Autophagy Defect Is Associated with Low Glucose-Induced Apoptosis in 661W Photoreceptor Cells

Delphine Balmer,Raphaël Roduit,Pénélope Andreux,Vanessa Ginet,Daniel F Schorderet,Johan Auwerx,Julien Puyal,Martine Emery,Kathrin Maedler

doi:10.1371/journal.pone.0074162

Abstract

Glucose is an important metabolic substrate of the retina and diabetic patients have to maintain a strict normoglycemia to avoid diabetes secondary effects, including cardiovascular disease, nephropathy, neuropathy and retinopathy. Others and we recently demonstrated the potential role of hypoglycemia in diabetic retinopathy. We showed acute hypoglycemia to induce retinal cell death both in vivo during an hyperinsulinemic/hypoglycemic clamp and in vitro in 661W photoreceptor cells cultured at low glucose concentration. In the present study, we showed low glucose to induce a decrease of BCL2 and BCL-XL anti-apoptotic proteins expression, leading to an increase of free pro-apoptotic BAX. In parallel, we showed that, in retinal cells, low glucose-induced apoptosis is involved in the process of autophagosomes formation through the AMPK/RAPTOR/mTOR pathway. Moreover, the decrease of LAMP2a expression led to a defect in the autophagosome/lysosome fusion process. Specific inhibition of autophagy, either by 3-methyladenine or by down-regulation of ATG5 or ATG7 proteins expression, increased caspase 3 activation and 661W cell death. We show that low glucose modifies the delicate equilibrium between apoptosis and autophagy. Cells struggled against low nutrient condition-induced apoptosis by starting an autophagic process, which led to cell death when inhibited. We conclude that autophagy defect is associated with low glucose-induced 661W cells death that could play a role in diabetic retinopathy. These results could modify the way of addressing negative effects of hypoglycemia. Short-term modulation of autophagy could be envisioned to treat diabetic patients in order to avoid secondary complications of the disease.

Highlights

Neural tissues, including retina, are totally dependent on glucose for normal metabolic activity
BAX protein expression was not changed (Fig. 1B), but B-cell lymphoma 2 (BCL2) and BCL-XL decrease leads to the release of active BAX, as showed by immunoprecipitation experiments and confirmed by immunostaining of active BAX in 661W cells exposed to low glucose (Fig. 1C)
We showed that low glucose conditions increased the expression of the autophagic marker, LC3-II, in 661W cells (Fig. 2A) and in retinal explants (Fig. 2B)

Summary

Introduction

Neural tissues, including retina, are totally dependent on glucose for normal metabolic activity. In both type I and II diabetes, normalization of blood glucose concentration is an important issue to avoid secondary long-term microvascular complications, including nephropathy, cardiovascular diseases, neuropathy and retinopathy [1]. We recently showed that hyperglycemia, and hypoglycemia, could be detrimental for the retina [2] Both in vivo short-term hypoglycemia, induced by a 5-hour hyperinsulinemic clamp, or the in vitro model of 661W photoreceptor cells cultured at low glucose condition, led to retinal cell death via an activation of the caspase 3 pathway and a decrease of glutathione (GSH) content. A recent publication, showing a decrease of central retinal function in human during acute hypoglycemia, strengthened the importance of glycemic excursion in patients [7]

Methods

Results

Conclusion