Abstract
Purpose:
Cells metabolize nutrients (glucose, fatty acids, and amino acids) to produce ATP and NAD(P)H via oxidative phosphorylation and glycolysis. The RPE is a post-mitotic layer of highly metabolic pigmented cells packed with mitochondria that actively engage in oxidative metabolism. Human RPE in vitro cell models, ARPE19 cells and RPE cultured from donor eyes, are important research tools used, with pros and cons depending on the experimental endpoint of interest. Herein we characterized the metabolism of these model systems and changes as a function of age.
Methods:
Seahorse Extracellular Flux (XF) assay was used to measure extracellular flux changes of oxygen (oxygen consumption rate, OCR, oxidative phosphorylation) and protons (extracellular acidification rate, ECAR, glycolysis). ARPE19, a spontaneously arising cell line from a 19 year-old donor, and RPE cells cultured from young (17 and 31 yrs) and old (62 and 67 yrs) donor eyes were grown to post-confluence and sequentially treated with glucose, oligomycin and 2-deoxyglucose (2-DOG). Glycolysis level, glycolytic capacity and glycolytic reserve were measured with the XF glycolysis stress test. With MitoTracker green relative cellular mitochondrial numbers were determined. mRNA levels of relevant genes in glucose metabolism (LDHA, PDHA and PHKA1) were determined by qPCR.
Results:
Basal levels of OCR and ECAR in ARPE19 cells were significantly lower than primary cells regardless of age. Glycolysis stress test results revealed an increased shift to glycolysis in young vs old (1.32 vs. 1.92 fold) and increased glycolytic capacity in young vs old (2.25 vs. 3.29 fold) cells relative to ARPE19 cells. Primary cells had a greater number of mitochondria compared to ARPE19 cells. Significant differences in expression of glucose metabolism genes between the cell models were not seen.
Conclusions:
The basal cell metabolisms of the RPE cell culture models are significantly different. Though primary RPE cells have more mitochondria than ARPE19 cells, they appear to be more glycolytic. The increased glycolysis level and glycolytic capacity of cells derived from older vs younger donors is consistent with aging studies showing a shift from oxidative metabolism to glycolysis with advanced age. Based on these findings, caution should be used in selecting an in vitro model for cell metabolism studies.
Keywords: 592 metabolism •
701 retinal pigment epithelium •
600 mitochondria