Abstract
Purpose :
Delayed corneal epithelial wound healing is a common complication of diabetes. Our laboratory has found that primary cultured diabetic human corneal epithelial cells show varying levels of mitochondrial depolarization and morphological changes compared to non-diabetic controls. In this study, we characterized mitochondrial and metabolic activity in corneal epithelial cells subject to acute and chronic hyperglycemic stress.
Methods :
Telomerase-immortalized human corneal epithelial (hTCEpi) cells were cultured in a defined serum-free keratinocyte growth media containing 6 mM D-glucose. To simulate hyperglycemia, cells were supplemented with additional glucose up to a final concentration of 25 mM. Mannitol was used as an osmotic control. Cells were cultured for 24 hours, 3, 5, 7, 9 or 14 days. Mitochondrial morphology and polarization were assessed using JC-1 and visualized using laser scanning confocal microscopy. Metabolic activity was quantified in real time using a Seahorse metabolic flux analyzer. Oxygen consumption rate (OCR, mitochondrial respiration) and extracellular acidification rate (ECAR, glycolysis) were analyzed using a Seahorse XFp Cell Mito Stress Test kit. Beta oxidation of fatty acids were further measured using a Substrate Oxidation Test kit.
Results :
Using JC-1, only cells cultured in high glucose for 14 days showed a loss of polarization and mitochondrial fragmentation. Metabolic activity was unchanged in cells cultured in high glucose for 24 hours. By day 3, cells showed a measurable drop in spare respiratory capacity that remained decreased through day 14. By day 5, glycolysis (ECAR) began to decrease and remained low through day 9. At days 7 and 9, OCR was increased, shifting cells towards a more respiratory phenotype. By day 14, consistent with JC-1 staining, OCR decreased and ECAR increased to normoglycemic levels.
Conclusions :
Chronic, not acute, exposure to high glucose negatively impacts mitochondrial structure and function. The decline in mitochondrial activity was associated with a recovery of glycolysis, indicative of metabolic adaptation. The ability of corneal epithelial cells subject to chronic hyperglycemia to rely on glycolysis as the primary energy source would account, in part, for the delayed wound healing seen in diabetes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.