Abstract
Purpose :
Prior studies have shown that corneal epithelial cells (CECs) do not require insulin for glucose uptake. Insulin, which is stored and secreted by the lacrimal gland, is however, an important hormone required for metabolic activity, proliferation, and the regulation of autophagy. The mechanism in which insulin regulates these effects in CECs is unknown. This study investigated the role of insulin and it's cognate receptor (INSR) in mediating metabolic activity, mitochondrial stability, and autophagy in CECs.
Methods :
Human telomerized corneal epithelial (hTCEpi) cells were cultured in defined keratinocyte growth medium (KGM) containing supplements, basal medium (KBM) without supplements, or KBM with 5 ug/ml of insulin for 24 or 48 hours. siRNA was used to knockdown INSR expression. Metabolic activity was measured using a Seahorse metabolic flux analyzer. Mitochondrial morphology and membrane potential were assessed using mitotracker, TMRE, and JC-1. Protein expression and localization of INSR, mitochondrial, and autophagic proteins were determined using western blot and immunofluorescence.
Results :
Under normal growth conditions, hTCEpi cells expressed a glycolytic phenotype. Culture of hTCEpi cells in KBM for 24 hours shifted them from a glycolytic to a respiratory phenotype due to a reduction in glycolysis. Co-incubation with insulin blocked this effect. Insulin treatment was also associated with a block in mitophagy. After 48 hours in KBM, there was a reduction in mitochondrial membrane polarization that was associated with a loss in mitochondrial respiration and a partial restoration of glycolysis. Western blotting further demonstrated a reduction in mitochondrial proteins, which was consistent with the release of cytochrome c. Co-incubation with insulin for 48 hours inhibited the loss in membrane polarization, mitochondrial respiration, and the release of cytochrome c. Insulin also restored mitochondrial protein levels.
Conclusions :
These data confirm a role for insulin in the regulation of glycolysis in CECs. These data further indicate that insulin is essential for maintaining mitochondrial stability in CECs during prolonged periods of stress. Additional studies are needed to elucidate the mechanism by which insulin mediates mitochondrial homeostasis in the corneal epithelium.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.