The activation of AMPK in cardiomyocytes at the very early stage of hypoxia relies on an adenine nucleotide-independent mechanism.
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 α(312) 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.
Institute of Burn Research, State Key Laboratory of Trauma, Burn and Combined Injury, Southwest Hospital, Third Military Medical University Chongqing, China. yanhong47@gmail.com
Hong
H
Yan
Dongxia
D
Zhang
Qiong
Q
Zhang
Pei
P
Wang
Yuesheng
Y
Huang
D000091162:AMP-Activated Protein Kinase Kinases; D000222:Adaptation, Physiological; D000244:Adenosine Diphosphate / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D032383:Myocytes, Cardiac / Q000201:enzymology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000096:biosynthesis*; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D011494:Protein Kinases; D000091162:AMP-Activated Protein Kinase Kinases