Mitochondrial damage in the cells comprising inner (retinal endothelial cells) and outer (retinal pigment epithelium (RPE)) blood–retinal barriers (BRB) is known to precede the initial BRB breakdown and further histopathological abnormalities in diabetic retinopathy (DR). We previously demonstrated that activation of acid sphingomyelinase (ASM) is an important early event in the pathogenesis of DR, and recent studies have demonstrated that there is an intricate connection between ceramide and mitochondrial function. This study aimed to determine the role of ASM-dependent mitochondrial ceramide accumulation in diabetes-induced RPE cell damage. Mitochondria isolated from streptozotocin (STZ)-induced diabetic rat retinas (7 weeks duration) showed a 1.64 ± 0.29-fold increase in the ceramide-to-sphingomyelin ratio compared to controls. Conversely, the ceramide-to-sphingomyelin ratio was decreased in the mitochondria isolated from ASM-knockout mouse retinas compared to wild-type littermates, confirming the role of ASM in mitochondrial ceramide production. Cellular ceramide was elevated 2.67 ± 1.07-fold in RPE cells derived from diabetic donors compared to control donors, and these changes correlated with increased gene expression of IL-1β, IL-6, and ASM. Treatment of RPE cells derived from control donors with high glucose resulted in elevated ASM, vascular endothelial growth factor (VEGF), and intercellular adhesion molecule 1 (ICAM-1) mRNA. RPE from diabetic donors showed fragmented mitochondria and a 2.68 ± 0.66-fold decreased respiratory control ratio (RCR). Treatment of immortalized cell in vision research (ARPE-19) cells with high glucose resulted in a 25% ± 1.6% decrease in citrate synthase activity at 72 h. Inhibition of ASM with desipramine (15 μM, 1 h daily) abolished the decreases in metabolic functional parameters. Our results are consistent with diabetes-induced increase in mitochondrial ceramide through an ASM-dependent pathway leading to impaired mitochondrial function in the RPE cells of the retina.

Oxygen consumption is a key metric of metabolism in aerobic organisms. Current respirometric methods led to seminal discoveries despite limitations such as high sample demand, exchange with atmospheric O₂, and cumulative titration protocols leading to limited choice of useable tissue, complex data interpretation, and restricted experimental design. We developed a sensitive and customizable method of measuring O₂ consumption rates by a variety of biological samples in microliter volumes without interference from the aerobic environment. We demonstrate that O₂ permeability of the photopolymer, VeroClear, is comparable to that of polyetheretherketone (0.125 vs. 0.143 barrer, respectively) providing an efficient barrier to oxygen ingress. Optical transparency of VeroClear, combined with high resolution 3D printing, allows for optode-based oxygen detection in enclosed samples. These properties yield a microrespirometer with over 100× dynamic range for O₂ consumption rates. Importantly, the enclosed respirometer configuration and very low oxygen permeability of materials makes it suitable, with resin pre-conditioning, for quantitative assessment of O₂ consumption rates at any desired [O₂], including hyperbaric, physiological or hypoxic conditions as necessary for each cell type. We characterized two configurations to study soluble enzymes, isolated mitochondria, cells in suspension, and adherent cells cultured on-chip. Improved sensitivity allows for routine quantitative detection of respiration by as few as several hundred cells. Specific activity of cell suspensions in the microrespirometer was in close agreement with that obtained by high-resolution polarographic respirometry. Adherent cell protocols allowed for physiologically relevant assessment of respiration in retinal pigment epithelial cells, ARPE-19, which displayed lower metabolic rates compared with those in suspension. By exchanging medium composition, we demonstrate that cells can be transiently inhibited by cyanide and that 99.6% of basal O₂ uptake is recovered upon its removal. This approach is amenable to new experimental designs and precision measurements on limited sample quantities across basic research and applied fields.

Summary: In diabetes, the retina, a tissue with unique metabolic needs, demonstrates dysregulation of the intricate balance between nutrient availability and utilization. This results in cholesterol accumulation, pro-inflammatory and pro-apoptotic changes, and consequently neurovascular damage. Sirtuin 1 (SIRT1), a nutrient sensing deacetylase, is downregulated in the diabetic retina. In this study, the effect of SIRT1 stimulation by fasting or by pharmacological activation using SRT1720, was evaluated on retinal cholesterol metabolism, inflammation and neurovascular damage. SIRT1 activation, in retinal endothelial cells (REC) and neuronal retinal progenitor cells (R28), led to Liver X Receptor alpha (LXRα) deacetylation and subsequent increased activity, as measured by increased ATP-binding cassette transporter (ABC) A1 and G1 mRNA expression. In turn, increased cholesterol export resulted in decreased REC cholesterol levels. SIRT1 activation also led to decreased inflammation. SIRT1 activation, in vivo , prevented diabetes-induced inflammation and vascular and neural degeneration. Diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response, was significantly improved as a result of SIRT1 activation. Taken together, activation of SIRT1 signaling is an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimes and prevention of diabetic retinopathy (DR).