Main Research Findings

1) Supplementation with NMN has been shown to improve both lipid profiles and glucose intolerance in mice experiencing age-induced diabetes.

2) Long-term administration of NMN has been shown to improve age-related physiological degeneration in mice.

Selected Data

1) Recent research has found that there has been a significant increase in cases of type 2 diabetes, worldwide, due to increases in sedentary lifestyle and calorie-rich diets. The primary mechanisms that are impaired in these cases are NAMPT-mediated NAD+ biosynthesis and the NAD+ dependent protein deacetylase SIRT1. These pathways play an important role in the regulation of metabolism, stress responses, differentiation, adaptive responses to energy intake, and the sleep-wake cycle. That being said, the research team of Yoshino et al assessed how administration of NMN affects glucose intolerance and lipid profiles in mice with age-induced type 2 diabetes [1].

For the purpose of this study, type 2 diabetes was induced in 3-6 month old mice by feeding them a high fat diet that contained ~42% of the total calories from fat. Diabetes in the animals was defined as having fasting glucose levels greater than 120 mg/dl or blood glucose levels at a 2 hour time period that was greater than 200 mg/dl. In order to assure that diabetes was properly induced both male and female mice were screened at 15-26 months of age. After criteria was met, NMN was intraperitoneally injected to the age-induced diabetic mice in doses of 500 mg/kg of body weight.

Both NAD+ levels and NMN levels were measured by the researchers using an HPLC system that included an LC-18 T column as well as a Hypercarb column for NAD+ and NMN, respectively. Next, primary hepatocytes were collected from the animals followed by isolation and culturing. The hepatocytes were cultured in DMEM containing 1.0% FBS and each reagent or reagent combination in order to measure changes following treatment with NMN, enzyme inhibitors, TNF-alpha, and menadione [1].

Frozen liver samples collected by the researchers were used to isolate total RNA from the control group of animals, the animals fed a high fat diet, and the animals fed a high fat diet and treated with NMN. The frozen liver samples were also used to prepare nuclear extracts. 100 ugs of the liver nuclear extract was then analyzed using an anti-acetyl NF-kB p65 antibody. The blots were re-probed with anti-NF-kB p65 antibody and the researchers utilized an ECL plus detection system to detect and quantify the signals [1].

2) The research team of Mills et al examined the potential of NMN to act as a potent anti-aging compound for the prevention of physiological decline related to advanced age. Previous research has found that short-term administration of NMN has the potential to elicit significant therapeutic benefits for various metabolic complications and disease conditions. Studies have shown that NMN improves glucose-stimulated insulin secretion in aged wild-type mice and various genetic mouse models. The compound also has the ability to enhance both insulin action and secretion in diet- and age-induced type 2 diabetes and obesity models [2].

Additionally, NMN protects the cardiovascular system from ischemia reperfusion injury by preventing ischemia-induced NAD+ depletion. The compound also assists in maintaining neural stem cell and progenitor cell populations, restoring mitochondrial function in skeletal muscle and arteries of aged mice, and improving mitochondrial function, neural survival, and cognitive performance in Alzheimer’s disease models. Increases in NAD+ levels have also been linked to longer lifespans in lower organisms such as yeast, worms, and flies. In rodents and humans, NAD+ levels typically decline with age across multiple organs, including the pancreas, adipose tissue, skeletal muscle, liver, skin, and brain [2].
Given this decline, enhancing NAD+ biosynthesis with NMN could potentially prevent age-related physiological deterioration. However, while short-term studies have shown therapeutic benefits, long-term administration studies in normal wild-type mice are necessary to determine whether these compounds can prevent age-related functional decline. To investigate the long-term effects of NMN administration, the research team conducted a 12-month study using male C57BL/6N mice housed in a controlled environment with a 12-hour light/dark cycle. Mice were given ad libitum access to a regular chow diet, and body weights were measured weekly.
Food and water intake were monitored monthly over several consecutive days, while blood samples were collected monthly to assess glucose, insulin, lipid levels, and blood cell counts. Intraperitoneal glucose tolerance tests and insulin tolerance tests were conducted before NMN administration and every three months throughout the experimental period. Other physiological parameters, including rectal body temperature, urine analysis, histopathology, and bone mineral density, were measured at various intervals during the experiment. NAD+ levels, gene expression profiles, and additional molecular analyses were performed on tissue samples collected every three months [2].
Before initiation of NMN administration, the animals water consumption was measured for two weeks to establish baseline intake levels. NMN was administered to the animals through the drinking water at either 100 or 300 mg/kg/day, based on prior water consumption data. Treatment began when mice were five months old and continued for 12 months, until they reached 17 months of age. NMN solutions were freshly prepared weekly, filtered for sterility, and tested by high-performance liquid chromatography to ensure stability, while water bottles and cages were changed twice weekly to maintain consistency [2].

Discussion

1) The results of the study conducted by the research team of Yoshino et al found that both male and female mice with age-induced type 2 diabetes experienced that NAMPT protein levels were reduced in the liver and white adipose tissue but not in the skeletal muscle. This was compared to the animals that were fed a regular chow diet rather than a high fat diet that contained 42% of total daily calories from fat. Similar findings were noted when observing levels of NAD+. With the reduction in NAMPT there was also a decrease in NAD+ levels in the liver and white adipose tissue, but not in the skeletal muscle. These findings indicate that there is an impairment in the biosynthesis of NAD+ in both the liver and white adipose tissue in mice with high-fat diet induced diabetic mice [1].

Based on the initial findings the researchers hypothesized that the reduction in NAMPT-mediated NAD+ biosynthesis could be resolved with the administration of NMN. When the activity of NMN was assessed it was found that the compound was immediately converted to NAD+ within 15 minutes. This resulted in a significant increase in overall NAD+ levels over the course of 60 minutes. There was also a 5-fold increase noted in levels of nicotinamide riboside levels, as well as a 15-fold increase in NMN. These findings indicate that NMN is potentially converted to nicotinamide riboside in order to enter the liver. Additionally, it was confirmed that NAD+ biosynthesis in primary hepatocytes was enhanced in a dose-dependent manner after the administration of NMN [1].
The results of intraperitoneal glucose tolerance tests reported that administration of NMN was shown to effectively normalize impaired glucose tolerance in diabetic female mice. Despite plasma insulin levels remaining unchanged before and after NMN treatment, insulin tolerance significantly improved in diabetic female mice. Comparatively, NMN also improved glucose tolerance in diabetic male mice, though the effect was not as significant. Additionally, glucose-stimulated insulin secretion was shown to improve at the 15- and 30-minute time points in males following NMN administration. This was confirmed in primary pancreatic islets isolated from diabetic males, where both NAD+ levels and glucose-stimulated insulin secretion was increased. However, insulin tolerance remained unchanged in males, suggesting sex-based differences in the primary tissues targeted by NMN. While the exact reason for this sex difference remains unclear, these findings highlight the ability of NMN to ameliorate glucose intolerance by enhancing either insulin sensitivity or insulin secretion in high-fat diet induced diabetic mice [1].
Considering that administration of NMN had the ability to restore normal NAD+ levels in diabetic livers, the research team investigated whether NMN improves hepatic insulin sensitivity in diabetic females. This was assessed by observing the phosphorylation status of AKT, a key downstream kinase in insulin signaling. The findings revealed that diabetic mice treated with NMN exhibited increased AKT phosphorylation, indicating improvements in hepatic insulin sensitivity. Furthermore, gene expression profiles of liver samples from regular chow-fed, high fat diet-fed, and NMN-treated high fat diet-fed mice were analyzed using parametric analysis of gene set enrichment. The results showed that NMN reversed the effects of a high fat diet on biological pathways, as well as various genes associated with oxidative stress, inflammation, immune response, and lipid metabolism that contribute to hepatic insulin resistance [1].
Additionally, glutathione S-transferases pathways that protect against lipid peroxidation products and support hepatic insulin sensitivity were shown to be suppressed in the animals fed a high-fat diet, however, they were restored with NMN administration. Specifically, the expression of the glutathione S-transferase alpha 2 gene was significantly reduced by a high-fat diet but recovered with NMN treatment. In a similar manner, Gsta1 and Gsta4 genes exhibited the same pattern of changes, although changes in their false discovery rates were not considered statistically significant.
On the other hand, pathways associated with damage, inflammation, and immune responses were upregulated by a high-fat diet and suppressed by administration of NMN. This was emphasized by changes in genes such as interleukin-1β , S100 calcium-binding proteins A8, and S100 calcium-binding proteins A9, all of which are direct NF-κB target genes linked to hepatic insulin resistance. Additional genes significantly altered by both HFD and NMN treatment included Lipin1 and pyruvate dehydrogenase kinase 4, both of which are implicated in insulin resistance. These findings suggest there is a notable connection between NAD+ biosynthesis, circadian rhythms, and metabolic disorders that reinforces the role of NAMPT-mediated NAD+ production in maintaining metabolic balance [1].
Apart from varying dietary factors, aging is another significant risk factor for developing type 2 diabetes. Researchers observed that NAD+ levels declined significantly in the pancreas, white adipose tissue, and skeletal muscle in older mice, with similar trends noted in the liver. NAMPT protein levels were also shown to decrease in the liver, white adipose tissue, and skeletal muscle with age. Based on these observations, the research team hypothesized that treatment with NMN may also be effective in models of age-induced type 2 diabetes [1].

Figure 1: Changes in A) NAMPT protein levels and B) NAD+ levels, in the liver, white adipose tissue, and skeletal tissue
Screening of regular chow-fed wild-type B6 mice of 15–26 months of age revealed that approximately 15% of males developed diabetes that was defined by glucose levels exceeding 200 mg/dL at the 2-hour mark in intraperitoneal glucose tolerance tests. These diabetic aged male mice were given a single intraperitoneal injection of NMN, which resulted in normalization of impaired glucose tolerance. In these mice, glucose-stimulated insulin secretion at both the 15- and 30-minute time points were shown to improve following administration of NMN, however, the differences were not deemed statistically significant. The researchers noted that NMN had no significant effects on aged, non-diabetic male mice, suggesting that the compound does not disrupt glucose homeostasis in health test subjects and is specifically beneficial for animals with age-induced diabetes [1].
Unlike males, aged female mice were more resistant to naturally developing diabetes. To induce type 2 diabetes, aged female mice were subjected to a high-fat diet for seven weeks, resulting in rapid development of severe diabetes. The glucose tolerance of these female mice were shown to completely normalize after they received 11 consecutive NMN injections. Additionally, aged females treated with NMN exhibited a significant increase in their respiratory quotient (RQ), indicating improved glucose utilization, however, overall oxygen consumption remained unchanged. Furthermore, a decrease in rectal body temperature was also noted, suggesting a metabolic shift favoring glucose utilization rather than fat utilization. NMN treatment was also shown to correct hyperlipidemia induced by a high-fat diet in aged female mice, reinforcing its therapeutic potential in age-related metabolic disorders [1].
2) The study conducted by the research team of Mills et al investigated the long-term effects of NMN administration on aging-associated physiological changes in mice. Lower doses of 100 mg/kg and 300 mg/kg of NMN were administered to the animals by adding it to drinking water to ensure feasible long-term treatment. It was confirmed that NMN remains stable in water for up to 10 days, and when administered via oral gavage, the compound was rapidly absorbed into the bloodstream within minutes. This resulted in increased hepatic NAD+ levels within 30 minutes. Tracer studies confirmed that orally ingested NMN was converted into NAD+ in the liver and skeletal muscle [2].
When NMN was administered to wild-type C57BL/6N mice from 5 to 17 months of age, the compound was shown to significantly suppress age-associated body weight gain in a dose-dependent manner. Overall weight was reduced by 4% in the group of animals receiving a 100 mg/kg dose of NMN and 9% in the group receiving a 300 mg/kg dose of NMN. NMN-treated mice maintained normal food and water intake, indicating that weight suppression was not due to appetite loss or growth defects. Additionally, the compound did not induce any obvious toxicity, as assessed through blood tests, urine analysis, and survival rates.
The study further examined the impact of NMN on glucose and lipid metabolism. Glucose tolerance remained unchanged across all groups, however, NMN administration tended to reduce both fasting and fed insulin levels, particularly in the 300 mg/kg/day group. After 12 months, NMN significantly improved insulin sensitivity compared to control mice, independent of body weight. Intrahepatic triglyceride levels, an indicator of insulin resistance, were lower in NMN-treated mice, supporting its beneficial role in metabolic health [2].
Additionally, plasma lipid analysis revealed that NMN administration helped stabilize free fatty acid levels, while preventing the age-associated increase that was observed in the control group of mice. The 300 mg/kg/day group showed the most stable free fatty acid levels over time. This effect was also noted with other metabolic improvements, such as lower intrahepatic triglycerides and enhanced insulin sensitivity. The study demonstrated that long-term NMN supplementation can counteract age-related weight gain and improve metabolic health without adverse effects. These findings suggest the potential of NMN to promote healthy aging and metabolic function in mammals [2].
Microarray analysis was also performed and the results revealed that administration of NMN prevented age-associated transcriptional changes in skeletal muscle, white adipose tissue, and liver. The control group of animals experienced significant changes in various genes, however, these genes remained stable in the mice treated with NMN, suggesting that the compound elicits protective effects. NMN administration was also found to mitigate age-related upregulation of immune and inflammatory pathways in white adipose tissue that are typically linked to obesity and insulin resistance.
Principal component analysis also showed that mice treated with NMN exhibited gene expression patterns that were similar to those of younger mice, particularly in skeletal muscle. Additionally, high-resolution respirometry demonstrated that administration of NMN significantly enhanced mitochondrial oxidative metabolism in skeletal muscle, as indicated by an increase in respiration rates and mitonuclear protein imbalance that acts as a hallmark of improved mitochondrial function [2].
In addition to metabolic benefits, administration of NMN was shown to reduce age-associated ocular degeneration. The C57BL/6N mouse strain has been found to carry a Crb1 gene mutation that results in the accumulation of subretinal microglia and retinal dysfunction. Treatment with NMN significantly reduced these pathological changes and preserved retinal function, which was measured by electroretinography. The mice treated with NMN displayed improved rod, cone, and bipolar cell responses, suggesting preserved visual function. Furthermore, NMN administration increased tear production in aged mice, reversing age-related declines in lacrimal gland function. Bone density also experienced minor increases in a dose-dependent manner. Blood analysis revealed that NMN administration reduced neutrophil counts while increasing lymphocytes. These findings suggest a potential immune modulation [2].
Overall, the results of the study indicate that long-term administration of NMN effectively counters age-associated physiological decline by preventing weight gain, improving insulin sensitivity, preserving mitochondrial function, and mitigating inflammatory and degenerative changes in metabolic tissues and the eye. These findings suggest that treatment with NMN has the potential to promote healthy aging and metabolic function in mammals. The compound appears to be a promising candidate for combating aging-related diseases and improving overall healthspan without adverse effects [2].

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

Citations

[1] Yoshino J, Mills KF, Yoon MJ, Imai S. Nicotinamide mononucleotide, a key NAD(+) intermediate, treats the pathophysiology of diet- and age-induced diabetes in mice. Cell Metab. 2011 Oct 5;14(4):528-36. doi: 10.1016/j.cmet.2011.08.014. PMID: 21982712; PMCID: PMC3204926.

[2] Mills KF, Yoshida S, Stein LR, Grozio A, Kubota S, Sasaki Y, Redpath P, Migaud ME, Apte RS, Uchida K, Yoshino J, Imai SI. Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice. Cell Metab. 2016 Dec 13;24(6):795-806. doi: 10.1016/j.cmet.2016.09.013. Epub 2016 Oct 27. PMID: 28068222; PMCID: PMC5668137.

Nicotinamide mononucleotide (NMN) is a naturally occurring nucleotide. In the body NMN is converted into the molecule nicotinamide adenine dinucleotide (NAD). NAD is crucial to many of the biological pathways related to energy production and metabolism, such as the cyclic acid cycle and the electron transport chain. NAD helps to regulate essential cellular functions. Utilization of NAD is based solely on the amount of NMN available, emphasizing the importance of its presence.

NMN levels tend to decrease with advanced age which results in a decrease in NAD and many residual side effects and potential health conditions. That being said, many of the known benefits of NMN supplementation are related to improved longevity and health. What’s interesting about this compound is that the observed benefits are related to NAD levels. However, as NAD has no way of passing through the cellular membrane the effects are elicited primarily by the conversion of NMN into NAD within the cells.

The majority of mammals tend to synthesize NMN from vitamin B3 in the form of nicotinamide. The key player in this reaction is the rate-limiting enzyme, nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the conversion of nicotinamide to NMN. NAMPT exists in both extracellular (eNAMPT) and intracellular (iNAMPT). However, eNAMPT is known for its higher enzymatic activity and wide availability as it is produced by fat cells, liver cells, white blood cells, heart cells, and brain cells.

Research has found that both brown and white adipose tissue actively secrete high levels of eNAMPT, indicating that adipose tissue may play a role in the regulation of NAD. In addition to secreting eNAMPT, adipose tissue has been shown to secrete extracellular vesicles (EVs) that can circulate through plasma. The performance of these EVs is found to be improved by the presence of NMN. Recent studies have identified an enzyme, Slc12a8, that is capable of transporting NMN through the cellular membrane directly into the cytoplasm. While EVs and eNAMPT are primarily secreted by adipose tissue, secretion of Slc12a8 is found to be 100 times higher in the small intestine. That being said, researchers have hypothesized that the gut microbiome could play a role in the production of NMN.

Effects of NMN on Anti-Aging and Overall Health

NMN is frequently referred to as the “fountain of youth” compound as many mouse models have emphasized the ability of NMN to enhance metabolism and performance, improve insulin sensitivity, and protect against ischemia and reperfusion injury. Specifically in terms of aging, NMN reduces the likelihood for age-related weight gain, restores skeletal muscle in aged mice, and inhibits age-related changes in gene expressions. Additionally, research has found that NMN reduces age-related increases in inflammation, also referred to as, inflammaging.
This is primarily due to NMN decreasing adipose tissue inflammation in aged mice. In fact, the cited study claims that older mice are often more receptive to the benefits of NMN than young mice are, in fact subjects exhibited higher energy levels and less fatigue when orally administered NMN (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238909/)

Further animal-based studies examined how irregular biosynthesis of NAD can lead to obesity and metabolic abnormalities such as insulin resistance. These studies report that since NMN is directly related to NAD, supplementation of NMN can lead to reduced rates of obesity and improved glucose tolerance and insulin sensitivity. Researchers Yoshino et. Al honed in their experimental groups and focused on the benefits of NMN in postmenopausal, prediabetic, overweight animals. The study included 25 test subjects. 12 of which were given a placebo while the remaining 13 received active NMN.

The study took place over a 10-week time period where each subject was given the same dose of either NMN or a placebo every day. Administration of NMN was seen to increase rates of insulin-stimulated glucose disposal which was measured through the hyperinsulinemic-euglycemic clamp. Additionally, by tracking the phosphorylation of AKT and mTOR, researchers were able to observe that NMN led to improvement of skeletal muscle insulin signaling. Results also reported that supplementation with NMN led to upregulated expression of various genes related to muscle remodeling, such as platelet-derived growth factor receptor 𝜷. Based on these results, the researchers were able to conclude that by administering NMN insulin sensitivity and skeletal muscle remodeling was improved specifically in prediabetic and overweight female subjects (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8550608/).

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