A viable hypomorphic Arnt2 mutation causes hyperphagic obesity, diabetes and hepatic steatosis.

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a member of the basic helix-loop-helix/PER-ARNT-SIM (bHLH/PAS) transcription factor family. ARNT2 heterodimerizes with several members of the family, including single-minded homolog-1 (SIM1) and neuronal PAS domain protein 4 (NPAS4), primarily in neurons of the central nervous system. We screened 64,424 third-generation germline mutant mice derived from N-ethyl-N-nitrosourea (ENU)-mutagenized great-grandsires for weight abnormalities. Among 17 elevated body weight phenotypes identified and mapped, one strongly correlated with an induced missense mutation in Arnt2 using a semidominant model of inheritance. Causation was confirmed by CRISPR/Cas9 gene targeting to recapitulate the original ENU allele, specifying Arg74Cys (R74C). The CRISPR/Cas9-targeted (Arnt2R74C/R74C) mice demonstrated hyperphagia and increased adiposity as well as hepatic steatosis and abnormalities in glucose homeostasis. The mutant ARNT2 protein showed decreased transcriptional activity when coexpressed with SIM1. These findings establish a requirement for ARNT2-dependent genes in the maintenance of the homeostatic feeding response, necessary for prevention of obesity and obesity-related diseases.

Effects of sequential exposure to water accommodated fraction of crude oil and chlorpyrifos on molecular and biochemical biomarkers in rainbow trout.

Fish can be simultaneously or sequentially exposed to various kinds of pollutants, resulting in combined effects. Polycyclic aromatic hydrocarbons induce cytochrome P450 monooxygenase 1A (CYP1A) expression, which catalyzes the conversion of the organophosphorus insecticide chlorpyrifos (CPF) into its most active derivative, CPF-oxon. CPF-oxon inhibits CYP1A and other enzymes, including carboxylesterases (CEs) and acetylcholinesterase (AChE). We studied the effects of an in vivo exposure to crude oil water accommodated fraction (WAF) followed by an ex vivo exposure of liver tissue to CPF on the expression of Cyp1a, AhR and ARNT mRNA, CYP1A protein and on the activity of biomarker enzymes in the rainbow trout (Oncorhynchus mykiss). Juvenile rainbow trout were exposed to WAF (62 µg L-1 TPH) for 48 h. Then, liver was dissected out, sliced and exposed to 20 µg L-1 CPF ex vivo for 1 h. Liver tissue was analyzed for mRNA and protein expression and for CEs, AChE, glutathione S-transferase (GST) and CYP1A (EROD) activity. WAF induced Cyp1a mRNA and CYP1A protein expression by 10-fold and 2.5-8.3-fold, respectively, with no effect of CPF. WAF induced AhR expression significantly (4-fold) in control but not in CPF treated liver tissue. ARNT mRNA expression was significantly lowered (5-fold) by WAF. CPF significantly reduced liver EROD activity, independently of WAF pre-treatment. CEs activity was significantly inhibited in an additive manner following in vivo exposure to WAF (42%) and ex vivo exposure to CPF (19%). CPF exposure inhibited AChE activity (37%) and increased GST activity (42%).

Emotion recognition associated with polymorphism in oxytocinergic pathway gene ARNT2.

The ability to correctly understand the emotional expression of another person is essential for social relationships and appears to be a partly inherited trait. The neuropeptides oxytocin and vasopressin have been shown to influence this ability as well as face processing in humans. Here, recognition of the emotional content of faces and voices, separately and combined, was investigated in 492 subjects, genotyped for 25 single nucleotide polymorphisms (SNPs) in eight genes encoding proteins important for oxytocin and vasopressin neurotransmission. The SNP rs4778599 in the gene encoding aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), a transcription factor that participates in the development of hypothalamic oxytocin and vasopressin neurons, showed an association that survived correction for multiple testing with emotion recognition of audio-visual stimuli in women (n = 309). This study demonstrates evidence for an association that further expands previous findings of oxytocin and vasopressin involvement in emotion recognition.

A Single Meal Containing Raw, Crushed Garlic Influences Expression of Immunity- and Cancer-Related Genes in Whole Blood of Humans.

BACKGROUND: Preclinical and epidemiologic studies suggest that garlic intake is inversely associated with the progression of cancer and cardiovascular disease. OBJECTIVE: We designed a study to probe the mechanisms of garlic action in humans. METHODS: We conducted a randomized crossover feeding trial in which 17 volunteers consumed a garlic-containing meal (100 g white bread, 15 g butter, and 5 g raw, crushed garlic) or a garlic-free control meal (100 g white bread and 15 g butter) after 10 d of consuming a controlled, garlic-free diet. Blood was collected before and 3 h after test meal consumption for gene expression analysis in whole blood. Illumina BeadArray was used to screen for genes of interest, followed by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on selected genes. To augment human study findings, Mono Mac 6 cells were treated with a purified garlic extract (0.5 µL/mL), and mRNA was measured by qRT-PCR at 0, 3, 6, and 24 h. RESULTS: The following 7 genes were found to be upregulated by garlic intake: aryl hydrocarbon receptor (AHR), aryl hydrocarbon receptor nuclear translocator (ARNT), hypoxia-inducible factor 1α (HIF1A), proto-oncogene c-Jun (JUN), nuclear factor of activated T cells (NFAT) activating protein with immunoreceptor tyrosine-based activation motif 1 (NFAM1), oncostatin M (OSM), and V-rel avian reticuloendotheliosis viral oncogene homolog (REL). Fold-increases in mRNA transcripts ranged from 1.6 (HIF1A) to 3.0 (NFAM1) (P < 0.05). The mRNA levels of 5 of the 7 genes that were upregulated in the human trial were also upregulated in cell culture at 3 and 6 h: AHR, HIF1A, JUN, OSM, and REL. Fold-increases in mRNA transcripts in cell culture ranged from 1.7 (HIF1A) to 12.1 (JUN) (P < 0.01). OSM protein was measured by ELISA and was significantly higher than the control at 3, 6, and 24 h (24 h: 19.5 ± 1.4 and 74.8 ± 1.4 pg/mL for control and garlic, respectively). OSM is a pleiotropic cytokine that inhibits several tumor cell lines in culture. CONCLUSION: These data indicate that the bioactivity of garlic is multifaceted and includes activation of genes related to immunity, apoptosis, and xenobiotic metabolism in humans and Mono Mac 6 cells. This trial is registered at clinicaltrials.gov as NCT01293591.

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.

Cloning and characterization of hypoxia-inducible factor-1 subunits from Ascaris suum - a parasitic nematode highly adapted to changes of oxygen conditions during its life cycle.

The parasitic nematode Ascaris suum successfully adapts to a significant decrease in oxygen availability during its life cycle by altering its metabolic system dramatically. However, little is known about the regulatory mechanisms of adaptation to hypoxic environments in A. suum. In multicellular organisms, hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor composed of HIF-1α and HIF-1ß subunits, is a master regulator of genes involved in adaptation to hypoxia. In the present study, cDNAs encoding HIF-1α and HIF-1ß were cloned from A. suum and characterized. The full-length A. suum hif-1α and hif-1ß cDNAs contain open reading frames encoding proteins with 832 and 436 amino acids, respectively. In the deduced amino acid sequences of A. suum HIF-1α and HIF-1ß, functional domains essential for DNA-binding, dimerization, and oxygen-dependent prolyl hydroxylation were conserved. The interaction between A. suum HIF-1α and HIF-1ß was confirmed by the yeast two-hybrid assay. Both A. suum hif-1α and hif-1ß mRNAs were expressed at all stages examined (fertilized eggs, third-stage larvae, lung-stage larvae, young adult worms, and adult muscle tissue), and most abundantly in the aerobic free-living third-stage larvae, followed by a gradual decrease after infection of the host. hif-1 mRNA transcription was not sensitive to the oxygen environment in either third-stage larvae or adult worms (muscle tissue), and was regulated in a stage-specific manner. High expression of hif-1 mRNAs in third-stage larvae suggests its contribution to pre-adaptation to a hypoxic environment after infection of their host. Sequence analysis of 5'-upstream regions of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) genes, which show stage-specific expression and play an important role in oxygen adaptation during the life cycle, revealed that all subunits except for the adult-type flavoprotein subunit (Fp) possess putative hypoxia-responsive elements (HREs), suggesting that they are hif-1 target genes.

Low-level laser therapy induces the expressions of BMP-2, osteocalcin, and TGF-ß1 in hypoxic-cultured human osteoblasts.

The aim of this study was to examine the effect of low-level laser therapy (LLLT) on the cell viability and the expression of hypoxia-inducible factor-1s (HIF-1s), bone morphogenic protein-2 (BMP-2), osteocalcin, type I collagen, transforming growth factor-ß1 (TGF-ß1), and Akt in hypoxic-cultured human osteoblasts. Human fetal osteoblast cells (cell line 1.19) were cultured under 1 % oxygen tension for 72 h. Cell cultures were divided into two groups. At the experimental side, low-level laser (808 nm, GaAlAs diode) was applied at 0, 24, and 48 h. After irradiation, each cell culture was incubated 24 h more under hypoxia. Total energy was 1.2, 2.4, and 3.6 J/cm(2), respectively. Non-irradiated cultures served as controls. Comparisons between the two groups were analyzed by t test; a p value <0.05 was considered statistically significant. Hypoxia resulted in a decrease in the expression of type I collagen, osteocalcin, and TGF-ß1 (p < 0.001, p < 0.001, and p < 0.01, respectively). Cell viability and BMP-2 expression were not decreased by hypoxic condition. On the other hand, LLLT on hypoxic-cultured osteoblast promoted the expression of BMP-2, osteocalcin, and TGF-ß1 (p < 0.05, p < 0.01, and p < 0.001, respectively). Cell proliferation was also increased time-dependently. However, hypoxia decreased in type I collagen expression (p < 0.001), and LLLT did not affect type I collagen expression in hypoxic-cultured osteoblasts. Furthermore, LLLT inhibited HIF-1 and Akt expression in hypoxic conditioned osteoblasts. We concluded that LLLT induces the expression of BMP-2, osteocalcin, and TGF- ß1 in 1 % hypoxic-cultured human osteoblasts.

Sim1a and Arnt2 contribute to hypothalamo-spinal axon guidance by regulating Robo2 activity via a Robo3-dependent mechanism.

Precise spatiotemporal control of axon guidance factor expression is a prerequisite for formation of functional neuronal connections. Although Netrin/Dcc- and Robo/Slit-mediated attractive and repulsive guidance of commissural axons have been extensively studied, little is known about mechanisms controlling mediolateral positioning of longitudinal axons in vertebrates. Here, we use a genetic approach in zebrafish embryos to study pathfinding mechanisms of dopaminergic and neuroendocrine longitudinal axons projecting from the hypothalamus into hindbrain and spinal cord. The transcription factors Sim1a and Arnt2 contribute to differentiation of a defined population of dopaminergic and neuroendocrine neurons. We show that both factors also control aspects of axon guidance: Sim1a or Arnt2 depletion results in displacement of hypothalamo-spinal longitudinal axons towards the midline. This phenotype is suppressed in robo3 guidance receptor mutant embryos. In the absence of Sim1a and Arnt2, expression of the robo3 splice isoform robo3a.1 is increased in the hypothalamus, indicating negative control of robo3a.1 transcription by these factors. We further provide evidence that increased Robo3a.1 levels interfere with Robo2-mediated repulsive axon guidance. Finally, we show that the N-terminal domain unique to Robo3a.1 mediates the block of Robo2 repulsive activity. Therefore, Sim1a and Arnt2 contribute to control of lateral positioning of longitudinal hypothalamic-spinal axons by negative regulation of robo3a.1 expression, which in turn attenuates the repulsive activity of Robo2.

Reduced cholesterol and triglycerides in mice with a mutation in Mia2, a liver protein that localizes to ER exit sites.

Through forward genetic screening in the mouse, a recessive mutation (couch potato, cpto) has been discovered that dramatically reduces plasma cholesterol levels across all lipoprotein classes. The cpto mutation altered a highly conserved residue in the Src homology domain 3 (SH3) domain of the Mia2 protein. Full-length hepatic Mia2 structurally and functionally resembled the related Mia3 protein. Mia2 localized to endoplasmic reticulum (ER) exit sites, suggesting a role in guiding proteins from the ER to the Golgi. Similarly to the Mia3 protein, Mia2's cytosolic C terminus interacted directly with COPII proteins Sec23 and Sec24, whereas its lumenal SH3 domain may facilitate interactions with secretory cargo. Fractionation of plasma revealed that Mia2(cpto/cpto) mice had lower circulating VLDL, LDL, HDL, and triglycerides. Mia2 is thus a novel, hepatic, ER-to-Golgi trafficking protein that regulates cholesterol metabolism.

Modified expression of aryl hydrocarbon receptor-related genes by deoxymiroestrol, a phytoestrogen, in mouse hepatocytes in primary culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Deoxymiroestrol (DM), a strong phytoestrogen from Pueraria candollei Wall. ex Benth. var. mirifica (family Leguminosae). This plant has long been used in traditional medicine for rejuvenation. MATERIALS AND METHODS: The expression of aryl hydrocarbon receptor-related genes in mouse hepatocytes in primary culture was quantified by real-time RT-PCR and hepatic microsomal P450 activity was assessed by using ethoxyresorufin O-dealkylation. RESULTS: The mRNA expression of the aryl hydrocarbon receptor (AhR), AhR nuclear translocator, and CYP1A1 was suppressed, whereas that of CYP1B1, estrogen receptor α (ERα), CYP2B9, and glutathione-S-transferase a2 (GSTa2) was increased. The effects of DM on the gene expression depended on treatment period and concentrations, and were similar to those of ß-estradiol (E2). DM and E2 at pharmacological concentrations had a marked synergistic effect on CYP1A1 expression after combined treatment with a typical CYP1 inducer, ß-naphthoflavone (ßNF), at the level of both transcription and enzymatic activity. DM enhanced the inducible mRNA expression of CYP1A1 and CYP1B1 similar to E2. Meanwhile, the expression of ERα mRNA was not affected by ßNF, which, on the contrary, completely eliminated the DM-induced mRNA expression of ERα, CYP2B9, and GSTa2. CONCLUSION: The findings that DM modified the expression of several metabolism-related genes suggest the need for caution when using health supplements having phytoestrogenic activity.

Curcumin decreases survival of Hep3B liver and MCF-7 breast cancer cells: the role of HIF.

BACKGROUND: Curcumin, a commonly used spice, affects the activities of cytokines, enzymes, and transcription factors that are linked to inflammation. Furthermore, curcumin has been assigned tumor growth inhibiting effects, possibly mediated by promoting hypoxia-inducible factor (HIF) degradation. HIFs are transcription factors that play a central role in the adaptation and response to low oxygen levels in metazoan cells. However, curcumin also exhibits properties of an iron chelator indicating its potential of inhibiting HIF-α prolyl hydroxylase (PHD) activity. METHODS: We were interested in clarifying these divergent actions of curcumin in due consideration of the effects on radio-therapy. Thus, concentration- and time-dependent effects of curcumin on HIF-α and -ß protein levels and activity in hepatoma and breast carcinoma cell cultures under normoxic and hypoxic conditions were studied. RESULTS: It was shown that HIF-1α accumulated in normoxia after the application of higher doses of the drug. Curcumin proved to lower HIF-1α and HIF-2α protein levels in hypoxia. HIF-1ß (ARNT; arylhydrocarbon nuclear translocator) protein levels and HIF transcriptional activity were reduced in normoxia and hypoxia after 4 h and 24 h incubation periods. Furthermore, curcumin treatment negatively impacted on clonogenic cell survival of Hep3B hepatoma and MCF-7 breast carcinoma cells. CONCLUSION: Effects of curcumin on cell growth and survival factor expression suggest its potential benefit in the treatment of cancer without a direct radiosensitizing influence of curcumin on these cells.

Curcumin inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon receptor nuclear translocator: a mechanism of tumor growth inhibition.

Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of HIF-1alpha and aryl hydrocarbon receptor nuclear translocator (ARNT), plays a key role in cell survival and angiogenesis in hypoxic tumors, and many efforts have been made to develop anticancer agents that target HIF-1alpha. However, although ARNT is also required for HIF-1 activity, ARNT has been disregarded as a therapeutic target. Curcumin is a commonly used spice and coloring agent with a variety of beneficial biological effects, which include tumor inhibition. In the present study, we tested the possibility that curcumin inhibits tumor growth by targeting HIF-1. The effects of curcumin on HIF-1 activity and expression were examined in cancer cell lines and in xenografted tumors. We found that curcumin inhibits HIF-1 activity and that this in turn down-regulates genes targeted by HIF-1. Moreover, of the two HIF-1 subunits, only ARNT was found to be destabilized by curcumin in several cancer cell types, and furthermore, ARNT expression rescued HIF-1 repression by curcumin. We also found that curcumin stimulated the proteasomal degradation of ARNT via oxidation and ubiquitination processes. In mice bearing Hep3B hepatoma, curcumin retarded tumor growth and suppressed ARNT, erythropoietin, and vascular endothelial growth factor in tumors. These results suggest that the anticancer activity of curcumin is attributable to HIF-1 inactivation by ARNT degradation.