Thyroid hormone activates adenosine 5'-monophosphate-activated protein kinase via intracellular calcium mobilization and activation of Calcium/Calmodulin-Dependent protein kinase kinase-β

AMP-activated protein kinase ( AMPK) is a key regulator of glucose and fatty acid homeostasis. In muscle cells, AMPK stimulates mitochondrial fatty acid oxidation and ATP production. The thyroid hormone T 3 increases cellular oxygen consumption and is considered to be a major regulator of mitochondrial activities. In this study, we examined the possible involvement of AMPK in the stimulatory action of T-3 on mitochondria. Treatment of C2C12 myoblasts with T-3 rapidly led to phosphorylation of AMPK. Acetyl-coenzyme A carboxylase, a direct target of AMPK, was also phosphorylated after T-3 treatment. Similar results were obtained with 3T3-L1, FRTL-5, and HeLa cells. Stable expression of T-3 receptor (TR)-alpha or TR beta in Neuro2a cells enhanced this effect of T-3, indicating the involvement of TRs. Because HeLa cells express only Ca2+/ calmodulin-dependent protein kinase kinase-beta(CaMKK beta), one of two known AMPK kinases, it was suggested that the effect of T-3 is mediated by CaMKK beta. Indeed, experiments using a CaMKK inhibitor, STO-609, and an isoform-specific small interfering RNA demonstrated the CaMKK beta-dependent phosphorylation of AMPK. Furthermore, T-3 was found to rapidly induce intracellular Ca2 + mobilization in HeLa cells, and a Ca2 + chelator, 1,2-bis(2-aminophenoxy) ethane-N,N,N,N'-tetraacetic acid (BAPTA), suppressed T-3- as well as ionomycin-dependent phosphorylation of AMPK. In addition, T-3-dependent oxidation of palmitic acids was attenuated by BAPTA, STO-609, and the small interfering RNA for CaMKK beta, indicating that T-3-induced activation of AMPK leads to increased fatty acid oxidation. These results demonstrate that T-3 nontranscriptionally activates AMPK via intracellular Ca2+ mobilization and CaMKK beta activation, thereby stimulating mitochondrial fatty acid oxidation.