Uncoupling protein 2 (UCP2) is a mitochondrial membrane protein that regulates 
energy metabolism and reactive oxygen species (ROS) production. We generated 
mouse carboxy- and amino-terminal green fluorescent protein (GFP)-tagged UCP2 
constructs to investigate the effect of UCP2 expression on cell proliferation 
and viability. UCP2-transfected Hepa 1-6 cells did not show reduced cellular 
adenosine triphosphate (ATP) but showed increased levels of glutathione. Flow 
cytometry analysis indicated that transfected cells were less proliferative than 
nontransfected controls, with most cells blocked at the G1 phase. The effect of 
UCP2 on cell cycle arrest could not be reversed by providing exogenous ATP or 
oxidant supply, and was not affected by the chemical uncoupler carbonyl 
cyanide-p-trifluoromethoxyphenylhydrazone (FCCP). However, this effect of UCP2 
was augmented by treatment with genistein, a tyrosine kinase inhibitor, which by 
itself did not affect cell proliferation on control hepatocytes. Western 
blotting analysis revealed decreased expression levels of CDK6 but not CDK2 and 
D-type cyclins. Examination of cell viability in UCP2-transfected cells with 
Trypan Blue and Annexin-V staining revealed that UCP2 transfection led to 
significantly increased cell death. However, characteristics of apoptosis were 
absent in UCP2-transfected Hepa 1-6 cells, including lack of oligonucleosomal 
fragmentation (laddering) of chromosomal DNA, release of cytochrome c from 
mitochondria, and cleavage of caspase-3. In conclusion, our results indicate 
that UCP2 induces cell cycle arrest at G1 phase and causes nonapoptotic cell 
death, suggesting that UCP2 may act as a powerful influence on hepatic 
regeneration and cell death in the steatotic liver.