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Aims/hypothesis: Hyper-reflective crystalline deposits found in retinal lesions have been suggested to predict the progression of diabetic retinopathy, but the nature of these structures remains unknown., Methods: Scanning electron microscopy and immunohistochemistry were used to identify cholesterol crystals (CCs) in human donor, pig and mouse tissue. The effects of CCs were analysed in bovine retinal endothelial cells in vitro and in db/db mice in vivo using quantitative RT-PCR, bulk RNA sequencing, and cell death and permeability assays. Cholesterol homeostasis was determined using 2 H 2 O and 2 H 7 -cholesterol., Results: We identified hyper-reflective crystalline deposits in human diabetic retina as CCs. Similarly, CCs were found in the retina of a diabetic mouse model and a high-cholesterol diet-fed pig model. Cell culture studies demonstrated that treatment of retinal cells with CCs can recapitulate all major pathogenic mechanisms leading to diabetic retinopathy, including inflammation, cell death and breakdown of the blood-retinal barrier. Fibrates, statins and α-cyclodextrin effectively dissolved CCs present in in vitro models of diabetic retinopathy, and prevented CC-induced endothelial pathology. Treatment of a diabetic mouse model with α-cyclodextrin reduced cholesterol levels and CC formation in the retina, and prevented diabetic retinopathy., Conclusions/interpretation: We established that cholesterol accumulation and CC formation are a unifying pathogenic mechanism in the development of diabetic retinopathy., (© 2023. The Author(s).)

The metabolically active retina obtains essential lipids by endogenous biosynthesis and from the systemic circulation. Clinical studies provide limited and sometimes conflicting evidence as to the relationships between circulating lipid levels and the development and progression of diabetic retinopathy in people with diabetes. Cardiovascular-system-focused clinical trials that also evaluated some retinal outcomes demonstrate the potential protective power of lipid-lowering therapies in diabetic retinopathy and some trials with ocular primary endpoints are in progress. Although triacylglycerol-lowering therapies with fibrates afforded some protection against diabetic retinopathy, the effect was independent of changes in traditional blood lipid classes. While systemic LDL-cholesterol lowering with statins did not afford protection against diabetic retinopathy in most clinical trials, and none of the trials focused on retinopathy as the main outcome, data from very large database studies suggest the possible effectiveness of statins. Potential challenges in these studies are discussed, including lipid-independent effects of fibrates and statins, modified lipoproteins and retinal-specific effects of lipid-lowering drugs. Dysregulation of retinal-specific cholesterol metabolism leading to retinal cholesterol accumulation and potential formation of cholesterol crystals are also addressed., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aims/hypothesis: Homo sapiens evolved under conditions of intermittent food availability and prolonged fasting between meals. Periods of fasting are important for recovery from meal-induced oxidative and metabolic stress, and tissue repair. Constant high energy-density food availability in present-day society contributes to the pathogenesis of chronic diseases, including diabetes and its complications, with intermittent fasting (IF) and energy restriction shown to improve metabolic health. We have previously demonstrated that IF prevents the development of diabetic retinopathy in a mouse model of type 2 diabetes (db/db); however the mechanisms of fasting-induced health benefits and fasting-induced risks for individuals with diabetes remain largely unknown. Sirtuin 1 (SIRT1), a nutrient-sensing deacetylase, is downregulated in diabetes. In this study, the effect of SIRT1 stimulation by IF, fasting-mimicking cell culture conditions (FMC) or pharmacological treatment using SRT1720 was evaluated on systemic and retinal metabolism, systemic and retinal inflammation and vascular and bone marrow damage., Methods: The effects of IF were modelled in vivo using db/db mice and in vitro using bovine retinal endothelial cells or rat retinal neuroglial/precursor R28 cell line serum starved for 24 h. mRNA expression was analysed by quantitative PCR (qPCR). SIRT1 activity was measured via histone deacetylase activity assay. NR1H3 (also known as liver X receptor alpha [LXRα]) acetylation was measured via western blot analysis., Results: IF increased Sirt1 mRNA expression in mouse liver and retina when compared with non-fasted animals. IF also increased SIRT1 activity eightfold in mouse retina while FMC increased SIRT1 activity and expression in retinal endothelial cells when compared with control. Sirt1 expression was also increased twofold in neuronal retina progenitor cells (R28) after FMC treatment. Moreover, FMC led to SIRT1-mediated LXRα deacetylation and subsequent 2.4-fold increase in activity, as measured by increased mRNA expression of the genes encoding ATP-binding cassette transporter (Abca1 and Abcg1). These changes were reduced when retinal endothelial cells expressing a constitutively acetylated LXRα mutant were tested. Increased SIRT1/LXR/ABC-mediated cholesterol export resulted in decreased retinal endothelial cell cholesterol levels. Direct activation of SIRT1 by SRT1720 in db/db mice led to a twofold reduction of diabetes-induced inflammation in the retina and improved diabetes-induced visual function impairment, as measured by electroretinogram and optokinetic response. In the bone marrow, there was prevention of diabetes-induced myeloidosis and decreased inflammatory cytokine expression., Conclusions/interpretation: Taken together, activation of SIRT1 signalling by IF or through pharmacological activation represents an effective therapeutic strategy that provides a mechanistic link between the advantageous effects associated with fasting regimens and prevention of microvascular and bone marrow dysfunction in diabetes.