Skeletal muscle cells generate energy for their functioning primarily through mitochondrial oxidative phosphorylation and glycolysis. Lactate, the main product of glycolysis, also serves as a potential energy substrate. In this thesis, we investigated which metabolic pathway predominates in ATP synthesis in rat skeletal muscle L6 cells. We further examined whether enhanced activity of the Na$^+$/K$^+$-ATPase—one of the largest cellular ATP consumers—induces a metabolic shift toward glycolysis and promotes lactate production. To characterize the metabolic phenotype of L6 cells, we analyzed the expression of lactate transporters (MCT1, MCT2, and MCT4) and employed the Seahorse Agilent analyzer to determine the effectiveness of different MCT inhibitors. The device was also used to quantify the contributions of oxidative phosphorylation and glycolysis to ATP production, both under basal conditions and in the presence of specific metabolic inhibitors (2-DG, GNE-140, MON, OM) as well as the MCT1 inhibitor AZD. Stimulation of the Na$^+$/K$^+$-ATPase was achieved with monensin to mimic increased ATP consumption. The expression profile of lactate transporters—predominantly MCT1, with low levels of MCT4 and negligible MCT2—suggested an oxidative metabolic phenotype, which was consistent with Seahorse measurements showing that most ATP in L6 cells is produced via oxidative phosphorylation. The LDH assay demonstrated that GNE-140 effectively inhibited the conversion of pyruvate to lactate at low concentrations, more strongly than OXA, a result further supported by Seahorse analysis. Inhibition of glycolysis (2-DG), LDH (GNE-140), and lactate transport (AZD) markedly reduced ECAR, while OCR remained largely unchanged. Upon monensin addition, ECAR increased significantly only in the presence of AZD; with 2-DG or GNE-140, the increase was modest, indicating that glycolysis plays an important role in fueling the Na$^+$/K$^+$-ATPase. In contrast, inhibition of oxidative phosphorylation with OM strongly reduced OCR, while Na$^+$/K$^+$-ATPase activation elevated ECAR. Taken together, these findings show that L6 cells rely mainly on oxidative phosphorylation under basal conditions but can shift toward glycolysis when energy demand increases, reflecting their metabolic flexibility.
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