Increased expression of HIF-1alpha, nNOS, and VEGF in the cerebral cortex of anemic rats

This study tested the hypothesis that specific hypoxic molecules, including hypoxia-inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF), are upregulated within the cerebral cortex of acutely anemic rats. Isoflurane-anesthetized rats underwent acute hemodilution by exchanging 50% of their blood volume with pentastarch. Following hemodilution, mean arterial pressure and arterial Pa(O(2)) values did not differ between control and anemic rats while the hemoglobin concentration decreased to 57 +/- 2 g/l. In anemic rats, cerebral cortical HIF-1alpha protein levels were increased, relative to controls (1.7 +/- 0.5-fold, P < 0.05). This increase was associated with an increase in mRNA levels for VEGF, erythropoietin, CXCR4, iNOS, and nNOS (P < 0.05 for all), but not endothelial NOS. Cerebral cortical nNOS and VEGF protein levels were increased in anemic rats, relative to controls (2.0 +/- 0.2- and 1.5 +/- 0.4-fold, respectively, P < 0.05 for both). Immunohistochemistry demonstrated increased HIF-1alpha and VEGF staining in perivascular regions of the anemic cerebral cortex and an increase in the number of nNOS-positive cerebral cortical cells (3.2 +/- 1.0-fold, P < 0.001). The nNOS-positive cells costained with the neuronal marker, Neu-N, but not with the astrocytic marker glial fibrillary acidic protein (GFAP). These nNOS-positive neurons frequently sent axonal projections toward cerebral blood vessels. Conversely, VEGF immunostaining colocalized with both neuronal (NeuN) and astrocytic markers (GFAP). In conclusion, acute normotensive, normoxemic hemodilution increased the levels of HIF-1alpha protein and mRNA for HIF-1-responsive molecules. nNOS and VEGF protein levels were also increased within the cerebral cortex of anemic rats at clinically relevant hemoglobin concentrations.     

Hyperbaric oxygen preconditioning alleviates myocardial ischemic injury in rats

It has been shown that after ischemia-reperfusion, application of hyperbaric oxygen (HBO) reduces cardiac injury. In this study we tested the hypothesis that HBO preconditioning reduces injury to the ischemic myocardium. One hundred and eight adult male Sprague-Dawley rats (250-280 g) were randomly divided into four groups: normoxia + sham surgery (CS), normoxia + permanent occlusion of the left anterior descending (LAD) coronary artery (CMI), HBO preconditioning + sham surgery (HS), and HBO preconditioning + permanent LAD occlusion (HMI). Rats receiving HBO preconditioning were intermittently exposed to 100% O(2) at 2.5 atmosphere absolute (ATA) for 60 min, twice daily for 2 days followed by 12 hrs of recovery in room air prior to the myocardial ischemic insult induced by LAD ligation. Rats in the normoxia group were time-matched with the HBO group and maintained under normoxic conditions prior to LAD occlusion. At 3 and 7 days after LAD occlusion, heart function parameters were measured by inserting a catheter into the left ventricle, infarct size was calculated using the method of TTC staining, myocardial capillary density was determined by immunohistochemical staining with a monoclonal anti-CD(31)/PECAM-1 antibody, and VEGF protein level was determined by Western blot analysis. At 3 days after LAD ligation, the infarct size of the HMI group was significantly smaller than that of the CMI group (26 +/- 2.5% vs. 38 +/- 3%, P < 0.05). The heart function parameters including left ventricular systolic pressure (LVSP), +dP/dt(max) and -dP/dt(max) were significantly improved in the HMI group compared to the CMI group at 3 and 7 days after LAD occlusion. Capillary density and VEGF protein levels were significantly increased in the ischemic myocardium pre-exposed to HBO. We conclude that HBO preconditioning alleviates myocardial ischemia in rat model.     

Intrinsic and extrinsic erythropoietin enhances neuroprotection against ischemia and reperfusion injury in vitro

This study was designed to investigate the neuroprotective effect of intrinsic and extrinsic erythropoietin (EPO) against hypoxia/ischemia, and determine the optimal time-window with respect to the EPO-induced neuroprotection. Experiments were conducted using primary mixed neuronal/astrocytic cultures and neuron-rich cultures. Hypoxia (2%) induces hypoxia-inducible factor-1alpha (HIF-1alpha) activity followed by strong EPO expression in mixed cultures and weak expression in neuron-rich cultures as documented by both western blot and RT-PCR. Immunoreactive EPO was strongly detected in astrocytes, whereas EPOR was only detected in neurons. Neurons were significantly damaged in neuron-rich cultures but were distinctly rescued in mixed cultures. Application of recombinant human EPO (rhEPO) (0.1 U/mL) within 6 h before or after hypoxia significantly increased neuronal survival compared with no rhEPO treatment. Application of rhEPO after onset of reoxygenation achieved the maximal neuronal protection against ischemia/reperfusion injury (6 h hypoxia followed 24 h reoxygenation). Our results indicate that HIF-1alpha induces EPO gene released by astrocytes and acts as an essential mediator of neuroprotection, prove the protective role of intrinsic astrocytic-neuronal signaling pathway in hypoxic/ischemic injury and demonstrate an optimal therapeutic time-window of extrinsic rhEPO in ischemia/reperfusion injury in vitro. The results point to the potential beneficial effects of HIF-1alpha and EPO for the possible treatment of stroke.     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

Prevention of HIF-1 activation and iNOS gene targeting by low-dose cadmium results in loss of myocardial hypoxic preconditioning in the rat

This study aimed to underline the interaction between hypoxia-inducible factor-1 (HIF-1) and the inducible nitric oxide synthase (iNOS) gene in vivo and their contribution to the delayed myocardial preconditioning induced by acute intermittent hypoxia (IH) in the rat using chromatin immunoprecipitation and pharmacological inhibition by low-dose cadmium. Langendorff-perfused hearts of Wistar rats exposed to normoxia or IH 24 h earlier were submitted to global ischemia and reperfusion. Effects of iNOS inhibition by aminoguanidine (100 microM) before ischemia or of low-dose injection of cadmium chloride (1 mg/kg) before normoxia or IH were tested. Myocardial HIF-1 and iNOS quantification and in vivo chromatin immunoprecipitation of HIF-1 bound to the iNOS gene promoter were performed. IH-induced delayed cardioprotection resulted in an improvement in coronary flow and functional recovery at reperfusion and a decrease in infarct size. Myocardial HIF-1 activity was increased with resulting targeting of the iNOS gene. Aminoguanidine abolished the cardioprotective effects of IH. Cadmium chloride treatment before IH prevented myocardial HIF-1 activation (72.3 +/- 4.0 vs. 42.1 +/- 9.7 arbitrary units after cadmium chloride; P < 0.05), targeting of the iNOS gene, iNOS expression, and preconditioning (infarct size: 15.9 +/- 5.6 vs. 30.1 +/- 5.4% after cadmium chloride; P < 0.05). This study is the first to demonstrate the interaction of HIF-1 with the myocardial iNOS gene in situ after hypoxic preconditioning. Prevention of HIF-1 activation and iNOS gene targeting by a single low dose of cadmium abolished the delayed cardioprotective effects, bringing insight into the cardiovascular consequences of cadmium exposure.     

Effect of combined therapy with hyperbaric oxygen and antioxidant on infarct volume after permanent focal cerebral ischemia

The aim of the present study was to evaluate the efficiency of combination of hyperbaric oxygen (HBO) and an antioxidant on permanent focal cerebral ischemia. Male Wistar rats underwent permanent middle cerebral artery occlusion (MCAO). Then, animals were randomly assigned to one of four groups: the control group (n=9) received no treatment, HBO group (n=9) was treated for 90 min at 2.5 absolute atmosphere for 3 days, the U-74389G group (n=8) received single U-74389G injection (3 mg/kg), the HBO + U-74389G group (n=8) received both HBO and U-74389G treatments. Treatments were initiated within the first 10 min after MCAO. After 3 days, the infarct volumes in rat brains were measured. The infarct ratios were 25.6+/-6.5 % for the control group, 21.9+/-6.4 % for the HBO group, 15.7+/-5.7 % for U-74389G group and 12.5+/-3.8 % for HBO + U74389G group. The infarct volumes were significantly reduced in rats treated with U-74389G (p<0.05) and combination therapy (p<0.05). HBO failed to reduce infarct volume significantly. We concluded that 1) U-74389G is more beneficial than HBO on permanent MCAO in rats, and 2) a combined therapy failed to significantly improve infarct volume more than either single treatment.     

Hypoxia-inducible factor 1 contributes to N-acetylcysteine’s protection in stroke

Stroke is a leading cause of adult morbidity and mortality with very limited treatment options. Evidence from preclinical models of ischemic stroke has demonstrated that the antioxidant N-acetylcysteine (NAC) effectively protects the brain from ischemic injury. Here, we evaluated a new pathway through which NAC exerted its neuroprotection in a transient cerebral ischemia animal model. Our results demonstrated that pretreatment with NAC increased protein levels of hypoxia-inducible factor-1α (HIF-1α), the regulatable subunit of HIF-1, and its target proteins erythropoietin (EPO) and glucose transporter (GLUT)-3, in the ipsilateral hemispheres of rodents subjected to 90 min middle cerebral artery occlusion (MCAO) and 24 h reperfusion. Interestingly, after NAC pretreatment and stroke, the contralateral hemisphere also demonstrated increased levels of HIF-1α, EPO, and GLUT-3, but to a lesser extent. Suppressing HIF-1 activity with two widely used pharmacological inhibitors, YC-1 and 2ME2, and specific knockout of neuronal HIF-1α abolished NAC’s neuroprotective effects. The results also showed that YC-1 and 2ME2 massively enlarged infarcts, indicating that their toxic effect was larger than just abolishing NAC's neuroprotective effects. Furthermore, we determined the mechanism of NAC-mediated HIF-1α induction. We observed that NAC pretreatment upregulated heat-shock protein 90 (Hsp90) expression and increased the interaction of Hsp90 with HIF-1α in ischemic brains. The enhanced association of Hsp90 with HIF-1α increased HIF-1α stability. Moreover, Hsp90 inhibition attenuated NAC-induced HIF-1α protein accumulation and diminished NAC-induced neuroprotection in the MCAO model. These results strongly indicate that HIF-1 plays an important role in NAC-mediated neuroprotection and provide a new molecular mechanism involved in the antioxidant’s neuroprotection in ischemic stroke.     

Nitric oxide and carbon dioxide in neurotoxicity induced by oxygen under pressure

Hyperbaric oxygen (HBO2) causes CO2 retention in the brain that leads to the increase in cerebral blood flow (CBF) by poorly understood mechanisms. We have tested the hypothesis that NO is implicated in CBF-responses to hypercapnia under hyperoxic conditions. Alert rats were exposed to HBO2 at 5 ata and blood flow in the striatum measured by H2 clearance every 10 min. Acetazolamide, the inhibitor of carbonic anhydrase, was used to increase brain PCO2. CBF responses to acetazolamide administration (30 mg/kg, i.p.) were assessed in rats breathing air at 1 ata or oxygen at 5 ata with and without NOS inhibition (L-NAME, 30 mg/kg, i.p.). In rats breathing air, acetazolamide increased CBF by 34 +/- 7.4% over 30 min and by 28 +/- 12% over 3 hours while NOS inhibition with L-NAME attenuated acetazolamide-induced cerebral vasodilatation. HBO2 at 5 ata reduced CBF during the first 30 min hyperoxia, after that CBF increased by 55 +/- 19% above pre-exposure levels. In acetazolamide-treated animals, no HBO, induced vasoconstricton was observed and striatal blood flow increased by 53 +/- 18% within 10 min of hyperbaric exposure. After NOS inhibition, cerebral vasodilatation in response to acetazolamide during HBO2 exposure was significantly attenuated. The study demonstrates that NO is implicated in acetazolamide (CO2)-induced cerebral hyperemia under hyperbaric oxygen exposure.     

Differential effects of HIF-1 inhibition by YC-1 on the overall outcome and blood-brain barrier damage in a rat model of ischemic stroke

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system.     

Transcription activator protein 1 mediates alpha- but not beta-adrenergic hypertrophic growth responses in adult cardiomyocytes

Acute systemic hypoxia induces delayed cardioprotection against ischemia (I)-reperfusion (R) injury via inducible nitric oxide synthase (iNOS)-dependent mechanism. Because CoCl2 is known to elicit hypoxia-like responses, we hypothesized that this chemical would mimic the delayed preconditioning effect in the heart. Adult male mice were pretreated with CoCl2 or saline. The hearts were isolated 24 h later and subjected to 20 min of global I and 30 min of R in Langendorff mode. Myocardial infarct size (% of risk area; mean +/- SE, n=6-8/group) was reduced in mice pretreated with 30 mg/kg CoCl2 (16.1 +/- 3.1% vs. 27.6 +/- 3.3% with saline control; P <0.05) without compromising postischemic cardiac function. Higher doses of CoCl2 failed to induce similar protection. Electrophoretic mobility gel shift assay demonstrated significant enhancement in DNA binding activity of hypoxia-inducible factor 1alpha (HIF-1alpha) and activator protein 1 (AP-1) in nuclear extracts from CoCl2-treated hearts. Activation of HIF-1alpha and AP-1 was evident at 30 min and sustained for the next 4 h after CoCl2 injection. In contrast, CoCl2-induced protection was independent of NF-kappaB activation because no DNA binding or p65 translocation was observed in nuclear extracts. Also, CoCl2-induced cardioprotection was preserved in p50 subunit NF-kappaB-knockout (KO) mice (11.1 +/- 3.0% vs. 25.1 +/- 5.0% in saline-treated p50-KO mice; P <0.05). The infarct-limiting effect of CoCl2 was absent in iNOS-KO mice (20.9 +/- 3.0%). We conclude that in vivo administration of CoCl2 preconditions the heart against I/R injury. The delayed protective effect of CoCl2 is achieved through a distinctive signaling mechanism involving HIF-1alpha, AP-1, and iNOS but independent of NF-kappaB activation.     

Ginkgolides mimic the effects of hypoxic preconditioning to protect C6 cells against ischemic injury by up-regulation of hypoxia-inducible factor-1 alpha and erythropoietin

Hypoxic preconditioning can play a significant neuroprotective role. However, it has not been employed clinically because of safety concerns. To find a safer preconditioning stimulus that is both practical and effective, we investigated whether ginkgolides are capable of preconditioning as hypoxia to protect C6 cells against ischemic injury. We demonstrated that both ginkgolides (37.5microg/mL) and hypoxia (1% O(2) for 16h) can significantly increase cell viabilities and expression of phosphorylated glycogen synthase kinase (p-GSK), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) in ischemic cells. The inhibitors of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3'-kinase (PI3K) significantly but not completely reduced the enhanced expression of these proteins and cell viabilities induced by ginkgolides and hypoxic preconditioning. These indicated that ginkgolides could mimic hypoxic preconditioning by increasing expression of HIF-1alpha as well as its target protein EPO and that the ginkgolides and hypoxic preconditioning role might be partly mediated by the activation of the p42/p44-mitogen-activated protein kinase and phosphatidylinositol 3'-kinase/AKT/glycogen synthase kinase 3beta pathways. The similar tendency in the changes of protein expression, cell viabilities and responses to MAPK or PI3K inhibitors of the cells treated with ginkgolides and hypoxia suggests that ginkgolides and hypoxic preconditioning might operate by similar mechanisms. The findings also imply that ginkgolides might have the potential for clinical use to prevent injury in high-risk conditions.     

Oxygen treatment after experimental hypoxia-ischemia in neonatal rats alters the expression of HIF-1alpha and its downstream target genes.

Recently, mounting evidence has emerged to suggest that hyperbaric oxygenation (HBOT)-induced neuroprotection after experimental global ischemia and subarachnoid hemorrhage entails a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Therefore, the purpose of this study was to test the hypothesis that oxygen-induced neuroprotection after neonatal hypoxia-ischemia involves alterations in the expression of HIF-1alpha. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O(2) at 37 degrees C). Pups were then treated with HBOT (2.5 ATA) or normobaric oxygenation treatment (NBOT) for 2 h. The expression and phosphorylation status of HIF-1alpha was evaluated at intervals up to 24 h after the insult, as was the expression of glucose transporter (GLUT)-1, GLUT-3, lactate dehydrogenase (LDH), aldolase (Ald), and p53. The protein-protein interaction of HIF-1alpha and p53 was also examined. An elevated expression of HIF-1alpha, GLUT-1, GLUT-3, Ald, and LDH was observed after the insult. An increase in the dephosphorylated form of HIF-1alpha was followed by an increase in the association of HIF-1alpha with p53 and an increase in p53 levels. Both HBOT and NBOT reduced the elevated expression of HIF-1alpha and decreased its dephosphorylated form. Furthermore, both treatments promoted a transient increase in the expression of GLUT-1, GLUT-3, LDH, and Ald, while decreasing the HIF-1alpha-p53 interaction and decreasing the expression of p53. Therefore, the alteration of the HIF-1alpha phenotype by a single oxygen treatment may be one of the underlying mechanisms for the observed oxygen-induced neuroprotection seen when oxygen is administered after a neonatal hypoxic-ischemic insult.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

Hypoxic preconditioning increases iron transport rate in astrocytes

The mechanisms involved in the neuroprotection induced by hypoxic preconditioning (HP) have not been fully elucidated. The involvement of hypoxia-inducible factor-1 alpha (HIF-1alpha) in such neuroprotection has been confirmed. There is also evidence showing that a series of genes with important functions in iron metabolism, including transferrin receptor (TfR1) and divalent metal transporter 1 (DMT1), are regulated by HIF-1alpha in response to hypoxia in extra-neural organs or cells. We therefore hypothesized that HP is able to affect the expression of iron metabolism proteins in the brain and that changes in these proteins induced by HP might be associated with the HP-induced neuroprotection. We herein demonstrated for the first time that HP could induce a significant increase in the expression of HIF-1alpha as well as iron uptake (TfR1 and DMT1) and release (ferroportin1) proteins, and thus increase tansferrin-bound iron (Tf-Fe) and non-transferrin-bound iron (NTBI) uptake and iron release in astrocytes. Moreover, HP could lead to a progressive increase in cellular iron content. We concluded that HP has the ability to increase iron transport speed in astrocytes. Based on our findings and the importance of astrocytes in neuronal survival in hypoxic/ischemic preconditioning, we proposed that the increase in iron transport rate and cellular iron in astocytes might be one of the mechanisms associated with the HP-induced neuroprotection. We also demonstrated that ferroportin1 expression was significantly affected by HIF-1alpha in astrocytes, implying that the gene encoding this iron efflux protein might be a hypoxia-inducible one.     

高压氧预处理对大鼠局灶性脑缺血再灌注损伤的保护作用

目的:研究高压氧预处理对大鼠脑缺血再灌注损伤的保护作用。方法:36只SD大鼠随机分为假手术组、模型组及高压氧预处理组,每组12只。高压氧预处理组大鼠在造模前5天给予高压氧预处理。采用线栓法建立大鼠脑缺血再灌注模型,观察高压氧预处理对脑缺血再灌注损伤大鼠神经功能缺损评分、脑梗死面积的影响,检测大鼠缺血脑组织COX-2 mRNA和蛋白的表达以及IL-1β、TNF-α、MDA的含量。结果:高压氧预处理可明显改善脑缺血再灌注大鼠神经功能缺损评分,减少脑梗死面积,降低COX-2m RNA和蛋白表达量,抑制IL-1β、TNF-α的表达,降低MDA水平。结论:高压氧预处理对大鼠脑缺血再灌注损伤具有明显的保护作用,其机制可能与抑制IL-1β、TNF-α、COX-2的表达以及减弱脂质过氧化反应有关。     

Ethane production rate in vivo is reduced with dietary restriction

Dietary restriction without malnutrition prolongs life and has a beneficial effect on age-related diseases and metabolic derangements. To test the effect of food restriction on ethane production rate, ethane exhalation was measured in rats with partial food restriction. Ethane production rate in room air in rats fed 60% of food consumed by ad libitum-fed animals for 2 wk was significantly reduced (3.50 +/- 0.25 vs. 5.21 +/- 0.34 pmol.min-1.100 g body wt-1, P less than 0.01). In 100% oxygen, ethane production in food-restricted rats was not different from that of ad libitum-fed rats (21.81 +/- 1.25 vs. 19.57 +/- 1.89 pmol.min-1.100 g-1). Fifteen hours of fasting compared with ad libitum feeding reduced ethane production modestly in room air (4.37 +/- 0.45 vs. 5.21 +/- 0.34 pmol.min-1.100 g-1) and more significantly in 100% oxygen (12.37 +/- 0.78 vs. 19.57 +/- 1.89 pmol.min-1.100 g-1). Thus, in 100% oxygen, 15 h of fasting, compared with ad libitum feeding, resulted in an approximately 40% decrease in ethane production rate. It is concluded that short-term food restriction significantly reduces ethane exhalation rate in rats when measured in room air.     

Chronic in vivo hypoxia in various organs: hypoxia-inducible factor-1alpha and apoptosis

We studied the in vivo persistence of hypoxia-inducible factor-1alpha (HIF-1alpha), main transducer of hypoxia, the differential response in organs exposed to the same degree of hypoxemia and the relationship with apoptosis. We measured HIF-1alpha (immunohistochemistry peroxidase and Western blot) and apoptosis (TUNEL) in heart, liver, kidney, gastrocnemius, and brain of rats exposed to chronic normobaric hypoxia (10% O2) or normoxia (21% O2) for 2 weeks. Despite same arterial O2 pressure and increased hemoglobin concentration (219 +/- 5 vs. 124 +/- 4 g/L), the organs responded differently. While marked in brain, muscle, and kidney cortex, HIF-1alpha was undetectable in heart and liver. In kidney medulla, HIF-1alpha was high in both normoxia and hypoxia. By contrast, apoptosis was marked in heart, slight in kidney medulla, and undetectable in other organs. We conclude that the HIF-1alpha response to chronic hypoxia can be a sustained phenomenon, but not in all organs, and that apoptosis responds differently from HIF-1alpha.