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PURPOSE. To determine plasma metabolite and metabolic pathway differences between patients with type 2 diabetes with diabetic retinopathy (DR) and without retinopathy (diabetic controls), and between patients with proliferative DR (PDR) and nonproliferative DR (NPDR). METHODS. Using high-resolution mass spectrometry with liquid chromatography, untargeted metabolomics was performed on plasma samples from 83 DR patients and 90 diabetic controls. Discriminatory metabolic features were identified through partial least squares discriminant analysis, and linear regression was used to adjust for age, sex, diabetes duration, and hemoglobin A1c. Pathway analysis was performed using Mummichog 2.0. RESULTS. In the adjusted analysis, 126 metabolic features differed significantly between DR patients and diabetic controls. Pathway analysis revealed alterations in the metabolism of amino acids, leukotrienes, niacin, pyrimidine, and purine. Arginine, citrulline, glutamic csemialdehyde, and dehydroxycarnitine were key contributors to these pathway differences. A total of 151 features distinguished PDR patients from NPDR patients, and pathway analysis revealed alterations in the b-oxidation of saturated fatty acids, fatty acid metabolism, and vitamin D 3 metabolism. Carnitine was a major contributor to the pathway differences. CONCLUSIONS. This study demonstrates that arginine and citrulline-related pathways are dysregulated in DR, and fatty acid metabolism is altered in PDR patients compared with NPDR patients.

ScopeVitamin D binding protein (VDBP) status has an effect on and can potentially improve the status of 25(OH) vitamin D and increase the metabolic actions of 25(OH) vitamin D under physiological and pathological conditions. Diabetes is associated with lower levels of glutathione (GSH) and 25(OH) vitamin D. This study examined the hypothesis that upregulation of GSH will also upregulate blood levels of VDBP and 25(OH) vitamin D in type 2 diabetic rats.Methods and resultsL‐cysteine (LC) supplementation was used to upregulate GSH status in a FL83B hepatocyte cell culture model and in vivo using Zucker diabetic fatty (ZDF) rats. Results show that LC supplementation upregulates both protein and mRNA expression of VDBP and vitamin D receptor (VDR) and GSH status in hepatocytes exposed to high glucose, and that GSH deficiency, induced by glutamate cysteine ligase knockdown, resulted in the downregulation of GSH, VDBP, and VDR and an increase in oxidative stress levels in hepatocytes. In vivo, LC supplementation increased GSH and protein and mRNA expression of VDBP and vitamin D 25‐hydroxylase (CYP2R1) in the liver, and simultaneously resulted in elevated blood levels of LC and GSH, as well as increases in VDBP and 25(OH) vitamin D levels, and decreased inflammatory biomarkers in ZDF rats compared with those in placebo‐supplemented ZDF rats consuming a similar diet.ConclusionLC supplementation may provide a novel approach by which to raise blood levels of VDBP and 25(OH) vitamin D in type 2 diabetes.

Aims: Vitamin D (VD) deficiency has become a worldwide epidemic, particularly affecting African Americans (AA). VD deficiency has been implicated in the excessive rate of complications associated with diabetes in AA. Blood levels of VD binding protein (VDBP) and glutathione (GSH) are lower in AA compared with those in Caucasians. This study tested the hypothesis that lower GSH levels are linked to VDBP and VD deficiency in AA-type 2 diabetic (AA-T2D) patients. Blood was analyzed from T2D and nondiabetic subjects (N). Experiments examining GSH deficiency and l-cysteine (LC) supplementation were performed using THP-1 monocytes. Results: Plasma levels of LC, GSH, VDBP, and VD were significantly lower in AA-T2D compared with age-matched AA-N or Caucasian-T2D. Lower levels of LC and GSH showed a significant positive correlation with lower VDBP and VD levels in AA-T2D. GSH deficiency investigated using an antisense approach depleted VDBP/vitamin D receptor (VDR); LC supplementation caused significant upregulation of GSH and of VDBP/VDR, while supplementation with VD+LC caused a significantly greater GSH and VDBP/VDR upregulation compared with that of VD alone in monocytes. Innovation and Conclusion: The reported observations suggest that VD deficiency may be linked to GSH and LC status and lead to a novel hypothesis that supplementation with LC in combination with VD will be effective in increasing VD levels and reducing health disparities in AA. Antioxid. Redox Signal. 23, 688–693.

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PurposeWe previously reported European mitochondrial haplogroup H to be a risk factor for and haplogroup UK to be protective against proliferative diabetic retinopathy (PDR) among Caucasian patients with diabetic retinopathy (DR). The purpose of this study was to determine whether these haplogroups are also associated with the risk of having DR among Caucasian patients with diabetes.MethodsDeidentified medical records for 637 Caucasian patients with diabetes (223 with DR) were obtained from BioVU, Vanderbilt University's electronic, deidentified DNA databank. An additional 197 Caucasian patients with diabetes (98 with DR) were enrolled from the Vanderbilt Eye Institute (VEI). We tested for an association between European mitochondrial haplogroups and DR status.ResultsThe percentage of diabetes patients with DR did not differ across the haplogroups (P = 0.32). The percentage of patients with nonproliferative DR (NPDR; P = 0.0084) and with PDR (P = 0.027) significantly differed across the haplogroups. In logistic regressions adjusting for sex, age, diabetes type, duration of diabetes, and hemoglobin A1c, neither haplogroup H nor haplogroup UK had a significant effect on DR compared with diabetic controls. Haplogroup UK was a significant risk factor (OR = 1.72 [1.13–2.59], P = 0.010) for NPDR compared with diabetic controls in the unadjusted analysis, but not in the adjusted analysis (OR = 1.29 [0.79–2.10], P = 0.20).ConclusionsMitochondrial haplogroups H and UK were associated with severity, but not presence, of DR. These data argue that the effect of these haplogroups is related to ischemia and neovascularization, the defining features of PDR.

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We previously demonstrated that the bile acid taurocholic acid (TCA) inhibits features of age-related macular degeneration (AMD) in vitro. The purpose of this study was to determine if the glycine-conjugated bile acids glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and glycoursodeoxycholic acid (GUDCA) can protect retinal pigment epithelial (RPE) cells against oxidative damage and inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in choroidal endothelial cells (CECs). Paraquat was used to induce oxidative stress and disrupt tight junctions in HRPEpiC primary human RPE cells. Tight junctions were assessed via transepithelial electrical resistance and ZO-1 immunofluorescence. GCA and GUDCA protected RPE tight junctions against oxidative damage at concentrations of 100–500 µM, and GDCA protected tight junctions at 10–500 µM. Angiogenesis was induced with VEGF in RF/6A macaque CECs and evaluated with cell proliferation, cell migration, and tube formation assays. GCA inhibited VEGF-induced CEC migration at 50–500 µM and tube formation at 10–500 µM. GUDCA inhibited VEGF-induced CEC migration at 100–500 µM and tube formation at 50–500 µM. GDCA had no effect on VEGF-induced angiogenesis. None of the three bile acids significantly inhibited VEGF-induced CEC proliferation. These results suggest glycine-conjugated bile acids may be protective against both atrophic and neovascular AMD.