Decrease of intracellular ATP content downregulated UCP2 expression in mouse hepatocytes.
Mitochondrial uncoupling protein 2 (UCP2) plays an important role in regulating energy metabolism. We previously reported that UCP2 expression in steatotic livers is increased which leads to diminished hepatic ATP stores and renders steatotic hepatocytes vulnerable to ischemic damage. In this study, reagents that inhibit the production of ATP were used to mimic an ischemic state in the liver in order to investigate the effects of decreased intracellular ATP levels on UCP2 expression in a murine hepatocyte cell line (HEP6-16). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), an oxidative phosphorylation uncoupler, was found to decrease intracellular ATP levels in a dose- and time-dependent manner. Relatively high concentrations of FCCP from 8 to 80 microM were required to reduce the intracellular concentration of ATP. The inhibitory effect of FCCP on intracellular ATP was significantly potentiated by 2-deoxy-D-glucose, an inhibitor of glycolysis that when administered alone had no negative effect on cellular ATP levels in mouse hepatocytes. Decreased intracellular ATP levels were accompanied by lower UCP2 mRNA expression. Upon removal of FCCP and/or 2-deoxy-D-glucose and reculture with normal medium, ATP and UCP2 mRNA levels returned to normal within a few hours. Mitochondrial membrane potential in HEP6-16 cells was dissipated by 80 microM FCCP but not 8 microM FCCP, suggesting that the downregulation of UCP2 expression by FCCP was not related to mitochondrial potential changes. Consequently, the in vitro manipulation of ATP stores is consistent with the in vivo observations associated with ischemia/reperfusion injury.
Cheng
Gang
G
Division of Transplant Surgery, Department of Surgery, Medical University of South Carolina, Charleston 29425, USA.
Polito
Carmen C
CC
Haines
Julia K
JK
Shafizadeh
Stephen F
SF
Fiorini
Ryan N
RN
Zhou
Xin
X
Schmidt
Michael G
MG
Chavin
Kenneth D
KD
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002460:Cell Line; D003847:Deoxyglucose / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005786:Gene Expression Regulation; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D007473:Ion Channels; D007700:Kinetics; D008564:Membrane Potentials; D026901:Membrane Transport Proteins; D051379:Mice; D008928:Mitochondria / Q000502:physiology; D024101:Mitochondrial Proteins; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D000071246:Uncoupling Protein 2
D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011506:Proteins; D012333:RNA, Messenger; C000604390:Ucp2 protein, mouse; D000071246:Uncoupling Protein 2; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D003847:Deoxyglucose