B-vitamins and NAD metabolism, or when vitamin B3's bioavailability is not enough

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Pharmacy

Abstract

The dietary vitamin B3, which encompasses nicotinamide, nicotinic acid and nicotinamide riboside, is precursor to the coenzyme nicotinamide adenine dinucleotide (NAD+), its phosphorylated parent (NADP+) and their respective reduced forms (NADH and NADPH). Once converted intracellularly to NAD(P)+, it is used as a co-substrate in two types of intracellular modifications, which control numerous essential signalling events (adenosine diphosphate ribosylation and deacetylation), and is a cofactor for over 400 redox enzymes, thus controlling metabolism. Critically, the NAD(P)(H)-cofactor family can promote mitochondrial dysfunction and cellular impairment if present in sub-optimal intracellular concentrations.
Vitamin B3, and other B-vitamins such as thiamine (vitamin B1), riboflavin (vitamin B2) and pyridoxine (vitamin B6) are extracted in their coenzyme forms from food stuff. During digestion, the coenzymes are catabolised to the free circulating vitamins, which are then passively or actively transported across membranes, and salvaged intracellularly to their respective cofactors. Mammals are entirely reliant on a dietary source of vitamin B1 and heavily dependent on the dietary supply of vitamin B3, B2, and B6. Of note, acute deficiencies in vitamin B1 and vitamin B3 effect identical organs, with identical outcomes if left untreated: dementia and death. Conditions such as diabetes and obesity, alcoholism, high fat diet and conditions where therapy impacts nutrition can compromise suitable absorption of these vitamins.
The bulk of intracellular NAD+ is regenerated via the effective salvage of nicotinic acid and nicotinamide (vitamin B3), while de novo NAD+ is obtained from tryptophan. Crucially, these salvage and de novo pathways depend on the functional forms of vitamin B1, B2 and B6 to generate NAD+ via a phosphoriboside pyrophosphate intermediate. Nicotinamide Riboside (NR) is the only form of vitamin B3 from which NAD+ can be generated in a vitamin B1, B2 and B6 independent manner and even though NR is a minor component of vitamin B3, the salvage pathway using NR for the production of NAD+ is expressed in most eukaryotes. While major strides have been made in the field of NAD+ biology and NAD+ metabolism, the role of this later pathway and the importance of the interplay between the bioavailability of vitamin B1, B2 and B6 and the pool of NAD(P)(H)-cofactors remain poorly explored.
Using our synthetic expertise in nucleotide and stable isotope labelling chemistry, we will generate isotopically labelled vitamin B1 and B3 derivatives. These entities will be used to determine the profile of the vitamin B3 metabolome quantified by mass spectroscopy, under vitamin B1, B2 and B6 depletion conditions, in genetically engineered yeast strains and mammalian (murine and human) hepatocytes. In mammalian cells, these metabolic profiles will be correlated to mitochondrial functions. With this information, we will be able to prioritise the mechanisms cells use to best maintain the NAD(P)(H) pool in time of shortage of vitamin B1, B2 or B6. We predict that the pathway, by which NR is converted to NAD+, provides the means to rapidly yet transiently elevate mitochondrial and cytosolic NAD(P)(H) levels to kick start mitochondrial functions. If demonstrated, this knowledge will help identify new, physiologically relevant, vitamin-B combinations that could better restore mitochondrial function through enhanced bioavailability, in cells and organs where metabolism has been compromised by imbalanced micronutrition. This knowledge will be particularly important in terms of understanding the impacts of a global or partial vitamin B deficiency and vitamin B supplementation on organ functions in relation to malnutrition and over-nutrition.

Technical Summary

In its functional forms as nicotinamide adenine dinucleotide (phosphate) NAD(P)+ and reduced forms NAD(P)H, vitamin B3 participates in more reactions than any other known vitamin-derived molecule, and is intimately implicated in essential cellular signalling, bioenergetics and metabolic pathways, with sub-optimal intracellular NAD+ levels [NAD+] promoting mitochondrial dysfunction and cellular impairment. As a consequence, [NAD+] is central to the overall homeostasis, growth and ageing of cells and organs. In the UK population, diet-linked NAD(P)+ deficiency is due to poor food choices, alcoholism, adverse drug reactions and infectious or autoimmune diseases. This is possibly compounded by excessive food intake and high fat diets which further promote the decline of oxidised form of the intracellular [NAD(P)+] pool. Critically, sustained sub-optimal intracellular [NAD+] has been shown to have long term physiological consequences, while health benefits associated with systemic vitamin B3 supplementation, with the view of boosting intracellular [NAD(P)+], remain in dispute.
Intracellular NAD(P)+ biosynthesis and recycling depend on the bioavailability of three other dietary vitamins B [thiamine (B1), riboflavin (B2) and pyridoxine (B6)] in addition to ATP. We hypothesise that a deficiency of vitamin B1 most decreases intracellular [NAD(P)+], resulting in an NAD+-deficiency signature at cellular levels, even when the dietary vitamin B3 is available, unless the nicotinamide riboside form of vitamin B3 is supplemented. We will investigate the effects of vitamin B1, B2, and B6 deficiency under a set of controlled supplementation conditions in both yeast and in mammalian hepatocytes using the abundance of the related metabolites and mitochondrial functions as measurable end-points. In this context, we will identify physiologically relevant combinations of B-vitamins that may restore mitochondrial function through enhanced bioavailability of these cofactors.

Planned Impact

There is a large body of evidence indicating that ageing, disease state as well as therapeutic treatment greatly changes our ability to absorb micro-nutrients, including vitamin B1, B2, B3 and B6 which are, in turn, critical to our long term well-being. Here, it is proposed that the functional intracellular NAD+ concentration not only depends of the bioavailability of vitamin B3 but critically relies on the functional form of vitamin B1, and to a lesser extent on that of vitamin B2 and B6. This knowledge is particularly important in terms of understanding the impacts of a global or partial vitamin B deficiency and vitamin B supplementation in disease in relation to malnutrition and over-nutrition. We hope to identify new, physiologically relevant, vitamin-B supplementation combinations that kick start cofactors' microbioavailability and restore mitochondrial function.

Food scientists, nutritionists and clinicians working in developing more efficient food stuff will particularly benefit from this research. This knowledge will be relevant to the field of micronutrition in disease and will help better inform nutritionists and clinicians of the importance of the vitamin B biosynthetic network, which will be critical to establishing a road-map to micronutrients supplementation to inform and educate patients:
- It will support evidence-based research towards the development of bioactive food components and tools to evaluate the impact of diet on disease prevention, for use by consumers and medical professionals. In terms of vitamins B bioavailability and nutritional impact on diseases progression, the work proposed here could be used to correlate the B1/B3-vitaminome to the risk of developing chronic diseases and to measure the impact of vitamins B's bioavailability on diseases progression.
- Alcoholism, its link to vitamin B deficiencies and psychological outcomes remain a primary area of research in the modern developed world. Studies have indicated that, in patients with alcoholic pellagra, vitamin B3 deficiency may be an important factor influencing both the onset and severity of the condition and patients with alcoholism are being recommended vitamin B1 as a supplement to minimise dementia and psychological episodes associated with alcohol abuse. Yet, combination supplementations are not yet considered as the pharmacological network between these two vitamins in terms of NAD bioavailability has never been demonstrated.
- A particularly critical food type which heavily relies on safe vitamin B content is infant formula, where macro and micronutrients supplementation that match to some extent the nutritional properties of a mother's milk are sought. Nicotinamide Riboside, vitamin B3 minor component, is found in breast milk but is not currently included in infant formula, unlike Nicotinamide. A better understanding of the roles of each component of the vitamin B3 in relation to the other vitamin Bs' supplements is therefore critical if one is to consider a betterment of this type of man-made product for the health of generations to come.

The time scale for impact can be anticipated to be quite short (2-3years) as the field of NAD+ biology research has been extremely active both in academia and in industry, with the leading pharmaceutical and food industries such as AbbVie and Nestle making headways in establishing the B-vitaminome as a key player in the future development of bio-active entities.

The work will be carried out by an expert synthetic chemist (Repdath) who through short placements in two internationally leading research groups in the field of NAD+ biology (Brenner-Iowa and Sobol-Alabama) will hone his skills in advanced analytical and biological techniques, which he will bring back to QUB for subsequent implementation. This will particularly match his aspirations in pursuing a broad-based scientific career in collaboration with the industry and the overall strategy of the Migaud group.
 
Description We have demonstrated beyond reasonable doubt that the nicotinamide riboside vitamin B3 is a bioavailable precursor to the cofactor NAD, key coenzyme in metabolism and cellular signalling, and that its physiological effect is different from that of other vitamin B3 precursors such as niacin, which require the phosphoribose pentose pathway and other vitamins to yield NAD. We have developed an assay to report on the methylation status of microRNA. methylation of microRNA is a rapid response by the cell to environmental and metabolic changes in the cell.
Exploitation Route commercial use of the tools we have developed for the studies thus reported in the literature
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.timelesslifemag.com/index.php/2016/10/31/star-research-team-maps-nr-and-nmn-conversion-path-to-cellular-nad/
 
Description Findings in animal studies establishing that Nicotinamide Riboside (NR) is a bioavailable precursor of the metabolic cofactor NAD and that it is safe for human use have spurred people to take commercially available NR supplements designed to boost NAD. However, these over-the-counter supplements have not undergone clinical trials to see if and how they work in people, as yet.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Chemicals,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description Prof. Rob Sobol, Mitchell Cancer Institute 
Organisation Mitchell Cancer Institute
Country United States 
Sector Hospitals 
PI Contribution We have synthesised NAD and ADPR precursors which the laboratory of Dr Sobol is particularly keen on investigating in relation to NAD consumption in response to BER.
Collaborator Contribution Dr Sobol's lab is evaluating our precursors and synthetic derivatives in a range of cancer cell lines to better understand BER response and impact on cellular metabolism and response to BER.
Impact A range of grant applications in the US and in the UK have been made to further explore this collaborative work. Publicaqtions are being prepared in addition to a book chapter on PARP in BER.
Start Year 2013
 
Description Professor C. Brenner- University of Iowa 
Organisation University of Iowa
Department Department of Biochemistry
Country United States 
Sector Academic/University 
PI Contribution Synthesis of chemical entities to probe the NAD metaoblome; Hosting of QUB team member at the university of Iowa; 2 x1 visiting fellows (QUB PhD students) hosted in the Brenner lab.
Collaborator Contribution Cell work; animal work; access to facilties and know-how.
Impact publication; fellowship; mobility ofresearchers
Start Year 2014
 
Description Professor M. Ziegler Collaboration 
Organisation University of Bergen
Country Norway 
Sector Academic/University 
PI Contribution Provide chemical entities to probe the NAD metaoblome
Collaborator Contribution evaluation in cell lines
Impact publication Sept 2015
Start Year 2015
 
Description collaboration with Innovative DNA Technology 
Organisation Integrated DNA Technologies
Country United States 
Sector Private 
PI Contribution Work on ionic liquids stabilised phosphoramidite
Collaborator Contribution New reagents on which to apply ionic liquids technology
Impact NDA in place. Only chemistry
Start Year 2014