Leptin is a hormone secreted by adipocytes that plays a pivotal role in regulating food intake, energy expenditure and neuroendocrine function. Leptin stimulates the oxidation of fatty acids and the uptake of glucose, and prevents the accumulation of lipids in nonadipose tissues, which can lead to functional impairments known as "lipotoxicity". The signalling pathways that mediate the metabolic effects of leptin remain undefined. The 5'-AMP-activated protein kinase (AMPK) potently stimulates fatty-acid oxidation in muscle by inhibiting the activity of acetyl coenzyme A carboxylase (ACC). AMPK is a heterotrimeric enzyme that is conserved from yeast to humans and functions as a 'fuel gauge' to monitor the status of cellular energy. Here we show that leptin selectively stimulates phosphorylation and activation of the alpha2 catalytic subunit of AMPK (alpha2 AMPK) in skeletal muscle, thus establishing a previously unknown signalling pathway for leptin. Early activation of AMPK occurs by leptin acting directly on muscle, whereas later activation depends on leptin functioning through the hypothalamic-sympathetic nervous system axis. In parallel with its activation of AMPK, leptin suppresses the activity of ACC, thereby stimulating the oxidation of fatty acids in muscle. Blocking AMPK activation inhibits the phosphorylation of ACC stimulated by leptin. Our data identify AMPK as a principal mediator of the effects of leptin on fatty-acid metabolism in muscle.

Inactivating mutations in the protein kinase LKB1 lead to a dominantly inherited cancer in humans termed Peutz-Jeghers syndrome. The role of LKB1 is unclear, and only one target for LKB1 has been identified in vivo [3]. AMP-activated protein kinase (AMPK) is the downstream component of a protein kinase cascade that plays a pivotal role in energy homeostasis. AMPK may have a role in protecting the body from metabolic diseases including type 2 diabetes, obesity, and cardiac hypertrophy. We previously reported the identification of three protein kinases (Elm1, Pak1, and Tos3 [9]) that lie upstream of Snf1, the yeast homologue of AMPK. LKB1 shares sequence similarity with Elm1, Pak1, and Tos3, and we demonstrated that LKB1 phosphorylates AMPK on the activation loop threonine (Thr172) within the catalytic subunit and activates AMPK in vitro [9]. Here, we have investigated whether LKB1 corresponds to the major AMPKK activity present in cell extracts. AMPKK purified from rat liver corresponds to LKB1, and blocking LKB1 activity in cells abolishes AMPK activation in response to different stimuli. These results identify a link between two protein kinases, previously thought to lie in unrelated, distinct pathways, that are associated with human diseases.

AMP-activated protein kinase (AMPK) is activated within the cell in response to multiple stresses that increase the intracellular AMP:ATP ratio. Here we show that incubation of muscle cells with the thiazolidinedione, rosiglitazone, leads to a dramatic increase in this ratio with the concomitant activation of AMPK. This finding raises the possibility that a number of the beneficial effects of the thiazolidinediones could be mediated via activation of AMPK. Furthermore, we show that in addition to the classical activation pathway, AMPK can also be stimulated without changing the levels of adenine nucleotides. In muscle cells, both hyperosmotic stress and the anti-diabetic agent, metformin, activate AMPK in the absence of any increase in the AMP:ATP ratio. However, although activation is no longer dependent on this ratio, it still involves increased phosphorylation of threonine 172 within the catalytic (␣) subunit. AMPK stimulation in response to hyperosmotic stress does not appear to involve phosphatidylinositol 3-phosphate kinase, protein kinase C, mitogen-activated protein (MAP) kinase kinase, or p38 MAP kinase ␣ or ␤. Our results demonstrate that AMPK can be activated by at least two distinct signaling mechanisms and suggest that it may play a wider role in the cellular stress response than was previously understood.

Hypothalamic AMP-activated protein kinase (AMPK) has been suggested to act as a key sensing mechanism, responding to hormones and nutrients in the regulation of energy homeostasis. However, the precise neuronal populations and cellular mechanisms involved are unclear. The effects of long-term manipulation of hypothalamic AMPK on energy balance are also unknown. To directly address such issues, we generated POMCα2KO and AgRPα2KO mice lacking AMPKα2 in proopiomelanocortin-(POMC-) and agouti-related protein-expressing (AgRP-expressing) neurons, key regulators of energy homeostasis. POMCα2KO mice developed obesity due to reduced energy expenditure and dysregulated food intake but remained sensitive to leptin. In contrast, AgRPα2KO mice developed an age-dependent lean phenotype with increased sensitivity to a melanocortin agonist. Electrophysiological studies in AMPKα2-deficient POMC or AgRP neurons revealed normal leptin or insulin action but absent responses to alterations in extracellular glucose levels, showing that glucose-sensing signaling mechanisms in these neurons are distinct from those pathways utilized by leptin or insulin. Taken together with the divergent phenotypes of POMCα2KO and AgRPα2KO mice, our findings suggest that while AMPK plays a key role in hypothalamic function, it does not act as a general sensor and integrator of energy homeostasis in the mediobasal hypothalamus.

Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that function upstream of GSK-3beta. Expression of Dvl-2 activated TCF-dependent transcription. This was blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevented GSK-3beta binding also generated a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localized within expressing cells. When Dvl-2 localization was altered using a C-terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggested that Dvl-association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta.