DO NOT TAKE MORE THAN ONE CAPSULE ON YOUR FIRST EXPERIENCE WITH NMN PLUS!
We include a very small dose of Niacin in the formula for NMN PLUS.
NIACIN is well known to cause an uncomfortable “Niacin Flush“ in some people.
We used a dosage that is about 1/2 of what research shows affects less than 1% of users. Read more about this, and what you can do to minimize niacin flush.
In addition to dosage, avoid taking NMN PLUS on an empty stomach to further minimize the chance you might experience a flush.
After the first trial or 2, you can increase to 2 capsules at a time if you desire.
Tolerance to Niacin Flush builds up rapidly. The few people that do experience it find it diminishes and no longer occurs after 4-7 days of continued use with a particular dosage, and can be increased further.
For those that want ONLY NMN, we offer NMN PURE, which contains 125 mg of NMN per capsule and nothing else, but that product will not be available to ship until Dec 8, 2017.
Time of Day
Humans have a natural Circadian rhythm with a peak NAD+ levels around Noon, and a second, smaller peak in the middle of the night.
It is not yet known if supplementing with 2 doses to emulate the 2 natural peaks is beneficial, or, just one peak in the middle of the day. Experts such as Dr Brenner, Dr Sinclair, and others are on both sides of this issue.
If you chose to take NMN Plus twice a day, we recommend:
1 Capsule between 8-10am
1 Capsule before bedtime
If you chose to take NMN Plus once per day, we recommend
1 Capsule between 8-10 am, until you are sure you don’t experience Niacin Flush
after acclimated for a few days
2 Capsules between 8-10 am
INGREDIENTS in NMN PLUS
In addition to NMN, which is the IMMEDIATE PRECURSOR used by our bodies to produce NAD+, we include the other 3 major precursors:
Obviously, it would be easier to produce only the single ingredient NMN PURE, but we are so convinced the proven NAD+ boosting ability of the secondary precursors make this product more effective, we have gone many months of research, development, and testing, in addition to the expense, to offer this Complete NAD+ Booster.
to read more about NMN and other NAD+ precursors Click here
In 2012, Research published by Dr Sinclair of Harvard stunned researchers by showing that short term supplementation with Nicotinamide Mono-Nucleotide (NMN) reversed many aspects of aging, making the cells of old mice resemble those of much younger mice, and greatly improving their health.
Since then Chromadex has been marketing a related product named Nicotinamide Riboside (NR or Niagen) that seems to work on the same cellular pathways as NMN.
1000s of articles have teased us with the possibility that taking a supplement called Niagen could halt or maybe even actually reverse some of the signs of aging. Of course there is a lot of skepticism with such claims.
Chromadex has been producing the only commercially available form of Niagen and supplies it to 20 or more different companies that put their own brand name on the bottle and sell to customers.
Elysium Health is one of these sellers, although they have fairly deep pockets and extensive connections among top researchers in the field.
To overcome the skepticism, Elysium Health decided they would enlist several Nobel Prize wining scientists as advisors and consultants.
This does add a lot of credibility to the company, even though the 7 nobel prize winning scientists Elysium pays had little or no input in creating this product, but are mostly used for marketing purposes.
Elysium Health makes a change to the formula for Basis
Basis contracted to buy Nicotinamide Riboside Chloride (often referred to as NR, or the brand name Niagen), and combine it with another off the shelf product from Chromadex, Pterostilbene.
But in June 2016, Elysium got into a contract dispute with Chromadex and later sued Chromadex for abusing the patent process.
Their goal seems to be to invalidate Chromadex patent(s) so they could produce their own version, which appears to be what they are doing now.
Elysium has changed the product information on their website to list “Nicotinamide Riboside”, without the Chloride. A key patent Chromadex has licensed the rights to involves adding the Chloride to make it more stable.
This new ingredient used by Basis likely has the same effects in the human body, but we don’t really know for sure.
It seems Elysium Health plans to go forward with this revised formula for now, and continue to use previous test results obtained with their prior formula that used Chromadex Nicotinamide Riboside Chloride. It will be interesting to see how that strategy holds up going forward.
Elysium Health has some very slick Marketing people
So now they have changed their product to use a slightly different ingredient, but want to smooth it over and not cause customers to worry about it.
Their answer is to portray the change as something they did on purpose – to MAKE THE PRODUCT BETTER.
When a customer questioned the change, this is how they answered on their Facebook page:
Thank you for reaching out to us with your questions. I’d be happy to provide additional information for you here!
From the start, Elysium has always been committed to bringing superior, high-quality supplements to market. As part of that effort, we have established a new supply chain, located in the United States, that utilizes a proprietary process to produce Nicotinamide Riboside — the first of its kind for the production of Basis, and this does fully meet the GMP standard as outlined by the FDA. We believe our vertically integrated supply chain benefits our customers as it enables us to better manage manufacturing, packaging, shipment and eventually the expansion of our new product line.
While the specific Basis formulation and the amount of each ingredient have not changed, this new production process has allowed us to take an exceptional product and make it even purer. This reflects our ongoing commitment to being a trusted source for our customers by continually exceeding the highest standards in the industry.
In regard to your question about GMO’s, this does not apply to Basis as we don’t have food products in our ingredients eligible for genetic modification. Basis is produced by nature identical synthesis, meaning that the active molecules are constructed to be nature identical. This process is preferable to attempting to distill down the ingredients from food as the final product is purer than what the bi-product would be via distillation.
If you have other questions or if there is anything we can do to help, my team can be reached directly here or by email at email@example.com or phone at 888-220-6436.
Pretty slick, I thought. No, they didn’t substitute something they just threw together to get around the patent and supply problem – they made something more pure and trustworthy.
I don’t know who is going to with the legal battles between Chromadex and Elysium Health, but I see Elysium as way ahead in the marketing department even if I don’t really trust their honesty.
(I have added the above update on the Elysium Health Basis onto an earlier review of the product below)
The two names are synonymous. Basis uses Niagen supplied by Chromadex.
NIAGEN PLUS PTEROSTILBENE
Pterostilbene is also manufactured by Chromadex.
Pterostilbene is described by Dr Guarente as “a close relative of resveratrol, but is potentially more potent and effective”.
So, Basis MIGHT be better than taking Nicotinamide Riboside plus resveratrol.
But there has been no testing at all to show any synergy from combining Nicotinamide Riboside with resveratrol or Pterostilbene.
The original research by Dr Sinclair with mice used NMN, but it is not commercially available yet. The recent excitement and dozens or research studies about slowing or halting the aging process is centered around Nicotinamide Riboside.
WHAT WE RECOMMEND
Since all Nicotinamide Riboside is supplied by Chromadex, we recommend the less expensive options below.
Note: If you do want to try NR with Pterostilbene, we have a listing for 1 bottle of Pterostilbene and 1 bottle of Nicotinamide Riboside.
These two bottles provide the exact SAME INGREDIENTS AND QUANTITIES AS 2 BOTTLES OF BASIS, AT HALF THE COST
AlivebyNature selected Vitaflavan®, an extract from the white grapes (less concentrated in tannins, high molar molecules) in the Bordeaux area that is locally prepared (100% made in France) from among all grape seed extracts on the market.
This is a guaranteed GMO-free extract prepared using an innovative process that produces a grape seed extract titrated to at least 96% in total polyphenols, including 75% minimum of proanthocyanidin (OPC), without pesticides or heavy metals . Most other brands do not generally distinguish between total polyphenols and OPC content even though this information is the most reliable measure for determining the active dose.
This product is also 100% guaranteed without exceptions. Each capsule contains only the dry grape seed extract (pure).
For each capsule:
dry extract of grape seed: 100 mg (concentrated 200/1)
>96% total polyphenols (>96 mg per capsule)
>75% proanthocyanidin (>75 mg per capsule)
>5% procyanidin B2 (>5mg per capsule)
antioxidant activity (ORAC): 19,000 (µM TEQ/L)
Grape seed extract with proven clinically health benefits
All grape seed extracts are not equal. AlivebyNature® grape seed extract Vitaflavan® contains a very high proportion (at least 75%) of low-mass procyanidins that are highly bioavailable (and therefore effective in the human body). The innovative extraction process extracts only the polyphenols and removes the tannins (large molecular weight molecules that are not bioavailable). It takes about 200 kg of grape seeds to make only 1 kg of this extract.
Unique phenolic content:
– monomers or flavan-3-ols (>21%)
– dimers (>17%)
– trimers (>16%)
– tetramers (>13%)
– oligomers (5–13 units; >31 %) of procyanidins
It is also the only one that provides more than 5% procyanidin B2
The purity of Vitaflavan® is used as a standard in the monograph of the US Pharmacopoeia.
The product we offer is the subject of clinical studies to ensure its effectiveness for health. Because of its rich antioxidant content, grape seed extract is also traditionally used as a first choice for fighting aging.
The only grape seed extract proven to increase NAD+
The Vitaflavan® specific phenolic content make it able to increase NAD+ in vivo by 3 fold as well as sirt1 activity. NAD+ is one of the most important biomarkers for aging.
In this study, we also see that this grape seed extract increased hepatic content of the NAD+ metabolite and precursor: niacin, niacinamide, NMN and tryptophan (the de novo precursor of NAD+)
Dietary proanthocyanidins boost hepatic NAD+ metabolism and SIRT1 expression and activity in a dose-dependent manner in healthy rats; Sci Rep. 2016; 6: 24977. Published online 2016 Apr 22. doi: 10.1038/srep24977
Dr. David Sinclair, Co-Director of the Biological Mechanisms of Aging at Harvard Medical School was named to the Times Magazine list of “Most Influential People in the World” after his research found a key cause of aging and a potential weapon to reverse it.
He and his team found that as we age, our cells become less and less efficient due to the lack of an essential metabolite called Nicotinamide Adenine Dinucleotide (NAD+).
WHAT IS NAD+
NAD+ is a key co-enzyme that the mitochondria in every cell of our bodies depend on to fuel all basic functions. (3,4)
NAD+ play a key role in communicating between our cells nucleus and the Mitochondria that power all activity in our cells (5,6,7,8)
Low NAD+ levels impair mitochondria function and are implicated in health problems such as cancer, diabetes, heart disease, immune problems, and perhaps even aging itself (5,6,7,9,10,11,13,14,15,16).
NAD+ LEVELS DECREASE WITH AGE
As we age, our bodies produce less NAD+ and the communication between the Mitochondria and cell nucleus is impaired. (5,8,10).
Over time, decreasing NAD+ impairs the cell’s ability to make energy, which leads to aging and disease (8, 5) and perhaps even the key factor in why we age (5).
“NAD+ levels drop by as much as 50% as we get older”
CAN INCREASED NAD+ REVERSE AGING?
There is reason to think that increased NAD+ can at least slow the aging process.
When taken orally, NAD+ does not survive the digestive system long enough to enter your cells (14)
The ground-breaking paper published by Dr Sinclair found that supplementation with Nicotinamide MonoNucleotide (NMN), the immediate precursor to NAD+, could boosts NAD+ levels in mice and resulted in the “equivalent of a human 60 year old becoming more like a 20 year old”(8).
More importantly, their research revealed that mitochondrial dysfunction is reversible with supplementation to boost NAD+
Ketosis is a metabolic state in which fat provides most of the fuel for the body. It occurs when there is limited access to glucose (blood sugar), which is the preferred fuel source for many cells in the body.
Ketosis can occur in many different diet plans whenever carb intake is low, but is most often associated the Ketogenic Diet, as that is a much easier method for restricting carbs (3, 4, 5, 6).
The Red bars in the chart at left show the increased levels of the Ketone Body BHB produced from a cyclical Keto diet, resulting in Increased NAD+ and greatly improved neurological function and health.
Researchers are finding that 2-3 short bouts of High Intensity Interval Training (HIIT) per week is far more effective at lowering inflammation (and increasing NAD+), especially among older adults (55)
Exercise is very effective at boosting AMPK and NAD+, especially when performed at times of low blood glucose levels ( more about HIIT ).
Short bouts of HIIT accomplishes the goal, while avoiding overtraining from endurance workouts which increases inflammation and consumes NAD+ (55).
Decline of NAD+ during Aging, Age-Related Diseases, and Cancer
Several evidences suggest a decline in NAD+ levels while we age, connecting NAD+ deficits to age-related diseases and cancer.
Inflammation increases during the aging process possibly due to the presence of senescent cells .
CD38 and bone marrow stromal cell antigen-1 (BST- 1) may provide explanations to NAD+ decline during aging.
CD38 is a membrane-bound hydrolase implicated in immune responses and metabolism. NAD+ can be degraded through its hydrolysis, deacetylation, or by NAD+ nucleosidases (also called NAD+ hydrolases or NADases) such as CD38.
Expression and activity of CD38 increase in older mice, promoting NMN degradation in vivo, responsible for NAD+ decline and mitochondrial dysfunctions .
Interestingly, loss of CD38 inhibits glioma progression and extends the survival of glioma- bearing mice.
Targeting CD38 in the tumor microenvironment may clearly serve as a novel therapeutic approach to treat glioma .
Daratumumab, a CD38 monoclonal antibody, rep- resents a first-in-class drug for the treatment of multiple myeloma. It promotes T cell expansion through inhibition of CD38+ immunosuppressive cells, improving patients’ responses .
These findings suggest that NAD+ boosters should be combined with CD38 inhibitors for a more efficient antiaging therapy.
NAD+ Biosynthesis Decreases during Aging, Age-Related Diseases, and Cancer
NAD+ increases can also occur independently of the Preiss–Handler route. NAM and NR are important NAD+ precursors first converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (NAMPT) and NR kinase (NRK), respectively. NMN is then transformed into NAD+ by NMN adenylyltransferase .
As we age, our bodies undergo changes in metabolism, and a key part of these processes may affect de novo NAD+ synthesis, also called the L-tryptophan/kynurenine pathway (see Figure IB in Box 1). In mammals, the use of the de novo NAD+ biosynthetic pathway is limited to a few specific organs.
Finally, dysregulation of the kynurenine pathway is also linked to genetic disorders and age-related diseases such as obesity and cancer [14,15]. These age-associated changes in de novo NAD+ biosynthesis may have the potential to impact several biological processes, and thus contribute to age-related diseases and cancer in the elderly.
Animal models mimicking downregulation of NAD+ biosyn-thesis are needed to modulate its activity and understand its pathophysiological relevance in age-related pathologies and cancer.
Boosting NAD+ with Niacin in Age-Related Diseases and Cancer
In humans, a lack of nicotinic acid (NA, also called niacin) in the diet causes the vitamin B3 deficiency disease pellagra, characterized by changes in the skin with very characteristic pigmented sunburn-like rashes developing in areas that are exposed to sunlight. Likewise, people with chronic L-tryptophan-poor diets or malnutrition develop pellagra.
Furthermore, several epidemiologic studies in human reported an association between incidence of certain types of cancers and niacin deficiency .
In this regard, low dietary niacin has also been associated with an increased frequency of oral, gastric, and colon cancers, as well as esophageal dysplasia.
In some populations, it was shown that daily supplementation of niacin decreased esophageal cancer incidence and mortality. Although the molecular mechanisms of niacin deprivation and cancer incidence are not well understood, it has been recently reported that NAD+ depletion leads to DNA damage and increased tumorigenesis, and boosting NAD+ levels is shown to play a role in the prevention of liver and pancreatic cancers in mice [19,28,29].
Thus, malnutrition through inadequate amounts and/or diversity of food may affect the intra- cellular pools of nicotinamide and NAD+ thereby influencing cellular responses to genotoxic damage, which can lead to mutagenesis and cancer formation [19,27]. NAD+ boosters are therefore essential in patients at risk of exposure to genotoxic and mutagenic agents, including ionizing or UV radiations or, DNA damaging chemicals.
In addition, niacin deficiency in combination with carcinogenic agents was described to induce and increase tumorigenesis in rats and mice.
For instance, in rats, the lack of niacin together with carcinogen treatment increased tumorigenesis and death of rats [30,31]. Additionally, in mice, the incidence of skin tumours induced by UV was significantly reduced by local application of NAM or by niacin supplementation in the diet .
Boosting NAD+ with NAM in Age-Related Diseases and Cancer
Recent research has focused on uncovering the consequences of a decrease in NAD+ during aging using age-related disease models. In PGC1a knockout mouse, a model of kidney failure, NAD+ levels are reportedly decreased, and boosting NAD+ by NAM improves kidney function .
NAM injections during four days re-establish local NAD+ levels via nicotinamide phos- phoribosyltransferase (NAmPRTase or NAMPT) activation and improve renal function in postischaemic PGC1a knockout mice .
Surgical resection of small renal tumors can induce kidney ischemia severely affecting the renal function. Therefore, NAD+ boosters can be beneficial to protect the organ from severe injury.
Moreover, in a model of muscular dystrophy in zebrafish, NAD+ increases, which functions as an agonist of muscle fiber–extracellular matrix adhesion, and corrects dystrophic phenotype recovering muscle architecture .
Boosting NAD+ with NR in Age-Related Diseases and Cancer
Further research has extensively used NR to ameliorate the effects of NAD+ deficits in pleiotropic disorders. NR naturally occurs in milk [35,36]. NR is converted to NAD+ in two step reactions by nicotinamide riboside kinases (NRKs)-dependent phosphorylation and adenylylation by nicotinamide mononucleotide adenylyl transferases (NMNATs) .
It is considered to be a relevant NAD+ precursor in vivo. Evidences demonstrate the beneficial effect of NR in skeletal muscle aging [37,38] and mitochondrial-associated disorders, such as myopathies [39,40] or those characterized by impaired cytochrome c oxidase biogenesis affecting the respiratory chain .
In line of these findings, a mouse model of Duchenne muscular dystrophy present significant reductions in muscle NAD+ levels accompanied with increased poly-ADP-ribose polymerases (PARP) activity, and reduced expression of NAMPT .
Replenishing NAD+ stores with dietary NR supplementation improved muscle function in these mice through better mitochondrial function .
Additionally, enhanced NAD+ concen- trations by NR are apparently beneficial for some neurodegenerative diseases , as well as in noise-induced hearing loss .
NR-mediated NAD+ repletion is also protective, and even therapeutic, in certain metabolic disorders associated with cancer, such as fatty liver disease [28,45] and type 2 diabetes [28,46]. Metabolic disorders characterized by defective mitochon- drial function could also benefit from an increase in NAD+ levels.
Indeed, stimulation of the oxidative metabolism in liver, muscle, and brown adipose tissue potentially protects against obesity . Interestingly, NAMPT protein levels are not affected in chow- and high fat diet (HFD)-treated mice fed with NR, arguing that in models of obesity, NR directly increases NAD+ levels without affecting other salvage reactions .
Recently, diabetic mice with insulin resistance and sensory neuropathy treated with NR reportedly show a better glucose toler- ance, reduced weight gain and liver damage, and protection against hepatic steatosis and sensory and diabetic neuropathy .
Boosting NAD+ with NMN in Age-Related Diseases and Cancer
NMN is also a key biosynthetic intermediate enhancing NAD+ synthesis and ameliorates various pathologies in mouse disease models [49,50].
Very recent research demonstrate that a 12- month-long NMN administration to regular chow-fed wild-type C57BL/6 mice during normal aging rapidly increases NAD+ levels in numerous tissues and blunts age-associated physio- logical decline in treated mice without any toxic effects . NMN is also beneficial in treating age- and diet-induced diabetes, and vascular dysfunction associated with aging in mice [51,52].
Administration of NMN also protects the heart of mice from ischemia-reperfusion injury  and restores mitochondrial function in muscles of aged mice [37,54].
It has been speculated that NMN is a circulating NAD+ precursor, due to the extracellular activity of NAMPT . However, the mechanisms by which extracellular NMN is converted to cellular NAD+ still remain elusive.
On the one hand, it is reported that NMN is directly trans- ported into hepatocytes . On the other hand, NMN can be dephosphorylated to NR to support elevated NAD+ synthesis [56–59].
It is recently shown that NAM can be metabolized extracellularly into NMN by extracellular NAMPT. NMN is then converted into NR by CD73 . Hence, NR is taken up by the cells and intracellularly phosphorylated firstly into NMN by NRKs and then, converted into NAD+ by NMNATs  (Figure 3).
Thus, mammalian cells require conversion of extracellular NMN to NR for cellular uptake and NAD+ synthesis. Consistent with these findings, in murine skeletal muscle specifically depleted for NAMPT, administration of NR rapidly restored muscle mass by entering the muscles and replenishing the pools of NAD+ through its conversion to NMN .
Interestingly, mice injected with NMN had increased NAM in their plasma that may come after initial conversion of NMN into NR . However, degradation of NR into NAM could only be observed when cells were cultured in media supplementing with 10% FBS .
Finally, it is important to note that NR is stably associated with protein fractions in milk with a lifetime of weeks .
Notably, as reported above, NMN may be degraded by CD38 in older mice promoting NAD+ decline and mitochondrial dysfunctions , suggesting that NR may be more efficient than NMN in elderly.
Yet, the beneficial synergistic activation of sirtuins and metabolic pathways to replenish NAD+ pools cannot be excluded. However, given its efficient assimilation and high tolerance, NR represents still the most convenient and efficient NAD+ booster.
Overall, these findings suggest that NAD+ decrease in disease models and NAD+ precursors (NAM, NR or NMN) may circumvent NAD+ decline to generate adequate levels of NAD+ during aging and thus be used as preventive and therapeutic antiaging supplements.
NMN and NR supplementations may be equivalent strategies to enhance NAD+ biosynthesis with their own limitations.
Side-Effects of Some NAD+ Boosters
Clearly, several intermediates of the salvage pathway can be considered to boost NAD+ levels but some have contraindications. High doses of NA given to rats are needed to robustly increase NAD+ levels .
Additionally, relevant and unpleasant side effects through NA-induced prostaglandin- mediated cutaneous vasodilation (flushing) affecting patient compliance are due to the activation of the G-protein-coupled receptor GPR109A (HM74A) and represent a limitation in the pharma- cological use of NA .
NAM is much less efficient than NA as a lipid lowering agent and has also several side effects; in particular, it causes hepatic toxicity through NAM-mediated inhibition of sirtuins .
The metabolism of these conventional compounds to NAD+ is also different, as NA is converted via the three-step Preiss–Handler pathway, whereas NAM is metabolized into NMN via NAMPT and then to NAD+ by NMNATs 
Manipulating NAD+ by Manipulating Enzyme Activity of Salvage Reactions
Enhancing the activity of enzymes that participate in salvage reactions can also be a strategic intervention to increase NAD+ concentrations. Different studies have addressed the importance of regulating the activity of NAMPT during disease, including metabolic disorders and cancer.
NAMPT is necessary in boosting NAD+ pools via the salvage pathway.
Consequently, NAMPT deletion provokes obesity-related insulin resistance, a phenotype rescued by boosting NAD+ levels in the white adipose tissue by giving NMN in drinking water .
Conversely, in a mouse model for atherosclerosis, NAMPT depletion promotes macro- phage reversal cholesterol transport, a key process for peripheral cholesterol efflux during atherosclerosis reversion .
Other recent reports suggest that NAMPT downregulation could be beneficial in treating pancreatic ductal adenocarcinoma [69,70] and colorectal cancer .
Recent findings show that Duchenne muscular dystrophy was accompanied by reduced levels of NAMPT in mice . Moreover, NAMPT knockout mice exhibit a dramatic decline in intramuscular NAD+ content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance.
NR treatment induced a modest increase in intra- muscular NAD+ pools but sufficient to rapidly restore muscle mass. Importantly, overexpres- sion of NAMPT preserves muscle NAD+ levels and exercise capacity in aged mice .
Inhibitors against NAMPT are being used in several phase II clinical trials as anticancer therapy.
Given that NAMPT activation is important to boost NAD+ levels, therapy involving NAMPT inhibition should be considered with caution. Although levels of NAD+ remain to be determined in models with NAMPT depletion, further investigation on the effects of NAMPT modulation is clearly required.
The specific mechanisms and actual benefits of regulation of NAMPT activity remain elusive, evidencing the need of more specific disease models.
Can Dietary Restriction and Protein Catabolism Maintain NAD+ Levels?
Among the questions that still remain not well understood is why DR profoundly increases lifespan? Can DR affect NAD+ levels?
It is well established that overfeeding and obesity are important risk factors for cancer in humans  and obesity-induced liver and colorectal cancer, among others, can shorten lifespan.
Earlier research has also shown that both increased physical activity and reduction in caloric intake (without suffering malnourishment) can extend lifespan in yeasts, flies, worms, fish, rodents, and primates [3–8].
Furthermore, a recent study pointed to the importance of the ratio of macronutrients more than the caloric intake as the determinant factor in nutrition-mediated health status and lifespan extension .
Although in humans it is difficult to measure the beneficial effects of DR and currently there is no reliable data that describe the consequences of significantly limiting food intake, some studies have assessed how DR affects health status.
People practicing DR seem to be healthier, at least based on risk parameters such as LDL cholesterol, triglycerides, and blood pressure .
Activation of the salvage pathways during DR could be turned on and glucose restriction can stimulate SIRT1 through activation of the AMPK-NAMPT pathway resulting in inhibition of skeletal myoblast differentiation .
Interestingly, effects of NMN supplementation and exercise on glucose tolerance in HFD-treated mice are very similar .
Even though these effects are tissue-specific since exercise predominantly affects muscle, whereas NMN shows major effects in liver, and that mechanism of action can be different, exercise and NMN predominantly affect mitochondrial functions and may both contribute to the boost of NAD+.
It is thus tempting to speculate that L-tryptophan concentrations and thus the de novo NAD+ biosynthesis could fluctuate during DR ameliorating the aging process.
Recent studies in humans and mice suggest that moderate exercise can increase blood NAD+ levels and decrease L-tryptophan levels .
A possible explanation for this phenomenon is that DR, and/or exercise, can induce autophagy and promote the release of several metabolites and essential amino acids .
Aging is proposed to be responsible for diverse pathologies, however, it should be considered as a disease among other diseases that appear in time while individuals age.
Although some questions still remain unclear, NAD+ precursors may present possible therapeutic solutions for the maintenance of NAD+ levels during aging and thus may provide prophylaxis to live longer and better.
Although more research is needed to understand the efficacy as well as potential adverse side effects of NAD+ Replacement Therapies in humans, recent studies already provided some pharmacological properties, showing low toxicity and high effectiveness.
RESTORNG NAD+ – RESEARCH
Experiments with mice have mostly focused on treating specific disease conditions, but in the process, some have noted increase in lifespan even though the NAD boosting therapies were commenced quite late in life (22).
Sirtuins are well-known longevity regulators, and their decreased function with age might at least be partially explained by a systemic decline in NAD+ levels upon aging (40). Rising NAD+ content, followed by sirtuin activation, has been reported to increase lifespan in yeast, worms, and mice (22).
Administration of NR, NMN, or NAM recovered NAD+ content and protected against aging-related complications, such as mitochondrial dysfunction (24, 41, 23), decline in physical performance (23,29) and muscle regeneration (22), arterial dysfunction (42), decline in vision (43, 23), including glaucoma (44), and age-associated insulin resistance (23).
The most striking benefits of NAD+ supplementation on aging were observed in several rare diseases linked to abnormal DNA repair that are typified by accelerated aging, such as the Cockayne syndrome group B (CSB), xeroderma pigmentosum group A (XPA), or ataxia-telangiectasia (A-T).
In a mouse model of CSB, neurons show mitochondrial defects, which have an impact on the cerebellum and inner ear. Administration of PARP inhibitors or the NAD+ precursor, NR, to csb / animals attenuated many of the phenotypes of CSB and restored altered mitochondrial function in their neurons.
Another DNA damage repair disorder is XPA, which is also characterized by mitochondrial alterations and reduced NAD+-SIRT1 signaling due to the overactivation of PARP1 (45).
Treatment with NAD+ precursors, NR and NMN rescued the XPA phenotype in cells and worms. Restoring the NAD+/SIRT1 pathway, by NR and NMN administration to C. elegans and mice, improved A-T neuropathology (45).
The importance of NAD+ as a metabolic regulator has been demonstrated by its efficacy to attenuate many features of the metabolic syndrome, a cluster of pathologies including insulin resistance, fatty liver, dyslipidemia, and hypertension, with increased risk of developing type 2 diabetes and heart failure.
Different approaches aiming to raise NAD+ levels were shown to provide protection against obesity, such as (i) inhibition of NAD+ consumers, PARPs (29) and CD38 (Barbosa et al, 2007), (ii) administration of NAD+ precursors, such as NR (49, 45T,51) or NMN (Yoshino et al, 2011), (iii).
NAD+ boosting was also efficient to improve glucose homeostasis in obese, prediabetic, and T2DM animals (29,46,51). Likewise, reestablishing NAD+ levels with NR or PARP inhibitors also protected from non-alcoholic steatohepatitis (NASH) (46) as well as alcoholic steatohepatitis (46).
Increase in muscle NAD+ content, resulting from NR administration or PARP inhibition, improved muscle function and exercise capacity in mice (49), including in aged animals (22).
Interestingly, muscular dystrophy is characterized by a dramatic drop in NAD+ in the muscle (Ryu et al, 2016). NR administration to the mdx mouse, a model for muscular dystrophy, improved muscle function by enhancing bioenergetics, attenuating inflammation and fibrosis (Ryu et al, 2016), as well as, by favoring regeneration and preventing the exhaustion and senescence of muscle stem cells, typical to the mdx mice (22).
The beneficial effects of improving muscle bioenergetics are also illustrated in models of mitochondrial myopathies. Increasing muscle NAD+ levels by the administration of NR or a PARP inhibitor preserved muscle function in two different models of mitochondrial myopathy (Cerutti et al, 2014; Khan et al, 2014).
Similar benefits on mitochondrial myopathy were seen with the AMPK agonist, AICAR (Viscomi et al, 2011), which may at least in part be due to the recovery of NAD+ content upon AMPK activation.,
Exposing the heart to different types of stresses was reported to result in a decline in cardiac NAD+ content (Pillai et al, 2005, 2010; Karamanlidis et al, 2013; Yamamoto et al, 2014). For instance, cardiomyocyte hypertrophy is characterized by a drop in cellular NAD+ levels. Supplementation with NAD+ was hence protective against cardiac hypertrophy in mice, and these anti-hypertrophic effects were in part attributed to the activation of SIRT3 (Pillai et al, 2010).
Cardiac ischemia is another condition causing a steep decrease in NAD+ levels. NMN administration protected the mice from ischemic injury via the recovery of cardiac NAD+ content and subsequent SIRT1 activation (Yamamoto et al, 2014).
Similarly, cardiac-specific overexpression of NAMPRT in mice increased NAD+ content and reduced the extent of myocardial infarction and apoptosis in response to prolonged ischemia and ischemia/reperfusion (Hsu et al, 2009).
Maintaining NAD+ levels in pressure-overloaded hearts is crucial for myocardial adaptation and protection from heart failure, as demonstrated by NMN administration to mice treated with the NAMPRT inhibitor FK866 (Yano et al, 2015) and to cardiac-specific mitochondrial complex I-deficient mice (Lee et al, 2016).
In a mouse model of heart failure caused by iron deficit, reconstituting NAD+ content also improved mitochondrial quality, protected cardiac function, and increased lifespan (Xu et al, 2015). Similarly, NR administration improved cardiac function in aged mdx mice, which, like muscular dystrophy patients, display cardiomyopathy (Ryu et al, 2016).
Multiple studies demonstrated the loss of SIRT1 and SIRT3 activity as a key feature of kidney dysfunction, including kidney abnormalities linked with aging (Koyama et al, 2011,53, Morigi et al, 2015; Ugur et al, 2015; Guan et al, 2017).
Acute kidney injury (AKI) is characterized by a reduction in NAD+ content and NAMPRT expression (Morigi et al, 2015; Ugur et al, 2015). Promoting NAD+ synthesis via NAM or NMN supplementation was reported to mitigate AKI in ischemia/reperfusionand cisplatininduced mouse models of AKI (Tran et al, 2016; Guan et al, 2017).
Furthermore, administration of the AMPK agonist, AICAR, which positively impacts on NAD+ levels (48), was protective against cisplatin-induced AKI in SIRT3-dependent manner (Morigi et al, 2015). Although no NAD+ quantification was performed in this particular study, the involvement of SIRT3, as well as the increase in Namprt expression detected upon AICAR administration, points toward a potential increase in NAD+ levels (Morigi et al, 2015).
Kidney mesangial cell hypertrophy is also characterized by a depletion of NAD+ content (53) and restoring intracellular NAD+ levels via supplementation with exogenous NAD+ prevented its onset by activating SIRT1 and SIRT3 (53z).
NAD+ boosting has also been shown to be neuroprotective. Raising NAD+ levels protects against neuronal death induced by ischemic brain (Klaidman et al, 2003; Sadanaga-Akiyoshi et al, 2003; Kabra et al, 2004; Feng et al, 2006; Kaundal et al, 2006; Zheng et al, 2012) or spinal cord injuries (Xie et al, 2017).
Axonal degeneration is considered as an early pathological mechanism in this type of neurodegeneration. An accumulating amount of data indicates that axonal degeneration is not only limited to ischemic brain and spinal cord injuries, but constitutes a hallmark process, preceding neuronal death, in a much larger spectrum of disease states, including traumatic brain injury, inflammatory disorders, like multiple sclerosis, and degenerative disorders, such as Alzheimer’s and Parkinson’s diseases (Lingor et al, 2012; Johnson et al, 2013).
Degenerating axons show a decrease in NAD+ content (Wang et al, 2005; Gerdts et al, 2015), while replenishing NAD+ by supplementing NAM (Wang et al, 2005), NR and NMN (Sasaki et al, 2006), and high doses of NAD+ (Araki et al, 2004), or overexpressing enzymes involved in NAD+ biosynthesis (Araki et al, 2004; Sasaki et al, 2006) delayed axonal degeneration.
In line with this, supplementation with NAM, NMN, or NR was neuroprotective in rodent models of Alzheimer disease (Qin et al, 2006; Gong et al, 2013; Liu et al, 2013; Turunc Bayrakdar et al, 2014; Wang et al, 2016a), and supplementation with NAM or LOF of PARP were protective in Drosophila models of Parkinson’s disease (Lehmann et al, 2017).
NAD+ depletion is also involved in the neurodegeneration induced by highly toxic misfolded prion protein (Zhou et al, 2015). Replenishment of intracellular NAD+ stocks, either by providing NAD+ or NAM, rescued the neurotoxic effects of protein aggregates (Zhou et al, 2015). Importantly, restoring NAD+ content is not exclusively protecting neurons, since it has also been reported to prevent the death of astrocytes (Alano et al, 2004).
P7C3, a compound that enhances neurogenesis (Pieper et al, 2010) and that was neuroprotective in mouse models of Parkinson’s disease (De Jesus-Cortes et al, 2012), amyotrophic lateral sclerosis (Tesla et al, 2012) and brain injury (Yin et al, 2014), was subsequently identified as an NAMPRT activator (Wang et al, 2014a). Therefore, the beneficial effects of P7C3 on neuron preservation seem at least in part to be due to a NAMPRT-mediated increase in NAD+ levels (Wang et al, 2014a).
Nicotinamide riboside supplementation recovered depressed sensory and motor neuron conduction velocities and thermal insensitivity in T2DM mice (51) and alleviated chemotherapy-induced peripheral neuropathy in rats (Hamity et al, 2017), indicating that NAD+ also is beneficial in the peripheral neuronal system.
NAD+ boosting was also able to protect mice from loss of vision and hearing (Shindler et al, 2007; Brown et al, 2014). Intravitreal injections of NR in mice attenuated optic neuritis in a dose-dependent manner (Shindler et al, 2007).
Even if no NAD+ quantification was performed in this study, SIRT1 activity was necessary for the neuroprotective effects of NR, since the protection was blunted in the presence of sirtinol, a SIRT1 inhibitor (Shindler et al, 2007). Furthermore, systemic administration of NAM and overexpression of Nmnat1 had spectacular effects on vision in DBA/2J mice, which are prone to glaucoma (44).
Noise exposure results in degeneration of the neurons innervating the cochlear hair cells. Increase in NAD+ levels induced by NR administration prevented against noise-induced hearing loss and neurite degeneration (Brown et al, 2014).
In line with this, CR was shown to protect against cochlear cell death and aging-associated hearing loss in a Sirt3-dependent manner (Someya et al, 2010). It is therefore tempting to speculate that this improvement could also be associated with increased NAD+ levels uponCR, though no direct measurements of NAD+ levels were performed in this study.
Manipulations of NAD+ concentrations have demonstrated multiple beneficial effects in a large spectrum of diseases in animal models . Translating these effects into clinical benefits now becomes one of the main challenges.
The fact that the long-term administration of the NAD+ precursor molecules showed no deleterious effects in animals should be considered promising.
As such, administration of NMN for 12 months demon- strated no toxicity in mice (23).
Similarly, administrtion of NR to mice for a duration of 5–6 months (Gong et al, 2013), 10 months (22), and 12 months (Tummala et al, 2014) showed no obvious adverse effects.
Another NAD+ precursor, NAM, has also been already tested in humans and protected b-cell function in type 1 diabetes patients.
Furthermore, a slow release form of NA (acipimox) was effective in inducing mitochondrial activity in skeletal muscle of type 2 diabetic patients (van de Weijer et al, 2015).
Inflammation influence on aging process itself, not just age related disease
We know NAD+ levels go down dramatically as we age. Many recent and ongoing research studies are investigating protocols for boosting NAD+ levels in elderly individuals to combat both disease and infirmities related to aging. Supplementing with Nicotinamide Riboside and/or NMN are 2 that are attracting a great deal of attention, especially after Dr David Sinclair demonstrated that raising NAD+ levels in elderly mice could “turn back the clock” leaving them with muscular function of young mice.
NAD+ is key for mitochondria to perform all functions within the body
NAD+ levels go down as we age
Lower NAD+ levels impair all functions in the body
Inflammation is a necessary part of our bodies defensive response to injury and disease, as it strives to eliminate the cause of irritation and is an essential initial stage of healing injured tissues. We could not survive without it. Unfortunately, if the cause of the irritation or illness is not eliminated, inflammation can often get out of control. Such Chronic Inflammation is now recognized to be both a cause and effect of nearly ALL age related conditions such as cancer, arthritis, metabolic syndrome, heart disease, osteoporosis, Alzheimers, IBS, asthma, COPD, depression, fatigue and more (4, 5, 6).
Anything that can help fight chronic inflammation is of potential importance in preventing and even treating these diseases. For example, Curcumin a powerful anti-inflammatory that matches the effectiveness of many prescription drugs in treating some chronic conditions, but without the side effects (7, 10, 11, 12, 13, 14). You can read more about some of the amazing benefits of Curcumin here.
Chronic inflammation, sometimes referred to as constitutive inflammation, other times as inflammaging, can persist over an extended period of weeks to months and even years. It is often associated with the presence of macrophages and lymphocytes, fibrosis, vascular proliferation, and tissue destruction. Moreover, chronic inflammation plays critical roles in many disease processes including cancers, dementias, diabetes, pulmonary diseases, cardiovascular diseases, atherosclorsis, sarcopenia, and anaemia. Chronic inflammation occurs in the case of incurable autoimmune diseases such as arthritis, lupus, scleroderma, asthma and chronic obstructive pulmonary disease (COPD).
The biological mechanisms of chronic inflammation can be very complex, Nuclear factor- B (NF-κB) is activated by more than 200 different stimuli has for good reason been thought of as the master activator of inflammation. It is a central topic in this blog entry. For example, during inflammation immune system macrophage cells could be activated by Toll-like receptors (TLRs), through the recognition of a pathogen endotoxin such as lipopolysaccharide (LPS). This event initiates a signaling pathway that releases NF-κB into the cell nucleus, activating genes associated with the transcription of proteins related to the inflammatory process, such as iNOS, responsible for NO synthesis, COXs, which synthetize prostaglandins, and cytokines like IL-6. The generation of ROS is also triggered by the TLR signaling pathway.
Among the highly technical topics related to chronic inflammation and its consequences are Activating protein-1 (AP-1), AGEs, RAGE receptor, PAMPs, DAMPs, RNS, leukotrienes, LOX, prostaglandins, COX1, COX2, Resolvins, Protectins, Maresins, the NLRP3 inflammasome, lipoxins, Ca++ induced inflammation, pyroptosis, cellular senescence-induced inflammation, roles of inflammation in aging, potassium efflux out of a cell, mitochondrial ROS, translocation of NLRP3 to the mitochondria, cytosolic release of mitochondrial DNA, cardiolipin release, release of lysosomal cathepsin D into the cytosol, extracellular LPS “priming” of NLRP3, amyloid-beta “triggering” of NLRP3 via TLR4, ATP, and pore-forming toxins. We expect to touch on most of these in this blog series on inflammation.
Inflammation and aging
Since chronic inflammation plays central roles in numerous deleterious health processes and in aging, it is often referred to as “inflammaging” and is the subject of much ongoing research. From the 2014 publication Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases: “Human aging is characterized by a chronic, low-grade inflammation, and this phenomenon has been termed as “inflammaging.” Inflammaging is a highly significant risk factor for both morbidity and mortality in the elderly people, as most if not all age-related diseases share an inflammatory pathogenesis. Nevertheless, the precise etiology of inflammaging and its potential causal role in contributing to adverse health outcomes remain largely unknown. The identification of pathways that control age-related inflammation across multiple systems is therefore important in order to understand whether treatments that modulate inflammaging may be beneficial in old people.”
The 2016 publication Inflammaging and Anti-Inflammaging: The Role of Cytokines in Extreme Longevity points out the central linkages between inflammation and aging. “Longevity and aging are two sides of the same coin, as they both derive from the interaction between genetic and environmental factors. Aging is a complex, dynamic biological process characterized by continuous remodeling. One of the most recent theories on aging focuses on immune response, and takes into consideration the activation of subclinical, chronic low-grade inflammation which occurs with aging, named “inflammaging.” Long-lived people, especially centenarians, seem to cope with chronic subclinical inflammation through an anti-inflammatory response, called therefore “anti-inflammaging.” In the present review, we have focused our attention on the contrast between inflammaging and anti-inflammaging systems, by evaluating the role of cytokines and their impact on extreme longevity. Cytokines are the expression of a network involving genes, polymorphisms and environment, and are involved both in inflammation and anti-inflammation. We have described the role of IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IL-22, IL-23, TNF-α, IFN-γ as pro-inflammatory cytokines, of IL-1Ra, IL-4, IL-10, TGF-β1 as anti-inflammatory cytokines, and of lipoxin A4 and heat shock proteins as mediators of cytokines. We believe that if inflammaging is a key to understand aging, anti-inflammaging may be one of the secrets of longevity.” This is an opinion I (Vince) hold. In a later blog entry in this series, I will share some of the approaches to anti-inflammaging I have been personally and professionally pursing.
Dr. Perricone’s years of research have shown that the inflammation-aging connection is the single greatest cause of aging and age-related diseases such as heart disease, diabetes, Alzheimer’s disease, arthritis, certain forms of cancer, diminished mental and physical energy, the loss of muscle mass and wrinkled, sagging skin.
This chronic inflammation goes on day after day, year in and year out, leading to disease states as well as the disease of aging. In fact, aging is a chronic, uniformly progressive, inflammatory disease that is always fatal.
Our food choices are critical when it comes to causing and controlling inflammation. This is good to know because it actually means we are in control of the situation!
This is the key to health, longevity, mental clarity, well-being and beautiful youthful skin. Foods that are pro-inflammatory, such as all forms of sugar, processed foods, pasta, breads, pastry, baked goods, and snack foods such as rice and corn cakes, chips, pretzels, etc., cause a highly destructive pro-inflammatory response in our bodies. If we choose sugary or starchy foods, we trigger this pro-inflammatory release of sugar into our bloodstream, which causes our body to store fat rather than burn it for energy.
The result? Acceleration of the aging process of all organ systems in our body, including the skin, causing an increased risk of degenerative disease and inflexible, wrinkled, sagging skin. In addition, by eating that muffin or couple of cookies, the resulting insulin response triggers our appetite—causing us to crave more and more of these types of carbohydrates, resulting in a vicious cycle of overeating.
That is the bad news! Now for the good news:
Fortunately we can control inflammation in our bodies. It starts with the very foods we eat. All we have to do is avoid foods that provoke a “glycemic” response in the body, i.e. cause a rapid rise in blood sugar.
Inflammation has been found to be associated with just about every health condition. Researchers are furiously investigating chronic inflammation’s effects on health and possible preventive medical applications.
It’s “an emerging field,” says UCLA’s Dr. David Heber. “It’s a new concept for medicine.” (1)
Why is it a new concept? Because modern medicine focuses on treating symptoms, not addressing the root cause of an issue.Arthritis is inflammation of the joints. Heart disease is inflammation of the arteries. Instead of taking a medication to reduce joint pain or lower cholesterol, we would be better served by reducing inflammation in the body.
Dr. Tanya Edwards, director of the Center for Integrative Medicine, writes that inflammation is now recognized as the “underlying basis of a significant number of diseases.”
Just to make sure that we’re all on the same page, I want to briefly explain what inflammation is.
I’m not going to get into much detail, because inflammation is extremelycomplicated.
It involves dozens of cell types and hundreds of different signalling molecules, all of which communicate in immensely complex ways.
Put simply, inflammation is the response of the immune system to foreign invaders, toxins or cell injury.
The purpose of inflammation is to affect the function of immune cells, blood vessels and signalling molecules, to initiate an attack against foreign invaders or toxins, and begin repair of damaged structures.
We’re all familiar with acute (short-term) inflammation.
For example, if you get bitten by a bug, or hit your big toe on the doorstep, then you will become inflamed.
The area will become red, hot and painful. This is inflammation at play.
Inflammation is generally considered to be a good thing. Without it, pathogens like bacteria and viruses could easily take over our bodies and kill us.
However, there is another type of inflammation that may be harmful, because it is inappropriately deployed against the body’s cells (7).
This is a type of inflammation that is active all the time, and may be present in your entire body. If is often called chronic inflammation, low-grade inflammation, or systemic inflammation (8).
For example, your blood vessels (like your coronary arteries) may be inflamed, as well as structures in your brain (9, 10).
It is now believed that chronic, systemic inflammation is one of the leading drivers of some of the world’s most serious diseases (11).
However, it is not known exactly what causes the inflammation in the first place.
Bottom Line: Inflammation is the response of the immune system to foreign invaders, toxins and cell injury. Chronic inflammation, involving the entire body, is believed to drive many killer diseases.
Why Care About Omega-6 and Omega-3 Fatty Acids?
Omega-6 and Omega-3 fatty acids are called polyunsaturated because they have many double bonds (poly = many).
Our bodies don’t have the enzymes to produce them and therefore we must get them from the diet.
If we don’t get any from the diet, then we develop a deficiency and become sick. That is why they are termed the “essential” fatty acids.
However, these fatty acids are different than most other fats. They are not simply used for energy or stored, they are biologically active and have important roles in processes like blood clotting and inflammation.
The thing is… Omega-6s and Omega-3s don’t have the same effects. Omega-6s are pro-inflammatory, while Omega-3s have an anti-inflammatory effect (1).
Of course, inflammation is essential for our survival. It helps protect our bodies from infection and injury, but it can also cause severe damage and contribute to disease when the inflammatory response is inappropriate or excessive.
In fact, excess inflammation may be one of the leading drivers of the most serious diseases we are dealing with today, including heart disease, metabolic syndrome, diabetes, arthritis, Alzheimer’s, many types of cancer, etc.
Put simply, a diet that is high in Omega-6 but low in Omega-3 increases inflammation, while a diet that includes balanced amounts of each reduces inflammation (2).
The problem today, is that people who eat a typical Western diet are eating way too many Omega-6s relative to Omega-3s.
Bottom Line: An Omega-6:Omega-3 ratio that is too high can contribute to excess inflammation in the body, potentially raising the risk of all sorts of diseases.
ROLE OF INTESTINAL FLORA
We may not realize that our intestinal flora can be a driver of inflammation. Gut-associated inflammation has been linked to insulin resistance, some forms of cancer, and even mental health concerns.
The trillions of bacteria that live within our gut have an intimate connection to our immune system, helping to strike a balance between tolerance and regulation. One type of bacteria that can cause inflammation is gram-negative bacteria.
GRAM-NEGATIVE BACTERIA INCITE INFLAMMATION
Some gram-negative bacteria exist naturally in a balance with gram-positive bacteria in our gut. But excessive or harmful gram-negative bacteria may appear due to an infection or in response to poor lifestyle choices, such as a high-fat, low-fibre diet.
Gram-negative bacteria have molecules in their cell walls called lipopolysaccharides (LPS), which are a little like a coat of gnarly armour. If the barrier function of the gut is diminished (which can result from a high-fat, high-sugar diet, stress, or other causes), these LPS can enter the bloodstream, where they incite an inflammatory response.
PROBIOTICS MAY REDUCE CHRONIC INFLAMMATION
While we see evidence that altered intestinal flora can lead to increased inflammatory markers, the ability of probiotics to reduce chronic inflammation is still being researched. In some studies, selected strains or blends of probiotic bacteria have outcompeted gram-negative bacteria. In addition, some probiotics have been shown to physically reinforce the gut barrier to prevent LPS passage. Through research to date, we see that certain probiotic strains within the Lactobacillus group are strong enough to act this way.
Some probiotic bacteria have also shown promise in reducing the production of messengers called pro-inflammatory cytokines. Probiotic bacteria produce substances known as short chain fatty acids, which can lower inflammatory markers in addition to strengthening the gut barrier.
You know what inflammation looks like: You get a cut or bruise, and the area around it soon turns red, gets warm, and swells up. This is called the acute inflammatory response, and it’s your immune system’s defensive reaction to infection or injury. A complex array of immune cells congregate at the site and release a variety of chemicals to deal with the infectious organisms or debris from the injury and to allow tissue repair to begin; normally the inflammation gradually subsides. This immune response is essential to life.
But there’s another way inflammation works—it can be chronic and cause a low-grade systemic reaction. Because it increases with aging, it has been dubbed “inflammaging.” Chronic systemic inflammation has been the focus of a great deal of scientific attention during the past two decades (especially the past few years) and is now viewed as a sort of “unified field” explanation for many, if not most, age-related chronic diseases.
Accordingly, factors (genetic, lifestyle, and environmental) that promote chronic inflammation or disrupt the body’s protective mechanisms against it may increase the risk of premature aging and the disorders that go with it. On the other hand, healthy aging and longevity may be related to reduced levels of inflammation and/or strong protective mechanisms that guard against its adverse effects.
This was suggested by the results of a study in the Canadian Medical Association Journal last year, which included 3,000 British civil servants. It found a strong link between higher levels of chronic inflammation (as measured by blood levels of an inflammatory marker) and a decreased likelihood of “successful aging,” defined as optimal physical and cognitive health and the absence of chronic diseases. In fact, elevated levels of inflammation appeared to reduce the odds of successful aging by half over the next decade and to markedly increase the odds of cardiovascular disease and death.
Many complex roles
It can be both a cause andan effect of some disorders—setting up a vicious cycle that helps explain their chronic nature.
For example, chronic inflammation plays reciprocal roles with obesity and insulin resistance. It contributes to the development of insulin resistance, which in turn may help promote obesity. Conversely, obesity worsens insulin resistance and increases chronic inflammation, partly because body fat (especially the type surrounding internal organs) releases pro-inflammatory compounds. In effect, inflammation, obesity, and insulin resistance reinforce one another, often resulting in type 2 diabetes. What’s more, many lifestyle factors that promote inflammation, such as being sedentary and having an unhealthy diet, also promote obesity and insulin resistance.
Hard to pin down
Chronic inflammation is a varied phenomenon that affects nearly every aspect of human physiology and disease development. Many different kinds of specialized cells and chemicals are involved in producing and regulating these inflammatory processes.
Since it is so complex, there is no way to measure chronic inflammation directly. Instead, researchers measure a variety of inflammatory chemical markers in the blood or tissue, notably interleukin-6, tumor necrosis factor (TNF), C-reactive protein, prostaglandins, and leukotrienes. Elevated levels of these factors are good indicators of disease activity for some conditions (such as inflammatory bowel disease). But it’s not clear whether measuring them adequately gauges inflammation and the resulting risks for some other disorders (such as cancer).
Time for CRP Testing?
C-reactive protein, or CRP, is produced by the liver in response to inflammation. Of all markers for inflammation, it has gotten the most attention because research has shown that elevated blood levels are strongly associated with an increased risk of cardiovascular disease, even in people otherwise at low risk.
This was seen in the well-known JUPITER study a few years ago, which focused on people with desirable cholesterol levels but elevated CRP. It found that they greatly reduced their risk of heart attacks and strokes when they took a statin drug. Besides lowering LDL (“bad”) cholesterol, statins have anti-inflammatory effects, as seen in reductions in CRP.
Subsequently the FDA approved rosuvastatin—the statin used in JUPITER—for people who have desirable levels of LDL but high CRP and at least one other coronary risk factor. And according to revised cholesterol guidelines released last year, in cases where there’s uncertainty about statin treatment, CRP level is one of several factors that doctors should consider in making the decision.
Most doctors do not routinely measure CRP, however. It’s not clear what cutoff should be used to define high CRP, nor is it certain that bringing down elevated CRP will, by itself, be beneficial. Still, if you’re at intermediate coronary risk, and you and your doctor are on the fence about starting drug therapy, you should consider CRP testing. A high result could tip the balance toward a statin.
The link to heart disease
For many years atherosclerosis was seen as a kind of plumbing problem—that is, merely a matter of plaque building up in the walls of coronary arteries and clogging them. But blood vessels are nothing like pipes—they are active tissue involved in complex processes. In simplest terms, cells lining the vessels absorb cholesterol (and other substances) from the blood, leading to the build-up of plaque. The body perceives this plaque as an injury and sends inflammatory cells into the vessel walls, where they set off a cascade of events that can ultimately cause plaque to rupture and a clot to form over it. If the clot breaks off or otherwise obstructs blood flow to the heart or brain, this can result in a heart attack or stroke.
It now appears that inflammation plays key roles in all stages of the development of cardiovascular disease. Bacterial or viral infection may also trigger the inflammatory process in blood vessels. Meanwhile, coronary risk factors such as obesity, high blood pressure, undesirable cholesterol levels, and smoking cause or worsen arterial inflammation. Having an inflammatory disorder, such as rheumatoid arthritis, diabetes, or inflammatory bowel disease, also increases coronary risk.
Some medications that help prevent heart attacks and strokes, notably statins, do so at least in part by reducing inflammation. The story is more complicated regarding aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). At the low doses used to protect the heart, aspirin has only a small effect on inflammation; its heart benefit comes primarily from its ability to reduce the risk of blood clots. No other NSAIDs are good for the heart. In fact, some NSAIDs, notably celecoxib (Celebrex), increase the risk of heart attacks slightly.
The cancer connection
As early as the mid-19th century, scientists found links between chronic inflammation (or its markers) and cancer. It’s now estimated that more than 20 percent of cancer cases are associated with inflammation.
Inflammation is involved with cancer development on many levels. Notably, it contributes to tumor initiation by inducing oxidative stress, DNA damage, and chromosomal instability. It promotes tumor cell proliferation and resistance to apoptosis (programmed cell death after a certain number of cell divisions, a good thing when it comes to cancer cells). Simply put, increased inflammation makes it easier for normal cells to transform into malignant cells.
The evidence is strongest concerning gastrointestinal cancers, including certain kinds of colon, liver, esophageal, and stomach cancer. It’s theorized that these organs are at high risk because they are exposed directly to pro-inflammatory dietary and environmental factors. Inflammation can also alter colonic microflora in ways that increase cancer risk.
On the positive side again, evidence is accumulating that aspirin, partly because of its anti-inflammatory effect, can reduce the risk of certain types of colon cancer and possibly certain other cancers.
What does all this mean for you?
What can you do to reduce chronic inflammation and the risks it entails? There is no magic food, pill, or treatment. But many of the same steps that help prevent cardiovascular disease may do so in part by helping to tamp down inflammation.
Eat a heart-healthy diet. Lab research has shown that many healthful foods, especially fatty fish, fruits, and vegetables (as well as chocolate, wine, and tea) have anti-inflammatory effects. Other studies have shown that the Mediterranean diet tends to reduce inflammation (as measured by CRP). On the other hand, saturated fats, trans fats, sugar, and other refined carbohydrates have pro-inflammatory effects in the body.
Aerobic exercise, done regularly and moderately, reduces chronic inflammation via a variety of complex mechanisms. In contrast, being sedentary or training very intensely both increase inflammation.
If you are very overweight, and especially if the extra pounds are in your abdomen, lose weight via a healthy diet and exercise. That will reduce inflammation and the risk of chronic diseases.
Don’t smoke—it’s a powerful cause of inflammation. Avoid secondhand smoke.
If you have had a heart attack or are at elevated risk for one, talk to your doctor about low-dose aspirin. If you have no history of cardiovascular disease, however, the risks of aspirin therapy (bleeding in the stomach or brain) may outweigh its small benefit. Similarly, if you are at high risk for colon cancer because of polyps or family history, discuss aspirin therapy with your doctor.
If you’re prescribed a statin, here’s an added reason to take it: It serves double duty—against cholesterol and inflammation.
Don’t drink more than moderate amounts of alcohol.
Get adequate sleep and try to find ways to deal with stress, anxiety, and depression. Social isolation can also increase chronic inflammation, as was seen in a study in the Journal of Health and Social Behavior last year, so increasing social activities may help.
As we age, our levels of the Co-enzyme Nicotinamide Adenine Dinucleotide NAD+ drop significantly in multiple organs in mice and humans (5,8,10).
NAD+ decrease is described as a trigger in age-associated decline(23), and perhaps even the key factor in why we age (5).
In 2013, research published by Dr David Sinclair demonstranted that short term supplementation with Nicotinamide MonoNucleotide (NMN) reversed many aspects of aging, making the cells of old mice resemble those of much younger mice, and greatly improving their health (8).
The quotes below are directly from that research:
NMN was able to mitigate most age-associated physiological declines in mice”
“treatment of old mice with NMN reversed all of these biochemical aspects of aging”
Since that landmark 2013 study, dozens of others have been published investigating the efficacy of supplementation with NMN in treatment and prevention of a wide range of disease including cancer, cardiovascular disease, diabetes, Alzheimers, Parkinsons, and more (5,6,7,9,10,11,13,14,15,16).
According to Dr Sinclair:
enhancing NAD+ biosynthesis by using NAD+ intermediates, such as NMN and NR, is expected to ameliorate age-associated physiological decline
WHAT IS NAD+
NAD+ is a key co-enzyme that the mitochondria in every cell of our bodies depend on to fuel all basic functions. (3,4)
NAD+ play a key role in communicating between our cells nucleus and the Mitochondria that power all activity in our cells (5,6,7)
NAD+ LEVELS DECREASE WITH AGE
As we age, our bodies produce less NAD+ and the communication between the Mitochondria and cell nucleus is impaired. (5,8,10).
Over time, decreasing NAD+ impairs the cell’s ability to make energy, which leads to aging and disease (8, 5) and perhaps even the key factor in why we age (5).
NAD+ METABOLISM IN HUMANS
NAD+ can be synthesized in humans from several different molecules (precursors), thru 2 distinct pathways: De Novo Pathway
Nicotinic Acid (NA)
NAM – Nicotinamide
NR – Nicotinamide Riboside
NMN – Nicotinamide MonoNucleotide
The NAD+ supply is constantly being consumed and replenished through the Salvage Pathway, with approximately 3g of NAM metabolized to NMN and then to NAD 2-4 times per day (14).
The salvage pathway sustains 85% or more of our NAD+ (14)
Nampt is the rate-limiting step in the salvage process (97).
As we age, Nampt enzyme activity is lower, resulting in less NAM recycling, less NAD+, more disease and aging (97,101).
ALL PRECURSORS BOOST NAD+ SIGNIFICANTLY IN LIVER
NAM, NA, NMN, NR, and Tryptophan ALL elevate levels of NAD+ significantly in the liver, which has many benefits for metabolic health.
This chart from the Trammell thesis shows the impact on liver NAD+ for mice given NR, NAM, and NA by oral gavage 0.25, 1, 2, 4, 6, 8 and 12 hours before testing.
Charts showing NMN impact on NAD+ levels in the liver are below.
* Note: These charts are somewhat deceptive. It shows NAM (green bar) elevated NAD+ nearly as much as NR (black bar)
However if they used equal mg of each supplement, which is how people actually purchase and use them, it would show NA about equal with NR and NAM far effective than NR at elevating NAD+ in the liver.
Mice in these experiments didn’t receive equal WEIGHTS of each precursor. Instead researchers chose to use quantity of molecules, which makes NR look “better” by comparison.
In this case, “185 mg kg−1 of NR or the mole equivalent doses of Nam and NA”(16).
Molecular weight for NR is 255 grams, NAM is 122 grams, and NA 123 grams. So this chart used a ratio of 255 grams of NR to 122 and 123 grams of NAM and NA.
“NMN makes its way through the liver, into muscle, and is metabolized to NAD+ in 30 minutes” (R)
Is much slower, taking 8 hours to reach peak NAD+ in humans (R)
Has very similar NAD+ profile to NR, taking 8 hours to reach peak NAD+ in humans (R)
Has been shown to increase NAD+ level in liver (47%), but was weaker in kidney (2%), heart (20%), blood (43%) or lungs (17%) (R)
Elevates NAD+ to peak levels in liver in 15 minutes (R)
raised NAD+ in liver (47%), and impressively raised kidney (88%), heart (62%), blood (43%) and lungs (11%) (R)
In the liver tryptophan is the preferable substrate for NAD+ production (R)
Administration of tryptophan, NA, or NAM to rats showed that tryptophan resulted in the highest hepatic NAD+ concentrations(R)
ONLY NMN BYPASSES THE NAMPT BOTTLENECK IN TISSUES THROUGHOUT THE BODY
Restoring NAD+ to youthful levels in ALL CELLS throughout the body is the goal.
However, many tissues cannot utilize NAD+ directly from the blood as NAD+ cannot readily pass through the cellular membrane.
Muscle tissue, for example, depends on cells internal recycling of NAD+ through the salvage pathway which is controlled by Nampt.
To restore depleted NAD+ levels in such cells, a precursor must:
Be available in the bloodstream
Once inside a cell, be able to bypass the Nampt bottleneck
NA and Tryptophan
NA and Tryptophan act through the De Novo pathway, which supplies a small percentage of our NAD+, primarily in the liver
NAM is abundant in the blood and easily carried into such cells throughout the body, but depends on Nampt, which is the rate limiting enzyme in the salvage pathway.
When taken orally as a supplement, most NR does not make it through the digestive system intact, but is broken down to NAM (97,98,99).
NMN levels in blood showed it is quickly absorbed from the gut into blood circulation within 2–3 min and then cleared from blood circulation into tissues within 15 min
NMN INCREASES NAD+ and SIRT1 DRAMATICALLY IN ORGANS
In this 2017 study, NMN supplementation for 4 days significantly elevated NAD+ and SIRT1, which protected the mice from Kidney damage.
NAD+ and SIRT1 levels were HIGHER in OLD Mice than in YOUNG Mice that did not receive NMN.
LONG TERM SUPPLEMENTATION WITH NMN
In a long-term experiment documented in the 2016 study (22) , mice were given 2 different doses of NMN over 12 months.
Testing revealed that NMN prevents some aspects of physiological decline in mice, noting these changes:
Decreased body weight and fat
Increased lean muscle mass
Increased energy and mobility
Improved visual acuity
Improved bone density
Is well-tolerated with no obvious bad side effects
Increased oxygen consumption and respiratory capacity
Improved insulin sensitivity and blood plasma lipid profile
Here are some quotes from the study:
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
NMN-administered mice switched their main energy source from glucose to fatty acids
These results strongly suggest that NMN has signiﬁcant preventive effects against age-associated impairment in energy metabolism
NMN effectively mitigates age-associated physiological decline in mice
LOWER FAT AND INCREASED LEAN MUSCLE MASS
Researchers found that NMN administration suppressed body weight gain by 4% and 9% in the 100 and 300 mg/kg/day groups.
Analyses of blood chemistry panels and urine did not detect any sign of toxicity from NMN.
Although health span was clearly improved, there was no difference in maximum lifespan observed.
These results suggest that NMN administration can significantly suppress body weight gain without side effects
INCREASED OXYGEN CONSUMPTION AND RESPIRATORY CAPACITY
Oxygen consumption significantly increased in both 100 and 300 mg/kg/day groups during both light and dark periods (Figure 3A).
Energy expenditure also showed significant increases (Figure 3B).
Respiratory quotient significantly decreased in both groups during both light and dark periods (Figure 3C),
This suggests that NMN-administered mice switched their main energy source from glucose to fatty acids.
The mice that had been receiving NMN for 12 months exhibited energy levels, food and water consumption equivalent to the mice in the control group that were 6 months younger.
NMN administration has significant preventive effects against age associated physical impairment
HUMAN STUDIES – LONG TERM SUPPLEMENTATION WITH NMN
The first clinical trial of NMN in humans is currently underway by an international collaborative team between Keio University School of Medicine in Tokyo and Washington University School of Medicine (33).
Participants are 50 healthy women between 55 and 70 years of age with slightly high blood glucose,BMI and triglyceride levels.
Using a dose of 2 capsules of 125mg NMN per day over a period of 8 weeks, researchers are testing for:
change in insulin sensitivity
change in beta-cell function
works to control blood sugar
blood vessels dilate
effects of NMN on blood lipids
effects of NMN on body fat
markers of cardiovascular and metabolic health
According to the study:
“Data from studies conducted in rodents have shown that NMN supplementation has beneficial effects on cardiovascular and metabolic health, but this has not yet been studied in people”
Testing of metabolic parameter will continue for 2 years after supplementation has ended, so final results will not be published for some time yet, but preliminary results are expected to be announced in early 2018.
FOODS THAT CONTAIN NMN
NMN is found in many food sources such as edamame, broccoli, cucumber,cabbage, avocado, tomato, beef and shrimp.
As such, it is likely free from serious side effects in humans, and has been available for purchase commercially for over 2 years.
In the long term (12 month) 2016 mouse study (22), both 100 and 300mg/kg per day improved oxygen consumption, energy expenditure, and physical activity more.
According to the FDA guidelines, an equivalent would be about 560 mg for a 150lb human.
It should be noted that NMN administration did not generate any obvious toxicity, serious side effects, or increased mortality rate throughout the 12-month-long intervention period, suggesting the long-term safety of NMN.
The current Human study uses a dosage of 2 capsules of 125 mg, which seems to be the most commonly used dosage.
NAD+ levels decrease throughout the body as we age, contributing to disease and aging.
Restoring NAD+ levels can ameliorate many age released health issues.
All the NAD+ precursors are effective at raising NAD+ levels in the liver.
Raising NAD+ in the liver has many benefits, but is not effective in tissues and organs that cannot access NAD+ directly from the bloodstream and so depend on internal cellular NAD+ recycling.
For these tissues, utilizing each cells internal Salvage Pathway is necessary to restore NAD+ levels.
NR is not stable in the body and not normally found in the bloodstream, so is not readily available as NR to many tissues. Once metabolized to NAD+ it cannot enter cells. If metabolized to NAM it cannot bypass the Nampt bottleneck.
NMN is the only precursor that is stable and available to cells through the bloodstream, and can bypass the Nampt bottleneck to quickly restore NAD+ throughout the body.
NAD(+) levels were increased significantly both in muscle and liver by NMN
NMN-supplementation can induce similar reversal of the glucose intolerance
NMN intervention is likely to be increased catabolism of fats
NMN-supplementation does mimic exercise
NMN could restore cognition in AD model rats.
The beneficial effect of NMN is produced by ameliorating neuron survival, improving energy metabolism and reducing ROS accumulation.
These results suggest that NMN may become a promising therapeutic drug for AD
In the first experiment, one Human subject was given a single dose 1,000 mg of NR each morning for 7 days. Blood levels of NAD+ and metabolites were 9 times the first day and every 24 hours thereafter.
From the results shown in chart above, we see NAD+ levels did not rise until 4 hours after ingesting, peaked at around 8 hours, and remained elevated up to 24 hours.
The second experiment involving human subjects included 12 individuals that were given 100,300, or 1,000 mg of NR with a washout period of 7 days between doses. Blood levels of NAD+ were recorded at 1, 2, 4, 8, and 24 hours.
100 mg per day
This chart shows 100mg per day (purple) elevates NAD+ levels around 4 hours, dropping significantly by 8 hours and continuing to decline throughout the 24 hours.
300 mg per day
The numbers in this line (red) are slightly elevated at 8 hours, then continue rising to 24 hours.
It appears that a dosage of 300mg achieved the same NAD+ increase as 1,000 mg at the 24 hour mark.
1,000 mg per day
This line (black) looks very similar to the first test with one subject given 1,000 mg daily.
Increased NAD+ noted at 4 hours, with maximum increase reached around 8 hour. It appears NAD+ levels remain at that maximum through 24 hours.
We can see that at all dosages the NAD+ levels were elevated somewhat within 4 hours.
It does appear an upper limit was reached after which, additional NR did not raise NAD+ any further.
Dr Brenner points to the increased NAAD levels that coincide with the peak of NAD+ and suggest NAAD acts as an “overflow pool”, that may later be converted to NAD+ if needed.
Do other Metabolites of NAD+ matter?
The author notes that supplementation with Nicotinamide Riboside elevates the level of many NAD+ metabolites at different rates:
“Because every NAD+ metabolite can be converted to one or more other metabolites, snapshots of the levels of NAD+ , nicotinamide (Nam) or any other NAD+ metabolite without assessment of the NAD+ metabolome on a common scale has the potential to be misleading.”
NAAD is much higher in the 1000mg subjects. However, the first study implies there is a limit to the possible increase of NAD+. Despite repeated usage over seven days, NAD+ tops out.
The second study shows that at 24 hours, NAD+ is elevated by approximately the same amount in the 300mg and 1,000mg test subjects.
[box]Conclusion: The maximum effect appears to be achieved at some dosage around 300mg per day.
Note: Subjects in this study were healthy and between 30-55 years of age. Older, sicker subjects might benefit from higher dosages. The Elysium Basis testing with older individuals (below) will hopefully shed more light on this.[/box]
SECOND STUDY OF NR EFFECT – ELDERLY PATIENTS TAKING ELYSIUM HEALTH BASIS
A single capsule of BASIS is 125 mg of Chromadex NIAGEN brand of Nicotinamide Riboside, along with 50 mg of Chromadex Pterostilbene.
Participants received either placebo, 1, or 2 capsules of BASIS
Elysium Health did issue a press release that states that 125 mg of NIAGEN resulted in a 40% increase in blood NAD+ levels that was maintained throughout the 8 weeks of the study.
The 250 mg dosage resulted in an increase that was “significantly higher” than the 125 mg dose, and reached 90% at one of the 4 checkpoints (4 weeks).
Since the increase from the 250 mg dosages reached a plateau at 4 weeks, and dropped afterwards, implies that a higher dosage probably would not be any more effective.
This rather speculative interpretation agrees with the results in Study #1 that the most effective dosage is higher than 125 mg, but has peaked out at 250mg a day
[box]Conclusion: Most people will likely get the maximum NAD+ increase at 250mg per day
NAD+ METABOLISM IN HUMANS
NAD+ is synthesized in humans by several different molecules (precursors), thru 2 different pathways: De Novo Pathway
Nicotinic Acid (NA)
NAM – Nicotinamide
NR – Nicotinamide Riboside
NMN – Nicotinamide MonoNucleotide
The NAD+ supply is not stagnant – it is constantly being consumed and replenished, with the entire NAD+ pool being turned over 2-4 times per day (14).
This recycling is through the salvage pathway, where the enzyme Nampt catalyzes NAM to NMN, which is then metabolized to NAD+.
Nampt is the rate-limiting step in the salvage process (97).
Many studies have confirmed the importance of Nampt in maintaining sufficient NAD+ levels, such as the quote below from a 2016 study that used mice lacking Nampt in muscle fiber:
“NAD content of muscle was decreased by ~85% confirmed the prevailing view that the salvage route of NAD synthesis from NAM sustains the vast majority of the NAD” (97)
These mice exhibited normal muscle strength and endurance while young, but deteriorated rapidly as they aged which confirmed Nampt is critical to maintaining NAD+ levels.
As we age, Nampt enzyme activity is lower, resulting in less NAM recycling, less NAD+, more disease and aging (97,101).
NMN and NR SUPPLEMENTS CAN BYPASS NAMPT
NR had been known for decades, but was not thought to be that important until 2004 when Dr. Charles Brenner discovered the enzyme NRK1 can phosphorylate NR directly to NMN, bypassing the Nampt “bottleneck” (100).
This newly discovered “shortcut” in the NAD+ salvage pathway found that NR can be metabolized directly to NMN to boost NAD+ levels more effectively than NAM.
MOST NR IS FIRST METABOLIZED TO NAM
When taken orally as a supplement, most NR does not make it through the digestive system intact, but is broken down to NAM (97,98,99).
Even when taken at very high dosages, NR has not been detected in the bloodstream in any research (97,98,99).
“This evidence indicates that NR is converted to NAM before absorption occurs and that this reaction is the rate-limiting step ” (98)
“NR has been shown be converted to Nam before being absorbed or reaching tissues” (99)
“we were surprised to find that NR exerts only a subtle influence on the steady state concentration of NAD in muscles. Our tracer studies suggest that this is largely attributable to breakdown of orally delivered NR into NAM prior to reaching the muscle. ” (97)
Note:NAM does elevate NAD+, but is on the “wrong” side of the Nampt bottleneck, which limits it’s effectiveness
HUMAN STUDY ON NR BIOAVAILABILITY
The following five charts are all from the thesis published by Samuel Alan Trammell in 2016 under supervision by Dr Brenner:
This chart above shows the impact on NAD+ metabolites over time for a 52 year old human after ingesting 1000mg of NR daily for 7 days.
NAD+ levels begin to rise at 4.1 hours, and peak at 8.1 hours.
NAM levels double at .6 hours and have a second peak at 7.7 hours, long before NAD+ levels are elevated.
This chart at right shows metabolites found in urine of the subject from the same experiment as above.
The red box shows NAM is elevated more than 10x baseline at the same time point that NAD+ is elevated, which implies that NR has elevated NAM to such an extent that excess NAM is excreted in urine.
This chart a left shows impact of NR, NA, and NAM supplementation on blood plasma NAD+ (b), and NAM (d) levels in 12 human subjects.
The red line at 2 hours shows NR supplementation increases NAM perhaps 3x (d), but has not yet elevated NAD+(b).
The 2 hour mark also is the point at which NAM supplementation begins to increase NAD+ levels (b).
The blue line at 8 hours is when both NR (b) and NAM (d) supplementation reach peak NAD+ increase.
Lastly, the green bar and black bar in chart b show that NAM elevates NAD+ slightly less than NR.
NR elevated NAD+ slightly more than NAM, but is much slower acting
MOUSE STUDIES ON NR BIOAVAILABILITY
The chart above shows the result on NAD+ metabolism of 15 mice fed NR by oral gavage at a dose of 185 mg/kg of bodyweight.
The NR was synthesized with heavy atoms of deuterium at the ribosyl C2 and 13C on the Nam side, to allow tracking.
The measurement at 2 hours shows 54% of the NAD+ has the single heavy molecule (white bar, M+1). This 54% was likely broken down to NAM first, losing the second labelled heavy atom.
At the same time point, 5% of the NAD+ had both labels (Grey bar, M+2).
This 5% of NR made it through the digestive tract intact and was metabolized through the shortcut from NR -> NMN -> NAD+, vs 54% that had been through NR -> NAM -> NMN -> NAD+.
The chart above shows the impact of the same double labeled NR on mouse liver, but this time after IP (Intraperitoneal) Injection.
Note the dramatic difference in the ratio of labelled M+2 over M+1. IP results in much higher levels of intact NR (M+2) being metabolized to NAD+, whereas Oral NR shows far more M+1 labelled NR to NAD+.
This different behavior in IP vs oral NR supplementation also implies oral NR is partially metabolized to NAM before conversion to NAD+.
The above chart shows the resultant increase in select NAD+ metabolites of mice fed NR (unlabeled) at 185 mg/kg of bodyweight.
As noted by the authors, NR and NAR are the only NAD+ precursors tested that did NOT result in elevated levels of the precursor in the liver.
Here is one last quote in discussion section from the Trammell thesis:
“NR has not been detected in the blood cell fraction nor in plasma …NR varied and displayed no response to NR administration … but was detected after IP of double labeled NR in liver (Figure 5.7) and muscle (Figure 5.9), revealing NR does circulate”
They are saying that NR is found in small quantities in the liver, but is not detectable in bloodstream. Oral supplementation with NR did not show any increase in NR in the body. However, Injection (IP) of NR does result in a detectable increase of NR in muscle and Liver. So NR does circulate in the bloodstream when injected, but has not yet been detected upon oral supplementation.
The timing and amplitude of the increases in metabolites noted above imply that:
Oral NR does not result in a detectable increase of NR in the body
It’s likely a majority of the increase in NAD+ is due to NR->NAM->NAD+.
Note: NAM does elevate NAD+, but is on the “wrong” side of the Nampt bottleneck, which limits it’s effectiveness
DOSAGE – SUMMARY
Further testing with larger sample sizes and more data points is underway that will give a much better estimate on the most effective dosage. For now, some conclusions on dosage we see are:
A single dose of NR does increase NAD+ levels
NAD+ levels remain elevated 24 hours after a single dose.
There is an upper limit on the increase of NAD+ levels with NR supplementation
Maximum NAD+ elevation is maintained at a dosage higher than 125 mg per day – likely close to 250mg per day
It appears that a single daily dose may be just as effective as 2 smaller dosages.
Most NR ends up as NAM after digestion, so is much slower and less effective than NMN.
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme found in the cells of all living creatures (3,4) and is critical for communication between the cell nucleus and the mitochondria that power the cells (5,6,7)
LOWER NAD+ LEVELS AS WE AGE
Everyone experiences lower NAD+ levels throughout the body as we age, effecting the communication within every cell of our bodies.
Scientists have known for some time that this loss of NAD+ leads to many different age related diseases as the cells lose ability to perform basic tasks and repair damage due to oxidative stress. (8)
[box]Conclusion: Declining NAD+ levels are implicated in many age related disease and chronic conditions[/box]
NAD+ DEFICIENCY IMPLICATED IN RETINAL DISEASE
In addition to the chronic age related diseases found to be related to declining NAD+ levels, the study below finds impaired NAD+ biosynthesis in many diverse retinal diseases among young and older mice.
Vision depends on the 2 classes of photoreceptors the rods and cones. Many different diseases such as Retinitis Pigments (RP), Age-Related Macular Degeneration (AMD), Rod and Cone Dystrophies, and Leber Congenital Amaurosis (LCA) all attack the photoreceptors thru diverse pathways that lead to photoreceptor death and blindness.
Photoreceptors are known to have high energy requirements, but limited reserves so are dependent on constant synthesis of NAD+ to meet energy needs. (Ames et al., 1992, Okawa et al., 2008)
The authors theorized that NAD+ biosynthesis plays a key role in healthy vision. They noted that the leading cause of blindness in children, LCA, is caused by a mutation in the enzyme NMNAT1 which results in impaired synthesis of NAD+.(Falk et al., 2012, Koenekoop et al., 2012, Sasaki et al., 2015)
In mammals, NAM is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT) as the first step in biosynthesis of NAD+.
In this study, researchers created knockout mice that lack NAMPt in rod photoreceptors which disrupts the normal NAD+ biosynthesis, resulting in a 26-43% reduction in retinal NAD+ levels.
Within 6 weeks, these mice exhibited significant photoreceptor death and vision loss. The results very closely matched the degeneration seen in patients with Retinitis Pigments and other degenerative vision disease.
According to the authors:
NAD+ deficiency caused metabolic dysfunction and consequent photoreceptor death…these findings demonstrate that NAD+ biosynthesis is essential for vision
SUPPLEMENTATION TO INCREASE NAD+ PREVENTS PHOTORECEPTOR DEGENERATION AND RESTORES VISION
To confirm the cause of vision loss, researchers supplemented knockout mice with daily injections of NMN, bypassing the need for NAMPT in the first step of NAD+ synthesis. Those mice receiving NMN experienced significant recovery of retinal function.
These data clearly demonstrate that NAMPT-mediated NAD+ biosynthesis is necessary for the survival and function of both rod and cone photoreceptors, as promoting NAD+ biosynthesis in the retina with NMN supplementation can compensate for Nampt deletion, thereby reducing photoreceptor death and improving vision.
NAD+ DEFICIENCY IS COMMON IN MANY RETINAL DISEASES
Researchers were able to determine that NAD+ deficiency is common in many vision problems.
Mice subjected to light exposure retinal damage had significant reduction in NAD+ levels (Figure 3H)
Similar reductions in NAD+ levels were found in mice with streptozotocin (STZ)-induced diabetic retinal dysfunction compared to non-hyperglycemic controls (Figure 3I).
Lastly, they compared 18-month-old vs 6 month old mice. As with the light exposed and diabetic induced mice, the older mice exhibited diminished vision along with decreased retinal NAD+ levels (Figure 3J).
These findings support the idea that NAD+ deficiency may be a shared feature of retinal dysfunction.
SUPPLEMENTATION TO INCREASE NAD+ PROTECTS VISION
After demonstrating that light induced retinal dysfunction was linked to decreased NAD+ levels, researchers were able to show that supplementation to increase NAD+ could protect against retinal damage.
Mice that were given injections of NMN for 6 days prior, and 3 days after light exposure exhibited improved retinal function vs those that did not receive NMN injections (Figures 4A–4C).
[box]Conclusion: Results from this study suggest that supplementation to increase NAD+ deficiencies can help repair macular damage and may be an effective treatment for many common degenerative vision problems.[/box]
IMPLICATIONS FOR HUMANS
This study was very specific for the impact of NAD+ on Retinal disease in Mice, but is also further evidence that the absence of sufficient NAD+ has dire consequences, and that replacement of NAD+ can repair damage.
Researchers are experimenting with various techniques for raising NAD+ levels in mice and humans such as:
The 2013 study by Dr David Sinclair that demonstrated increased levels of NAD+ reverses age related degeneration in mice also used injections of NMN (25).
Raising NAD+ levels back to youthful levels to combat aging is an exciting new field of research.
Supplementing with NAD+ precursors such as Nicotinamide Riboside and NMN are getting a lot of the attention, and recent research is proving it is effective.
Rather than boosting NAD+ levels, an alternative and/or complementary approach seeks to remedy the cause of WHY NAD+ levels drop as we age.
Recent studies have found the enzyme CD38 RISES at the same time NAD+ levels decline, and seems to destroy NAD+. They found that inhibiting CD38 results in much higher NAD+ levels.
Other studies have found Flavonoids like Quercetin and Apigenin are effective at inhibiting CD38, resulting in higher NAD+ levels.
WHAT IS QUERCETIN
Quercetin is a flavonoid and known anti-inflammatory agent (1) that has been shown to have beneficial effects against cancer (2), and atherosclerosis (3).
It is found in many vegetable, tea, coffee and red wine.
Quercetin supplements are highly bioavable with a half-life of 11-28 hours which means that supplementation results in greatly increased blood plasma levels.
Possible health benefits: anti-carcinogenic, anti-inflammatory, antiviral, antioxidant, psychostimulant, capillary permeability, mitochondrial biogenesis
Quercetin is a natural product reputed and used traditionally for its beneficial effects on health. It is categorized as a flavonol, one of the six subclasses of flavonoid compounds. The name Quercetin is derived from quercetum (oak forest), after Quercus.
Quercetin has unique biological properties that offer potential benefits to overall health and disease resistance, including anti-carcinogenic, anti-inflammatory, antiviral, antioxidant, psychostimulant activities, capillary permeability and stimulation of mitochondrial biogenesis. Quercetin is present in various vegetables as well as in tea and red wine.
In a typical Western diet the daily intake of quercetin is estimated to be in the range of 0 and 30 mg.
Quercetin is also one of the most complex flavonol compounds to understand due to its metabolism. It involves intestinal uptake and/or deglycosylation, glucuronidation, sulfation, methylation, possible deglucuronidation and so on.
From the various quercetin metabolites generated in the body it has been recently demonstrated that quercetin “3-O-β-D-glucuronide (Q3GA)” and quercetin “3′-sulfate” are the dominant quercetin conjugates in human plasma. Typically, the human quercetin plasma concentration is in the order of nanomolar, but upon quercetin supplementation it may increase upto the low micromolar range.
This together with a half-life of the atom and its metabolites in the range of 11-28h it can be assumed that continuous supplementation leads to an increased plasma concentration.
In human studies, quercetin has been mostly well tolerated.
Doses up to 1,000 mg/day for several months did not produce adverse effects on blood parameters, liver and kidney function,hematology, or serum electrolytes. In general, there is plentiful available evidence that support the safety of quercetin for addition to food. A few items should be noted however.
At high dosing, quercetin has been shown to inhibit topoisomerase II which is an essential enzyme in DNA replication and inhibition leads to diplochromosomes.
The second note is with regards to other drug interactions. Due to is complexity it may interfere with these prescription medications.
Therefore, it is great to see the continuing research into characterizing the promising benefits and also potential side effects of this novel dietary supplement.
Quercetin the key behind Rutin’s many benefits
The more calories you store over time — the more you’ll be stuck feeling:
Bloated and sleepy
Hungry even after you just ate
But there’s good news. Doctors and scientists are now rethinking EVERYTHING they know about weight loss, thanks to a new discovery. No, we’re not talking about Garcinia Cambogia – that is so 2016…
An ingredient that burns BIG amounts of fat… even if you’re just sitting down!
I know… when I looked at the research I almost couldn’t believe it myself. But this incredible ingredient is really catching the attention of the scientific community…
… and could start making a HUGE difference in people’s lives sooner than you think.
It’s called Rutin, an all-natural compound that’s theorized to kickstart your Brown Adipose Tissue fat — or “Brown fat.”
You may have heard of this before. Brown fat is the incredible “permanent fat” that keeps you warm by using your regular calorie-packed fat as fuel.
So in a study published just last month by The FASEB Journal,scientists tested Rutin’s fat-burning effects on obese mice…
And incredibly, Rutin didn’t just work…
It significantly boosted metabolism and weight loss in 100% of the obese mice… without any exercise!1
Brown Fat Is GOOD
So can activating Brown fat work on YOU?
Well, unfortunately, the older humans get — the less Brown fat they have…
In fact, the humans with the most Brown fat are actually babies…
Luckily, adults still keep a tiny fraction of their original Brown fat…
And cutting-edge research now shows activating this tiny amount of “helpful” fat could work on HUMANS at any age.
So to test this, researchers from the elite University of Sherbrooke “activated” the Brown fat of healthy human subjects.
And over the course of a three-hour “sitting” period, the researchers couldn’t believe the numbers they were seeing…
The human subjects with “activated Brown fat” burned an incredible 1.8 times more calories compared to the others! 2
That’s right — by activating their own Brown fat, the human subjects nearly DOUBLED their fat-burn… by just sitting there.
So how can you start feeling the powerful effects of Rutin?
Well, one of the BIGGEST sources of Rutin is actually in mulberries — a flavorful fruit jam packed with lots of other vitamins and minerals, too…
And conveniently, this potent berry is available everywhere — so you can start feeling the powerful effects of Rutin right now.
I’d say the best thing to do is go stock up on mulberries at your local grocery store.
Not all mulberries are the same. In fact, the mulberries with the highest levels of healthy vitamins, antioxidant content, and Rutin are the mature, dark berries. So when you’re at the grocery store DO NOT get the hard, colorless mulberries. Because they aren’t RIPE and won’t give you nearly as much good stuff as the dark, mature mulberries!
CD38 INCREASES, NAD+ DECLINES AS WE AGE
This June 2016 study shows that CD38 increases dramatically with age and plays a key role in destroying Nicotinamide MonoNucleotide (NMN), a NAD+ precursor.
The authors show that protein levels of CD38 increased in multiple tissues during aging.
They then compared the relationship of CD38 levels to NAD+ of normal mice vs CD38 Knockout mice (Mice bred without the gene to product CD38).
The NAD+ levels of normal mice aged 32 months are about 1/2 that of young mice, whereas the CD38 knockout mice showed no decrease in NAD+ levels at the same age.
This study did not suggest a cause for the rapid increase in CD38 activity but other studies have shown a link to CD38 increase with inflammation from disease and injury as we age.
The exponential rise suggests that CD38 may deplete NAD+ needed for other processes and directly relate to the aging process.
Finally, the authors addressed how CD38 may affect therapies designed to raise NAD+ levels. Currently, the favored approach in mouse and humans is to treat with NAD+ precursors, such as nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN).
Interestingly, CD38 not only degrades NAD+ in vivo, but also NMN. When CD38 knockout mice were given injections of NAD+, NMN, or NR (which is converted to NMN), circulating levels of NAD metabolites remained stable after 150 min, long after they began to fall in the wild-type animals.
Furthermore, when compared to the wild-type, CD38 knockout mice on a high-fat diet exhibited a much larger improvement in glucose tolerance when given NR.
Conclusion:CD38 degrades NAD+ and its precursors. Inhibiting CD38 can lead to increased NAD+ levels
NAD+ DECLINE LINKED TO AGING
Prior research shows that declining NAD+ levels is linked to many age related diseases and metabolic disorders such as diabetes, and a possible contributing factor to aging (4).
A landmark 2013 study by Dr David Sinclair demonstrated that increased levels of NAD+ have been shown to reverse age related degeneration in mice, giving older mice the muscle capacity, endurance and metabolism of much younger mice (5).
Other studies have shown that supplementing old mice with NAD+ precursors can greatly improve metabolic health such as increased insulin sensitivity, improved mitochondrial function, reduced stem cell senescence, and increased lifespan (6,7,8)
This suggests that many of the the normal age related conditions are at least partly driven by decreased mitochondrial functioning, and that increasing NAD+ levels can restore mitochondrial functioning and reverse many age related problems (9).
Conclusion: Top researchers have been investigating raising NAD+ levels for treatment of disease and degenerative aging.
QUERCETIN INHIBITS CD38
While many of the health benefits of Quercetin are well documented, the mechanisms of action are not completely understood.
In this study published April 2013, obese mice that received Apigenen or Quercetin showed improved glucose homeostasis, glucose tolerance, and lipid metabolism.
The authors theorized the mechanism may be the anti-inflammatory properties inhibit CD38 which results in increased NAD+ levels in tissues.
First, they measured the effect of quercetin on endogenous cellular CD38 activity and found that quercetin promotes an increase in intracellular NAD+ in a dose-dependent manner (Fig. 4B).
They also compared NAD+ levels of normal mice with CD38 knockout mice after supplementation with Quercetin and found it promotes an increase in NAD+ in normal mice but does not further increase NAD+ levels in CD38 knockout mice (Fig. 4D), indicating that the effect of quercetin on NAD+ levels is CD38 dependent.
They were able to demonstrate that Quercetin inhibits CD38 and promotes an increase in NAD+ levels.
Conclusion: The researchers concluded that apigenin and quercetin as well as other CD38 inhibitors may be used to raise NAD+ levels and treat metabolic syndrome and obesity-related diseases.
QUERCETIN INCREASES EFFECTIVENESS OF NICOTINAMIDE RIBOSIDE
Supplementation with NAD+ precursors such as Nicotinamide Riboside is generating a lot of excitement as recent research is proving it is effective at raising NAD+ levels in humans, which has tremendous therapeutic potential to treat metabolic and age-related disease.
Dr Sinclair is at the forefront of research on NAD+ levels and their effects on aging. In this video (18 minute mark), he demonstrates recent research supplementing with NAD+ precursors to return old mice to youthful states.
Results from this study need to be folded into the article above
Several epidemiologic studies have indicated that coffee consumption has a neuroprotective effect against Parkinson disease (PD) and Alzheimer disease (AD). Liu et al. (2012) analyzed 304,980 participants in the National Institute of Health (NIH) study and found a mild protective effect of coffee against PD. Palacios et al. (2012) found a similar effect in a smaller cohort, again attributing it to caffeine intake. Qi and Li (2014) did a meta-analysis of 13 articles on coffee, tea, and caffeine consumption and concluded there was a linear dose-response effect reaching a maximum at approximately 3 cups of coffee per day. As far as AD is concerned, Eskelinen and Kivipelto (2010) reported that caffeine and other factors in coffee were possible protective factors. On the basis of the Cardiovascular Risk Factors, Aging and Dementia study they concluded that drinking 3e5 cups of coffee at midlife was associated with a decreased risk of dementia/AD of about 65% in late life, and this was due to caffeine and/or other mechanism such as antioxidant capacity by other components. Eskelinen et al. (2009) showed that drinking 3e5 cups of decaffeinated coffee at midlife decreases the risk of AD.
Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson’s disease and Alzheimer’s disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-g or interferon-g released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-a and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFkB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/ nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson’s disease and Alzheimer’s disease.
Attributing these effects to caffeine in coffee was recently challenged by the findings of Ding et al. (2015). They examined the effects of consuming total, caffeinated, and decaffeinated coffee in 3 very large cohorts of men and women. They found significant inverse associations with coffee consumption and deaths attributed to cardiovascular disease, neurological diseases, and suicide. There was no significant difference between consuming caffeinated or decaffeinated coffee. This latter report indicated that other constituents of coffee than caffeine must be responsible for the protective effect. For example, flavonoids are polyphenolic bioactive compounds that are found in foodstuffs of plant origin (Babu et al., 2013). Flavonoids are classified into subgroups based on their chemical structure: flavanones, fla- vones, flavonols, flavan-3-ols, anthocyanins, and isoflavones. Flavonoids have a backbone of 2-phenylchromen-4-one (2-phenyl-1-benzopyran-4-one). Specifically, adding hydroxyl groups to the backbone generates flavonols including quercetin and flavones such as apigenin and luteolin. It is known that they functions generally to remove oxidant and to inhibit inflamma- tion (Marzocchella et al., 2011).
In this report, we examined the effects of caffeine and other well-known coffee components, such as quercetin, flavone, and chlorogenic acids (CGAs), on neuroinflammation and neurotoxicity mediated by toxic factors from activated microglia and astrocytes. We also investigated changes in oxidative stress markers caused by quercetin and caffeine because depletion of glutathione (GSH) in glial cells induces neuroinflammation resulting in neuronal death (Lee et al., 2010a).
We found that CGA, flavone, quercetin, and caffeine reduced the release of proinflammatory cytokines such as tumor necrosis factor- a (TNFa) and interleukin-6 (IL-6) from lipopolysaccharide/inter- feron-g (LPS/IFNg)-stimulated microglia and THP-1 cells, as well as from IFNg-stimulated astrocytes and U373 cells. The toxicity was attenuated in a concentration and incubation-time dependent manner. This was due to reduced activation of intracellular inflammatory pathways such as P38 mitogen-activated protein ki- nase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFkB) proteins and decreased release of proin- flammatory cytokines such as TNFa and IL-6. We found that quer- cetin was the most potent anti-inflammatory and neuroprotective coffee component. In addition, quercetin has antioxidative prop- erties, but caffeine does not.
The data indicate that quercetin, but not caffeine, is a major component reducing the risk of pathogenesis in degenerative neurological diseases such as PD and AD. It may prove to be a useful therapeutic agent.
All reagents were purchased from Sigma (St. Louis, MO, USA) unless stated otherwise. The following substances were applied to the cell cultures: bacterial LPS (Escherichia coli 055:B5) and human recombinant IFNg (Bachem California, Torrance, CA, USA).
2.2. Cell culture and experimental protocols
The human monocyte THP-1 and astrocytoma U373 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The human neuroblastoma SH-SY5Y cell line was a gift from Dr R. Ross, Fordham University, NY, USA. These cells were grown in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) medium containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 100 IU/mL penicillin and 100 mg/mL streptomycin (Invitrogen) under humidified 5% CO2 and 95% air.
Human astroglial and microglial cells were isolated from sur- gically resected temporal lobe tissue as described earlier (Lee et al., 2010b). Briefly, tissues were rinsed with a phosphate-buffered saline (PBS) solution and chopped into small (<2 mm3) pieces with a sterile scalpel. They were treated with 10 mL of a 0.25% trypsin solution at 37 C for 20 minutes. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d’Urfé, PQ, Canada) was added to reach a final concentration of 50 mg/mL. Tissues were incubated for an additional 10 minutes at 37 C. After centrifugation at 275g for 10 minutes, the cell pellet was re-suspended in the serum-containing medium and passed through a 100-mm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ, USA). The cell sus- pension was centrifuged again (275g for 10 minutes) and re- suspended in 10 mL of DMEM/F12 with 10% FBS containing gentamicin (50 mg/mL), and plated onto tissue culture plates (Becton Dickinson) in a humidified 5% CO2, 95% air atmosphere at 37 C for 2 hours. This achieved adherence of microglial cells. The nonadherent astrocytes along with myelin debris were transferred into new culture plates. Astrocytes adhered slowly and were allowed to grow by replacing the medium once a week. New pas- sages of cells were generated by harvesting confluent astrocyte cultures using a trypsineEDTA solution (0.25% trypsin with EDTA,
Invitrogen). Human astrocytes from up to the fifth passage from 4 surgical cases were used in the study.
For estimating the purity of astrocytic and microglial cell cul- tures, aliquots of the cultures were placed on glass slides at 37 C for 48 hours. The attached cells were then fixed with 4% para- formaldehyde for 1 hour at 4 C and made permeable with 0.1% Triton X-100 for 1 hour at room temperature. After washing twice with PBS, the astrocytic culture slides were treated with a mono- clonal anti-glial fibrillary acidic protein antibody (1/4,000, DAKO) and the microglial slides with the polyclonal anti-ionized calcium- binding adapter molecule 1 (Iba-1) antibody (1/500, Wako Chem- icals, Richmond, VA, USA) for 3 hours at room temperature. The slides were then incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG antibody (Invitrogen, 1:500) and Alexa Fluor 546-conjugated goat anti-rabbit IgG antibody (Invitrogen, 1:500) in the dark for 3 hours at room temperature to yield a positive red fluorescence. To visualize all cells, the slides were washed twice with PBS and counterstained with the nuclear dye 40,6-diamidino- 2-phenylindole, dihydrochloride (DAPI) (100 mg/mL, Sigma) to give a blue fluorescent color. Images were acquired using an Olympus BX51 microscope and a digital camera (Olympus DP71). Fluorescent images were colocalized with ImagePro software (Improvision Inc, Waltham, MA, USA). The purity of microglia and astrocytes were more than 99% (2.54 ` 0.54 astrocytes in 500 total cells in micro- glial culture and 3.17 ` 0.62 microglia in 500 total cells in astrocytic culture, n 1⁄4 30).
To achieve SH-SY5Y differentiation, the undifferentiated cells were treated for 4 days with 5-mM retinoic acid (RA) in DMEM/F12 medium containing 5% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin (Singh et al., 2003). The RA-containing medium was changed every 2 days. Differentiated SH-SY5Y cells demonstrated neurite extension, indicative of their differentiation (Lee et al., 2013).
2.3. Experimental protocols
2.3.1. Protocol 1
Human astrocytes, U373 astrocytoma cells and THP-1 cells (5 105 cells), and human microglial cells (5 104 cells) were seeded into 24-well plates in 1 mL of DMEM/F12 medium con- taining 5% FBS. Caffeine, chlorogenic acid, quercetin, and flavones (Sigma, St. Louis, MO, USA) were then introduced at concentra- tions of 100 ng/mL to 1 mg/mL. The stock solutions were prepared with organic solvents; dimethyl sulfoxide (DMSO) for quercetin, caffeine, and CGA and acetone for flavone. Incubation of the mixtures was carried out for 2, 4, 8, or 12 hours. One set of cells was then incubated at 37 C for 2 days in the presence of in- flammatory stimulants. For microglia and THP-1 cells, the stimu- lants were LPS at 1 mg/mL and IFNg at 333 U/mL. For astrocytes and U373 cells, the stimulant was IFNg alone at 150 U/mL. A compa- rable set of cells was incubated in media without inflammatory stimulants. After incubation, the supernatants (400 mL) were transferred to differentiated human neuroblastoma SH-SY5Y cells (2 105 cells per well). The SH-SY5Y cells were incubated for further 72 hours, and MTT assays were performed as described in the following section.
2.3.2. Protocol 2
Because it is perceived that caffeine, chlorogenic acid, quercetin, and flavones could be useful as pharmaceutical agents, they must be shown to be nontoxic to humans. To determine whether they were directly affecting SH-SY5Y cell viability in the presence of LPS/ IFNg-stimulated THP-1 conditioned medium (CM) or IFNg-stimu- lated U373 CM, each compound was added to a glial cell superna- tant (400 mL) just before the supernatants were added to the SH-SY5Y cells. The glial cell supernatants were from THP-1 cells or
U373 cells that had been activated for 2 days with the inflammatory stimulants previously described. The subsequent procedures were the same as in protocol 1.
2.4. SH-SY5Y cell viability assays
The viability of SH-SY5Y cells after incubation with glial cell supernatants was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assays as previously described (Lee et al., 2011). Briefly, the viability was determined by adding MTT to the SH-SY5Y cell cultures to reach a final concentration of 1 mg/mL. After a 1 hour incubation at 37 C, the dark crystals formed were dissolved by adding an SDS/DMF extraction buffer (300 mL, 20% sodium dodecyl sulfate, 50% N, N-dimethylformamide, pH 4.7). Subsequently plates were incubated overnight at 37 C, and optical densities at 570 nm were measured by transferring 100 mL aliquots to 96-well plates and using a plate reader with a corre- sponding filter. Data are presented as a percentage of the values obtained from cells incubated in fresh medium only.
2.5. Measurement of TNFa and IL-6 release
Cytokine levels were measured in cell-free supernatants after 48-hour incubation of THP-1 cells, U373 cells, microglial cells, and astrocytes. The cell stimulation protocols in these experiments were the same as that used in protocol 1. Quantitation was per- formed with enzyme-linked immunosorbent assay (ELISA) detec- tion kits (Peprotech, NJ, USA) following protocols described by the manufacturer.
2.6. Activation of P38 MAPK and NFkB protein by Western blotting
Western blotting on cell lysates was performed as described previously (Lee et al., 2011). Briefly, microglia and astrocytes were exposed to quercetin and caffeine at 10 mg/mL each for 8 hours and were subsequently exposed to stimulants for 2 hours. Human microglia and astrocytes were treated with a lysis buffer (150 mm NaCl, 12 mm deoxycholic acid, 0.1% Nonidet P-40, 0.1% Triton X-100, and 5 mm Tris-EDTA, pH 7.4). The protein concentration of the cell lysates was then determined using a BCA protein assay reagent kit (Pierce, Rockford, IL, USA). Proteins in each sample were loaded onto gels and separated by 10% sodium dodecyl sulfate-poly- acrylamide gel electrophoresis (SDS-PAGE) (150 V, 1.5 hours). The loading quantities of lysate proteins were 100 mg. Following SDS- PAGE, proteins were transferred to a polyvinylidene fluoride membrane (Bio-Rad, CA, USA) at 30 mA for 2 hours. The mem- branes were blocked with 5% milk in PBS-T (80 mm Na2HPO4, 20 mm NaH2PO4, 100 mm NaCl, 0.1% Tween 20, pH 7.4) for 1 hour and incubated overnight at 4 C with a polyclonal anti-phospho- P38 MAP kinase antibody (9211, Cell Signaling, Beverly, MA, USA; 1/2000) or anti-phospho-P65 NFkB antibody (3031, Cell Signaling; 1/1000). The membranes were then treated with a horseradish peroxidase-conjugated anti-IgG (P0448, DAKO, Mississauga, Ontario, CA, USA; 1:2000) or the secondary antibody anti-mouse IgG (A3682, Sigma, 1/3000) for 3 hours at room temperature, and the bands were visualized with an enhanced chemiluminescence system and exposure to photographic film (Hyperfilm ECL, Amer- sham Biosciences). Equalization of protein loading was assessed independently using a-tubulin as the housekeeping protein. The primary antibody was anti-a-tubulin (T6074, Sigma, 1/2000) and the secondary antibody was anti-mouse IgG (A3682, Sigma, 1/ 3000). Primary antibody incubation was overnight at 4 C, and the secondary antibody incubation was for 3 hours at room temperature.
2.7. Glutathione level
The GSH level was assessed by the method of Hissin and Hilf (1976) and Lee et al. (2010a). This assay detects reduced GSH by its reaction with o-phthalaldehyde at pH 8.0. Cells (106) in 1.5-mL tubes were washed twice with PBS, and 200 mL of 6.5% trichloro- acetic acid (TCA) was added. The mixture was incubated on ice for 10 minutes and centrifuged (13,000 rpm, 1 minute). The superna- tant was discarded, and the pellets were re-suspended in 200 mL of ice-cold 6.5% TCA and centrifuged again (13,000 rpm, 2 minutes). Supernatants (7.5 mL) were transferred to 96-well plates containing 277.5-mL phosphate-EDTA buffer (pH 8.0) in 1 M NaOH solution. Then 15 mL o-phthalaldehyde (1 mg/mL in methanol) was added. The reaction mixture was incubated in the dark at room tempera- ture for 25 minutes. The fluorescence at 350 nm excitation/420 nm emission was measured in a multiwell plate reader. The concen- tration was calculated from a standard curve using serial dilutions of reduced GSH. The concentration was expressed as mmol/g protein.
2.8. Protein carbonyls
Protein carbonyl content was determined by a modification of the procedure of Lyras et al. (1996). Cells (3 106) were lysed with a buffer containing 0.2% Triton X-100, 60 mL of protease inhibitor cocktail, and 1 mL of phenylmethylsulfonyl fluoride in 100 mM KH2PO4/K2HPO4 (pH 7.4). The lysate was incubated at 37 C for 2 hours, centrifuged at 8000 g, and the supernatant collected. A 10% (w/v) streptomycin sulfate solution was added to the supernatant at a final concentration of 1% to remove remaining nucleic acid. The solution was mixed at room temperature for 10 minutes and centrifuged for 10 minutes at 8000g. The supernatant was removed and 800 mL divided equally into 2 12 mL plastic centrifuge tubes. For each 1 mL of supernatant, 400 mL of 10-mM 3, 4-dinitrophenylhydrazine in 2 M-HCl was added to one tube and 400 mL of 2-M HCl to the other tube. The tubes were then incubated for 1 hour, and the protein was precipitated by adding an equal volume of 20% (w/v) TCA. The tubes were centrifuged at 8000g, the supernatants discarded, and the pellets washed several times with 1.5 mL of an ethyl acetate ethanol mixture (1:1) to remove excess 3, 4-dinitrophenylhydrazine. The final protein pellets were dissolved in 1 mL 6-M guanidium hydrochloride and the absorbance of both solutions measured at 280 nm and 370 nm as per Reznick and Packer (1994).
2.9. Measurements of 8-OHdG, lipid peroxide and 3-nitrotyrosine by ELISA assays
Levels of 8-hydroxy-2’-deoxyguanosine (OHdG; JaICA, Shi- zuoka, Japan), lipid peroxide (Cayman Chemical, Ann Arbor, MI, USA) and 3-nitrotyrosine (Nitrotyrosine assay kit, Millipore, Temecula, CA, USA) were measured in cell extracts after 2e3 days of incubation. The protocols were the same as previously described measuring cell viability. Quantitation was performed with ELISA detection kits following protocols described by the manufacturer.
2.10. Data analysis
The significance of differences between data sets was analyzed by 1-way or 2-way analysis of variance tests. Multiple group com- parisons were followed by a post hoc Bonferroni test. p values are given in the figure legends.
We purchased small amounts (10 oz 1⁄4 approximately 280 mL) of coffee from 3 different companies and dried it completely at room temperature. Dried weights were found to average 140 mg. To assess the neuroprotective effect of various coffee constituents, we dissolved the dried coffee in sterilized water. We then added supernatants from stimulated THP-1 cells and U373 cells and incubated the mixture for 2 hours (final concentrations: 140 mg/ 280 mL 1⁄4 0.5 mg/mL). The stimulants for THP-1 cells were LPS/IFNg and for U373 cells, IFNg was applied for 2 days. The resultant CM from both THP-1 cells and U373 cells was transferred to RA-differentiated SH-SY5Y cells (experimental protocol 1). MTT assays were performed after 3 days. Data are shown in Fig. 1. There were significant increases in SH-SY5Y cell survival after treatment with CMs from LPS/IFNg-stimulated THP-1 cells and IFNg-stimu- lated U373 cells that had been exposed to coffee and decaffeinated coffee.
In coffee beans, the constituents most likely to exert anti- inflammatory and antioxidative function are caffeine, quercetin, flavonoids, and CGA as well as their derivatives (Kreicbergs et al., 2011). The amounts of the 4 compounds included in 100 grams of coffee beans are 280 mg for CGA, 200 mg for quercetin, 60 mg for flavones (a representative of flavonoids), and 40 mg for caffeine. We calculated the amount of each compound in 140 mg dried weight of coffee (CGA: 392 mg, quercetin: 280 mg, flavones: 84 mg and caffeine: 56 mg). We found that mixtures of the 4 components protected SH-SY5Y cells from toxicity of LPS/IFNg-stimulated THP-1 CM and IFNg-stimulated U373 CM just as much as both types of coffee (Fig. 1). The data indicate that the protective function of coffee beans must come almost entirely from these 4 compounds.
We then investigated the protective effects of caffeine, CGA, flavones, or quercetin (10 mg/mL each), alone, or in combination, against the toxicity of CMs toward RA-differentiated SH-SY5Y cells. The CMs were obtained by 2 days treatment of LPS/IFNg-activated
Fig. 2. Effects of caffeine (CAF in X-axis), chlorogenic acid (CGA in X-axis), flavone (FLA in X-axis), and quercetin (QUE in X-axis) alone (10 mg/mL each) or their combination on SH- SY5Y cell viability changes mediated by (A) LPS/IFNg-stimulated THP-1 and (B) IFNg-stimulated U373 CMs. THP-1 cells and U373 cells were pretreated with each compounds or their mixtures for 2 h and were exposed to stimulants for 2 d. Then the cell-free supernatants were transferred to RA-differentiated SH-SY5Y cells (Experimental protocol 1). MTT assays were carried out in 3 d. Values are mean ` standard error of the mean, n 1⁄4 4. Two-way analysis of variance was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests. *p < 0.01 for LPS/IFNg-activated THP-1 (A) or IFNg-activated U373 groups (B) compared with control (CON) groups, **p < 0.01 for CGA and FLA groups compared with LPS/IFNg-activated THP-1 (A) or IFNg-activated U373 groups (B) or CAF groups, ***p < 0.01 for QUE groups compared with CGA and FLA groups, #p < 0.01 for CAF þ CGA and CAF þ FLA groups compared with CAF groups, ##p < 0.01 for CAF þ QUE groups compared with CAF þ CGA and CAF þ FLA groups, $p < 0.01 for CGA þ FLA groups compared with CAF þ CGA and CAF þ FLA groups, and $$p < 0.01 for CGA þ QUE, FLA þ QUE groups compared with FLA þ CGA groups. Note that quercetin was most potent anti-inflammatory and neuroprotective compounds in coffee components and caffeine was not effective. Abbreviations: CGA, chlorogenic acid; CM, conditioned medium; IFNg, interferon-g; LPS, lipopolysaccharide.
THP-1 cells or IFNg-activated U373 cells and were then transferred to differentiated SH-SY5Y cells. MTT assays were performed after 3 days (see experimental protocol 1). It was found that CGA, flavones, and quercetin, but not caffeine, attenuated SH-SY5Y cell viability loss by THP-1 CM (Fig. 2A) as well as U373 CM (Fig. 2B). Quercetin demonstrated the most protective effect. It was also the most protective when it was added to any of the 3 other compounds.
3.1. Neuroprotectiveeffectofquercetin,caffeine,chlorogenicacid,or flavones against microglial, astrocytic, THP-1, and U373 cell toxicity
The effects of pretreatment with quercetin, caffeine, CGA, or flavones (100 ng/mLd1 mg/mL each) for 2, 4, 8, or 12 hours before incubation on the toxicity of LPS/IFNg-stimulated THP-1 CM toward SH-SY5Y cells were investigated (experimental protocol 1). It was found that quercetin, caffeine, CGA, or flavones attenuated SH-SY5Y cell viability loss by THP-1 CM in a concentration and preincubation time-dependent manner [Fig. 3AeD; (A): 2 hours before incuba- tion, (B): 4 hours before incubation, (C): 8 hours before incubation, and (D): 12 hours before incubation]. Quercetin was the most protective [p < 0.01 compared with untreated group from 3 mg/mL and 2 hours before incubation (A), from 1 mg/mL and 4 hours before incubation (B), from 0.3 mg/mL and 8 hours before incubation (C), and from 0.1 mg/mL and 12 hours before incubation (D)]. Caffeine was the least effective [p < 0.01 compared with untreated group from 100 mg/mL and 2 hours before incubation (A), from 30 mg/mL and 4 hours before incubation (B), from 10 mg/mL and 8 hours before incubation (C), and from 3 mg/mL and 12 hours before in- cubation (D)]. CGA and flavone were also effective but less so than
quercetin [p < 0.01 for quercetin group compared with CGA or flavone groups from 10 mg/mL and 2 hours preincubation (A), from 3 mg/mL and 4 hours preincubation (B), from 1 mg/mL and 8 hours preincubation (C), and from 0.3 mg/mL and 12 hours preincubation (D)]. At the shortest time interval of 2 hours and at the lowest concentration of 3 mg/mL, the protective effect of quercetin was minimal. But the toxicity was reduced to about half at a concen- tration of 1 mg/mL (p < 0.01 from 1 mM). By 12 hours the protective effect had increased to the point where the lowest concentration reduced the toxicity by about 1/4 while the highest concentration reduced it by about 4 fold.
We also investigated the effect of supernatants from LPS/IFNg- stimulated microglia and IFNg-stimulated astrocytes on SH-SY5Y cell viability after treatment of both types of glia with the agents in coffee. Owing to the limited availability of microglia and astrocytes, we only examined the protective effects of quercetin against glial-mediated neurotoxicity. Caffeine was used for com- parison. Both microglia and astrocytes were pre-exposed to the compounds at only 1 time period (8 hours) before treatment with stimulatory agents (experimental protocol 1). For measuring SH-SY5Y cell viability, MTT assays were utilized. It was observed that both quercetin and caffeine reduced the toxicity of both LPS/ IFNg-stimulated microglia (Fig. 5A) and IFNg-stimulated astro- cytes (Fig. 5B) toward SH-SY5Y cells (quercetin: for microglia p < 0.01 compared with control at 0.1 mg/mL and for astrocytes p < 0.01 compared with control at 0.3 mg/mL; and caffeine: for microglia and astrocytes p < 0.01 compared with control at 10 mg/ mL). Again, quercetin had a greater neuroprotective effect than caffeine (for microglia: p < 0.01 compared with caffeine group at the same concentration [0.1 mg/mL] and for astrocytes: p < 0.01 compared with caffeine group at the same concentration [0.3 mg/ mL]). These data demonstrate that the effects observed with cultured microglia and astrocytes are comparable to those observed with the THP-1 and U373 cell lines.
However, although CGA, flavones, quercetin, and caffeine had a protective effect against neuronal SH-SY5Y cells, they had no pro- tective effect against glial cells. Treatment of THP-1 cells or U373 cells for 12 hours, and microglia and astrocytes for 8 hours, with stimulated CM did not change the viability of any glial cells (Supplemental Fig. 1). Fig. 6A and B demonstrates that quercetin and caffeine acted indirectly and were not directly protective of SH-SY5Y cells. When they were added to the CM after stimulation had taken place (experimental protocol 2), they had no effect (A: THP-1 cells and B: U373 cells). As the figure shows, there was no difference between the agents and there was no effect of concen- tration. This establishes that the agents were working by inhibiting
The table summarized the IC50 results of the studies in Figs. 3 and 4. Values are mean ` standard error of the mean, n 1⁄4 4. Two-way analysis of variance was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests. Note that there was a significant reduction in IC50 values between any pre- incubation time groups and that there was a significant reduction in IC50 values of quercetin compared with those of CGA and flavone in the same incubation time groups and of CGA and flavones compared with those of caffeine.
the glial inflammatory response. In summary, quercetin, caffeine, CGA, and flavone were not toxic to the glial cells in the presence of inflammatory stimuli. The viability of SH-SY5Y cells treated with the CM from LPS/IFNg-stimulated THP-1 and IFNg-stimulated U373 cells was unchanged.
3.2. Release of inflammatory cytokines
Inflammatory stimulation of microglia or THP-1 cells causes them to release the inflammatory cytokines TNFa and IL-6 (Hashioka et al., 2007; Klegeris et al., 1999). Fig. 7 shows the effect on TNFa release of treatment of glial cells with quercetin and caffeine (100 ng/mL to 1 mg/mL for 8 hours before incubation). THP-1 release of TNFa (Fig. 7A) and IL-6 (Fig. 7B) and human microglial release of TNFa (Fig. 7C) and IL-6 (Fig. 7D) are illustrated. The release of TNFa (Fig. 7A) and IL-6 (Fig. 7B) was reduced by quercetin and caffeine in a concentration-dependent manner (for quercetin: p < 0.01 for 100 ng/mL or higher and for caffeine: p < 0.01 for 10 mg/mL or higher). The inhibitory effects of quercetin were more powerful than caffeine (p < 0.01 for 300 ng/mL or higher for TNFa and p < 0.01 for 3 mM or higher for IL-6). The pattern was similar in microglia (Fig. 7C and D). LPS/IFNg stimulation caused a 9.5-fold increase of TNFa and IL-6. Treatment with quercetin and caffeine reduced this release (for quercetin: p < 0.01 for 100 ng/mL or higher and for caffeine: p < 0.01 for 300 ng/mL or higher).
For astrocytes, IL-6 is the main inflammatory mediator that is generated (Van Wagoner et al., 1999). Fig. 8 shows comparable data for IL-6 release from U373 cells (Fig. 8A) and cultured astrocytes (Fig. 8B). When cells were activated with IFNg, U373 cells and hu- man primary-cultured astrocytes release IL-6 (7-fold increase in U373 cells and 15-fold increase in astrocytes). However, both quercetin and caffeine reduced the release of IL-6 from IFNg-acti- vated U373 cells or astrocytes in a concentration-dependent manner (for quercetin: p < 0.01 for 100 ng/mL or higher in both cells and for caffeine: p < 0.01 for 30 mg/mL or higher in U373 cells and for 10 mg/mL in astrocytes). Again, quercetin is stronger in reducing IL-6 release than caffeine in both cell types (p < 0.01 for 100 ng/mL or higher in both).
3.3. Activation of intracellular inflammatory pathway in microglia and astrocytes
We investigated the effects of pretreatment with quercetin and caffeine at 10 mg/mL for 8 hours on activation of intracellular inflammatory pathway such as phospho-P38 MAPK and phospho- NFkB production. The data are shown in Fig. 9. On exposure to the stimulants, both microglia and astrocytes showed an increase in these proteins. For microglia, P38 MAPK was increased 8-fold and NFkB 9-fold. For astrocytes, both proteins were increased 8- to 10-fold. Both quercetin and caffeine at 10 mg/mL for 8 hours before
Fig. 5. Effect of pretreatment with caffeine or quercetin for 8 h on SH-SY5Y cell viability changes induced by (A) LPS/IFNg-activated microglial CM and (B) IFNg-activated astrocytic CM as followed by MTT assays. See experimental protocol 1 in Section 2. Values are mean ` standard error of the mean, n 1⁄4 4. One-way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated (A) or IFNg-activated (B) groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg-activated (A) or IFNg-activated groups (B) #p < 0.01 for quercetin groups compared with caffeine groups at the same concentration. Abbreviations: CM, conditioned medium; IFNg, interferon-g; LPS, lipopolysaccharide.
Fig. 6. Effect of treatment with caffeine, CGA, flavones, and quercetin on SH-SY5Y cell viability changes induced by LPS/IFNg-activated THP-1 cell CM (A) or IFNg-activated U373 cell CM (B) as followed by MTT assays. (A) THP-1 cells and (B) U373 cells. After THP-1 cells and U373 cells were stimulated for 2 d with LPS/IFNg or IFNg, respectively their supernatants were transferred to SH-SY5Y cells. Then caffeine, CGA, flavones, and quercetin were added. MTT tests were performed after 3 d (See experimental protocol 2 in Section 2). Values are mean ` standard error of the mean, n 1⁄4 4. One-way analysis of variance was carried out to test significance. Multiple comparisons were followed with post hoc Bonferroni tests where necessary. Note that there are no viability changes when all the compounds were exposed to SH-SY5Y cells after LPS/IFNg-activated THP-1 cell CM (A) or IFNg-activated U373 cell CM (B) were transferred. Abbreviations: CGA, chlorogenic acid; CM, conditioned medium; IFNg, interferon-g; LPS, lipopolysaccharide.
Fig. 7. Effect of pretreatment with caffeine or quercetin on released levels of TNFa (A, C) or IL-6 (B, D) from LPS/IFNg-activated THP-1 cells (A and B) or LPS/IFNg-activated microglia (C and D). 8 h preincubation with caffeine or quercetin was performed before LPS/IFNg was exposed to the cells for 2 d. Values are mean ` standard error of the mean, n 1⁄4 4. One- way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg-activated groups, and #p < 0.01 for quercetin groups compared with caffeine groups at the same concentration. Abbreviations: IFNg, interferon-g; IL-6, interleukin-6; LPS, lipopolysaccharide; TNFa, tumor necrosis factor-a.
incubation attenuated these increases (quercetin: 65%e75% decrease in both phospho-P38 MAPK and phospho-NFkB proteins, and caffeine: 30%e40% decrease in both proteins). Quercetin was again more powerful than caffeine (approximately 50%e60% less than caffeine, p < 0.01).
3.4. Alteration in oxidative stress markers by quercetin and caffeine
In the final set of experiments, we measured the antioxidant properties of quercetin and caffeine on oxidative stress in SH- SY5Y cells caused by LPS/IFNg-stimulated microglial CM and
IFNg-stimulated astrocytic CM. For these experiments, microglia and astrocytes were pretreated with 10 mg/mL caffeine and quercetin for 8 hours and then treated with stimulants (LPS/IFNg for microglia and IFNg for astrocytes). After 2 days incubation, microglial and astro- cytic CMs were transferred to SH-SY5Y cells. Levels of GSH and oxidative damages to DNA, proteins, and lipids were assessed in SH-Sy5Y cells. Data are shown in Figs. 10 and 11. Treatment with quercetin increased intracellular GSH levels by 37% (p < 0.01) but caffeine did not (Fig. 10). Exposure of SH-SY5Y cells to microglial CM and astrocytic CM for 3 days decreased GSH levels by 85%e90%. Quercetin, but not caffeine, attenuated this reduction (p < 0.01).
Fig. 8. Effect of pretreatment with caffeine or quercetin on released levels of IL-6 from IFNg-activated U373 cells (A) or IFNg-activated astrocytes (B). 8 h preincubation with quercetin and caffeine was performed before IFNg was exposed to the cells for 2 d. Values are mean ` standard error of the mean, n 1⁄4 4. One-way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg- activated groups, #p < 0.01 for quercetin groups compared with caffeine groups at the same concentration. Abbreviations: IFNg, interferon-g; IL-6, interleukin-6; LPS, lipopolysaccharide.
Fig. 9. Effect of pretreatment with caffeine or quercetin (10 mg/mL each) for 8 h on levels of phospho-P38 MAPK and phospho-P65-NFkB in LPS/IFNg-activated human microglia (A, left panel) and IFNg-activated astrocytes (A, right panel). Cell extracts were prepared and the proteins separated by SDS-PAGE. Representative blots are shown in (A) and quantitative results in (B). To ensure equal loading, the densitometric value of each band was normalized to the corresponding band for a-tubulin. Values are mean ` standard error of the mean, n 1⁄4 3. One-way analysis of variance was carried out to test significance. $p < 0.01 for LPS/IFNg-activated microglial or IFNg-activated astrocytic groups compared with CON groups in each condition, *p < 0.01 for quercetin or caffeine groups compared with LPS/IFNg-activated microglial or IFNg-activated astrocytic groups, #p < 0.01 for quercetin groups compared with caffeine groups. Abbreviations: IFNg, interferon-g; LPS, lipopolysaccharide.
Fig. 11AeD demonstrates production of the oxidative damage products, 8-OHdG (A), protein carbonyl (B), lipid peroxide (C), and 3-nitrotyrosine (D). These products were substantially generated under normal conditions and were significantly reduced by quercetin (p < 0.01). Again, caffeine was not effective. When SH- SY5Y cells were exposed to microglial and astrocytic CM for 3 days, these damage products were increased (8-OHdG: 2 fold increase, protein carbonyl: 2-fold increase, lipid peroxide: 4.2- to 4.5-fold increase and 3-nitrotyrosine: 7.5- to 9-fold increase, p < 0.01). Treatment with quercetin attenuated the increase (8-OHdG: 35%e40% decrease, protein carbonyl: 35% decrease, lipid peroxide: 40%e50% decrease, and 3-nitrotyrosine: 40% decrease, p < 0.01). Caffeine was without effect.
Brain injury results in neuroinflammation by activating micro- glia and astrocytes to release proinflammatory factors such as cytokines, toxic free radicals, and proteases. Oxidative stress in neuronal cells plays important direct and indirect roles in their death (Cobb and Cole, 2015). This can be reduced by the simple expedient of drinking coffee. Several epidemiologic studies attest to
Fig. 10. Effect of pretreatment with caffeine or quercetin (10 mg/mL each) for 8 h on changes in levels of intracellular GSH concentration in SH-SY5Y cells induced by LPS/ IFNg-activated microglial CM and IFNg-activated astrocytic CM. Microglia and astro- cytes were pretreated with caffeine and quercetin and exposed to stimulants (LPS/IFNg for microglia and IFNg for astrocytes) 2 d. Cell-free supernatants were transferred to RA-differentiated SH-SY5Y cells. The cells were collected to measure the 4 damage parameters in 3 d. One-way analysis of variance was carried out to test significance. *p < 0.01 for quercetin groups compared with CON groups in normal condition, $p < 0.01 for LPS/IFNg-activated microglial or IFNg-activated astrocytic groups compared with CON groups in normal condition, and **p < 0.01 for quercetin groups in LPS/IFNg-activated microglial or IFNg-activated astrocytic CM treated condition compared with LPS/IFNg-activated microglial or IFNg-activated astrocytic groups. Abbreviations: CM, conditioned medium; GSH, glutathione; IFNg, interferon-g; LPS, lipopolysaccharide; RA, retinoic acid.
a reduction in the risk of developing PD and AD in late life by developing the habit of drinking coffee in early midlife. Caffeine was assumed to be the active agent (Arendash and Cao, 2010; Eskelinen and Kivipelto, 2010; Hernán et al., 2002). However, this assumption was not supported by results of Ding et al. (2015), who found there was no difference in protective effects between caffeinated and decaffeinated coffee. These studies all assessed the protective effects over the long term. Such protective effects may not be revealed by short term studies. For example, Laitala et al. (2009) studied coffee consumption in 2006 middle-aged Finnish twins between 1975 and 1981. They found that coffee consumption over this 6-year interval was not an independent predictor of cognitive performance in old age. Similarly van Boxtel et al. (2003) measured cognitive performance in 1376 individuals from the Maastricht Aging Study over the same 6-year interval. They found no significant difference in cognitive decline from ages 24 to 81.
In this study, we have shown that quercetin, not caffeine, is the major constituent in coffee which inhibits glial-mediated toxicity against neuronal SH-SY5Y cells. In a standard cup of coffee, caffeine occurs in smaller amount than quercetin, CGA, and flavonoids (40 mg/100 grams of dried coffee) (Barone and Roberts, 1996). However, it needs very high concentrations to show anti- inflammatory and neuroprotective properties (at least 100 mg/mL in both THP-1 and U373 cells, Figs. 3 and 4). We found there was no significant difference in a coffee mixture of compounds with and without caffeine in our experimental condition (Fig. 1). This finding is consistent with the epidemiologic study of Ding et al. (2015).
Quercetin protected against SH-SY5Y cell loss after exposure to LPS/IFNg-stimulated microglia and IFNg-stimulated astrocytes. The effect was concentration and incubation time-dependent (Figs. 3e5). Quercetin also inhibited activation of proin- flammatory pathways such as P38 MAP kinase and NFkB stimula- tion (Fig. 8). This led to a reduction in the release of proinflammatory factors such as TNFa and IL-6 from LPS/IFNg- stimulated microglia and their surrogate THP-1 cells and IFNg-stimulated astrocytes and their surrogate U373 astrocytoma cells (Figs. 6 and 7). It was demonstrated that caffeine has these prop- erties but only very weakly. The other major coffee components, CGA and flavones, are more effective than caffeine (Fig. 2).
Quercetin also has antioxidative properties, which significantly increased intracellular GSH levels ([GSH]i) in SH-SY5Y cells under normal conditions. It also attenuated the reduction in [GSH]i in SH-SY5Y cells caused by glial CMs (Fig. 9). This phenomenon reflected a decrease in 8-OHdG, a biomarker of oxidative damage to DNA (Kasai, 1997) (Fig. 10). Quercetin also attenuated the increase in protein carbonyls, a general marker of oxidative damage to amino acids in proteins (Stadtman and Burlett, 1998). It reduced lipid peroxide, a product of the attack of reactive oxygen species on unsaturated fatty acids in lipids (Adibhatla and Hatcher, 2010), as well as 3-nitrotyrosine, a product of the attack of reactive nitrogen species such as peroxinitrite on tyrosine in proteins (Ischiropolous, 1998). Caffeine did not show any of these antioxidant functions.
Previously, we reported that depletion of GSH in both microglia and astrocytes induces neuroinflammation and results in neuro- toxicity (Lee et al., 2010a). Therefore, it can be concluded that quercetin protected SH-SY5Y cells not only by reducing the release of proinflammatory factors from glial cells but also by inhibiting attack by reactive oxygen species/reactive nitrogen species in glial CMs.
In our studies, we found that CGA and flavones also have anti- inflammatory and neuroprotective properties although they are weaker than quercetin (Figs. 2e4). It is possible that other unidentified coffee constituents could also exert antioxidative, anti- inflamatory, and neuroprotective effects via other mechanisms such as by anti-amyloid, anti-caspase, or other mechanisms.
Chronic neuroinflammation is closely associated with the pathogenesis of several neurodegenerative diseases, including AD and PD (McGeer and McGeer, 2002; McGeer et al., 2003; Whitton, 2007). Quercetin is a readily available protective. It may have potential to be a useful therapeutic agent in AD, PD, and possibly other neurodegenerative disorders.
Several experiments were carried testing response to NAD+ levels in both human and mouse test subjects.
In one, a human subject was given 1000 mg of NR daily for 7 days, and blood NAD+ levels were tested. It was found that the single dose of Nicotinamide Riboside raises NAD+ levels up to 270%.
NAD+ levels were raised approximately 4 hours after ingesting NR, and peaked at approximately 8 hours. NAD+ remained elevated 24 hours later.
Further testing involved 6 male and 6 female adults. Subject received 100 (purple), 300 (red), or 1000mg (black) of NR, with a 7 day washout period between doses to monitor the response on NAD+ levels.
Subjects showed the increase to NAD+ levels was dose dependent.
The average NAD+ increases were 30% at 100mg, and 50% at the higher dosages.
The lead researcher, Charles Brenner, PhD, was somewhat surprised that a single dose of 100 mg was able to raise NAD+ levels, however it seems the 300mg dose was much more effective over a 24 hour span.
Optimum dosage of NR?
The 300 and 1000mg dosages resulted in similar maximum increase in NAD+ levels at 24 hours. However the 1,000 mg dose resulted in higher increase at 8 hours.
As participants were given a single dose, with a washout period of 7 days between dosages, it is not known what the cumulative effect of daily dosages would achieve.
It is quite possible the 300 mg dosage would be even higher at the 8 hour point on days 2,3,4 and on.
The optimum dose for maximum NAD+ elevation with minimum NR dosage is likely closer to 300 mg than 1,000 mg. A cost effective guess would be 300 to 500mg per day.
Among the dozens of studies underway currently, one involves NR taken daily for eight consecutive weeks is underway to measure the long term NAD+ increase from NR and determine the most effective dosage.
Why NAD+ levels matter
Prior research has shown supplementation with NMN can raise NAD+ levels and result in more youthful cellular function thru improved mitochondria health. NAD+ levels naturally drop as we age and is thought to be a key driver in many age related diseases and health problems. Restoring NAD+ levels to youthful levels has proven to work in restoring youthful energy and performance in mouse studies.
NAD+ is a key factor in cellular health as it is required to power all activity in every cell of our bodies.
Increased NAD+ levels is key to maintaining healthy cellular activity and mitochondrial function. Besides the decrease in NAD+ levels due to aging, they are also used up when battling chronic problems such as cancer. When this happens, the mitochondria are less effective which causes many health problems.
RESEARCH SHOWS BENEFITS OF INCREASED NAD+
One example is shown in a mouse study published in November 2014 that showed NR was effective at restoring NAD+ levels in mitochondria in mice that suffered from the accelerated aging disease known as Cockayne Syndrome (CS). NR was described to show promise as a therapy for the disease in addition to other chronic health problems that result in lower NAD+ levels.
Below are some of the benefits that have been proven to result from increased NAD+ levels in studies with mice.
Less muscles soreness
Improved muscle endurance
have been completed showing:
hearing damage protection
NAD+ levels restored to youthful levels
Testing in humans for any potential Anti-Aging benefits is further down the road, due to the extreme cost and time involved. Also, the FDA does not recognize aging as a disease, so any proof that it may slow or even reverse aging cannot be claimed.
However, more short term testing for specific age-related disease and conditions is ongoing in Humans and Mice.
About NIAGEN brand Nicotinamide Riboside (NR)
Niagen is manufactured by Chromadex and is the only supplier of NR, with multiple patents from Dartmouth College, Cornell University and Washington University.