Nicotinamide adenine dinucleotide phosphate

Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NADPH as a reducing agent ('hydrogen source'). NADPH is the reduced form of NADP, the oxidized form. NADP is used by all forms of cellular life. NADP differs from NAD by the presence of an additional phosphate group on the 2' position of the ribose ring that carries the adenine moiety. This extra phosphate is added by NAD+ kinase and removed by NADP+ phosphatase. In general, NADP+ is synthesized before NADPH is. Such a reaction usually starts with NAD+ from either the de-novo or the salvage pathway, with NAD+ kinase adding the extra phosphate group. ADP-ribosyl cyclase allows for synthesis from nicotinamide in the salvage pathway, and NADP+ phosphatase can convert NADPH back to NADH to maintain a balance. Some forms of the NAD+ kinase, notably the one in mitochondria, can also accept NADH to turn it directly into NADPH. The prokaryotic pathway is less well understood, but with all the similar proteins the process should work in a similar way. NADPH is produced from NADP+. The major source of NADPH in animals and other non-photosynthetic organisms is the pentose phosphate pathway, by glucose-6-phosphate dehydrogenase (G6PDH) in the first step. The pentose phosphate pathway also produces pentose, another important part of NAD(P)H, from glucose. Some bacteria also use G6PDH for the Entner–Doudoroff pathway, but NADPH production remains the same. Ferredoxin-NADP reductase, present in all domains of life, is a major source of NADPH in photosynthetic organisms including plants and cyanobacteria. It appears in the last step of the electron chain of the light reactions of photosynthesis. It is used as reducing power for the biosynthetic reactions in the Calvin cycle to assimilate carbon dioxide and help turn the carbon dioxide into glucose.

The role of nicotinamide riboside metabolism in pancreatic beta cells

Angélique Satoko Cercillieux

Pancreatic beta cells play a major role in the regulation of glucose homeostasis by secreting insulin in response to elevated blood glucose levels. A gradual deterioration of functional beta cell mass and the chronic elevation of blood glucose levels are central to development of type 2 diabetes. While the etiologies of Type 2 diabetes and age-related metabolic complications are complex, they are often associated with a decline of intracellular nicotinamide adenine dinucleotide (NAD+). NAD+ levels are maintained through the balance between its synthesis from NAD+ precursors and its degradation by several families of enzymes, including the sirtuin family of protein deacetylases, the ADP-ribosyl transferase enzymes (ARTDs or PARPs) and cADP ribose hydrolases (CD38). NAD+ declines are mostly associated with increased NAD+ consumption, which can be counteracted by dietary supplementation of NAD+ precursors. However, our knowledge on how different tissues utilize and respond to different NAD+ precursors for NAD+ synthesis is still incomplete. This, in turn, limits our ability to provide therapeutic benefits in clinical settings. In this project, we aim to understand the relevance of a particular NAD+ precursor, nicotinamide riboside (NR), in beta cell physiology and its potential use to treat beta cell dysfunction. To do so, we have used mice that are deficient for nicotinamide riboside kinase 1 (NRK1), the first and rate-limiting enzyme for the transformation of NR into NAD+. We demonstrate that NRK1 whole body knockout mice exhibit impaired insulin secretion after a meal or upon glucose challenge when fed a high fat diet. However, NRK1 deficiency in beta cell was not the causality for beta cell failure as differences observed in the NRK1 whole body KO mice were not phenocopied in NRK1 beta cell specific KO mice. Aged NRK1 whole body KO mice also exhibited dysfunctional beta cells and significant loss of insulin content and beta cell mass. Furthermore, NRK1 deficiency exacerbates the decline of NAD+ bioavailability and mitochondria function with age, even promoting fibrosis in multiple tissues. Together, our data suggests that endogenous NR metabolism is critical in maintaining whole body NAD+ homeostasis and to protect against mitochondria dysfunction and stress-related cell responses in pancreatic beta cells.

EPFL2022

Semisynthetic Biosensors for the Quantification of Nicotinamide Adenine Dinucleotides in Live Cells

Olivier Vincent Sallin

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are crucial redox cofactors regulating energy metabolism, reductive biosynthesis and the antioxidant defense system. Besides their role as redox cofactors, NAD(P)s have more recently also been described as important signaling molecules, acting for example as cosubstrate of different enzymes such as the sirtuins and poly(ADP-ribose) polymerases (PARPs), involved in the control of important cellular functions. Highlighting their important role, an imbalance of their intracellular concentration has been associated with aging and different pathologies. Therefore, the development of methods enabling the accurate subcellular quantification of NAD(P) in live cells is of great importance. This thesis describes the development and application of two FRET-based semisynthetic biosensors for NADPH/NADP+ and NAD+ in live cells. These sensors, named NAD(P)-Snifits, possess a high dynamic range, high specificity, are pH insensitive and can be excited at long wavelenghts. By using ratiometric imaging and fluorescence lifetime imaging, we quantified free NADPH/NADP+ and NAD+ in different subcellular compartments in live cells. We report here, for the first time, that free NADPH/NADP+ is significantly higher in the mitochondria than in the cytosol and observed moderately higher NAD+ level in the mitochondrial matrix consistent with previous report. We have also demonstrated that NADP-Snifit can be used to monitor the dynamics of NADPH/NADP+ caused by oxidative stress using conventional widefield fluorescence microscopy. The easy handing and use of these sensors make it possible to study not only the kinetics of cellular H2O2 scavenging and NADPH regeneration rate, but also to monitor drug-related side effects. Finally, we measured the effect of different conditions and drug treatment mimicking calorie restriction on free cytosolic and mitochondrial NADPH/NADP+ and NAD+ by flow cytometry. We observed that all these treatments increased free cytosolic and mitochondrial NAD+ concentrations with various levels of efficiency, but had divergent effects on the different subcellular NADPH/NADP+. NAD(P)-Snifits have the potential of becoming valuable tools to study the NAD(P) metabolism in physiological relevant conditions. Their wide applicability, in particular with high-throughput methods such as flow cytometry opens the possibilities for their use in metabolic and drug screening of large libraries.

EPFL2018