June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Mitochondrial and metabolic alterations in diabetic corneal epithelial cells
Author Affiliations & Notes
  • Natalia Mussi
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
    Ophthalmology, Universidade Federal de Sao Paulo, Sao Paulo, São Paulo, Brazil
  • Whitney Stuard
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Melis Kabaalioğlu Güner
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Danielle M Robertson
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Footnotes
    Commercial Relationships   Natalia Mussi, None; Whitney Stuard, None; Melis Kabaalioğlu Güner, None; Danielle Robertson, None
  • Footnotes
    Support  Capes-Print (Brazil), NIH/NEI R01 EY024546 (DMR), NIH/NEI R01 EY029258 (DMR), NIH/NEI F30 EY031559 (WLS), NIH/NEI P30 EY030413, Scientific Research Award, Eye Bank Association of America Scientific Research Award, Lions Foundation for Sight Unrestricted grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 877. doi:
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    • Get Citation

      Natalia Mussi, Whitney Stuard, Melis Kabaalioğlu Güner, Danielle M Robertson; Mitochondrial and metabolic alterations in diabetic corneal epithelial cells. Invest. Ophthalmol. Vis. Sci. 2021;62(8):877.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Diabetic keratopathy affects up to 70% of patients with diabetes. Mitochondrial dysfunction is considered a central feature underlying diabetic complications. The purpose of this study was to characterize mitochondrial and metabolic alterations in primary cultured diabetic human corneal epithelial cells (HCECs) and in telomerase-immortalized human corneal epithelial (hTCEpi) cells exposed to hyperglycemic and hyperosmotic stress.

Methods : To determine the effects of diabetes on mitochondria and metabolism, donor human corneas from diabetics were obtained from Tissue Transplant Services at UT Southwestern Medical Center. Primary cultures were generated using an established lab protocol. HCECs and hTCEpi cells were cultured in a defined serum-free keratinocyte growth media containing 6 mM glucose. To determine the effects of hyperglycemia on hTCEpi cells, cells were cultured for 24 hours, 7 or 14 days in media containing an additional 19 mM glucose. Cells supplemented with 19 mM mannitol were used as an osmotic control. Mitochondrial morphology and polarization were assessed using MitoTracker and TMRE, respectively. Metabolic changes were quantified in real time using a Seahorse metabolic flux analyzer. Cell cycle was determined by staining with Propidium Iodide and analyzed using a Celigo imaging cytometer. Reactive oxygen species were measured using Amplex red. Expression of the mechanistic target of rapamycin (mTOR) and downstream effectors were assessed by western blot.

Results : Diabetic HCECs demonstrated variable growth rates in culture. Similarly, mitochondrial morphology varied greatly among diabetic HCECs ranging from mild to severe with robust fragmentation and depolarization. Exposure of hTCEpi cells to high glucose shifted cells towards a respiratory phenotype. Cell cycle was unaltered at 24 hours and at day 7. After 14 days of culture, hTCEpi cells in mannitol arrested in G2/M and this was associated with a decrease in mTOR expression.

Conclusions : These findings confirm the presence of mitochondrial and metabolic abnormalities in the diabetic corneal epithelium. Further studies are needed to determine the molecular mechanisms that underly these changes.

This is a 2021 ARVO Annual Meeting abstract.

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