Implication de la créatine pour la thermogénèse?

[Nutrimuscle-Conseils]

Mechanism of futile creatine cycling in thermogenesis
Lawrence Kazak Am J physiol 12 NOV 2020

INTRODUCTION
The forward and reverse phosphotransfer reactions of phosphocreatine–creatine in most cells occur in a strict 1:1 stoichiometry with the ATP/ADP couple (9). However, mitochondria in thermogenic adipocytes liberate a large molar excess of ADP with respect to added creatine (2, 10). Based on bioenergetics principles and the established stoichiometry of the P/O ratio (ATP molecules synthesized per oxygen atom consumed) (23), we proposed two models wherein creatine might support this super-stoichiometric regeneration of ADP to drive thermogenic respiration in fat (10, 12, 18).

The first and simplest model is that a mitochondrial creatine kinase will use mitochondrial ATP to phosphorylate creatine to phosphocreatine (PCr), and the ensuing liberation of ADP locally within the organelle would be a powerful respiratory stimulus. Next, PCr would be the direct substrate of a phosphatase, which would replenish the mitochondrial creatine pool. The regenerated creatine from direct hydrolysis of PCr would act as a substrate for another round of this futile creatine cycle. Since we identified creatine-elicited respiration in isolated mitochondria (2, 10), but because crude mitochondrial preparations are not 100% pure, PCr hydrolase activity could be present within mitochondria themselves or on organelles that co-purify with them. Thus, either a pool of PCr and creatine circulate within the intermembrane space (IMS) or PCr is channeled out of the IMS toward PCr phosphatase activity that is external from mitochondria. The experimental data are consistent with either scenario (2, 10). We also proposed a second model where multiple phosphotransfer reactions might occur before phosphate hydrolysis from a phosphometabolite that lies downstream of PCr (10, 12). These variations on the futile creatine cycle have recently been extensively reviewed (12, 18).

Based on theoretical points founded in non-adipocyte work, Wallimann and colleagues (22) hypothesize that thermogenic adipocytes utilize the PCr/creatine kinase circuit (3) to fuel thermogenesis. In this circuit, mitochondrial creatine kinase generates PCr, which can diffuse throughout the cell to maintain high ATP/ADP ratios locally near sites of ATP consumption. In their hypothetical model, Wallimann et al. propose a creatine-driven calcium cycle wherein the PCr/creatine kinase circuit would support calcium cycling by sustaining a high ATP/ADP ratio at the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump (20). Operation of a PCr/creatine kinase circuit in adipocytes is not mutually exclusive with thermogenic futile creatine cycling in mitochondria. Nevertheless, the experimental evidence from studies conducted directly in thermogenic adipocytes eliminates the PCr/creatine kinase circuit as the molecular mechanism regulating creatine-driven thermogenesis.

We now provide point-by-point responses to the claims of Wallimann and colleagues:

Claim
1) Creatine kinase is the only confirmed enzyme that can use PCr as substrate.
At present, only creatine kinase has been identified to utilize PCr as substrate. However, we must refrain from using arguments from ignorance; absence of evidence is not evidence of absence. The super-stoichiometric action of creatine in purified mitochondria from thermogenic adipocytes suggests that these preparations exhibit unique properties with respect to the chemistry of PCr. The appropriate experimental system and cell type required to identify non-creatine kinase proteins that metabolize PCr has, to our knowledge, never been implemented. A PCr phosphatase would hydrolyze PCr to creatine and Pi. All assays that monitor creatine kinase activity use indirect measurements of NAD and NADH absorbance and rely on phosphotransferase activity of creatine kinase. However, the direct measurement of products derived from the metabolism of PCr is required to study hydrolytic activity, and this has not yet been employed, thus obviating the ability to identify non-creatine kinase proteins that hydrolyze PCr.

Claim
2) The PCr/creatine kinase circuit supports calcium cycling.
The SERCA pump harnesses energy from ATP to pump Ca2+ into the SR/ER lumen against a steep concentration gradient, in a 2:1 stoichiometry of Ca2+:ATP. Deviation from the 2:1 stoichiometry of Ca2+:ATP is necessary for calcium cycling. Maintaining high local ATP/ADP ratios near SERCA is not a sufficient explanation for a futile calcium cycle. In muscle, a single-pass transmembrane peptide, sarcolipin, interacts with SERCA to decrease the Ca2+:ATP stoichiometry (21). However, sarcolipin is not expressed in fat (14, 19). A molecular factor akin to sarcolipin that modulates the stoichiometry of the SERCA-mediated Ca2+/ATPase influx relationship in thermogenic adipocytes has yet to be identified. Nevertheless, Wallimann and colleagues hypothesize that the PCr/creatine kinase circuit buffers ATP levels locally at the SERCA pump to support calcium cycling. A mechanism such as this would sustain a high ATP/ADP ratio near SERCA to increase the thermodynamic efficiency of ATP hydrolysis. However, calcium is not added to our mitochondrial preparations. Therefore, calcium cycling as an explanation for creatine-stimulated thermogenic respiration lacks experimental support and is therefore eliminated by published data (2, 10). Furthermore, SERCA inhibition only reduces the respiration of Ucp1−/−, not wild-type, adipocytes(8). In contrast, the futile creatine cycle does not require Ucp1 deletion (2, 10, 11, 13). Together, these data are not consistent with the idea that calcium cycling is fueled by the PCr/creatine kinase circuit and rule out the hypothesis that creatine and calcium support thermogenesis through the same pathway.

Claim
3) A PCr phosphatase would disrupt cell energetics.
Wallimann and colleagues hypothesize that a PCr phosphatase would disrupt cellular energetics. However, the release of free energy as heat is the defining feature of brown and beige adipocyte thermogenesis. Contrary to their opinion, we take the viewpoint that attacking the ATP pool directly by calcium cycling would be more dangerous for cellular energetics than attacking PCr, which will primarily form when ATP reaches sufficient levels.

Wallimann and colleagues conclude that creatine-driven thermogenesis does “not necessarily require the presence of a hypothetical, novel PCr phosphatase.” However, given the strong bioenergetics data in support of a PCr phosphatase, the current lack of molecular identification of this enzyme is a weak argument against its existence. From a historical perspective, mitochondria were the first organelles to be linked with calcium handling, an activity that was measured around the time that the chemi-osmotic theory was put forth (6, 15). Although this process was thoroughly investigated, it took half a century to identify the channel responsible for mitochondrial calcium accumulation (1, 5). Similarly, 40 years spanned between the earliest proposition of a mitochondrial pyruvate transporter and its molecular identification (4, 7, 16). Furthermore, lactate was once thought to be a cellular waste product but now, through utilization of isotope tracing techniques, is being recognized as a key nutrient, even rivaling glucose under specific physiological contexts (17).

Conclusion
The experimental data in thermogenic fat point to a two-enzyme system that utilizes mitochondrial-derived ADP phosphorylation to support cycling between creatine and PCr. A mitochondria-localized creatine kinase triggers the phosphorylation of creatine from mitochondrial ATP, which liberates local ADP to powerfully trigger respiration. Next, PCr is hydrolyzed to creatine by a PCr phosphatase to initiate another round of the futile creatine cycle. There is one point where we do agree with Wallimann and colleagues: diligent experimentation is required. We would further add that investigating creatine biology with modern molecular tools and diverse cell types has the potential to reveal functions for this ancient metabolite that until now have been overlooked. Finally, we will conclude our counterpoint by stating that we have now identified the creatine kinase isoenzyme and the PCr phosphatase of the futile creatine cycle. This work is currently in preparation.

[Nutrimuscle-Conseils]

Role of creatine and creatine kinase in UCP1-independent adipocyte thermogenesis
Theo Wallimann, Am J Physiol 12 NOV 2020

Targeting thermogenesis in brown and beige adipocytes for treating obesity and metabolic disease has sparked enormous interest even beyond the scientific community (4). Several more recent publications have added a new player in this field: the well-studied enzyme creatine kinase (CK) together with its substrates phosphocreatine (PCr) and creatine (Cr). These are best known for their eminent bioenergetic role in energy buffering and energy channeling via the so-called CK/PCr shuttle (3) or circuit (31) that link sites of ATP generation (e.g., mitochondria and glycolysis) with sites of ATP utilization (e.g., cytosolic and membrane bound ATPases) in tissues with high energy turnover, such as skeletal, cardiac and smooth muscle, brain, neural tissue, spermatozoa, inner ear hair cells, etc. (25, 27, 30, 31). As highly energy-dependent cells, adipocytes also express CK isoenzymes and contain the CK substrates PCr and Cr (for review, see Ref. 28). In fact, a series of elegant and very comprehensive studies by Spiegelman, Kazak, and colleagues (2, 15, 16, 18), well summarized in a recent review (17), provides ample genetic and functional evidence for a direct involvement of the CK system in adipocyte thermogenesis with an increased Cr/PCr turnover being thermogenic. In beige or brown adipocytes, such Cr-dependent heat generation would work in parallel to uncoupling of mitochondrial respiration by uncoupling protein 1 (UCP1), a futile proton cycle that represents the canonical pathway of nonshivering thermogenesis. Since noncanonical UPC1-independent, but ATP- and Cr-dependent, thermogenesis is not only operational during short-term cold response and long-term cold acclimation, but also in diet-induced thermogenesis (16, 18), these recent advances open exciting new avenues for targeting obesity and metabolic disease.

What then is the exact molecular mechanism underlying Cr-dependent thermogenesis? For heat generation, increased Cr/PCr turnover has to be linked to a futile cycle, i.e., a metabolic pathway that is not generating useful work, but dissipates free energy as heat. The mechanism put forward so far has been futile Cr-cycling (15). Here, PCr generated by mitochondrial CK in the mitochondrial intermembrane space (IMS) (31) would be immediately hydrolyzed by a postulated PCr phosphatase, colocalizing in the IMS or eventually outside mitochondria, e.g., at the endoplasmic reticulum (ER) (17). However, CK is the only confirmed enzyme that can use PCr and Cr as substrates, while the existence and subcellular localization of the proposed PCr-phosphatase are still uncertain and ill-defined, respectively. Importantly, the presence of such a phosphatase would potentially disrupt PCr-based cell energetics by depleting cellular PCr and thus lowering local PCr/ATP and ATP/ADP ratios. This would be detrimental for proper Ca2+ homeostasis that depends on highly energy-demanding Ca2+ pumps (10, 12, 23, 29–31).

Based on our extensive research on the molecular structure, function, and localization of CK isoforms at specific subcellular microcompartments (6, 7, 24, 25, 27, 29–31), we propose here an alternative model for CK/PCr/Cr-mediated thermogenesis in adipocytes (Fig. 1). It combines the classical CK/PCr shuttle (3, 25, 31) with futile Ca2+ cycling at the ER. The latter has been described in detail in recent years in beige and brown adipocytes (10, 12). In this model, PCr fulfills its classical function by shuttling energy from mitochondria to the ER (1, 5, 8, 26, 29), where it is converted by membrane-associated cytosolic CK into ATP. Since the CK/PCr shuttle can also use glycolytic ATP for PCr generation, this pathway would also work in case the mitochondrial ATP would become limiting, which seems the case in adipocytes (11, 19). In fact, there is evidence for additional, noncanonical UCP1-independent nonshivering thermogenesis, fueled by enhanced glycolytic ATP generation (9). ER-localized, cytosolic CK then locally fuels the SR/ER Ca2+-ATPase2b (SERCA2b) (23), which is also expressed in adipocytes (12), for efficient Ca2+ uptake into the ER lumen. Requiring a very high ATP/ADP ratio for its function, this Ca2+ pump has a particular requirement for PCr-dependent fueling via CK (23, 29–31). Although such local ATP supply has not been specifically analyzed in adipocytes, it is well known and documented for skeletal and cardiac muscle, as well as for heater organs of fish (20, 31).

Futile Ca2+ cycling in brown/beige adipocytes is then induced by coupling Ca2+ uptake via SERCA2b to immediate Ca2+ release via different routes that may operate in parallel: adipocyte ryanodine receptor (RyR2), inositol 1,4,5-triphosphate receptor type 1 (IP3-R1), or more so type 3 (IP3-R3). All these constituents have been identified in beige/brown adipocytes, and they all rely on the stimulation of adrenergic receptors (α1, β3) (10, 12, 13). Adipocyte Ca2+ cycling between SERCA2b and RyR2 occurs under cold exposure and/or adrenergic receptor stimulation (13), is accompanied by a significant increase of oxygen consumption rate (OCR) and heat generation, and improves cold tolerance and metabolic status (12, 13). Furthermore, evidence for an involvement of IP3-R in futile Ca2+ cycling is given by the fact that RyR2 blockage by high dose ryanodine or ruthenium red only partially but not completely disrupts norepinephrine-induced thermogenesis in UCP1-knockout adipocytes (12). This is supported by earlier data (10) showing that elevated cytosolic Ca2+, caused by mitochondrial Ca2+ release, plus elevated IP3 levels, after α1-adrenergic stimulation of adipocytes together result in a Ca2+ release from the ER/SR via IP2 receptor (22). This event is likely to happen in parallel with Ca2+ release via RyR2 (12). There is also evidence that the CK system and Ca2+ cycling via SERCA2b operate in the same pathway for UCP1-independent thermogenesis. When using UCP1-knockout beige adipocytes, both SERCA2b depletion and CK inhibition by β-guanidinopropionic acid (β-GPA) reduced OCR, but β-GPA treatment had no additional effect on OCR in already SERCA2b-depleted adipocytes (12).

In conclusion, the role of Cr and CK in thermogenesis of beige and brown adipocyte has attracted much attention. Their role in UCP1-independent, but ATP-dependent nonshivering thermogenesis is clearly emerging (15–18, 21), but the detailed molecular nature of the thermal energy generator is less evident. It is our contention, however, that it does not necessarily require futile Cr cycling and the presence of a hypothetical, novel PCr phosphatase. It is proposed here that Cr and PCr may just operate as part of the classical energy shuttle, providing ATP to other thermogenic pathways, in particular for futile Ca2+ cycling at the ER. For the latter, ample evidence for its functioning in adipocytes is available (10, 12, 13). Much diligent experimentation, however, is still needed to clarify these issues before they can be harnessed for mechanism-based therapeutic approaches to combat obesity, type 2 diabetes, and metabolic syndrome

[Nutrimuscle-Diététique]

Traduction de l’étude

Rôle de la créatine et de la créatine kinase dans la thermogenèse adipocytaire indépendante de l'UCP1
Theo Wallimann, Am J Physiol 12 NOV 2020

Le ciblage de la thermogenèse dans les adipocytes bruns et beiges pour traiter l'obésité et les maladies métaboliques a suscité un énorme intérêt même au-delà de la communauté scientifique (4). Plusieurs publications plus récentes ont ajouté un nouvel acteur dans ce domaine: l'enzyme créatine kinase (CK) bien étudiée avec ses substrats phosphocréatine (PCr) et créatine (Cr). Celles-ci sont surtout connues pour leur rôle bioénergétique éminent dans la mise en tampon de l'énergie et la canalisation de l'énergie via la navette (3) ou circuit (31) appelé CK / PCr qui relie les sites de génération d'ATP (par exemple, les mitochondries et la glycolyse) aux sites d'utilisation de l'ATP. (par exemple, les ATPases cytosoliques et liées à la membrane) dans les tissus à fort renouvellement énergétique, tels que les muscles squelettiques, cardiaques et lisses, le cerveau, les tissus neuraux, les spermatozoïdes, les cellules ciliées de l'oreille interne, etc. (25, 27, 30, 31). En tant que cellules fortement dépendantes de l'énergie, les adipocytes expriment également les isoenzymes CK et contiennent les substrats CK PCr et Cr (pour une revue, voir la référence 28). En fait, une série d'études élégantes et très complètes par Spiegelman, Kazak et ses collègues (2, 15, 16, 18), bien résumées dans une revue récente (17), fournit de nombreuses preuves génétiques et fonctionnelles d'une implication directe du Système CK dans la thermogenèse adipocytaire avec un renouvellement accru de Cr / PCr étant thermogénique. Dans les adipocytes beiges ou bruns, une telle génération de chaleur dépendante du Cr fonctionnerait en parallèle avec le découplage de la respiration mitochondriale par découplage de la protéine 1 (UCP1), un cycle de protons futile qui représente la voie canonique de la thermogenèse sans frisson. Étant donné que la thermogenèse non-canonique indépendante de l'UPC1, mais dépendante de l'ATP et du Cr, est non seulement opérationnelle pendant la réponse au froid à court terme et l'acclimatation au froid à long terme, mais aussi dans la thermogenèse induite par l'alimentation (16, 18), ces avancées récentes de nouvelles voies pour cibler l'obésité et les maladies métaboliques.

Quel est alors le mécanisme moléculaire exact qui sous-tend la thermogenèse dépendante du Cr? Pour la production de chaleur, l'augmentation du renouvellement du Cr / PCr doit être liée à un cycle futile, c'est-à-dire à une voie métabolique qui ne génère pas de travail utile, mais dissipe l'énergie gratuite sous forme de chaleur. Le mécanisme mis en avant jusqu'à présent a été un cycle de Cr futile (15). Ici, la PCr générée par la CK mitochondriale dans l'espace intermembranaire mitochondrial (IMS) (31) serait immédiatement hydrolysée par une phosphatase PCr postulée, se colocalisant dans l'IMS ou éventuellement à l'extérieur des mitochondries, par exemple au niveau du réticulum endoplasmique (ER) (17). Cependant, CK est la seule enzyme confirmée qui peut utiliser le PCr et le Cr comme substrats, tandis que l'existence et la localisation subcellulaire de la PCr-phosphatase proposée sont encore incertaines et mal définies, respectivement. Surtout, la présence d'une telle phosphatase perturberait potentiellement l'énergie cellulaire à base de PCr en appauvrissant le PCr cellulaire et en abaissant ainsi les ratios PCr / ATP et ATP / ADP locaux. Cela serait préjudiciable à une bonne homéostasie du Ca2 + qui dépend de pompes Ca2 + très gourmandes en énergie (10, 12, 23, 29–31).

Sur la base de nos recherches approfondies sur la structure moléculaire, la fonction et la localisation des isoformes CK dans des microcompartiments subcellulaires spécifiques (6, 7, 24, 25, 27, 29–31), nous proposons ici un modèle alternatif pour CK / PCr / Cr- thermogenèse médiée dans les adipocytes (Fig. 1). Il combine la navette classique CK / PCr (3, 25, 31) avec un vélo Ca2 + futile aux urgences. Ce dernier a été décrit en détail ces dernières années dans des adipocytes beiges et bruns (10, 12). Dans ce modèle, le PCr remplit sa fonction classique en faisant passer l'énergie des mitochondries au RE (1, 5, 8, 26, 29), où il est converti par la CK cytosolique associée à la membrane en ATP. Étant donné que la navette CK / PCr peut également utiliser de l'ATP glycolytique pour la génération de PCr, cette voie fonctionnerait également au cas où l'ATP mitochondrial deviendrait limitant, ce qui semble le cas dans les adipocytes (11, 19). En fait, il existe des preuves d'une thermogenèse supplémentaire, non-canonique indépendante de l'UCP1, sans frissons, alimentée par une génération améliorée d'ATP glycolytique (9). La CK cytosolique localisée ER alimente ensuite localement le SR / ER Ca2 + -ATPase2b (SERCA2b) (23), qui est également exprimé dans les adipocytes (12), pour une absorption efficace de Ca2 + dans la lumière ER. Exigeant un rapport ATP / ADP très élevé pour son fonctionnement, cette pompe Ca2 + a un besoin particulier pour un ravitaillement dépendant du PCr via CK (23, 29–31). Bien qu'un tel apport local d'ATP n'ait pas été spécifiquement analysé dans les adipocytes, il est bien connu et documenté pour les muscles squelettiques et cardiaques, ainsi que pour les organes chauffants des poissons (20, 31).
Le cyclage futur du Ca2 + dans les adipocytes bruns / beiges est ensuite induit en couplant la captation de Ca2 + via SERCA2b à la libération immédiate de Ca2 + via différentes voies qui peuvent fonctionner en parallèle: récepteur adipocyte ryanodine (RyR2), récepteur d'inositol 1,4,5-triphosphate de type 1 (IP3 -R1), ou plus type 3 (IP3-R3). Tous ces constituants ont été identifiés dans les adipocytes beiges / bruns, et ils reposent tous sur la stimulation des récepteurs adrénergiques (α1, β3) (10, 12, 13). Le cyclage des adipocytes Ca2 + entre SERCA2b et RyR2 se produit sous exposition au froid et / ou stimulation des récepteurs adrénergiques (13), s'accompagne d'une augmentation significative du taux de consommation d'oxygène (OCR) et de la génération de chaleur, et améliore la tolérance au froid et le statut métabolique (12, 13) . En outre, la preuve d'une implication de l'IP3-R dans le cyclage futile du Ca2 + est donnée par le fait que le blocage de RyR2 par de la ryanodine à haute dose ou du rouge de ruthénium ne perturbe que partiellement mais pas complètement la thermogenèse induite par la noradrénaline dans les adipocytes knock-out UCP1 (12). Ceci est corroboré par des données antérieures (10) montrant qu'une élévation du Ca2 + cytosolique, causée par la libération de Ca2 + mitochondrial, ainsi que des niveaux élevés d'IP3, après une stimulation α1-adrénergique des adipocytes entraînent ensemble une libération de Ca2 + par l'ER / SR via le récepteur IP2 (22) . Cet événement est susceptible de se produire en parallèle avec la sortie de Ca2 + via RyR2 (12). Il existe également des preuves que le système CK et le cycle Ca2 + via SERCA2b fonctionnent dans la même voie pour la thermogenèse indépendante de l'UCP1. Lors de l'utilisation d'adipocytes beiges knock-out UCP1, la déplétion en SERCA2b et l'inhibition de la CK par l'acide β-guanidinopropionique (β-GPA) réduisaient l'OCR, mais le traitement β-GPA n'avait aucun effet supplémentaire sur l'OCR dans les adipocytes déjà appauvris en SERCA2b (12).

En conclusion, le rôle du Cr et de la CK dans la thermogenèse des adipocytes beiges et bruns a beaucoup retenu l'attention. Leur rôle dans la thermogenèse non frissonnante indépendante de l'UCP1, mais dépendante de l'ATP, émerge clairement (15–18, 21), mais la nature moléculaire détaillée du générateur d'énergie thermique est moins évidente. Cependant, nous affirmons que cela ne nécessite pas nécessairement un cyclage du Cr futile et la présence d'une hypothétique nouvelle phosphatase PCr. Il est proposé ici que le Cr et le PCr puissent simplement fonctionner dans le cadre de la navette d'énergie classique, fournissant de l'ATP à d'autres voies thermogéniques, en particulier pour le cyclage inutile de Ca2 + au RE. Pour ce dernier, de nombreuses preuves de son fonctionnement dans les adipocytes sont disponibles (10, 12, 13). Cependant, beaucoup d'expérimentation diligente est encore nécessaire pour clarifier ces problèmes avant qu'ils ne puissent être exploités pour des approches thérapeutiques basées sur des mécanismes pour lutter contre l'obésité, le diabète de type 2 et le syndrome métabolique.