23 REDUCTION OF MITOCHONDRIAL FUNCTION, PROLIFERATION, AND GENE EXPRESSION IN FIBROBLAST DONOR CELLS FOR USE IN SOMATIC CELL NUCLEAR TRANSFER BY CPI-613 AND PS48

Abstract

The morphology (spherical and without cristae) and metabolism (lowly functional) of mitochondria in early embryos and other rapidly proliferating cells exhibit a Warburg effect (WE)-like metabolism. A hallmark of the WE is the predominate use of glycolysis for energy production as opposed to the tricarboxylic acid cycle used by differentiated cells. Additionally, increased signalling of the PI3K pathway is correlated with an increase in glucose metabolism within cancer cells and is consistent with the WE. PS48 stimulates the PI3K pathway, and CPI-613 inhibits pyruvate dehydrogenase. The goal was to achieve a WE-like effect in donor cells before NT. Day 35 porcine fetal fibroblasts were treated as controls (CON, 0 μM) or with CPI (25, 50, or 100 μM) or PS48 (1, 5, 10 μM) for 7 days. Cytometry data were processed using SUMMIT software and analysed via GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA); all variables were analysed for the main effect of drug concentration. Trypan blue cell viability measures were analysed using GLM. For each collection day (i.e. Day 3, 5, and 7), all variables were analysed for the main effect of treatment, duration of culture, and their interaction. All mRNA expression as measured via the ΔΔ-ct method by qPCR was analysed using CT values in GLM for the main effect of drug treatment. Total number of cells and live cells at 120 h was decreased (P ≤ 0.03) in all PS48 treatments compared with CON cells (total cells: CON = 8.95 × 10⁶ v. PS48 treatments ≤6.98 × 10⁶; live cells: CON = 8.39 × 10⁶ v. PS48 treatments ≤6.50 × 10⁶). While the percentage live cells in CPI and CON cells did not differ (P ≥ 0.09), 100 μM decreased the number of total cells and live cells from that of CON for every time point by ~50% (P ≤ 0.02), whereas the other CPI treatments 25 and 50 μM were intermediate. Expression of PDK2 was reduced with 10 μM PS48 treatment compared with CON, and 50 and 100 μM CPI treated cells (P < 0.001; PS48 10 μM: 0.335 v. ≤1.012 other treatments). The CPI 100 μM and 10 μM PS48 concentrations decreased PKM M1 variant expression compared with CON and 50 μM CPI cells (P < 0.001; CPI 100 μM and PS48 10 μM <0.44; CPI 50 μM and CON >0.68). To determine the mitochondrial membrane potential, JC-10 was used. The percentage of cells with high mitochondrial membrane potential decreased (P = 0.04) with PS48 treatment (PS48 treatments ≤19.6%, control = 25.6%). Treatment with CPI also decreased (P ≤ 0.01) membrane potential and the percentages of cells (high function: CPI treatments ≤12.7 v. 25.6% in control; low function: CPI treatments ≥80.3 v. 74.3%). Because PS48 or CPI decrease mitochondrial membrane potential and the abundance of PKM M1, the metabolism of these potential donor cells may be more blastomere like. Experiments are underway to determine whether cells treated with PS48 or CPI will result in better development after somatic cell NT.

This study was funded by Food for the 21st Century and NIH R01HD080636.