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 normoglycemia is an essential challenge for diabetic patients. Chronic, exaggerated, glycemic excursions could lead to cardiovascular diseases, nephropathy, neuropathy and retinopathy. We recently showed that hypoglycemia 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 was modified by hypoglycemia and we discuss the potential implication of those genes in retinal cell death. In addition, we identify by gene set enrichment analysis, three important pathways, including lysosomal function, GSH metabolism and apoptotic pathways. Then we tested the effect of recurrent hypoglycemia (three successive 4h periods of hypoglycemia spaced by 48 h recovery) on retinal cell death. Interestingly, exposure to multiple hypoglycemic events prevented GSH decrease and retinal cell death, or adapted 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, represents a therapeutic challenge in order to avoid side effects of diabetes, especially diabetic retinopathy.

Fig 1. Identification of differentially expressed genes during hyperinsulinemic/hypoglycemic clamp.

A) Mouse plasma glucose concentration before, during and after the clamp. *p<0.0001 Hypo (n = 18) vs. Eugly (n = 16). B) Heatmap of significantly differentially regulated genes (p≤0.01) at 4 hours and 48 hours, showing mean normalized intensities (n = 3) at each time-point and condition.

Fig 2. Heatmaps of selected pathways that were significantly enriched for hypoglycemia up-regulated genes at 48 hours identified by GSEA.

A) KEGG Lysosome pathway; B) KEGG Glutathione metabolism; C) REACTOME Apoptosis. The genes shown in the heatmaps are the leading edge subsets (the enriched genes) from the GSEA analysis [20]. GSEA score plots are shown alongside the heatmaps: Here a score peak towards the left indicates enrichment of pathway genes at the top of the list of genes ranked by fold-change. All three pathways shown were significantly enriched (corrected p-value ≤ 1e-4) based on 10000 permutations.

Fig 3. Increase expression of proteins involved in GSH metabolism and in the establisment of the extracellular matrix.

Immunostaining of GPX3 and GSTO-1 proteins (A), and VERSICAN and FIBULIN-1 (B) in the retina of mice submitted to a hypoglycemic (Hypo) or euglycemic (Eugly) clamp (white arrows showed accumulation of proteins of interest). This experiment is representative of 3 retinas for each group.

Fig 4. Insulin-induced multiple and single hypoglycemia in C57BL/6 mouse.

A) Graphic representation of plasma glucose levels during hyperinsulinemic clamps. Three successive hypoglycemic (mHypo; n = 6) and euglycemic (mEugly; n = 4) clamps (right part of the graph) spaced by 48 h were performed; during the last clamp, single hypoglycemic (sHypo; n = 6) and euglycemic (sEugly; n = 4) clamps were performed additionally (left part of the graph). B) Graphic representation of glucose infusion rates during all hyperinsulinemic/hypoglycemic clamps (black circle for mHypo and black square for sHypo) and the control hyperinsulinemic/euglycemic clamp (white circle for mEugly and white square for sEugly). Multiple clamps are showed in the left part of the graph, while single clamps are showed in the right par of the graph C) Mouse characteristics before and during the clamp. *p<0.005 Hypo vs. Eugly.

Fig 5. Multiple successive hypoglycemias did not exacerbate cell death in mouse retina but protect against retinal apoptosis.

A) Flat-mounted retinas were isolated 48 h after the clamp, stained for cell death by TUNEL assay and DAPI counter coloration was performed. White arrows show TUNEL positive cells in single hypoglycemic condition (sHypo) while no TUNEL positive cells were detected when multiple successive hypoglycemia (mHypo) was applied. Control situation with multiple euglycemia (mEugly) or single euglycemia (sEugly) showed, as expected, no TUNEL positive cells. Results were representative of two (sEugly/mEugly) to three (sHypo/mHypo) observed flat-mount retinas for each group. B) Cleaved Caspase 3 antibody showed positive cells (white arrow) in the outer nuclear (ONL) of the hypoglycemic mice retina, while no positive cells were observed in all other conditions. Counterstaining with DAPI was performed to identify the retinal cell layers. Results were representative of four (sEugly), one (mEugly), four (sHypo) or two (mHypo) observed retinas for each group.

Fig 6. GSH homeostasis was modulated in the retina of mice suffered single hypoglycemia but not after multiple successive hypoglycemic events.

A) GSH content was measured in protein lysates obtained from retina of single euglycemic (sEugly: white inverted triangle), multiple euglycemic (mEugly: white triangle), single hypoglycemic (sHypo: black square) and multiple hypoglycemic (mHypo: black circle) mice. Results were expressed as percent of sEugly and as mean ± SEM of 2 to 4 samples. *p<0.01 sHypo vs. sEugly. B) We tested the expression of two genes, Gpx3 and Gsto1, in the retina of hypoglycemic and euglycemic animals 48 h after the clamp. We were able to show, by RT-qPCR, the induction of both genes in single hypoglycemic conditions, but not after multiple successive hypoglycemic or euglycemic. RL8 (ribosomal protein L8) was used as internal control to normalize RNA expression and results are expressed as percent of respective control (sEugly, mEugly) and as mean ± SEM of 3 retinas, *p<0.04.