June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Insulin in Combination with N-Acetylcysteine Protects Hypoxia Induced Toxicity in 661W Cells
Author Affiliations & Notes
  • Martin Garcia
    Molecular Science, The University of Texas Rio Grande Valley School of Medicine, Edinburg, Texas, United States
  • Laura Leanne Valdez
    Molecular Science, The University of Texas Rio Grande Valley School of Medicine, Edinburg, Texas, United States
  • Andrew T C Tsin
    Molecular Science, The University of Texas Rio Grande Valley School of Medicine, Edinburg, Texas, United States
  • Footnotes
    Commercial Relationships   Martin Garcia None; Laura Valdez None; Andrew Tsin None
  • Footnotes
    Support  NIH-T32, UTRGV/SOM
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 3610 – A0065. doi:
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    • Get Citation

      Martin Garcia, Laura Leanne Valdez, Andrew T C Tsin; Insulin in Combination with N-Acetylcysteine Protects Hypoxia Induced Toxicity in 661W Cells. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3610 – A0065.

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

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Abstract

Purpose : Proliferative diabetic retinopathy (PDR) is the leading cause of blindness among working-age adults. Photoreceptors are the most common and metabolically demanding cells in the retina thus oxygen is vital for retinal function. Hypoxia induced metabolic stress leads to photoreceptor atrophy and retinopathy. Furthermore, photoreceptor cell death is known to occur mainly via apoptosis. However, the protection of hypoxia-induced-cytotoxicity in cone photoreceptor cells has not been investigated extensively. The aim of this study was to determine whether co-treatment of insulin and the N-Acetyl-L-Cysteine (NAC) efficiently protects against hypoxia-induced cytotoxicity in 661W cells.

Methods : 661W were cultured at 5% CO2 at 37 C in DMEM with 10% FBS, 1% a penicillin/streptomycin. Cobalt (II) Chloride hexahydrate (CoCl2) was used to induce hypoxia. Insulin was suspended in sterile water, and NAC was diluted in the culture medium. For recovery experiment, cells were pretreated with CoCl2 for 24hrs, followed by replacing of medium with 100nM insulin and 3mM NAC alone, or with a combination of the two reagents for another 24hrs. Cell viability was determined by MTT assay in a 96 well plate. Morphological changes of the cells were observed and photographed under phase-contrast microscope and protein expression was measured by Western blot analysis. Statistical analysis was undertaken using independent two-tailed Students t-test using SPSS Statistics software.

Results : Treatment with CoCl2 significantly inhibited cell proliferation, reduced the number of viability cells, and induced apoptosis, initiated PARP cleavage, and increased caspase 3 activation. In addition, CoCl2 treatment led to oxidative stress, autophagy, and ubiquitination in the 661W cells. These effects, including cell proliferations were significantly reversed by the combination treatment of Insulin and NAC. In contrast, treatment with Insulin alone did not result in a similar protective effect and NAC partially protects against hypoxia induced toxicity.

Conclusions : Hypoxia induces significant apoptosis, oxidative stress, and protein ubiquitination in 661W cone photoreceptors. A combination treatment of Insulin and NAC completely reversed such hypoxia-induced cytotoxicity. Additional research on a combination therapy employing insulin and NAC may provide a promising therapeutic strategy for hypoxia-mediated cone photoreceptor cell damage.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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