Abstract
Purpose :
Mitochondrial dysfunction is a known complication of diabetes. Our prior studies indicate that the insulin-like growth factor binding protein-3, IGFBP-3, is secreted by corneal epithelial cells in response to hyperglycemia and is increased in diabetic human tears. Importantly, the increase in tear levels of IGFBP-3 correlates with severity of diabetes. The role of IGFBP-3 in the normal, non-diabetic corneal epithelium is unknown. The purpose of this study was to investigate a potential role for IGFBP-3 in regulating mitochondrial homeostasis in the corneal epithelium.
Methods :
Human telomerized corneal epithelial (hTCEpi) cells were cultured in serum-free keratinocyte growth (KGM) medium containing growth factor supplements, keratinocyte basal (KBM) medium devoid of supplements, and KBM treated with recombinant human (rh)IGFBP-3. Protein and mRNA levels for genes that regulate mitochondrial fusion, respiration and mitophagy were analyzed using real time PCR, subcellular fractionation, western blot, and immunofluorescence. Mitochondrial fragmentation and depolarization were further assessed using tetramethylrhodamine, ethyl ester (TMRE) and mitotracker labeling. A Seahorse metabolic flux analyzer was used to measure cellular respiration and glycolysis.
Results :
Culture in KBM increased secretion of IGFBP-3 and induced a shift in mitochondrial morphology and activity. Compared to KGM, KBM also showed a shift IGF-1R and insulin receptor (INSR) expression and localization in hTCEpi cells that was altered by IGFBP-3. In addition, expression of mitochondrial proteins NADH dehydrogenase 1, ATP Synthase, and Acyl-CoA Synthetase were altered and varied temporally. Superoxide Dismutase 2 was unchanged. Treatment with rhIGFBP-3 was associated with changes in mitochondrial depolarization, mitochondrial respiratory protein expression, and mitophagy.
Conclusions :
Out data indicate that IGFBP-3 mediates mitochondrial activities by inducing the trafficking of key survival proteins during stress. This suggests that IGFBP-3 may direct stress-related mitochondrial-to-nuclear communication in the corneal epithelium. Further studies to elucidate the role of IGFBP-3 in mitochondrial homeostasis in the normal and diseased cornea are needed.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.