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Int J Clin Exp Pathol 2012;5(8):770-776

Original Article
The activation of AMPK in cardiomyocytes at the very early stage of hypoxia relies on an
adenine nucleotide-independent mechanism

Hong Yan, Dongxia Zhang, Qiong Zhang, Pei Wang, Yuesheng Huang

Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest Hospital, Third Military Medical University,
Chongqing 400038, China

Received July 22, 2012; Accepted August 29, 2012; Epub October 1, 2012; Published October 15, 2012

Abstract: The energy status of a cell plays a key role in its survival, and the exposure of eukaryotic cells to the hypoxia that accompanies the
depletion of intracellular ATP triggers specific systemic adaptive responses. AMP-activated protein kinase (AMPK) has emerged as a key
regulator of energy metabolism in the heart and plays a critical role in inducing these responses. However, the specific mechanism
responsible for AMPK activation in cardiomyocytes at very early stages of hypoxia remain unclear. The goals of this study were to assess the
relative contribution to AMPK activation of phosphorylation by AMPK kinase (AMPKK) and of positive allosterism due to AMP:ATP ratios in the
early stages of hypoxia. Our results demonstrated that, compared with normoxic controls, neither intracellular AMP concentrations nor AMP:ATP
ratios significantly increased within 1h of hypoxia onset. In contrast, a SAMS peptide phosphorylation assay and an immunoblot analysis
revealed significant increases in both AMPK activity and ACC phosphorylation within 5min of hypoxic treatment. Furthermore, exposure of
cardiomyocytes to hypoxia significantly increased AMPK phosphorylation within 5min, by 3- to 4-fold compared with controls (P<0.01), while
overall levels of AMPKα protein did not differ between aerobic and anoxic cardiomyocytes. We also observed increased AMPKK activity in anoxic
cardiomyocytes, through use of an α312 substrate. Taken together, our findings demonstrate that in the early stage of hypoxia in
cardiomyocytes, increases in AMPK activity occur prior to and independently of increases in AMP concentration or in the AMP:ATP ratio. Instead,
under these circumstances, AMPK is primarily activated by phosphorylation of the conserved Thr-172 residue in its activation loop by its
upstream kinase AMPKK. (IJCEP1207018).

Keywords: Cardiomyocytes, hypoxia, AMPK, AMP, AMPK kinase

Address all correspondence to:
Dr. Hong Yan, Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest Hospital, Third Military Medical
University, Chongqing 400038, China. Tel: +86-23-68754148; Fax: +86- 23-65461696; E-mail: yanhong47@gmail.com