Pyruvate dehydrogenase kinase 1 (PDK1) regulates the pyruvate dehydrogenase complex and thus glucose metabolism, its dysregulation is linked to various diseases. Dichloroacetate (DCA) inhibits PDK1 activity and simultaneously lowers its expression. The lysosome is responsible for degradation of intracellular material delivered through autophagy. Lysosomal degradation via autophagy can be prevented by inhibiting autophagosome-lysosome fusion, raising lysosomal pH, or inhibiting lysosomal enzymes.
We investigated the effect of lysosomal inhibitors on PDK1 degradation in cultured L6 rat skeletal muscle cells. We summarized the aim in three hypotheses: 1) chloroquine, ammonium chloride and leupeptin increase PDK1 levels in cultured L6 rat skeletal muscle cells; 2) in combination chloroquine, ammonium chloride and leupeptin increase PDK1 levels more effectively than individual inhibitors in cultured L6 rat skeletal muscle cells; 3) ammonium chloride and leupeptin reduce the inhibitory effect of DCA on PDK1 expression and activity in cultured L6 rat skeletal muscle cells. Experiments were performed on cultured L6 rat skeletal muscle cells. The expression of selected proteins was determined by the western blot method, followed by statistical evaluation of results.
Ammonium chloride and leupeptin increased PDK1 levels, while chloroquine had no effect. Combinations of lysosomal inhibitors did not show greater effects on PDK1 levels than individual inhibitors. DCA inhibited PDK1 activity, while co-treatment with ammonium chloride or leupeptin reduced PDK1 levels. Lysosomal inhibition was confirmed by elevated levels of autophagy markers microtubule-associated protein light chain 3 (LC3B II) and sequestosome-1 (SQSTM1/p62), induced by ammonium chloride and leupeptin, while DCA activity was confirmed by reduced phosphorylation of the pyruvate dehydrogenase α1 subunit. The results partially support the first hypothesis of lysosomal PDK1 degradation, but not the second or third hypotheses.
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