Replacing glucose with galactose reduces glycolysis and upregulates mitochondrial respiration. (A) Cell viability was not altered by 1 or 5 days of galactose culture (n = 6). Five days of galactose culture (B) significantly reduced lactate production (n = 6) and (C) trended toward a decrease in lactate dehydrogenase (LDH) activity (n = 4), indicating a reduction in non-oxidative glycolytic flux. The reduction in glycolysis after 5 days in galactose culture was offset by (D) an increase in the basal rate of cellular oxygen consumption (n = 6), which was associated with a near maximal rate of oxygen consumption, as indicated by (E) the ratio of coupled to uncoupled respiration approaching 100 (n = 4). The increase in mitochondrial respiration did not correspond to (F) an increase in the expression of genetic mediators of mitochondrial biogenesis (TFAM and PGC1A) after 1 day of galactose culture (n = 4) or an (G) increased abundance of mitochondrial proteins (ATP5B and VDAC1) after 5 days in galactose culture (n = 3). (H) However, 5 days of galactose culture significantly increased the expression of the mitochondrial electron transport chain and Krebs cycle enzyme succinate dehydrogenase (SDH) (n = 4). These metabolic changes were not able to maintain cellular metabolic homeostasis after 5 days of galactose culture, as indicated by (I) an increased ratio of NAD+ to NADH (n = 4) and (J) a decrease in the cellular energy charge (n = 3). Bars represent mean ± standard error of the mean. *P <0.05 and **P <0.01 between glucose and galactose.