Muscarinic Receptor Activation of AMP-activated Protein Kinase Inhibits Orexigenic Neuropeptide mRNA Expression

Abstract

AMP-activated protein kinase (AMPK) plays a crucial role in both cellular and whole body energy homeostasis. Here we demonstrate that the muscarinic receptor agonist carbachol activates AMPK␣1-containing complexes in the human SH-SY5Y cell line via a mechanism specific for the AMPK upstream kinase, Ca 2؉ /calmodulin-dependent protein kinase kinase ␤. Activation of AMPK inhibits mRNA expression of the orexigenic neuropeptides Agouti-related peptide and melanin-concentrating hormone but surprisingly has no effect on neuropeptide Y mRNA, a neuropeptide previously shown to be regulated by AMPK. Rather than restoring mRNA levels to baseline, pharmacological inhibition of Ca 2؉ /calmodulin-dependent protein kinase kinase ␤ or AMPK greatly increases Agouti-related peptide and melanin-concentrating hormone mRNA expression. These data support a hypothesis that modulating basal AMPK activity in the hypothalamus is essential for maintaining tight regulation of pathways contributing to food intake. AMP-activated protein kinase (AMPK)2 is a key metabolic enzyme in the regulation of energy homeostasis (1). At a cellular level, AMPK is activated in response to diverse physiological and pathological stimuli that cause ATP depletion. Subsequently, AMPK acts to maintain the AMP:ATP ratio through inhibition of pathways that catabolize ATP and promotion of ATP-generating pathways (2).AMPK is a heterotrimeric serine/threonine kinase, comprising a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. There are two ␣, two ␤, and three ␥ isoforms, and all 12 ␣␤␥ combinations have been identified in vivo. Activation of AMPK requires the action of upstream kinases that phosphorylate threonine residue 172 within the T-loop activation domain of the ␣ subunit (2). Three AMPK kinases have been identified to date. LKB1 is a tumor suppressor kinase linked to the rare hereditary cancer predisposition, Peutz-Jeghers syndrome (3).In complex with regulatory proteins STE20-related adaptor (STRAD␣) and MO25, LKB1 phosphorylates and activates AMPK (4, 5), possibly in response to changes in the AMP:ATP ratio. In addition, Ca 2ϩ /calmodulin-dependent protein kinase kinase ␤ (CaMKK␤) has also been identified as an AMPK kinase (6 -8), phosphorylating and activating AMPK in response to elevated intracellular Ca 2ϩ concentrations. Finally, transforming growth factor-␤-activated kinase 1 (TAK1) was very recently implicated in the regulation of AMPK activity in cells and in the heart, although its regulation remains unknown (9, 10). Thus, AMPK is well equipped to respond to a diverse range of stimuli.AMPK is expressed ubiquitously, and recent findings have stimulated interest in roles for neuronal AMPK (11). Although prolonged pharmacological activation of AMPK in neuroblastoma cells is reported to induce apoptosis (12), increasing evidence points toward a role for AMPK in neuroprotection. AMPK is activated in response to excitotoxic stimuli in hippocampal neurons, and inhibiting AMPK potentiates neurodegeneration (13). More recently, AMPK was shown to intera...

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