Metabolic Hypoxia Research Articles

Metabolic programs drive function of therapeutic NK cells in hypoxic tumor environments.

Limited oxygen (hypoxia) in solid tumors poses a challenge to successful immunotherapy with natural killer (NK) cells. NK cells have impaired cytotoxicity when cultured in hypoxia (1% oxygen) but not physiologic (>5%) or atmospheric oxygen (20%). We found that changes to cytotoxicity were regulated at the transcriptional level and accompanied by metabolic dysregulation. Dosing with interleukin-15 (IL-15) enhanced NK cell cytotoxicity in hypoxia, but preactivation with feeder cells bearing IL-21 and 4-1BBL was even better. Preactivation resulted in less perturbed metabolism in hypoxia; greater resistance to oxidative stress; and no hypoxia-induced loss of transcription factors (T-bet and Eomes), activating receptors, adhesion molecules (CD2), and cytotoxic proteins (TRAIL and FasL). There remained a deficit in CD122/IL-2Rβ when exposed to hypoxia, which affected IL-15 signaling. However, tri-specific killer engager molecules that deliver IL-15 in the context of anti-CD16/FcγRIII were able to bypass this deficit, enhancing cytotoxicity of both fresh and preactivated NK cells in hypoxia.

Hypoxia impairs blood glucose homeostasis in naked mole-rat adult subordinates but not queens.

Naked mole-rats (NMRs) are among the most hypoxia-tolerant mammals and metabolize only carbohydrates in hypoxia. Glucose is the primary building block of dietary carbohydrates, but how blood glucose is regulated during hypoxia has not been explored in NMRs. We hypothesized that NMRs mobilize glucose stores to support anaerobic energy metabolism in hypoxia. To test this, we treated newborn, juvenile and adult (subordinate and queen) NMRs in normoxia (21% O2) or hypoxia (7, 5 or 3% O2), while measuring metabolic rate, body temperature and blood [glucose]. We also challenged animals with glucose, insulin or insulin-like growth factor-1 (IGF-1) injections and measured the rate of glucose clearance in normoxia and hypoxia. We found that: (1) blood [glucose] increases in moderate hypoxia in queens and pups, but only in severe hypoxia in adult subordinates and juveniles; (2) glucose tolerance is similar between developmental stages in normoxia, but glucose clearance times are 2- to 3-fold longer in juveniles and subordinates than in queens or pups in hypoxia; and (3) reoxygenation accelerates glucose clearance in hypoxic subordinate adults. Mechanistically, (4) insulin and IGF-1 reduce blood [glucose] in subordinates in both normoxia but only IGF-1 impacts blood [glucose] in hypoxic queens. Our results indicate that insulin signaling is impaired by hypoxia in NMRs, but that queens utilize IGF-1 to overcome this limitation and effectively regulate blood glucose in hypoxia. This suggests that sexual maturation impacts blood glucose handling in hypoxic NMR queens, which may allow queens to spend longer periods of time in hypoxic nest chambers.

MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia

Hypoxia requires metabolic adaptations to sustain energetically demanding cellular activities. While the metabolic consequences of hypoxia have been studied extensively in cancer cell models, comparatively little is known about how primary cell metabolism responds to hypoxia. Thus, we developed metabolic flux models for human lung fibroblast and pulmonary artery smooth muscle cells proliferating in hypoxia. Unexpectedly, we found that hypoxia decreased glycolysis despite activation of hypoxia-inducible factor 1α (HIF-1α) and increased glycolytic enzyme expression. While HIF-1α activation in normoxia by prolyl hydroxylase (PHD) inhibition did increase glycolysis, hypoxia blocked this effect. Multi-omic profiling revealed distinct molecular responses to hypoxia and PHD inhibition, and suggested a critical role for MYC in modulating HIF-1α responses to hypoxia. Consistent with this hypothesis, MYC knockdown in hypoxia increased glycolysis and MYC over-expression in normoxia decreased glycolysis stimulated by PHD inhibition. These data suggest that MYC signaling in hypoxia uncouples an increase in HIF-dependent glycolytic gene transcription from glycolytic flux.

Overexpression of c-MYC Promoter Binding Protein-1 Enhances Proliferation and Glucose Metabolism of Melanoma Cells Lines.

c-MYC promoter binding protein (MBP-1) is a product of alternatively translated mRNA encoding alpha-enolase (ENO1). In contrast to ENO1, MBP-1 possesses no enzymatic activity but acts as a transcriptional repressor of c-MYC. Ectopic over-expression of MBP-1 in tumor cells was shown to reduce cell proliferation and tumorigenicity, thus making it an attractive target for anticancer strategies. This study aimed to assess the effects of MBP-1 over-expression on human cutaneous melanoma cell lines. We overexpressed the full-length MBP-1 or its C-terminal truncated variant (MBP-1ΔC), in two human melanoma cell lines (A375, WM9) and assessed their subcellular localization. qPCR was then used to quantitate c-MYC transcription. Further, 5-ethynyl-2'-deoxyuridine incorporation assay was used to measure cell proliferation and a lactate assay was performed to measure the glycolysis rate of cells in normoxia and hypoxia. Finally, an in vitro wound-healing assay was performed to evaluate cell migration. The overexpressed MBP-1 variants predominantly localized in the cytoplasm and barely decreased c-MYC expression. Unexpectedly, the proliferation rate of MBP-1- transduced cells increased in comparison to controls, as did the rate of glucose metabolism in hypoxia. Furthermore, over-expression of MBP-1, but not MBP-1ΔC, led to a substantial decrease in the cell migration capacity of metastatic WM9 cells but not A375 cells from the primary tumor lesion. Misslocalization of over-expressed MBP-1 in the cytoplasm of two melanoma cell lines resulted in an unexpected tumor promoting activity by increasing cell proliferation and glycolysis rates in hypoxia.

PECULIARITIES OF THE ORGANISM'S REACTION TO DOSED HYPOXIA IN ELDERLY PEOPLE WITH IMPAIRED GLUCOSE TOLERANCE

Background. The incidence of carbohydrate metabolism disorders increases with age, resulting in a significantly increased risk of cardiovascular disease. Hypoxia plays an important role in the pathogenesis of carbohydrate metabolism disorders. The aim – to establish the features of carbohydrate metabolism in hypoxia in the elderly with impaired carbohydrate tolerance.
 Materials and methods. 20 elderly people with impaired glucose tolerance and 23 elderly people with preserved glucose tolerance were examined. Determined the concentration of glucose and insulin in the blood when breathing air with an oxygen content of 12%.
 Results. With hypoxia in people with impaired glucose tolerance, the decrease in saturation is more significant than in people with preserved glucose tolerance. Shifts in glucose concentration in hypoxia in people with impaired glucose tolerance are greater, but the ratio of Δ glucose concentration/ΔSpO2 is higher than in people with preserved glucose tolerance. Shifts in the concentration of insulin in the blood during hypoxia did not differ in people with different glucose tolerance, but the ratio of Δinsulin concentration /ΔSpO2 in people with impaired glucose tolerance is less. In people with impaired glucose tolerance, a relationship was found between glucose levels after 120 minutes of glucose tolerance test and SpO2 shifts in hypoxia, as well as a relationship between HOMA index and SpO2 shifts in hypoxia.
 Conclusions. Elderly people with impaired glucose tolerance have reduced resistance to hypoxia and increased utilization of glucose in hypoxia, due to smaller changes in insulin concentrations. At the same time at them disturbance of a carbohydrate metabolism depends on resistance to a hypoxia.

Response of some indicators of the respiratory system to dosed hypoxia in elderly people with impaired glucose tolerance

Abstract. The response of the respiratory system to dosed hypoxia (breathing with a gas mixture of 12% oxygen for 20 min) in the elderly with impaired (n = 35) and preserved glucose tolerance (n = 33) was studied. It is shown that the increase in lung ventilation occurs regardless of the state of carbohydrate metabolism in hypoxia. In people with impaired glucose tolerance, changes in lung ventilation in hypoxia are less significant than in people with persistent glucose tolerance. In persons with impaired glucose tolerance, an inverse relationship was found between the increase in pulmonary ventilation during hypoxia and insulin resistance (r = -0.26, p = 0.035), as well as between the increase in pulmonary ventilation during hypoxia and plasma glucose concentration due to 2 hours of standard glucose tolerance test (r = -0.31, p = 0.012). It is concluded that there is a causal relationship between impaired glucose tolerance and insufficient response of pulmonary ventilation to hypoxia in the elderly. Keywords: elderly; impaired glucose tolerance; hypoxia; ventilation.

Cancer Cell Metabolism in Hypoxia: Role of HIF-1 as Key Regulator and Therapeutic Target.

In order to meet the high energy demand, a metabolic reprogramming occurs in cancer cells. Its role is crucial in promoting tumor survival. Among the substrates in demand, oxygen is fundamental for bioenergetics. Nevertheless, tumor microenvironment is frequently characterized by low-oxygen conditions. Hypoxia-inducible factor 1 (HIF-1) is a pivotal modulator of the metabolic reprogramming which takes place in hypoxic cancer cells. In the hub of cellular bioenergetics, mitochondria are key players in regulating cellular energy. Therefore, a close crosstalk between mitochondria and HIF-1 underlies the metabolic and functional changes of cancer cells. Noteworthy, HIF-1 represents a promising target for novel cancer therapeutics. In this review, we summarize the molecular mechanisms underlying the interplay between HIF-1 and energetic metabolism, with a focus on mitochondria, of hypoxic cancer cells.

Regulation of glycolysis by the hypoxia-inducible factor (HIF): implications for cellular physiology.

Under conditions of hypoxia, most eukaryotic cells can shift their primary metabolic strategy from predominantly mitochondrial respiration towards increased glycolysis to maintain ATP levels. This hypoxia-induced reprogramming of metabolism is key to satisfying cellular energetic requirements during acute hypoxic stress. At a transcriptional level, this metabolic switch can be regulated by several pathways including the hypoxia inducible factor-1α (HIF-1α) which induces an increased expression of glycolytic enzymes. While this increase in glycolytic flux is beneficial for maintaining bioenergetic homeostasis during hypoxia, the pathways mediating this increase can also be exploited by cancer cells to promote tumour survival and growth, an area which has been extensively studied. It has recently become appreciated that increased glycolytic metabolism in hypoxia may also have profound effects on cellular physiology in hypoxic immune and endothelial cells. Therefore, understanding the mechanisms central to mediating this reprogramming are of importance from both physiological and pathophysiological standpoints. In this review, we highlight the role of HIF-1α in the regulation of hypoxic glycolysis and its implications for physiological processes such as angiogenesis and immune cell effector function.

The hypoxia tolerance of eight related African mole-rat species rivals that of naked mole-rats, despite divergent ventilatory and metabolic strategies in severe hypoxia.

Burrowing mammals tend to be more hypoxia tolerant than non-burrowing mammals and rely less on increases in ventilation and more on decreases in metabolic rate to tolerate hypoxia. Naked mole-rats (Heterocephalus glaber, NMRs), eusocial mammals that live in large colonies, are among the most hypoxia-tolerant mammals, and rely almost solely on decreases in metabolism with little change in ventilation during hypoxia. We hypothesized that the remarkable hypoxia toleranceof NMRs is an evolutionarily conserved trait derived from repeated exposure to severe hypoxia owing to their burrow environment and eusocial colony organization. We used whole-body plethysmography and indirect calorimetry to measure the hypoxic ventilatory and metabolic responses of eight mole-rat species closely related to the NMR. We found that all eightspecies examined had a strong tolerance to hypoxia, with most species tolerating 3kPa O2 , Heliophobius emini tolerating 2kPa O2 and Bathyergus suillus tolerating 5kPa O2 . All species examined employed a combination of increases in ventilation and decreases in metabolism in hypoxia, a response midway between that of the NMR and that of other fossorial species (larger ventilatory responses, lesser reductions in metabolism). We found that eusociality is not fundamental to the physiological response to hypoxia of NMRs as Fukomys damarensis, another eusocial species, was among this group. Our data suggest that, while the NMR is unique in the pattern of their physiological response to hypoxia, eight closely related mole-rat species share the ability to tolerate hypoxia like the current "hypoxia-tolerant champion," the NMR.

Distinct metabolic adjustments arise from acclimation to constant hypoxia and intermittent hypoxia in estuarine killifish (Fundulus heteroclitus).

Many fish experience daily cycles of hypoxia in the wild, but the physiological strategies for coping with intermittent hypoxia are poorly understood. We examined how killifish adjust O2 supply and demand during acute hypoxia, and how these responses are altered after prolonged acclimation to constant or intermittent patterns of hypoxia exposure. We acclimated killifish to normoxia (∼20 kPa O2), constant hypoxia (2 kPa) or intermittent cycles of nocturnal hypoxia (12 h:12 h normoxia:hypoxia) for 28 days, and then compared whole-animal O2 consumption rates (ṀO2 ) and tissue metabolites during exposure to 12 h of hypoxia followed by reoxygenation in normoxia. Normoxia-acclimated fish experienced a pronounced 27% drop in ṀO2 during acute hypoxia, and modestly increased ṀO2 upon reoxygenation. They strongly recruited anaerobic metabolism during acute hypoxia, indicated by lactate accumulation in plasma, muscle, liver, brain, heart and digestive tract, as well as a transient drop in intracellular pH, and they increased hypoxia inducible factor (HIF)-1α protein abundance in muscle. Glycogen, glucose and glucose-6-phosphate levels suggested that glycogen supported brain metabolism in hypoxia, while the muscle used circulating glucose. Acclimation to constant hypoxia caused a stable ∼50% decrease in ṀO2 that persisted after reoxygenation, with minimal recruitment of anaerobic metabolism, suggestive of metabolic depression. By contrast, fish acclimated to intermittent hypoxia maintained sufficient O2 transport to support normoxic ṀO2 , modestly recruited lactate metabolism and increased ṀO2 dramatically upon reoxygenation. Both groups of hypoxia-acclimated fish had similar glycogen, ATP, intracellular pH and HIF-1α levels as normoxic controls. We conclude that different patterns of hypoxia exposure favour distinct strategies for matching O2 supply and O2 demand.

Hypoxia causes reductions in birth weight by altering maternal glucose and lipid metabolism

Hypoxia of residence at high altitude (>2500 m) decreases birth weight. Lower birth weight associates with infant mortality and morbidity and increased susceptibility to later-in-life cardiovascular and metabolic diseases. We sought to determine the effects of hypoxia on maternal glucose and lipid metabolism and their contributions to fetal weight. C57BL6/NCrl mice, housed throughout gestation in normobaric hypoxia (15% oxygen) or normoxia, were studied at mid (E9.5) or late gestation (E17.5). Fetal weight at E17.5 was 7% lower under hypoxia than normoxia. The hypoxic compared with normoxic dams had ~20% less gonadal white adipose tissue at mid and late gestation. The hypoxic dams had better glucose tolerance and insulin sensitivity compared with normoxic dams and failed to develop insulin resistance in late gestation. They also had increased glucagon levels. Glucose uptake to most maternal tissues was ~2-fold greater in the hypoxic than normoxic dams. The alterations in maternal metabolism in hypoxia were associated with upregulation of hypoxia-inducible factor (HIF) target genes that serve, in turn, to increase glycolytic metabolism. We conclude that environmental hypoxia alters maternal metabolism by upregulating the HIF-pathway, and suggest that interventions that antagonize such changes in metabolism in high-altitude pregnancy may be helpful for preserving fetal growth.

PPARα-independent effects of nitrate supplementation on skeletal muscle metabolism in hypoxia

Hypoxia is a feature of many disease states where convective oxygen delivery is impaired, and is known to suppress oxidative metabolism. Acclimation to hypoxia thus requires metabolic remodelling, however hypoxia tolerance may be aided by dietary nitrate supplementation. Nitrate improves tissue oxygenation and has been shown to modulate skeletal muscle tissue metabolism via transcriptional changes, including through the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator of fat metabolism. Here we investigated whether nitrate supplementation protects skeletal muscle mitochondrial function in hypoxia and whether PPARα is required for this effect. Wild-type and PPARα knockout (PPARα−/−) mice were supplemented with sodium nitrate via the drinking water or sodium chloride as control, and exposed to environmental hypoxia (10% O2) or normoxia for 4 weeks. Hypoxia suppressed mitochondrial respiratory function in mouse soleus, an effect partially alleviated through nitrate supplementation, but occurring independently of PPARα. Specifically, hypoxia resulted in 26% lower mass specific fatty acid-supported LEAK respiration and 23% lower pyruvate-supported oxidative phosphorylation capacity. Hypoxia also resulted in 24% lower citrate synthase activity in mouse soleus, possibly indicating a loss of mitochondrial content. These changes were not seen, however, in hypoxic mice when supplemented with dietary nitrate, indicating a nitrate dependent preservation of mitochondrial function. Moreover, this was observed in both wild-type and PPARα−/− mice. Our results support the notion that nitrate supplementation can aid hypoxia tolerance and indicate that nitrate can exert effects independently of PPARα.

Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance.

Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.

Ecophysiological limits to aerobic metabolism in hypoxia determine epibenthic distributions and energy sequestration in the northeast Pacific ocean

AbstractExpansion of oxygen deficient waters (hypoxia) in the northeast Pacific Ocean (NEP) will have marked impacts on marine life. The response of the resident communities will be a function of their ecophysiological constraints in low oxygen, although this remains untested in the NEP due to a lack of integrative studies. Here, we combine in situ surveys and lab‐based respirometry experiments were conducted on three indicator species (spot prawnPandalus platyceros, slender soleLyopsetta exilis, squat lobsterMunida quadrispina) of seasonally hypoxic systems in the NEP to test if metabolic constraints determine distributions and energy sequestration in a hypoxic setting. These experiments were integrated with a global review of critical oxygen levels (; lower threshold of aerobic metabolism) for crustaceans to determine if‐based hypoxia thresholds are different among ocean basins. Our results show that species‐specific differences inand standard metabolic rates (1) determine the lowest environmental oxygen ([O2]env) at which in situ populations occur, (2) result in disproportionate shifts in distributions among co‐occurring species during summer hypoxia expansion events, and (3) characterize shifts in megafaunal community respiration rates due to marked spatio‐temporal variability in [O2]env. Our results show that‐based hypoxia thresholds are significantly lower in the East Pacific Ocean relative to other major ocean basins, which suggests that the physiological response of local fauna to deoxygenation can be determined by the natural variability and oxygen exposure in a region. In order to establish realistic predictions on the biological consequences of marine deoxygenation, we suggest integrating metabolism‐based traits to calculate hypoxia thresholds for marine ecosystems.

Synergistic Inhibitory Effects of Hypoxia and Iron Deficiency on Hepatic Glucose Response in Mouse Liver.

Hypoxia and iron both regulate metabolism through multiple mechanisms, including hypoxia-inducible transcription factors. The hypoxic effects on glucose disposal and glycolysis are well established, but less is known about the effects of hypoxia and iron deficiency on hepatic gluconeogenesis. We therefore assessed their effects on hepatic glucose production in mice. Weanling C57BL/6 male mice were fed an iron-deficient (4 ppm) or iron-adequate (35 ppm) diet for 14 weeks and were continued in normoxia or exposed to hypoxia (8% O2) for the last 4 weeks of that period. Hypoxic mice became hypoglycemic and displayed impaired hepatic glucose production after a pyruvate challenge, an effect accentuated by an iron-deficient diet. Stabilization of hypoxia-inducible factors under hypoxia resulted in most glucose being converted into lactate and not oxidized. Hepatic pyruvate concentrations were lower in hypoxic mice. The decreased hepatic pyruvate levels were not caused by increased utilization but rather were contributed to by decreased metabolism from gluconeogenic amino acids. Pyruvate carboxylase, which catalyzes the first step of gluconeogenesis, was also downregulated by hypoxia with iron deficiency. Hypoxia, and more so hypoxia with iron deficiency, results in hypoglycemia due to decreased levels of hepatic pyruvate and decreased pyruvate utilization for gluconeogenesis. These data highlight the role of iron levels as an important determinant of glucose metabolism in hypoxia.

A New Hydroxy Metabolite of 2-Oxoglutarate Regulates Metabolism in Hypoxia

Two articles in this issue (Intlekofer et al., 2015; Oldham et al., 2015) show a new metabolic pathway regulated by hypoxia, but independently of HIF1 or HIF2. L-2-hydroxyglutarate, produced in hypoxia by malate dehydrogenases and LDHA, is a potent inhibitor of KDM4C, and through redox stress reduces glycolysis.

254 CHRONIC HYPOXIA CAUSES NO CHANGE IN CARDIAC PYRUVATE DEHYDROGENASE FLUX IN THE CONTROL OR DIABETIC RAT: AN IN VIVO STUDY

Introduction In the healthy heart, approximately 30% of the cells’ energy comes from glucose oxidation and the remaining 70% from fatty acids. On exposure to hypoxia, the balance is altered, with increased reliance on glycolysis due to reduced oxygen availability. Pyruvate dehydrogenase (PDH) is the enzyme that stands as the gatekeeper between glycolysis and the Krebs’ cycle. It has previously been demonstrated that hypoxia causes increased expression of pyruvate dehydrogenase kinase (PDK) 1, an enzyme that inhibits PDH. Upregulation of PDK1 enhances inhibition, and it can therefore be hypothesised that PDH flux will be decreased. In the diabetic heart there is a preference for fatty acid use even in the presence of elevated circulating glucose. Exposure to hypoxia and the associated upregulation of glycolysis, may to some extent rebalance fuel selection. However, it is not known if this would be the case as it has also been suggested that the diabetic heart is compromised in terms of its ability to respond to hypoxia. The advent of hyperpolarized carbon-13 ( 13 C) magnetic resonance spectroscopy (MRS) has enabled the visualisation of metabolism in real-time and in vivo . 13 C-labelled pyruvate can be injected into an anaesthetised animal in an MRS system, and spectra acquired (one per second over one minute) to show the metabolism of pyruvate and therefore the flux through PDH. Using hyperpolarized 13 C MRS, this study has investigated in vivo alterations in glucose metabolism in hypoxia in comparison with normoxia, and how these alterations are affected by diabetes. Methods A rat model of late-stage type 2 diabetes was induced by high fat feeding and injection of streptozotocin (25mg/kg). Diabetic and control (chow-fed) animals (n=8) were housed for 3 weeks either in hypoxia (one week gradual acclimatisation and two further weeks at 11% oxygen) or normoxia. Animals were then anaesthetised and scanned in a 7T MRS system, with an injection of hyperpolarized [1- 13 C]-labelled pyruvate and cardiac spectra acquired over ∼1 min. Animals were terminated after scanning, blood samples taken and tissue samples freeze-clamped for later analysis. Data were analysed using jMRUI and the AMARES package, and the conversion of pyruvate into bicarbonate taken as a measure of PDH flux. Results There were no alterations in cardiac PDH flux (Kpyr-bic) observed in hypoxic control animals when compared to normoxic controls. There were also no changes in 13 C label transfer to lactate (Kpyr-lac). Diabetic animals in both normoxia and hypoxia demonstrated decreased PDH flux when compared to controls, however there was no interaction in terms of PDH flux between the hypoxic and diabetic conditions (Figure 1). Conclusions We have observed no alteration in cardiac PDH flux caused by three weeks of chronic hypoxia. In addition, whilst there was a significant reduction in PDH flux in diabetes, exposure to chronic hypoxia had no further metabolic effect on PDH flux.

Haem oxygenase-1 induction reverses the actions of interleukin-1β on hypoxia-inducible transcription factors and human chondrocyte metabolism in hypoxia

HO-1 (haem oxygenase-1) catalyses the degradation of haem and possesses anti-inflammatory and cytoprotective properties. The role of inflammatory mediators in the pathogenesis of OA (osteoarthritis) is becoming increasingly appreciated. In the present study, we investigated the effects of HO-1 induction in OA and healthy HACs (human articular chondrocytes) in response to inflammatory cytokine IL-1 β (interleukin-1β) under hypoxic conditions. Hypoxia was investigated as it is a more physiological condition of the avascular cartilage. Hypoxic signalling is mediated by HIFs (hypoxia-inducible factors), of which there are two main isoforms, HIF-1α and HIF-2α. Normal and OA chondrocytes were stimulated with IL-1β. This cytokine suppresses HO-1 expression and exerts both catabolic and anti-anabolic effects, while increasing HIF-1α and suppressing HIF-2α protein levels in OA chondrocytes in hypoxia. Induction of HO-1 by CoPP (cobalt protoporphyrin IX) reversed these IL-1β actions. The hypoxia-induced anabolic pathway involving HIF-2α, SOX9 [SRY (sex determining region Y)-box 9] and COL2A1 (collagen typeII α1) was suppressed by IL-1β, but importantly, levels were restored by HO-1 induction, which down-regulated TNFα (tumour necrosis factor α), MMP (matrix metalloproteinase) activity and MMP-13 protein levels. Depletion of HO-1 using siRNA (small interfering RNA) abolished the CoPP effects, further demonstrating that these were due to HO-1. The results of the present study reveal the different mechanisms by which HO-1 exerts protective effects on chondrocytes in physiological levels of hypoxia.

Abstract 2799: Unexpected sustained hypoxic glutamine metabolism and therapeutic opportunities

Abstract Rationale: As the result of genetic alterations and tumor hypoxia, cancer cells re-program metabolism to meet increased energy demand for enhanced anabolism, cell proliferation, and protection from oxidative damage and cell death signals. Whereas glycolysis and its regulation by the MYC oncogene product (Myc) have been intensively studied, glutaminolysis and anapleurosis, especially under hypoxia, have not been defined. Objectives: We sought to determine whether glutamine, a key source of nitrogen and anabolic carbon skeleton for mammalian cells, is still utilized under hypoxic condition and how it is regulated by MYC. Methods: We used NMR and FT-ICR-MS to resolve 13C and 15N labeling patterns of metabolites derived from labeled glutamine or glucose in the P493 human B lymphoma model that bear a tetracycline-repressible MYC, under normoxic and hypoxic conditions. Levels of specific enzymes, which are involved in glucose and glutamine metabolism, were determined by immunoblotting and LC-MRM-MS and compared with the metabolomic profiles. Measurements and Main Results: Myc regulates almost the entire glycolytic pathway. As expected for hypoxia, many glycolytic enzymes levels and lactate production were elevated, and 13C- glucose catabolism by the TCA cycle was significantly diminished. Myc elevated glutaminase (GLS, which converts glutamine to glutamate and NH4+), the glutamine transporter SLC1A5, and the transaminases GOT1 and GPT2, suggesting that transamination is favored with Myc activation. While GLS-mediated NH4+ release persisted under hypoxia, the Myc-induced transaminases favor the production of α-ketoglutarate without additional NH4+ production. The 13C labeling pattern of Ala is consistent with active mitochondrial GPT2 that transaminates 13C-pyruvate derived from 13C- glucose. Given that mitochondrial function is generally believed to be diminished by hypoxia, glutamine metabolism unexpectedly persisted in hypoxia with continued oxidation of glutamine carbons by the TCA cycle. The key enzymes and main products of 13C labeled glutamine were not decreased by hypoxia, and glutamine contributed to substantial de novo glutathione production in hypoxia. Because glutamine metabolism is sustained in hypoxia, which is pervasive in the tumor microenvironment, we sought to target glutaminase in vivo. We found that small molecule inhibition of glutaminase could diminish lymphoma and pancreatic cancer xenograft growth in vivo. Conclusions: Our studies reveal a previously unsuspected sustained glutamine metabolism in hypoxia. Glutamine contributes to TCA cycle carbons as well as enhances glutathione synthesis in hypoxia, suggesting that cancer cells reprogram metabolism via both glucose and glutamine to adapt to the tumor microenvironment. Our findings indicate that key nodal points in glutamine metabolism in addition to those in glycolysis could be targeted for therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2799. doi:10.1158/1538-7445.AM2011-2799

Energy metabolism in hypoxia: reinterpreting some features of muscle physiology on molecular grounds

An holistic approach for interpreting classical data on the adaptation of the animal and, particularly, of the human body to hypoxic stress was promoted by the discovery of HIF-1, the "master regulator" of cell hypoxic signaling. Mitochondrial production of ROS stabilizes the O(2)-regulated HIF-1α subunit of the HIF-1 dimer promoting transaction functions in a large number of potential target genes, activating transcription of sequences into RNA and, eventually, protein production. The aim of the present preliminary study is to assess whether adaptive changes in oxygen sensing and metabolic signaling, particularly in the control of energy turnover known to occur in cultured cells exposed to hypoxia, are detectable also in the muscles of animals and man. For the present analysis, data obtained from the proteome of the rat gastrocnemius and of the vastus lateralis muscle of humans together with functional measurements were compared with homologous data from hypoxic cultured cells. In particular, the following variables were assessed: (1) the role of stress response proteins in the maintenance of ROS homeostasis, (2) the activity of the PDK1 gene on the shunting of pyruvate away from the TCA cycle in rodents and in humans, (3) the COX-4/COX-2 ratio in hypoxic rodents, (4) the overall efficiency of oxidative phosphorylation in humans during exercise in hypoxia, (5) some features of muscle mitochondrial autophagy in humans undergoing subchronic and chronic altitude exposure. Despite the limited number of observations and the differences in the experimental approach, some initial interesting results were obtained encouraging to pursue this innovative effort.