Oxygen sensing and metabolic homeostasis.

ARTICLE IN PRESS Molecular and Cellular Endocrinology ■■ (2014) ■■–■■

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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e

Review

Oxygen sensing and metabolic homeostasis Biff F. Palmer a,*, Deborah J. Clegg b a b

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA Biomedical Research, Cedars-Sinai Medical Center, Beverly Hills, California, USA

A R T I C L E

I N F O

Article history: Available online Keywords: Hypoxia inducible factor (HIF) Hypobaric hypoxia Adipose tissue Sexual dimorphism Warburg effect Oxygen sensing

A B S T R A C T

Oxygen-sensing mechanisms have evolved to maintain cell and tissue homeostasis since the ability to sense and respond to changes in oxygen is essential for survival. The primary site of oxygen sensing occurs at the level of the carotid body which in response to hypoxia signals increased ventilation without the need for new protein synthesis. Chronic hypoxia activates cellular sensing mechanisms which lead to protein synthesis designed to alter cellular metabolism so cells can adapt to the low oxygen environment without suffering toxicity. The master regulator of the cellular response is hypoxia-inducible factor (HIF). Activation of this system under condition of hypobaric hypoxia leads to weight loss accompanied by increased basal metabolic rate and suppression of appetite. These effects are dose dependent, gender and genetic specific, and results in adverse effects if the exposure is extreme. Hypoxic adipose tissue may represent a unified cellular mechanism for variety of metabolic disorders, and insulin resistance in patients with metabolic syndrome. © 2014 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Introduction ............................................................................................................................................................................................................................................................. Hypoxia inducible factor ..................................................................................................................................................................................................................................... HIF’s influence on cellular fuel utilization .................................................................................................................................................................................................... HIF’s influence on basal metabolic rate (BMR) ............................................................................................................................................................................................ HIF’s influence on muscle metabolism ........................................................................................................................................................................................................... Adverse effects of HIF activation on exercise performance ..................................................................................................................................................................... Cellular adaptation to altitude .......................................................................................................................................................................................................................... HIF-induced alterations in cellular energetics in cancer ......................................................................................................................................................................... Genetic variability in HIF activation with altitude ..................................................................................................................................................................................... Sexual dimorphism in HIF activation at altitude ........................................................................................................................................................................................ Adipose tissue and hypoxia ................................................................................................................................................................................................................................ Hypoxia inducible factor in diabetes mellitus: effects of hyperglycemia .......................................................................................................................................... Summary ................................................................................................................................................................................................................................................................... References ................................................................................................................................................................................................................................................................

1. Introduction Oxygen is a vital metabolic substrate for cellular functions. Oxygen sensing occurs at many levels and is critical for successful adaption to abnormal oxygen levels due either to changes in ambient oxygen levels or because of disease processes. A global oxygen sensing mech-

* Corresponding author. Address: Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA. Tel.: +1 214 648 7848; fax: +1 214 648 2071. E-mail address: [email protected] (B.F. Palmer).

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anism is represented by the ability of the glomus cells in the carotid body to signal increased ventilation in response to hypoxemia. When an imbalance between oxygen supply and demand results in hypoxemia a cascade of physiologic and biochemical events is triggered potentially resulting in deleterious effects on enzyme activities, mitochondrial function, cytoskeletal structure, and membrane transport. At the cellular level adaptation to hypoxia is an essential cellular response controlled by the oxygen-sensitive transcription factor hypoxia-inducible factor 1 (HIF-1). Studies in normal subjects exposed to hypoxia at altitude illustrate the metabolic consequences of HIF activation. At altitude hypoxia results from the reduction in barometric pressure causing a decrease in the in-

http://dx.doi.org/10.1016/j.mce.2014.08.001 0303-7207/© 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Biff F. Palmer, Deborah J. Clegg, Oxygen sensing and metabolic homeostasis, Molecular and Cellular Endocrinology (2014), doi: 10.1016/ j.mce.2014.08.001

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Glucose GLUT 1

GLUT 3

Glucose

CO2 + H2O

H+ MCT4

NHE1

(+) (+)

Lactate

Na+

H+

CO2

HIF-1

Glycolysis

HCO3 + H+ CA IX

2

(+)

Pyruvate

Lactate dehydrogenase A NADH

Lactate Electron transport chain

NAD+

Citrate Acetyl CoA

(-)

Pyruvate dehydrogenase (PDH)

Pyruvate dehydrogenase kinase-1 (PDK1)

Isocitrate

Oxaloacetate

NAD+

NADH

Malate

NADH

NAD+

α-Ketoglutarate

(+)

HIF-1

NAD+ NADH

Fumarate

FADH2

Succinyly-CoA

FAD

Succinate Fig. 1. Under condition of limited oxygen supply upregulation of hypoxia inducible factor leads to a shift in cell metabolism favoring glycolysis so as to limit generation of potentially harmful reactive oxygen species via oxidative phosphorylation in the mitochondria. A critical step in this shift is HIF-mediated activation of pyruvate dehydrogenase kinase-1 (PDK-1). This enzyme inactivates pyruvate dehydrogenase which is the mitochondrial enzyme responsible for converting pyruvate to acetyl-CoA. In combination with activation of lactate dehydrogenase A (LDHA) which converts pyruvate to lactate, there is less delivery of acetyl-CoA into the Krebs cycle and therefore a reduction of flavin adenine dinucleotide (FADH2) and nicotinamide adenine dinucleotide (NADH) delivered to the electron transport chain. HIF upregulates the expression of GLUT1 and GLUT3 on the cell membrane so as to facilitate glucose entry into the cell. HIF also upregulates monocarboxylate transporter 4 which is a proton-lactate symporter. This transporter creates an avenue for lactate exit from the cell. The cotransport of the proton contributes to the maintenance of intracellular pH. Cell pH is also defended by HIF-induced expression of carbonic anhydrase-9 and increased expression of the Na+/H+ antiporter, NHE1.

spired partial pressure of oxygen. This paper will focus on the cellular response to hypoxemia giving particular emphasis to how HIF activation alters metabolic homeostasis in both favorable as well as unfavorable ways depending on the cause and duration of hypoxemia. 2. Hypoxia inducible factor HIF is a DNA-binding transcription factor which in association with specific nuclear cofactors transactivates a variety of genes triggering an adaptive response aimed to optimize the utilization of available oxygen under conditions of hypoxia (Greer et al., 2012; Semenza, 2012). The three members of the family (HIF-1, HIF-2 and HIF-3) are heterodimers consisting of an oxygen-sensitive α-subunit and a constitutively expressed β-subunit. In the presence of oxygen, HIFα is extremely unstable due to an oxygen-dependent hydroxylation which targets it for proteosomal degradation. This hydroxylation is mediated by three prolyl hydroxylases (PHD1-3) and requires oxygen as well as Fe2+, 2-oxoglutarate, and ascorbate for their catalytic activity (Appelhoff et al., 2004). Prolyl-hydroxylated HIFα is bound by the von Hippel-Lindau (VHL) tumor suppressor protein and subsequently ubiquitylated by the elongin C–elongin B–cullin 2–E3-ubiquitin–ligase complex marking HIFα for proteosomal degradation. The requirement of oxygen for the catalytic activity of PHD1-3 allows for HIFα to escape recognition by VHL under conditions of hypoxia. In this setting HIFα is stabilized and accumulates in the nucleus where it dimerizes with HIFβ and subsequently binds to the hypoxia response element in target genes.

Activation of HIF-induced genes give rise to a myriad of effects designed to promote survival in low-oxygen conditions. These include increases in red cell mass brought about by stimulation of erythropoietin production and promotion of angiogenesis through stimulation of vascular endothelial growth factor (VEGF). HIFinduced gene activation also causes a coordinated shift in metabolism from oxidative phosphorylation to a less oxygen requiring production of ATP via the glycolytic pathway (Hamanaka and Chandel, 2010) (Fig. 1). A critical step in this shift is HIF-mediated activation of pyruvate dehydrogenase kinase-1 (PDK-1). This enzyme inactivates pyruvate dehydrogenase which is the mitochondrial enzyme responsible for converting pyruvate to acetyl-CoA. In combination with activation of lactate dehydrogenase A (LDHA) which converts pyruvate to lactate, there is less delivery of acetyl-CoA into the Krebs cycle and therefore a reduction of flavin adenine dinucleotide (FADH2) and nicotinamide adenine dinucleotide (NADH) delivered to the electron transport chain. This shift away from mitochondrial respiration serves to reduce the production of potentially harmful reactive oxygen species which occurs as a function of decreasing oxygen tension (Wheaton and Chandel, 2011). Under normoxic conditions reduction of molecular oxygen to water by the mitochondrial electron transport chain enables the conversion of ADP into ATP providing the energy source for normal cellular function. In this setting generation of reactive oxygen species (ROS) is minimal (Turrens, 2003). When oxygen becomes limiting, however, there is increased production of ROS which can lead to cellular damage and dysfunction. Increased production of ROS is the result of electrons being delivered to oxygen prior to the reduc-



tion of oxygen to water at cytochrome c oxidase, a phenomenon referred to as electron leak. In addition to lack of oxygen, accumulation of ROS in the cytosol specifically generated by mitochondrial complex III directly inhibits HIF-1α prolyl hydroxylation and degradation. This effect provides an additional trigger leading to the stabilization of HIFα (Guzy et al., 2005) thereby assuring redirection of metabolism away from mitochondrial respiration. Once activated HIF also induces a switch in subunits from COX4-1 to COX42, the latter of which is more efficient at facilitating the transfer of electrons to oxygen (Fukuda et al., 2001). This effect helps to ensure generation of ROS is minimized from any remaining aerobic metabolism. 3. HIF’s influence on cellular fuel utilization The switch to glycolysis and lactate production in the hypoxic cell requires an adequate supply of glucose. HIF up regulates the expression of GLUT1 and GLUT3 on the cell membrane so as to facilitate glucose entry into the cell (Porporato et al., 2011). Some of this glucose is directed toward glycogen synthesis under the dictates of HIF thereby ensuring an additional source of substrate for ongoing glycolysis (Pescador et al., 2010). This shift to a greater dependency on glucose uptake is required from an energetic standpoint since only two ATPs are generated for every mol of glucose metabolized. Glucose kinetic studies using radiolabeled tracers show high altitude is associated with increased rates of glucose flux as demonstrated by increased rates of glucose appearance, disappearance, and oxidation both at rest and exercise compared to sea level consistent with a change in metabolism favoring increased cellular glucose uptake (Brooks et al., 1991; Roberts et al., 1996a, 1996b). The resting plasma glucose concentration tends to decrease upon altitude exposure indicative of higher glucose flux. This shift in metabolism represents an adaptive response to limited oxygen availability since metabolism of glucose via glycolysis requires less oxygen as compared to oxidative phosphorylation. Additionally, hypoxia increases glucose output in hepatocytes in association with transcriptional activation of phosphoenolpyruvate carboxykinase (PEPCK), the rate limiting enzyme for gluconeogenesis (Choi et al., 2005). HIF directly binds to the promoter region of PEPCK providing a molecular mechanism by which hypoxia can stimulate hepatic gluconeogenesis. Gluconeogenesis in the kidney is also markedly increased in rats chronically exposed to a simulated altitude of approximately 5400 meters (Ou, 1974). Cori cycle activity supports the redirection of glucose metabolism toward production of lactate at high altitude. HIF upregulates monocarboxylate transporter 4 which is a proton-lactate symporter. This transporter creates an avenue for lactate exit from the cell. The cotransport of the proton contributes to the maintenance of intracellular pH. Cell pH is also defended by HIF-induced expression of carbonic anhydrase-9 and increased expression of the Na+/H+ antiporter, NHE1 (Brahimi-Horn et al., 2007). Once exported, cell lactate is taken up by the liver and kidney in a more efficient manner where it can then serve as a substrate for increased gluconeogenesis. In this regard, both hepatic lactate uptake and glucose output increase several fold when normal subjects performing submaximal exercise are switched from a normoxic to a hypoxic breathing mixture (Rowell et al., 1984). It is possible that increased gluconeogenesis under conditions of hypoxia may serve as a mechanism to minimize accumulation of lactate in the setting of increased cellular production and provide adequate amounts of glucose substrate in order to support the switch in fuel preference (Fig. 2). 4. HIF’s influence on basal metabolic rate (BMR) When lowlanders are taken to high altitude the BMR increases by 6–27% and then tends to slowly decline with acclimatization.

3

Glucose GLUT 1

Glucose

GLUT 3

Glucose Glucose Phosphoenolpyruvate carboxykinase (PEPCK)

↑ Glycolysis

6 ATP 2 Pyruvate

(+)

2 ATP

HIF-1 2 Pyruvate ↓ Mitochondrial respiration

2 Lactate

2 Lactate ↑ Hepatic Lactate uptake

↑ Gluconeogenesis

34 ATP

Skeletal Muscle Cell

Hepatocyte

Fig. 2. An increase in Cori cycle activity under conditions of hypoxia can supply the necessary substrate to fuel the greater dependency on blood glucose brought about by increased HIF activity. Under conditions of hypoxia both hepatic lactate uptake and glucose production are increased. HIF upregulates the gene expression of phosphoenolpyruvate carboxykinase which is the rate limiting enzyme for gluconeogenesis. This change in metabolism is energy inefficient since only two ATPs are formed for each mol of glucose metabolized and six ATPs are consumed for every two molecules of lactate converted to glucose. This energy wasting may play a role in the increase in basal metabolic rate which occurs at altitude and in some cancers. This energy inefficiency may also be detrimental to exercise performance at altitude do to the shift away from mitochondrial respiration where up to 34 ATPs are formed for each mol of glucose metabolized.

During prolonged stays up to 5000 m the BMR tends to return toward normal values but remains persistently increased at more extreme elevations (Kellogg et al., 1957; Stock et al., 1978). The initial increase in BMR is directly related to the altitude attained. At 3650 m BMR increases by 6% whereas increases of 10% and 27% occur at 3800 and 4300 m respectively (Butterfield et al., 1992; Pugh, 1962; Rose et al., 1988). In some reports the BMR declines back to normal after several weeks whereas others report a sustained elevation. Part of this variability may be due to differences in the magnitude of weight loss. In those reports where BMR returns to sea-level values, loss of metabolically active tissue may be more pronounced accounting for the normalization. However, when energy balance is maintained so as to prevent weight loss, the increase in BMR is persistent. For example, in one study acute altitude exposure to 4300 m increased basal metabolic rate by 27% over that of sea level and remained elevated by 17% after 3 weeks of acclimatization. The shift in metabolism away from oxidative phosphorylation to glycolysis mediated by HIF activation creates an energy wasting state which accounts for the persistent increase in energy expenditure despite weight loss under conditions of hypobaric hypoxia. The maximal increase in BMR soon after arrival to altitude followed by a slow decline toward baseline values with acclimatization mirrors the pattern of HIF activity upon exposure to hypoxia. Upregulation of HIF activity upon acute exposure to hypoxia tends to decline towards a lower steady state value with chronic exposure suggesting the presence of a feedback mechanism. In this regard both PHD2 and PHD3 are HIF-dependent genes and retain some level of activity under conditions of hypoxia (Marxsen et al., 2004). In addition HIF-1α has been shown to upregulate the microRNA miR155 which in turn exerts an inhibitory effect on HIF-1α mRNA translation (Bruning et al., 2011). The presence of an effective negative feedback loop provides a means to limit excessive HIF signaling which could prove detrimental to cells over the long term as well accelerate the degradation of HIF following re-oxygenation. Activation of HIF has also been implicated in the reduction in appetite which typically occurs at altitude. Anorexia tends to be maximal in the first several days upon arrival to altitude when protein and caloric intake can decrease as much as 30 and 40% respectively. Below 5000 m food intake tends to return toward normal


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following several days of acclimatization (Westerterp-Plantenga et al., 1999). At more extreme altitudes anorexia is more pronounced and becomes persistent. Hypoxia is a potent stimulus for increased leptin gene expression and subsequent secretion by fat cells. Leptin crosses the blood– brain barrier where it activates its receptors in the hypothalamus to control body weight by reducing food intake and increasing energy expenditure (Yingzhong et al., 2006). The leptin gene contains eight HIF response elements in the promoter region (Ambrosini et al., 2002). Measurement of leptin levels with ascent to altitude has provided conflicting results (Sierra-Johnson et al., 2008; Tschop et al., 1998). Some of this discrepancy may be methodological in nature, since leptin is secreted in a diurnal fashion and is subject to feedback regulation; therefore, values are likely to vary due to timing of the sample collection and whether the subject was at altitude acutely or chronically. In two independent studies ascent to altitude was associated with increased leptin levels and this was more pronounced in those subjects with the greatest weight loss (Tschop et al., 1998). Importantly, weight loss per se reduces leptin levels due to the preferential loss in body fat noted following altitude exposure. Since leptin is secreted by fat cells, loss of body fat can mask the stimulatory effect of hypoxia on leptin production. In this regard, leptin levels are likely to be greater in subjects suffering weight loss at altitude as compared to subjects who have lost a comparable amount of weight at sea level. In addition to HIF-induced stimulation of leptin, decreased appetite in hypobaric hypoxia may also be the result of increased HIF activity in the hypothalamus. The HIF-2α isoform is expressed in the arcuate nucleus of the hypothalamus and has been shown to function as a nutrient sensor and regulate POMC neurons (Zhang et al., 2011). HIF-2α leads to upregulation of POMC through a direct effect on gene transcription, providing a mechanism of HIF-induced reductions in energy intake. When HIF-2α is specifically deleted in the hypothalamus with use of lox/cre methodology, animals develop positive energy balance and obesity. By contrast, over expressing HIF-2α in the arcuate nucleus using a lentiviral co-expression system, animals develop a hypermetabolic phenotype with resistance to the development of obesity upon challenged with a high fat diet. 5. HIF’s influence on muscle metabolism At moderate altitude the most significant change in body composition is a loss of body fat which accounts for 70% of body weight reduction on a trek to 5400 m. Whereas, extreme elevations muscle protein catabolism becomes the dominant change and loss of fat accounts for only 27% of any further weight loss (Boyer and Blume, 1984). These changes underlie the decision to place base camps at altitudes no higher than 5000–5500 m during high altitude mountaineering expeditions. At this altitude weight loss from a reduction in fat and muscle can be minimized by maintaining adequate dietary intake. The muscle wasting which occurs at extreme altitude is accompanied by a substantial decrease in mitochondrial volume density in skeletal muscle (Ferretti, 2003; Howald and Hoppeler, 2003; Levett et al., 2012). Skeletal muscle biopsies taken from climbers on Mt Everest show a decrease in mitochondrial volume by up to 30% primarily accounted for by a reduction in the subsarcolemmal subpopulation of mitochondria. The change in mitochondrial volume is accompanied by significant decreases in the activity of enzymes responsible for aerobic oxidative metabolism, a signature of increased HIF activity. Similar effects on muscle and tissue oxidative capacity are found in subjects exposed to simulated altitude. Upregulation in HIF activity may play a role in the change in skeletal muscle mitochondria at altitude. Peroxisome proliferatoractivated receptor (PPAR)-γ coactivator (PGC)-1α and its homolog PGC-1β are transcriptional co-activators abundant in skeletal muscle

and stimulate mitochondrial biogenesis (Liang and Ward, 2006). HIF leads to a downregulation of PGC-1α potentially accounting for the reduction in mitochondrial volume noted on muscle biopsies taken from climbers at high altitude (Krishnan et al., 2012; Zhang et al., 2007). In this regard levels of PGC-1α are decreased by 35% in climbers after extended stay at altitude with ascent beyond 6400 m (Levett et al., 2012). PGC-1α also normally induces a remodeling of skeletal muscle fiber composition such that the ratio of more oxidative type I fibers to the glycolytic type IIb fibers is increased. A reduction in this ratio due to HIF-mediated suppression of PGC-1α would fit with the body wide shift toward glycolytic metabolism. HIF has also been shown to induce mitochondrial autophagy by upregulating the pro-apoptotic protein BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) (Zhang et al., 2008). BNIP3 disrupts the interaction of Beclin-1 with Bcl2 leading to Atg-5 dependent mitochondrial autophagy. In mice exposed to a simulated altitude of 4300 m muscle protein levels of HIF-1α increased by 70% over control values but returned to baseline when measured after 1 week of exposure (Le Moine et al., 2011). A similar pattern was seen in the muscle protein level of PDK1. When animals were exercised on a treadmill, blood and muscle lactate levels were significantly greater in mice exposed to 24 h of hypoxia but returned toward normoxic levels when exercise was repeated at 1 week of hypoxic exposure. These data suggest chronic hypoxic exposure is associated with a downregulation of HIF and a secondary reduction of PDK1 levels. These changes allow exercising muscle to assume a phenotype more reminiscent of normoxia where pyruvate conversion to acetylCoA is preferred and conversion to lactate is reduced. 6. Adverse effects of HIF activation on exercise performance HIF-induced changes in cellular energetics come at the expense of less ATP production and lead to rapid fatigue in exercising muscle. The adverse effects of a persistent increase in HIF activity on exercise performance is seen in patients with Chuvash polycythemia (Formenti et al., 2010; McClain et al., 2013). This autosomal recessive disorder is due to a mutation in the VHL gene resulting in an inability of VHL to bind to HIF-α resulting in stabilization of HIF and increased expression of HIF-target genes under normoxic conditions. Clinically these patients are polycythemic and have increased pulmonary artery pressures and reduced systemic arterial pressure. Upon exercise, these patients demonstrate early and marked depletion of phosphocreatinine and develop acidosis in skeletal muscle as measured with 31P magnetic resonance spectroscopy (Formenti et al., 2010). When compared to normal controls, lactate accumulation in blood is significantly greater and maximum exercise capacity is significantly reduced. In muscle biopsy specimens mRNA expression for PDK1 is increased along with increased expression of phosphofructokinase and pyruvate kinase. Upregulation of these enzymes are consistent with a shift in energy metabolism away from oxidative phosphorylation suggesting these patients are limited in the capacity to utilize oxygen in response to exercise. In a separate study random glucose levels and glycosylated hemoglobin A1c levels were reduced in these subjects when compared to normal subjects from the same geographical region (McClain et al., 2013). Mice with the same VHL mutation also demonstrate a reduction in glucose as well as lower glucose excursions consistent with increased skeletal muscle uptake and glycolysis. To further demonstrate the role of HIF on exercise capacity in skeletal muscle, mice have been generated in which the HIF gene is specifically deleted. These animals have greater exercise endurance as measured by a swimming endurance test or runtime on a treadmill when compared to wild type controls (Mason et al., 2004). Measurement of glycolytic and mitochondrial enzyme activity as



well as decreased amounts of lactate in the serum of exercising HIF1α knockout mice suggest these animals have increased activity of oxidative pathways in muscle. Furthermore the respiratory exchange ratio and the capillary to muscle fiber ratio in these animals indicate removal of skeletal muscle HIF-1α results in adaptations reminiscent of endurance training without the training (Mason et al., 2007).

7. Cellular adaptation to altitude Altitude acclimatization has been reported to blunt the accumulation of lactate during periods of exertion despite the persistence of hypoxia (West, 2007). Although controversial, this phenomenon has been referred to as the lactate paradox. It is interesting to speculate that in a manner similar to that observed in experimental animals, downregulation of HIF with chronic hypobaric hypoxia may decrease pyruvate to lactate flux and account for this metabolic change. Changes in HIF activity during acute and chronic hypoxic exposure may also explain the pattern of erythropoietin secretion measured at altitude. Circulating levels of erythropoietin increase rapidly with peak levels occurring 48–72 h after arrival to altitude. This rise is short lived however as levels return toward baseline values over the subsequent 5–10 days of acclimatization (Heinicke et al., 2003; Milledge and Cotes, 1985). With further ascent erythropoietin levels once again increase and depending on the degree of elevation may remain significantly elevated. Using erythropoietin levels as a biomarker of HIF activity, this pattern suggests feedback inhibition of HIF activity can be overridden by more extreme hypobaric hypoxia which accompanies higher altitudes. In this setting, high steady state levels of HIF activity may lead to untoward effects particularly with regards to the characteristics of weight loss.

8. HIF-induced alterations in cellular energetics in cancer The mRNA levels of both HIF-1α and HIF-2α increase several fold as compared to sea level values in skeletal muscle biopsies taken after 7–9 days at 4559 m (Robach et al., 2007). This situation is analogous to what has been described in cancer patients. As a tumor develops and grows, a hypoxic environment is created because of the extreme energy demands of the numerous, rapidly dividing cells (Tisdale, 2009). In these zones HIF is stabilized and results in a shift to glycolysis as the primary means for ATP synthesis. This phenomena known as the Warburg effect leads to increased lactic acid production and glucose uptake in cancer cells which are known indicators of tumor aggressiveness and poor patient prognosis. This metabolic shift is energy inefficient since only 2 ATPs are formed for each mol of glucose as compared to 38 ATPs formed when glucose is metabolized via oxidative phosphorylation (Fig. 2). It is estimated tumors require a 40-fold greater amount of glucose to provide the energy for growth because of this shift in metabolism (Eden et al., 1984). In a manner similar to what was described previously, lactate produced in tumors is delivered to the liver where it is resynthesized into glucose. Hepatic gluconeogenesis contributes to the energy wasting in that six ATPs are consumed in order to generate 1 mol of glucose from 2 mol of lactic acid. The increase in Cori cycle activity has been estimated to account for 300 kcal/day of additional energy loss in cancer patients (Holroyde et al., 1975). Cori cycle activity and glucose production and turnover rates are significantly greater in cancer patients with progressive weight loss in comparison to those whose weight is stable (Young, 1977). All of these effects are similar to that which occur following altitude exposure and increases in HIF activity.

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9. Genetic variability in HIF activation with altitude Variability in HIF-mediated changes in oxygen delivery and utilization influence performance at altitude. HIF-regulated genes contribute to the adaptive changes indicative of high-altitude populations. For example, lowlanders ascending to altitude have increases in their hemoglobin and hematocrit which are higher than basal levels of native highlanders (Erzurum et al., 2007). Tibetans living at high altitude have hemoglobin levels similar to lowlanders, and upon further ascent to higher altitudes develop much less of an increase when compared to those residing at lower elevations. Genomic and candidate gene comparisons of Tibetan highlanders and lowland Han Chinese indicate an evolutionary adaption in HIF-mediated erythropoiesis (Beall et al., 2010; Simonson et al., 2010, 2012). In these studies a significant divergence between Tibetans and lowlanders was noted in the allelic frequency of singlenucleotide polymorphism (SNP) alleles located in or near the HIF2α/EPAS1 and PHD2/EGLN1 genes which was correlated with lower hemoglobin levels in the Tibetans. Activation and stabilization of HIF-2α mediates hypoxia-induced increases in erythropoietin production, therefore, HIF-2α/EPAS1 and PHD2/EGLN1 allelic differences are responsible for the reduced hemoglobin concentration in the Tibetans thereby conferring a survival advantage at high altitude by facilitating a blunted hematologic response to hypobaric hypoxia. This blunted rise in hemoglobin is a favorable adaptation since polycythemia increases blood viscosity and adversely affects microcirculatory blood flow (Martin et al., 2009). Tibetan highlanders are more resistant to chronic mountain sickness or Monge’s disease, which is characterized by excessive erythrocytosis resulting in adverse effects on cardiovascular function due to increased blood viscosity when compared to the Han Chinese. There is also a correlation between hemoglobin concentration and haplotype variation in the nuclear receptor peroxisome proliferator activated receptor-α gene (PPARA) in Tibetan highlanders (Simonson et al., 2012). Hypoxia decreases and increases PPARα activity depending on the tissue examined. PPARα interacts with the HIF pathway to influence proteins associated with fatty acid oxidation. In a manner analogous to the effect on hemoglobin, the shift toward anaerobic metabolism under the dictates of HIF may be attenuated such that aerobic metabolism via oxidative phosphorylation is better preserved in Tibetans. This difference would allow a greater amount of ATP to be generated for exercising muscle and in part account for the enhanced climbing performance Sherpas exhibit on high altitude expeditions. Enzyme activity measured in Sherpa muscle biopsy specimens demonstrate low activity of LDH relative to pyruvate kinase, indicative of muscle metabolism shifted toward burning of carbohydrate to CO2 and H2O as opposed to lactate (Hochachka et al., 1992). In addition, the intensity of exercise at which point lactate begins to accumulate in the blood (lactate threshold) is greater in Sherpas when compared to lowlander populations. Body weight change in Sherpas, despite prolonged stays at high altitude, is minimal suggesting an attenuation of HIF effects on metabolism. Lowlanders ascending to altitude typically lose fat mass followed by muscle wasting at more extreme elevations (Boyer and Blume, 1984). In the American Medical Research Expedition to Everest (AMREE) study, Sherpas who arrived at base camp with half as much body fat as Western counterparts were able to maintain body weight during prolonged durations above 5400 m. Limb circumference remained the same in the Sherpas as compared to a fall in the Westerners indicating a preservation of muscle mass in the Sherpas (Table 1). 10. Sexual dimorphism in HIF activation at altitude There is a sexual dimorphism with respect to metabolism at altitude suggestive of hormonal influences of HIF activity. HIF shifts



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Table 1 Beneficial and adverse effects of HIF activation: influence of altitude. Parameter

5000 m

HIF activity

Persistent increase

Muscle mass

Initial increase followed by decline to lower steady state level (Ferretti, 2003) Initial increased with decline toward lower steady state level (Brahimi-Horn et al., 2007; Choi et al., 2005; Ou, 1974) Reduced followed by improvement with acclimatization Reduced with preferential loss of fat Stable

Leptin

Increased

ATP production

Mild reduction

Basal metabolic rate

Appetite

Body weight

Sustained increase

Persistent reduction

Reduced with loss of fat and lean muscle mass Reduced with decrease in mitochondrial volume Increased when factored for weight loss Severe reduction

metabolism to favor glucose utilization, and in two separate studies of men taken to 4300 m, whole body glucose uptake and glucose extraction was significantly increased when compared to sea level values (Brooks et al., 1991; Roberts et al., 1996b). This increase was detected within hours of arrival and remained elevated after 21 days of exposure. By contrast, in women taken to 4300 m blood glucose utilization rates were lower when compared to values measured at sea level (Braun et al., 2000). Along with a respiratory exchange ratio less than 1 these data suggest when women are exposed to altitude there is a reliance on fatty acids and a shift away from carbohydrate utilization which is unlike what happens in men. Similar effects are seen in female rats under conditions of hypobaric hypoxia (McClelland et al., 1998). This shift in metabolism toward fatty acid oxidation provides greater amounts of ATP and translates into improved exercise performance when compared to men under hypoxic conditions. Comparisons of men and women undergoing high-intensity intermittent static contractions of the adductor pollicis muscle at sea level and shortly after arrival to 4300 m demonstrate less altitudeinduced impairment in women (Fulco et al., 2001). In particular, women did not display the increase in overall fatigue rate and decrease in endurance time to exhaustion found in men. Altitude-induced changes in body weight are less in women as compared to men. Eight college women residing at 4300 m for 2.5 months had a decrease in skin fold thickness and reduction in limb circumference, but little change in body weight (Hannon et al., 1969). The authors concluded that women required no more energy intake at high altitude to maintain body weight as compared to sea level. When 12 women were taken to 5050 m for 21 days, mean body mass did not change and there was no significant difference in fat or fat free mass compared to baseline values (Ermolao et al., 2011). Measurement of basal metabolic rate is increased by 6.9% above sea level by day 3 at an altitude of 4300 m but falls to sea level values by day 6 (Mawson et al., 2000). This transient and short lived increase is in contrast to the greater and more prolonged increase in basal metabolic rate described in men at a similar altitude. Attenuation in HIF activation in response to hypobaric hypoxia in woman is an attractive way to account for the gender differences in glucose utilization, exercise performance, body weight change, and change in basal metabolic rate noted earlier. In a number of settings estrogen has been shown to downregulate HIF activity. In ovariectomized (OVX) female rats there is increased protein expression of HIF-1α in periaortic fat as compared to controls (Xu et al., 2012). Western blot and immunohistochemical staining show HIF-1α is reduced to control values in the periaortic fat of estro-

gen treated OVX females. In a rat model of obstructive sleep apnea in which animals are exposed to intermittent bouts of hypoxemia, administration of estrogen was found to significantly attenuate the fatigue measured in the genioglossus muscle (Resta et al., 2001). Both mRNA and protein levels of HIF-1α were increased in intermittent hypoxia animals as compared to controls; however, administration of estradiol was found to decrease both gene expression and protein levels of HIF-1α in a dose dependent manner. Physiologic doses of 17-β-estradiol attenuates hypoxia-induced erythropoietin gene expression by interfering with hypoxiainduced increases in HIF-1α levels and activity (Jia and Liu, 2010; Mukundan et al., 2004). Estradiol’s inhibitory effect on HIF-1α can be blocked in the presence of an estrogen receptor antagonist. Chronic mountain sickness is rare in premenopausal women and increases in frequency after menopause (Gonzales and Villena, 2000; Leon-Velarde et al., 1997), and this may be secondary to estrogeninduced reductions in HIF-mediated erythrocytosis. In animal models of hypoxemia, females develop less severe pulmonary hypertension, right ventricular hypertrophy, and polycythemia when compared to males (Jia and Liu, 2010).

11. Adipose tissue and hypoxia There is substantial evidence demonstrating white adipose tissue (AT) becomes hypoxic as adipocyte size and tissue mass expand in obesity. When faced with chronic, excessive energy intake, AT rapidly expands to provide increased lipid storage capacity. As the tissue expands, its growth velocity outpaces that of its supporting vasculature (Sun et al., 2011; Ye, 2009). Moreover, the adipocytes themselves become larger than the typical effective diffusion distance of oxygen in body tissue. The net result is that AT in obese individuals experiences a state of chronic hypoxia. Several mouse models have verified the existence of chronic hypoxia in adipose tissue using oxygen sensing probes. For example, the interstitial partial pressure of oxygen (PO2) of the epididymal fat pad of obese ob/ob mice was reported to be 70% lower than lean controls, while perigonadal fat pads of diet-induced obese C57BL/6 mice were reported to be 50% lower than lean controls (Halberg et al., 2009; Ye et al., 2007). The development of hypoxia underlies the development of inflammation and cellular dysfunction characteristic of adiposity. Hypoxic areas within adipose tissue are colocalized with macrophages suggesting an immediate link between hypoxia and the inflammatory response in adipose tissue. HIF-1 subunits have been directly linked to adiposity, as well as to the response to hypoxia, with increased levels of HIF-1α being evident in adipose tissue of obese mice (He et al., 2011). Transgenic mice in which HIF-1β is selectively lacking in adipose tissue show reduced weight gain relative to wild-type controls and resist the development of obesity when fed a high-fat diet (Lee et al., 2011). On the other hand transgenic overexpression of HIF-1α in adipose tissue leads to elevated body fat on a normal diet and increased obesity on a high-fat diet (Ichiki and Sunagawa, 2014). In hypoxic, dysfunctional AT, HIF-1 has been shown to upregulate mediators of fibrosis, inflammation and insulin resistance, including Col-6, IL6, NF-kB and TNFα (Sun et al., 2013). Table 2 lists key adipokines influenced by hypoxia (Famulla et al., 2012; Zhang et al., 2008). On balance the changes in these proteins would be expected to exert both pro-inflammatory and pro-angiogenic effects. In an attempt to maintain adipose tissue function under conditions of hypoxia, these effects may be counterbalanced by a reduction in NF-κB signaling (Famulla et al., 2012). In this regard hypoxia treated adipocytes show an attenuated response to TNFα as measured by macrophage chemotactic protein (MCP-1) and other NF-κB related



Table 2 Effects of hypoxia on gene expression of selected adipokines in fat cells. Increased expression

Reduced expression

Hypoxia inducible factor Leptin Vascular endothelial growth factor (VEGF) Matrix metalloproteinase MMP1 and MMP9 Angiopoietin-like protein 4 (ANGPTL4) Interleukin 6

Adiponectin Haptoglobin

cytokines. Moreover, downregulation of HIF-1 activation has been shown to improve AT insulin resistance in an obese mouse model (Ichiki and Sunagawa, 2014). Thus, convincing evidence exists implicating HIF-1 as a critical player in the cycle of hypoxia and inflammation leading to dysfunction in AT. Glucose utilization through the glycolytic pathway is strongly upregulated in adipocytes by hypoxia reflecting a switch from aerobic to anaerobic metabolism under the dictates of HIF-1 activation. Lactate production increases in hypoxic adipose tissue with the amount released increasing in obesity (Trayhurn, 2013). Accumulation of lactate has been implicated in adipose tissue dysfunction by stimulating inflammation and contributing to the antilipolytic action of insulin (Ahmed et al., 2010). 12. Hypoxia inducible factor in diabetes mellitus: effects of hyperglycemia Recent evidence suggests hyperglycemia can promote the degradation of HIF through pathways independent of the PHD system and in the presence of ongoing hypoxia. Since activation of HIF promotes cell survival in low-oxygen conditions such pathways could prove detrimental to cell function and survival under conditions of low oxygen levels (Bento and Pereira, 2011). Down regulation of HIF has been implicated in poor would healing, decreased arteriogenic response to myocardial ischemia, and development of cardiomyopathy in the setting of diabetes (Botusan et al., 2008; Marfella et al., 2002; Romana Bohuslavova et al., 2014). Down regulation of HIF1α impairs translocation of the glucose transporter 4 (GLUT-4) into the plasma membrane of skeletal muscle further contributing to impaired glucose metabolism (Sakagami et al., 2014). Strategies designed to maintain HIF activity under conditions of hyperglycemia could prove useful as a therapeutic approach for the treatment of diabetic complications associated with hypoxia. 13. Summary Activation of HIF is a critical step in promoting cell survival with minimal dysfunction in the setting of limited oxygen availability. Depending on the cause of hypoxemia HIF activation alters metabolic homeostasis in both favorable as well as unfavorable ways. Under condition of hypobaric hypoxia activation leads to weight loss accompanied by increased basal metabolic rate and suppression of appetite. These effects are dose dependent, gender and genetic specific, and results in adverse effects if the exposure is extreme. In the setting of obesity hypoxia is more regional and largely confined to adipose tissue. In this setting activation contributes to inflammation of adipose tissue contributing to various manifestations of the metabolic syndrome. In the setting of hyperglycemia, downregulation of HIF impairs cellular adaptation to low oxygen levels and may contribute to end organ injury in patients with diabetes. A greater insight into the factors regulating HIF activation and effect will allow newer therapies to be developed tailored to the underlying disease state giving rise to hypoxia.

7

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