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Miyuki Kubota, Carla Siegfried, Ying-Bo Shui, Fang Bai, Andrew Huang, David Beebe, Beebe Lab; Oxygen Metabolism in Primary Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5749.
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The hypoxic environment around the lens is important for lens transparency. Lens epithelial cells (LECs) play a key role in lens metabolism. We measured oxygen consumption, lactate production and mitochondrial function to assess the role of LECs in maintaining hypoxia around the lens.
Baseline cellular respiration was measured in rabbit LECs (NN1003A), kidney epithelial cells (MDCK), trabecular meshwork cells (TM-5), and corneal endothelial cells using a XF96 Analyzer (Seahorse Bioscience), which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in 96-well plates. Following appropriate written consent, capsule-epithelial cells were obtained from patients during cataract surgery and were divided into small explants in 96-well plates. Capsules were removed when LECs became confluent. OCR and ECAR were normalized to the number of cells per well. Mitochondrial function was assessed using a “stress test” which measures oxygen-dependent ATP production, maximal and non-mitochondrial oxygen consumption. The effect on oxygen consumption of patient age, sex, race, and the presence of diabetes or glaucoma was assessed.
NN1003A cells had the lowest oxygen consumption rate among the cell lines tested. Inhibiting mitochondrial ATP synthesis reduced OCR by 26% in LECs and by 63% (± 4%) in the other cells. Thus, 26% and 74%, respectively, of cellular oxygen consumption was coupled to ATP synthesis. LECs also had the highest OCR (22 ± 0.42 fmol/min/cell) and ECAR (0.0038 ± 0.0009 mpH/min/cell) when mitochondria were uncoupled, stimulating maximal oxygen consumption. These initial studies used primary LECs from patients of mean age of 69 (± 11) years (23 women, 8 men). The OCR from donors aged 60-70 years was higher than in those aged 80-90 years (4.11 ± 1.02 vs. 1.96 ± 0.79 fmol/min/cell; p<0.05). Diabetic patients had lower OCR than non-diabetic patients (1.73 ± 0.62 vs. 3.83 ± 1.25 fmol/min/cell; p<0.01).
Oxygen consumption and proton production differ in cells derived from different ocular tissues, with the lowest rate in a LEC line. Most oxygen consumption in lens cells is not used for ATP synthesis and the mitochondria in these cells have high “reserve capacity.” The lower O2 consumption in older donors and diabetics could contribute to cataract. Ongoing studies examine pO2 at the anterior surface of the lens in vivo and oxygen consumption in the patient’s LECs.
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