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Biological characterization of gene response to insulin-induced hypoglycemia in mouse retina

ABSTRACT: Glucose is the most important metabolic substrate of the retina and maintenance of nor-moglycemia is an essential challenge for diabetic patients. Glycemic excursions could lead to cardiovascular disease, nephropathy, neuropathy and retinopathy. We recently showed that hy-poglycemia induced retinal cell death in mouse via caspase 3 activation and glutathione (GSH) decrease. Ex vivo experiments in 661W photoreceptor cells confirmed the low-glucose induction of death via superoxide production and activation of caspase 3, which was concomitant with a decrease of GSH content. We evaluate herein retinal gene expression 4 h and 48 h after insulin-induced hypoglycemia. Microarray analysis demonstrated clusters of genes whose expression is modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we highlight, by gene set enrichment analysis, three important pathways, including KEGG lysosomes, KEGG GSH metabolism and REACTOME apoptosis pathways. We tested the effect of recurrent hypoglycemia (three successive 5h periods of hypoglycemia separated by 48 h recovery) on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevents retinal cell death and GSH decrease, or adapts the retina to external stress by restoring GSH level comparable to control situation. We hypothesize that scavenger GSH is a key compound in this apoptotic process, and maintaining “normal” GSH level, as well as a strict glycemic control, may represent a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.

ORGANISM(S): Mus musculus

PROVIDER: GSE67523 | GEO | 2017/01/03

SECONDARY ACCESSION(S): PRJNA280156

REPOSITORIES: GEO

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Project description:Purpose. Patients with diabetic retinopathy may experience severe vision loss due to macular edema and neovascularization secondary to vascular abnormalities. However, before these abnormalities become apparent, there are functional deficits in contrast sensitivity, color perception, and dark adaptation. The goals of this study are to evaluate early changes (up to 3 months) in retinal gene expression, selected visual cycle proteins, and optokinetic tracking (OKT) in streptozotocin (STZ)-induced diabetic rats.Methods. Retinal gene expression in diabetic Long Evans rats was measured by whole genome microarray 7 days, 4 weeks, and three months after onset of hyperglycemia. Select gene and protein changes were probed by PCR and immunohistochemistry respectively, and OKT thresholds were measured using a virtual optokinetics system. Results. Microarray analysis showed that the most consistently affected molecular and cellular functions were cell-to-cell signaling and interaction, cell death, cellular growth and proliferation, molecular transport, and cellular movement. Further analysis revealed reduced expression of several genes encoding visual cycle proteins including lecithin:retinol acyltransferase (LRAT), retinal pigment epithelium (RPE)-specific protein 65kDa (RPE65) and RPE retinal G protein coupled receptor (RGR). Immunohistochemistry revealed a decrease in RPE65 in the RPE layer of diabetic rats. These molecular changes occurred simultaneously with a decrease in OKT thresholds by 4 weeks of diabetes. Conclusions. The data presented here are further evidence that inner retinal cells are affected by hyperglycemia prior to vasculopathy suggesting that glial and neuronal dysfunction may underlie some of the early visual deficits in diabetics. At each of the three timepoints (day 7, day 28, and day 84) one retina each from three diabetic rats were pooled for analysis on a single microarray chip. Three independent experiments were conducted for each group (n=9 animals/group). Each timepoint contained a hyperglycemic (STZ) and a control (buffer injection only) group. Additionally, on day 7 gene changes in the retina of rats which received a single injection of STZ, but did not develop hyperglycemia (STZ-non-c) were analyzed.

Project description:Diabetes is a disease now affecting more than 20 million people in the United States alone. When left untreated it has numerous debilitating effects including diabetic retinopathy, which is characterized by the death of retinal neurons as well as a breakdown of the retinal vascularization. This leads to nearly 24,000 cases of blindness each year among diabetic patients. Studies have shown the importance of both nitric oxide and the gene VEGF in retinopathy, however little is known about the condition's progression. This study would determine which genes are affected early in diabetes and more generally, give insite into how neural degeneration occurs in the retina. We aim to show the gene-level changes in the diabetic mouse retina five weeks after induction of diabetes. Our laboratory's primary focus is on NO production through nNOS, iNOS and endothelial (eNOS). We hope to be able to correlate some of the transcript level changes with genes known to be involved in the NO signalling pathways. Previous data by our lab and others have shown that neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) are upregulated under diabetic conditions in the retina. Additional work in our lab has shown that the lack of nitric oxide (NO) produced by nNOS may affect neurotransmitter production, particularly with GABA. We have also shown that mice with induced diabetes show neurochemical changes in the retina after only one to three weeks. Thus, we expect to see gene level changes associated with the retina before psysological changes in newly diabetic mice. Adult CD1 mice of random sexes are injected with the drug streptozotocin on three successive days while fasting 8 hours before the injection. This drug selectivley destroys the beta-islet cells of the pancrease. Their blood sugar levels are then tested after a resting period. Mice with glucose levels above 250mg/dL are considered diabetic from the date of the last injection. Mice are then sacrificed after the waiting period by breif isoflourine inhalation followed by decapitation. Retinas are immediately removed in cold HBSS and RNA is extracted using Trizol extraction. To get adequate amounts of RNA, we will pool four retinas (2 animals) together.

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Biological characterization of gene response to insulin-induced hypoglycemia in mouse retina

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