by quantitative PCR and Western blot analysis. Gene silencing of ASMase and NSMase was obtained by siRNA treatment. RESULTS: Inflammatory cytokines TNFalpha and IL-1beta induced cellular adhesion molecule (CAM) expression and rapid increase in ASMase and NSMase activity in HRECs. DHA decreased basal and cytokine-induced ASMase and NSMase expression and activity and the upregulation of CAM expression. Anti-inflammatory effects of DHA on cytokine-induced CAM expression were mimicked by inhibition/gene silencing of ASMase and NSMase. The sphingomyelinase pathway rather than ceramide de novo synthesis pathway was important for inflammatory signaling in HRECs. CONCLUSIONS: This study provides a novel potential mechanism for the anti-inflammatory effect of DHA in HRECs. DHA downregulates the basal and cytokine-induced ASMase and NSMase expression and activity level in HRECs, and inhibition of sphingomyelinases in endothelial cells prevents cytokine-induced inflammatory response.

Remodeling of retinal Fatty acids in an animal model of diabetes: a decrease in long-chain polyunsaturated fatty acids is associated with a decrease in fatty acid elongases Elovl2 and Elovl4.

OBJECTIVE: The results of the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study revealed a strong association between dyslipidemia and the development of diabetic retinopathy. However, there are no experimental data on retinal fatty acid metabolism in diabetes. This study determined retinal-specific fatty acid metabolism in control and diabetic animals. RESEARCH DESIGN AND METHODS: Tissue gene and protein expression profiles were determined by quantitative RT-PCR and Western blot in control and streptozotocin-induced diabetic rats at 3-6 weeks of diabetes. Fatty acid profiles were assessed by reverse-phase high-performance liquid chromatography, and phospholipid analysis was performed by nano-electrospray ionization tandem mass spectrometry. RESULTS: We found a dramatic difference between retinal and liver elongase and desaturase profiles with high elongase and low desaturase gene expression in the retina compared with liver. Elovl4, an elongase expressed in the retina but not in the liver, showed the greatest expression level among retinal elongases, followed by Elovl2, Elovl1, and Elovl6. Importantly, early-stage diabetes induced a marked decrease in retinal expression levels of Elovl4, Elovl2, and Elovl6. Diabetes-induced downregulation of retinal elongases translated into a significant decrease in total retinal docosahexaenoic acid, as well as decreased incorporation of very-long-chain polyunsaturated fatty acids (PUFAs), particularly 32:6n3, into retinal phosphatidylcholine. This decrease in n3 PUFAs was coupled with inflammatory status in diabetic retina, reflected by an increase in gene expression of proinflammatory markers interleukin-6, vascular endothelial growth factor, and intercellular adhesion molecule-1. CONCLUSIONS: This is the first comprehensive study demonstrating diabetes-induced changes in retinal fatty acid metabolism. Normalization of retinal fatty acid levels by dietary means or/and modulating expression of elongases could represent a potential therapeutic target for diabetes-induced retinal inflammation.