- Email: [email protected]
NADH redox ratio in cell metabolism
Archives of Biochemistry and Biophysics 595 (2016) 176e180
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Role of NADþ/NADH redox ratio in cell metabolism A tribute to Helmut Sies and Theodor Bücher and Hans A. Krebs ~ a*, Guillermo T. Saez, Juan Gambini, Mari Carmen Gomez-Cabrera, Jose Vin s** Consuelo Borra Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain
a r t i c l e i n f o Article history: Received 2 October 2015 Accepted 4 October 2015
1. Our cousin, Helmut Sies Sir Hans Krebs, in a very interesting, classical paper written in Nature in 1962 stressed the idea of the genealogies of scientists [1]. He made the point that scientists are not born, rather they are bred. His idea of the making of a scientist is that when a young person would like to become a scientist, should be attached to a distinguished scientist in a centre of excellence, if at all possible, and then try and learn from the attitudes of such person. In his memoirs [2] Krebs stated that he had been immensely lucky to be associated with Otto Warburg in a critical moment of his career. Moreover, in the same book of memoirs he stressed that his own group was really more than a professional body had become a family. We (JV and GTS) were lucky enough to have experienced the joy of belonging to that family. And extending the concept of the “family of scientists” two cousins are persons who have a common grandfather, their parents thus being brothers. If we think of scientific groups as families, then Helmut Sies and we (JV and GTS) are cousins because our parents were brothers. Indeed, Helmut started with Theodor Bücher and we started with Hans Krebs. Krebs and Bücher were pupils of one of the towering personalities in the 20th century biology and even science in general, Otto H. Warburg [3]. So in this sense we feel a very special relationship with our cousin Helmut Sies. See the “family” relationship of the authors of this
* Corresponding author. Department of Physiology. Faculty of Medicine. Avenida ~ ez 15, 46010, Valencia, Spain. Blasco Iban ** Corresponding author. Department of Physiology. Faculty of Medicine. Avenida ~ ez 15, 46010, Valencia, Spain. Blasco Iban ~ a), [email protected] (C. Borr E-mail addresses: [email protected] (J. Vin as). http://dx.doi.org/10.1016/j.abb.2015.11.027 0003-9861/© 2015 Elsevier Inc. All rights reserved.
paper in Fig. 1. Over the course of the years, we have experienced what it is like to work in a very similar field from very different backgrounds (the Mediterranean side of Spain and Germany) but with the same aim, i.e., advancing the science of biochemistry and trying to imitate our forebears. It is a great pleasure for us to have the opportunity of writing this review paper on a topic that was of the highest interest to the splendid tutors we had. Indeed, Theodor Bücher opened up the idea that the NADþ/NADH ratio could not be determined directly but had to be calculated from the concentrations of metabolites of reactions taken to be at equilibrium in vivo [4]. Krebs and his group fully developed this idea and wrote a classical paper where they determined very accurately the NADþ/ NADH ratio in different cell compartments [5]. A revision of these ideas underlining the importance of metabolite determinations to calculate the nicotinamides redox ratio is the major aim of this short review. 2. The difficulty in the determination of free adenine nucleotides levels in cells Levels of NADþ or those of NADH are of course very important because they are co-enzymes of dehydrogenases. NADþ and NADH are really co-substrates of the dehydrogenases in that they are not regenerated in the dehydrogenase reaction itself, but mainly NADH is oxidised in the respiratory chain. However, because many classical papers, including those we are reviewing (see [5]) use the term coenzymes, we shall refer to NADþ and NADH as coenzymes. The concentration of these free co-enzymes very seriously affects cell metabolism. However, the free concentrations of these two co-enzymes are very difficult to measure because it presents two major problems. One is that a significant amount of both of them is bound to the dehydrogenases themselves but is released upon acid extraction therefore making the free levels in cells impossible to determine. The second reason is that these coenzymes are very heavily compartmentalised. The usual methods of determination of these substrates always involved extraction of either the whole cell or the subcellular organelles. Isolation of these organelles, for instance mitochondria, is likely to cause significant changes in the concentration of the redox-dependent NADþ/NADH. Both difficulties can be overcome if one measures the ratio of
~ a et al. / Archives of Biochemistry and Biophysics 595 (2016) 176e180 J. Vin
177
Fig. 1. Our “cousin” Helmut Sies. This scientific genealogy starting from the towering figure Otto Warburg shows the “familial” relationship between Helmut Sies and the authors of this paper. It is ment to be a tribute to our outstanding mentors Sir Hans A. Krebs and Prof. Dr. Theodor Bücher.
NADþ/NADH by determining the concentrations of the oxidised and reduced substrates of dehydrogenases that are likely to be at equilibrium in vivo. This problem was overcome when work from the laboratory of Feodor Linen and from Theodor Bücher and Klingenburg showed that one could measure the ratio of the concentration of oxidised and reduced substrates of NAD-linked dehydrogenases. The chemical assumption is that the catalysed reactions are at equilibrium in the cell based on the chemical characteristics of the reaction and on the high activity of the enzymes involved. The concept that dehydrogenases like lactate dehydrogenase might not be at equilibrium in vivo was proposed by a group of scientists in China [6]. However, the majority of the research groups support the “classical” view. In any case, the estimation of the free NADþ/NADH ratio remains a very important issue to understand the regulation of metabolic reactions involving redox changes. That being said, we will accept as a general principle that the metabolite determination method to estimate NADþ/NADH ratio is
the best approach we have to understand the NADþ-dependent redox status of cells. 3. Estimation of NADþ/NADH redox ratio by metabolite measurements The general idea of this method lies in determining the concentration of suitable NAD-linked dehydrogenases. One has to be able to determine accurately both the oxidised and the reduced substrate. Since we know the pH at which these reactions operate and the equilibrium constant of the reaction in case, we can easily estimate the NADþ/NADH ratio by introducing values of the reduced and oxidised substrate in the equations described in Fig. 2. For instance, in the case of the lactate dehydrogenase one has to determine accurately lactate and pyruvate and by using the equilibrium reaction of the lactate dehydrogenase one can estimate the NADþ/NADH ratio. This was discussed in detail in Krebs’ classical paper on the redox state of free NADþ in cytoplasm [5].
178
~a et al. / Archives of Biochemistry and Biophysics 595 (2016) 176e180 J. Vin
dehydrogenase catalysing the reaction shown in Fig. 3. The principle is similar to that discussed for the lactate dehydrogenase reaction. Indeed, by using the equilibrium constant of the beta hydroxybutyrate dehydrogenase, and knowing the pH, one can estimate the NADþ/NADH ratio in mitochondria. Williamson et al. calculated that the NADþ/NADH in livers from well-fed rats is very different between cytosol and mitochondria. The value obtained for cytosol is around 700 and that of mitochondria is around 7, i.e., there is a two orders of magnitude difference between the redox ratio in mitochondria and cytosol. This underpins the importance of taking into account the very heavy compartmentation observed. One cannot just measure NADþ/NADH in whole cell extracts and assume that they are going to be similar in all cell compartments rate because they differ in at least two orders of magnitude. Isolation procedures, on the other hand, may affect the redox sate of the organelles studied. This is why we believe that the metabolite concentration method that we review here gives us the best estimate of the redox rate, at least in cytosol and mitochondria. The value of the NADþ/NADH redox ratio in nuclei is similar to that obtained in cytosol [8]. No clear estimates of the endoplasmic reticulum values have been published so far. We can therefore conclude that nucleus and cytoplasm have an NADþ/NADH ratio of around 700, and that the value for mitochondria is around 7.
Fig. 2. Equilibrium reactions and equations for the calculation of the NADH/NADþ ratio. a) In cytosol; b) In mitochondria. (HBDH: Hydroxybutyrate deshydrogenase).
4. The role of the NADþ/NADH ratio in the regulation of metabolism
The view that the pyruvate and lactate redox exchange predominantly is solely cytosolic was challenged by George Brooks and co-workers who described a role of mitochondrial lactate dehydrogenase in lactate oxidation. This is a major paper that sheds light on the critical role of lactate as an oxidisable substrate in muscle tissue [7]. However, because lactate dehydrogenase activity is significantly higher in cytosol than in mitochondria, we can assume that the ratio NADþ/NADH can still be calculated from the total values of lactate and pyruvate measured in whole tissue homogenates. This does not contradict the critical contribution of George Brooks and his team in showing that lactate can be readily oxidised in mitochondria and that therefore it has to be considered as a major oxidisable substrate of physiological importance in mitochondria [7]. The question of mitochondrial NADþ/NADH ratio can be solved by measuring the concentrations of two ketone bodies i.e. acetoacetate and beta hydroxybutyrate. These are interconverted by the activity of the mitochondrial enzyme beta hydroxybutyrate
The general idea is that it is useful to understand the regulation of metabolism and cell function to separate reactions that require energy (in the form of ATP) from those which occur spontaneously. The idea being that these reactions occur spontaneously because they tend to establish equilibrium. The major factors for these considerations is that the reactions must have enzymes that are of a high activity; that the permeability of the tissues is sufficient for the given metabolites; and finally, that there is a tendency of the reactions to become at equilibrium between the metabolites. When one set of reactions is at equilibrium, the general flow of the first reaction is driven normally by the removal of one of the products or by the accumulation of one of the substrates. Normally it is the removal of products that drives the reaction. For instance, the reaction of glutamate dehydrogenase is pulled away from equilibrium because the ammonium that is formed is removed from solutions and eventually is converted to urea in the liver. Glutamate dehydrogenase is taken to be at equilibrium. This idea was originally based on the fact that the NADþ/NADH ratio is very similar when it is calculated from the acetoacetate-
Fig. 3. Sirtuin deacetylase reaction. Taken from [10].
~ a et al. / Archives of Biochemistry and Biophysics 595 (2016) 176e180 J. Vin
179
hydroxybutirate reaction as well as from that of the glutamate dehydrogenase reaction. As stated before, the glutamate dehydrogenase reaction does not take place unless the equilibrium is displaced by withdrawal of ammonia or oxoglutarate. Importantly, in acidosis, renal gluconeogenesis is activated and oxoglutarate levels fall and therefore the glutamate reaction is activated. There are many other reactions in which the equilibrium of NADþ/NADH is critical for cell function. Our idea is to point out that one must take into account this ratio to understand several reactions of importance in cell function. Much more recent evidence has come from the case of sirtuins. 5. NADþ/NADH ratio and the case of sirtuins Sirtuins are protein deacetylases that are involved in many cell functions [9]. The chemical reaction is indicated in Fig. 3 [10]. It shows that the nicotinamide acetyl group that is bound to a lysine residue in proteins is transferred to the ribose moiety of NADþ, releasing as product nicotineamide. Thus, the chemical reaction of the sirtuins depends on the concentration of NADþ. In turn, NADþ concentrations (given a relatively constant NADþ NADH pool) depend on the NADþ/NADH ratio. This therefore links sirtuin activity to the redox state of cell as determined by the ratio we have just mentioned. Moreover, we reasoned that because NADþ/NADH is of such an importance for the chemical reaction of sirtuins that its expression might also be regulated by the redox couple NADþ/NADH and this turned out to be the case. Expression of sirtuin1 was increased when the NADþ/NADH ratio was decreased in both ethanol cells (in which we modified that ratio by incubating the cells with alcohol) and with whole mice in which we modified this ratio by subjecting the animals to physical exercise, the reason being that exercise increases lactate production and therefore shifts the NADþ/NADH reaction. Therefore one must consider that sirtuins are a group of enzymes in which the NADþ/NADH ratio is important for both the enzyme activity and its expression [11]. The most important findings in this work are summarized in Fig. 4. 6. Twenty-five years of oxidative stress in Valencia The term oxidative stress was coined by Helmut Sies and Enrique Cadenas in 1985 [12]. It invokes one of the most productive concepts in field of free radicals in biology and medicine. The number of times it has been quoted in the literature is counted by the thousands. To commemorate the twenty-fifth anniversary of the concept,
Fig. 5. Participants of the commemorative meeting of the 25 years of oxidative , Dr. Jose Vin ~ a, Dr. Malcolm Jackson, stress. From the left to the right: Dr. Lisardo Bosca ez, Dr. Giuseppe Poli, Dr. Manuel Dr. Helmut Sies, Dra. Josiane Cillard, Dr. Guillermo Sa Escolano and Dr. Santiago Grisolía.
one of us (GTS) organised a commemorative meeting in Valencia at n Valenciana de Estudios Avanzados. The general idea the Fundacio being that Helmut Sies’ and some people who have developed the concept and worked within the light of that concept could explain their views after twenty-five years of using it (Fig. 5). We gathered in Valencia on 26th October 2010 and some of the participants are shown in the picture. One can see, apart from the central figure of Helmut Sies, Malcolm Jackson, Josiane Cillard, Santiago Grisolia, Lisardo Bosca, and Giuseppe Poli, amongst others. Helmut Sies then proposed his new views of the definition of oxidative stress that he had developed together with Dean Jones as “an imbalance between oxidants and antioxidants in favour of the oxidants leading to a disruption of redox signalling and control and/ or molecular damage” [13]. Five years have elapsed since this refined definition and meeting took place and the concept of oxidative stress is still as fresh as ever. 7. Conclusions The cell is controlled by many redox systems. One of the earliest ones studied was precisely that of NADþ and NADH. The general idea that was originally proposed by Theodor Bücher and developed by Krebs was that this ratio could not be obtained by determining NADþ and NADH, but that it should be calculated by using relevant metabolites of reactions taken to be at equilibrium in vivo. In this short review we have attempted to just summarise these concepts, which are often overlooked in the recent literature, and to pay tribute not only to Helmut Sies but also to the pioneering work in this field of Theodor Bücher and Hans Krebs, whose ideas have caused an enormous impact on cell metabolism and function. Acknowledgments
Fig. 4. Principal findings from our laboratory ([11]).
This work was supported by grants SAF2010-19498, from the Spanish Ministry of Education and Science (MEC); ISCIII2006RED13-027 and ISCIII2012-RED-43-029 from the “Red Tematica de investigacion cooperativa en envejecimiento y fragilidad” (RETIn, Cultura CEF), PROMETEO2010/074 from “Conselleria de Educacio y Deporte”, 35NEURO GentxGent from “Fundacio Gent Per Gent de la Comunitat Valenciana”, RS2012-609 intramural grant from INCLIVA and EU Funded CM1001 and FRAILOMIC-HEALTH. 2012.2.1.1-2. This study has been co-financed by FEDER funds from the European Union (to J.V.) and PI13/01848 project, integrated into n the Plan Estatal I+D+I 2013-2016 funded by the ISCIII-Subdireccio
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n y el Fondo Europeo de Desarrollo Regional General de Evaluacio (FEDER) (to G.S.T.). References [1] H.A. Krebs, Nature 215 (1967) 1441e1445. [2] H.A.S. Krebs, A. Martin, Reminiscences and Reflections, Clarendon Press, Oxford, 1981. [3] H.A. Krebs, Biogr. Mem. Fellows R. Soc. 18 (1972) 629e699. [4] T. Bucher, B. Brauser, A. Conze, F. Klein, O. Langguth, H. Sies, Eur. J. Biochem. 27 (1972) 301e317. [5] D.H. Williamson, P. Lund, H.A. Krebs, Biochem. J. 103 (1967) 514e527.
[6] F. Sun, C. Dai, J. Xie, X. Hu, PLoS One 7 (2012) e34525. [7] G.A. Brooks, H. Dubouchaud, M. Brown, J.P. Sicurello, C.E. Butz, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 1129e1134. [8] M. Fulco, R.L. Schiltz, S. Iezzi, M.T. King, P. Zhao, Y. Kashiwaya, E. Hoffman, R.L. Veech, V. Sartorelli, Mol. Cell 12 (2003) 51e62. [9] L. Guarente, Nat. Genet. 23 (1999) 281e285. [10] P. Bheda, C. Wolberger, Nature 496 (2013) 41e42. [11] J. Gambini, M.C. Gomez-Cabrera, C. Borras, S.L. Valles, R. Lopez-Grueso, V.E. Martinez-Bello, D. Herranz, F.V. Pallardo, J.A. Tresguerres, M. Serrano, J. Vina, Arch. Biochem. Biophys. 512 (2011) 24e29. [12] H. Sies, E. Cadenas, Oxidative stress: damage to intact cells and organs, Philos. Trans. R Soc. Lond. B Biol. Sci. 311 (1985) 617e631. [13] D.P. Jones, H. Sies, The redox code. Antioxid, Redox Signal 23 (2015) 734e746.
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Role of NADþ/NADH redox ratio in cell metabolism A tribute to Helmut Sies and Theodor Bücher and Hans A. Krebs ~ a*, Guillermo T. Saez, Juan Gambini, Mari Carmen Gomez-Cabrera, Jose Vin s** Consuelo Borra Department of Physiology, Faculty of Medicine, University of Valencia, Valencia, Spain
a r t i c l e i n f o Article history: Received 2 October 2015 Accepted 4 October 2015
1. Our cousin, Helmut Sies Sir Hans Krebs, in a very interesting, classical paper written in Nature in 1962 stressed the idea of the genealogies of scientists [1]. He made the point that scientists are not born, rather they are bred. His idea of the making of a scientist is that when a young person would like to become a scientist, should be attached to a distinguished scientist in a centre of excellence, if at all possible, and then try and learn from the attitudes of such person. In his memoirs [2] Krebs stated that he had been immensely lucky to be associated with Otto Warburg in a critical moment of his career. Moreover, in the same book of memoirs he stressed that his own group was really more than a professional body had become a family. We (JV and GTS) were lucky enough to have experienced the joy of belonging to that family. And extending the concept of the “family of scientists” two cousins are persons who have a common grandfather, their parents thus being brothers. If we think of scientific groups as families, then Helmut Sies and we (JV and GTS) are cousins because our parents were brothers. Indeed, Helmut started with Theodor Bücher and we started with Hans Krebs. Krebs and Bücher were pupils of one of the towering personalities in the 20th century biology and even science in general, Otto H. Warburg [3]. So in this sense we feel a very special relationship with our cousin Helmut Sies. See the “family” relationship of the authors of this
* Corresponding author. Department of Physiology. Faculty of Medicine. Avenida ~ ez 15, 46010, Valencia, Spain. Blasco Iban ** Corresponding author. Department of Physiology. Faculty of Medicine. Avenida ~ ez 15, 46010, Valencia, Spain. Blasco Iban ~ a), [email protected] (C. Borr E-mail addresses: [email protected] (J. Vin as). http://dx.doi.org/10.1016/j.abb.2015.11.027 0003-9861/© 2015 Elsevier Inc. All rights reserved.
paper in Fig. 1. Over the course of the years, we have experienced what it is like to work in a very similar field from very different backgrounds (the Mediterranean side of Spain and Germany) but with the same aim, i.e., advancing the science of biochemistry and trying to imitate our forebears. It is a great pleasure for us to have the opportunity of writing this review paper on a topic that was of the highest interest to the splendid tutors we had. Indeed, Theodor Bücher opened up the idea that the NADþ/NADH ratio could not be determined directly but had to be calculated from the concentrations of metabolites of reactions taken to be at equilibrium in vivo [4]. Krebs and his group fully developed this idea and wrote a classical paper where they determined very accurately the NADþ/ NADH ratio in different cell compartments [5]. A revision of these ideas underlining the importance of metabolite determinations to calculate the nicotinamides redox ratio is the major aim of this short review. 2. The difficulty in the determination of free adenine nucleotides levels in cells Levels of NADþ or those of NADH are of course very important because they are co-enzymes of dehydrogenases. NADþ and NADH are really co-substrates of the dehydrogenases in that they are not regenerated in the dehydrogenase reaction itself, but mainly NADH is oxidised in the respiratory chain. However, because many classical papers, including those we are reviewing (see [5]) use the term coenzymes, we shall refer to NADþ and NADH as coenzymes. The concentration of these free co-enzymes very seriously affects cell metabolism. However, the free concentrations of these two co-enzymes are very difficult to measure because it presents two major problems. One is that a significant amount of both of them is bound to the dehydrogenases themselves but is released upon acid extraction therefore making the free levels in cells impossible to determine. The second reason is that these coenzymes are very heavily compartmentalised. The usual methods of determination of these substrates always involved extraction of either the whole cell or the subcellular organelles. Isolation of these organelles, for instance mitochondria, is likely to cause significant changes in the concentration of the redox-dependent NADþ/NADH. Both difficulties can be overcome if one measures the ratio of
~ a et al. / Archives of Biochemistry and Biophysics 595 (2016) 176e180 J. Vin
177
Fig. 1. Our “cousin” Helmut Sies. This scientific genealogy starting from the towering figure Otto Warburg shows the “familial” relationship between Helmut Sies and the authors of this paper. It is ment to be a tribute to our outstanding mentors Sir Hans A. Krebs and Prof. Dr. Theodor Bücher.
NADþ/NADH by determining the concentrations of the oxidised and reduced substrates of dehydrogenases that are likely to be at equilibrium in vivo. This problem was overcome when work from the laboratory of Feodor Linen and from Theodor Bücher and Klingenburg showed that one could measure the ratio of the concentration of oxidised and reduced substrates of NAD-linked dehydrogenases. The chemical assumption is that the catalysed reactions are at equilibrium in the cell based on the chemical characteristics of the reaction and on the high activity of the enzymes involved. The concept that dehydrogenases like lactate dehydrogenase might not be at equilibrium in vivo was proposed by a group of scientists in China [6]. However, the majority of the research groups support the “classical” view. In any case, the estimation of the free NADþ/NADH ratio remains a very important issue to understand the regulation of metabolic reactions involving redox changes. That being said, we will accept as a general principle that the metabolite determination method to estimate NADþ/NADH ratio is
the best approach we have to understand the NADþ-dependent redox status of cells. 3. Estimation of NADþ/NADH redox ratio by metabolite measurements The general idea of this method lies in determining the concentration of suitable NAD-linked dehydrogenases. One has to be able to determine accurately both the oxidised and the reduced substrate. Since we know the pH at which these reactions operate and the equilibrium constant of the reaction in case, we can easily estimate the NADþ/NADH ratio by introducing values of the reduced and oxidised substrate in the equations described in Fig. 2. For instance, in the case of the lactate dehydrogenase one has to determine accurately lactate and pyruvate and by using the equilibrium reaction of the lactate dehydrogenase one can estimate the NADþ/NADH ratio. This was discussed in detail in Krebs’ classical paper on the redox state of free NADþ in cytoplasm [5].
178
~a et al. / Archives of Biochemistry and Biophysics 595 (2016) 176e180 J. Vin
dehydrogenase catalysing the reaction shown in Fig. 3. The principle is similar to that discussed for the lactate dehydrogenase reaction. Indeed, by using the equilibrium constant of the beta hydroxybutyrate dehydrogenase, and knowing the pH, one can estimate the NADþ/NADH ratio in mitochondria. Williamson et al. calculated that the NADþ/NADH in livers from well-fed rats is very different between cytosol and mitochondria. The value obtained for cytosol is around 700 and that of mitochondria is around 7, i.e., there is a two orders of magnitude difference between the redox ratio in mitochondria and cytosol. This underpins the importance of taking into account the very heavy compartmentation observed. One cannot just measure NADþ/NADH in whole cell extracts and assume that they are going to be similar in all cell compartments rate because they differ in at least two orders of magnitude. Isolation procedures, on the other hand, may affect the redox sate of the organelles studied. This is why we believe that the metabolite concentration method that we review here gives us the best estimate of the redox rate, at least in cytosol and mitochondria. The value of the NADþ/NADH redox ratio in nuclei is similar to that obtained in cytosol [8]. No clear estimates of the endoplasmic reticulum values have been published so far. We can therefore conclude that nucleus and cytoplasm have an NADþ/NADH ratio of around 700, and that the value for mitochondria is around 7.
Fig. 2. Equilibrium reactions and equations for the calculation of the NADH/NADþ ratio. a) In cytosol; b) In mitochondria. (HBDH: Hydroxybutyrate deshydrogenase).
4. The role of the NADþ/NADH ratio in the regulation of metabolism
The view that the pyruvate and lactate redox exchange predominantly is solely cytosolic was challenged by George Brooks and co-workers who described a role of mitochondrial lactate dehydrogenase in lactate oxidation. This is a major paper that sheds light on the critical role of lactate as an oxidisable substrate in muscle tissue [7]. However, because lactate dehydrogenase activity is significantly higher in cytosol than in mitochondria, we can assume that the ratio NADþ/NADH can still be calculated from the total values of lactate and pyruvate measured in whole tissue homogenates. This does not contradict the critical contribution of George Brooks and his team in showing that lactate can be readily oxidised in mitochondria and that therefore it has to be considered as a major oxidisable substrate of physiological importance in mitochondria [7]. The question of mitochondrial NADþ/NADH ratio can be solved by measuring the concentrations of two ketone bodies i.e. acetoacetate and beta hydroxybutyrate. These are interconverted by the activity of the mitochondrial enzyme beta hydroxybutyrate
The general idea is that it is useful to understand the regulation of metabolism and cell function to separate reactions that require energy (in the form of ATP) from those which occur spontaneously. The idea being that these reactions occur spontaneously because they tend to establish equilibrium. The major factors for these considerations is that the reactions must have enzymes that are of a high activity; that the permeability of the tissues is sufficient for the given metabolites; and finally, that there is a tendency of the reactions to become at equilibrium between the metabolites. When one set of reactions is at equilibrium, the general flow of the first reaction is driven normally by the removal of one of the products or by the accumulation of one of the substrates. Normally it is the removal of products that drives the reaction. For instance, the reaction of glutamate dehydrogenase is pulled away from equilibrium because the ammonium that is formed is removed from solutions and eventually is converted to urea in the liver. Glutamate dehydrogenase is taken to be at equilibrium. This idea was originally based on the fact that the NADþ/NADH ratio is very similar when it is calculated from the acetoacetate-
Fig. 3. Sirtuin deacetylase reaction. Taken from [10].
~ a et al. / Archives of Biochemistry and Biophysics 595 (2016) 176e180 J. Vin
179
hydroxybutirate reaction as well as from that of the glutamate dehydrogenase reaction. As stated before, the glutamate dehydrogenase reaction does not take place unless the equilibrium is displaced by withdrawal of ammonia or oxoglutarate. Importantly, in acidosis, renal gluconeogenesis is activated and oxoglutarate levels fall and therefore the glutamate reaction is activated. There are many other reactions in which the equilibrium of NADþ/NADH is critical for cell function. Our idea is to point out that one must take into account this ratio to understand several reactions of importance in cell function. Much more recent evidence has come from the case of sirtuins. 5. NADþ/NADH ratio and the case of sirtuins Sirtuins are protein deacetylases that are involved in many cell functions [9]. The chemical reaction is indicated in Fig. 3 [10]. It shows that the nicotinamide acetyl group that is bound to a lysine residue in proteins is transferred to the ribose moiety of NADþ, releasing as product nicotineamide. Thus, the chemical reaction of the sirtuins depends on the concentration of NADþ. In turn, NADþ concentrations (given a relatively constant NADþ NADH pool) depend on the NADþ/NADH ratio. This therefore links sirtuin activity to the redox state of cell as determined by the ratio we have just mentioned. Moreover, we reasoned that because NADþ/NADH is of such an importance for the chemical reaction of sirtuins that its expression might also be regulated by the redox couple NADþ/NADH and this turned out to be the case. Expression of sirtuin1 was increased when the NADþ/NADH ratio was decreased in both ethanol cells (in which we modified that ratio by incubating the cells with alcohol) and with whole mice in which we modified this ratio by subjecting the animals to physical exercise, the reason being that exercise increases lactate production and therefore shifts the NADþ/NADH reaction. Therefore one must consider that sirtuins are a group of enzymes in which the NADþ/NADH ratio is important for both the enzyme activity and its expression [11]. The most important findings in this work are summarized in Fig. 4. 6. Twenty-five years of oxidative stress in Valencia The term oxidative stress was coined by Helmut Sies and Enrique Cadenas in 1985 [12]. It invokes one of the most productive concepts in field of free radicals in biology and medicine. The number of times it has been quoted in the literature is counted by the thousands. To commemorate the twenty-fifth anniversary of the concept,
Fig. 5. Participants of the commemorative meeting of the 25 years of oxidative , Dr. Jose Vin ~ a, Dr. Malcolm Jackson, stress. From the left to the right: Dr. Lisardo Bosca ez, Dr. Giuseppe Poli, Dr. Manuel Dr. Helmut Sies, Dra. Josiane Cillard, Dr. Guillermo Sa Escolano and Dr. Santiago Grisolía.
one of us (GTS) organised a commemorative meeting in Valencia at n Valenciana de Estudios Avanzados. The general idea the Fundacio being that Helmut Sies’ and some people who have developed the concept and worked within the light of that concept could explain their views after twenty-five years of using it (Fig. 5). We gathered in Valencia on 26th October 2010 and some of the participants are shown in the picture. One can see, apart from the central figure of Helmut Sies, Malcolm Jackson, Josiane Cillard, Santiago Grisolia, Lisardo Bosca, and Giuseppe Poli, amongst others. Helmut Sies then proposed his new views of the definition of oxidative stress that he had developed together with Dean Jones as “an imbalance between oxidants and antioxidants in favour of the oxidants leading to a disruption of redox signalling and control and/ or molecular damage” [13]. Five years have elapsed since this refined definition and meeting took place and the concept of oxidative stress is still as fresh as ever. 7. Conclusions The cell is controlled by many redox systems. One of the earliest ones studied was precisely that of NADþ and NADH. The general idea that was originally proposed by Theodor Bücher and developed by Krebs was that this ratio could not be obtained by determining NADþ and NADH, but that it should be calculated by using relevant metabolites of reactions taken to be at equilibrium in vivo. In this short review we have attempted to just summarise these concepts, which are often overlooked in the recent literature, and to pay tribute not only to Helmut Sies but also to the pioneering work in this field of Theodor Bücher and Hans Krebs, whose ideas have caused an enormous impact on cell metabolism and function. Acknowledgments
Fig. 4. Principal findings from our laboratory ([11]).
This work was supported by grants SAF2010-19498, from the Spanish Ministry of Education and Science (MEC); ISCIII2006RED13-027 and ISCIII2012-RED-43-029 from the “Red Tematica de investigacion cooperativa en envejecimiento y fragilidad” (RETIn, Cultura CEF), PROMETEO2010/074 from “Conselleria de Educacio y Deporte”, 35NEURO GentxGent from “Fundacio Gent Per Gent de la Comunitat Valenciana”, RS2012-609 intramural grant from INCLIVA and EU Funded CM1001 and FRAILOMIC-HEALTH. 2012.2.1.1-2. This study has been co-financed by FEDER funds from the European Union (to J.V.) and PI13/01848 project, integrated into n the Plan Estatal I+D+I 2013-2016 funded by the ISCIII-Subdireccio
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n y el Fondo Europeo de Desarrollo Regional General de Evaluacio (FEDER) (to G.S.T.). References [1] H.A. Krebs, Nature 215 (1967) 1441e1445. [2] H.A.S. Krebs, A. Martin, Reminiscences and Reflections, Clarendon Press, Oxford, 1981. [3] H.A. Krebs, Biogr. Mem. Fellows R. Soc. 18 (1972) 629e699. [4] T. Bucher, B. Brauser, A. Conze, F. Klein, O. Langguth, H. Sies, Eur. J. Biochem. 27 (1972) 301e317. [5] D.H. Williamson, P. Lund, H.A. Krebs, Biochem. J. 103 (1967) 514e527.
[6] F. Sun, C. Dai, J. Xie, X. Hu, PLoS One 7 (2012) e34525. [7] G.A. Brooks, H. Dubouchaud, M. Brown, J.P. Sicurello, C.E. Butz, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 1129e1134. [8] M. Fulco, R.L. Schiltz, S. Iezzi, M.T. King, P. Zhao, Y. Kashiwaya, E. Hoffman, R.L. Veech, V. Sartorelli, Mol. Cell 12 (2003) 51e62. [9] L. Guarente, Nat. Genet. 23 (1999) 281e285. [10] P. Bheda, C. Wolberger, Nature 496 (2013) 41e42. [11] J. Gambini, M.C. Gomez-Cabrera, C. Borras, S.L. Valles, R. Lopez-Grueso, V.E. Martinez-Bello, D. Herranz, F.V. Pallardo, J.A. Tresguerres, M. Serrano, J. Vina, Arch. Biochem. Biophys. 512 (2011) 24e29. [12] H. Sies, E. Cadenas, Oxidative stress: damage to intact cells and organs, Philos. Trans. R Soc. Lond. B Biol. Sci. 311 (1985) 617e631. [13] D.P. Jones, H. Sies, The redox code. Antioxid, Redox Signal 23 (2015) 734e746.