Steatotic livers are not used for transplantation because they have a reduced tolerance for ischemic events with reduced ATP levels and greater levels of cellular necrosis, which ultimately result in total organ failure. Mitochondrial uncoupling protein-2 (UCP2) is highly expressed in steatotic livers and may be responsible for liver sensitivity to ischemia through mitochondrial and ATP regulation. To test this hypothesis, experiments were conducted in lean and steatotic (ob/ob), wild-type, and UCP2 knock-out mice subjected to total warm hepatic ischemia/reperfusion. Although ob/ob UCP2 knock-out mice and ob/ob mice have a similar initial phenotype, ob/ob UCP2 knock-out animal survival was 83% when compared with 30% in ob/ob mice 24 h after reperfusion. Serum alanine aminotransferase concentrations and hepatocellular necrosis were decreased in the ob/ob UCP2 knock-out mice when compared with ob/ob mice subjected to ischemia. Liver ATP levels were increased in the ob/ob UCP2 knock-out animals after reperfusion when compared with the ob/ob mice but remained below the concentrations from lean livers. Lipid peroxidation (thiobarbituric acid-reactive substances) increased after reperfusion most significantly in the steatotic groups, but the increase was not affected by UCP2 deficiency. These results reveal that UCP2 expression is a critical factor, which sensitizes steatotic livers to ischemic injury, regulating liver ATP levels after ischemia and reperfusion.Complications from liver steatosis represent a significant clinical concern, especially for liver surgeries including resection and transplantation. This is of escalating importance as nonalcoholic fatty liver disease is independently correlated to obesity and insulin resistance, which are both epidemic in the Unites States (1, 2). Steatotic livers are considerably more sensitive to acute stressors including ischemia/reperfusion (I/R) 2 as experienced in transplantation, and organs meeting this criterion are routinely turned down for donation (3-6). Under I/R conditions, steatotic livers are ATP-depleted, and the predominant hepatocellular fate is shifted from apoptosis to oncotic necrosis, strongly implicating inappropriate energy homeostasis as the primary cause of liver sensitivity (7-10).In a state of energy substrate abundance and forward shift in cellular redox potential, hepatocytes are thought to combat mitochondrial electron transport chain-derived reactive oxygen species (ROS) production through mitochondrial uncoupling. In normal lean livers, mitochondrial uncoupling protein-2 (UCP2) is confined to Kupffer cells; however, hepatocellular concentrations of UCP2 greatly increase with steatosis (7,11,12). Although the mechanism is not known, UCP2 facilitates passive proton conductance across the mitochondrial inner membrane into the matrix during respiration (13,14). Maximum employment of electrochemical potential at ATP synthase is sacrificed by UCP2, and heat, rather than ATP, is produced. Proton conductance is thought to require posttranslational activ...

Prostate-specific antigen (PSA) is expressed primarily by both normal prostate epithelium and the vast majority of prostate cancers. Increases in serum PSA during endocrine therapy are generally considered as evidence for prostate cancer recurrence or progression to androgen independence. The mechanisms by which PSA upregulation occurs in androgen-refractory prostate cancer cells are unknown. In this study, by using LNCaP and its lineage-derived androgen-independent PSA-producing subline, C4-2, we identified two cis-elements within the 5.8-kilobase pair PSA promoter that are essential for the androgen-independent activity of PSA promoter in prostate cancer cells. First, a previously reported 440-bp androgen-responsive element enhancer core (AREc) was found to be important for the high basal PSA promoter activity in C4-2 cells. Both mutation analysis and supershift experiments demonstrated that androgen receptor (AR) binds to the AREs within the AREc and activate the basal PSA promoter activity in C4-2 cells under androgen-deprived conditions. Second, a 150-bp pN/H region was demonstrated to be a strong AR-independent positive-regulatory element of the PSA promoter in both LNCaP and C4-2 cells. Through DNase I footprinting and linker scan mutagenesis, a 17-bp RI site was identified as the key cis-element within the pN/H region. Data from electrophoretic mobility shift analysis and UV cross-linking experiments further indicated that a 45-kDa (p45) cell-specific transcription factor associates with RI in prostate cancer cells and may be responsible for driving the PSA promoter activity independent of androgen and AR. Furthermore, by juxtaposing AREc and pN/H, we produced a chimeric PSA promoter (supra-PSA) that exhibits 2-3-fold higher activity than the wild type PSA promoter in both LNCaP and C4-2 cells.

Material Supplementary 4.DC1http://www.jimmunol.org/content/suppl/2010/04/16/jimmunol.090202

Steatotic donors are routinely rejected for transplantation because of their increased rate of primary nonfunction. These grafts are more sensitive to ischemia/reperfusion (I/R) during transplantation. Removal of endotoxin before reperfusion improves liver performance post-I/R. We hypothesize that the main modality of injury in steatotic livers is toll-like receptor 4 (TLR4) signaling. We fed 4-week-old control and TLR4-deficient (TLR4KO) mice a normal diet (ND) or a 60% high-fat diet (HFD) for 4 weeks to induce steatosis. Mice were subjected to total hepatic ischemia (35 minutes) and reperfusion (1 or 24 hours). Survival improved and liver pathology decreased at 24 hours in TLR4KO HFD animals compared to control HFD animals. An investigation of infiltrates showed that neutrophils and CD4ϩ cells were increased at 24 hours in control HFD animals, whereas TLR4KO HFD animals were similar to ND controls. Messenger RNA levels of interleukin 6 (IL-6), IL-12, and interferon gamma were elevated at 1 hour in control HFD animals, whereas TLR4KO HFD animals were similar to ND controls. IL-10 levels at 1 hour of reperfusion in control HFD and TLR4KO animals were decreased versus control ND animals. In conclusion, these improvements in liver function in TLR4KO HFD animals implicate TLR4 as a mediator of steatotic graft failure after I/R. Liver Exacerbating the already short supply of donor livers for transplantation is the usability of steatotic donor organs. Livers, whose parenchyma contains more than 30% of fat, have a dramatically increased chance of primary nonfunction, 1 and the vast majority of these organs are discarded. Furthermore, it has been shown that the degree of steatosis correlates with higher postoperative liver enzymes and increased long-term mortality.2 These livers are more sensitive to the stresses of ischemia/reperfusion (I/R) injury, especially the stresses of endotoxin exposure, than normal, lean livers. 3,4 Endotoxin from intestinal microflora is translocated across the intestinal barrier during periods of gut hypoperfusion and during periods of mesenteric congestion associated with the anhepatic phase of liver transplantation. 5 Research has shown that steatotic animals are much more sensitive to the hepatic effects of endotoxin than their lean counterparts. 6 In addition, previous research in our laboratory has shown that neutralization of translocated endotoxin with a monoclonal antibody, when administered intravenously before I/R, dramatically improves the survival and liver function of overtly steatotic animals after a period of warm I/R. The main signaling receptor for endotoxin is toll-like receptor 4 (TLR4).So far, 13 TLRs have been identified in rodents, and each of these recognizes a specific pathogen-associated molecular pattern. 8 In addition to several endogenous ligands, TLR4 specifically recognizes bacterial cell wall

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