A Combination NAD+ Treatment Has Benefits for Mice
- The combination had stronger effects than either individual approach.
- Administering NMN to cells directly increases NAD+, while inhibiting CD38 through apigenin prevents its depletion.
- This combination restored function to cells and improved multiple aspects of musculoskeletal and gut health in aged wild-type mice.
Researchers have found that simultaneously supplying NAD+ through NMN and reducing its loss through apigenin restores muscle function and bone structure to aged mice.
A well-documented issue
NAD+ is one of the most well-documented compounds in biology, including within the context of aging. Precursor supplementation has been repeatedly documented to have measurable benefits; for example, a human clinical trial found that one precursor, nicotinamide riboside (NR) reduces mortality in chronic obstructive pulmonary disorder [1], and another found that a different precursor, nicotinamide mononucleotide (NMN), restores insulin senstivity [2].
However, CD38, an enzyme that consumes NAD+, increases with age [3]. Its inhibitor, apigenin [4], has also been researched and found to have beneficial effects in various organisms.
Combining NMN and apigenin into a formulation known as N + A has been previously researched as well. In muscle precursor cells, N + A was found to fight inflammation and senescence [5]. This study builds upon that work by administering this formulation to other types of cells along with in vivo experimentation.
Restoring NAD+ restores cellular function
At first, the researchers used biopsy data from publicly available datasets to measure four types of cells: myofibers, muscle stem cells, chondrocytes, and osteoblasts. As expected, samples derived from older people had fewer muscle stem cells than samples derived from younger people did. A gene expression analysis revealed that a broad swath of genes related to NAD+ were downregulated in the older samples.
The researchers then performed their own experiment, driving three types of musculoskeletal cells senescent through oxidative stress, doxocirubin, or replication. All three approaches reduced the prevalence of NAD+ within these cells while reducing the amount of available ATP and suppressing their differentiation capabilities.
Their next experiment involved administering NMN, apigenin, or their combination to cells that had been driven senescent through oxidative stress. Individually, NMN slightly restored NAD+ in treated cells while apigenin may have restored slightly more; their combination was stronger than either individually, restoring NAD+ levels nearly to that of a control group that had never been driven senescent.
In all three cell types, senescence increased levels of the pro-inflammatory chemokine CXCL8; NMN did little to alleviate this, but apigenin reduced it, and the combination appeared to reduce it down to nearly normal levels. Both NMN and apigenin reduced levels of the DNA damage marker γ-H2ax, while their combination reduced it further in these cells. There were benefits for mitochondrial respiration as well, as the combination restored ATP synthesis and promoted proper mitochondrial membrane potential.
N + A was also found to assist in cellular differentiation. Cell precursors of cartilage, bone, and muscle tissue all had their cell-specific differentation markers upregulated by the treatment.
Broad benefits in mice
The researchers then turned to mice. With natural aging, the muscles of mice visibly shrink, and senescent cells accumulate throughout musculoskeletal tissues. Fibrosis also becomes visible, and the gaits of the animals become noticeably impaired.
According to this study, N + A administration alleviated all of these age-related issues, although not quite to the levels of young mice. More vigorous voluntary movement was restored, and the treated mice had better limb strength. Like with the cellular study, the N + A combination was found to be stronger than either compound individually.
These benefits were found to be partially dependent on the sirtuin SIRT3. Aged mice that had been genetically modified to be deficient in SIRT3 enjoyed significantly fewer benefits from taking N + A than wild-type mice did, including in senescence biomarkers, serum NAD+, and muscle strength.
N + A also provided benefits for the gut. Treated wild-type mice had more diversity in the gut microbiome. Ferroptosis, a form of cellular death caused by iron, was reduced in the gut tissues of the treated group. Furthermore, administering fecal bacteria from aged mice that had been treated with N + A was found to have benefits in untreated aged mice, similarly to fecal bacteria derived from young mice. The intestinal metabolite phytosphingosine (PHS) was found to confer some of these benefits, and the researchers hold that it deserves further study.
Of course, these experiments were only performed in cells and mice. It is not known whether administering a combination of NMN and apigenin has side effects in human beings that outweigh any benefits. However, as the researchers contend, this “increasing income and reducing expenditure” approach has “potential clinical translational value” in restoring muscle, cartilage, and bone tissue; trials on larger animals, and possibly human clinical trials, can determine if this is indeed the case.
Literature
[1] Norheim, K. L., Ben Ezra, M., Heckenbach, I., Andreasson, L. M., Eriksen, L. L., Dyhre-Petersen, N., … & Scheibye-Knudsen, M. (2024). Effect of nicotinamide riboside on airway inflammation in COPD: a randomized, placebo-controlled trial. Nature aging, 4(12), 1772-1781.
[2] Yoshino, M., Yoshino, J., Kayser, B. D., Patti, G. J., Franczyk, M. P., Mills, K. F., … & Klein, S. (2021). Nicotinamide mononucleotide increases muscle insulin sensitivity in prediabetic women. Science, 372(6547), 1224-1229.
[3] Camacho-Pereira, J., Tarragó, M. G., Chini, C. C., Nin, V., Escande, C., Warner, G. M., … & Chini, E. N. (2016). CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell metabolism, 23(6), 1127-1139.
[4] Escande, C., Nin, V., Price, N. L., Capellini, V., Gomes, A. P., Barbosa, M. T., … & Chini, E. N. (2013). Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome. Diabetes, 62(4), 1084-1093.
[5] Wu, J., Singh, K., Lin, A., Meadows, A. M., Wu, K., Shing, V., … & Sack, M. N. (2022). Boosting NAD+ blunts TLR4-induced type I IFN in control and systemic lupus erythematosus monocytes. The Journal of clinical investigation, 132(5).
About the author
Josh Conway
