Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor

Hypoxia sensing is the generic term for pO₂-sensing in humans and other higher organisms. These cellular responses to pO₂ are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the factor inhibiting HIF (FIH-1), which couples O₂-activation to the hydroxylation of the hypoxia inducible factor α (HIFα). Uncoupled O₂-activation by human FIH-1 was studied by exposing the resting form of FIH-1 (αKG + Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λ_max = 583 nm) arising from enzyme auto-hydroxylation of Trp²⁹⁶, forming an Fe(III)-O-Trp²⁹⁶ chromophore [Y.-H. Chen, L.M. Comeaux, S.J. Eyles, M.J. Knapp, Chem. Commun. (2008), doi:10.1039/B809099H]; the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue (oxo)Met²⁷⁵. The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however, product yield was saturable with increasing [αKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate + Fe)FIH-1, or by glycerol addition to (αKG + Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1 (αKG + Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O₂-activation by loss of an H₂O ligand from the metal center.     

Protein modulation in mouse heart under acute and chronic hypoxia

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1α), energy balance (AMPK), mitochondrial biogenesis (PGC-1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1α, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.     

Measurement of alpha-keto acids in plasma using an amino acid analyzer

A method for measuring keto acid concentrations in physiological fluids using an amino acid analyzer was developed. After preliminary deproteinization and removal of amino acids, reduction with sodium cyanoborohydride at 105 degrees C resulted in efficient conversion of the keto acids to their corresponding amino acids. In applying the technique to plasma samples, the use of MeOH for deproteinization was necessary to avoid the large losses of keto acids that occurred during precipitation of proteins with perchloric acid. The method was used to follow plasma ketoisocaproate concentrations in rat plasma after administration of leucine, and was sufficiently sensitive to detect concomitant changes in other branched-chain keto acid concentrations.     

Dynamic changes in expression of heme oxygenases in mouse heart and liver during hypoxia

Heme oxygenase cleaves heme to form biliverdin, carbon monoxide (CO), and iron, and consists of two structurally related isozymes, HO-1 and HO-2. HO-2 is also known as a potential oxygen sensor. Here we show that the relative CO content in arterial blood, which reflects the total amount of endogenous heme degradation, dynamically changes in mice during acclimatization to normobaric hypoxia (10% O2), with the two peaks at 1 day and 21 days of hypoxia. The expression levels of HO-1 and HO-2 proteins were decreased by 20% and 40%, respectively, in the mouse liver at 7 days of hypoxia, which returned to the basal levels at 14 days. On the other hand, HO-1 and HO-2 proteins were increased 2-fold and 1.3-fold, respectively, in the heart at 28 days of hypoxia. Thus, hypoxia induces or represses the expression of HO-1 and HO-2 in vivo, depending on cellular microenvironments.     

Effects of hypoxia on plasma amino acids of fetal sheep

Summary. Secondary amino acid disturbances from circulatory responses during hypoxia may cause problems in interpreting plasma amino acid profiles of sick babies investigated for possible inherited defects. Systematic studies to characterise them are difficult in man. We investigated the effects of hypoxia on plasma amino acids by studying 9 late gestation fetal sheep in utero during 11 one hour episodes of moderately severe isocapnic hypoxia. In 6 experiments, maternal plasma amino acids were also monitored. Fourteen fetal plasma amino acids increased significantly, with the largest proportionate changes in alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, ornithine and lysine. Maternal amino acids did not increase. Probable explanations were reflex peripheral vasoconstriction in skeletal muscle beds and decreased hepatic blood flow. The findings extend our knowledge of the fetal response to hypoxic stress, demonstrate the importance of skeletal muscle in branched-chain amino acid metabolism, and should help with interpretation of postnatal plasma amino acid disturbances. Received January 29, 1999, Accepted February 22, 1999     

Activation of hepatic branched-chain alpha-keto acid dehydrogenase complex by tumor necrosis factor-alpha in rats

Tumor necrosis factor-alpha (TNFalpha) promotes oxidation of branched-chain amino acids (BCAA). BCAA catabolism is regulated by branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, which is regulated by phosphorylation-dephosphorylation of the E1alpha subunit at Ser293. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined the effects of TNFalpha administration on hepatic BCKDH complex and kinase in rats. Rats were intravenously administered with 25 or 50 microg TNFalpha/kg body weight 4 h prior to sacrifice. The TNFalpha treatment at both doses elevated the activity state (percentage of the active form) of BCKDH complex from 22% to 69% and 86%, respectively, and the amount of phospho-Ser293 on the E1alpha subunit in each group of rats corresponded inversely to the activity state of BCKDH complex. The TNFalpha treatment of rats significantly decreased the activity as well as the bound form of BCKDH kinase. These results suggest that the decrease in the bound form of kinase is involved in the mechanism responsible for TNFalpha-induced activation of the BCKDH complex.     

Hypergravity induces expression of cyclooxygenase-2 in the heart vessels

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme for prostaglandin biosynthesis, is induced by various stimuli including mechanical stress and plays important roles in pathophysiological conditions. For example, gravitational stress has been shown to induce expression of COX-2 in bone tissues, which is essential for bone homeostasis. To investigate whether COX-2 is induced by gravitational loading in other tissues than bone, we exposed mice to hypergravity at 2G and 3G for 4 h. We demonstrate here that COX-2 is induced in the mouse heart vessels by hypergravity. Moreover, hypoxia-inducible factor (HIF)-1alpha and its downstream genes such as inducible nitric oxide synthase, vascular endothelial growth factor, and heme oxygenase-1 were induced in the heart simultaneously, while none of these genes were induced in the COX-2(-/-) mouse heart. Therefore, COX-2 induced in the heart helps protect the heart function against hypoxia under hypergravity condition through HIF-1alpha induction.     

Fluorescent analysis of alpha-keto acids in serum and urine by high-performance liquid chromatography

A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases.     

Suppression of hypoxia inducible factor-1alpha (HIF-1alpha) by YC-1 is dependent on murine double minute 2 (Mdm2)

Inhibition of HIF-1alpha activity provides an important strategy for the treatment of cancer. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) has been identified as an anti-HIF-1alpha drug in cancer therapy with unclear molecular mechanism. In the present study, we aimed to investigate the effect and mechanism of YC-1 on HIF-1alpha in a hepatocellular carcinoma cell line under hypoxic condition, which was generated by incubating cells with 0.1% O(2). The phenotypic and molecular changes of cells were determined by cell proliferation assay, apoptosis assay, luciferase promoter assay, and Western blot analysis. YC-1 arrested tumor cell growth in a dose-dependent manner, whereas it did not induce cell apoptosis. Hypoxia-induced upregulation of HIF-1alpha was suppressed by YC-1 administration. YC-1 inhibited HIF-1alpha protein synthesis under normoxia and affected protein stability under hypoxia. YC-1 suppressed the expression of total and phosphorylated forms of murine double minute 2 (Mdm2), whereas this inhibitory effect was blocked by overexpression of Mdm2. In conclusion, YC-1 suppressed both protein synthesis and stability of HIF-1alpha in HCC cells, and its inhibitory effects on HIF-1alpha were dependent on Mdm2.     

Effects of hypoxia on contents of essential elements in pika and rat heart

The effects of hypoxia on the levels of essential macroelements and trace elements (K, Na, Ca, Mg, Cu, Zn, Fe, and Mn) in the heart muscles of Wistar rats and plateau pikas (Ochotona curzoniae) were studied by atomic absorption spectrometry. Unlike the rat, the plateau pika is tolerant to hypoxia. The levels of K, Na, and the trace element Mn were not significantly changed in rat or pika hearts after exposure to hypoxia for 1, 10, or 25 d at simulated altitudes of 5000 and 7000 m. Other minerals (Ca, Mg, Cu, Zn, and Fe) were significantly affected by hypoxia and the levels followed different time-courses under different hypoxic regimes in these two animals. There were marked differences between the rat and pika in myocardial accumulation of essential elements such as Ca, which was increased to high levels in the rat but not affected in the pika. The results suggest that hypoxia affects animal physiological mechanisms by regulating the levels of essential elements.     

Effects of endogenous carbon monoxide on collagen synthesis in pulmonary artery in rats under hypoxia

To study the role of endogenous carbon monoxide (CO) in collagen metabolism during hypoxic pulmonary vascular remodeling, a total of 18 Wistar rats were used in the study and they were randomly divided into three groups: hypoxia group (n = 6), hypoxia with zinc protoporphyrin-IX (ZnPP-IX) group (n = 6) and control group (n = 6). The measurement of mean pulmonary artery pressure (mPAP) and carboxyhemoglobin (HbCO) formation in lung tissue homogenates was measured. A morphometric analysis of pulmonary vessels was performed, in which the percentage of muscularized arteries (MA); partially muscularized arteries (PMA) and nonmuscularized arteries (NMV) in small and median pulmonary vessels, relative medial thickness (RMT) and relative medial area (RMA) of pulmonary arteries were analyzed. Collagen type I and III and transforming growth factor-beta3 (TGF-beta3) expressions were detected by immunohistochemical assay. The expressions of procollagen type I and III and TGF-beta3 mRNA were detected by in situ hybridization. The results showed that ZnPP-IX significantly increased mPAP and markedly decreased HbCO formation in lung tissue homogenates in rats under hypoxia (P < 0.01). In the hypoxia rats treated with ZnPP-IX, the percentage of muscularized arteries of small and median pulmonary vessels was obviously increased, and RMT and RMA of intra-acinar muscularized pulmonary arteries were markedly increased compared with hypoxic rats. Ultrastructural changes, such as hyperplasia and hypertrophy of endothelial cells (ECs) and smooth muscle cells (SMCs) and the increased number of SMCs in synthetic phenotype were found in intra-acinar pulmonary muscularized arteries of hypoxic rats treated with ZnPP-IX. Meanwhile, ZnPP-IX promoted the expression of collagen type I and III and TGF-beta3 protein in pulmonary arteries of rats under hypoxia (P < 0.01). Furthermore, ZnPP-IX elevated obviously the expressions of procollagen type I and III mRNA, and TGF-beta3 mRNA in pulmonary arteries of rats under hypoxia (P < 0.01). The results of this study suggested that ZnPP-IX played an important role in promoting collagen synthesis in pulmonary arteries of rats with hypoxic pulmonary structural remodeling by increasing the expression of TGF-beta3. The above findings also suggested a possible role of endogenous CO in the pathogenesis of chronic hypoxic pulmonary hypertension.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Diurnal variations of amino acids and organic acids in xylem fluid from Lagerstroemia indica: an endogenous circadian rhythm

Diurnal variations in the concentrations of major organic compounds occurred in xylem fluid extracted from Lagerstroemia indica L. The concentration of amino acids and the N/C ratio was at a maximum and that of organic acids was at a minimum between 1230 and 2030 h. Since the concentrations of total organic nitrogen, total amino acids and most individual amino acids (but not organic acids or sugars) were also proportional to xylem tension two experiments were performed to discern whether variations in chemistry were a consequence of diurnal changes in moisture stress. In the first experiment, L. indica , exposed to variable levels of moisture stress during midday, manifested an increase in organic acids and a reduction in the N/C ratio. In the second experiment, chemical profiles of xylem fluid were collected and compared for plants exposed to a natural photoperiod, constant darkness or continuous light at noon and midnight. After 1 day amino acids increased in concentration during midday for all treatments; the variation was greatest (10-fold) for plants in constant darkness where xylem tension varied from 0.20 to 0.25 MPa. Only plants exposed to continuous light lost a diurnal rhythm after 3 days. Thus, the circadian rhythm was endogenous, terminated in continuous light and was not mediated by changes in moisture stress. Glutamine accounted for most of the diurnal variation in total amino acids, organic nitrogen and the N/C ratio in xylem fluid.     

Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.     

Age-related compensatory activation of pyruvate dehydrogenase complex in rat heart

Mitochondrial uptake and beta-oxidation of long-chain fatty acids are markedly impaired in the aging rat heart. While these alterations would be expected to adversely affect overall pyridine nucleotides, NADH levels do not change significantly with age. This conundrum suggests that specific compensatory mechanisms occur in the aging heart. The comparison of cardiac pyruvate dehydrogenase complex (PDC) kinetics in 4- and 24- to 28-month-old F344 rats revealed a 60% significant increase in V(max) with no change in PDC expression, and a 1.6-fold decrease in the Michaelis constant (K(m)) in old compared to young rats. The observed kinetic adjustments were selective to PDC, as neither the V(max) nor K(m) of citrate synthase changed with age. PDC kinase-4 mRNA levels decreased by 57% in old vs young rat hearts and correlated with a 45% decrease in PDC phosphorylation. We conclude that PDC from old rat hearts catabolizes pyruvate more efficiently due to an adaptive change in phosphorylation.     

Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10–18 areas, and increased in only 1–3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.     

Storage and oxidation of long-chain fatty acids in the C57/BL6 mouse heart as measured by NMR spectroscopy

Triglyceride turnover in the isolated C57/BL6 mouse heart was measured by dynamic 13C edit-(1)H observe NMR and the rate of fatty acid oxidation was determined by 13C NMR isotopomer analysis. In the presence of a physiological mixture of substrates, energy was produced in the citric acid cycle by oxidation of long-chain fatty acids (18%), ketones (34%), lactate (24%), pyruvate (7%), and other sources (17%). Exogenous fatty acids appeared in the triglyceride pool at 0.24 micromol/g dry wt/min, similar to the rate of oxidation of long-chain fatty acids, 0.16 micromol/g dry wt/min. Isoproterenol decreased the rate of de novo triglyceride synthesis and increased the rate of fatty acid oxidation.     

Regulation of the rate of synthesis of nitric oxide by Mg(2+) and hypoxia. Studies in rat heart mitochondria

Summary.  In isolated rat heart mitochondria, L-arginine is oxidized by a nitric oxide synthase (mtNOS) achieving maximal rates at 1 mM L-arginine. The NOS inhibitor N^G-nitro-L-arginine methyl ester (NAME) inhibits the increase in NO production. Extramitochondrial free magnesium inhibited NOS production by 59% at 3.2 mM. The mitochondrial free Mg²⁺ concentration increased to different extents in the presence of L-arginine (29%), the NO donor (S-nitroso-N-acetylpenicillamine) (105%) or the NOS inhibitors L-NAME (48%) or N^G-nitro-L-arginine methyl ester, N^G-monomethyl-L-arginine (L-NMMA) (53%). Under hypoxic conditions, mtNOS activity was inhibited by Mg²⁺ by up to 50% after 30 min of incubation. Reoxygenation restored the activity of the mtNOS to pre-hypoxia levels. The results suggest that in heart mitochondria there is an interaction between Mg²⁺ levels and mtNOS activity which in turn is modified by hypoxia and reoxygenation. Received April 2, 2001 Accepted September 21, 2001