Glucose uptake and adenoviral mediated GLUT1 infection decrease hypoxia-induced HIF-1alpha levels in cardiac myocytes.
Hypoxia causes a large array of adaptive and physiological responses in all cells including cardiac myocytes. In order to elucidate the molecular effects of increased glucose flux on hypoxic cardiac myocytes we focused on the basic helix-loop-helix transcription factor, hypoxia inducible factor 1 alpha (HIF-1alpha), which is rapidly upregulated in hypoxic cells and elicits a number of responses including augmentation of glucose uptake. Primary cultures of neonatal rat cardiac myocytes as well as embryonic rat heart-derived myogenic H9c2 cells demonstrated a significant upregulation of HIF-1alpha when subjected to hypoxia of 6-8h in the absence of glucose. Re-addition of extracellular glucose to the medium resulted in a decrease of HIF-1alpha levels by almost 50%. This glucose effect was blocked by addition of glycolytic inhibitors. In addition, glucose uptake and glycolysis resulted in substantial decreased levels of p53, which is regulated by HIF-1alpha. Adenoviral infection of cultures of cardiac myocytes with the facilitative glucose transporter, GLUT1 followed by hypoxia of 24h also resulted in a significant reduction in the protein expression of HIF-1alpha compared to control vector-infected cultures. GLUT1 infected cultures also demonstrated fewer apoptotic cells and a reduction in the release of cytochrome c after hypoxia. Inhibition of the ubiquitin-proteasomal pathway by a variety of 26S proteasomal inhibitors increased HIF-1alpha to similar levels under both normoxic and hypoxic conditions and in the presence or absence of glucose. This result suggested that glucose induces HIF-1alpha degradation via a proteasomal pathway. This conclusion was substantiated by immunoprecipitation experiments of total cell extracts, which demonstrated an increase of ubiquitinated HIF-1alpha relative to total HIF-1alpha in the presence of glucose during hypoxia. Thus, glucose as well as GLUT1 overexpression diminishes hypoxia-induced HIF-1alpha protein via an ubiquitin-proteasomal pathway in hypoxic cardiac myocytes. This represents a novel feedback mechanism that may play an important role in adaptation of cardiac myocytes to hypoxia and ischemia.
Malhotra
Ricky
R
Department of Internal Medicine, University of Michigan Medical Center, 1150 W Medical Center Drive, 1560 MSRB II, Ann Arbor, MI 48109-0676, USA. rmalhotr@umich.edu
Tyson
David G W
DG
Sone
Hirohito
H
Aoki
Kasunori
K
Kumagai
Arno K
AK
Brosius
Frank C
FC
D000256:Adenoviridae; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D003574:Cytochrome c Group / Q000378:metabolism; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism*; D051272:Glucose Transporter Type 1; D000860:Hypoxia / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis
D003574:Cytochrome c Group; D051272:Glucose Transporter Type 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009004:Monosaccharide Transport Proteins; C495679:Slc2a1 protein, rat; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D005947:Glucose