AIMS: General purpose of doctoral dissertation was to study molecular mechanisms that regulate AMP-activated protein kinase (AMPK) activity in skeletal muscles in physiological and pathophysiological conditions related to induction of immune response. Also, the aim was to elucidate molecular mechanisms by which pharmacological modulators of metabolism affect AMPK in skeletal muscles. Specific aims of doctoral thesis were: (1) to determine molecular mechanisms through which dichloroacetate (DCA) affects pyruvate dehydrogenase kinase 1 (PDK1) and AMPK in metabolically different cell types; (2) to investigate how the activation of STING-TBK1 signaling pathway affects cell’s metabolic status and AMPK activity, and (3) to examine the role of AMPK in the effect of STING-TBK1 signaling pathway on insulin action in skeletal muscle cells.
HYPOTHESES: We tested three hypotheses: (1) Dichloroacetate decreases pyruvate dehydrogenase kinase level and induces the activation of AMPK in skeletal muscle cells. (2) STING-TBK1 signaling pathway suppresses AMPK activity in skeletal muscle cells. (3) Pharmacologic activation of AMPK decreases TBK1-induced insulin resistance in skeletal muscle cells.
METHODS: In vitro models of the skeletal muscle (primary human skeletal muscle cells (hSkMCs), induced pluripotent stem cells (iPSCs)-derived human skeletal muscle cells (id-SkMCs), and rat (L6) skeletal muscle cells) were used to investigate molecular processes associated with metabolic responses. The differentiation of skeletal muscle cells developed from iPSCs was monitored with RT-qPCR, immunofluorescence and western blot. In order to study target protein response and cell signalization, we used molecular methods (western blot, RT-qPCR, gene silencing with siRNA, ELISA and Magnetic Luminex Assay). Effects of pharmacological agents on metabolic processes were determined by analysis of lactate production and glucose uptake.
RESULTS: Ad H1: Dysfunction of PDK is associated with metabolic disorders, type 2 diabetes, obesity and cancer. By directly inhibiting PDK and indirectly suppressing PDK gene transcription, dichloroacetate (DCA) promotes the activity of pyruvate dehydrogenase complex (PDC) and improves metabolic homeostasis. Treatment with DCA selectively decreased PDK1 protein abundance in metabolically different cell types, which was partially independent of the regulation at the transcription level. Activated AMPK was not required for additional post-transcriptional mechanisms underlying DCA action. Ad H2: Obesity and type 2 diabetes are associated with chronic inflammation. In adipocytes, the inflammation is likely to develop, also, because obesity promotes release of mitochondrial DNA into the cytosol and, consequently, the activation of the cGAS-STING-TBK1 signaling pathway. In human SkMCs and L6 myotubes the activation of STING-TBK1 was induced with exogenous 2'3'-cGAMP or with STING activator DMXAA, respectively. STING-TBK1 activation increased AMPK activity. Ad H3: Obesity-related chronic inflammation contributes to the development of insulin resistance. cGAMP and TBK1 promote the activity of protein kinase B (Akt), the major kinase within the insulin signaling pathway. Treatment of id-SkMCs with 2'3'-cGAMP increased Akt activity, while DMXAA suppressed insulin-induced Akt activity and glucose uptake in L6 myotubes. AMPK activator AICAR further enhanced, whereas indirect inhibition of AMPK activity with STO-609 prevented TBK1-mediated effects.
CONCLUSIONS: (1) DCA selectively decreases PDK1 and PDK2 protein levels and induces the activation of AMPK in L6 myotubes, which supports our first hypothesis. (2) Skeletal muscle cells have functional cGAS-STING-TBK1 signaling pathway which promotes AMPK activity, which does not support the second hypothesis. (3) 2'3'-cGAMP and DMXAA exhibit divergent effects on insulin signaling, but functional AMPK is required for TBK1 downstream effects in skeletal muscle cells. The pharmacologic activation of AMPK further enhances TBK1-mediated effects, which does not support the third hypothesis.
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