Lysosomal SLC46A3 modulates hepatic cytosolic copper homeostasis.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.

Enzymatic browning inhibition and antioxidant activity of pear juice from a new cultivar of asian pear (Pyrus pyrifolia Nakai cv. Sinhwa) with different concentrations of ascorbic acid.

Different ascorbic acid (AA) concentrations of 0.16, 0.20, and 0.24% (w/v) were added to pear juice from the new cultivar Pyrus pyrifolia Nakai cv. "Sinhwa". Enzymatic browning reduction and antioxidant activity were analyzed after 24 h at 37°C. Juices with 0.20% added AA showed the highest inhibition of 78.8% of polyphenol oxidase (PPO) activity. L* values of juices a with 0.20 and 0.24% added AA decreased more slowly than controls lacking AA addition and juice with 0.16% added AA after storage for 24 h. Browning indices of juices with added AA were lower than for controls. However, indices increased after storage for 24 h. The DPPH radical-scavenging activity, reducing power, and nitrite scavenging activity of all juices with added AA were higher than for controls and decreased after storage for 24 h. Addition of 0.20% AA to pear juice from the new "Sinhwa" cultivar showed the highest browning activity reduction.

Functional characterization of purified pear protease and its proteolytic activities with casein and myofibrillar proteins.

This study was performed to characterize pear protease proteolytic activity and investigate the use of pear protease as a meat tenderizer. Pear protease was purified and stabilized by 5% dextrin during lyophilization (dry) or concentration (liquid). Pear protease was further characterized with respect to pH, thermodynamics, and enzyme kinetics. Pear protease was stable at a pH range of 5-8 with an optimum pH of 6.5. From Arrhenius plots, liquid protease showed higher temperature dependency (23.49 kJ/mol) than dry protease (18.62 kJ/mol) due to its higher activation energy. The kcat/Km, catalytic efficiency of enzyme, was similar with 2.9 and 2.7 µM/min with dry and liquid proteases. Pear protease was evaluated for its proteolytic activities with casein and beef myofibrillar proteins by individually and combination with fig and kiwifruit proteases. These result indicated that pear and kiwifruit proteases could be complementary to be a desirable product for meat tenderization.

Changes in total phenolic and flavonoid content and antioxidative activities during production of juice concentrate from Asian pears (Pyrus pyrifolia Nakai).

The total phenolic and flavonoid content and antioxidative activities during production of pear juice concentrate (PJC) from two cultivars, Hwasan and Niitaka, were investigated. The main processing steps in PJC production are washing, pressing, pasteurization, clarification, filtration, evaporation, and packaging. Total phenolic and flavonoid content of end-product PJC from Niitaka decreased by 53.11 and 46.47%, respectively, while those from Hwasan decreased by 55.46 and 36.09%, respectively, compared to the phenolic and flavonoid content of original fresh fruit. DPPH radical-scavenging activities, reducing power and nitrate radical-scavenging activities showed a similar tendency as total phenolic and flavonoid content; that is, they decreased in juice concentrate made from both cultivars. Also, antioxidant activities of press cake waste (skin and seeds) from Niitaka and Hwasan pears were higher than fresh pears. In conclusion, antioxidant levels were significantly affected during processing of PJC, especially during the pressing step in which press cake waste retains the seeds and skin.

The aryl hydrocarbon receptor nuclear translocator (Arnt) is required for tumor initiation by benzo[a]pyrene.

Benzo[a]pyrene (B[a]P) is a ligand for the aryl hydrocarbon receptor (Ahr). After binding ligand, Ahr dimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) protein, and the dimer upregulates the transcription of Cyp1a1, Cyp1b1 and other enzymes involved in the metabolic activation of B[a]P. Arnt null mice die in utero. Mice in which Arnt deletion occurs constitutively in the epidermis die perinatally. In the current study, mice were developed in which the Arnt gene could be deleted specifically in adult skin epidermis. This deletion had no overt pathological effect. Homozygosity for a null reduced nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase allele was introduced into the above mouse strain to render it more susceptible to tumor initiation by B[a]P. Deletion of Arnt in the epidermis of this strain completely prevented the induction of skin tumors in a tumor initiation-promotion protocol in which a single topical application of B[a]P acted as the tumor-initiating event, and tumor promotion was provided by repeated topical applications of 12-O-tetradecanoyl phorbol-13-acetate (TPA). In contrast, deletion of Arnt did not prevent the induction of skin tumors in a protocol also using TPA as the promoter but using as the initiator N-methyl-N'-nitro-N-nitrosoguanidine, whose activity is unlikely to be affected by the activity of Ahr, Arnt or their target genes. These observations demonstrate that Arnt is required for tumor initiation by B[a]P in this system.

Intestinal hypoxia-inducible transcription factors are essential for iron absorption following iron deficiency.

Iron deficiency and iron overload are among the most prevalent nutritional disorders worldwide. Duodenal cytochrome b (DcytB) and divalent metal transporter 1 (DMT1) are regulators of iron absorption. Their expression is increased during high systemic requirements for iron, but the molecular mechanisms that regulate DcytB and DMT1 expression are undefined. Hypoxia-inducible factor (HIF) signaling was induced in the intestine following acute iron deficiency in the duodenum, resulting in activation of DcytB and DMT1 expression and an increase in iron uptake. DcytB and DMT1 were demonstrated as direct HIF-2alpha target genes. Genetic disruption of HIF signaling in the intestine abolished the adaptive induction of iron absorption following iron deficiency, resulting in low systemic iron and hematological defects. These results demonstrate that HIF signaling in the intestine is a critical regulator of systemic iron homeostasis.

Hypoxia-inducible factor augments experimental colitis through an MIF-dependent inflammatory signaling cascade.

BACKGROUND & AIMS: Colon epithelial cells are critical for barrier function and contain a highly developed immune response. A previous study has shown hypoxia-inducible factor (HIF) as a critical regulator of barrier protection during colon epithelial injury. However, the role of HIF signaling in colon mucosal immunity is not known. METHODS: With the use of cre/loxP technology, intestinal-specific disruption of von Hippel-Lindau tumor suppressor protein (Vhl), hypoxia-inducible factor (Hif)-1alpha, and aryl hydrocarbon nuclear translocator (Arnt) was generated. Colon inflammation was induced using a dextran sulfate sodium (DSS)-induced colitis model, and the mice were analyzed by histologic analysis, Western blot analysis, and quantitative polymerase chain reaction. RESULTS: In mice, colonic epithelium disruption of Vhl resulted in constitutive expression of HIF, which initiated an increase in inflammatory infiltrates and edema in the colon. These effects were ameliorated in mice by disruption of both Vhl and Arnt/Hif1beta (which inactivates HIF). In a DSS-induced colitis model, increased HIF expression correlated with more severe clinical symptoms and an increase in histologic damage, while disruption of both Vhl and Arnt in the colon epithelium inhibited these effects. Furthermore, colons with constitutive activation of HIF displayed increased expression of proinflammatory mediators that were synergistically potentiated following DSS administration and reduced by inhibition of the proinflammatory and direct HIF target gene macrophage migration inhibitory factor. CONCLUSIONS: The present study shows that a chronic increase in HIF signaling in the colon epithelial cells initiates a hyperinflammatory reaction that may have important implications in developing therapeutic strategies for inflammatory bowel disease.

Hepatocyte nuclear factor 4alpha in the intestinal epithelial cells protects against inflammatory bowel disease.

BACKGROUND: Hepatocyte nuclear factor 4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily expressed in liver and intestine. While HNF4alpha expression is critical for liver function, its role in the gut and in the pathogenesis of inflammatory bowel disease (IBD) is unknown. METHODS: Human intestinal biopsies from control and IBD patients were examined for expression of mRNAs encoding HNF4alpha and other nuclear receptors. An intestine-specific HNF4alpha null mouse line (Hnf4alpha(DeltaIEpC)) was generated using an Hnf4alpha-floxed allele and villin-Cre transgene. These mice and their control floxed counterparts (Hnf4alpha(F/F)), were subjected to a dextran sulfate sodium (DSS)-induced IBD colitis protocol and their clinical symptoms and gene expression patterns determined. RESULTS: In human intestinal biopsies, HNF4alpha was significantly decreased in intestinal tissues from Crohn's disease and ulcerative colitis patients. HNF4alpha expression was also suppressed in the intestine of DSS-treated mice. In Hnf4alpha(DeltaIEpC) mice, disruption of HNF4alpha expression was observed in the epithelial cells throughout the intestine. In the DSS-induced colitis model Hnf4alpha(DeltaIEpC) mice showed markedly more severe changes in clinical symptoms and pathologies associated with IBD including loss of body weight, colon length, and histological morphology as compared with Hnf4alpha(F/F) mice. Furthermore, the Hnf4alpha(DeltaIEpC) mice demonstrate a significant alteration of mucin-associated genes and increased intestinal permeability, which may play an important role in the increased susceptibility to acute colitis following an inflammatory insult. CONCLUSIONS: While HNF4alpha does not have a major role in normal function of the intestine, it protects the gut against DSS-induced colitis.

Dietary phytochemicals regulate whole-body CYP1A1 expression through an arylhydrocarbon receptor nuclear translocator-dependent system in gut.

Cytochrome P450 1A1 (CYP1A1) is one of the most important detoxification enzymes due to its broad substrate specificity and wide distribution throughout the body. On the other hand, CYP1A1 can also produce highly carcinogenic intermediate metabolites through oxidation of polycyclic aromatic hydrocarbons. We describe what we believe to be a novel regulatory system for whole-body CYP1A1 expression by a factor originating in the gut. A mutant mouse was generated in which the arylhydrocarbon receptor nuclear translocator (Arnt) gene is disrupted predominantly in the gut epithelium. Surprisingly, CYP1A1 mRNA expression and enzymatic activities were markedly elevated in almost all non-gut tissues in this mouse line. The induction was even observed in early-stage embryos in pregnant mutant females. Interestingly, the upregulation was CYP1A1 selective and lost upon administration of a synthetic purified diet. Moreover, the increase was recovered by addition of the natural phytochemical indole-3-carbinol to the purified diet. These results suggest that an Arnt-dependent pathway in gut has an important role in regulation of the metabolism of dietary CYP1A1 inducers and whole-body CYP1A1 expression. This machinery might be involved in naturally occurring carcinogenic processes and/or other numerous biological responses mediated by CYP1A1 activity.

Loss of ARNT/HIF1beta mediates altered gene expression and pancreatic-islet dysfunction in human type 2 diabetes.

beta cell dysfunction is a central component of the pathogenesis of type 2 diabetes. Using oligonucleotide microarrays and real-time PCR of pancreatic islets isolated from humans with type 2 diabetes versus normal glucose-tolerant controls, we identified multiple changes in expression of genes known to be important in beta cell function, including major decreases in expression of HNF4alpha, insulin receptor, IRS2, Akt2, and several glucose-metabolic-pathway genes. There was also a 90% decrease in expression of the transcription factor ARNT. Reducing ARNT levels in Min6 cells with small interfering RNA (siRNA) resulted in markedly impaired glucose-stimulated insulin release and changes in gene expression similar to those in human type 2 islets. Likewise, beta cell-specific ARNT knockout mice exhibited abnormal glucose tolerance, impaired insulin secretion, and changes in islet gene expression that mimicked those in human diabetic islets. Together, these data suggest an important role for decreased ARNT and altered gene expression in the impaired islet function of human type 2 diabetes.

Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes.

To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.