MINI REVIEW published: 31 January 2022 doi: 10.3389/fendo.2021.815565 NAD+-Increasing Strategies to Improve Cardiometabolic Health? Francisco Blanco-Vaca 1,2,3†, Noemi Rotllan 3,4†, Marina Canyelles 3,4, Didac Mauricio 3,4,5, Joan Carles Escolà-Gil 3,4 and Josep Julve 3,4* 1Servei de Bioquímica, Hospital de la Santa Creu i Sant Pau i Institut d’Investigació Biomèdica Sant Pau, Barcelona, Spain, 2Departament de Bioquı´mica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Madrid, Spain, 4 Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau i Institut d’Investigació Biomèdica de l’Hospital de la Santa Creu i Sant Pau, Barcelona, Spain, 5 Servei de Endocrinologia i Nutrició, Hospital de la Santa Creu i Sant Pau i Institut d’Investigació Biomèdica Sant Pau, Barcelona, Spain Depleted nicotinamide adenine dinucleotide (NAD+) is a common hallmark of metabolic disorders. Therefore, NAD+-increasing strategies have evolved as a potential therapeutic venue to combat cardiometabolic diseases. Several forms of vitamin B3, i.e., nicotinamide Edited by: and nicotinamide mononucleotide, and especially nicotinamide riboside, have attracted Alexandre Gabarra Oliveira, most interest as potentially safe and efficacious candidates for NAD+ restoration. Herein, São Paulo State University, Brazil we dissected the characteristics of the latest clinical trials testing the therapeutic potential Reviewed by: Mal Evans, of different vitamin B3 molecules to improve cardiometabolic health, with a special focus KGK Science Inc., Canada on randomized, placebo-controlled clinical trials performed in the context of obesity or *Correspondence: other pathologies, mainly linked to cardiovascular system and skeletal muscle Josep Julve functionality. The favorable outcomes via NAD+-increasing strategies found in the jjulve@santpau.cat † different studies were quite heterogeneous. NAD+-increasing interventions improved These authors share first authorship capacity to exercise, decreased blood pressure, increased the anti-inflammatory profile Specialty section: and insulin-stimulated glucose disposal, and reduced the fat-free mass. Except for the This article was submitted to decreased blood pressure, the significant results did not include many hard clinical end Obesity, a section of the journal points, such as decreases in weight, BMI, fasting glucose, or HbA1c percentage. Frontiers in Endocrinology However, the analyzed trials were short-term interventions. Overall, the accumulated Received: 15 November 2021 clinical data can be interpreted as moderately promising. Additional and long-term studies Accepted: 08 December 2021 Published: 31 January 2022 will be needed to directly compare the doses and duration of treatments among different Citation: vitamin B3 regimes, as well as to define the type of patients, if any, that could benefit from Blanco-Vaca F, Rotllan N, these treatments. In this context, a major point of advancement in delineating future Canyelles M, Mauricio D, clinical trials would be to identify subjects with a recognized NAD+ deficiency using novel, Escolà-Gil JC and Julve J (2022) NAD+-Increasing Strategies to appropriate biomarkers. Also, confirmation of gender-specific effect of NAD+-increasing Improve Cardiometabolic Health? treatments would be needed. Front. Endocrinol. 12:815565. doi: 10.3389/fendo.2021.815565 Keywords: vitamin B3, clinical trials, obesity, diabetes mellitus, nicotinamide Frontiers in Endocrinology | www.frontiersin.org 1 January 2022 | Volume 12 | Article 815565 Blanco-Vaca et al. Vitamin B3 Therapies in Cardiometabolic Health INTRODUCTION NAM, NR, or NMN (5, 6). However, these NAD+ intermediates can exhibit a distinct behavior due to the different tissue Nicotinamide adenine dinucleotide (NAD+) is a dual molecule. distribution and relative abundance of enzymes or transporters Apart from its well-established role as a cofactor for redox involved in NAD+ metabolism. reactions and, in its reduced form, as an electron donor to the mitochondrial oxidative phosphorylation system for the synthesis NA of ATP, NAD+ has also been described as a signaling molecule. NA has been the most widely used form of vitamin B3 in clinical Indeed, NAD+ is also a substrate for NAD+-consuming enzymes practice due to its hypolipemic properties, i.e., triglyceride- involved in the metabolic adaptations that govern cell metabolism reducing and high-density lipoprotein cholesterol (HDL-C)- and survival. raising effects (5). Besides such lipid effects, NA also interacts It is known that low levels of NAD+ result from altered NAD+ with the GPR109A receptor expressed in immune cells, thus homoeostasis. Impaired NAD+-mediated signaling and blunting immune activation (5). Although such NA properties concurrent alterations in dysfunctional mitochondria are beneficial to treat hyperlipidemia, NA administration is commonly underlie cardiometabolic disorders, such as type 2 frequently associated with some serious adverse effects, i.e., diabetes, non-alcoholic fatty liver, and aging. flushing, deterioration of insulin resistance, and hepatic and NAD+ precursors are naturally found in food, and their use gastrointestinal toxicity (5), which reduce medication has emerged as a strategy for NAD+ replenishment and hence to adherence. Moreover, early reports supporting efficacy of NA favorably influence NAD+ dependent pathways and rescue in secondary cardiovascular prevention were not confirmed by tissues from the adverse consequences of aging and metabolic more recent, large prospective trials, including 29,087 subjects, diseases (1). NAD+ content in animal and human tissues can be the AIM-HIGH (9), and the HPS2-THRIVE (10), which used increased via the supplementation of different precursors (2–4). extended-released NA and laropiprant, a selective antagonist of Although NAD+ can be synthesized directly from tryptophan prostaglandin D2 receptors, to control adverse effects (5). Since (de novo pathway), it is more efficiently generated from other NA also ameliorated wound healing and cardiac function after precursors, such as nicotinic acid (NA) (Preiss-Handler myocardial infarction via prostaglandin D2 receptor subtype 1- pathway) and nicotinamide (NAM) (Salvage pathway). mediated M2 macrophage polarization, the inhibition of Nicotinamide phosphoribosyltransferase (NAMPT), which prostaglandin D2 signaling could have attenuated NA converts NAM into nicotinamide mononucleotide (NMN), is cardiovascular benefits (11). The latter finding would be the key step for NAD+ synthesis (5). Another source of NAD+ consistent with data from earlier clinical trials using NA, comes from nicotinamide riboside (NR). showing a reduction of mortality in subjects nine years after NAD+ precursors that are used to elevate NAD+ availability stopping NA therapy (12). It is also possible that the protective in target tissues have demonstrated efficiency in improving effect of NA is no longer significant in the context of previous insulin sensitivity and reducing diabetes burden and associated statin treatments (9). However, NA may also be a potential metabolic derangements in preclinical models. Actually, a source for NAD+ synthesis (13), and could present beneficial growing number of studies have successfully used different effects raising tissue NAD+ levels in case of deficiency. In this vitamin B3 forms to boost NAD+ production and positively regard, the effect of NA in 5 subjects (4 females) with adult-onset influence aging and cardiometabolic diseases in experimental in mitochondrial myopathy subjects and systemic NAD+ deficiency vivo models, including our studies performed in the context of and 10 healthy controls (8 females) was recently assessed (14). accelerated atherosclerosis or obesity (4, 6, 7). However, less NA (up to 750–1,000 mg/day) or placebos were administered in a consistent results have been obtained in clinical studies. non-randomized, open-able parallel assignment to subjects and This review analyzes the accumulating evidence from the their matched controls for 10 or 4 months, respectively. The NA latest clinical studies that used different NAD+-increasing administration resulted in blood elevations of NAD+ in all strategies to improve cardiometabolic health, with a special subjects, up to eightfold, also restoring the muscle NAD+ focus on randomized, double-blind, placebo-controlled clinical levels of the subjects (14). Noteworthy was that muscle trials, since they are considered the “gold standard” for testing strength and mitochondrial biogenesis increased in all subjects. clinical intervention-based studies (8). Particularly, a Pubmed Furthermore, the muscle metabolome in the subjects with adult- search on clinical reports published in the last 5 years on this onset mitochondrial myopathy shifted toward that of the healthy topic using “nicotinamide” and "randomized clinical trials" as controls, whereas liver fat accumulation decreased by 50%. keywords was performed. Adiponectin concomitantly increased, a finding consistent with previous studies in subjects treated with a high dose of NA (i.e., 1,500 mg/day) (15, 16). Important, the blood analysis was RESULTS OF THERAPEUTIC revealed as a useful sample to identify NAD+ deficiency in INTERVENTIONS WITH DIFFERENT subjects with myopathy (14). FORMS OF VITAMIN B3 NAM A plethora of pathways require NAD+ as a coenzyme. The Unlike NA, NAM is not considered a GPR109A agonist, thus easiest strategy to increase NAD+ in vivo is via the provision avoiding prostaglandin-related vasodilatory side effects. NAM is of NAD+ precursors, which include NA (also termed niacin), a widely available dietary supplement, which, at doses of no more Frontiers in Endocrinology | www.frontiersin.org 2 January 2022 | Volume 12 | Article 815565 Blanco-Vaca et al. Vitamin B3 Therapies in Cardiometabolic Health than 3 g/day, has been proven to be safe (17). In the ENDIT In relation to the impact of NMN intervention on dynamic study, 276 subjects with type 1 diabetes mellitus were metabolic adaptations, NMN supplementation enhanced aerobic administered with NAM in daily doses of 1.2 g/m2 (25–50 mg/ capacity in amateur runners in a 6-week randomized, double- kg), up to a maximum of 3 g/day for 5 years, with minimal side blind, placebo-controlled, four-arm clinical trial (Table 1) (23). effects (17). However, this clinical trial with NAM failed to In this study, up to 48 young and middle-aged recreational prevent the progression to overt of autoimmune type 1 runners were included (40 males and 8 females, aged between diabetes (18). Importantly, higher doses of NAM have been 27–50 years, with a previous history of 1–5 years of regular reported to produce severe but reversible hepatotoxicity (19). exercise). The participants were randomized into three groups Effects on glucose kinetics and insulin sensitivity are (with a ratio of 10 males to 2 females per group), taking a low inconsistent, but minor degrees of insulin resistance have also (300 mg/day), medium (600 mg/day), and a high dosage group been reported (17). (1,200 mg/day). A control group (placebos) followed the same NAD+ deficiency is also a risk factor for acute kidney injury male to female ratio. The runners trained 5–6 times/week in (20). Recently, a phase 1 placebo-controlled study of oral NAM sessions of 40–60 min. The participants did not show any demonstrated that a dose-related increase in circulating NAD+ obvious side effects. Exercise combined with NMN intake did was associated with less acute kidney injury (20). Similarly, not change the body composition or BMI, but analysis of the although minimal, NAD+ biosynthesis from tryptophan, at the change from baseline over the 6-week treatment showed that the expense of the quinolinate phosphoribosyltransferase, also oxygen uptake (VO2), the percentage of maximum oxygen prevents renal NAD+ depletion and mediates resistance to uptake (VO2 max), and the power at the first and second acute kidney injury. Overall, these data support the concept ventilatory thresholds increased in the medium- and high-dose that increasing NAD+ levels could be beneficial for the treatment NMN groups compared with the placebo. of some forms of kidney disease. In a very recent report (24), 25 overweight or obese women Consistent experimental observations have suggested similar (BMI ranged from 25.3 to 39.1 kg/m2, mean age of 61.5) and NAD+-increasing benefits by NAM for the treatment of heart prediabetes completed a randomized, double-blind, parallel- failure with preserved ejection fraction (21). In the former study, assigned treatment with 250 mg of NMN or placebos over 10 it was found that the dietary intake of NAD+ precursors was consecutive weeks (Table 1). There were no major adverse side negatively associated with decreased blood pressure and effects. Plasma concentrations of N-methyl-2-pyridone-5- cardiovascular mortality in a 20-year follow-up of the Bruneck carboxamide and N-methyl-4-pyridone-5-carboxamide Study, and persisted after adjusting for caloric intake, age, BMI, metabolites of NMN and NAD+ content in peripheral blood sex, smoking, diabetes, alcohol intake, and categories of mononuclear cells (PBMC) increased in subjects with NMN food items. supplementation, but not in those taking placebo. Neither body weight nor body composition differed on NMN treatment. In NMN contrast, insulin-stimulated glucose disposal, as assessed by The safety of NMN administration (100, 250, and 500 mg) was hyperinsulinemic-euglycemic clamp, and skeletal muscle recently investigated in 10 healthy men, aged from 40 to 60, in a insulin signaling (AKT and mTOR total protein and the single-arm, non-randomized intervention clinical trial (22). In phosphorylated forms) increased with NMN, thus revealing this study, single oral administrations of NMN did not cause any improved insulin signaling in treated subjects. In line with this, significant clinical symptoms or changes in heart rate, blood NMN also upregulated the expression of platelet-derived growth pressure, oxygen saturation, or body temperature 5 h after NMN factor receptor b (which enhances insulin-stimulated AKT administration. The plasma concentrations of NAM metabolites phosphorylation and glucose transport) and other genes in (N-methyl-2-pyridone-5-carboxamide and N-methyl-4- skeletal muscle that are involved in tissue remodeling. pyridone-5-carboxamide) were dose-dependently increased in However, no changes in muscle mitochondrial oxidative treated subjects. capacity or muscle function were observed. NMN could TABLE 1 | Recently (from 2018) published randomized, double-blind, placebo-controlled clinical trials that tested NMN administration on healthy individuals or in individuals with overweight and obesity. Country, Dose, duration of treatment, number, and main clinical Main results Reference NCT code characteristics China NMN or placebos (300, 600, and 1,200 mg) were No major adverse effects. Evidence of increased plasma metabolites. Medium (23) 2000035138 administered during 6 weeks to 48 subjects (8 women), and high doses presented increased exercise capacity (i.e., increased oxygen aged 27–50. The recreational runners, training 5–6 times a uptake). No effect on cardiac function, BMI, or fat-free mass. week, were divided into four groups. USA NMN or placebo per day (250 mg) over 10 weeks, given to No major adverse effects. Increased insulin-stimulated glucose disposal and (24) 03151239 25 women with mean age of 61, BMI of 33.5 kg/m2, skeletal muscle insulin signaling. Up-regulated expression of platelet-derived plasma glucose of 5.65 mmol/L, and HbA1c of 5.55%, growth factor receptor b and other genes related to muscle remodeling. No change in body weight or composition. BMI, body mass index; NMN, nicotinamide mononucleotide; NCT, clinical trial identifier (ClinicalTrials.gov). Frontiers in Endocrinology | www.frontiersin.org 3 January 2022 | Volume 12 | Article 815565 Blanco-Vaca et al. Vitamin B3 Therapies in Cardiometabolic Health therefore be a useful intervention approach in obese, prediabetic In another study, 12 healthy old men, aged from 70 to 80, and subjects, which often present muscle loss with impaired glucose BMI 20–30 kg/m2 (able to discontinue aspirin for 3 days prior to a metabolism (25). Further studies are warranted to clarify the muscle biopsy, statins and vitamin D one week before the potential contribution of NMN in the latter condition. beginning of the study) were supplemented with 500 mg of NR, twice a day, for 21 days, in a placebo-controlled, randomized, NR double-blind, crossover trial (Table 2) (30). NR supplementation NR is also a source for NAD+ synthesis. Consistently, human decreased levels of circulating inflammatory cytokines (30) but did blood NAD+ levels rose 2.7-fold in a pilot study of one healthy not produce favorable changes in body weight, blood pressure, 52-year-old male individual, receiving an oral daily dose of NR lipid profile, fasting glucose and insulin, or homeostatic model (1,000 mg/day) for 7 consecutive days (26). More recently, the assessment of insulin resistance (HOMA-IR). Although the different daily doses of NR, administered orally (i.e., 100, 300, supplementation of NR elevated the muscle NAD+ metabolome, and 1,000 mg) in 12 healthy subjects (6 male and 6 female) with it did not influence mitochondrial bioenergetics or whole-body ages from 30–55 and BMI 18.5–29.9 kg/m2, were investigated in glucose homeostasis. Despite the hand-grip strength values in the context of a randomized, double-blind, three-arm crossover these subjects being consistent with muscle aging, targeted NAD+ pharmacokinetic study design. The NR produced concomitant metabolome analysis likely revealed NAD+ sufficiency. Overall, dose-dependent elevations in the blood NAD+ metabolome. The these findings suggest that chronological age per se may not be a rise in nicotinic acid adenine dinucleotide (NAAD) was a highly major factor impairing NAD+ metabolism. sensitive biomarker of effective NAD+ replenishment (26). In an early phase I study of 5 patients admitted with a class IV In an independent, non-randomized, open-label New York Heart Failure Classification to demonstrate NR safety pharmacokinetic study made in 8 healthy volunteers (6 female, and feasibility, blood samples were obtained before and after 5–9 2 male, age range 21–50 years), 250 mg of NR was orally days of oral NR administration (NR was up-titrated over 3 days administered on days 1 and 2, and then increased to 1,000 mg to a final NR dose of 1,000 mg twice daily) (31). The treatment twice daily on days 7 and 8. On the morning of day 9, subjects enhanced PBMC respiration and reduced pro-inflammatory completed a 24-h pharmacokinetics study after receiving 1,000 cytokine gene expression in 4 male subjects, as one of the mg of NR (27). The treatment was well tolerated with no subjects did not complete the study. apparent adverse side events. Significant increases from Several clinical trials have focused on searching for the baseline to mean NR blood concentrations at a steady state beneficial effects of NR in individuals with overweight or were observed for both NR and NAD+, with a 100% increase in obesity. Data from the first of these clinical trials were the NAD+ levels. Absolute changes from baseline to day 9 in NR published in three different reports (32–34). This clinical trial and NAD+ levels were highly correlated (27). consisted of a 12-week randomized, double-blind, placebo- In another study, a 2- × 6-week randomized, double-blind, controlled, parallel-group trial conducted in 40 non-smoking, placebo-controlled, crossover clinical trial, 24 healthy lean medication-free middle-aged males (40–70 years) with obesity (average BMI = 24 ± 4 kg/m2) men (n = 11) and women (n = (BMI mean of 32.85 kg/m2), sedentarism (< 30 min of daily 13), aged 55 to 79, received 500 mg of NR twice a day (Table 2) exercise), with a mean fasting glucose of 5.6 mmol/L and HbA1c (28). NR supplementation was well tolerated by all participants. of 5.7% (Table 2) (32). They were administered NR at 1,000 mg, The NR increased the NAD+ and related metabolite twice a day (n = 20) or a placebo (n = 20). After 12 weeks of concentrations in PBMC. Importantly, treatment with NR treatment, increased concentrations of NAD-derived metabolites significantly decreased blood pressure, aortic pulse wave were detected in the urine of NR-treated subjects, thereby velocity, and carotid artery compliance. There were no changes showing that the oral NR was readily absorbed, metabolized, in total energy expenditure or energy expenditure from fat and excreted. No serious adverse events were observed upon NR oxidation under resting conditions, blood glucose control, supplementation, and the safety blood tests were normal. HbA1c, insulin sensitivity, aerobic exercise capacity, or motor function. insulin sensitivity, endogenous glucose production, glucose It is worth noting that this study was conducted in non-obese, disposal and oxidation, resting energy expenditure, lipolysis, healthy middle-aged, and older adults without baseline metabolic oxidation of lipids, and body composition did not change with dysfunction (28), and this could have limited the improved NR supplementation (32). The NR supplementation did not metabolic outcomes. affect fasting, the post-glucose challenge concentrations of The effect of different doses of NR (100, 300, and 1,000 mg/ glucose, insulin, C-peptide, glucagon, glucagon-like peptide-1 day) on NAD+ metabolite concentration in urine and blood was or gastric inhibitor polypeptide, or the beta-cell function (as investigated in 133 healthy males and females (54%–66%, revealed by the oral glucose test tolerance testing) (33). Protein depending on the group), aged 40–60, and overweight (BMI levels of NAMPT in skeletal muscle decreased by 14% with NR, 25–30 kg/m2), in an independent 8-week randomized, double- while NAD+ levels, as well as gene expression and protein blind, placebo-controlled parallel clinical trial (29). The NR abundance of other NAD+ biosynthetic enzymes, remained increased, dose-dependently and significantly, whole blood unchanged between the groups. The respiratory capacity of NAD+ (from 22% to 142%) and other NAD+ metabolites skeletal muscle mitochondria, abundance of mitochondrial within the first 2 weeks. The NAD+ elevations were associated proteins, mitochondrial fractional area, or network maintained thereafter, without any adverse effects (29). morphology in the skeletal muscle of NR-treated participants did Frontiers in Endocrinology | www.frontiersin.org 4 January 2022 | Volume 12 | Article 815565 Blanco-Vaca et al. Vitamin B3 Therapies in Cardiometabolic Health TABLE 2 | Recently (from 2018) published randomized, double-blind, placebo-controlled clinical trials that tested NR administration on individuals either healthy, aged, or overweight or obese. Country, Dose, duration of treatment, number, and Main results Reference NCT code main clinical characteristics USA, Administered 1,000 mg per day, 6 weeks in a No major adverse effects. Increased NAD+ and related metabolites in peripheral blood (28) 02921659 crossover design, n = 24 (13 women), mononuclear cells. Treatment decreased blood pressure, aortic pulse wave velocity, and described as 55–79-year-old healthy, aged, carotid compliance. No change in total energy intake and expenditure, BMI, % body fat, lean subjects (BMI 24 ± 4 kg/m2). glucose and insulin metabolism, or exercise capacity. USA Administered 100 mg, 300 mg, 1,000 mg per No major adverse effects. Dose-dependent increase of NAD+ whole blood and urine (29) 0271593 day, 8 weeks parallel study, 133 (85 women); metabolome. No changes in blood pressure, mean heart rate, weight, or resting energy 40–60 years old, healthy overweight (BMI 25– expenditure. 30 kg/m2) in a parallel study. UK, USA, Single center, double blind, placebo-controlled, Muscle RNA sequencing revealed that NR down-regulated energy metabolism and (30) Australia and crossover study on 12 male aged mitochondria pathways without altering mitochondrial bioenergetics or whole-body glucose 02950441 volunteers, recruited from the Birmingham homeostasis, decreasing circulatory cytokines (especially IL-6, IL-5 and IL-2). NR did not 1,000 Elders group (https://www.birmingham. alter hand-grip strength. ac.uk/research/activity/mds/centres/healthy- ageing/elders.aspx), age 70–80 years, BMI 20– 30 kg/m2 receiving 1,000 mg of NR or placebos during 21 days Denmark, 2,000 mg per day, 12 weeks: 40 men, 40–70- No major adverse effects. No changes in carbohydrate metabolism, resting energy (32–34) 2303483 year-old, insulin resistant, with mean BMI of expenditure, lipolysis, lipid oxidation, or body composition. No impact on glucose tolerance, 38.5 kg/m2, glucose of 5.6 mmol/L and HbA1c b-cell secretory capacity, a-cell function or incretin secretion, or bile acid levels. Moreover, of 5.7%. there were no changes in NAD+ metabolite concentration in skeletal muscle. Netherlands 1,000 mg per day, 6 weeks, 13 (7 women), No major adverse effects. Increased markers of NAD+ synthesis in skeletal muscle. Minor (37) 02835664 age 59 ± 5, healthy overweight or obese (BMI increase in fat free mass, especially in women (6 out of 7). Minor increase in sleeping 30.2 ± 2.6 kg/m2) metabolic rate that could be related to the change in fat free mass. Acetylcarnitine concentrations in skeletal muscle and the capacity to form acetylcarnitine upon exercise were increased in NR in respect to placebo. No effects of NR were found on insulin sensitivity, mitochondrial function, hepatic and intramyocellular lipid accumulation, cardiac energy status, cardiac ejection fraction, ambulatory blood pressure, plasma markers of inflammation, or energy metabolism. BMI, body mass index; NAD, nicotinamide adenine dinucleotide; NR, nicotinamide riboside; NCT, clinical trial identifier (ClinicalTrials.gov). not differ from the placebo group (34). Overall, these data do not also affected by NR supplementation. The NR-treated subjects support the hypothesis that dietary NR supplementation has a also showed concomitantly higher metabolic rates than the significant impact on skeletal muscle mitochondria in obese and placebo subjects. In contrast, insulin sensitivity was not insulin-resistant men. This could at least partly be explained by improved in NR-treated men. However, NR did increase the the fact that, despite having elevated urine levels of NAD+- fat-free mass in 6 out of 7 women, whereas such an increase was derived metabolites, the tissue content of NAD+, NADH, NADP only observed in 1 out of 6 men; thus, there might be a gender- +, or NADPH in the skeletal muscle in men receiving NR did not specific response pattern in the effect of NR supplementation. differ from those receiving placebos (35, 36). In another randomized, double-blinded, placebo-controlled, crossover intervention study, 13 healthy overweight or obese DISCUSSION men and women (age: 59 ± 5; BMI: 30.2 ± 2.6; n = 7 women) receiving either NR (1,000 mg/day) or placebo supplementation Thanks to the extensive research already performed with some during 6 consecutive weeks were followed by broad metabolic vitamin B3 forms, its considerable safety has been confirmed. phenotyping, including hyperinsulinemic-euglycemic clamps, The analyzed clinical trials were built on this concept. However, magnetic resonance spectroscopy, muscle biopsies, ex vivo special consideration should be given to the fact that some mitochondrial function, and in vivo energy metabolism enzymes determining NAD+ levels have been related to cancer assessment (Table 2) (37). NR supplementation resulted in progression, at least at an experimental level (38). elevations of NAD+ synthesis, such as NAAD and methyl- Data from different clinical trials are somewhat difficult to nicotinamide (me-NAM), in skeletal muscle. In particular, the compare and interpret, mainly due to differences in the vitamin percentage of fat-free mass (62.65% ± 2.49% compared with B3 forms, doses, duration of treatment, gender, and 61.32% ± 2.58%), the skeletal muscle acetylcarnitine (4,558 ± 749 cardiometabolic characteristics of the studied subjects. compared with 3,025 ± 316 pmol/mg dry weight), and the One of the emerging interpretations is that gender could be a capacity to form acylcarnitine upon exercise were increased determinant of positive response to treatment, since most reports upon NR supplementation (37). To our knowledge, such an that obtained significant cardiometabolic differences in vitamin B3 effect of NR on body composition in humans has not been treatment comprised a higher proportion of women (24, 28, 37) reported before. Consistently, the sleeping metabolic rate was than those in which no such differences were found (32–34). Frontiers in Endocrinology | www.frontiersin.org 5 January 2022 | Volume 12 | Article 815565 Blanco-Vaca et al. Vitamin B3 Therapies in Cardiometabolic Health However, this was not without exceptions. Elhassaen et al. (30) adaptations provided by restored NAD+ homeostasis, found some differences in men only, and Conze et al. (2019) did concurrent with improved mitochondrial function. Moreover, not find such differences, even though the study featured a future clinical trials should focus on subjects with selective majority of women (29). Interestingly, the percentage of men in biomarkers of recognized NAD+ deficiency (26) and analyzing the AIM-HIGH (9) and HPS-THRIVE (10) trials was around 85%. the gender-effect in order to maximize the chances of success. Apart from gender-specific differences, cardiometabolic status may also differ among studies. In this regard, subjects with obesity were excluded from these two latter studies (29, 30), whereas the analysis of the effect of NR was conducted in subjects CONCLUSION with obesity and prediabetes in other clinical trials (24, 37). In Although NAD+-increasing strategies have the potential to the study of Remie et al. in 2020, basically only the women in the improve overweight, obesity, and cardiometabolic health in study showed a response to the NR (37). In contrast, the main vivo, additional larger and long-term studies are needed to difference in the clinical trials that failed to find response to shed light on its precise clinical indications, preferred vitamin vitamin B3 treatment was directed only to men (32–34), even B3 form, doses, and treatment duration. though it should also be considered that it also used twice the dose of the other studies. Furthermore, circadian rhythms influence the action of at least some of the key enzymes involved in the bioavailability of the currently used NAD+ AUTHOR CONTRIBUTIONS precursors, and this could also be considered to be minimizing the variability among future clinical trials (39). Last, in the Conceptualization: FB-V, NR, and JJ. Writing-Original Draft clinical trial of Dollerup et al. (32–34) there were some Preparation: FB-V and JJ. Writing-Review and Editing: MC, DM, indications of limited NR tissue bioavailability. and JE-G. Funding Acquisition: FB-V, NR, JE-G, and JJ. All As mentioned, the favorable outcomes in response to NAD+ authors contributed to the article and approved the precursor supplementation found in the different studies were submitted version. quite heterogeneous. Thus, NAD+-increasing interventions increased capacity to exercise (23), decreased blood pressure (28), decreased anti-inflammatory circulating cytokines (30, 31), FUNDING increased insulin-stimulated glucose disposal (24), or decreased fat-free mass (37). Most of these concrete findings were not This work was partly funded by the Instituto de Salud Carlos III reproduced in the rest of the clinical trials. Most of the positive and FEDER “Una manera de hacer Europa” grant PI19/00136 findings were not very hard clinical end points (except the (JE-G), PI18/00164 (to FB-V), and PI17/00232 (JJ). This work decreased blood pressure). No significant reductions in body was also funded by Ministerio de Ciencia, Innovació n y weight or its surrogate, BMI, fasting glucose, or %HbA1c were Universidades (PID2019-104367RB-100), as well as from the observed in response to NAD+ precursors. This could be due to Agencia Estatal de Investigació n (AEI/10.13039/501100011033) the short duration of the clinical trials analyzed, which obviously within the Subprograma Ramó n y Cajal (RYC-201722879) to impacted the probability of the change in HbA1c percentage. NR. JJ was recipient of a Miguel Servet Type 2 contract (CPII18/ The health potential of targeting NAD+ homeostasis is still 00004; ISCIII). CIBERDEM is an Instituto de Salud Carlos III an underexplored field for future interventions of non- project. JE-G and JJ are members of Red de Investigació n en communicable disorders. Given the contribution of NAD+- “Enfermedades Metabó licas y Cá ncer” (RED2018-102799-T), sirtuin signaling on mitochondrial and metabolic homeostasis, Ministerio de Economı́a y Competitividad (MINECO), Madrid, an interesting proposal for future clinical trials could be the study Spain. Institut de Recerca de l’Hospital de la Santa Creu i Sant of the effects of vitamin B3 interventions in combination with Pau is accredited by the Generalitat de Catalunya as Centre de moderate exercise (40) to directly assess the metabolic Recerca de Catalunya (CERCA). 4. Mendez-Lara KA, Rodriguez-Millan E, Sebastian D, Blanco-Soto R, Camacho REFERENCES M, Nan MN, et al. Nicotinamide Protects Against Diet-Induced Body Weight 1. Abdellatif M, Baur JA. NAD+ Metabolism and Cardiometabolic Health: The Gain, Increases Energy Expenditure, and Induces White Adipose Tissue Human Evidence. Cardiovasc Res (2021) 117:e106–9. doi: 10.1093/cvr/ Beiging. Mol Nutr Food Res (2021) 65(11):e2100111. doi: 10.1002/mnfr. cvab212 202100111 2. Yoshino J, Mills KF, Yoon MJ, Imai S. Nicotinamide Mononucleotide, a Key 5. Elhassan YS, Philp AA, Lavery GG. Targeting NAD+ in Metabolic Disease: NAD(+) Intermediate, Treats the Pathophysiology of Diet- and Age-Induced New Insights Into an Old Molecule. J Endocr Soc (2017) 1:816–35. Diabetes in Mice. Cell Metab (2011) 14:528–36. doi: 10.1016/ doi: 10.1210/js.2017-00092 j.cmet.2011.08.014 6. Yoshino J, Baur JA, Imai SI. NAD(+) Intermediates: The Biology and 3. Canto C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y, et al. Therapeutic Potential of NMN and NR. Cell Metab (2018) 27:513–28. The NAD(+) Precursor Nicotinamide Riboside Enhances Oxidative doi: 10.1016/j.cmet.2017.11.002 Metabolism and Protects Against High-Fat Diet-Induced Obesity. Cell 7. Mendez-Lara KA, Letelier N, Farre N, Diarte-Anazco EMG, Nieto-Nicolau N, Metab (2012) 15:838–47. doi: 10.1016/j.cmet.2012.04.022 Rodriguez-Millan E, et al. Nicotinamide Prevents Apolipoprotein B- Frontiers in Endocrinology | www.frontiersin.org 6 January 2022 | Volume 12 | Article 815565 Blanco-Vaca et al. Vitamin B3 Therapies in Cardiometabolic Health Containing Lipoprotein Oxidation, Inflammation and Atherosclerosis in (NR) and Its Effects on Blood NAD+ Levels in Healthy Volunteers. PloS Apolipoprotein E-Deficient Mice. Antioxid (Basel) (2020) 9(11):1162. One (2017) 12:e0186459. doi: 10.1371/journal.pone.0186459 doi: 10.3390/antiox9111162 28. Martens CR, Denman BA, Mazzo MR, Armstrong ML, Reisdorph N, 8. Misra S. Randomized Double Blind Placebo Control Studies, the “Gold McQueen MB, et al. Chronic Nicotinamide Riboside Supplementation Is Standard” in Intervention Based Studies. Indian J Sex Transm Dis AIDS Well-Tolerated and Elevates NAD(+) in Healthy Middle-Aged and Older (2012) 33:131–4. doi: 10.4103/0253-7184.102130 Adults. Nat Commun (2018) 9:1286. doi: 10.1038/s41467-018-03421-7 9. Boden WE, Probstfield JL, Anderson T, Chaitman BR, Desvignes-Nickens P, 29. Conze D, Brenner C, Kruger CL. Safety and Metabolism of Long-Term Koprowicz K, et al. Niacin in Patients With Low HDL Cholesterol Levels Administration of NIAGEN (Nicotinamide Riboside Chloride) in a Receiving Intensive Statin Therapy. N Engl J Med (2011) 365:2255–67. doi: Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Healthy 10.1056/NEJMoa1107579 Overweight Adults. Sci Rep (2019) 9:9772. doi: 10.1038/s41598-019-46120-z 10. Landray MJ, Haynes R, Hopewell JC, Parish S, Aung T, Tomson J, et al. Effects 30. Elhassan YS, Kluckova K, Fletcher RS, Schmidt MS, Garten A, Doig CL, et al. of Extended-Release Niacin With Laropiprant in High-Risk Patients. N Engl J Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD(+) Med (2014) 371:203–12. doi: 10.1056/NEJMoa1300955 Metabolome and Induces Transcriptomic and Anti-Inflammatory Signatures. 11. Kong D, Li J, Shen Y, Liu G, Zuo S, Tao B, et al. Niacin Promotes Cardiac Cell Rep (2019) 28:1717–1728 e1716. doi: 10.1016/j.celrep.2019.07.043 Healing After Myocardial Infarction Through Activation of the Myeloid 31. Zhou B, Wang DD, Qiu Y, Airhart S, Liu Y, Stempien-Otero A, et al. Boosting Prostaglandin D2 Receptor Subtype 1. J Pharmacol Exp Ther (2017) NAD Level Suppresses Inflammatory Activation of PBMCs in Heart Failure. 360:435–44. doi: 10.1124/jpet.116.238261 J Clin Invest (2020) 130:6054–63. doi: 10.1172/JCI138538 12. Canner PL, Berge KG, Wenger NK, Stamler J, Friedman L, Prineas RJ, et al. Fifteen 32. Dollerup OL, Christensen B, Svart M, Schmidt MS, Sulek K, Ringgaard S, et al. Year Mortality in Coronary Drug Project Patients: Long-Term Benefit With A Randomized Placebo-Controlled Clinical Trial of Nicotinamide Riboside in Niacin. J Am Coll Cardiol (1986) 8:1245–55. doi: 10.1016/s0735-1097(86)80293-5 Obese Men: Safety, Insulin-Sensitivity, and Lipid-Mobilizing Effects. Am J 13. Romani M, Hofer DC, Katsyuba E, Auwerx J. Niacin: An Old Lipid Drug in a Clin Nutr (2018) 108:343–53. doi: 10.1093/ajcn/nqy132 New NAD(+) Dress. J Lipid Res (2019) 60:741–6. doi: 10.1194/jlr.S092007 33. Dollerup OL, Trammell SAJ, Hartmann B, Holst JJ, Christensen B, Moller N, 14. Pirinen E, Auranen M, Khan NA, Brilhante V, Urho N, Pessia A, et al. Niacin et al. Effects of Nicotinamide Riboside on Endocrine Pancreatic Function and Cures Systemic NAD(+) Deficiency and Improves Muscle Performance in Incretin Hormones in Nondiabetic Men With Obesity. J Clin Endocrinol Adult-Onset Mitochondrial Myopathy. Cell Metab (2020) 31:1078–1090 Metab (2019) 104:5703–14. doi: 10.1210/jc.2019-01081 e1075. doi: 10.1016/j.cmet.2020.04.008 34. Dollerup OL, Chubanava S, Agerholm M, Sondergard SD, Altintas A, Moller 15. Westphal S, Luley C. Preferential Increase in High-Molecular Weight AB, et al. Nicotinamide Riboside Does Not Alter Mitochondrial Respiration, Adiponectin After Niacin. Atherosclerosis (2008) 198:179–83. doi: 10.1016/ Content or Morphology in Skeletal Muscle From Obese and Insulin-Resistant j.atherosclerosis.2007.09.036 Men. J Physiol (2020) 598:731–54. doi: 10.1113/JP278752 16. Westphal S, Borucki K, Taneva E, Makarova R, Luley C. Extended-Release 35. Moore MP, Mucinski JM. Impact of Nicotinamide Riboside Supplementation on Niacin Raises Adiponectin and Leptin. Atherosclerosis (2007) 193:361–5. Skeletal Muscle Mitochondria and Whole-Body Glucose Homeostasis: Challenging doi: 10.1016/j.atherosclerosis.2006.06.028 the Current Hypothesis. J Physiol (2020) 598:3327–8. doi: 10.1113/JP279749 17. Knip M, Douek IF, Moore WP, Gillmor HA, McLean AE, Bingley PJ, et al. 36. Leduc-Gaudet JP, Dulac M, Reynaud O, Ayoub MB, Gouspillou G. Nicotinamide Safety of High-Dose Nicotinamide: A Review. Diabetologia (2000) 43:1337– Riboside Supplementation to Improve Skeletal Muscle Mitochondrial Health and 45. doi: 10.1007/s001250051536 Whole-Body Glucose Homeostasis: Does It Actually Work in Humans? J Physiol 18. Gale EA, Bingley PJ, Emmett CL, Collier T. European Nicotinamide Diabetes (2020) 598:619–20. doi: 10.1113/JP279280 Intervention Trial (ENDIT): A Randomised Controlled Trial of Intervention 37. Remie CME, Roumans KHM, Moonen MPB, Connell NJ, Havekes B, Before the Onset of Type 1 Diabetes. Lancet (2004) 363:925–31. doi: 10.1016/ Mevenkamp J, et al. Nicotinamide Riboside Supplementation Alters Body S0140-6736(04)15786-3 Composition and Skeletal Muscle Acetylcarnitine Concentrations in Healthy 19. Winter SL, Boyer JL. Hepatic Toxicity From Large Doses of Vitamin B3 Obese Humans. Am J Clin Nutr (2020) 112:413–26. doi: 10.1093/ajcn/nqaa072 (Nicotinamide). N Engl J Med (1973) 289:1180–2. doi: 10.1056/ 38. Parsons RB, Facey PD. Nicotinamide N-Methyltransferase: An Emerging NEJM197311292892208 Protagonist in Cancer Macro(R)Evolution. Biomolecules (2021) 11(10):1418. 20. Poyan Mehr A, Tran MT, Ralto KM, Leaf DE, Washco V, Messmer J, et al. De doi: 10.3390/biom11101418 Novo NAD(+) Biosynthetic Impairment in Acute Kidney Injury in Humans. 39. Benedict C, Shostak A, Lange T, Brooks SJ, Schioth HB, Schultes B, et al. Diurnal Nat Med (2018) 24:1351–9. doi: 10.1038/s41591-018-0138-z Rhythm of Circulating Nicotinamide Phosphoribosyltransferase (Nampt/visfatin/ 21. Abdellatif M, Trummer-Herbst V, Koser F, Durand S, Adao R, Vasques- PBEF): Impact of Sleep Loss and Relation to Glucose Metabolism. J Clin Novoa F, et al. Nicotinamide for the Treatment of Heart Failure With Endocrinol Metab (2012) 97:E218–222. doi: 10.1210/jc.2011-2241 Preserved Ejection Fraction. Sci Transl Med (2021) 13(580):eabd7064. 40. Fluharty NT, Brenner C. Fat Mobilization Without Weight Loss is a Potentially doi: 10.1126/scitranslmed.abd7064 Rapid Response to Nicotinamide Riboside in Obese People: It’s Time to Test 22. Irie J, Inagaki E, Fujita M, Nakaya H, Mitsuishi M, Yamaguchi S, et al. Effect of With Exercise. Am J Clin Nutr (2020) 112:243–4. doi: 10.1093/ajcn/nqaa109 Oral Administration of Nicotinamide Mononucleotide on Clinical Parameters and Nicotinamide Metabolite Levels in Healthy Japanese Men. Endocr J Conflict of Interest: The authors declare that the research was conducted in the (2020) 67:153–60. doi: 10.1507/endocrj.EJ19-0313 absence of any commercial or financial relationships that could be construed as a 23. Liao B, Zhao Y, Wang D, Zhang X, Hao X, Hu M. Nicotinamide potential conflict of interest. Mononucleotide Supplementation Enhances Aerobic Capacity in Amateur Runners: A Randomized, Double-Blind Study. J Int Soc Sports Nutr (2021) Publisher’s Note: All claims expressed in this article are solely those of the authors 18:54. doi: 10.1186/s12970-021-00442-4 and do not necessarily represent those of their affiliated organizations, or those of 24. Yoshino M, Yoshino J, Kayser BD, Patti GJ, Franczyk MP, Mills KF, et al. the publisher, the editors and the reviewers. Any product that may be evaluated in Nicotinamide Mononucleotide Increases Muscle Insulin Sensitivity in this article, or claim that may be made by its manufacturer, is not guaranteed or Prediabetic Women. Science (2021) 372:1224–9. doi: 10.1126/science.abe9985 endorsed by the publisher. 25. Hepler C, Bass J. Supplements to Treat Prediabetes. Science (2021) 372:1147– 8. doi: 10.1126/science.abj0764 Copyright © 2022 Blanco-Vaca, Rotllan, Canyelles, Mauricio, Escolà-Gil and Julve. 26. Trammell SA, Schmidt MS, Weidemann BJ, Redpath P, Jaksch F, Dellinger This is an open-access article distributed under the terms of the Creative Commons RW, et al. Nicotinamide Riboside Is Uniquely and Orally Bioavailable in Mice Attribution License (CC BY). The use, distribution or reproduction in other forums is and Humans. Nat Commun (2016) 7:12948. doi: 10.1038/ncomms12948 permitted, provided the original author(s) and the copyright owner(s) are credited and 27. Airhart SE, Shireman LM, Risler LJ, Anderson GD, Nagana Gowda GA, that the original publication in this journal is cited, in accordance with accepted Raftery D, et al. An Open-Label, Non-Randomized Study of the academic practice. No use, distribution or reproduction is permitted which does not Pharmacokinetics of the Nutritional Supplement Nicotinamide Riboside comply with these terms. Frontiers in Endocrinology | www.frontiersin.org 7 January 2022 | Volume 12 | Article 815565