Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration

• Published in: American journal of physiology. Heart and circulatory physiology 2007-07, Vol.293 (1), p.H457-H466
• Main Authors: Zhang, Li, He, Huamei, Balschi, James A
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - metabolism; Adenosine triphosphatase; Adenylate Kinase - metabolism; Animals; Biochemistry; Cellular biology; Cytosol - metabolism; Dose-Response Relationship, Drug; Enzyme Activation - drug effects; Heart; Hypoglycemic Agents - administration & dosage; Kinases; Male; Metformin - administration & dosage; Myocardium - metabolism; Phenformin - administration & dosage; Rats; Rats, Sprague-Dawley; Signal Transduction - drug effects; Signal Transduction - physiology; Spectrum analysis
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00002.2007

NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts Submitted 2 January 2007 ; accepted in final form 15 March 2007 AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts ( n = 5–7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and 31 P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr 172 on AMPK- , and phosphorylation of Ser 79 on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not. 31 P-NMR spectroscopy; HPLC; total AMP content; AMP/ATP; biguanides Address for reprint requests and other correspondence: J. A. Balschi, 221 Longwood Ave., BLI 247, Boston, MA 02115 (e-mail: jbalschi{at}rics.bwh.harvard.edu )