Sarcopenia and frailty are urgent socio-economic problems worldwide. Here we demonstrate a functional connection between the lateral hypothalamus (LH) and skeletal muscle through Slc12a8, a recently identified nicotinamide mononucleotide transporter, and its relationship to sarcopenia and frailty. Slc12a8-expressing cells are mainly localized in the LH. LH-specific knockdown of Slc12a8 in young mice decreases activity-dependent energy and carbohydrate expenditure and skeletal muscle functions, including muscle mass, muscle force, intramuscular glycolysis, and protein synthesis. LH-specific Slc12a8 knockdown also decreases sympathetic nerve signals at neuromuscular junctions and β2-adrenergic receptors in skeletal muscle, indicating the importance of the LH-sympathetic nerve-β2-adrenergic receptor axis. LH-specific overexpression of Slc12a8 in aged mice significantly ameliorates age-associated decreases in energy expenditure and skeletal muscle functions. Our results highlight an important role of Slc12a8 in the LH for regulation of whole-body metabolism and skeletal muscle functions and provide insights into the pathogenesis of sarcopenia and frailty during aging.

Old animals display significant alterations in sleep-wake patterns such as increases in sleep fragmentation and sleep propensity. Here, we demonstrated that PR-domain containing protein 13 (Prdm13)+ neurons in the dorsomedial hypothalamus (DMH) are activated during sleep deprivation (SD) in young mice but not in old mice. Chemogenetic inhibition of Prdm13+ neurons in the DMH in young mice promotes increase in sleep attempts during SD, suggesting its involvement in sleep control. Furthermore, DMH-specific Prdm13-knockout (DMH-Prdm13-KO) mice recapitulated age-associated sleep alterations such as sleep fragmentation and increased sleep attempts during SD. These phenotypes were further exacerbated during aging, with increased adiposity and decreased physical activity, resulting in shortened lifespan. Dietary restriction (DR), a well-known anti-aging intervention in diverse organisms, ameliorated age-associated sleep fragmentation and increased sleep attempts during SD, whereas these effects of DR were abrogated in DMH-Prdm13-KO mice. Moreover, overexpression of Prdm13 in the DMH ameliorated increased sleep attempts during SD in old mice. Therefore, maintaining Prdm13 signaling in the DMH might play an important role to control sleep-wake patterns during aging.

Nicotinamide mononucleotide (NMN) is a biosynthetic precursor of NAD+ known to promote cellular NAD+ production and counteract age-associated pathologies associated with a decline in tissue NAD+ levels. How NMN is taken up into cells has not been entirely clear. Here we show that the Slc12a8 gene encodes a specific NMN transporter. We find that Slc12a8 is highly expressed and regulated by NAD+ in the murine small intestine. Slc12a8 knockdown abrogates the uptake of NMN in vitro and in vivo. We further show that Slc12a8 specifically transports NMN, but not nicotinamide riboside, and that NMN transport depends on the presence of sodium ion. Slc12a8 deficiency significantly decreases NAD+ levels in the jejunum and ileum, which is associated with reduced NMN uptake as traced by doubly labeled isotopic NMN. Finally, we observe that Slc12a8 expression is upregulated in the aged murine ileum, which contributes to the maintenance of ileal NAD+ levels. Our work identifies the first NMN transporter and demonstrates that Slc12a8 has a critical role in regulating intestinal NAD+ metabolism.

Aging is a significant risk factor for impaired tissue functions and chronic diseases. Age-associated decline in systemic NAD+ availability plays a critical role in regulating the aging process across many species. Here, we show that the circulating levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) significantly decline with age in mice and humans. Increasing circulating eNAMPT levels in aged mice by adipose-tissue-specific overexpression of NAMPT increases NAD+ levels in multiple tissues, thereby enhancing their functions and extending healthspan in female mice. Interestingly, eNAMPT is carried in extracellular vesicles (EVs) through systemic circulation in mice and humans. EV-contained eNAMPT is internalized into cells and enhances NAD+ biosynthesis. Supplementing eNAMPT-containing EVs isolated from young mice significantly improves wheel-running activity and extends lifespan in aged mice. Our findings have revealed a novel EV-mediated delivery mechanism for eNAMPT, which promotes systemic NAD+ biosynthesis and counteracts aging, suggesting a potential avenue for anti-aging intervention in humans.

The mammalian Sir2 ortholog Sirt1 plays an important role in metabolic regulation. However, the role of Sirt1 in the regulation of aging and longevity is still controversial. Here we demonstrate that brain-specific Sirt1-overexpressing (BRASTO) transgenic mice show significant life span extension in both males and females, and aged BRASTO mice exhibit phenotypes consistent with a delay in aging. These phenotypes are mediated by enhanced neural activity specifically in the dorsomedial and lateral hypothalamic nuclei (DMH and LH, respectively), through increased orexin type 2 receptor (Ox2r) expression. We identified Nk2 homeobox 1 (Nkx2-1) as a partner of Sirt1 that upregulates Ox2r transcription and colocalizes with Sirt1 in the DMH and LH. DMH/LH-specific knockdown of Sirt1, Nkx2-1, or Ox2r and DMH-specific Sirt1 overexpression further support the role of Sirt1/Nkx2-1/Ox2r-mediated signaling for longevity-associated phenotypes. Our findings indicate the importance of DMH/LH-predominant Sirt1 activity in the regulation of aging and longevity in mammals.

We tested the hypothesis that supplementation of nicotinamide mononucleotide (NMN), a key NAD(+) intermediate, increases arterial SIRT1 activity and reverses age-associated arterial dysfunction and oxidative stress. Old control mice (OC) had impaired carotid artery endothelium-dependent dilation (EDD) (60 ± 5% vs. 84 ± 2%), a measure of endothelial function, and nitric oxide (NO)-mediated EDD (37 ± 4% vs. 66 ± 6%), compared with young mice (YC). This age-associated impairment in EDD was restored in OC by the superoxide (O2-) scavenger TEMPOL (82 ± 7%). OC also had increased aortic pulse wave velocity (aPWV, 464 ± 31 cm s(-1) vs. 337 ± 3 cm s(-1) ) and elastic modulus (EM, 6407 ± 876 kPa vs. 3119 ± 471 kPa), measures of large elastic artery stiffness, compared with YC. OC had greater aortic O2- production (2.0 ± 0.1 vs. 1.0 ± 0.1 AU), nitrotyrosine abundance (a marker of oxidative stress), and collagen-I, and reduced elastin and vascular SIRT1 activity, measured by the acetylation status of the p65 subunit of NFκB, compared with YC. Supplementation with NMN in old mice restored EDD (86 ± 2%) and NO-mediated EDD (61 ± 5%), reduced aPWV (359 ± 14 cm s(-1) ) and EM (3694 ± 315 kPa), normalized O2- production (0.9 ± 0.1 AU), decreased nitrotyrosine, reversed collagen-I, increased elastin, and restored vascular SIRT1 activity. Acute NMN incubation in isolated aortas increased NAD(+) threefold and manganese superoxide dismutase (MnSOD) by 50%. NMN supplementation may represent a novel therapy to restore SIRT1 activity and reverse age-related arterial dysfunction by decreasing oxidative stress.

Recently, it has become a consensus that systemic decreases in NAD+ are a critical trigger for age-associated functional decline in multiple tissues and organs. The hypothalamus, which contains several functionally distinct subregions called nuclei, functions as a high-order control center of aging in mammals. However, due to a technical difficulty, how NAD+ levels change locally in each hypothalamic nucleus during aging remains uninvestigated. We were able to establish a new combinatorial methodology, using laser-captured microdissection (LCM) and high-performance liquid chromatography (HPLC), to accurately measure NAD+ levels in small tissue samples. We applied this methodology to examine local NAD+ changes in hypothalamic nuclei and found that NAD+ levels were decreased significantly in the arcuate nucleus (ARC), ventromedial hypothalamus (VMH), and lateral hypothalamus (LH), but not in the dorsomedial hypothalamus (DMH) of 22-month-old mice, compared to those of 3-month-old mice. The administration of nicotinamide mononucleotide (NMN) significantly increased NAD+ levels in all these hypothalamic nuclei. Interestingly, the administration of extracellular nicotinamide phosphoribosyltransferase-containing extracellular vesicles (eNampt-EVs) purified from young mice increased NAD+ levels in the ARC and DMH. These results reveal the unique specificity of NAD+ regulation in the hypothalamus during aging.

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite for fundamental biological phenomena, including aging. Nicotinamide mononucleotide (NMN) is a key NAD+ intermediate that has been extensively tested as an effective NAD+-boosting compound in mice and humans. However, the accurate measurement of NMN in biological samples has long been a challenge in the field. Here, we have established an accurate, quantitative methodology for measuring NMN by using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) with double isotopic NMN standards. In this new methodology, the matrix effects of biological samples were properly adjusted, and the fate of NMN could be traced during sample processing. We have demonstrated that this methodology can accurately quantitate NMN levels in mouse plasma and confirmed quick, direct NMN uptake into blood circulation and cells. This double isotope-mediated LC-MS/MS (dimeLC-MS/MS) can easily be expanded to other NAD+-related metabolites as a reliable standard methodology for NAD+ biology.

Recent studies have shown that the hypothalamus functions as a control center of aging in mammals that counteracts age-associated physiological decline through inter-tissue communications. We have identified a key neuronal subpopulation in the dorsomedial hypothalamus (DMH), marked by Ppp1r17 expression (DMHPpp1r17 neurons), that regulates aging and longevity in mice. DMHPpp1r17 neurons regulate physical activity and WAT function, including the secretion of extracellular nicotinamide phosphoribosyltransferase (eNAMPT), through sympathetic nervous stimulation. Within DMHPpp1r17 neurons, the phosphorylation and subsequent nuclear-cytoplasmic translocation of Ppp1r17, regulated by cGMP-dependent protein kinase G (PKG; Prkg1), affect gene expression regulating synaptic function, causing synaptic transmission dysfunction and impaired WAT function. Both DMH-specific Prkg1 knockdown, which suppresses age-associated Ppp1r17 translocation, and the chemogenetic activation of DMHPpp1r17 neurons significantly ameliorate age-associated dysfunction in WAT, increase physical activity, and extend lifespan. Thus, these findings clearly demonstrate the importance of the inter-tissue communication between the hypothalamus and WAT in mammalian aging and longevity control.

The effects of two different mitochondrial-targeted drugs, SS-31 and NMN, were tested on Old mouse hearts. After treatment with the drugs, individually or Combined, heart function was examined by echocardiography. SS-31 partially reversed an age-related decline in diastolic function while NMN fully reversed an age-related deficiency in systolic function at a higher workload. Metabolomic analysis revealed that both NMN and the Combined treatment increased nicotinamide and 1-methylnicotinamide levels, indicating greater NAD+ turnover, but only the Combined treatment resulted in significantly greater steady-state NAD(H) levels. A novel magnetic resonance spectroscopy approach was used to assess how metabolite levels responded to changing cardiac workload. PCr/ATP decreased in response to increased workload in Old Control, but not Young, hearts, indicating an age-related decline in energetic capacity. Both drugs were able to normalize the PCr/ATP dynamics. SS-31 and NMN treatment also increased mitochondrial NAD(P)H production under the higher workload, while only NMN increased NAD+ in response to increased work. These measures did not shift in hearts given the Combined treatment, which may be owed to the enhanced NAD(H) levels in the resting state after this treatment. Overall, these results indicate that both drugs are effective at restoring different aspects of mitochondrial and heart health and that combining them results in a synergistic effect that rejuvenates Old hearts and best recapitulates the Young state.

NAD+ availability decreases with age and in certain disease conditions. Nicotinamide mononucleotide (NMN), a key NAD+ intermediate, has been shown to enhance NAD+ biosynthesis and ameliorate various pathologies in mouse disease models. In this study, we conducted a 12-month-long NMN administration to regular chow-fed wild-type C57BL/6N mice during their normal aging. Orally administered NMN was quickly utilized to synthesize NAD+ in tissues. Remarkably, NMN effectively mitigates age-associated physiological decline in mice. Without any obvious toxicity or deleterious effects, NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies. Consistent with these phenotypes, NMN prevented age-associated gene expression changes in key metabolic organs and enhanced mitochondrial oxidative metabolism and mitonuclear protein imbalance in skeletal muscle. These effects of NMN highlight the preventive and therapeutic potential of NAD+ intermediates as effective anti-aging interventions in humans.

Nicotinamide adenine dinucleotide (NAD(+)) is an essential coenzyme/cosubstrate for many biological processes in cellular metabolism. The rate-limiting step in the major pathway of mammalian NAD(+) biosynthesis is mediated by nicotinamide phosphoribosyltransferase (Nampt). Previously, we showed that mice lacking Nampt in forebrain excitatory neurons (CamKIIαNampt(-/-) mice) exhibited hyperactivity, impaired learning and memory, and reduced anxiety-like behaviors. However, it remained unclear if these functional effects were accompanied by synaptic changes. Here, we show that CamKIIαNampt(-/-) mice have impaired induction of long-term depression (LTD) in the Schaffer collateral pathway, but normal induction of long-term potentiation (LTP), at postnatal day 30. Pharmacological assessments demonstrated that CamKIIαNampt(-/-) mice also display dysfunction of synaptic GluN2B (NR2B)-containing N-methyl-d-aspartate receptors (NMDARs) prior to changes in NMDAR subunit expression. These results support a novel, important role for Nampt-mediated NAD(+) biosynthesis in LTD and in the function of GluN2B-containing NMDARs.

Neuromuscular decline occurs with aging. The neuromuscular junction (NMJ), the interface between motor nerve and muscle, also undergoes age-related changes. Aging effects on the NMJ components-motor nerve terminal, acetylcholine receptors (AChRs), and nonmyelinating terminal Schwann cells (tSCs)-have not been comprehensively evaluated. Sirtuins delay mammalian aging and increase longevity. Increased hypothalamic Sirt1 expression results in more youthful physiology, but the relationship between NMJ morphology and hypothalamic Sirt1 was previously unknown. In wild-type mice, all NMJ components showed age-associated morphological changes with ~80% of NMJs displaying abnormalities by 17 months of age. Aged mice with brain-specific Sirt1 overexpression (BRASTO) had more youthful NMJ morphologic features compared to controls with increased tSC numbers, increased NMJ innervation, and increased numbers of normal AChRs. Sympathetic NMJ innervation was increased in BRASTO mice. In contrast, hypothalamic-specific Sirt1 knockdown led to tSC abnormalities, decreased tSC numbers, and more denervated endplates compared to controls. Our data suggest that hypothalamic Sirt1 functions to protect NMJs in skeletal muscle from age-related changes via sympathetic innervation.

The dorsomedial hypothalamus (DMH) controls a number of essential physiological responses. We have demonstrated that the DMH plays an important role in the regulation of mammalian aging and longevity. To further dissect the molecular basis of the DMH function, we conducted microarray-based gene expression profiling with total RNA from laser-microdissected hypothalamic nuclei and tried to find the genes highly and selectively expressed in the DMH. We found neuropeptide VF precursor (Npvf), PR domain containing 13 (Prdm13), and SK1 family transcriptional corepressor (Skor1) as DMH-enriched genes. Particularly, Prdm13, a member of the Prdm family of transcription regulators, was specifically expressed in the compact region of the DMH (DMC), where Nk2 homeobox 1 (Nkx2-1) is predominantly expressed. The expression of Prdm13 in the hypothalamus increased under diet restriction, whereas it decreased during aging. Prdm13 expression also showed diurnal oscillation and was significantly upregulated in the DMH of long-lived BRASTO mice. The transcriptional activity of the Prdm13 promoter was upregulated by Nkx2-1, and knockdown of Nkx2-1 suppressed Prdm13 expression in primary hypothalamic neurons. Interestingly, DMH-specific Prdm13-knockdown mice showed significantly reduced wake time during the dark period and decreased sleep quality, which was defined by the quantity of electroencephalogram delta activity during NREM sleep. DMH-specific Prdm13-knockdown mice also exhibited progressive increases in body weight and adiposity. Our findings indicate that Prdm13/Nkx2-1-mediated signaling in the DMC declines with advanced age, leading to decreased sleep quality and increased adiposity, which mimic age-associated pathophysiology, and provides a potential link to DMH-mediated aging and longevity control in mammals.

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases.

Inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) are promising cancer drugs currently in clinical trials in oncology, including APO866, CHS-828 and the CHS-828 prodrug EB1627/GMX1777, but cancer cell resistance to these drugs has not been studied in detail.

Intracellular nicotinamide phosphoribosyltransferase (iNampt) is an essential enzyme in the NAD biosynthetic pathway. An extracellular form of this protein (eNampt) has been reported to act as a cytokine named PBEF or an insulin-mimetic hormone named visfatin, but its physiological relevance remains controversial. Here we show that eNampt does not exert insulin-mimetic effects in vitro or in vivo but rather exhibits robust NAD biosynthetic activity. Haplodeficiency and chemical inhibition of Nampt cause defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. These defects are corrected by administration of nicotinamide mononucleotide (NMN), a product of the Nampt reaction. A high concentration of NMN is present in mouse plasma, and plasma eNampt and NMN levels are reduced in Nampt heterozygous females. Our results demonstrate that Nampt-mediated systemic NAD biosynthesis is critical for beta cell function, suggesting a vital framework for the regulation of glucose homeostasis.