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.

Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0

“…'After a 5-h fast (7:30-12:30am), awake and freely moving mice were randomized and subjected to three consecutive clamps performed in the same mice as described above, with a 2 days recovery after each hyperinsulinemic/hypoglycemic (mHypo, n = 6) or hyperinsulinemic/euglycemic (mEugly, n = 4) clamps' [136].…”

Glucose Variability: How Does It Work?

“…A gene response to insulin-induced hypoglycemia was estimated in the mouse retina by an array. Genes whose expression was modified by low glucose were enriched in lysosomal function, glutathione metabolism and apoptotic pathways and potentially involved in retinal cell death [158]. A set of genes specifically activated by recurrent hypoglycemia was revealed in a study of whole genome expression profiling after recurrent hypoglycemia and acute hypoglycemia in the adrenal medulla of normal Sprague Dawley rats.…”

GSH-Independent Induction of ER Stress during Hypoglycaemia in the Retinal Cells of Mice

“…Hypoglycaemia for five hours is harmful to mouse retinas as it causes an increase in ROS production, caspase-3 activation, and ultimately leads to apoptosis. GSH, the most important antioxidant defence against ROS in the cell, is also decreased after acute hypoglycaemia [ 33 ], and microarray analysis has shown a deregulation in the expression of some of the enzymes involved in GSH metabolism after acute hypoglycaemia in mice [ 34 ]. GSH has a central protective role against oxidative stress in the retina; when GSH is decreased by BSO injections, apoptosis increases in several cell types in mouse retinas [ 40 ] and in 661W photoreceptors [ 33 ].…”

“…As a consequence of increased oxidative stress, GSH levels were found to decrease [ 33 ]. Moreover, microarray analysis of retinas collected 4 h and 48 h after the clamp showed broad changes in the expression of genes involved in lysosomal function, GSH metabolism, and apoptotic pathways [ 34 ].…”