Abstract
Purpose :
Since circadian clock proteins have been identified in the rat lens, this study aimed to determine whether glutathione homeostasis is controlled in a circadian manner.
Methods :
Lenses and aqueous humor were collected every 4 hours over a 24-hour period from 6-week male Wistar rat lenses (n=8 for each time point) and glutathione levels (reduced and oxidised GSH) measured using HPLC. Western blotting and immunohistochemistry were used to confirm and localize the rhythmic expression of circadian clock core proteins (BMAL1 & CLOCK) and confirm if this expression pattern was linked to the rhythmic expression of the glutathione synthesis enzyme, gamma- glutamylcysteine ligase (GCL), the glutathione regeneration enzyme, glutathione reductase (GR) and NRF2, a transcription factor involved in regulating the expression of genes related to GSH homeostasis.
Results :
GSH levels in the lens and aqueous humor oscillated throughout the 24-hour period. Western blotting confirmed protein expression of BMAL1, CLOCK and NRF2 in the lens. Immunohistochemistry revealed differences in the localisation and sub-cellular distribution of BMAL1, CLOCK and NRF2. During the day, BMAL1 and CLOCK were present in the germinative zone of the lens with strong labelling present in the nuclei of epithelial and fiber cells. On the other hand, NRF2 labelling was present in the germinative zone but was not co-localised with the nuclei and instead appeared cytoplasmic. These differences in localisation patterns may correlate with fluctuations in GSH synthesis and GSH levels in the lens at different times of the day to protect against oxidative stress.
Conclusions :
Collectively, these findings provide preliminary evidence that glutathione levels may be under the control of a circadian clock. In the future, work will be performed on older rats to determine if decreased circadian rhythmicity alters glutathione levels and predisposes lenses to cataract formation.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.