Academic literature on the topic 'Energy metabolism; Hypoxia; Erythropoietin'

Recent studies have indicated that regulatory mechanisms underlying the oxygen-dependent expression of the haematopoietic growth factor erythropoietin are widely operative in non-erythropoietin-producing cells and are involved in the regulation of other genes. An important characteristic of this system is that the inducible response to hypoxia is mimicked by exposure to particular transition metals such as cobaltous ions, and by iron chelation. We have investigated the extent of operation of this system in the regulation of a range of genes concerned with energy metabolism. The effects of hypoxia (1% oxygen), cobaltous ions and desferrioxamine on gene expression in tissue-culture cells was studied using RNase protection assays. Hypoxia induced the expression of glucose transporters in an isoform-specific manner; GLUT-1 and GLUT-3 were induced by hypoxia, whereas expression of GLUT-2 was decreased. Isoenzyme-specific regulation by hypoxia was also observed for genes encoding phosphofructokinase, aldolase and lactate dehydrogenase. For all of these genes, responses to cobaltous ions and desferrioxamine correlated in both direction and magnitude with the response to hypoxia. In contrast, a reduction in mitochondrial transcripts was observed in hypoxia, but these changes were not mimicked by either cobaltous ions or desferrioxamine. These findings indicate that similarities with erythropoietin regulation extend to the oxygen-dependent regulation of genes encoding glucose transporters and glycolytic enzymes but not to the regulation of mitochondrial transcripts, and they show that in glucose metabolism regulation by this system is isoenzyme- or isoform-specific.

A great many aspects of the anatomy and physiology of large animals are constrained by the need to match oxygen supply to cellular metabolism and appear likely to involve the regulation of gene expression by oxygen. Some insight into possible underlying mechanisms has been provided by studies of erythropoietin, a haemopoietic growth factor which stimulates red cell production in response to hypoxia. Studies of hypoxia-inducible cis-acting sequences from the erythropoietin gene have led to the recognition of a widespread transcriptional response to hypoxia based on the activation of a DNA-binding complex termed hypoxia-inducible factor-1 (HIF-1). Perturbation of the transcriptional response by particular transition metal ions, iron chelators and certain redox-active agents have suggested a specific oxygen sensing mechanism, perhaps involving a haem protein in a flavoprotein/cytochrome system. In addition to erythropoietin, HIF-1-responsive genes include examples with functions in cellular energy metabolism, iron metabolism, catecholamine metabolism, vasomotor control and angiogenesis, suggesting an important role in the coordination of oxygen supply and cellular metabolism. In support of this, we have demonstrated an important role for HIF-1 in tumour angiogenesis. HIF-1 itself consists of a heterodimer of two basic-helix-loop-helix proteins of the PAS family, termed HIF-1alpha and HIF-1beta, although other closely related members of this family may also contribute to the response to hypoxia. We have fused domains of HIF-1 genes to heterologous transcription factors to assay for regulatory function. These experiments have defined several domains in HIF-1alpha which can independently confer the hypoxia-inducible property, and they suggest a mechanism of HIF-1 activation in which post-translational activation/derepression of HIF-1alpha is amplified by changes in HIF-1alpha abundance most probably arising from suppression of proteolytic breakdown. Pursuit of the mechanism(s) underlying these processes should ultimately lead to better definition of the oxygen-sensing process.

Hypoxia is a physiological cue that impacts diverse physiological processes, including energy metabolism, autophagy, cell motility, angiogenesis, and erythropoiesis. One of the key cell-autonomous effects of hypoxia is as a modulator of cell proliferation. For most cell types, hypoxia induces decreased cell proliferation, since an increased number of cells, with a consequent increase in O2 demand, would only exacerbate hypoxic stress. However, certain cell populations maintain cell proliferation in the face of hypoxia. This is a common pathological hallmark of cancers, but can also serve a physiological function, as in the maintenance of stem cell populations that reside in a hypoxic niche. This review will discuss major molecular mechanisms by which hypoxia regulates cell proliferation in different cell populations, with a particular focus on the role of hypoxia-inducible factors.

ABSTRACT In mammals, the liver integrates nutrient uptake and delivery of carbohydrates and lipids to peripheral tissues to control overall energy balance. Hepatocytes maintain metabolic homeostasis by coordinating gene expression programs in response to dietary and systemic signals. Hepatic tissue oxygenation is an important systemic signal that contributes to normal hepatocyte function as well as disease. Hypoxia-inducible factors 1 and 2 (HIF-1 and HIF-2, respectively) are oxygen-sensitive heterodimeric transcription factors, which act as key mediators of cellular adaptation to low oxygen. Previously, we have shown that HIF-2 plays an important role in both physiologic and pathophysiologic processes in the liver. HIF-2 is essential for normal fetal EPO production and erythropoiesis, while constitutive HIF-2 activity in the adult results in polycythemia and vascular tumorigenesis. Here we report a novel role for HIF-2 in regulating hepatic lipid metabolism. We found that constitutive activation of HIF-2 in the adult results in the development of severe hepatic steatosis associated with impaired fatty acid β-oxidation, decreased lipogenic gene expression, and increased lipid storage capacity. These findings demonstrate that HIF-2 functions as an important regulator of hepatic lipid metabolism and identify HIF-2 as a potential target for the treatment of fatty liver disease.

Tissue hypoxia not only occurs under pathological conditions but is also an important microenvironmental factor that is critical for normal embryonic development. Hypoxia-inducible factors HIF-1 and HIF-2 are oxygen-sensitive basic helix-loop-helix transcription factors, which regulate biological processes that facilitate both oxygen delivery and cellular adaptation to oxygen deprivation. HIFs consist of an oxygen-sensitive α-subunit, HIF-α, and a constitutively expressed β-subunit, HIF-β, and regulate the expression of genes that are involved in energy metabolism, angiogenesis, erythropoiesis and iron metabolism, cell proliferation, apoptosis, and other biological processes. Under conditions of normal Po2, HIF-α is hydroxylated and targeted for rapid proteasomal degradation by the von Hippel-Lindau (VHL) E3-ubiquitin ligase. When cells experience hypoxia, HIF-α is stabilized and either dimerizes with HIF-β in the nucleus to form transcriptionally active HIF, executing the canonical hypoxia response, or it physically interacts with unrelated proteins, thereby enabling convergence of HIF oxygen sensing with other signaling pathways. In the normal, fully developed kidney, HIF-1α is expressed in most cell types, whereas HIF-2α is mainly found in renal interstitial fibroblast-like cells and endothelial cells. This review summarizes some of the most recent advances in the HIF field and discusses their relevance to renal development, normal kidney function and disease.

Hypoxia-inducible factor-1α (HIF-1α), a member of the transcription family characterized by a basic helix-loop-helix (bHLH) domain and a PAS domain, regulates the transcription of hypoxia-inducible genes involved in erythropoiesis, vascular remodelling and glucose/energy metabolism. It contains bHLH/PAS domains in the N-terminal half, and a nuclear localization signal (NLS) and two transactivation domains (TADs) in the C-terminal half. It also has an oxygen-dependent degradation (ODD) domain, which is required to degrade HIF-1α protein by the ubiquitin—proteasome pathway. In this study, we identified a new alternatively spliced variant of human HIF-1α mRNA, which lacked both exons 11 and 12, producing a frame shift and giving a shorter form of HIF-1α. In the corresponding protein, a part of the ODD domain, both TADs and the C-terminal NLS motif were missing. Expression of endogenous HIF-1α variant protein was identified using immunoprecipitation and immunoblotting methods. The expressed HIF-1α variant exhibited neither the activity of transactivation nor hypoxia-induced nuclear translocation. In contrast with HIF-1α, the variant was strikingly stable in normoxic conditions and not up-regulated to such an extent by hypoxia, cobalt ions or desferrioxamine. It was also demonstrated that the HIF-1α variant competed with endogenous HIF-1α and suppressed HIF-1 activity, resulting in the down-regulation of mRNA expression of hypoxia-inducible genes. The association of the variant and arylhydrocarbon receptor nuclear translocator in the cytoplasm may be related to the inhibition of HIF-1 activity. It is assumed that this isoform preserves the balance between aerobic and anaerobic metabolism by counteracting the overaction of HIF-1α.

Abstract Insufficient oxygen availability under stress conditions including hypoxia and anemia is a major stimulus for stress erythropoiesis. Adenosine is known to be induced under hypoxia and energy depletion. Increased adenosine signaling via its specific receptors regulates multiple cellular functions including anti-inflamation, anti-vascular leakage and vasodilation. However, its function in stress erythropoiesis and underlying mechanisms are enigmatic. Among four adenosine receptors, we report that adenosine A2B receptor (ADORA2B) is expressed at a significant higher level in megakaryocyte-erythroid progenitor (MEP) compared to common pluoripotent progenitors (CMP) or granulocyte-erythroid progenitor (GMP) in undifferentiated human CD34+. To determine the function role of ADORA2B in stress erythropoiesis, we generated erythroid Adora2b specific knockouts by crossing Adora2bf/fmice with EpoR-Cre+mice. First, we demonstrated that EpoR specifically ablated ADORA2B gene only in MEP but not in CMP or GMP lineages. Next, we challenged EpoR-Cre+mice (control) and Adora2bf/fEpoR-Cre+ mice (erythroid specific ablation of Adora2b genes) with hypoxia. We discovered that genetic deletion of ADORA2B at MEP stage blocked erythroid vs myeloid commitment under hypoxia-induced stress erythropoiesis. Further metabolic profiling revealed that ADORA2B activation regulated erythroid lineage commitment by promoting glucose uptake and erythroid metabolic reprogramming channelling glucose metabolism toward the pentose phosphate pathway (PPP) rather than glycolysis to generate more ribose phosphate as well as facilitate glutamine uptake to serve as a nitrogen donor for de novo nucleotide biosynthesis. Meanwhile, ADORA2B-stimulated glutaminolysis increased TCA cycle intermediates and thus increased energy production under stress erythropoiesis. We further demonstrated that ADORA2B on MEP is also important for erythroid commitment in response to PHZ-induced mouse model. Followup studies revealed that HIF-1a is induced in erythroid progenitors in a ADORA2B-dependent manner and ablation of HIF-1a only in MEP but not in CMP or GMP attenuated erythroid lineage commitment in both hypoxia-induced and anemia-induced stress erythropoiesis mouse models. Moreover, we showed that ADORA2B-triggered metabolic reprogramming depended on HIF-1a-preferentially upregulated gene expression of transporters for glucose and glutamine and key enyzmes of PPP and glutaminolysis over glycolytic enzymes. Similar to mouse studies, in cultured Epo-stimulated human CD34+ hematopoietic stem progenitor cells, enhancing ADORA2B signaling induced gene expression of the transporters for glucose and glutamine, key enzymes of PPP and glutaminolysis over glycolysis and thus controlled the commitment to erythrioid versus myeloid lineage and in turn promoted erythroid colony formation including BFU-E, CFU-E versus CFU-GM. Further studies showed that inhibition of HIF-1a by Chrysin significantly attenuated ADORA2B activation-induced upregulation of gene expression of the transporters of glucose and glutamine, metabolic enzymes and thus reduces erythroic commitment and BFU-E and CFU-E in Epo-stimualted CD34+ HPSCs. Overall, using multidisciplinary approaches including mouse genetics, metabolomics, isotopically labelled glucose and glutamine flux analysis, human CD34+ HPSCs and pharmacological studies, we provide both mouse and human evidence that ADORA2B is a missing cofactor controlling erythroid lineage commitment in stress erythropoiesis via HIF-1a-dependent upregulation of key genes to promote metabolic reprogramming. These findings add significant new insights to erythroid commitment and immediately provide new strategies for regulating stress erythropoiesis. Disclosures Nemkov: Omix Technologies inc: Equity Ownership.

Hypoxia-inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis that controls the expression of genes encoding proteins that play key roles in angiogenesis, erythropoiesis, and glucose/energy metabolism. The stability of the HIF-1α subunit is regulated by ubiquitination and proteasomal degradation. In aerobic cells, O2-dependent prolyl hydroxylation of HIF-1α is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, which then recruits the Elongin C ubiquitin-ligase complex. SSAT2 (spermidine/spermine N-acetyltransferase-2) binds to HIF-1α and promotes its ubiquitination/degradation by stabilizing the interaction of VHL and Elongin C. Treatment of cells with heat shock protein HSP90 inhibitors induces the degradation of HIF-1α even under hypoxic conditions. HSP90 competes with RACK1 for binding to HIF-1α, and HSP90 inhibition leads to increased binding of RACK1, which recruits the Elongin C ubiquitin-ligase complex to HIF-1α in an O2-independent manner. In this work, we demonstrate that SSAT1, which shares 46% amino acid identity with SSAT2, also binds to HIF-1α and promotes its ubiquitination/degradation. However, in contrast to SSAT2, SSAT1 acts by stabilizing the interaction of HIF-1α with RACK1. Thus, the paralogs SSAT1 and SSAT2 play complementary roles in promoting O2-independent and O2-dependent degradation of HIF-1α.

Abstract Chuvash polycythemia (CP) is a monogenic disorder characterized by an upregulated hypoxic response at normoxia. Homozygosity for the VHLR200W mutation leads to decreased degradation of the a subunits of hypoxia inducible factor (HIF)-1 and HIF-2 by the hypomorphic variant of VHL, the principal negative regulator of HIFs. An array of HIF-regulated genes, including the principal regulators of erythropoiesis and iron metabolism, have altered expression. Previous studies in CP using peripheral blood mononuclear cells (PBMCs), a heterogeneous mixture of cells, identified significant gene expression differences from wild type controls, but the cell linage specificity of these hypoxia-regulated genes remains unknown. In this study, we systematically analyzed gene expression by unbiased deep RNA sequencing in purified reticulocytes, granulocytes and platelets of CP and control individuals living at the same altitude of ~200 meters. Thirty-one samples passed quality control: reticulocytes from 10 individuals (5 VHLR200W homozygotes and 5 wild type controls), platelets from 7 individuals (3 VHL homozygotes and 4 controls) and granulocytes from 14 individuals (5 VHL homozygotes, 1 heterozygote and 8 controls). The samples were analyzed for expression differences (VHL homozygote/heterozygote versus wild type) in each cell type. We found abundant gene expression differences in these three cell types. The differential genes detected in the three cell types showed no more overlap than expected by random (Binomial test P=1 for all pairings of the three cell types), suggesting cell lineage specificity of hypoxic gene expression in CP. At 5% false discovery rate (FDR, i.e., <5 false positives in 100 detected genes), 737 of 7238 analyzed genes (10%) were altered in the reticulocytes of VHLR200W homozygotes, 271 up-regulated and 466 down-regulated. The up-regulated genes were enriched in pathways of "Telomere maintenance", "Oxidative phosphorylation", "Parkinson's disease", "Ribosome", "Systemic lupus erythematosis", "Apoptosis", "Influenza Infection", "Metabolism of proteins", "Huntington's disease", and "Integration of energy metabolism". The down-regulated genes were enriched in pathways of "Cell cycle" and "Ubiquitin mediated proteolysis". At 5% FDR, 3646 of 12,334 analyzed genes (30%) were differentially expressed in the platelets of VHLR200W homozygotes, 1830 up-regulated and 1816 down-regulated. The up-regulated genes were enriched in pathways of "Lysosome" and "Signaling in immune system". The down-regulated genes were enriched in pathways of "Hemostasis" and "Opioid signaling". At 5% FDR, 3423 of 11,274 analyzed genes (30%) were differentially expressed in the granulocytes of VHLR200W homozygotes, 1490 up-regulated and 1933 down-regulated. The up-regulated genes were enriched in pathways of "Gene Expression", "Metabolism of nucleotides", "Metabolism of proteins", and "Aminoacyl-tRNA biosynthesis". The down-regulated genes were highly enriched in immune pathways including "Chemokine signaling pathway", "Fc gamma R-mediated phagocytosis", "Endocytosis", "Neurotrophin signaling pathway", "B cell receptor signaling pathway", "Fc epsilon RI signaling pathway", as well as several cancer-related pathways. The relative abundance of alternative transcript isoforms differed in VHLR200W homozygotes relative to wild type controls for many genes in these three blood lineages indicating a role for HIFs in regulation of mRNA processing. At 1% FDR, 3121 of 12,514 analyzed genes (25%) in platelets, 233 of 7342 analyzed genes (3%) in reticulocytes, and 224 of 11,306 analyzed genes (2%) in granulocytes contained alternative exon(s) in VHLR200W homozygotes compared to wild type controls. In conclusion, we report marked gene expression variation in three blood cell lineages from individuals with CP, the first described disorder of congenital augmentation of hypoxia sensing. Dysregulated expression of genes not known to be transcriptionally regulated by HIFs may be due to the well-known but poorly defined effects of HIFs on epigenetic regulation of transcription. Our results demonstrate extensive cell lineage specificity in blood gene expression variations induced by augmented signaling of HIFs caused by the VHLR200W mutation. This provides novel insights to our understanding of clinical complications in CP and more broadly of hypoxic gene regulation. Disclosures No relevant conflicts of interest to declare.

Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators that play crucial roles in angiogenesis, erythropoiesis, energy metabolism, and cell fate decisions. The group of enzymes that can catalyse the hydroxylation reaction of HIF-1 is prolyl hydroxylase domain proteins (PHDs). PHD inhibitors (PHIs) activate the HIF pathway by preventing degradation of HIF-αvia inhibiting PHDs. Osteogenesis and angiogenesis are tightly coupled during bone repair and regeneration. Numerous studies suggest that HIFs and their target gene, vascular endothelial growth factor (VEGF), are critical regulators of angiogenic-osteogenic coupling. In this brief perspective, we review current studies about the HIF pathway and its role in bone repair and regeneration, as well as the cellular and molecular mechanisms involved. Additionally, we briefly discuss the therapeutic manipulation of HIFs and VEGF in bone repair and bone tumours. This review will expand our knowledge of biology of HIFs, PHDs, PHD inhibitors, and bone regeneration, and it may also aid the design of novel therapies for accelerating bone repair and regeneration or inhibiting bone tumours.

This thesis is concerned with a very common disorder of iron homeostasis: iron deficiency. The specific focus is the manner in which iron deficiency influences physiological responses to hypoxia in humans. This work is predicated on observations made over many decades in vitro and in vivo, suggesting that variations in the bioavailability of iron have important consequences for certain biological processes known to depend on oxygen availability. Three separate but related studies together form the basis for this thesis. The first two, Study A and Study B, adopt a similar approach in recruiting healthy volunteers who differ according to iron status, yielding iron-deficient and iron-replete groups in both cases. In Study A, the behaviour of the pulmonary circulation is investigated during a sustained hypoxic exposure, before and after an intravenous infusion of iron. In Study B, skeletal muscle metabolism is explored, both at the level of high-energy phosphate metabolism and the integrated physiological responses to exercise on a cycle ergometer. In the third study, Study C, a different approach is taken, recruiting patients with chronic obstructive pulmonary disease (COPD), and exploring the prevalence and associations of iron deficiency in this condition. Chapters 2 and 3 describe experiments using sustained hypoxia in a normobaric chamber, during which the pulmonary circulation is assessed non-invasively using Doppler echocardiography. These reveal augmented hypoxic pulmonary vasoconstriction (HPV) in iron-deficient individuals, who also exhibit greater sensitivity to the effects of an infusion of intravenous iron. Additionally, the way in which certain circulating mediators important for iron haemostasis change over the course of these hypoxic exposures, and how iron status influences these responses, is explored. Chapter 4 reports the findings of experiments using 31P-magnetic resonance spectroscopy and cardiopulmonary exercise testing, which demonstrate abnormal whole-body metabolism in iron-deficient individuals during large muscle-mass exercise, despite the absence of a clear defect in mitochondrial oxidative phosphorylation. Intravenous iron is found to have significant effects to alter the lactate threshold in healthy individuals, but the effects are more striking in iron-deficient individuals. Collectively, these experiments imply that iron deficiency promotes a more glycolytic phenotype. Chapter 5 explores iron deficiency in COPD, a condition in which pulmonary vascular disease, hypoxia and skeletal muscle dysfunction coexist, and examines some of the difficulties in assessing iron status in the setting of a chronic inflammatory disorder. Iron deficiency is found to be common, and unexpectedly associated with significantly more severe hypoxaemia, in patients with COPD. Possible reasons for these findings, and their clinical implications, are considered. Chapter 6 provides a summary of the main conclusions to be drawn from the studies presented in this thesis.

Understanding the respiratory physiology and energy metabolism is important for establishing the dissolved oxygen (DO) requirements of freshwater mussels, determining the metabolite(s) indicative of environmental stress, and interpreting environmental conditions based on physiological indicators of mussels. Three studies were undertaken to address these questions. The first study was conducted with seven mussel species collected from reservoir and riverine habitats. The two objectives were to determine the diurnal patterns of valve gaping of freshwater mussels from different habitats, and to monitor heart rate changes of a mussel species that exhibited the diurnal gaping. The results showed that night gaping is evident for the mussels collected from lentic areas, but not for those collected from lotic areas. The heart rate of Pyganodon grandis increased when they gaped. The second study was conducted with nine species of freshwater mussels from different habitats. The three objectives were to determine the patterns (i.e., regulator and conformer) of oxygen consumption (OC) rate under declining DO, evaluate the effects of temperature on ability to regulate OC under declining DO, and finally to use this information to predict DO criteria for maintaining freshwater mussels in captivity. The results showed that mussels living in the habitats subjected to low DO have a better ability to regulate the OC and were more tolerant to hypoxia. The third study assessed three mussel species from different habitats with different abilities to regulate OC under low DO. The two objectives in this study were to identify the energetic metabolite changes under different DO levels and air exposure for the three species, and to determine the appropriate tissue(s) and metabolite(s) to use for estimating the stress in mussels. The results showed that different biochemical strategies were used by Villosa iris, Elliptio complanata, and Pyganodon grandis. Villosa iris had the lowest anaerobic capacity. Elliptio complanata had a more efficient anaerobic metabolism, and P. grandis reduced energy metabolism under low DO and air exposure. posterior adductor muscle, gill and mantle were good tissues for evaluating hypoxic stress. The mantle tissue had the highest glycogen store and was the best tissue for non-lethal study of physiological condition.
Ph. D.

INTRODUÇÃO: Apesar do amplo uso da medida da saturação central de oxigênio como meta terapêutica em pacientes de terapia intensiva, diferenças absolutas em relação à saturação venosa mista existem. As causas desses gradientes, bem como o comportamento das mesmas ao longo do tempo nas doenças graves não foram completamente esclarecidas. Considerando que a maioria das intervenções atualmente empregadas para reverter desequilíbrios de oxigenação tecidual presentes nos pacientes graves é direcionada, direta ou indiretamente, ao coração; a situação particular de elevada taxa de extração de oxigênio basal do miocárdio e a ausência de ferramentas de monitorização do impacto miocárdico dessas intervenções, o presente estudo, diante da possibilidade teórica da participação do efluente do seio coronário nessas diferenças centrais para pulmonares, não só para a saturação de oxigênio (SO2), analisou o comportamento da SO2, pressão parcial de dióxido de carbono (PCO2), lactato e glicose, em diferentes modelos de hipóxia e compartimentos vasculares, com ênfase na avaliação do metabolismo miocárdico e seu impacto nos gradientes centrais para pulmonares. MÉTODOS: 37 porcos, machos, com peso em torno de 35 Kg, sedados e ventilados mecanicamente, foram estudados após indução de quatro diferentes tipos de injúria hipóxica (hipóxia anêmica, estagnante, hipóxica e sepse), sendo 8 animais por grupo e mais 5 controles. Além de variáveis hemodinâmicas e de oxigenação, SO2, PCO2, lactato e glicose foram medidos, em diferentes momentos, em 9 compartimentos vasculares distintos, incluindo o seio coronário (artéria femoral, veia cava inferior e superior, átrio direito, ventrículo direito, artéria pulmonar, veia suprahepática direita e veia porta). PRINCIPAIS RESULTADOS: As concentrações de O2, lactato e glicose no efluente do seio coronário apresentaram padrões distintos entre os grupos: troca de substrato energético de lactato por glicose nos grupos hipóxia hipóxica e anêmica, aumento no consumo de ambos os substratos na sepse e ausência de tendência clara no grupo da hipóxia estagnante. Os gradientes de PCO2 entre seio coronário e artéria femoral mantiveram-se estáveis com tendência de alargamento tardio em todos os modelos. Na análise dos demais gradientes regionais, o seio coronário apresentou a menor SO2 do organismo, as menores concentrações de lactato, os maiores níveis de PCO2, e esses padrões variaram ao longo do tempo. Mesma tendência evolutiva foi percebida entre os gradientes centrais para pulmonares de O2, lactato, CO2 e glicose e a medida desses mesmos parâmetros no seio coronário. CONCLUSÕES: As concentrações de O2, lactato e glicose no efluente do seio coronário estão relacionadas ao tipo de injúria e não apenas à disponibilidade de substrato energético. Padrões de gravidade, comuns às fases tardias de todos os grupos, puderam ser identificados: qualquer redução da SO2 coronariana; incremento do metabolismo de glicose; produção de lactato pelo miocárdio e surgimento de igualdade ou inferioridade dos níveis da PCO2 coronariana em relação aos valores dos demais compartimentos vasculares do organismo (independentemente da trajetória). A tendência dos gradientes de PCO2 transmiocárdicos seguiu a do débito cardíaco e, certamente, deve refletir fluxo coronariano. A análise dos gradientes regionais se mostrou capaz de permitir a avaliação de contextos orgânicos regionais específicos, como na avaliação do metabolismo hepático, na qual foi possível demonstrar que na hipóxia, a produção de glicose hepática é mantida até o óbito, diferentemente do padrão descrito para a sepse. Por fim, com a análise dos dados do grupo sepse, foi possível demonstrar que: a) assim como os gradientes centrais para pulmonares de SO2 e lactato já foram descritos, gradientes de glicose e PCO2 também existem; b) o seio coronário participa, significativamente, na formação desses gradientes de lactato, CO2 e glicose
INTRODUCTION: Despite of the widespread use of the central venous oxygen saturation measurement as a therapeutic goal in critically ill patients, absolute differences between this measurement and the mixed venous oxygen exist. Causes of these differences, as well the behavior of these gradients in critical illness, are not completely understood. Considering current therapeutic interventions aimed to reverse tissue oxygenation impairment are mediated by increases in cardiac output; the particular scenario in which the heart is not physiologically able to further increase oxygen extraction and the absence of tools to monitoring the myocardium impact of those interventions, the present study, facing the theoretical possibility of the coronary sinus effluent participation in those central to mixed venous differences, has analyzed the oxygen saturation (SO2), carbon dioxide partial pressure (PCO2), lactate and glucose concentrations behaviors over time, in different models of tissue hypoxia and in different vascular sites. Emphasis on the myocardial energetic metabolism and its impact over central to mixed venous gradients was placed. METHODS: 37 pigs, males, weighting about 35 Kg, sedated and mechanically ventilated, were studied after induction of four different hypoxic injury models (sepsis, and anemic, stagnant, hypoxic hypoxia), eight for group and five controls. In addition to hemodynamic and oxygen variables, SO2, PCO2, lactate and glucose were measured in different phases, in 9 distinct vascular sites, including coronary sinus (femoral artery, inferior and superior vena cava, right atria, right ventricle, pulmonary artery, right suprahepatic vein and portal vein). MAIN RESULTS: Concentrations of O2, lactate and glucose in the coronary sinus effluent presented distinctive patterns among groups: shift from lactate to glucose consumption in hypoxic hypoxia and anemic hypoxia groups, increase in both glucose and lactate consumption in sepsis and absence of clear trend in stagnant hypoxia group. PCO2 gradients from systemic artery to coronary sinus presented late enlargement trend in all groups. In the regional gradients analysis comparisons, coronary sinus presented the lowest SO2, the lowest lactate concentrations, the highest PCO2 levels, and these patterns changed over time. Similar evolution trends were observed between central to mixed venous O2, PCO2, lactate and glucose gradients and the same parameters measured in coronary sinus. CONCLUSIONS: Different concentrations of O2, PCO2, lactate and glucose in coronary sinus are related to the type of hypoxic injury and not only to energetic substrate availability. Severity-related patterns, common to all groups in late phases, were identified: any reduction of coronary SO2, shift to glucose consumption, net lactate myocardial production and equality or inferiority of PCO2 levels related to other vascular compartments (independently of trend). Trends in transmyocardial PCO2 gradients followed cardiac output ones and, certainly, should mirror coronary blood flow. Regional gradients analysis showed suitable to explore specific regional metabolic settings, as in the described example of liver metabolism, in which production of glucose were maintained in all phases by this organ in hypoxic hypoxia groups, differently from the impaired production described in literature for sepsis. At last, data from sepsis group have showed: a) as to the previously known central to mixed venous SO2 and lactate gradients, PCO2 and glucose gradients also exist; b) coronary sinus has participated significantly in formation of central to mixed venous lactate, PCO2 and glucose gradients

Ce travail etait destine a etudier les effets de l'hypoxie chronique (fo2 = 10% ; pendant 3 semaines), sur le metabolisme energetique et la fonction du muscle cardiaque de rat. - une etude in situ de la fonction contractile des ventricules droit (vd) et gauche (vg), grace a l'introduction d'un microcapteur de pression millar dans les cavites ventriculaires, a revele une legere depression de la fonction du vg, alors que l'ensemble des variables fonctionnelles enregistrees au niveau du vd (pression developpee, dp/dt max et min) sont fortement augmentees. Ces variations sont associees a une augmentation de la masse du ventricule droit. L'evaluation de la fonction ventriculaire gauche sur un modele de cur isole perfuse ne revele aucune limitation des capacites fonctionnelles dans le groupe hypoxique, la fonction apparaissant meme augmentee lors de stimulations inotropes par le calcium et l'isoprenaline. - les principales observations sur le metabolisme energetique des curs isoles de rats hypoxiques sont les suivantes: (1) la balance energetique myocardique est preservee en hypoxie chronique et le taux d'atp intracellulaire est identique a celui des curs temoins. (2) la consommation d'oxygene myocardique du cur isole est globalement reduite, pour une activite contractile egale ou superieure a celle des curs temoins. (3) la production de lactate des curs isoles, perfuses en presence de glucose 11 mm, est augmentee. (4) la variation des concentrations des composes phosphoryles (phosphate inorganique pi, phosphocreatine pcr, et atp), detectee par la spectroscopie rmn du p-31, lors de situations de perturbations de la balance energetique (stimulations inotropes ou hypoxie aigue), est attenuee. De plus, apres -stimulation (isoprenaline 10#-#6 m), on observe une augmentation de la vitesse de resynthese de la pcr dans les curs d'animaux adaptes a l'hypoxie. (5) l'addition d'hexanoate dans le milieu de perfusion modifie profondement le comportement metabolique des curs de rats temoins, alors qu'elle est sans effet dans le cas du groupe hypoxique. - l'etude des caracteristiques de la respiration mitochondriale, sur une preparation de fibres cardiaques permeabilisees, a permis de montrer, pour les curs de rats exposes a l'hypoxie, une reduction de l'effet stimulant de la creatine sur la vitesse de respiration. Cette donnee traduit une modification des systemes de canalisation de l'energie dans la cellule myocardique, entre les sites d'utilisation (atpases myofibrillaires) et de production (mitochondries) = la navette creatine-phosphocreatine. Les resultats obtenus evoquent une plus grande sollicitation de la voie glycolytique, associee a une efficacite amelioree des processus oxydatifs apres acclimatation a l'hypoxie. Ces donnees conduisent a l'hypothese d'une reorganisation des systemes cellulaires responsables du transfert de l'energie dans le cardiomyocyte

L'etude de l'echange chimique par resonance magnetique nucleaire (rmn) est un outil unique pour suivre in vivo des cinetiques enzymatiques a l'etat stationnaire. Nous proposons d'utiliser la spectroscopie d'echange bidimensionnelle (2d exsy) du 31p pour etudier le metabolisme energetique des pointes de racines de mais excisees et perfusees. Le systeme de perfusion utilise est decrit. Nous mesurons d'abord les vitesses de relaxation longitudinale des aimantations 31p in vivo, puisque l'exsy en depend de facon critique. Nous perfectionnons la methode de mesure, et nous montrons par des mesures precises comment elles dependent des parametres physiologiques (croissance, oxygenation, temperature). L'exsy 31p permet d'identifier 10 reactions enzymatiques unidirectionnelles : synthese d'atp par phosphorylation oxydative, hydrolyse d'atp, formation en deux etapes de phosphoenolpyruvate a partir de 3-phosphoglycerate lors de la glycolyse et vice versa, tous les echanges entre hexoses phosphate (uridinediphosphoglucose, glucose-1-phosphate, glucose-6-phosphate et fructose-6-phosphate) lors du metabolisme des sucres. Les parametres experimentaux sont optimises en fonction des vitesses de relaxation precedemment mesurees afin d'obtenir le meilleur rapport signal-sur-bruit : temps de melange, temps de repetition, effet noe1h-31p et filtrage adapte de la dimension indirecte pendant l'acquisition. Nous abordons la quantification des flux d'echange a partir de spectres exsy. Celle-ci necessite un developpement theorique de l'echange chimique hors-equilibre, mais en etat stationnaire avec un flux net, tel que nous l'observons in vivo. Nous utilisons des courbes de croissance pour verifier le modele et nous demontrons la possibilite de quantifier des echanges a partir d'un seul spectre en utilisant des parametres exterieurs (vitesses de relaxation ou aimantations a l'equilibre). Enfin, les resultats des mesures de flux d'echange chimique en fonction de parametres physiologiques (oxygenation, temperature) sont presentes et la question d'une enzymologie in vivo est discutee.

碩士
中臺科技大學
藥物科技研究所
104
Abstract Fenofibrate (FF) is a lipid-lowering agent that can suppress tumorigenesis in many cancers. In the present study, we demonstrated that treatment of human U87MG glioma cells with FF decreased the protein levels of HIF-1α under normoxia and hypoxia conditions. The suppression of HIF-1α was reversed by pretreatment with proteasome inhibitor. The reduction of HIF-1α level was associated with decreased expression levels of glucose transporter Glut-1, hexokinase-2 (HK-2) and lactate dehydrogenase A (LDHA). On the other hand, treatment with FF also decreased the expression of pyruvate dehydrogenase kinase-1 (PDK-1) and pyruvate dehydrogenase phosphorylation (p-PDH), which reactivated mitochondria flux of acetyl CoA and TCA cycle. However, FF decreased mitochondria membrane potential presumably by reactive oxygen species (ROS) induced mitochondrial damage. These results suggest that FF may not only suppress the Warburg effect and reactive mitochondria oxidative phosphorylation but also increase ROS accumulation which leads to mitochondrial damage and subsequent cell death. Key word: Fenofibrate (FF), Warburg effect , HIF-1α, energy metabolism, ROS

The kidneys are responsible for maintaining the constant chemical composition of body fluids. This process begins with high-pressure filtration in specialized glomerular capillaries located in the renal cortex. The pressure filtration produces an ultrafiltrate of plasma made up of the water and smaller molecules. As the fluid passes along the renal tubules, water, electrolytes, and non-electrolytes are reabsorbed in the required amounts by a process of selective reabsorption. Some active secretion of unwanted substances also occurs. Following this reabsorption the remaining tubule fluid is passed to the renal pelvis and then down the ureters to the bladder for storage until voided. The effort involved in all this is quite staggering. One-fifth of the daily cardiac output, about 1400 litres of whole blood, including 840 litres of plasma, passes through the kidneys. Of the 540 litres of plasma (the effective renal plasma flow) passing each day through the glomerular capillaries, one-fifth of the plasma water and small molecules are freely filtered at the glomeruli to produce about 170–180 litres per day of glomerular filtrate for the renal tubules. Since typically only 1–2 litres of urine are passed each day (that is about 1 ml per minute) 99 % of the initial filtrate is reabsorbed as the fluid passes along the renal tubules. In oliguria, urine production can fall below 300ml per day, as in severe dehydration. In situations causing polyuria, urine output can rise to several litres per day, or more, as in excessive water intake or untreated diabetes mellitus or diabetes insipidus. The kidney’s main functions are osmoregulation, acid–base balance, and the excretion of waste products of metabolism, notably urea. Osmoregulation is mostly under endocrine control by antidiuretic hormone and the renin–angiotensin–aldosterone system. Acid–base balance is driven mainly by the carbon dioxide partial pressure in renal tubule cells, although kidneys work together with lungs and the control of breathing in overall acid–base balance. The kidney has important endocrine functions. It is the source of erythropoietin, the hormone that stimulates red blood cell production in hypoxia.

Hypoglycaemia is defined as a blood glucose level less than 2.6 mmol/l. This is based on the consistent impairment of central nervous system function observed in subjects when blood glucose levels are below this (1). Glucose homeostatic mechanisms should maintain blood glucose level to preserve cognitive function. Hypoglycaemia triggers protective glucose homeostatic mechanisms and persistent hypoglycaemia is the result of a failure of homeostasis. This is a medical emergency with serious short- and long-term consequences, which result from a reduced supply of glucose to the brain. Recurrent and persistent hypoglycaemia does cause significant morbidity and death due to brain damage. In an adult, after recovery of glucose levels, neurological impairment usually recovers over minutes to hours. In children, the duration of hypoglycaemia leading to permanent damage is not known, but is presumed to depend on the age of the child, the frequency of hypoglycaemia, the degree and the rapidity of the fall in glucose, concurrent circumstances such as infection, trauma and hypoxia, the degree of resilience of the brain tissue at the current stage of development. and the energy demands of the particular parts of the brain. The reasons for the increased sensitivity in children appear to relate to the higher energy requirements and immaturity of the homeostatic mechanisms of the brain. In congenital hyperinsulinism of infancy (CHI) the rates of severe neurological impairment remain high at 20–50%, permanent neurological impairment with damage occurring mainly in the cerebral cortex, hippocampus, and caudate putamen. Appropriate long term management of hypoglycaemia requires the correct diagnosis, and this depends on obtaining ‘critical blood and urine samples’ during a hypoglycaemic episode. In the first 48 h of life 20% of normal full–term infants have a blood glucose level <2.6 mmol/l (2), after this it is relatively uncommon in infancy and childhood with the incidence of various underlying diagnoses varying with age. The causes of hypoglycaemia can be classified into five groups: ◆ excess insulin (or insulin-like factors) for the given circumstances ◆ lack of one or more of the counter regulatory hormones (cortisol, growth hormone) ◆ disturbance of intermediate metabolism causing impairment of gluconeogenesis and/or glycogenolysis ◆ disturbance of fat breakdown or ketone body formation or utilization ◆ lack of nutrient sufficient for current energy demands

Tissue hypoxia is a common and important feature of rapidly growing malignant tumors and their metastases. Tumor cells mainly depend on energy production thru anaerobic glycolysis rather than aerobic oxidative phosphorylation in mitochondria [1]. Intervening the tumor metabolic process via thermal energy infusion is worthy attempting. And hyperthermia, mildly elevated local temperature above the body temperature, is one of such kind. Previously, after being heated for a short period of time, tumor glucose and lactate level increased and ATP level decreased, which suggested energy metabolism was modified following hyperthermia through increased ATP hydrolysis, intensified glycolysis and impaired oxidative phosphorylation [2]. Many researchers designed experiments to determine thermal dose in hyperthermia [3], but few focused on the relationship between tumor and energy, especially for a long-term local hyperthermia treatment. One clinical trial indicated the effective long-term hyperthermo-therapy for maintaining performance status, symptomatic improvement, and prolongation of survival time in patients with peritoneal dissemination [4].