letters to nature NATURE | VOL 415 | 17 JANUARY 2002 | www.nature.com 339 into pCRII-TOPO (Invitrogen) or pAMPI (Strategene), and individual transformed colonies were subjected to T7/SP6 dideoxy sequencing. Peptide-binding assays Peptide-binding assays were performed by measuring inhibition of iodinated p11C peptide binding to 721.221 cells that express the MHC class I molecule Mamu-A*01. 2 ´ 10 6 cells were incubated overnight at 26 8C with 3 mg ml -1 human b 2 m; 1.5 ´ 10 5 c.p.m. iodinated p11C peptide and serial log dilutions of unlabelled test peptides were then incubated with washed cells for 4 h at 20 8C. Cells were then washed three times, and the radioactivity of the cell pellet was measured with a scintillation counter. Per cent inhibition of binding by the test peptides was calculated as 1 - (c.p.m. with competitor peptide/c.p.m. without competitor peptide). ELISPOT assays We coated 96-well multiscreen plates overnight with 100 ml per well of 10 mg ml -1 anti- human interferon-g (IFN-g) (B27; BD Pharmingen) in endotoxin-free Dulbecco's PBS (D-PBS). The plates were then washed three times with D-PBS containing 0.25% Tween- 20 (D-PBS/Tween), blocked for 2 h with D-PBS containing 5% FBS at 37 8C, washed three times with D-PBS/Tween, rinsed with RPMI 1640 containing 10% FBS to remove the Tween-20, and incubated with 8, 0.8, 0.08 or 0.008 mg ml -1 peptide and 2 ´ 10 5 PBMCs in triplicate in 100-ml reaction volumes. Following an 18-h incubation at 37 8C, the plates were washed nine times with D-PBS/Tween and once with distilled water. The plates were then incubated with 2 mg ml -1 biotinylated rabbit anti-human IFN-g (Biosource) for 2 h at room temperature, washed six times with Coulter Wash (Beckman-Coulter), and incubated for 2.5 h with a 1:500 dilution of streptavidin-AP (Southern Biotechnology). 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Competing interests statement The authors declare that they have no competing ®nancial interests. Correspondence and requests for materials should be addressed to D.H.B. (e-mail: dan_barouch@hotmail.com). ................................................................. Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase Yasuhiko Minokoshi*, Young-Bum Kim*, Odile D. Peroni*, Lee G. D. Fryer², Corinna Mu È ller*, David Carling² & Barbara B. Kahn* * Division of Endocrinology, Diabetes and Metabolism, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA ² The Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, London W12 0NN, UK .............................................................................................................................................. Leptin is a hormone secreted by adipocytes that plays a pivotal role in regulating food intake, energy expenditure and neuroen- docrine function 1 . Leptin stimulates the oxidation of fatty acids 2 and the uptake of glucose 3,4 , and prevents the accumulation of lipids in nonadipose tissues, which can lead to functional impair- ments known as ``lipotoxicity'' 5 . The signalling pathways that mediate the metabolic effects of leptin remain unde®ned. The 59-AMP-activated protein kinase (AMPK) potently stimulates fatty-acid oxidation in muscle by inhibiting the activity of acetyl coenzyme A carboxylase (ACC) 6,7 . 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 6 . Here we show that leptin selectively stimulates phosphorylation and acti- vation of the a2 catalytic subunit of AMPK (a2 AMPK) in skeletal muscle, thus establishing a previously unknown signalling path- way 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. Impaired fuel metabolism is an early pathogenic factor in obesity and type 2 diabetes 8 . Skeletal muscle is a principal site of glucose and fatty-acid use, and is one of the primary tissues responsible for © 2002 Macmillan Magazines Ltd