Metabolic and nutritional consequences of chronic hypoxia

Clinical Nutrition (1998) 17(6): 241-251 © 1998 Harcourt Brace & Co. Ltd

REVIEW

Metabolic and nutritional consequences of chronic hypoxia* X. LEVERVE Service d'Accueil, d'Urgences et de R~animation M~dicale, Unit~ de Nutrition Parent~rale, CHU, Grenoble, Laboratoire de Bio~nerg~tique Fondamentale et Appliqu~e, Universit6 J. Fourier, Grenoble, France (Correspondence to: X. L., Laboratoire de Bio6nerg6tique Fondamentale et Appliquee, Universit~ J. Fourier, BP 53X, F-38041 Grenoblecedex 09, France)

Key words: hypoxia, oxygen, metabolism

is the oxygen consumption still low despite normal cardiac index and oxygen delivery? The third kind of observation concerns the efficacy of oxygen therapy in COPD patient's outcome. Indeed, it has been shown that intermittent oxygen therapy very significandy improves the patient's outcome (4) and, furthermore, that continuous therapy (24 h/day) is more effective than intermittent administration (12-h/day) (5). Such a result is undoubtedly proof for the beneficial effect of oxygen but it does not mean that the 'substrate oxygen' was lacking. Indeed, since there is a very limited storage of oxygen in humans, 3-4 rain of autonomy, this excludes any effect of intermittent therapy by an 'oxygen storage' mechanism. Moreover, if oxygen autonomy is so low, we might expect a very quick positive effect of oxygen supply in the case where oxygen is supposed to be lacking while it takes almost 1 year to significantly affect the mortality rate. Hence (i) COPD is a disease due to a lack of oxygen but where oxygen consumption is normal, (ii) impairment of oxygen consumption due to a low cardiac output and oxygen delivery is not corrected by the correction of cardiac output and (iii) oxygen therapy improves the survival rate after 1 year and only when delivered either in a continuous or in intermittent manner while oxygen storage is limited to 3-4 minutes. These facts clearly show that oxygen is not a simple metabolic substrate which can be replaced like other kinds of substrates like glucose. In fact, oxygen is so important that it cannot lack for a sustained period without leading to death. Oxygen is much more that a substrate, it is a signal.

Introduction

The relationship between life and oxygen is so close that the term 'air' (aero-) is contained in the words defining the two types of life energy metabolism: aerobic or anaerobic. This is due to its unique property as electron acceptor: oxygen is one of the most powerful oxidizing agents and the couple oxygen-water has the lowest reducing potential permitting to 'extract almost entirely' the energy content of the electrons. Despite a multitude of effects including several biosynthetic pathways (cholesterol synthesis, for example) and free radical production, oxygen is often confined to its role as respiratory-chain substrate in energy metabolism of aerobic life. In this sense, oxygen is mainly viewed as a substrate but when considering several classical or common clinical situations, it is possible to show that oxygen is in fact much more than a metabolic substrate. Indeed, human life as a whole is dependent on oxygen and any change of its concentration and/or availability (often defined as hyperor hypoxia) is potentially extremely harmful. As a first example, if we consider a patient suffering from a severe chronic obstructive pulmonary disease (COPD), the severity of the disease is often related to the degree of hypoxia evidenced by the low PaO2, implying, in fact, that a lack of oxygen is responsible for the disease. Most of the time, however, the energy expenditure (i. e. oxygen consumption) of such a patient is either normal or even slightly increased (1-3). Hence, we may address the following question: how can we explain that such a patient is actually lacking oxygen while his oxygen consumption is normal? In the second example, a patient with a severe cardiac failure, due to a progressive dilated cardiomyopathy, has a very deep limitation of oxygen consumption which is clearly due to the severity of the circulatory failure and then to a very limited oxygen delivery. When such a patient undergoes a successful heart transplantation, however, leading to a normalization of the heart function, he has an abnormal and limited oxygen consumption rate for a long time. Why

Oxygen and energy metabolism

Energy transduction: the role of oxygen Life may be viewed as a transduction of energy through several forms, i. e. the succession of different energy potentials. For example, the chemical energy which is contained in one molecule of glucose can be transferred to another form of energy (Fig. 1A): the phosphate potential (ATP/ ADP.Pi) in the fermentation pathway (glycolysis). Because of the presence of the Na ÷, K + ATPase, this form of energy can be further converted by an active electrogenic sodium/ potassium exchange leading to a new form of energy: the transmembrane potential (Ap), which in turns permits some

*Based on the Sir David Cuthbertson lecture, 20th ESPEN Congress, Nice, 16-19 September 1998 241

242

METABOLIC AND NUTRITIONALCONSEQUENCES OF CHRONIC HYPOXIA

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Fig. 1 Examples of living energy transducing systems. A: Anaerobic system. Energy source is provided by organic nutrients (i.e. made by a living system) converted through a pathway (fermentation) resulting in ATP generation (phosphate potential or ATP/ADP.Pi ratio). The plasma-membrane ATPase (sodium/potassiumATPase) permits to convert this phosphate potential into a membrane potential (Ap)because of the electrogenic exchange of 3 Na+ with 2 K÷ and permitting to the cell to generate some works. Erythrocyte represents a good example of a completely anaerobic cell where the unique source of energy is glucose and the unique energy wastage is lactate with a stoichiometry of 2 moles of ATP for one mole of glucose. B: Phototrophicsystem. Energy source is the light which permits to extract hydrogen from water. The transduction of energy into ATP and then to membrane is similar except the presence of a sodium/potassium ATPase which is replaced by a proton ATPase. C: Aerobic system. The source of energy is the redox power contained in the nutrients (carbohydrates or lipids), electrons from hydrogen being transferred in fine to oxygen permitting the formation of water. The successive energy transduction processes are similar to those of the anaerobic system (see A) but the production of ATP is much higher. The stoichiometry of ATP synthesis (see table 1) depends of the substrates used and the wastage products are water and CO>

work, transport for instance (6). In this example, energy transduction occurs even in absence of oxygen, the substrate being glucose and the wastage product lactate. Of course, one the main questions is the origin o f the initial energy source since glucose must have been synthesized by a living organism. Four billion years ago, the atmosphere surrounding the earth was totally lacking oxygen: free oxygen was the result of life! Later, 1.5-2 billion years ago, new organisms appeared able to use the energy of the light to extract hydrogen from water (phototrophy). In this case (Fig. 2B) the initial substrate is water, the energy source comes from the light, and the wastage product is oxygen. Hence, due to a progressive increase in oxygen production from life, the atmosphere was progressively (and quite rapidly as compared to the duration o f evolution) enriched from 0% to 20% between 2 and 1.5 billion years ago. Because of the extreme toxicity of the free radicals coming from oxygen, an actual 'holocaust by oxygen' (Christian de Duwe) occurred which caused possibly the greatest disappearance of life ever seen on Earth (7). Survival of some organisms was probably linked to the presence of several protective antioxidant compounds as well as to the emer-

H

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Fig. 2 Schematicrepresentation of the oxidative phosphorylation pathway. Respiratory chain is a succession of enzymatic complexes catalysing oxidoreductions from substrates with a high degree of reducing potential (NADH or FADH2coming from the Krebs cycle or from other dehydrogenases of intermediary metabolism) to water, the compound with the lowest reducing potential. By allowing a vectorial export of proton, three complexes (I, II, IV) catalyse an energy transduction into another form of energy: the chimioosmotic potential (protonmotiveforce, i.e. the proton gradient across the inner membrane). This potential is converted into phosphate potential (ATP/ADP.Pi)by the ATPsynthase (complex V). A leakage of proton into the matrix results in an uncoupling of oxidative phosphorylation, the energy resulting from the successive oxidoreductions being dissipated as heat.

gence of new pathways reducing the toxic free oxygen to the non-toxic form of water. This was achieved by a new enzyme activity, i.e. the cytochrome oxidase and it is possible that the selection of this activity was initially more related to its property for lowering oxygen concentration than for leading to A T P synthesis. Progressively, during the millions of years of subsequent evolution, a complete pathway was organized, i.e. the mitochondrial respiratory chain, leading to the synthesis of ATP through the oxidative phosphorylation coupling (Fig. 1C & Fig. 2) (6). Hence, this pathway is able not only to protect against oxygen toxicity but also to use it in combination with the nutrients as an almost inexhaustible supply of energy since it comes indirectly from sunlight. O f course, in very complex organisms such as mammals and humans, the co-existence of high oxygen needs for energy supply with the severe toxicity of high oxygen concentration has required very efficient and sophisticated physiological systems such as hemoglobin, myoglobin, ventilation, circulation, etc. It must be kept in mind that even if the data concerning the actual oxygen concentration in the vicinity of mitochondria is very difficult to assess precisely, it is believed that it is extremely low, i.e. in the range of 1% of atmospheric oxygen, i.e. 0.2 kPa or 2 p M (8). As a matter of comparison, most of the other metabofic substrates (glucose, fatty acids, ketone bodies, amino acids, etc) are 100-1000 fold more concentrated. Mammals energy metabolism: aerobic, anaerobic or both?

The size of total A T P turnover and o f total oxygen consumption are very close but they are not identical. Indeed, it is well known that a part of A T P synthesis is actually achieved from the 'anaerobic' glycolysis while a part of oxygen consumption is not devoted to A T P synthesis but to other pathways requiring free oxygen (biosynthetic pathways and free radical production) (6). Depending on

CLINICAL NUTRITION

the different tissues and the physiological situations, the part of non-aerobic ATP production is generally estimated to be around 10% of the total value and that of nonphosphorylating oxygen consumption is within the same range. Hence, this would lead us to think that 90% of oxygen consumption is responsible for 90% of ATP synthesis and therefore that 10% of ATP synthesis in our body is independent of oxygen. As shown previously (Fig. 1A) a purely anaerobic pathway is characterized by the use of glucose as substrate and a release of lactate (2 lactates for 1 glucose) for the wastage of both carbons and reducing equivalents since in absence of oxygen, NADH can only be reoxidized by converting the glycolytic end-product pyruvate to lactate. This is achieved in the erythrocyte, a mitochondria-lacking cell, where anaerobic ATP production is the unique source of energy (Fig. 3). There is no doubt that some anaerobic metabolism occurs in humans. Conversely, (Fig. 3) liver energy metabolism is aerobic since oxygen consumption is very active, the reducing substrate coming predominantly from fatty acid B-oxidation. Moreover, since there is no net lactate release, it is possible to say that liver is a totally aerobic organ. If we consider erythrocyte and liver metabolisms together as a whole, it is clear that lactate coming from the erythrocyte is metabolized by the liver into glucose (Coil's cycle). Hence, in the couple liver plus erythrocyte there is oxygen and fatty acid consumption and no net lactate release from glucose. This is the metabolic feature of a fully aerobic metabolism: the liver respires for the red blood cells. It is possible, therefore, to draw the following conclusion: that as long as there is no net lactate accumulation or excretion, i.e. in all steady state situations, energy metabolism in humans, as in every mammal, is fully aerobic (9, 10). Even in the case of a very exhausting physical effort, there is some anaerobic metabolism during the phase of lactate accumulation, but during the subsequent phase of recovery the accumulated lactate is metabolized in such a way that the net resulting effect of both phases is a fully aerobic

Glucose --

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Combination of anaerobic and aerobic pathways results in a fully aerobic life. The glucose recycled via lactate creates a futile cycle between 3 and 6 carbon compounds. On one hand this dissipates directly 2/3 of the energy as heat (since 6 ATP are needed to build one glucose from 2 lactate whereas 2 ATP only are produced when splitting glucose into lactate), but on the other hand, the source of energy in liver is mainly coming from fatty acid oxidation. Hence, glucose recycling provides 'glycolytic ATP' to several peripheral cells (erythrocytes for instance) while this ATP is formed from energy coming actually from lipid oxidation. In other words, one can say that 'liver respires for anaerobic tissues like erythrocytes'.

243

energy metabolism. Similarly, in pathological situations, even at very severe stages, as long as lactate concentration is stable (whatever its concentration) and not quantitatively excreted, energy metabolism is aerobic.

Production and distribution of energy: cellular strategy and priorities

Control of oxygen consumption and A TP production It is clear that ATP synthesis flux is tightly connected to the oxygen consumption rate: oxygen reduction into water (i. e. respiratory chain flux) being linked to matrix proton translocation connected to ATP synthesis and conversely ATP synthesis being only possible when a sustained membrane potential is maintained due to the activit~ of the respiratory chain (Fig. 2). Hence, in absence of uncoupling (see later) oxygen can be reduced to water only when there is a need for ATP synthesis, i. e. in presence of ADP and Pi: oxidation is dependent on phosphorylation and vice versa. Because there is no possibility for storage of substrates of oxidative phosphorylation (oxygen and ADP) nor of product (ATP), one may realize that this pathway is indeed an example of a very tight metabolic control between oxidation rate and ATP production and utilization. Furthermore, since oxygen accumulation might be very toxic for the mitochondria, the rate of oxygen delivery to the cell must be precisely adjusted to that of oxygen consumption, in order to avoid excessive free radical production. These considerations raise the question of the control of oxygen consumption and ATP synthesis as represented in Fig. 4. The sophisticated machinery of the oxidative phosphorylation pathway can be simplified as a single step catalyzing the following reaction: NADH + 02 + ADP --~ NAD

+

H20 + ATP

As in any biochemical reaction, the rate through this step is dependent on kinetic as well as thermodynamic parameters. The kinetic parameters are represented by enzyme(s) characteristics: activity and affinity. The resulting thermodynamic strength applied to a given step is the difference between upstream and downstream potentials. In the case of oxidative phosphorylation (Fig. 4), if we neglect the OzPrI20 potential as it is almost constant, the upstream potential which 'pushes' the flux is represented by the redox potential (NADH/NAD) while the downstream potential which 'fimits' it is represented by the phosphate potential (ATP/ADP.Pi). Thus, an increased NADH/NAD ratio and/or a decreased ATP/ADP.Pi ratio increases the flux whereas a decrease in NADH/NAD and/or an increased ATP/ADP.Pi ratio results in a decreased oxidative phosphorylation flux. Of course, the upstream potential (redox potential) depends on the activity of the pathways leading to NADH production (among them the Krebs cycle plays a main role) while the downstream potential (phosphate potential) depends on ATP hydrolysis, i.e. the cellular work. From these considerations it appears that a large change in flux of ATP synthesis must be accompanied by a large change in the

244 METABOLIC AND NUTRITIONAL CONSEQUENCES OF CHRONIC HYPOXIA

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Fig. 4 Regulation of oxidative phosphorylation flux. The control of oxidative phosphorylation is exerted by two different factors: kinetic parameters (i.e. kinetic properties of the different enzymes) and thermodynamic factors (i.e. forces exerted on the system, see text). A dramatic change in the flux without large change in either redox potential or phosphate potential implies subtle co-ordinated mechanisms as it is done for instance by calcium or fatty acids which affect the oxidative phosphorylation pathway simultaneously upstream and downstream.

related forces. This leads to a paradox: a sustained high flux of ATP production can be achieved only if ATP concentration is very low! Hence, it is clear that a much more subtle mechanism of regulation is needed in order to achieve large changes in ATP synthesis flux at a nearly constant forces (11, 12). The first mechanism showing this possibility of regulation clearly was related to the effect of calcium on energy metabolism. Indeed, it has been known for long time that a calcium rise was responsible for an increase in energy dissipating processes, e.g. myofibrillar contraction. It has also been shown that several dehydrogenases (and particularly those from the Krebs cycle) are also activated by calcium. Hence, the increase in calcium results in coordinated changes affecting energy metabolism via a simultaneous increase of both NADH supply system (Krebs cycle) and ATP consuming processes (muscle contraction) in such a way that this large change in oxygenconsumption/ATP-synthesis pathway does not affect the related forces: redox or phosphate potentials (13). There are probably many other coordinated mechanisms of regulation permitting to regulate the flux at constant forces, fatty acid metabolism has been shown to stimulate also the pathway simultaneously upstream and downstream of the oxidative phosphorylation (14, 15).

Modification of the yield of oxidative ATP synthesis From the considerations previously discussed, it seems that ATP synthesis from oxidative pathway is achieved in a fixed stoichiometry: a given respiratory rate results in a given ATP synthesis flux. The ratio between these two fluxes, the yield of oxidative phosphorylation pathway, is expressed as the ATP/O ratio, i.e. the number of moles of ATP synthesized with the formation of one mole of water from one atom of oxygen. Classically, this ratio is assumed to be equal to 3. In fact, there are many factors affecting this stoichiometry. Assessment of ATP turnover is extremely

III) Effect of respiratory chaine coupling (slipping)

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Fig. 5 Modulation of the yield of ATP synthesis (ATP/O ratio). The yield of ATP synthesis can be modulated by three different factors. I - the nature of the respiratory substrates (NADH or FADH2) since they involve either two or three coupling sites, the difference in ATP/O being 1/3. II - the proton permeability of the inner membrane (membrane proton conductance or leak) by dissipating some energy in heat affects the yield of ATP synthesis. Such uncoupling can be related to membrane composition and/or uncoupling protein (UCP) I I I - the efficiency of the coupling of the mitochondrial proton pumps (i.e. the slipping between oxidoreduction and vectorial proton transport).

difficult in steady-sate living systems, such as intact cells and of course organs or organisms, explaining that the experimental values are mainly coming from isolated mitochondria, i.e. far from the physiological conditions (11, 12, 16). There are three different factors affecting the ratio of ATP/O (Fig. 5): 1) The nature of the substrate: from one mole of glucose metabolized through the glycolytic pathway two moles of ATP (and three when the precursor is glycogen) are produced in the absence of any oxygen consumption. This is responsible for an apparent increase in the ATP/O ratio when compared with other substrates. Moreover, the relative proportions of NADH and FADH 2 affect the ATP/O stoichiometry since NADH enters the chain at the first complex (involving three coupling sites) while FADH 2 enters at the second complex (involving then only two coupling sites) therefore the difference in yield is 1/3 (Fig. 2). This phenomenon is of importance for the [3-oxidation of fatty acids which produces a higher proportion of FADH2 as compared to the Krebs cycle. A similar phenomenon occurs in hyperthyroidism since thyroid hormones are responsible for an increased activity of the mitochondrial glycerol,3phosphate dehydrogenase which provides reducing equivalents directly to the complex 3. 2) The proton permeability of the inner mitochondrial membrane: it is clear that the degree of permeability (membrane conductance) of the inner mitochondrial membrane is directly related to the efficiency of the system; the more protons can enter into the matrix by shunting the ATP synthase the more oxygen reduction lead to heat production instead of ATP synthesis (17, 18). This phenomenon is known as 'proton leak' or 'proton uncoupling'. It has been extensively studied since it plays a major role in energy homeostasis: heat production and basal metabolism

CLINICAL NUTRITION

(19-21), control of obesity (22, 23), control of redox state and of mitochondrial production of free radicals (24), etc. The change in the membrane conductance can be due either to a change in membrane lipid composition (14, 15, 17, 25) or to the presence of uncoupling proteins (UCP) (19, 21, 23, 26-30). Since the discovery of 'thermogenin' (UCP-1) and its major role in heat production in brown adipose tissues, two other uncoupling proteins (UCP-2 and UCP-3) have been found in several tissues including muscle, spleen, thymus, white adipose tissue, macrophages, etc. (20, 22-24, 30-33). The physiological meaning of these regulatory mechanisms in different tissues is not yet fully understood but it is clear that it is probably a very important point in physiology and probably in pathology as well. 3) The intrinsic efficiency of the coupling of the proton pumps: the proton pumps of the oxidative phosphorylation pathway comprise a vectorial transduction of energy, e.g. a proton extrusion from the mitochondrial matrix coupled to a redox transfer for the enzymes of the chain or a proton entry into the matrix coupled to ATP synthesis for the mitochondial ATP synthase. The decrease in coupling efficiency, called 'slipping' was first proposed to explain the effect of general and local anesthetics, it occurs also in the case of polyunsaturated fatty acid deficiency in rats (12, 16, 25, 34-36). Although, in general, the needs for oxidation and phosphorylation appear to be parallel, it is probably necessary to slightly change one of these two parameters without modifying the other. Both protonophore uncoupling and enzyme slipping allow to obtain a fine and separate regulation of oxidation and phosphorylation fluxes, but in one case (uncoupling) the membrane potential is decreased by the proton leak while in the second case the respiratory rate is modified despite a constant membrane potential. Cellular strategy and priorities

The mechanism by which ATP is distributed to the different cellular sites of energy dissipation (e.g. biosynthetic pathways, muscle contraction, metabolite transport and membrane potential maintenance, gene transcription and translation, protein synthesis and degradation, etc.) is not well understood (37, 38). It is often supposed that in the cytosol, ATP diffuses freely, specific carriers being involved only for passing through organelle membranes. Such a simple view cannot hold if we consider that in this case all pathways would be in direct competition between each others for ATP supply. Furthermore, a diffusion through the cytoplasm would prevent any specific information or energy supply to a specific step. During the last decade, numerous works have extensively studied this problem principally on muscle and cardiac cells by investigating the role of the phosphocreatine/creatine shuttle in the cellular transfer of energy (37, 39-43). From these works it seems that cytosolic and mitochondrial creatine kinase serve mainly as energy shuttle (Fig. 6) permitting the channeling of energy from one cellular location (a part of the mitochondrion membrane) to a precise site of energy utilization

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Fig. 6 Channelling of energy in cardiac cell. The pathway for intra cellular energy transport and the corresponding feedback signal conduction in muscle, brain and many other types of cells (but excluding liver cells) is the phosphocreatine pathway (energy circuit or energy shuttle). This canalisafion permits to transfer a 'wave of energy' from a precise location in the mitochondfial membrane to a precise site of dissipation (myofibrils, Ca++ATPase in the sarcoplasmic reticulum, sodium ATPase, etc.). Conversely, the signal given by a rise in ADP, due to a mechanical work for instance, is channelled to the mitochondfial matrix without effective transportation of the ADP molecule. From this picture it is clear that the meaning of an averaged cellular ATP or ADP concentration becomes limited. From Valdur Saks (39) with permission.

(myofibrillar ATPase, NA +, K+ ATPase, endoplasmic reticulum ATPase, biosynthetic pathway, etc.). It is of interest to note that in such a view the 'energy rich bound' is transported rather than ATP or creatine phosphate molecules per se. Hence, by playing with the location of creatine kinase (for instance by the transcription of different isoforms) the cell can build new tracks for energy channeling, expressing by this way new priorities. Recently, there was a growing interest in the literature concerning the role of mitochondrial metabolism in some major cellular strategies like modulation of calcium signaling (44-47) or cellular death by apoptosis (48-50). The finding of a probable physiological role for a permeability transition pore (PTP) in these cellular phenomena suggest a physiological link between calcium, redox state, phosphate potential and mitochondrial proton driving force (51-56). Thus, oxygen as well as oxidative phosphorylation flux are probably connected to the regulation to these major cellular events.

Lacking oxygen: how to know, how to face? How to know ?

As proposed previously, the lack of oxygen in vivo is much more complex than it is the case of any other metabolite. Indeed, this substrate is so crucial for cell surviving that energy metabolism has to adapt always precisely to oxygen supply and ATP production rate. In fact, in order to better adapt, there is some evidence showing that the cell can even anticipate a possible lack of oxygen. This phenomenon is shown in Fig. 7. In this work by P. Schumacker, isolated

246 METABOLIC AND NUTRITIONAL CONSEQUENCES OF CHRONIC HYPOXIA Oxygen consumptionand cellularATP as a function ofPO2 14 o-d~ 2

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Fig. 7 Oxygen sensing: kinetic effect of progressive oxygen impoverishment. Hepatocytes were exposed to rapid (q < 40 min) or slow (1 > 2 hr) deoxygenation, P Q of the media being reduced from 100 to 0 ton-. At 100, 70, 40, 15 and 0 ton-, samples were taken out for measurements of oxygen consumption (left panel) and ATP concentration (right panel). Fast decrease in oxygen content of the media does not affect oxygen consumption nor ATP concentration except of course when oxygen tension was 0. Conversely, when oxygen impoverishment was slower, it was a clear relationship between oxygen consumption or ATP concentration and oxygen tension. Adapted from Schumacker et al. (57).

hepatocytes respond to the oxygen tension in the medium according to the time course of the changes (57). It is clear from Figure 7 that changes of oxygen in the medium ranging from 100 to 20% over a short period (40 rain) did not affect both oxygen consumption and ATP concentration while when it occurs over several hours there is a significant effect on both parameters. It must be added that between 90 and 20% of oxygen, cells are of course never hypoxic. It appears, therefore, that cells do sense the oxygen tension. If the complete pathway of the cellular signaling related to oxygen sensing is not fully understood some steps are already evidenced as well as their effects on gene expression. This signaling pathway is schematized on Figure 8 (58-60). Cellular metabolism is able to sense" oxygen in order to anticipate several metabolic changes allowing to protect the cell (or the body) against the dramatic effect of a lack of oxygen (61-70). These changes are oriented in the direction of an increased efficiency together with changes in cellular priorities. Hochachka has recently summarized these adaptive changes to hypoxia in two different phases: defense and rescue (61). The defense phase occurs after a decline in oxygen has been sensed by the cell, the signaling pathway is activated leading to major metabolic changes: channel arrest and decreases in Na + K + ATPase, urea synthesis, gluconeogenesis and finally in protein synthesis and proteolysis (a highly ATP consuming process) in such a way that ATP demand equals ATP production which at the maximum is near zero. Then follows the rescue phase involving the transcriptional consequences of the signaling pathway: (1) activation of genes involved in transcriptional factors (Hypoxia Induced Factor or HIF), (2) HIF mediated activation of genes for sustained survival at low ATP turnover (increased glycolytic enzyme, decreased enzyme involved in aerobic-linked metabolism) and finally production of tertiary messengers (fos and jun, Fig. 8).

3' end

Fig. 8 Cellular oxygen sensing pathway. Cellular oxygen sensing pathway involves plasma-membrane oxidoreductiou sensing followed by a cellular signalling amplification leading to several effect located at the gene transcription regulation level. From Hochachka et al. (61).

How to adapt? The adaptation to hypoxia has been largely studied both in animals and in humans either in physiological conditions (hypobaric hypoxia due to altitude) or to normobaric hypoxia as it is the case in patients with severe pulmonary disease. These adaptive mechanisms can be divided in behavioral and metabolic changes. 1) behavioral and physiological changes: a strong initial decrease in food intake associated with a clear decrease in body weight, a drop in physical activity and body temperature, a hypertrophy of the right ventricle associated with an increased hematocrit which is related to an increased erythropoietin secretion by kidney. Actually, the first oxygen-sensing signaling system was described on renal cell and erythropoietin pathway (58, 60). 2) The metabolic changes related to hypoxia exposure are very broad and affect probably several pathways, many of these effects remaining to be described. For instance, the negative effect of hypoxia on nitrogen balance is well known (71-75), but the precise effect on protein synthesis and degradation is not clearly known (69). During recent years we have been interested in lactate metabolism during experimental hypoxia in rat (9, 76-78). Indeed, due to its pivotal role in energy metabolism, lactate is probably a key intermediate in the metabolic adaptation toward hypoxia. There is a well known increase in lactate production, in muscle for instance, as well as in plasma concentration leading to an increase in the lactate-glucose Coil's recycling. Thus, we have investigated the liver gluconeogenesis from lactate with the hypothesis that in vivo hypoxia would lead to an enhanced liver lactate uptake (76). Surprisingly, we found a very clear inhibition which was located at the phosphoenolpyruvate carboxykinase step: its transcription was depressed by hypoxia. As summarized in Figure 9, the net result of hypoxia on lactate metabolism is an increase in the steady state blood lactate concentration due to an increase in peripheral lactate production associated with an inhibition of lactate metabolism in the gluconeogenic pathway because of the decreased PEPCK

CLINICAL NUTRITION 247

A) NORMOXIA GLUCOSE

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Fig. 9 Effect of hypoxia on lactate metabolism. Hypoxia is responsible for a simultaneous increase in lactate formation in some tissue due to the limitation of aerobic energy supply and also to an inhibition of liver ghiconeogenesis due to decrease in PEPCK activity. The resulting effect is an increase in lactate concentration but without parallel increase in the energy wastage process lactate-glucose-recycling. Hence the increase in lactate may result in no or only minor increase in glucose flux (i.e. Cori's cycle).

B)

HYPOXIA

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activity. Since in vivo liver gluconeogenesis is not at its maximal rate (i.e. saturating flux), an increase in lactate concentration can compensate for the decreased PEPCK activity in such a way that the resulting glucose-lactate turnover can be normal or even increased despite the inhibition of gluconeogenesis, depending on the lactate concentration. It appears, therefore, that the main resulting effect is an increased plasma lactate concentration and then the question arises of the metabolic meaning of such an increase in lactate. As shown in Fig. 10, the increase in lactate might be viewed as a key metabolic event permitting to modify the metabolism according to the new condition of hypoxia. We would propose a similar metabolic picture as has been proposed for ketone bodies metabolism during fasting. In this particular situation, the very large increase in plasma ketone body concentration is probably a major event permitting to spare glucose oxidation in brain, acetoacetate being predominantly oxidized (79, 80). A similar picture can be proposed concerning hypoxia but with lactate instead of ~-hydroxybutyrate as competitive substrate to glucose. In normoxic condition (Fig. 10A) plasma lactate is low and its metabolic fate is mainly recycling via liver gluconeogenesis, while in peripheral tissues, like muscle, the main substrate is the glucose which is oxidized to CO2, a small part being possibly released as lactate. During hypoxia (Fig. 10B) due to the high lactate concentration, the competition between lactate and glucose as carbohydrate source for oxidation is in favor of lactate. This permits to spare some glucose for privileged tissues such as heart (62). It has been shown in humans that chronic hypoxia led to an increased glucose oxidation in the heart (79, 80). According to this, the increased lactate is a metabolic tool permitting to oxidize either lactate or glucose as aerobic substrates according to a 'metabolic priority' (9). Indeed, glucose oxidation becomes a clear advantage in terms of oxygen energetic yield (ATP/O ratio) because of the non-aerobic ATP provided by the glycolysis pathway (Table 1).

Fig. 10 Competition between glucose and lactate as oxidative carbohydrate source. A Under normoxia, glucose is the main source for carbohydrate oxidation in several peripheral tissues, muscle for instance. Lactate is mainly produced from anaerobic cells (erythrocytes or cells of lens, cornea, renal medulla, etc.), it might be also released from many other kinds of cells but in limited amount. Lactate is mainly metabolised through liver gluconeogenesis while it can also be oxidised in some organs. B In the case ofhypoxia, leading to a high lactate plasma concentration (see figure 9), there is a competition between lactate and glucose for carbohydrate supply as substrate for mitochondrial oxidation. Due to its high plasma concentration, lactate could be oxidised preferentially in some tissues (like muscle), the glucose being then 'spared' for privileged tissues (like heart). Hence, the heart benefits from the glycolytic ATP whereas the oxidative tissues have only access to the purely oxidative part of the pathway (i.e. from pyruvate to CO2).

Table 1 Oxygen consumption, ATP synthesis and wastage production for glucose, palrnitic acid and a standard protein

Glucose Palmitic acid Standard protein Molar mass (g) 180 256 O z consumed (l/g) 0.747 2.013 CO2 produced (l/g) 0.747 1.4 1-I20 produced (g/g) 0.6 1.125 Respiratory Quotient 1.00 0.70 Energy potential (Kcal/g) 3.87 9.69 02 energy equivalent (Kcal/1) 5.19 4.81 CO2 energy equivalent (Kcal/1) 5.19 6.92 Synthesized ATP, tool/tool 38 129 Synthesized ATP, Kcal/mol 456 1548 Yield of synthesized ATP 0.65 0.62

2257.4 1.045 0.864 0.427 0.83 4.704 4.50 5.44 450 5400 0.51

ATP production was calculated by using a fixed mitochondrial stoichiometry i.e. 3 ATP/NADH and 2ATP/FADH z.

248

METABOLIC AND NUTRITIONAL CONSEQUENCES OF CHRONIC HYPOXIA

Nutritional implications of chronic hypoxia

Treatment of undernutrition ?

Effect of COPD on nutritional status

Whatever the cause of hypoxia is (living at high altitude or chronic pulmonary disease), in any case oxygen delivery must meet oxygen needs, i.e. energy demand. Two major mechanisms are involved to reach such balance: (i) oxygen delivery is increased (hyperventilation, cardiac hypertrophy with increased cardiac output, increased erythrocyte number, etc.) and (ii) oxygen consumption is decreased (decrease in physical activity and in lean body mass for instance). These adaptive changes permit to reach a new steady state where more oxygen is consumed in some tissues and less in others. Table 1 shows that the different substrates (carbohydrate, lipid or protein) are not equivalent as energetic substrates. It is very clear that, if on one hand lipids contain much more energy per unit of mass than glucose, on the other hand the energy equivalent per liter of oxygen is less for lipids than it is for carbohydrates. As already discussed above, in chronic hypoxia as in normal life, energy metabolism of the body as a whole is entirely aerobic. The use of lipids, therefore, has two advantages: very large energy storage and high energy content per gram, and two drawbacks: low oxygen yield and wastage energy due to carbohydrate recycling. Similarly, the use of carbohydrates has also two main advantages: high oxygen yield and no need for substrate recycling, and two drawbacks: very low storage and high cost in terms of muscle protein for neosynthesis, but they are opposite to those of lipids. Hence a new compromise between organs and substrates must be found.

The nutritional consequence of chronic hypoxia has been studied in patients suffering from COPD. The main metabolic alterations leading to the nutritional status changes are: • a decreased lipid storage due to a high rate of lipid oxidation and to a low triglyceride reesterification. The advantage of the high energy content of lipid is limited by the low ATP/O efficiency as well as high rate of carbohydrate recycling (glucose-lactate cycle, see below). • an increased glucose utilization in either anaerobic (recycling) and aerobic pathways. • a strong decrease in protein content probably due to a low rate of protein synthesis together with a high rate of amino acid utilization for gluconeogenesis. Many studies have shown that COPD was responsible for undernutrition which associates weight loss and muscle wasting (1-3, 74, 75, 81-86). The nutritional changes are clearly related to the severity of the disease and probably also to the prognosis of these patients. Indeed muscle wasting is responsible for a decreased muscle performance and a depressed respiratory muscle function which is related to the outcome of the patients (87). Beside the metabolic events already described, several pathological changes must be considered in the pathogenesis of undernutrition. An increased basal energy expenditure is often reported (1-3, 74, 81, 83, 86, 88). Although the exact significance of this increased metabolic rate is not completely clear, it is undoubtedly an additive factor leading to a state of undernutrition. Additionally, patients often complain about anorexia which might be related to many pathological changes (where cytokine increase probably plays a dominant role) but the effect of hypoxia per se must be also considered since the increase in energy expenditure due to the post-prandial thermogenesis might be responsible for a deep hypoxia, preventing any further food intake (89). This situation is even further complicated by the fact that the metabolic adaptation to the very early stage of fasting appears in animal studies to be impaired by hypoxia (77). On one hand these patients are unable to take large amounts of food, but on the other hand even a short fasting may have a deleterious effect. It is probably of great importance, then, to feed them frequently with small amounts of food. The last important point in the pathogenesis of the nutritional disorders of such patients is related to the inflammatory status (87). Even if the complete proof for such causal relationship is not yet available, many facts are in favor of such a relation. For instance, it has been shown that T N F a was significantly higher in undernourished compared to obese COPD patients (90). In fact, the link between the inflammatory status and the type, the severity and the reversibility of the nutritional defect is probably a general feature in many other undernourished states such as renal failure for instance.

Summary In hypoxic patients, metabolic and nutritional disorders are due to various causes; among them the low P O 2 is probably a major determinant. The low PaO z may have detrimental effects in two different ways: by affecting the energy metabolism regulation and by changing the cellular phenotype of several organs because of effects on gene transcription. Since oxygen consumption is generally not decreased (and even increased in many cases), the major determinant is probably not the lack of oxygen availability per se but rather the cellular exposure to a low oxygen tension. In other words, the organism anticipates the possibility of a real lack of oxygen, which would be fatal in all cases, by several adaptive mechanisms such as muscle wasting and decreased activity. These adaptive mechanisms are actually responsible for the nutritional depletion and its deleterious consequences: in this view undernutrition is an adaptive mechanism to face hypoxia. In addition, nutritional depletion can also be the consequence of an inflammatory status either chronic or with a succession of acute or subacute phases. Such a mechanism can be of major importance in these hypoxic patients since they are less adapted to fasting and less efficient in restoring a normal body composition during the anabolic response to nutrition. In this case, undernutrition is also the conse-

CLINICAL NUTRITION

quence of an inflammatory state and then it might be deleterious per se in these hypoxic patients.

Epilogue There is a dilemma, therefore, in the therapeutic approach: on one hand losing body mass is an adaptive mechanism allowing one to face chronic hypoxia, but on the other hand the loss of body mass, and especially of lean body mass, is related to a poor outcome. Hence, the question arises as to whether we should correct the undernourished state of such patients or not. This debate is a major issue since a non-adapted correction of the lean body mass to the aerobic capacity of the body may actually worsen the situation as it was shown with the use of growth hormone (91). Conversely, it is tempting to try to correct the nutritional status (84, 88) in the framework of a rehabilitation program (92, 93) if it is thought that it may make a significant difference to the outcome. Very recently, it was shown by A. Schols and the Maastficht group that when a correction of the undernourished state of COPD patients was achieved it was significantly linked to an improvement of the patient outcome (94). But, in addition, it was clear that similar therapeutic tools (nutrient supply associated with a rehabilitation program) did not permit to achieved a similar correction of the nutritional depletion in all patients, some are resistant and they exhibit a significantly worse outcome. Thus, we propose the following hypothesis. When body composition improvement cannot be achieved, the defect in the nutritional status is directly and mainly related to the limited aerobic capacity: the body energy metabolism is not able to sustain a higher lean body mass. The hypoxic disease is very severe and the decreased nutritional status illustrates the upper limit of adaptation. Conversely, when the correction of the nutritional depletion can be achieved, this could mean that the disorder was caused by a mechanism associated to chronic hypoxia and related to previous episodes of inflammatory responses. In such cases, the efficacy of the therapeutical approach in improving the nutritional status is proof that the body energy metabolism is able to maintain a higher lean body mass. In this case the depletion is not a sign of an upper limit of adaptation toward hypoxia but rather of the involvement of some additive adverse events, and its correction will improve the patient outcome.

Acknowledgment The author would like to thank all co-workers who have participated in this work: Clinical unit: M. Guignier, F. Carpentier, D. Barnoud, H. Roth Research unit: C. Pison, C. Keriel, E. Fontaine, V. Saks, F. Prronnet, M-A Piquet, B. Sibille, C. Filippi, S. Hamant Outside collaborations: M. Rigoulet, R. Chioldro, L. Tappy, R. VenturaClapier, I. Mustafa, L. Bourdon.

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Submission date: 24 November 1998; Accepted." 24 November 1998

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