NAD+ Intermediates: The Biology and Therapeutic Potential of NMN and NR | HeadWinds Lab

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NAD+ Intermediates: The Biology and Therapeutic Potential of NMN and NR

NMN is synthesized from nicotinamide, a form of water-soluble vitamin B3, and 50-phosphoribosyl-1-pyrophosphate (PRPP), by NAMPT, the rate-limiting NAD+ biosynthetic enzyme in mam- mals (Figure 1A) (Imai and Yoshino, 2013; Revollo et al., 2004; Wang et al., 2006). NMN is also synthesized from NR via an NRK-mediated phosphorylation reaction, which is discussed further in the next section. Conversion of NMN into NAD+ is cata- lyzed by NMNATs (Figure 1B). Evidence accumulating from studies conducted in rodents has demonstrated that systemic NMN administration effectively enhances NAD+ biosynthesis in various peripheral tissues, including pancreas (Yoshino et al., 2011), liver (Peek et al., 2013; Yoshino et al., 2011), adipose tissue (Stromsdorfer et al., 2016; Yoshino et al., 2011), heart (Karamanlidis et al., 2013; Martin et al., 2017; North et al., 2014; Yamamoto et al., 2014), skeletal muscle (Gomes et al., 2013), kidney (Guan et al., 2017), testis (North et al., 2014), eyes (Lin et al., 2016), and blood vessel (aorta) (de Picciotto et al., 2016), under normal and pathophysiological conditions. Although it remains unclear whether NMN can cross the blood- brain barrier (BBB), intraperitoneal NMN administration rapidly (within 15 min) increases NAD+ levels in brain regions such as hippocampus and hypothalamus (Stein and Imai, 2014; Yoon et al., 2015), suggesting that NMN could pass through the BBB and contribute to NAD+ biosynthesis in the brain. Finally, it was recently reported that long-term (1-year) oral administration of NMN (up to 300 mg/kg) is safe and well tolerated and does not cause any obvious deleterious or toxic effects in normal wild- type C57BL/6 mice (Mills et al., 2016).

These results suggest that NMN could offer broad applica- tions and therapeutic potential. Indeed, there is a growing body of evidence showing that NMN has beneficial effects on a diverse array of key physiological functions and therapeutic im- plications in various disease models (Table 1). Of particular note are the remarkable metabolic benefits from NMN administration. Pioneering studies have suggested that pancreatic b cells are very sensitive to systemic NAD+ decline and NMN administra- tion. A single bolus injection of NMN (500 mg/kg) enhances glucose-stimulated insulin secretion and thus improves glucose tolerance in age- and diet-induced diabetic mice (Caton et al., 2011; Yoshino et al., 2011), Nampt heterozygous knockout mice (Revollo et al., 2007), and aged wild-type and b cell-specific Sirt1-overexpressing (BESTO) mice (Mills et al., 2016; Ramsey et al., 2008; Yoshino et al., 2011). NMN enhances not only insulin secretion but also insulin action: NMN treatment ameliorates high-fat-diet-induced hepatic insulin resistance by restoring NAD+ biosynthesis, SIRT1 activity, and gene expression related to inflammation, oxidative stress, and circadian rhythms (Yosh- ino et al., 2011). Oral NMN administration also increases adipose tissue NAD+ biosynthesis and SIRT1 activity and normalizes

severe hypoadiponectinemia and multi-organ insulin resistance in adipocyte-specific Nampt knockout (ANKO) mice (Stromsdor- fer et al., 2016). In addition, long-term NMN administration suppresses age-associated adipose tissue inflammation and improves whole-body insulin sensitivity independently from ef- fects on body weight in regular chow-fed wild-type C57BL/6 mice (Mills et al., 2016). Given that adipose tissue NAD+ biosyn- thesis is severely impaired in obese and aged mice (Camacho- Pereira et al., 2016; Yoshino et al., 2011), these findings suggest that adipose tissue NAD+ could be a good therapeutic target for insulin resistance, which is an important risk factor of type 2 diabetes and cardiovascular disease (DeFronzo and Ferrannini, 1991; Reaven, 1988; Yamaguchi and Yoshino, 2017). NMN has also been reported to improve mitochondrial function in various metabolic organs, including skeletal muscle (Gomes et al., 2013; Mills et al., 2016), liver (Peek et al., 2013; Uddin et al., 2016), heart (Lee et al., 2016), and eyes (Lin et al., 2016). NMN-treated mice have increased mitochondrial oxidative phosphorylation in skeletal muscle (Gomes et al., 2013; Mills et al., 2016), likely contributing to weight loss by increasing whole-body energy expenditure (Mills et al., 2016). NMN increases SIRT3 activity and hepatic mitochondrial lipid oxidation in circadian mutant mice (Peek et al., 2013) and hepatic citrate synthase activity in high-fat-diet-fed obese mice (Uddin et al., 2016). An important aspect of the above findings is that aged animals appear to be more responsive to NMN treatment, compared with young ani- mals. NMN improves high-fat-diet-induced glucose intolerance and dyslipidemia (Yoshino et al., 2011), skeletal muscle mito- chondrial oxidative metabolism (Gomes et al., 2013), and endothelial function (de Picciotto et al., 2016) better in aged mice than young mice. It is conceivable that age-associated decline in NAD+ availability (Braidy et al., 2011; Camacho-Per- eira et al., 2016; Frederick et al., 2016; Gomes et al., 2013; Mas- sudi et al., 2012; Stein and Imai, 2014; Yoshino et al., 2011) could sensitize aged mice to NAD+ replenishment by NMN administra- tion. Therefore, it will be of great interest to carefully compare the metabolic effects of NMN in aged mice with those in young mice.

Recent studies have suggested that NMN improves numerous neuronal functions in the brain. NMN administration improves cognition and memory in mouse and rat models of Alzheimer’s disease (Long et al., 2015; Wang et al., 2016; Yao et al., 2017). NMN protects neurons from cell death after ischemia (Park et al., 2016) or intracerebral hemorrhage (Wei et al., 2017a) and ameliorates the loss of BBB integrity and tissue plasminogen- activator-induced hemorrhagic transformation in brain ischemia (Wei et al., 2017b). NMN also restores severe retinal degenera- tion, through mitochondrial sirtuins SIRT3 and SIRT5, in rod or cone photoreceptor-specific Nampt knockout mice (Lin et al., 2016). Furthermore, long-term NMN administration prevents age-associated loss of the neural stem/progenitor pool in the dentate gyrus in wild-type C57BL/6 mice (Stein and Imai, 2014). Interestingly, data obtained from dose-response studies have indicated that lower doses of NMN may be more beneficial, compared with larger doses for some neuronal outcomes. For example, 100 mg/kg of NMN improves physical activity better than 300 mg/kg (Mills et al., 2016); 62.5 mg/kg of NMN more effectively treats ischemia-induced brain damage than higher doses (125–500 mg/kg) (Park et al., 2016). Therefore, it is possible that local NMN accumulation, due to administration of

high doses of NMN, has negative impacts in a brain region- or function-specific manner. Indeed, data obtained from in vitro experiments suggest that NMN promotes Wallerian degenera- tion, although this notion was recently challenged (Di Stefano et al., 2015; Gerdts et al., 2016). Future studies are awaited to fully elucidate the pharmacokinetics of NMN in the brain and determine the optimal dosing regimen of NMN in each key neuronal function and brain region.

In addition to its anti-diabetic and neurological effects, NMN administration inhibits acute renal injury in a SIRT1-dependent manner (Guan et al., 2017), heart failure (Karamanlidis et al., 2013; Lee et al., 2016; Martin et al., 2017; Yamamoto et al., 2014), and radiation-induced DNA damage (Li et al., 2017), further demonstrating pleiotropic benefits of NMN. Given that NMN is effective in aged mice and suppresses age-associated physiological decline (Mills et al., 2016), it will be of great impor- tance to evaluate the effects of NMN administration on other age-associated diseases such as cancer and sarcopenia, and aging and lifespan per se in rodents.

Nicotinamide Riboside

The direct contribution of NR to NAD+ metabolism was first recognized by Bieganowski and Brenner in 2004 (Bieganowski and Brenner, 2004). This report described a class of enzymes known as NRKs that can convert NR directly to NMN, bypassing the need for NAMPT in the salvage pathway (Figure 1). Since the reaction catalyzed by NAMPT is rate limiting, requires the use of energetically costly PRPP, and is subject to feedback inhibition by NAD+ (Dietrich et al., 1968), NR also offers an interesting op- portunity to boost NAD+ levels beyond what is achievable through conventional B vitamin metabolism. There is a substan- tial body of evidence supporting metabolic benefits for NR, as well as efficacy in a variety of disease models and a modest life- span increase in aged mice (Table 2).

The first major study of NR in mice examined metabolic health after high-fat-diet feeding. Mice receiving NR at a dose of 400 mg/kg/day in the diet were protected from weight gain, were more insulin sensitive, and had increased mitochondrial content in skeletal muscle and brown adipose tissue compared with untreated controls (Canto et al., 2012). Accordingly, NR was found to confer increased endurance and improved cold toler- ance in the high-fat-diet-fed mice. Whether NR has significant benefits in lean, healthy muscle is less clear, as only a nonsignif- icant trend toward increased endurance was observed in regular chow-fed mice (Canto et al., 2012), and a subsequent study in rats showed a trend toward decreased endurance in a forced swim test (also not significant) (Kourtzidis et al., 2016).

NR has been shown to have therapeutic effects in a number of muscle disorders. Despite not correcting the underlying genetic defects, NR improved mitochondrial abundance and function in two different mitochondrial myopathies (Cerutti et al., 2014; Khan et al., 2014). NR also increased survival time, induced auto- phagy, and decreased the mitochondrial unfolded protein response in a model of heart failure induced by cardiac-specific deletion of the transferrin receptor (Xu et al., 2015). Mice lacking Nampt in skeletal muscle exhibit a progressive wasting syn- drome that is reversed by providing NR in the drinking water, with endurance restored in as little as 1 week of treatment (Fred- erick et al., 2016). These mice histologically and transcriptionally resemble the mdx model of Duchenne’s muscular dystrophy, which has also been reported to display decreased NAD+ con- tent in skeletal muscle (Chalkiadaki et al., 2014). Intriguingly, NR treatment has since been shown to improve stem cell func- tion and partially ameliorate the muscle wasting phenotype in mdx mice, leading to hope for a rapidly translatable therapy for the human condition (Ryu et al., 2016; Zhang et al., 2016). Improvement in stem cell function appears to be a general phe- nomenon during NR treatment and has been suggested to un- derlie a small, but significant extension of lifespan in mice treated beginning from 2 years of age (Zhang et al., 2016).

NR improves liver health in a variety of contexts. It potently re- duces fat accumulation through a mechanism that involves in- duction of the mitochondrial unfolded protein response (Gariani et al., 2016). It further reduces inflammation at least in part through decreased activity of the NLRP3 inflammasome and lessens the development of fibrosis (Lee et al., 2015). Moreover, NAD+ biosynthesis was found to be impaired in a mouse model of hepatocellular carcinoma, and both DNA damage and tumor- igenesis were prevented when NR was provided in the diet to restore NAD+ (Tummala et al., 2014). In the regenerating liver (following partial hepatectomy), NR reduces lipid accumulation, promotes hepatocyte replication, increases hepatic ATP con- tent, and leads to faster regain of liver weight (Mukherjee et al., 2017). Together, these observations suggest that enhancing NAD+ content by NR is a promising therapeutic strategy to improve liver health.

Like NMN, NR has further been reported to have a number of intriguing benefits in the nervous system. One of the first studies to examine the effects of NR in vivo revealed a striking improve- ment in the progression of Alzheimer’s disease pathology in the Tg2576 model of the disease (Gong et al., 2013). NR has been shown to prevent noise-induced hearing loss and neurite retrac- tion from hair cells in the inner ear through a SIRT3-dependent mechanism (Brown et al., 2014). NR also protects against dia- betic and chemotherapy-induced neuropathy in mice (Hamity et al., 2017; Trammell et al., 2016b), raising hopes that it could have a role in the management of chronic pain. It has recently been recognized that PARP-mediated NAD+ depletion plays a major role in the pathogenesis of neurodegenerative disorders that involve DNA repair defects, including Cockayne syndrome, xeroderma pigmentosum, and ataxia telangiectasia. While NR is unable to correct the primary DNA repair deficiencies, it dramat- ically improves phenotypes in mouse models of each of these conditions, most remarkably more than tripling survival time in ataxic mice (Fang et al., 2014, 2016; Scheibye-Knudsen et al., 2014).

Limitations of Our Current Understanding and Possible Detrimental Effects of Enhancing NAD+ Biosynthesis
As outlined above, it is clear that both NMN and NR have beneficial effects in multiple conditions in rodents. Indeed, there are a number of pathophysiological conditions that show significant de- creases in tissue NAD+ levels (Table 3). Although both compounds have been tested in some models, no side-by-side comparisons have been conducted between NMN and NR. Therefore, even though both compounds are capable of enhancing NAD+ biosyn- thesis, there might be certain interesting differences in their effects on these pathophysiological conditions. In addition, in the vast majority of cases in which NMN and NR are effective, it still remains unclear what downstream mechanisms mediate their beneficial ef- fects. NAD+ is required as a cosubstrate for PARPs, sirtuins, ADP- ribosyl cyclases, and mono-ADP ribosyltransferases but also serves as a redox cofactor for countless enzymes (Figure 1B). In several cases, deletion of sirtuins has been shown to block key benefits of NAD+ supplementation, supporting a role for these en- zymes (Brown et al., 2014; Gomes et al., 2013; Guan et al., 2017; Martin et al., 2017). In contrast, pharmacological or genetic inhibi- tion of other key NAD+-consuming enzymes, such as PARP1/2 and CD38, is sufficient to confer beneficial effects that have been attributed to increased tissue NAD+ availability (Bai et al., 2011a, 2011b; Camacho-Pereira et al., 2016). Given the wide range of benefits that have been reported, and the potentially complex in- teractions between NAD+-dependent processes, a great deal of work will be required to define the precise mechanisms acting downstream of NAD+ supplementation to promote health.

It is also critical to carefully assess potentially detrimental side effects for these NAD+ intermediates. Although NAD+ appears to promote certain pathways related to DNA repair and suppres- sion of inflammation, NAD+-depleting drugs are currently under development as cancer chemotherapeutics, and there might be a risk that boosting NAD+ could drive tumor growth (Gujar et al., 2016). Similarly, one of the downstream targets that is hy- pothesized to mediate many of NMN and NR’s effects, SIRT1, has been shown to have pro-carcinogenic or anti-carcinogenic effects in different contexts (Chalkiadaki and Guarente, 2015). In addition, we cannot exclude the possibility that increased accumulation of nicotinamide, a potent inhibitor of sirtuins (Ava- los et al., 2005; Bitterman et al., 2002), could have detrimental effects in NMN- and NR-treated mice. It is also clear that high- dose supplementation greatly exceeds the body’s requirement for niacin equivalents and thus will result in substantial elimina- tion through the urine. One major route to nicotinamide elimina- tion is methylation via nicotinamide N-methyltransferase, and there has been little investigation of whether this can lead to methyl donor depletion over time. Thus, further preclinical and clinical studies are needed to establish the long-term safety of NMN and NR as human therapeutics.