Eva Drevet Mulard

La phosphatase alcaline non spécifique d’un tissu (TNAP), une enzyme ancrée aux membranes cellulaires et libérée dans la circulation sanguine, est principalement connue pour son rôle crucial dans la minéralisation du squelette par l’hydrolyse du pyrophosphate inorganique (PPi), le principal inhibiteur de la minéralisation. Cependant, la TNAP déphosphoryle potentiellement un large éventail de substrats extracellulaires, contribuant ainsi à diverses fonctions physiologiques. En utilisant la métabolomique par résonance magnétique nucléaire (RMN), le laboratoire a récemment identifié la phosphocholine comme un substrat possible in vivo, suggérant un lien hypothétique entre la TNAP, la production hépatique de phosphatidylcholine, et le métabolisme lipidique. Dans ce mécanisme, la TNAP serait nécessaire pour la synthèse hépatique de phosphatidylcholine, indispensable à l’assemblage des lipoprotéines et l’export des triglycérides. Les travaux réalisés dans le cadre de cette thèse avaient pour objectif d’explorer ce lien hypothétique in vivo chez la souris, dans des sérums de souris et humains, en culture de cellules et in vitro. Les résultats montrent tout d’abord que les souris déficientes en TNAP développent une stéatose hépatique et présentent une réduction du taux de triglycérides sériques, mimant les effets d’une carence en choline. Dans des souris sauvages, la métabolomique par RMN a montré que l’inhibition de la TNAP par l’inhibiteur SBI-425 entraîne une diminution significative des niveaux de choline dans le sang et dans le foie et nous a permis de confirmer deux substrats potentiels de la TNAP : la phosphocholine et la phosphoéthanolamine. Des expériences d’enzymologie par RMN montrent que l’inhibition de la TNAP bloque l’hydrolyse de la phosphocholine et de la phosphoéthanolamine dans le sérum de souris et d’humains. Dans les hépatocytes humains HuH6, l’inhibition de la TNAP bloque l’hydrolyse de la phosphocholine et altère la prolifération cellulaire lorsque la phosphocholine est la seule source de choline, suggérant un impact direct sur le métabolisme lipidique cellulaire. En outre, nous avons déterminé que l’enzyme recombinante hydrolyse la phosphocholine et la phosphoéthanolamine avec une efficacité similaire, voire supérieure, à celle du PPi. La détermination par nos collaborateurs de l’UMR CNRS 5086 de la structure 3D de la TNAP par diffraction des rayons X et cryo-EM nous a permis de déterminer les résidus d’acides aminés du site actif interagissant avec les substrats et l’inhibiteur de la TNAP. Collectivement, ces résultats suggèrent fortement que la TNAP hydrolyse la phosphocholine et phosphoéthanolamine pour participer au métabolisme lipidique. En parallèle de cette première étude, une expérience de métabolomique par RMN sur des souris ApoE-KO (modèle d’athérosclérose) a suggéré que la TNAP est également impliquée dans la déphosphorylation du nicotinamide mononucléotide (NMN). Nous montrons ici que la TNAP et l’ectonucléotidase CD73 hydrolysent toutes deux le NMN dans le sérum, ainsi qu’en culture d’ostéoblastes humains. In vitro, nous montrons que la TNAP et la CD73 recombinantes de souris hydrolysent le NMN, et que l’hydrolyse du NMN par la TNAP a une efficacité catalytique comparable à l’hydrolyse de la phosphocholine, de la phosphoéthanolamine et du PPi. Enfin, l’implication d’ENPP1 dans le métabolisme du NAD+ étant proposée dans la littérature, nous montrons sa capacité à hydrolyser le NAD+ in vitro. ENPP1, puis la TNAP et CD73 semblent donc participer à l’hydrolyse extracellulaire du NAD+ en nicotinamide. En conclusion, cette étude a permis d’identifier de nouvelles fonctions enzymatiques de la TNAP. La TNAP semble donc importante pour la captation cellulaire de choline, son absence ou inhibition perturbant le métabolisme lipidique. La TNAP pourrait également être impliquée dans le métabolisme du NAD ; l’impact d’une déficience ou d’une inhibition de la TNAP sur ce métabolisme reste à approfondir.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical approach that enables both the structural determination and precise quantification of small molecules, such as metabolites. However, achieving precise quantification with NMR involves more than simply comparing integrals derived from NMR peaks to a concentration reference; quantitative NMR (qNMR) is a distinct and specialized application within the field. To obtain absolute quantitative results, spectra must be acquired under strict experimental conditions. Unfortunately, these acquisition parameters can be challenging to implement experimentally and often require trade-offs that compromise high throughput or practicality. In such situations, alternative strategies based on relative quantification and advanced software tools offer valuable solutions. This review aims to provide non-specialists with the key concepts and methodologies required for accurate NMR-based quantification in biomedical research, focusing on practical guidelines and experimental considerations. Unlike prior reviews, it prioritizes accessibility and practical implementation for researchers outside the field, emphasizing key experimental workflows and applications in biological and clinical studies. It clarifies the distinctions between absolute and relative concentration determinations and emphasizes the critical importance of sample preparation, pulse sequence selection, and rigorous control of experimental parameters. Recent technological advancements, such as high-field spectrometers and cryoprobes, have significantly enhanced the sensitivity and accuracy of NMR, enabling the reliable detection of low-concentration metabolites. Quantitative NMR thus offers critical potential in elucidating metabolic processes, supporting drug development, and aiding disease diagnosis.

Phosphatases are enzymes that catalyze the hydrolysis of phosphate esters. They play critical roles in diverse biological processes such as extracellular nucleotide homeostasis, transport of molecules across membranes, intracellular signaling pathways, or vertebrate mineralization. Among them, tissue-nonspecific alkaline phosphatase (TNAP) is today increasingly studied, due to its ubiquitous expression and its ability to dephosphorylate a very broad range of substrates and participate in several different biological functions. For instance, TNAP hydrolyzes inorganic pyrophosphate (PP_i) to allow skeletal and dental mineralization. Additionally, TNAP hydrolyzes pyridoxal phosphate to allow cellular pyridoxal uptake, and stimulate vitamin B6-dependent reactions. Furthermore, TNAP has been identified as a key enzyme in non-shivering adaptive thermogenesis, by dephosphorylating phosphocreatine in the mitochondrial creatine futile cycle. This latter recent discovery and others suggest that the list of substrates and functions of TNAP may be much longer than previously thought. In the present review, we sought to examine TNAP within the alkaline phosphatase (AP) superfamily, comparing its sequence, structure, and evolutionary trajectory. The AP superfamily, characterized by a conserved central folding motif of a mixed beta-sheet flanked by alpha-helices, includes six subfamilies: AP, arylsulfatases (ARS), ectonucleotide pyrophosphatases/phosphodiesterases (ENPP), phosphoglycerate mutases (PGM), phosphonoacetate hydrolases, and phosphopentomutases. Interestingly, TNAP and several ENPP family members appear to participate in the same metabolic pathways and functions. For instance, extra-skeletal mineralization in vertebrates is inhibited by ENPP1-mediated ATP hydrolysis into the mineralization inhibitor PP_i, which is hydrolyzed by TNAP expressed in the skeleton. Better understanding how TNAP and other AP family members differ structurally will be very useful to clarify their complementary functions. Structurally, TNAP shares the conserved catalytic core with other AP superfamily members but has unique features affecting substrate specificity and activity. The review also aims to highlight the importance of oligomerization in enzyme stability and function, and the role of conserved metal ion coordination, particularly magnesium, in APs. By exploring the structural and functional diversity within the AP superfamily, and discussing to which extent its members exert redundant, complementary, or specific functions, this review illuminates the evolutionary pressures shaping these enzymes and their broad physiological roles, offering insights into TNAP’s multifunctionality and its implications for health and disease.

Obesity is a risk factor for many diseases, such as type 2 diabetes and cardiovascular diseases. In line with the need for precision medicine, the search for biomarkers reporting the progression of obesity- and diet-associated disorders is urgent. We used NMR to determine the metabolomics profile of key organs (lung, liver, heart, skeletal muscle, kidney, and brain) and serum from male C57Bl/6J mice (5 weeks old) fed for 6, 10, and 14 weeks on a high-fat and high-sucrose diet (HFHSD) vs. a standard diet (STD). We determined metabolite concentrations in the organs at each time point, which allowed us to discriminate age- and diet-related effects as well as the interactions between both, highlighting the need to evaluate the influence of age as a confounding factor on metabolic signatures. Notably, the analysis revealed the influence of time on metabolite concentrations in the STD condition, probably reflecting the juvenile-to-adult transition. Variations impacted the liver and lung metabolites, revealing the strong influence of the HFHS diet on normal metabolism maturation during youth.

Interactions between mitochondria and the endoplasmic reticulum, known as MAMs, are altered in the liver in obesity, which contributes to disruption of the insulin signaling pathway. In addition, the plasma level of glycine is decreased in obesity, and the decrease is strongly correlated with the severity of insulin resistance. Certain nutrients have been shown to regulate MAMs; therefore, we tested whether glycine supplementation could reduce insulin resistance in the liver by promoting MAM integrity. Glycine (5 mM) supported MAM integrity and insulin response in primary rat hepatocytes cultured under control and lipotoxic (palmitate 500 µM) conditions for 18 h. In contrast, in C57 BL/6 JOlaHsd mice (male, 6 weeks old) fed a high-fat, high-sucrose diet (HFHS) for 16 weeks, glycine supplementation (300 mg/kg) in drinking water during the last 6 weeks (HFHS-Gly) did not reverse the deleterious impact of HFHS-feeding on liver MAM integrity. In addition, glycine supplementation worsened fasting glycemia and glycemic response to intraperitoneal pyruvate injection compared to HFHS. The adverse impact of glycine supplementation on hepatic gluconeogenesis was further supported by the higher oxaloacetate/acetyl-CoA ratio in the liver in HFHS-Gly compared to HFHS. Although glycine improves MAM integrity and insulin signaling in the hepatocyte in vitro, no beneficial effect was found on the overall metabolic profile of HFHS-Gly-fed mice.