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Enhancing brown adipose tissue through NAD+ precursors: a new approach to treat cardiometabolic disease

Periodic Reporting for period 1 - eBAT (Enhancing brown adipose tissue through NAD+ precursors: a new approach to treat cardiometabolic disease)

Reporting period: 2019-06-01 to 2021-05-31

Cardiometabolic disease comprises a group of disorders including obesity, hypertension, dyslipidemia, hypercholesterolemia and glucose intolerance. These are the underlying cause of the majority of cardiovascular diseases, which represent the leading cause of mortality in the Western World. The discovery of active brown adipose tissue (BAT) in adult humans in 2009 has increased the interest on the role of this tissue in the development of these disorders, and in the potential of its metabolic activation for their treatment. It has been demonstrated that BAT activation can be achieved by means of supplementation with NAD+ precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR).
However, the promising results obtained in preclinical studies have been difficult to translate to humans, where NMN and NR have only shown very mild effects. This lack of beneficial effects in humans could be due to the limitations that these molecules have, including maximal NAD+-enhancing effects of 2-fold, the need of high doses, or their rapid degradation in plasma to nicotinamide (NAM). Therefore, we are now in need for more potent NAD+ boosting molecules.
This MSCA IF project (eBAT) is focused on the use of a previously undescribed NAD+ precursor, namely reduced nicotinamide mononucleotide (NMNH), as a powerful NAD+ enhancer in vitro and in vivo, with the long-term goal of inducing BAT activation and fight metabolic disease in humans.
During the course of the eBAT project, I have identified NMNH as an NAD+ precursor for the first time, studied its impact on the cellular NAD+ metabolome thanks to the development of a dedicated mass spec method, and elucidated the molecular mechanisms behind this molecule’s great potential to boost NAD+ levels. Moreover, I have conducted a mouse study in which I have identified NMNH as an active molecule also in vivo, with greater NAD+-enhancing effects in every tissue tested, including BAT, than its oxidized counterpart NMN.
The results obtained in this project are of outstanding relevance for the future pharmacological use of NMNH as an NAD+ enhancer in mouse models of metabolic disease and, in the long-term, for its clinical application.
Through the work carried out in this project, I have identified NMNH as an NAD+ precursor and a powerful NAD+ enhancer for the first time, and elucidated its metabolic route to NAD+. NMNH proved to be a faster and more potent NAD+ precursor than NMN or NR in different cell lines, including brown adipocytes. Moreover, thanks to my contribution to the development of a dedicated mass spectrometry method for the identification of NAD+-related metabolites, I could also determine the impact of NMNH supplementation at the NAD+ metabolome level.
By blocking the activity of the different enzymes involved in NAD+ recycling, I deciphered NMNH metabolic pathway to NAD+. This adenosine kinase (AK)-dependent and nicotinamide phosphoribosyl transferase (NAMPT)-independent metabolic route constitutes a novel pathway, so far only described for reduced NAD+ precursors.
Finally, I successfully completed the animal experimentation course and obtained the corresponding license to design and carry out animal experiments in the Netherlands. This allowed me to conduct a mouse study in which I injected NMN and NMNH at the same dose. After 24 hours, I determined NAD+ content in several tissues, including blood, liver, muscle, adipose tissue (WAT and BAT), kidney, brain and heart. These results confirmed that NMNH is a much more powerful NAD+ booster than NMN also in vivo, and is able to raise and sustain NAD+ levels in blood for much longer than its oxidized counterpart. Moreover, NMNH significantly increased NAD+ levels in brown adipose tissue in only 24 hours, while NMN could not.
In conclusion, I have identified NMNH as a new and potent NAD+ precursor in vitro, and elucidated its metabolic route to NAD+. More importantly, I have demonstrated that NMNH can act as a powerful NAD+ enhancer in mice, greatly surpassing the effects of NMN in every tissue.
These results have been published in The FASEB Journal (doi:10.1096/fj.202001826R). Moreover, I have presented my work in several conferences, including the Keystone eSymposium “Metabolic Decisions in Development and Disease” and the NovoNordisk Foundation “Metabolism Month”. The expertise acquired during the development of this project and the relevance of the results obtained with NMNH have also allowed me to publish a review on this topic in EMBO Molecular Medicine (doi:10.15252/emmm.202113943) a News&Views article in Nature Metabolism (doi:10.1038/s42255-020-0177-x) and to collaborate with several top European and American groups working on the NAD+ field. This fruitful collaboration network has led to the publication of four articles in the journals Molecular Metabolism (doi:10.1016/j.molmet.2019.09.013) The American Journal of Clinical Nutrition (doi: 10.1093/ajcn/nqaa072) The Journal of Nutrition (doi:10.1093/jn/nxab193) and the European Journal of Nutrition (In press).
During my stage as an MSCA fellow, I also brought my enthusiasm about the NAD+ field to BSc and Masters students by giving lectures and by supervising literature reviews on this topic. Moreover, I have actively discussed about these results and other aspects of NAD+ metabolism in my personal blog (https://enhancingnad.wordpress.com/) and in my social media network (Twitter: @RZapataPerez, LinkedIn: https://www.linkedin.com/in/rubén-zapata-pérez-76ab58127).
Cardiometabolic disease is the leading cause of mortality in Western societies, with the subsequent impact that this has for healthcare systems. Therefore, pharmacological interventions aimed to alleviate the consequences and, especially, prevent the onset of this group of diseases, are of especial interest. In this sense, NAD+ repletion therapies have come under the spotlight mostly because of the promising results obtained in animal models. However, the beneficial effects of NAD+ repletion obtained in preclinical studies have been difficult to translate to humans; something that could be partially caused by the limited effect of the current repertoire of NAD+-enhancing molecules on cellular NAD+ content.
Through this project, I have expanded this repertoire with a new NAD+ precursor, termed reduced nicotinamide mononucleotide (NMNH), which has shown an unprecedented potential to raise NAD+ levels in cells and in mice. Interestingly, NMNH supplementation led to an increase in brown adipose tissue NAD+ levels in mice, while NMN at the same concentration did not achieve statistical significance. Therefore, NMNH arises as a great alternative to oxidized NAD+ precursors, with the potential to overcome their limitations.
The identification of this new molecule has attracted the attention of not only the scientific community, but also of potential industrial partners. At this moment, I am establishing new collaborations with the academic and non-academic sectors to explore the therapeutic potential of NMNH in animal models of disease, with the ultimate goal of bringing this molecule to the clinic. As a first step in this process, I am currently investigating whether longer-term supplementation with NMNH via oral administration in mice is effective and safe. The results obtained in this study will be of special relevance to bring NMNH to the public and, hopefully, to metabolic disease patients in the future.
Summary for publication