SUMMARY A decline in capillary density and blood flow with age is a major cause of mortality and morbidity. Understanding why this occurs is key to future gains in human health. NAD+ precursors reverse aspects of aging, in part, by activating sirtuin deacylases (SIRT1-7) that mediate the benefits of exercise and dietary restriction (DR). We show that SIRT1 in endothelial cells is a key mediator of pro-angiogenic signals secreted from myocytes. Treatment of mice with the NAD+ precursor nicotinamide mononucleotide (NMN) improves blood flow and increases endurance in elderly mice by promoting SIRT1-dependent increases in capillary density, an effect augmented by exercise or increasing the levels of hydrogen sulfide (H2S), a DR mimetic and regulator of endothelial NAD+ levels. These findings have implications for improving blood flow to organs and tissues, increasing human performance, and reestablishing a virtuous cycle of mobility in the elderly.

Graphical Abstract Highlights d Declining NAD(P)H is associated with oocyte dysfunction during reproductive aging d Oocyte quality and fertility can be restored by NMN treatment in aged mice d Supplementation of embryo media with NMN improves developmental milestones d SIRT2 overexpression mimics benefits of NMN but is unlikely to mediate its effects SUMMARYReproductive aging in female mammals is an irreversible process associated with declining oocyte quality, which is the rate-limiting factor to fertility.Here, we show that this loss of oocyte quality with age accompanies declining levels of the prominent metabolic cofactor nicotinamide adenine dinucleotide (NAD + ). Treatment with the NAD + metabolic precursor nicotinamide mononucleotide (NMN) rejuvenates oocyte quality in aged animals, leading to restoration in fertility, and this can be recapitulated by transgenic overexpression of the NAD + -dependent deacylase SIRT2, though deletion of this enzyme does not impair oocyte quality. These benefits of NMN extend to the developing embryo, where supplementation reverses the adverse effect of maternal age on developmental milestones. These findings suggest that late-life restoration of NAD + levels represents an opportunity to rescue female reproductive function in mammals.

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2Female infertility is a common and devastating condition with life-long health, emotional and 3 social consequences. There is currently no pharmacological therapy for preserving oocyte 4 quality during aging, which is the strongest risk factor for infertility. This leads to an age 5 dependent decline in natural conception and IVF success rates (1). Here, we show that this is 6 due in part to declining levels of the metabolic cofactor nicotinamide adenine dinucleotide 7 (NAD + ), and that restoring NAD + levels with its metabolic precursor nicotinamide 8 mononucleotide (NMN) rejuvenates oocyte quality and quantity in aged animals, leading to 9 improved fertility. These benefits extend to the developing embryo, where NMN 10 supplementation in embryo culture media following IVF enhances blastocyst formation in 11 older mice. The NAD + dependent deacylase SIRT2 is sufficient, but not essential, to 12 recapitulate the benefits of in vivo NMN treatment, and transgenic overexpression of SIRT2 13 maintains oocyte spindle assembly, accurate chromosome segregation, decreased oxidative 14 stress and overall fertility with ageing. Pharmacological elevation of NAD + may be an 15 effective, non-invasive strategy for restoring and maintaining female fertility during ageing, 16 and for improving the success of IVF.17 18 19 risks (4), is expensive and has a limited success rate. Repeated IVF failures are a substantial 1 source of emotional distress, and failure to conceive offspring is a substantial source of 2 relationship breakdown (5). 4The rate-limiting factors for successful pregnancies in IVF are oocyte quantity and quality, 5 both of which start to decline from the middle of the third decade of life in humans (1, 3). 6 Despite the enormous need, there are no clinically viable strategies to either preserve or 7 rejuvenate oocyte quantity or quality during ageing. There is a major need in reproductive 8 medicine for a non-invasive, pharmacological treatment to maintain or restore oocyte quantity 9 and/or quality during ageing. The effect of such a therapy would be to alleviate a rate-limiting 10 barrier to IVF success, or increase the chances of unaided conception, without having to resort 11 to IVF. 12 13The molecular basis for the decline in oocyte quality with advancing age is not clear but is 14 certainly multifactorial. The key factors thought to be involved include genome instability, 15 reduced mitochondrial bioenergetics, increased reactive oxygen species (ROS), and impaired 16 fidelity during meiotic chromosome segregation due to disrupted spindle assembly and 17 compromised function of the spindle assembly checkpoint (SAC) surveillance system (6). This 18 latter hypothesis is evidenced by an increased rate of aneuploidy in embryos with increased 19

Almost 40% of adults worldwide are classified as overweight or obese. Exercise is a beneficial intervention in obesity, partly due to increases in mitochondrial activity, and subsequent increases in nicotinamide adenine dinucleotide (NAD+), an important metabolic cofactor. Recent studies have shown that increasing NAD+ levels through pharmacological supplementation with precursors such as nicotinamide mononucleotide (NMN) improved metabolic health in high fat diet (HFD) fed mice. However, the effects of combined exercise and NMN supplementation are unknown. Thus here we examined the combined effects of NMN and treadmill exercise in female mice with established obesity after 10 weeks of diet. Five-week old female C57BL/6J mice were exposed to control diet (n=16) or HFD. Sedentary mice fed HFD were either untreated (HFD; n=16), received NMN in drinking water (400mg/kg; HNMN; n=16), were exposed to treadmill exercise 6 days/week (HEx; n=16) or exercise combined with NMN (HNEx; n=16). Whilst some metabolic benefits of NMN have been described, at this dose, NMN administration impaired several aspects of exercise-induced benefits in obese mice, including glucose tolerance, glucose stimulated insulin secretion from islets and reduced hepatic triglyceride accumulation. HNEx mice also exhibited increased antioxidant and reduced prooxidant gene expression in both islets and muscle, suggesting that altered redox status is associated with the loss of exercise-induced health benefits with NMN co-treatment. Our data show that NMN treatment impedes the beneficial metabolic effects of exercise in a mouse model of diet-induced obesity in association with disturbances in redox metabolism.