The association of dyslipidemia with the development of diabetic retinopathy has been underscored by the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications cohort study (8
). Despite this evidence, the experimental data on diabetes-induced changes in lipid profile and lipid metabolism in the retina are not available. This is the first comprehensive study to analyze retinal-specific fatty acid profiles and metabolism and to compare them to liver and blood plasma in control and diabetic animals.
In this study utilizing STZ-induced diabetic rats, we found a decreased level of DHA22:6n3
, the major retinal long-chain PUFA, in diabetic retina. This finding confirmed earlier studies showing a decrease in relative percentage of DHA22:6n3
in the diabetic retina (34
). In addition to DHA22:6n3
, VLCPUFAs including 32:6n3 and 34:6n3 were detected as substituents of retina GPCho. VLCPUFAs were not detected in lipid classes other than GPCho and were only detected in retina. Three weeks of diabetes reduced retinal levels of 32:6n3-GPCho compared with controls. As a result of these changes, the diabetic retina had a lower unsaturation index and lower long-chain–to–short-chain PUFA ratio. Moreover, there was a shift toward n3 PUFA–deficient, n6 PUFA–rich, profile in the diabetic retina.
In general, n6 PUFAs induce, while n3 PUFAs inhibit, inflammation, and the relative amount of these PUFAs plays an important role in the regulation of immunity (36
). Our previous studies indicated that treatment of a cell type affected by diabetic retinopathy, HRECs, with n6 PUFA leads to a lipoxygenase-dependent increase in ICAM-1/vascular cell adhesion molecule-1 expression (37
). Conversely, we have demonstrated that DHA22:6n3
inhibited cytokine-induced activation of the NFκB signaling pathway and adhesion molecule expression in HRECs (28
). Thus, a decrease in the n3-to-n6 PUFA ratio in the diabetic retina observed in this study would create proinflammatory conditions potentially contributing to the development of diabetic retinopathy. Indeed, previous studies demonstrated an upregulation in a number of inflammatory markers in the retina early in diabetes: VEGF (4
), ICAM-1 (6
), TNF-α (8
), and IL-6 (9
). ICAM-mediated leukostasis was detected within 1 week of diabetes in rats (38
). VEGF was shown to increase ICAM expression in retinas of nondiabetic mice (40
), and vitreal VEGF levels were found to be correlated with that of IL-6 and severity of diabetic retinopathy in diabetic patients (41
In this study, we chose a cytokine (IL-6), a growth factor (VEGF), and an adhesion molecule (ICAM-1) as readout of an inflammatory status in the retinas of diabetic animals with decreased n3/n6 PUFAs. mRNA levels of all three markers were elevated in diabetic retinas compared with controls.
Importantly, diabetes induced the most pronounced changes in the retinal fatty acid profile, whereas liver fatty acid profile was only slightly affected, indicating that the disruption of retinal fatty acid metabolism in diabetes might not simply be a result of altered liver metabolism. Moreover, VLCPUFA-containing phospholipids detected in the retina were not present in the liver or erythrocyte total lipids. The fatty acid profile in a particular peripheral tissue depends on two factors: 1
) the profile in circulation due to the diet and liver metabolism and 2
) the ability of a local tissue to remodel fatty acids. Retina has a unique fatty acid profile characterized by one of the highest levels of DHA22:6n3
in the body and by the presence of VLCPUFAs (27
). While the expression level of retinal desaturases was relatively low compared with retinal elongases, it has been reported that retina can synthesize DHA22:6n3
acid and EPA20:5n3
). Although retina may obtain additional DHA22:6n3
by uptake from the circulation, changes in the retinal fatty acid profiles of diabetic animals did not mirror changes observed in liver and plasma fatty acid profiles. Thus, a retina-specific decrease in DHA22:6n3
in diabetes is likely to be due to changes in retinal fatty acid metabolism.
To determine the effect of diabetes on retinal fatty acid metabolism, we analyzed the level of fatty acid elongase and desaturase gene expression in control and diabetic animals. Retinas had a very high expression level of the retinal-specific elongase, Elovl4, as well as high expression levels of long-chain PUFA elongase Elovl2. Δ5-, Δ6-, and Δ9-desaturase levels were low compared with the liver expression levels. The high levels of Elovl4 and Elovl2 and low levels of desaturases suggest that the retina is preferentially involved in production of very-long-chain fatty acids and exhibits a low level of de novo lipogenesis. The retinal elongase expression profile that we observed likely explains the high level of long-chain PUFAs in the retina compared with liver and blood plasma levels. Elovl2 elongates C20–22
fatty acids (44
). Elovl4 was recently shown to be involved in VLCPUFA synthesis with substrate specificity for C26–36
fatty acids (15
). The role of VLCPUFAs is not known. Because of their specific presence in tissues with high membrane curvature and their ability to span both leaflets of the lipid bilayer, VLCPUFAs are suggested to play the role of an anchor stabilizing high curvature cellular membranes (15
). In the retina, VLCPUFAs are mainly present in the rod outer-segment membrane (15
), where they are suggested to play a role in stabilizing the rims of photoreceptor disks. This specific localization might explain low abundance of VLCPUFAs in the total retinal lipids extracted in this study. At the same time, specific localization suggests that VLCPUFAs might play an important role in photoreceptor function. This study provides the first direct evidence that a significant decrease in Elovl4 in diabetic retina is indeed associated with a decrease in VLCPUFA (i.e., 32:6n3) synthesis. Despite lower abundance, diabetes-induced decrease in 32:6n3 containing GPCho (24%) was similar to the decrease in DHA22:6n3
containing GPChos (15–34%). Elovl4 protein expression in diabetic retina was inhibited to a higher degree (73%) compared with mRNA expression (40%), suggesting control of Elovl4 expression at both transcriptional and translational levels. Although decrease of VLCPUFAs is most likely to arise from Elovl4 loss, another plausible explanation could be that this reduction was due to reduction in VLCPUFA precursor lipids, EPA20:5n3
, and/or DHA22:6n3
. This possibility can be tested in the future by determining whether downregulation of VLCPUFAs in diabetes persists in animals supplemented with high-EPA20:5n3
Another possibility could be that high level of reactive oxygen species in diabetic retina leads to degradation of a highly oxidation-prone DHA molecule. Previous studies using the same STZ-induced diabetic model of similar duration, however, did not find oxidized DHA products in diabetic retina (47
Several Elovl4 gene mutations have been recently identified in pathogenesis of another retinal disease, Stargardt-like macular dystrophy (19
). Stargardt-like macular dystrophy is an autosomal-dominant disorder due to a dominant-negative effect of the mutated Elovl4 on wild-type protein (19
). As Elovl4 is highly expressed in the photoreceptors (19
), it is not surprising that mutant Elovl4 transgenic mice are characterized by lipofuscin accumulation, abnormal electrophysiology, and photoreceptor degeneration (20
). Although photoreceptors are not the primary site of diabetic retinopathy, several abnormalities in neural retina have been associated with the development of diabetic retinopathy (48
). The decrease in Elovl4 observed in this study would not be expected to have as dramatic an effect on photoreceptor viability as the dominant-negative mutation in Elovl4. However, the reduction in Elovl4 in diabetic retina could be responsible for more subtle changes in photoreceptor/RPE cell function that could lead to metabolic changes in the whole retina and eventually contribute to the pathology characteristic of diabetic retinopathy.
In conclusion, a decrease in the expression level of retinal fatty acid elongases Elovl2 and Elovl4 and concomitant decrease in the major n3 PUFA, DHA22:6n3, as well as the VLCPUFA32:6n3, results in an increased n6-to-n3 PUFA ratio in the diabetic retina that likely creates a proinflammatory state contributing to the development of diabetic retinopathy. Increasing the gene expression of fatty acid elongases in the retina represents a potential therapeutic strategy for modulating fatty acid metabolism and altering the pathogenesis of diabetic retinopathy.