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Several interventions have recently emerged to reverse rather than just attenuate aging.

By Jonathan D. Grinstein, Ph.D.

There’s a lot of talk these days about stopping or slowing down aging. But what about rejuvenation — the reversal of aging? Can we actually turn back the clock to become biologically younger?

Anti-aging vs. Rejuvenation

First things first: what constitutes rejuvenation, and how does it differ from anti-aging?

The line between rejuvenation therapies and other longevity interventions, such as those that slow down or prevent aging, is hazy, and these strategies are frequently used interchangeably.  Anti-aging indicates the maintenance or preservation of aging biomarker status.  This is typically measured by biomarkers of aging, such as clocks based on DNA modification patterns or the length of telomeres — the protective DNA caps of chromosomes.

Rejuvenation goes one step further and requires a strong, prolonged, and systemic reduction in biological age.  A more important feature common to the existing and potential rejuvenation interventions is the exceptional enhancement of regenerative capacity.

The reversal of aging is inherently multidimensional: it may include a reduction in damage at the molecular level, renewed cell functionality at the cellular level, and meaningful physiological improvement at the organismal level.  Some age reversal therapies may also induce lifespan extension.  Along these lines, the effect at one level of biological organization is usually accompanied by connected effects at other levels.

Rejuvenation Strategies

Rejuvenation strategies have much in common with existing longevity interventions.  Although most longevity interventions cannot systemically reverse biological age like rejuvenation therapies, they attenuate certain age-related hallmarks, with essential effects such as the reduced presence of senescent cells and increased stem cell pool size and functionality.

Some rejuvenation strategies have been shown to reverse epigenetic age, increase stem cell function, reverse age-related loss of eyesight, and increase the lifespan of mouse models. Interventions usually influence age-related characteristics across multiple levels, and robust measures (biomarkers) of age-related damage at one level could be used to identify putative rejuvenation interventions.

There are all sorts of strategies for rejuvenation, ranging from the relatively innocuous like exercise and taking supplements to the more “daring” like gene therapy and organ transplantation.

(Bouchard and Villeda 2015 | J Neurochem.)

Systemic manipulations that promote rejuvenation. Extrinsic systemic manipulations like exercise, caloric restriction, and changing blood composition by heterochronic parabiosis or young plasma administration can partially counteract aging in many organs, such as the brain.

Exercise & Other Physiological Regimens

Physical exercise increases blood delivery to most tissues and leads to changes in the systemic environment. Interestingly, numerous studies have documented rejuvenating effects of exercise on the functional and regenerative capacity of peripheral tissues and central nervous system (CNS) in animal models.  

Outside the CNS, exercise can promote hematopoiesis (regeneration of blood cells) in the aging systemic environment, and increase the proliferative capacity of aged skeletal muscle stem cells.  There is research showing that exercise can enhance telomerase activity to protect and even extend telomere length.

For people who cannot exercise, there are other activities that can influence systemic rejuvenation. For example, another study showed that hyperbaric oxygen therapy – exposure to 100% oxygen at elevated environmental pressure to optimize body tissue oxygen absorption – extends telomeres 20-38% in different immune cell types and reduced the senescent cell population 11-37%, depending on the type of cell.

Caloric restriction

Another systemic manipulation shown to counteract the age-induced effects on tissue regeneration is caloric restriction, a reduction of 20–40% of caloric intake without malnutrition.  Caloric restriction has been shown to rejuvenate tissue regeneration in aged organisms, similar to the effects of exercise.  A number of studies have shown rejuvenating effects of caloric restriction on the decline of hematopoietic stem cell function.

Rejuvenation of regeneration was also observed in skeletal muscle and on intestinal stem cells.  The effects of both short-term and long-term caloric restriction on rejuvenation of regeneration are also found in the central nervous system – the brain and spinal cord.

NAD+ Homeostasis

Supplementation through NAD+ precursors in specific settings may also reverse hallmarks of aging, such as improving telomere length.  For example, Increasing NAD+ levels has been shown to not only protect but also improve telomere length.  One study in mice showed that the NAD+ precursor nicotinamide mononucleotide (NMN) treatment improved the length of telomeres in mice.

Senolytics

There is increasing evidence of the detrimental role of senescent cells in aging.  Some of the hallmarks of aging (mitochondrial dysfunction, deregulated nutrient-sensing, loss of proteostasis, epigenetic alterations, telomere attrition, and genomic instability) induce normal cells to become senescent, which in turn can induce paracrine senescence in nearby normal cells through senescence-associated secretory phenotype (SASP).  Senescence-promotion through SASP together with a decline in the immune system activity, converge to induce organismal accumulation of senescent cells.

In aged individuals, chronic accumulation of senescent cells contributes to tissue dysfunction and increased risk of age-associated diseases development.  Clearance of senescent cells has improved age-associated pathologies in animal models, leading to promising new clinical trials.  Different mechanisms of senescent cells can be exploited pharmacologically to develop new therapeutic targets.  Senescent cells elimination with different senotherapeutic approaches can improve healthspan in aged individuals.

(Borghesan 2020 | Trends Cell Biol.)

Senescence-Centric View of Aging. Cellular senescence is a state of stable cell cycle arrest associated with macromolecular alterations and secretion of proinflammatory cytokines and molecules.  Senescent cells are potential contributors to the age-associated loss of regenerative potential and are implicated as a central regulatory mechanism of the aging process.  Exploiting mechanisms by which senescent cells drive aging and diseases can serve as therapeutic targets. 

Telomere Extension

Aging is linked to shortening telomeres.  This happens, in part, because of insufficient activity of an enzyme called telomerase reverse transcriptase (TERT) that maintains telomere length.  Animals deficient in TERT have shorter telomeres, shorter lifespans, and an increased risk of age-related diseases like heart disease. Recent studies on animal models have shown the therapeutic efficacy of TERT in increasing healthy longevity and reversing the aging process.

Telomeres can be extended in various genetic, pharmacological, and physiological means, typically by activating TERT.  For example, one screen identified small molecules that activate TERT and extend telomeres.

Another major route to elongate telomeres has been through gene therapy with TERT, either by injection or even intranasal administration.  For example, a study that used safe and effective viral TERT gene therapy strategies increased telomere length in the heart, liver, kidney, brain, lung, and muscle in 2-year-old-mice by six times than in control mice of the same age.  Notably, the mice treated with TERT gene therapy demonstrated healthier aging and extended longevity.

(Boccardi and Herbig 2012 | EMBO Mol Med.)

Rejuvenation in mice using a telomerase gene therapy. Delivery of telomerase (TERT) using adeno-associated viruses (rAAV) suppresses aging-associated telomere erosion and extends short telomeres in various mouse tissues.  Consequently, animals display rejuvenation, improving healthspan and extending lifespan.

Cellular reprogramming

On a microscopic scale, the most extreme case of heterochronic transplantation is somatic cell nuclear transfer, which has emerged as the basis for modern-day cloning approaches (Gurdon et al., 1958).  By transferring an adult cell nucleus to a de-nucleated oocyte, a new individual can be generated.  This technique encapsulates the full potential of reversing the biological age of a somatic cell to that of the new embryo.  Interestingly, this implies that methylation patterns in the transferred nucleus are likely to reset by cytosolic components in the oocyte, suggesting another potential mechanism for rejuvenation.

To recapitulate this effect without the introduction of complex microscopic procedures, scientists discovered four critical “reprogramming factors” (Yamanaka factors), which, when expressed in somatic cells, could effectively reverse the developmental status to that of early embryos, generating induced pluripotent stem cells (iPSCs).

Interestingly, low epigenetic ages around zero were predicted when epigenetic clocks were applied to iPSC samples.  Almost all clocks showed considerable epigenetic age decreases compared with dermal fibroblasts used as the source of fully reprogrammed iPSCs.

Similar characteristics were observed in mice.  In the case of mice, clocks reported a range of epigenetic ages of the identical iPSCs.  Taken together, most human and mouse clocks reach a consensus in establishing age reversal that occurs as a result of reprogramming, although consistent predictions of age in these cells across different models remain a challenge.

(Zhang et al., 2021 | Aging Cell)

Reprogramming approaches for rejuvenation. Schematic of reprogramming approaches for rejuvenation in cell culture (in vitro) and animals (in vivo).  Full reprogramming of cells in vitro can reverse biological age to that of the embryo, but this approach can be tumorigenic in vivo.  Partial reprogramming could reverse the cell’s biological age without an irreversible change of cell identity, and the in vivo approach may be promising to achieve rejuvenation.

Heterochronic transplantation

Since the 1960s, and perhaps even earlier, researchers have been transplanting tissues and organs from animals of one age to animals of different ages.  These “heterochronic” age chimeras have shown rejuvenating properties.

Of the potential rejuvenation therapies that remain to be thoroughly characterized, linking the circulatory systems between a young and old organism – heterochronic parabiosis – is one of the most notable.  This surgical procedure has been performed for years on rodents, and it was shown that mouse lifespan could be extended by linking the circulatory system of an old mouse with that of a young mouse.  

Heterochronic parabiosis was rediscovered as one of the most promising rejuvenation interventions in 2005.  By briefly connecting the circulatory system of young and aged mice, old mice exhibited youthful features in the brain, muscle, and liver, characterized by increased cognitive function, replenished stem cell pools, and augmented regenerative capacity.

(Zhang et al., 2021 | Aging Cell)

Damage dilution in rejuvenation. In heterochronic transplantation, the damage accumulated with age is likely diluted by donor tissues (i.e., young blood), resulting in lower DNA age, as calculated by DNA methylation (DNAm) readouts.  Bulk and single-cell clocks may be used to assess biological age readouts resulting from these phenomena.

Following up on this study, researchers have focused on blood components for biological age reversal, while others have investigated the transplantation of other organs to replace aged tissues.  With bone marrow transplantation, the blood epigenetic age of the recipients generally matches the age of the donors, although whether this effect is systemic has yet to be established.  Of note, bone marrow transplantation has also shown a 12% increase in mouse lifespan.

Additionally, a study using an undisclosed plasma fraction demonstrated robust reversal of epigenetic age, further suggesting that heterochronic transplantation may be a potential rejuvenation intervention.  Recent work has also revealed that young splenocyte transplantation ameliorates the aging features of progeroid animals.  In addition, transplantation of embryonic brain tissues has shown potential for neuronal repair, and ovarian transplantation was reported to improve health parameters and result in an extended lifespan.

(Zhang et al., 2021 | Aging Cell)

Potential heterochronic transplantation interventions for rejuvenation in mice. Schematic of potential heterochronic transplantation interventions for rejuvenation in mice.

The Future of Rejuvenation 

Overall, rejuvenation procedures provide promising prospects to reverse people’s biological ages, thereby prolonging life and improving health.  However, many of the biological underpinnings of rejuvenation remain unknown, and the present adverse effects generated by some of these therapies (especially reprogramming) prohibit their widespread implementation.  Existing methods, such as lowering the dose and regularly monitoring key statistics, can assist to mitigate these side effects.

However, in the future, extensive multi-modal assessments of the common aspects of present and emerging rejuvenation therapies will be important to remove these detrimental consequences.  It will eventually be feasible to assess and harness the underlying links between age-reversing techniques as more of these therapies are created and independently validated based on molecular and physiological aging biomarkers.  

Finally, our work at the intersection of aging, molecular profiling, high-resolution methods, and physiological assessments may pave the way for the safe and successful use of systemic rejuvenation medicines in people.

References

Boccardi V, Herbig U. Telomerase gene therapy: a novel approach to combat aging. EMBO Mol Med. 2012 Aug;4(8):685-7. doi: 10.1002/emmm.201200246. Epub 2012 May 15. PMID: 22585424; PMCID: PMC3494068.

Borghesan M, Hoogaars WMH, Varela-Eirin M, Talma N, Demaria M. A Senescence-Centric View of Aging: Implications for Longevity and Disease. Trends Cell Biol. 2020 Oct;30(10):777-791. doi: 10.1016/j.tcb.2020.07.002. Epub 2020 Aug 13. PMID: 32800659.

Bouchard J, Villeda SA. Aging and brain rejuvenation as systemic events. J Neurochem. 2015 Jan;132(1):5-19. doi: 10.1111/jnc.12969. Epub 2014 Dec 5. PMID: 25327899; PMCID: PMC4301186.

Zhang B, Trapp A, Kerepesi C, Gladyshev VN. Emerging rejuvenation strategies-Reducing the biological age. Aging Cell. 2021 Dec 31:e13538. doi: 10.1111/acel.13538. Epub ahead of print. PMID: 34972247.

Research shows that taking nicotinamide mononucleotide (NMN) improves intestine bacteria composition and telomere length.

By Brett J. Weiss.    

Highlights

As people get older, the repeated DNA sequences at the ends of our chromosomes called telomeres shorten.  The erosion of telomeres leads to disorders associated with aging, like metabolic disease and heart complications.

Along these lines, inflammation from aging disrupts the balance between helpful and harmful gut bacteria, leading to inflammation that precedes age-related diseases (inflammaging).  Previous studies have indicated that the nicotinamide adenine dinucleotide (NAD+) precursor, NMN, lengthens telomeres and restores healthy gut microbes.  Whether NMN provides these benefits for people has not yet been examined thoroughly with clinical trials.

Wu and colleagues from the Tianjin Institute of Industrial Biotechnology have shown in a recent publication from a clinical trial that administering 500 mg/L of NMN in drinking water to 16-month-old pre-aging mice (equivalent to 45-60 year-old people) for 40 days shifted gut microbiome diversity and increased telomere length.

The China-based team also found that oral NMN supplementation doubles telomere length over 90 days in human blood cells called peripheral blood mononuclear cells (PBMCs).  Given these findings, it is possible that increasing telomere length may inhibit the onset of age-related diseases to thus enhance healthspan, which may also potentially increase lifespan.

NMN Boosts Metabolism in Mice

To establish the benefits of NMN given to mice, Wu and colleagues measured thermogenesis, the output of body heat, which correlates to increased cell energy production.  Thermogenesis is a biomarker, a biological indicator, of metabolism — the generation of energy through cellular processes that produce the energy packet molecule adenosine triphosphate (ATP).  The assessment of metabolism through body heat showed that administering NMN over a 40-week time course increases thermogenesis by about 10%, confirming that the compound improves metabolic function.

NMN Alters Intestinal Bacteria Diversity

Wu and colleagues next measured fecal bacteria composition in mice and found that NMN promotes a healthy gut by improving bacterial composition.  Intestinal bacteria health is a biomarker of overall physiological well-being.  The Tianjin researchers observed that short-term NMN administration reduced the diversity of bacterial species in the intestine.  Notably, several species of healthy gut-associated bacteria species increased in abundance, like Mucispirillum, Colidextribacter, and Candidatus_Saccharimonas.  On the other hand, bacteria often found in patients with age-related diseases like Staphylococcus, Corynebacterium, and Paenalcaligenes diminished in prevalence.

Oral Usage of NMN Drives Telomere Extension

Wu and colleagues then measured the length of telomeres in mice and humans.  The team found that administering NMN for short time periods increases telomere lengths 20-25% in mice.  The researchers also found that taking NMN for short periods of time (90 days) almost doubles the length of telomeres in humans, indicating potentially breakthrough health benefits and lifespan extension effects.

telomere 1
telomere 1

(Wu et al., 2021 | Frontiers in Nutrition)

Administering NMN to mice and people substantially extends telomere length in immune cells.  The left figure shows telomere length increased by about 20% in peripheral blood mononuclear cells (PBMCs).  The figure on the right illustrates that telomere lengths linearly increase such that by 90 days of orally taking NMN, telomere length almost doubled.

The evidence gathered from this research study provides insight that NMN improves gut microbial composition in mice and telomere length in mice along with people, and these effects may drastically improve healthspan.  Whether or not NMN promotes a longer lifespan, the study indicates that it might help people extend the number of years they live in health and wellbeing.

Limitations to the study include that long-term NMN supplementation was not examined.  The way that the body metabolizes NMN, especially in higher dosages, must be checked, also.  Furthermore, what effects a lower diversity of gut flora may have during aging will require further investigation.  In essence, a concern that researchers should confront soon is “how much NMN is too much?”  Until the research community has a better understanding of the recommended dosages for NMN and appropriate clinical guidance, the proper amount of NMN to be consumed in humans for certain clinical indications has yet to and is currently being explored.

References

Niu KM, Bao T, Gao L, Ru M, Li Y, Jiang L, Ye C, Wang S, Wu X. The Impacts of Short-Term NMN Supplementation on Serum Metabolism, Fecal Microbiota, and Telomere Length in Pre-Aging Phase. Front Nutr. 2021 Nov 29;8:756243. doi: 10.3389/fnut.2021.756243. PMID: 34912838; PMCID: PMC8667784.

Just like the genomes in the nuclei of our cells, these energy-generating structures have their own set of DNA, which is a proxy measure for mitochondrial function and has been associated with several aging-related diseases.

By Brett J. Weiss

Highlights

Our cells contain more than one set of DNA: one in our nucleus encodes most cellular processes and another in the energy-generating structure of the cell known as the mitochondria.  Just like the DNA in the nuclei of every cell, the replication and integrity of mitochondrial DNA (mtDNA) is critical for cells to function and flourish and for us to fulfill a healthy and long life. But we have a lot to learn about the link between mtDNA and mitochondrial function in the context of aging and longevity.  Doing so can enable us to bolster the activity of this crucial cell structure and integrity of its separate genetic hard drive for improving healthspan and lifespan.

Kang and colleagues from Kyushu University in Japan reported in the Journal of Biochemistry that nicotinamide mononucleotide (NMN) enhances mtDNA replication.  The researchers, who wrote on behalf of the Japanese Biochemical Society, found that treating human kidney cells with NMN activated and increased the rate of mtDNA replication by increasing the number of building blocks of DNA (nucleotides) in mitochondria while decreasing their degradation products (nucleosides).  These findings suggest a mechanism for how NMN benefits mitochondria, metabolism, and, ultimately, aging.

The Role of NAD+ in mtDNA Replication

In cooperation with many nuclear-encoded proteins, mtDNA genes contain the instructions to components for essential cell processes, such as generating energy.  Mitochondrial DNA copy number, a measure of the number of mitochondrial genomes per cell, is a minimally invasive proxy for mitochondrial function and has been associated with several aging-related diseases and all-cause mortality.  However, we still only know a little about how metabolism inside mitochondria affects mtDNA maintenance and replication, let alone how these processes may underlie aging and longevity.

To address this problem, Kang and colleagues created a new method for measuring metabolism within mitochondria to understand how mitochondrial metabolism regulates mtDNA replication.  With this method, which they called SLO.  The Japanese research team genetically manipulated levels of an enzyme called ‘twinkle’ — a component of mtDNA replication machinery that can increase the number of mitochondria.

By increasing mtDNA copy number through twinkle manipulation, Kang and colleagues found that several components related to metabolism changed, including increases in nucleotides and NAD+ — a vital molecule that serves as an essential factor for innumerable cell processes, including mitochondrial energy generation.  Kang and colleagues speculate that these results suggest that nucleotide and NAD+ increases may result from increased demand for components required for mtDNA replication.

mitochondrial 1
mitochondrial 1

(Kang et al., 2021 | Journal of Biochemistry)

Mitochondrial DNA replication alters the levels of nucleotides and NAD+.  This plot shows how the level of metabolites changed during controlled activation of mitochondrial DNA (mtDNA) replication through a gene called Twinkle.  Some notable nucleotide metabolites in these results are related to adenosine (AMP, ADP) and uridine (UTP, UDP) as well as phosphoribosyl diphosphate (PRPP), which is essential for the generation of nucleotides.  Red, large significant changes; blue, small significant changes; green, insignificant changes; black, no change.

NMN Modulates Mitochondrial Replication

Kang and colleagues then examined the nature of the relationship between mtDNA replication and NAD+.  To show that NAD+ causally influenced mtDNA and was not just correlative, the researchers treated human kidney cells with NMN, a precursor of NAD+, for three consecutive days and then quantified the amount of mtDNA.

In NMN treated cells, the amount of mtDNA increased to levels comparable to a condition activating mtDNA replication by controlling the ‘twinkle’ protein that executes this mtDNA copying process.  After establishing that NMN administration can increase the amount of mtDNA, Kang and colleagues showed that NMN also increases the rate of mtDNA replication.  Combining NMN administration and twinkly activation enhanced the beneficial effects on mtDNA replication and copy number.

NMN enhances mtDNA replication.  Kang and colleagues compared the amount of mitochondrial DNA (mtDNA copy number) in conditions where the mtDNA replication protein twinkle was activated (DOX), NMN was administered, and the combination of these two.  These results show that relative to untreated cells, all conditions show increases in mtDNA number.  The actions of twinkle activation together with NMN have a combined effect.

Kang and colleagues conclude the article by discussing how these data show that NMN and mtDNA replication are functionally linked through the regulation of nucleotide pool synthesis.  They indicate that they are further investigating this issue to clarify the underlying mechanism.

References

Nomiyama T, Setoyama D, Yasukawa T, Kang D. Mitochondria Metabolomics Reveals a Role of β-Nicotinamide Mononucleotide Metabolism in Mitochondrial DNA Replication. J Biochem. 2021 Dec 4:mvab136. doi: 10.1093/jb/mvab136. Epub ahead of print. PMID: 34865026.

Taking NMN by under-the-skin injections or oral consumption improves immune cells that attack cancer and virally infected cells in mice.

By Brett J. Weiss

Highlights

As the average human lifespan increases, people are experiencing poor health and age-related diseases longer, incurring major financial and societal costs.  One substantial contributing factor to age-related diseases and their progression is degraded immune function.  Specifically, NK cells that terminate cancer and virally infected cells become less effective.  At present, no remedy exists to restore the function of NK cells during aging.  So, compounds that stimulate NK cell activation may prevent age-related ailments or slow their advancement.

Okumura and colleagues from Juntendo University in Japan treated mice with nicotinamide mononucleotide (NMN) to restore NK cell cytotoxicity — the ability to destroy malfunctioning cells.  Published in Biomedical Research, NK cell stimulation in mice occurred when NMN was injected (313, 625, or 1250 mg/kg) or orally administered (625 mg/kg) for four days.  Notably, the boosted NK cell cytotoxicity didn’t come from their enhanced proliferation or higher NK cell numbers but rather improved NK cell abilities to destroy cancer cells.  These findings support that NMN supplementation may boost NK cell immunity and overall tissue health by curtailing the accumulation of precancerous and viral-infected cells as people age.

NAD+ Levels and Immune Function Drop With Age

Many aspects of immunity decline with age, and NK immune cell function is no exception to this phenomenon.  The age-related deterioration of immune function with age is linked to levels of an essential molecule called nicotinamide adenine dinucleotide (NAD+), which also declines with age.  However, boosting NAD+ levels has been shown to improve immune function in mice.  For example, studies have shown that increasing NAD+ levels by inhibiting an NAD+-consuming enzyme improves immunity to attenuate tumors in mice.

The NAD+ precursor NMN helps with age-related disorders like insulin insensitivity and metabolic impairments like obesity in aged mice. Also, since boosting NAD+ levels have been shown to work against tumors in mice, it may also potentially enhance anti-tumor NK cell activity.  Figuring out whether NMN has these immunity-restoring effects in NK cells is critical for determining if it is the compound we’ve been looking for to restore their cytotoxicity.

cancer 1
cancer 1

(Paul & Lal, 2017 | Frontiers in Immunology)  

The natural killer (NK) immune cell cytotoxic response targets cancer and viral-infected cells.  In the first step (A), the NK cell recognizes the target cell and clusters toward it.  In the second step (B), the cell reorganizes its internal structure to orient towards the target cell.  The third step (C) encompasses the NK cell docking and moving closer to the target cancer cell.  The fourth and final step (D) entails the NK cell destroying the target cancer cell so that it bursts open.

Injecting NMN Enhances Mouse Immune Cell Activity

To analyze NMN’s influence on NK cells, Takeda and Okumura injected mice at various ages with NMN and evaluated the activation and cytotoxicity of NK cells.  The research duo injected varying doses of 1250, 625, 313, or 125 mg/kg/day of NMN for four days into 36-week old mice and then harvested NK cells from the liver and spleen.  These NK cells were then put into a dish with cancer cells at different ratios and were analyzed for cytotoxicity based on the percentage of destroyed cancer cells.  These findings indicate that injecting 313 mg/kg or more of NMN enhances aged NK cell capabilities to destroy dysfunctional cells in mice.

cancer 2
cancer 2

(Takeda & Okumura, 2021 | Biomedical Research)

NMN injections (313 mg/kg/day or greater) significantly enhanced NK cell cytotoxicity in 36-week old mice.  The effector/target ratio represents the proportion of NK cells to cancer cells.  The cytotoxicity percentage – the percentage of destroyed cancer cells among those analyzed – varied with different effector/target ratios.  An effector/target ratio of 10 and 5 showed substantial enhancements of cytotoxicity with NMN in the liver, and an effector/target ratio of 100 was optimal for NMN’s efficacy in the spleen.  The upside-down solid triangles represent 125 mg/kg/day doses, the upright and solid triangles show 313 mg/kg/day doses, the solid square signifies 625 mg/kg/day doses, and the solid circle shows 1250 mg/kg/day dose values.  These results indicate that NMN’s beneficial cytotoxicity-promoting benefits have tissue-specific effects that optimize at different effector/target cell ratios depending on tissue type.

Oral NMN Dosing Improves Mouse Immune Cell Function

Using the same cytotoxicity measurement method, the research team then sought to determine whether similar benefits come from administering NMN orally.  In the 32-week-old mice Takeda and Okumura used for their analysis, 625 mg/kg of orally-supplemented NMN significantly enhanced NK cell cytotoxicity.

Neither injecting or orally dosing NMN increased the NK cell numbers.  This result strongly supports that NMN supplementation improves overall NK cell cytotoxicity in mice, because NK cells show improved, potent cancer cell-destroying abilities without becoming more abundant.

cancer 3
cancer 3

(Takeda & Okumura, 2021 | Biomedical Research)  

Oral NMN supplementation with 625 mg/kg/day doses enhances cytotoxicity in the spleen and liver.  The optimal effector/target ratios – the proportion of NK cells to cancer cells – were 5, 10, and 20 for the liver and 200 for the spleen.  At these ratios, the percentage of destroyed cancer cells, the cytotoxicity percentage, increased substantially at 625 mg/kg/day doses (solid squares on the graphs).  These findings show that orally dosing with NMN as well as injections can improve NK cell cytotoxicity in two different mouse strains.

“We have herein demonstrated that intraperitoneal or oral administration of NMN, a key NAD+ intermediate in mammals, augments the cytotoxicity of NK cells,” said Okumura and colleagues in their publication.

Studies continue to show the translatability of NMN’s health benefits from mice to humans in conditions like reduced insulin sensitivity, running endurance, and aged muscle function.  So, although how and whether these results apply to humans remain up in the air, the study offers hope for the tantalizing prospect that NMN improves aged human immunity through improved NK cell cytotoxicity.

Some study limitations include that NMN boosts NK cell cytotoxicity optimally at different ratios of NK cells to cancer cells, but the significance of this finding wasn’t explored.  For example, the optimal proportion of NK cells to cancer cells with oral dosages is 10 to 20 in the liver but 200 in the spleen.  The precise meaning of this finding remains uncertain. 

Another limitation is that the cytotoxicity analyses were done in laboratory dishes, which don’t always translate to live tissue. Future experiments can utilize cytotoxicity measurements in live animal tissues to further support that N MN boosts NK cell cytotoxicity.

Takeda K, Okumura K. Nicotinamide mononucleotide augments the cytotoxic activity of natural killer cells in young and elderly mice. Biomed Res. 2021;42(5):173-179. doi: 10.2220/biomedres.42.173. PMID: 34544993. 

Mid-aged mice treated for 20 weeks with nicotinamide mononucleotide (NMN) – a precursor to a vital molecule – show improved fertility potential and reduced aging of ovaries.

By Jonathan D. Grinstein, Ph.D.

Highlights

The relationship between female fertility and aging is complex.  We know for sure that the number of eggs (ovum) and the window for their fertilization are limited by ovarian aging.  As women get older, the ability to support conception shrinks like a reverse snowball: the quantity and quality of structures that can support the ovum called ovarian follicles gradually decline, ending with menopause.  Since menopause and the preceding decline in oocyte quality seem to have a fixed time interval, treatments that expand the female reproductive lifespan are highly sought after.

Researchers from Jiangsu University in China demonstrate that long-term treatment with nicotinamide mononucleotide (NMN) has anti-aging effects on ovaries.  In an article published in The Journal of Nutritional Biochemistry, Huang and colleagues show that 20 weeks of oral NMN treatment (0.5 mg/mL/day) could improve mid-aged ovary aging, preserving the ovarian reserve.  In mid-aged mice, this NMN treatment regimen improved the structure of ovaries and reduced senescence — a phenomenon characterized by the cessation of cell replication, which is linked to aging.

“Our work expounds a theoretical basis for application of NMN to improve the fertility of aged women,” said Huang and colleagues.

The menstrual cycle for dummies

A major aspect of reproductive capacity in women is its cyclical activity, a feature strikingly reflected in the growth and development of dominant follicles.  Ovarian follicles are tiny sacs filled with fluid that secrete hormones to influence menstrual cycle stages and have the potential to release an egg (ovum) for fertilization.

Typically, the human ovaries produce a single dominant follicle that results in each menstrual cycle’s single ovulation.  These reproductive structures progress through several distinct phases before it releases an ovum, starting from a reserve of quiescent primordial follicles set up in early life and ending with either ovulation or follicular death.

During each menstrual cycle, several follicles develop in the ovaries.  Each follicle contains an egg, but only one follicle continues to develop.  Next, this follicle releases an egg (ovulation), and the ruptured follicle closes after releasing the egg and forms a corpus luteum.  If the egg is not fertilized, the corpus luteum degenerates, and, depending on ovarian age, a new menstrual cycle begins.

Phases of the human menstrual cycle.  

The normal human menstrual cycle is 28 days.  On this basis the cycle is described as starting with about five days of menstruation, followed by a proliferative phase that lasts to about the 14th day, and then a secretory phase that lasts until the next menstruation.  The external manifestation of menstruation depends upon cyclical change in the lining of the body of the uterus called endometrium.  First, an ovarian follicle (occasionally more than one) ripens in one of the ovaries.  

This ovarian follicle contains the ovum, surrounded by a group of smaller cells, called granulosa cells.  At about mid-cycle ovulation occurs:  The ovum is discharged out of the follicle and from the surface of the ovary, to be received into the fallopian tube, down which it is carried to the uterus.

After ovulation the granulosa cells lining the follicle from which the ovum has been extruded accumulate yellow lipid and are therefore called lutein cells, from the Latin word luteus, “saffron-yellow.”

What happens during ovarian aging?

The number of follicles in the ovary is limited and critical for assessing ovarian function. Women experience a gradual decline in the number and quality of ovarian follicles, a key component of ovarian aging.  At birth, females have approximately 1-2 million primordial follicles – dormant follicles that each contain an oocyte.

The number of primordial follicles continues to decline during the reproductive years until the critical threshold at menopause, leaving only about 1,000 follicles.

NMN improves follicle development of mid-aged mice

In the present study, Huang and colleagues investigated the effects of long-term NMN treatment on ovarian aging.  The Jiangsu University researchers tested the effect of NMN on the ovarian aging of 40-week-old mice, which correlates to 38-year-old women.  After 20 weeks of treatment with NMN, the number of follicles at several key phases — from the progression of several small primordial follicles into large preovulatory follicles and through corpus luteum — was elevated.  These findings indicate that NMN can maintain the ovarian reserve of middle-aged mice.

NMN administration enhances follicle development.  The number of primordial follicles and corpus luteums span the ovarian cycle at different ages in female mice.

NMN decreases senescence marker in ovaries of mid-aged mice

The p16 protein slows cell divisions and proliferation during the progression of senescence.  Recently, elevated p16 levels have been considered one of the indicators of ovarian aging.  The present study showed high levels of p16 in ovarian follicles at 60 weeks.  Huang and colleagues found that NMN diminished the levels of p16 in mouse ovaries.  The drop in the ovary’s levels of p16 suggests that NMN alleviates ovary tissue senescence and protects it during aging.

The Jiangsu University researchers then show that the improvement to ovarian senescence by NMN treatment is likely due to the upregulation of critical cellular processes.  For example, Huang and colleagues show increased mitochondria function and the recycling of cellular damage and debris (autophagy) in the ovaries of mid-aged mice exposed to long-term administration of NMN.  In addition, the protein balance and the antioxidant capacity of follicles were restored, thereby keeping follicles relatively young.

ovaries 3
ovaries 3

(Huang et al., 2021 | J Nutr Biochem.)  

NMN administration decreases the levels of P16 protein.  The protein level of p16 increases with age, reaching a peak in the 60-week control (60WC) group.  Mice treated with NMN for 20 weeks beginning at 40 weeks of age (60WN) showed significantly decreased levels of p16.   

These results indicate that the ovarian status of mice in the 60WN group was relatively younger after NMN supplementation.

Can a supplement a day keep ovarian aging at bay?

The importance of issues related to ovarian aging has been increasing progressively over the past several decades since, increasingly, more couples in all developed countries choose to postpone parenthood to more advanced female ages.  This trend affects the likelihood of fertilization and conception, whether resulting from natural conception, conventional in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI).

Since advanced male age seems to have little effect on the success rate for IVF/ICSI attempts using oocytes from young donors, much of the attention to improving the chances of parenthood has pointed to improving ovarian aging.  Though treatment options need to be fine-tuned to reflect each woman’s condition, it is possible that supplementing with NMN can go a long way for improving the chances to experience some of life’s most miraculous adventures: pregnancy, childbirth, and parenthood.

References

Huang P, Zhou Y, Tang W, Ren C, Jiang A, Wang X, Qian X, Zhou Z, Gong A. Long-term treatment of Nicotinamide mononucleotide improved Age-related Diminished Ovary Reserve through enhancing the mitophagy level of granuloas cells in mice. J Nutr Biochem. 2021 Nov 18:108911. doi: 10.1016/j.jnutbio.2021.108911.

Nicotinamide mononucleotide (NMN) has been shown as safe in the short-term (up to six weeks); however, longer-term studies are required to prove safety.

Article first appears in NMN.com

Highlights

Research shows that nicotinamide mononucleotide (NMN) boosts physiological function to confer numerous health benefits.  For example, NMN increased mitochondrial replication and genesis (mitogenesis), likely stemming from restoring more youthful patterns of gene regulation.  

In doing so, NMN can also increase the activation of a protein deacetylase called Sirtuin1 (SIRT1), which improves the function of cell-rejuvenating proteins to reduce oxidative stress.  Along those lines, some researchers have referred to NMN as the “holy grail of reversing aging.”[1]

The questions that continually pops into mind are “how safe is NMN for daily usage,” and furthermore, “what are the effects of taking NMN daily for months on end?”  Well, the data acquired up to this point appear to show that NMN is safe for mice and for people to take.  

Moreover, taking NMN for up to six weeks has shown no adverse health-related effects.  Examples for these prospective conclusions include that dosages up to 1200 mg/day over a six-week time course confer no adverse health effects.

Twelve Weeks of NMN Was Proven Safe Without Side Effects

More evidence shows that NMN has no negative side effects, coming from data gathered from a clinical study testing the effects of NMN on skeletal muscle study over 12 weeks.  Using 250 mg per day over a six-week time course in men over age 65 had no negative safety-related effects.  Over a 12-week time course, 250 mg NMN daily enhanced muscle function and mobility.  The physiological parameters measured in the study were blood chemistry markers, including liver enzymes and indicators of kidney function.

“We report that supplementation of 250 mg per day NMN for 12 weeks in healthy old men was safe, well tolerated, and significantly increased NAD+ and NAD+ metabolites in whole blood,” said Yamauchi and colleagues in their study.  “Additionally, NMN induced improvements in muscle strength and performance.  Thus, chronic oral administration of NMN could be an effective strategy for the prevention of age-related muscle disorders.”

In another study, Klein and colleagues from the Mayo Clinic in Rochester showed that administering 250 mg per day to women over the age of 65 for 10 weeks substantially improved metabolism.  “NMN supplementation at 250 mg/day increases skeletal muscle insulin signaling, insulin sensitivity, and muscle remodeling in postmenopausal women with prediabetes who are overweight or obese,” said Klein and colleagues.

Ongoing Clinical Trials for NMN Safety

Currently, there are three ongoing clinical trials on the US Government-run clinicaltrials.gov; however, in other countries like China and Japan, no publicized safety trials are underway.  One of the clinical trials titled “Safety and Pharmacokinetics of Nicotinamide Mononucleotide (NMN) in Healthy Adults” is active with healthy volunteers.  The study is being conducted by Vitalab Clinics in Toronto, Ontario, Canada, and results have not yet been posted.  The estimated completion of this study is set for March 15, 2022.

Another study published on clinicaltrials.gov is the safety evaluation titled “Evaluate the Efficacy and Safety of Uthever NMN (Nicotinamide Mononucleotide, a Form of Vitamin B3).”  This study from the Swasthiye Clinic and Research Center in India on the effect of NMN on aging adults has been completed, but the results have not yet been published the results.

The third and final study published to the Clinical Trials website is titled “To Evaluate the Efficacy and Safety of NMN as an Anti-ageing Supplement in Middle Aged and Older (45-69 Years) Adults.”  The study evaluated the safety of 900 mg of NMN per day over the course of 60 days.  Although this clinical trial was completed in September 2021, results have not yet been made available.

Long-Term NMN Safety Trials Are Required

The bottom line is that, to date, no single study of NMN’s safety has shown that the compound has any adverse effects when it comes to humans’ blood chemistry or cardiovascular health.  Not only that, but NMN has been shown to improve muscle function, immunity, and metabolism.  The only possible issue that scientists need to confront is what the long-term effects of taking NMN have on the body.  

For example, we do not yet understand if any negative health impacts may come from using NMN over the course of a year or more.

References

  1. Shade C. The Science Behind NMN-A Stable, Reliable NAD+Activator and Anti-Aging Molecule. Integr Med (Encinitas). 2020 Feb;19(1):12-14. PMID: 32549859; PMCID: PMC7238909.
  2. https://www.nmn.com/news/metabolic-benefits-of-nmn-shown-for-first-time-in-humans
  3. https://clinicaltrials.gov/ct2/show/results/NCT04910061?term=nmn+safety&draw=2&rank=1
  4. https://clinicaltrials.gov/ct2/show/NCT04228640?term=nmn+safety&draw=2&rank=2
  5. https://clinicaltrials.gov/ct2/show/NCT04823260?term=nmn+safety&draw=2&rank=3