Decreased genetic variation in metabolic variables of Litopenaeus vannamei shrimp after exposure to acute hypoxia

Concentration of proteins, carbohydrates, lactate, total lipids, acylglycerides, and carotenoids in shrimp were evaluated for their changes under acute hypoxia, and for their genetic variation under normoxic and hypoxic conditions. Proteins and lactate concentrations in muscle and hepatopancreas were significantly higher and carbohydrates in hepatopancreas were decreased in the hypoxic group. Family variances were significantly different only for proteins and carbohydrates in hepatopancreas in the normoxic group, indicating the existence of genetic variation for these traits. When family variances for each biochemical component were compared between normoxic and hypoxic groups, it was seen that most decreased. However, total variance was not significantly changed in response to hypoxia except for lactate (increased) and carotenoids (decreased) in hepatopancreas. The decrease in genetic variance without an increase in phenotypic variances in an acute response to hypoxia might be related to the known suppression of metabolic pathways that either use or produce ATP, which could result in a decreased expression of additive genes.     

Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions.

This study was performed to explore changes in gene expression as a consequence of exercise training at two levels of intensity under normoxic and normobaric hypoxic conditions (corresponding to an altitude of 3,850 m). Four groups of human subjects trained five times a week for a total of 6 wk on a bicycle ergometer. Muscle biopsies were taken, and performance tests were carried out before and after the training period. Similar increases in maximal O(2) uptake (8.3-13.1%) and maximal power output (11.4-20.8%) were found in all groups. RT-PCR revealed elevated mRNA concentrations of the alpha-subunit of hypoxia-inducible factor 1 (HIF-1) after both high- (+82.4%) and low (+78.4%)-intensity training under hypoxic conditions. The mRNA of HIF-1alpha(736), a splice variant of HIF-1alpha newly detected in human skeletal muscle, was shown to be changed in a similar pattern as HIF-1alpha. Increased mRNA contents of myoglobin (+72.2%) and vascular endothelial growth factor (+52.4%) were evoked only after high-intensity training in hypoxia. Augmented mRNA levels of oxidative enzymes, phosphofructokinase, and heat shock protein 70 were found after high-intensity training under both hypoxic and normoxic conditions. Our findings suggest that HIF-1 is specifically involved in the regulation of muscle adaptations after hypoxia training. Fine-tuning of the training response is recognized at the molecular level, and with less sensitivity also at the structural level, but not at global functional responses like maximal O(2) uptake or maximal power output.     

Molecular characterization of the glutamine synthetase gene in the Pacific oyster Crassostrea gigas: expression study in response to xenobiotic exposure and developmental stage

In this study, we characterized the full-length cDNA and genomic sequence of the gene encoding cytosolic glutamine synthetase (CgGSII) in the Pacific oyster, Crassostrea gigas. A phylogenetic analysis of GS sequences showed that CgGS clustered with the invertebrate group as expected. We analyzed the expression of mRNA CgGSII using RT-PCR to follow the expression of this gene in gills and digestive gland of oysters exposed, under experimental conditions, to hypoxia and to several contaminants (hydrocarbons and two pesticide treatments, glyphosate and a mixture of atrazine, diuron and isoproturon). We also investigated the expression of CgGSII in different developmental stages of C. gigas. Our results show that CgGSII expression was highly regulated in xenobiotic-exposed oysters compared to the control for all the treatments. Likewise, CgGSII expression was highly regulated according to the developmental stage of C. gigas. Finally, use of CgGSII as a possible marker to monitor xenobiotic exposure in disturbed ecosystems is discussed.     

Chronic hypoxia opposes pregnancy-induced increase in uterine artery vasodilator response to flow

We tested the hypotheses that pregnancy increases the uterine artery (UA) vasodilator response to flow and that this increase is impaired under conditions of chronic hypoxia (30 days, simulated elevation 3,960 m). UA were isolated from 24 normoxic or chronically hypoxic midpregnant guinea pigs and studied with the use of pressure myography. Normoxic pregnancy increased UA flow vasodilator response and protected against a rise in wall shear stress (WSS). Chronic hypoxia opposed these effects, prompting vasoconstriction at high flow and increasing WSS above levels seen in normoxic pregnant UA. The nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA) eliminated the pregnancy-associated increase in flow vasodilation in normoxic UA, suggesting that increased nitric oxide production was responsible. The considerable residual vasodilation after nitric oxide synthase and cyclooxygenase inhibition implicated endothelial-derived hyperpolarizing factor (EDHF) as an additional contributor to flow vasodilation. l-NNA increased flow vasodilation in UA from chronically hypoxic animals, suggesting that chronic hypoxia may have lowered EDHF or elevated peroxynitrite production. In conclusion, flow is an important physiological vasodilator for the acute and more chronic UA dimensional changes required to increase uteroplacental blood flow during normal pregnancy. Chronic hypoxia may be a mechanism that opposes the pregnancy-associated rise in UA flow vasodilation, thereby increasing the incidence of preeclampsia and intrauterine growth restriction at a high altitude.     

Genetic screening of new genes responsible for cellular adaptation to hypoxia using a genome-wide shRNA library

Oxygen is a vital requirement for multi-cellular organisms to generate energy and cells have developed multiple compensatory mechanisms to adapt to stressful hypoxic conditions. Such adaptive mechanisms are intricately interconnected with other signaling pathways that regulate cellular functions such as cell growth. However, our understanding of the overall system governing the cellular response to the availability of oxygen remains limited. To identify new genes involved in the response to hypoxic stress, we have performed a genome-wide gene knockdown analysis in human lung carcinoma PC8 cells using an shRNA library carried by a lentiviral vector. The knockdown analysis was performed under both normoxic and hypoxic conditions to identify shRNA sequences enriched or lost in the resulting selected cell populations. Consequently, we identified 56 candidate genes that might contribute to the cellular response to hypoxia. Subsequent individual knockdown of each gene demonstrated that 13 of these have a significant effect upon oxygen-sensitive cell growth. The identification of BCL2L1, which encodes a Bcl-2 family protein that plays a role in cell survival by preventing apoptosis, validates the successful design of our screen. The other selected genes have not previously been directly implicated in the cellular response to hypoxia. Interestingly, hypoxia did not directly enhance the expression of any of the identified genes, suggesting that we have identified a new class of genes that have been missed by conventional gene expression analyses to identify hypoxia response genes. Thus, our genetic screening method using a genome-wide shRNA library and the newly-identified genes represent useful tools to analyze the cellular systems that respond to hypoxic stress.     

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.     

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.     

δ-Opioid Receptor Activation Modified MicroRNA Expression in the Rat Kidney under Prolonged Hypoxia

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.     

Effects of PACAP and VIP on cyclic AMP formation in rat neuronal and astrocyte cultures under normoxic and hypoxic condition

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) concentration (0.001-1000 nM)-dependently stimulated cyclic AMP production in rat primary neuronal and glial cell (astrocyte) cultures. The actions of both peptides were much more pronounced in astrocytes than in neuronal cultures. Stimulatory effects of PACAP and VIP on cyclic AMP formation were significantly smaller in cell cultures subjected to 24h lasting hypoxic conditions, induced either chemically (100 microM cobalt chloride) or by low 3% oxygen hypoxia, compared to the normoxic condition (95% air and 5% CO(2)). This picture contrasted with the effects of forskolin that were similar under normoxic and hypoxic conditions. It is suggested that hypoxia leads to changes in PACAP- and VIP-driven cyclic AMP-dependent signaling in the rat brain by influencing molecular processes likely occurring at the level of receptor protein or receptor-Gs protein coupling.     

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure.

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.     

长时间低氧对大鼠颈动脉体培养细胞的细胞内pH和膜电位的影响

本文的目的是研究长时间低氧对离体培养的大鼠颈动脉体球细胞（ｇｌｏｍｕｓｃｅｌｌ）的影响。对实验组Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ（ＳＤ）大鼠,首先将其置于模拟５０００ｍ高度低氧环境的低压舱中饲养７—１０ｄ,然后麻醉动物,取出颈动脉体,将其分离成单个细胞和细胞群体（ｃｌｕｓｔｅｒｓ）。这些细胞在低氧条件（１１％Ｏ２,５％ＣＯ２,８４％Ｎ２）下培养２—３ｄ。取自正常ＳＤ大鼠的颈动脉体细胞被分为两组,分别将其培养在常氧（２１％Ｏ２,５％ＣＯ２,７４％Ｎ２）或低氧环境中。球细胞的细胞内ｐＨ（ｐＨｉ）和膜电位（ＭＰ）分别用Ｈ＋选择性微电极和常规微电极同时测量。结果表明：长时间低氧降低球细胞的ｐＨｉ,增加ＭＰ,其变化程度远远大于急性低氧的影响,而且当将细胞置于常氧中测量时其值不恢复。