A role for uncoupling protein (UCP) homologues in mediating the proton leak in mammalian mitochondria is controversial. We subjected insulinoma (INS-1) cells to adenoviral expression of UCP2 or UCP1 and assessed the proton leak as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential. Cells were infected with different amounts of rat UCP2, and, in other experiments, with either UCP2 or UCP1. The relative molar expression of these subtypes was quantified through comparison with histidinetagged UCP1 or UCP2 proteins engineered by expression in Escherichia coli. Adenoviral infection with UCP2, compared with ␤-galactosidase, resulted in a dosedependent shift in kinetics indicating increased H ؉ flux at any given membrane potential. UCP1 also enhanced H ؉ flux, but, on a relative molar basis, the overexpression of the endogenous protein, UCP2, was more potent than UCP1. These results were not due to nonspecific overexpression of mitochondrial protein since UCP1 activity was inhibited by GDP and because overexpression of another membrane carrier protein, the oxoglutarate malate carrier had no effect. UCP2-mediated H ؉ conduction was not GDP sensitive. These data suggest that the UCP homologue, UCP2, mediates the proton leak in mitochondria of a mammalian cell wherein UCP2 is the native subtype.It appears that the proton leak, at least in rodent brown adipose tissue (BAT), 1 is a catalytic property of a specific mitochondrial uncoupling protein termed UCP1. The active protein dissipates the proton electrochemical gradient generated by electron transport by movement of hydrogen ions across the inner mitochondrial membrane to the matrix side. Although the exact mechanism of UCP1-mediated transport remains controversial, fatty acids play a role and proton transfer is inhibited by guanidine or adenine nucleotides (1, 2). More recently UCP1 homologues including UCP2 and long and short forms of UCP3 (UCP3 L and UCP3 S ) have been identified. There is evidence that these proteins also function to dissipate the proton electrochemical gradient, however, this issue is controversial (3, 4).The issue of whether UCP homologues mediate the proton leak is particularly uncertain for mammalian cells since studies of expressed UCP homologues have largely been carried out in yeast and proteoliposomes (3, 4). In the current studies, we assessed the effect of adenoviral overexpression of UCP2 on the proton leak in mitochondria isolated from rat insulinoma (INS-1) cells, a mammalian cell line in which UCP2 is present as a native protein. To accomplish this, the proton leak was measured as the kinetic relationship between oxygen use and the inner mitochondrial membrane potential under conditions wherein oxygen use is leak-dependent and proportional to H ϩ transport. To our knowledge, these are the first cellular studies to use this approach for assessment of the effect of an overexpressed UCP homologue on leak-dependent H ϩ conductance in mammalian mitochondria.INS-1 cells were selected for these studies ...

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.

Heme oxygenase-1 (HO-1) is the rate limiting enzyme in heme catabolism and degrades heme to carbon monoxide, biliverdin, and ferrous iron. HO-1 transcriptional induction occurs in response to multiple forms of chemical, physical stress and cytokines. HO-1 confers cytoprotection by inhibiting apoptosis, oxidative stress, and inflammation. Hepatitis C virus (HCV) is a major cause of cirrhosis and hepatocellular carcinoma. It has been shown that HO-1 induction and HO-1 products interfere with replication of HCV and markedly decreased HCV replication. However, a growing body of evidence indicates that induction of HO-1 may be involved in carcinogenesis and can play a role in the metastasis and growth of tumors. The antioxidant, antiviral activity of HO-1 makes it the cytoprotective enzyme for liver tissue in HCV infection, and induction of HO-1 can be suggested as a future therapeutic approach. However, the role of HO-1 in tumor growth should not be ignored.

The mechanisms responsible for dysregulation of iron metabolism in response to ethanol ingestion are poorly understood. Relatively brief ethanol exposures in rodents are associated with reduced hepatic hepcidin expression without increases in hepatic iron content. This study evaluated the effects of long‐term ethanol treatment on hepatic iron metabolism in two mouse strains. Ethanol was administered in the drinking water to C57BL/6 and BALB/c mice for up to 11 months. Hepatic histology and iron concentrations (HIC) were assessed, along with expression of relevant genes and proteins by real‐time RT‐PCR and western blot, respectively. The livers of ethanol‐consuming mice of both strains showed mild steatosis without inflammation or fibrosis. Stainable hepatocyte iron was modestly increased in both strains ingesting ethanol, although hepatic iron concentrations were significantly higher only in C57BL/6 mice. Long‐term ethanol did not affect hepcidin mRNA (Hamp1 or Hamp2) in either strain, nor was the expression of several oxidative stress‐responsive genes (glutamate cysteine ligase, gamma‐glutamyl transpeptidase, heme oxygenase‐1 and growth differentiation factor 15) altered in response to ethanol, suggesting that oxidative stress and suppression of hepcidin expression in short‐term ethanol feeding models may be transient phenomena that resolve as mice adapt to ethanol exposure. This murine model of chronic ethanol ingestion demonstrates modest increases in hepatic iron without changes in hepcidin expression, markers of oxidative stress or significant histologic liver injury. Further investigations are needed to characterize the mechanisms of dysregulated iron metabolism resulting from chronic ethanol ingestion.