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.
NAD+ is a co-enzyme found in all animals that enable the mitochondria to perform their role in powering the basic functioning of every cell in our bodies. NAD+ levels naturally drop as we age and is thought to be a key driver in many age related diseases and health problems.
Prior research has shown that increased NAD+ levels in older mice results in improved energy and muscle performance similar to that of young animals.
This study was performed using Niagen, which is the only commercially available brand of Nicotinamide Riboside (NR). They also compared the effects of NR with that of similar dosage of Nicotinamide (also referred to as niacinamide) , a more common and better studied form of B3 vitamin with a long record of safe usage.
In the first phase of the study, mice that were fed 5000 mg per kg of bodyweight exhibited no mortality.
The second phase was a 90 day assessment comparing dosages of 300, 1000, and 3000 mg/kg of bodyweight per day.
Researchers compared the effects on liver, kidneys, ovaries, and testes and found no toxicity at any of the dosages tested.
Their was no adverse effects noted at the 300 mg level. Minor effects at 1000 and 3000 mg levels were similar to that of Nicotinamide.
There are guidelines used by the FDA to translate the dosages used in mice to the equivalent dosage for the Human Equivalent Dose (HED).
The 300 mg/kg dosage equals 48mg/kg HED, which would be approximately 2880 mg a day for a 132 lb human, which is well over the dosage recommended. However, the FDA requires a 10x safety factor which would result in a dosage of 288 mg, which is at the lower end of recommended dosage.
Comparing results on animals given NR vs Niacinamide, the researchers concluded that NR has a similar toxicity to niaciniamide.
Niacinamide has a long track record of safe use in humans, and is rated as LIKELY SAFE at normal dosages. At dosages above 3 grams per day, Niacinamide can result in serious side effects including liver problems, gout, vision, elevated blood sugar, and other serious problems.
3 grams of Niacinamide per day equals roughly 7 grams per day of Nicotinamide Riboside.
Based partly on the results of this study, Chromadex was able to obtain GRAS status for Niagen.
Niagen (Nicotinamide Riboside) recognized as GRAS (Generally Recognized as Safe)
The company called ChromaDex Corporation, an innovator of nutritional and health ingredients, announced that an independent scientific panel of experts determined that NIAGEN , a patented formula of Nicotinamide Riboside (NR) is Generally Recognized As Safe (GRAS).
Generally recognized as safe (GRAS) is an American Food and Drug Administration (FDA) designation that a chemical or substance added to food is considered safe by experts, and so is exempted from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) food additive tolerance requirements.
It is a very strong testimony that Nicotinamide Riboside supplementation is safe without side effect. It should be noted here that the GRAS status was only given to Chromadex’s commercially available form of nicotinamide riboside (NR) called Niagen. This formula is protected by five patents issued and several pending and so far it appears this is the only NR form that is known to be commercially available. Hence when buying NR it seems prudent to ensure oneself of the source to avoid buying false formula’s.
Frank Jaksch Jr., founder and CEO of ChromaDex, commented, “Receiving GRAS status immediately allows NIAGEN to be included as an ingredient in both food and beverages –applications that we believe presents a very substantial opportunity for ChromaDex. Coupled with the recent NDI status, we believe the stage is set for widespread commercialization of NIAGEN as an innovative and compelling ingredient across a myriad of consumer products.”
As schematized in Figure 1, the reason that a poor diet can produce a requirement for Na and Nam is that Trp, Na, and Nam are all NAD+ precursors (7). Trp is converted to NAD+ through an eight-step de novo pathway (Figure 2), so termed because the Nam base is essentially made from scratch. In contrast, Na and Nam are considered “salvageable precursors” that require only three steps and two steps, respectively, to rebuild NAD+. Nicotinamide riboside (NR) is an additional salvageable NAD+ precursor vitamin with a two-step pathway (14) and a three-step pathway (8) to form NAD+. As schematized in Figures 1 and 2, cells require ongoing NAD+ synthesis because NAD+ and NADP are not only coenzymes, which are recycled back and forth between oxidized (NAD+ and NADP) and reduced (NADH, NADPH) forms by hydride transfer enzymes, but are also substrates of NAD+ -consuming enzymes that break the glycosidic bond between the Nam moiety and the ADPribose moiety. NAD+consuming enzymes transfer ADPribose and/or ADPribose polymers, form signaling compounds from NAD+ and NADP, and reverse the acetyl modification of protein lysine residues. Each of these reactions consumes an NAD+ equivalent to a salvageable Nam product plus an ADPribosyl product (7).
NAD+-consuming enzymatic activities are induced, in part, by stresses such as DNA damage and inflammation. Many of these stresses are accompanied by specifically induced biosynthetic pathways, which appear to function to maintain NAD+ homeostasis. The term NAD+ homeostasis should be used cautiously, however, because it is not clear that cells are always in NAD+ homeostasis. Mammalian NAD+ biosynthesis is not a closed, cell-autonomous system, and there appear to be situations in which cells actively increase and/or reduce the concentration of NAD+ and NAD+ metabolites to promote vital and/or regulatory functions, including cell death.
RECOMMENDED DAILY REQUIREMENTS OF THE NAD+ PRECURSORS
Despite the fact that the biosynthetic pathways are not the same, in the literature of human and animal nutrition, Nam and Na are collectively termed niacin and/or vitamin B3 . To protect against pellagra that can develop with Trp deficiency, recommended daily allowances (RDAs) of niacin are 16 and 14 mg
per day for adult men and women, respectively (79). Because all plant, animal, and fungal inputs in our diet contain cellular NAD+ and NAD+ metabolites, foods provide NAD+ , NADH, NADP, and NADPH, which are considered nutritional “niacin equivalents,” in addition to Nam and Na. Whereas Nam and Na are the fully broken down NAD+ metabolites from animals and plants/fungi/bacteria respectively, NR and nicotinic acid riboside (NaR) can be considered partly broken down niacin equivalents. In the genetically tractable yeast system, all of the salvageable precursors (Na, Nam, NR, and NaR) support the growth of cells inactivated for de novo NAD+ synthesis (83).
A single yeast cell deficient in de novo synthesis or undergoing a biological process that requires more than the minimum vital concentration of NAD+ must convert an available vitamin precursor to NAD+ in a cell-autonomous fashion. In contrast, humans exhibit the complexity of systemic NAD+ metabolism in which particular cells may utilize an NAD+ precursor to produce an excess of NAD+ and export salvageable precursors to other cells. Accordingly, dietary Trp is also classified as a niacin equivalent. However, because of the protein and other biosynthetic uses of Trp, 60 mg of Trp is considered the equivalent of 1 mg of niacin (79). This physiological fact, that high levels of dietary Trp result in circulation and excretion of Na (9, 10, 60, 70), has resulted in claims in textbooks and reviews that Na is derived from Trp. Whereas this is true in vertebrate organisms, there may not be a vertebrate intracellular pathway that is responsible. As illustrated in Figure 2, Trp can be converted to Nam but not Na in any vertebrate cell expressing a de novo pathway and an NAD+-consuming enzyme such as poly(ADPribose)polymerase (PARP) or a sirtuin. Nam can then be converted in the intestinal lumen by bacterial nicotinamidase to Na. The most reasonable pathway by which a single vertebrate cell might convert Trp to Na was depicted by Magni and coworkers (53). Magni’s scheme of human NAD+ metabolism depicts the activity of Na phosphoribosyltransferase (NAPRT1 gene product) and several other enzymes as bidirectional. Indeed, if there is sufficient pyrophosphate and NaMN in cells, then Naprt1 could catabolize nicotinic acid mononucleotide (NaMN) to Na, thereby creating a cell-autonomous route from Trp to Na. This is an interesting possibility that has not been demonstrated in vivo.
NR is a newly discovered salvageable precursor of NAD+ that occurs in cow’s milk (14). Studies in Saccharomyces cerevisiae have shown that, like Na and Nam, NR is an NAD+ precursor that contributes to maintaining intracellular NAD+ concentration and improves NAD+ dependent activities in the cell including Sir2-dependent gene silencing and longevity (8, 14). NR can either be converted to NAD+ by the Nrk pathway (14), which is induced by axotomy in dorsal root ganglion (DRG) neurons (71), or by the action of nucleoside phosphorylase and nicotinamide salvage (8). It has also been shown that the same two pathways required for NR salvage in yeast cells can also be used for NaR salvage (83). In yeast cells, NR clearly qualifies as a vitamin by virtue of rescuing growth of strains deficient in de novo synthesis (14, 83), improving Sir2 functions (8), and utilizing a dedicated transporter (8a). Additionally, because cells deleted for the NR/NaR salvage enzymes have a significant deficiency in intracellular NAD+ when not supplemented with these compounds, it appears that NR and/or NaR are also normal metabolites (8).
Five lines of reasoning support designation of NR as an authentic NAD+ precursor vitamin in vertebrates. First, Haemophilus influenza, a flu-causing bacterium, which has no de novo pathway and cannot utilize Na or Nam, is strictly dependent on NR, NMN, or NAD+ for growth in the host bloodstream (22). Second, milk is a source of NR (14). Third, NR protects murine DRG neurons in an ex vivo axonopathy assay via transcriptional induction of the nicotinamide riboside kinase (NRK) 2 gene (71). Fourth, exogenously added NR and derivatives increase NAD+ accumulation in a dose-dependent fashion in human cell lines (94). Fifth, Candida glabrata, an opportunistic fungus that depends on NAD+ precursor vitamins for growth, utilizes NR during disseminated infection (51).
It should be realized that not every cell is capable of converting each NAD+ precursor to NAD+ at all times. Expression of the eightstep de novo pathway is required to utilize trp. Expression of the Nampt pathway is required to utilize Nam. Expression of either the Nrk pathway or nucleoside phosphorylase and the Nampt pathway is required to utilize NR. Finally, expression of the Preiss-Handler pathway is required to utilize Na. Because tissue and celltype specific enzyme expression differences exist, the precursors are differentially utilized in the gut, brain, blood, and organs. Understanding the unique aspects of metabolism of each precursor is necessary to define the mechanisms underlying the physiological effects and side effects of each.
NAD+ FUNCTIONS AND THE REQUIREMENT FOR NAM SALVAGE
NAD+ is classically known as a coenzyme for hydride-transfer enzymes. As a coenzyme, NAD+ is essential to a variety of diverse biological processes including energy production and synthesis of fatty acids, cholesterol, and steroids. NAD+ participates in oxidationreduction (redox) reactions as hydride donor (NADH and NADPH) and acceptor (NAD+ and NADP). NAD+ most commonly functions in energy-producing catabolic reactions, such as the degradation of carbohydrates, fats, proteins, and alcohol, whereas NADP functions in anabolic reactions, such as the synthesis of cellular macromolecules including fatty acids and cholesterol. As depicted in Figure 1, coenzymatic activities of NAD+ and its reduced and phosphorylated derivatives interconvert but do not alter total cellular levels of NAD+.
In recent years, multiple enzyme-mediated, nonredox roles for NAD+ have been discovered. NAD+-consuming enzymes break down NAD+ to Nam and an ADPribosyl product (7). These enzymes fall into three classes. The first class consists of ADPribose transferases (ARTs) and PARPs, which transfer and/or polymerize NAD+-derived ADPribose, frequently as a post-translational modification. PARP activity, which is upregulated by DNA strand breaks (54), may be the major source of intracellular NAD+ consumption. The second class of NAD+-consuming enzymes consists of cADPribose synthases, which are membranebound ecto-enzymes also known as CD38 and CD157, that produce and hydrolyze the Ca2+-mobilizing second messenger cADPribose from NAD+ (38, 39, 43). Additionally, CD38 catalyzes a base exchange between NADP and Na to form nicotinic acid adenine dinucleotide phosphate (NaADP) (1), which is also a hydrolytic substrate (30). The products of these reactions have distinct and important roles in Ca2+ mobilization and signaling. The third class of NAD+-consuming enzymes consists of sirtuins, named for their sequence similarity to the yeast Sir2 genesilencing protein. Sirtuins exhibit protein lysine deacetylase and, occasionally, ADPribose transferase activities. Sirtuin deacetylation reactions proceed by binding an acetyl-modified lysine on a target protein and NAD+ in distinct pockets. Deacetylation of the modified lysine side chain is coupled to the cleavage of the glycosidic bond in NAD+ such that the products are the deacetylated lysine, acetylated ADPribose, and Nam (72). Sirtuin-dependent deacetylation of histones and other proteins results in reprogrammed gene expression, mitochondrial synthesis and function, cell survival, and longevity (91). Sirtuins have been recently reviewed as master switches of metabolism (18a).
Nam regulates the activity of NAD+ consuming enzymes both by direct enzyme inhibition and by its role as an NAD+ precursor. Nam inhibits NAD+ -consuming enzymes by binding a conserved pocket that participates in NAD+ binding and catalysis (4). Recycling of Nam back to NAD+ raises NAD+ levels, increasing substrate availability and relieving Nam inhibition. In systems such as yeast in which NAD+ concentration has been determined carefully, basal intracellular NAD+ concentration is approximately 0.8 mM and can be elevated with vitamin precursors by 1 mM or more (8). At first glance, it would seem that 0.8 mM NAD+ should be saturating for virtually all sirtuins, which exhibit Km values for NAD+ between 5 μM and 500 μM (72). However, the first 0.8–1 mM of intracellular NAD+ concentration may largely be bound by redox enzymes such that salvage-derived synthesis of NAD+ may generate the majority of free NAD+ to drive sirtuin functions.
The first step in Nam salvage is catalyzed by Nam phosphoribosyltransferase (Nampt), which is encoded by the PBEF1 gene. This polypeptide, first identified as an extracellular protein termed pre B-cell colony enhancing factor (PBEF), was characterized as cytokine that enhances the maturation of B-cell precursors in the presence of IL-7 and stem cell factor (69). The same polypeptide was also termed
Visfatin, due to reported insulin mimetic functions and regulation of systemic metabolism (26). However, the intracellular form of the molecule was shown to be induced in activated lymphocytes and function simply as Nampt (68).
Because of the disparate functions ascribed to the same polypeptide, it had been assumed that intracellular Nampt functions as an NAD+ biosynthetic enzyme, whereas extracellular Nampt has cytokine and insulin-mimetic roles. However, it has now been reported that both intraand extracellular Nampt exhibit robust phosphoribosyltransferase activities and that inhibiting NAD+ biosynthesis through the Nampt pathway, either genetically or pharmacologically, causes impaired glucose tolerance and reduced insulin secretion in mice, a defect that can be corrected by administering nicotinamide mononucleotide (NMN) (66). Moreover, murine plasma contains high concentrations of Nampt and NMN, which are reduced in nampt heterozygous animals. These new findings strongly indicate that the primary role for extracellular PBEF/Visfatin/Nampt is to catalyze NMN production from Nam, and that NMN has an important role in maintaining β-cell function (66). In a recent review, we speculated that a partially extracellular NAD+ cycle might consist of a Nampt step, followed by extracellular dephosphorylation of NMN to NR, intracellular transport of NR, and conversion of NR to NAD+ (7).
NAD+ PRECURSORS IN FOODS
Consistent with Goldberger’s studies (29), niacin is abundant in meat, eggs, fish, dairy, some vegetables, and whole wheat. Notably, corn contains abundant Na and Nam, largely present in bound forms that are not bioavailable. Treatment with alkali is used to increase bioavailability, a practice that protected native and South American populations from deficiency. Untreated corn is considered “pellagragenic,” causing increased sensitivity to low dietary niacin concentrations in animal studies (46, 47). Milk, now known to be a natural source of NR (14), was shown to counteract the growth defect seen in corn-fed animals (46). In meats, Na and Nam are scarce and NAD+ and NADP are the abundant sources of niacin (34, 86). Nam is produced by mucosal enzymes that cleave NAD+ (86), and Na is produced from Nam by deamination by bacterial nicotinamidase in the gut (13). Both Na and Nam are absorbed from the alimentary canal and enter the bloodstream for distribution to tissues (40, 82, 86). Studies indicate that Nam is the dominant absorbed form of niacin when the dietary sources are NAD+ and NADP (16, 35, 36, 81). However, it has also been reported that NAD+ is digested by pyrophosphatases to NMN and hydrolyzed to NR, which was found in the lumen of the upper small intestine (32). We surmise that NR is incorporated into the cellular NAD+ pool via the action of Nrk pathway (14) or via Nam salvage after conversion to Nam by phosphorolysis (8).
OF NAD+ BIOSYNTHESIS
Qprt, Nampt, Naprt1, and Nrk1,2 are the committed enzymes in the synthesis of NAD+ from Trp, Nam, Na, and NR. As such, by examining the expression of each enzyme and by following the metabolic fates of dietary inputs, one can describe tissue-specific pathways of NAD+ biosynthesis.
Animals on diets containing sufficient amounts of both Trp and niacin have a measurable concentration of each in liver (9, 10). Nam and Na, however, are thought to supply only a fraction of the NAD+ produced in the liver, with much niacin circulating to other tissues. Trp is thought to be the principal NAD+ precursor utilized in liver (9). In addition to producing quinolinate for entry into NAD+ biosynthetic pathways, Trp is incorporated into protein, utilized to generate energy through total oxidation, and utilized to form kynurenic acid. Inducible enzymes of Trp utilization regulate the flux of Trp through different pathways depending on diet and cellular metabolic state. Under conditions of low Trp consumption, circulating levels of Trp decrease and enzymes that direct Trp to non-NAD+ biosynthetic routes are down-regulated, suggesting a shift of all possible Trp catabolism to NAD+ generation (75). Supplementing high amounts of Trp allows more flux to the oxidative branch and allows increased levels of Nam to be released into the vasculature (6, 9, 10). All Trp that reaches quinolinate in the liver is thought to be converted to NAD+ via subsequent enzyme reactions of Qprt, Nmnat1-3, and Nadsyn1, which are highly expressed in liver (24, 58).
In addition to liver (27), Qprt is expressed in human and rat brain and plays a critical role in protection against the neurotoxic effects of quinolinate (23, 45, 58, 93). Quinolinate is a potent endogenous neurotoxin, and elevated levels in brain are associated with neurodegenerative disorders including epilepsy and Huntington’s disease (73, 74). The normal concentration of quinolinate in the brain was found to be in the lowto mid-nanomolar range (37), and Qprt activity increases in response to increased levels of quinolinate (25), suggesting a protective role. The highest levels of quinolinate are found in spleen, lymph nodes, thymus, and many specific immune cell types and are increased following stimulation by immune activators (57).
Activated lymphocytes induce expression of Nampt (68), which is also expressed in smooth muscle cells with loss of expression in senescence (87). In the mouse, Nampt has been shown to circulate and to be highly expressed intracellularly in brown adipose tissue, liver, and kidney, with fat as the source of extracellular Nampt. Human fat is also a source of circulating Nampt (66). From classical feeding studies, the testes were found to utilize Nam rather than Na or quinolinate (50), and blood and liver were also found to be major sites of Nam utilization (41). Nam crosses the blood-brain barrier and is converted to NAD+ in brain, though it is not known whether Nam is a precursor of NAD+ in neuronal or non-neuronal cells (78). Studies with DRG neurons suggest that Nampt is not neuronally expressed (71). Nam and NR are also taken up by intestinal epithelial cells and both are utilized by Nam salvage (15).
By global analysis of mRNAs, Naprt1 is expressed in most tissues of the adult mouse, including colon, heart, kidney, and liver, suggesting the presence and utilization of the substrate Na as an NAD+ precursor in these tissues (21). Classical feeding studies showed that exogenously added Na is a better NAD+ precursor than Nam in liver, intestine (16), and kidney (50). Similarly, rats fed Na showed elevated levels of NAD+ in the heart and kidney in addition to blood and liver, which are sites of Na and Nam utilization (41). Classical studies have been corroborated by a recent report that mouse Naprt1 is expressed in intestine, liver, kidney, and heart. In addition, human kidney cell lines are able to use Na to increase intracellular NAD+ concentration in a manner that depends on the NAPRT1 gene. Moreover, Naprt1 expression decreases vulnerability to oxidative damage from NAD+ depletion. The use of Na as an NAD+ precursor in normal and stress conditions implicates the presence of Na as a normal cellular metabolite in humans (33). As discussed earlier, we consider the bacterial flora of the intestinal lumen to be the first major site in a vertebrate for production of Na, though bacterial and fungal degradation of cellular NAD+ in food and direct Na supplementation will also produce supplies of Na in the alimentary canal for distribution to tissues through the vasculature. True intracellular production of Na in a vertebrate cell (53) would require high levels of Trp and/or NAD+ and substantial reverse flux through what are usually considered anabolic pathways.
The use of NR as a precursor in mammalian cell types was first demonstrated in DRG neurons, which induce the NRK2 transcript when damaged by axotomy (71). The ubiquitous expression of Nrk1 in mammalian tissues (80) suggests utilization of NR and/or NaR (83) in a diverse array of cell types. However, Nrk2 is present in heart, brain, and skeletal muscle, and is notably absent in kidney, liver, lung, pancreas, and placenta (48, 71). The fact that DRG neurons cannot be protected from damage
induced neuropathy by Na or Nam without concurrent gene expression of Na or Nam salvage genes suggests that NR is a uniquely useful precursor to the nervous system (71) when de novo synthesis of NAD+ from Trp is not sufficient.
Available data summarizing the system-wide use of NAD+ precursors are summarized in Figure 3. The data are more static than one would like, such that it will be important to determine how gene expression and precursor utilization changes as a function of nutrition, age, stress, and disease state. It is particularly striking that two enzymes, namely Nampt and Nrk2, were first identified as highly regulated proteins involved in immune cell (69) and muscle cell (48) development. Thus, developmental regulation of NAD+ synthesis and utilization remains on the forefront of NAD+ biology.
ALTERATION OF NAD METABOLISM BY CALORIC RESTRICTION
Caloric restriction (CR) is the most effective intervention to extend the lifespan of multiple model organisms including mammals. CR is defined as a 20% reduction versus ad libitum feeding without compromising adequate nutrition (56). Although the mechanisms of CR remain elusive, it is thought that CR modulates fat and carbohydrate metabolism, attenuates oxidative damage, and activates a stress-induced hormetic response that mediates improved vitality and disease resistance (55). Among these three major mechanisms, modulation of fat and carbohydrate utilization is the most direct response to reduced dietary inputs, and hormesis is potentially the mechanism most influenced by the “signaling” aspect of CR.
The hormetic theory is supported by experiments in which model organisms exhibit extended lifespan when placed in a variety of sublethal stress conditions including high temperature, high salt, or osmotic stress. In yeast, such conditions increase expression of nicotinamidase, thereby altering NAD+ metabolism in a manner that favors Sir2 activity (2). The principal mechanism by which Sir2 extends lifespan in a wild-type yeast cell is repression of formation of aging-associated extrachromosomal ribosomal DNA circles (76). Though this mechanism is unique to yeast, there are substantial data showing that sirtuins are conserved from fungi to metazoans to mediate some of the beneficial effects of CR (77). Sir2 is not the only mediator of CR-induced lifespan extension in yeast (42), nor are sirtuins necessarily the only targets of nicotinamide inhibition (84). Nonetheless, there is excellent evidence that NAD+ metabolism is altered in vertebrate systems by CR and that increased activity of sirtuins may mediate beneficial brain and liver physiology under CR conditions.
In CR-treated mice, brain NAD+ levels are increased and Nam levels are decreased, and these changes accompany neuronal Sirt1 activation, which reduces Alzheimer’s neuropathology (63). In fasted mice, NAD+ levels are increased in liver, which is accompanied by Sirt1 activation, PGC1α deacetylation, and increased mitochondrial biogenesis (67). The mechanisms by which lower food inputs increase NAD+ levels in brain and liver are completely unknown. Two potential mechanisms that may account for this phenomenon are systemic mobilization of NAD+ precursors to the brain and liver and reduced NAD+ breakdown. Among the potential precursors that could mediate this phenomenon, Na and Trp seem unlikely because one would expect that increased food consumption would be required to increase their availability. Analysis of CR-induced systemic metabolites should permit the detection of either Nam or NR as candidate mediators of increased brain and liver NAD+ levels. Reduced NAD+ breakdown is another mechanism by which CR might increase NAD+ levels in particular tissues. This could occur if an NAD+-consuming activity such as PARP is negatively regulated by CR. The pathways that produce NR or NaR at the expense of NAD+ (8) are not known. These, too, might be negatively regulated by CR in order to elevate brain and liver NAD+.
LESSONS FROM THE wlds MOUSE
Wallerian degeneration refers to the ordered process of axonal degeneration. Wallerian degeneration occurs when an axon is severed from the cell body, and proceeds via characteristic fragmentation of cellular components initiated by a factor or factors intrinsic to the neuron (28, 31). The distal part of a severed or damaged axon usually undergoes Wallerian degeneration within 24–48 hours of injury (88). This type of axonopathy is thought to be a critical, early event in neurodegenerative conditions including multiple sclerosis, Alzheimer’s, and Parkinson’s diseases and in polyneuropathies associated with diabetes and acute chemotherapy use (64, 90). Remarkably, a mouse mutant, termed wlds, with delayed Wallerian degeneration has been identified in the C57BL/6 background. Axons from the wlds mouse survive several weeks after transection (61, 62).
The dominant neuroprotective gene in the wlds mouse is an in-frame fusion of the Nterminal 70 amino acids of a ubiquitin assembly factor (Ube4b/Uf2a) with the entire coding sequence of Nmnat1 (17). Transgenic mice expressing the Ube4b/Nmnat fusion gene showed that the nuclear protein protected from axonopathy in a manner that depended on the level of protein expression, indicating the activity of a nuclear-derived factor (52). Although Nmnat in flies can protect against degeneration of optic neurons in an active site-independent manner (95, 96), the protective factor for DRG neurons appears simply to be NAD+. The evidence is as follows. Lentiviral expression of Nmnat1 protects DRG neurons from axonopathy in an active site-dependent manner (3). Overexpression of wlds or Nmnat1 prevents NAD+ and ATP decline in response to mechanical and chemical damage (89). Nam and Na also protect against axonopathy as long as Nampt or Naprt1 are concomitantly expressed in DRG neurons, whereas NR protects without engineered gene expression of a biosynthetic gene (71). Nrk2 mRNA levels following axonopathy are induced approximately 20-fold, indicating a preferential use of NR as a precursor in maintaining intracellular NAD+ levels in DRG neurons (71). Whether sufficient oral NR supplementation might protect against diabetic or chemotherapy-induced neuropathy or protect against age-associated neurodegenerative conditions remains to be determined.
NAD+ SYNTHESIS IN CANDIDA GLABRATA
Candida glabrata, the second leading cause of candidiasis, does not encode genes for de novo synthesis of NAD+, such that it is a Na auxotroph. Because the C. glabrata Sir2 homolog represses transcription of a set of adhesin genes, Na limitation leads to adhesin gene expression and host colonization (19). Recent data establish that NR is also utilized by C. glabrata as the primary vitamin precursor in disseminated infection in mouse (51). The Nrk pathway (14) and NR to Nam salvage (8) are both components of NR utilization in C. glabrata (51).
NICOTINIC ACID MECHANISMS OF ACTION IN DYSLIPIDEMIA
Na has been used to treat dyslipidemias in humans since the 1950s. Gram dosages reduce triglycerides and low-density lipoprotein (LDL, i.e., “bad”) cholesterol and raise highdensity lipoprotein (HDL, i.e., “good”) cholesterol levels. As a monotherapy, Na is one of the most effective means to improve cardiovascular risk factors and, in combination with statins and bile acid treatments, can enhance therapeutic effects (18). In addition to lowering circulating cholesterol levels, Na prevents establishment of lipid deposits and the progression of atherosclerosis in a cholesterol-fed rabbit model (59). However, high-dose Na utilization produces a painful flushing response that limits use.
The mechanism of action of Na in treatment of dyslipidemias is not clear. Na is an agonist of the G-protein-coupled receptor Gpr109A (PUMAG in mice) (85, 92). However, it is not
clear that activation of this receptor, which is not expressed in the liver, can account for the clinical efficacy of Na. Activation of Gpr109A in adipocytes inhibits the liberation of free fatty acids from stored triglycerides. However, activation of GPR109A in epidermal Langerhans cells is directly responsible for flushing (11). The lack of expression of Gpr109A in the liver and the finding that Gpr109A mediates flushing cast serious doubt on the receptor model of Na function in dyslipidemia.
We have hypothesized that the beneficial effects of high-dose Na derive simply from NAD+ biosynthesis (7, 14). The fact that Nam is not beneficial in promoting reverse cholesterol transport can be explained in two ways. First, Na is a better NAD+ precursor than Nam in liver (16). Second, if the requirement for elevated NAD+ biosynthesis for improved reverse cholesterol transport depends on sirtuin function, one would expect Nam to be inhibitory.
Sirt1 has been identified as a positive regulator of liver X receptor (LXR), which in turn is a regulator of cholesterol and lipid homeostasis. Sirt1 deacetylates LXR at conserved lysine residues, resulting in LXR activation. sirt1−/− animals show reduced expression of LXR target genes, SREBP1, and ABCA1, in macrophages and in liver, which play important roles in HDL biogenesis (49). These data would appear to make the simple model of Na as an NAD+ precursor highly reasonable.
PROSPECTS FOR NR AS A SUPPLEMENT
The most fundamental use of NAD+ precursor molecules, Na and Nam, is in the prevention of pellagra. Like Na and Nam, NR is a natural product found in milk (14), which is incorporated into the intracellular NAD+ pool (94), and thus could be used as a general supplement, potentially for people who have adverse reactions to Na or Nam. More significantly, however, the specific utilization of NR by neurons may provide qualitative advantages over niacins in promoting function in the central and peripheral nervous system.
NR may also find uses related to the pharmacological uses of Na or Nam, which are limited by the side effects of each. Because Gpr109A is specific for the acid and not the amide (85, 92), one would not expect NR to cause flushing. Similarly, the side effects associated with high-dose use of Nam in the prevention and treatment of diabetic disorders (65) raise substantial health and safety concerns (44). In light of the inhibitory effects of Nam on sirtuins and the protective roles of sirtuins in normal cellular metabolism (18a, 91), NR may represent an alternative supplement. Though uncertainties as to the mechanisms of action of therapeutic doses of Na and Nam exist, positive results with NR would clarify the mechanisms of action of Na and Nam.
Because of the prevalence of PARP activation in neuropathies, inflammation, and neurodegeneration and the association of C. glabrata adherence with low NAD+, NR has great potential as a supplement or therapeutic agent that would elevate or maintain NAD+ in specific tissues. Future work will evaluate the pharmacokinetics, safety, and efficacy in animal and human systems to maintain health and to prevent disease.
Researchers at the Wageningen University in the Netherlands took a different approach to understand the effect of NAD+ pre-cursers and their influence on health. They presented a poster publication during the “10th World Congress on Polyphenols Applications” about an experiment to test the effects of marginal dietary supplementation with Nicotinamide Riboside (NR) on metabolic flexibility and anti-oxidant response in mice exposed to a high fat diet. This is in particular interesting because most studies with NR have been done at higher therapeutic levels as opposed to levels more typical for daily supplementation with an aim to maintain health.
The researchers fed two groups of adult male mice with a high fat diet containing dosing of 5 or 30 mg NR/kg. They measured body weight, lean mass, fat mass and feed intake weekly. After 14 weeks the metabolic flexibility was assessed with a fasting and re-feeding challenge using indirect calorimetry. With indirect calorimetry a measurement of respiratory exchanges takes place. Oxidation of the energetic substrates by the body is associated with oxygen consumption, carbon dioxide production, and heat release specific to the nature of the energetic substrates being oxidized. In the following week (so at 15 weeks) the researchers determined the anti-oxidant response in epididymal white adipose tissue.
The results showed that in the fasting and re-feeding challenge, the delta respiratory exchange ratio (ΔRER) was higher when comparing 30 to 5 mg/kg NR dosing thus indicating an increase in metabolic flexibility. In addition the expression levels of anti-oxidant genes were enhanced in the 30 mg/kg group compared to the 5 mg/kg group. The body physiological parameters were not affected by the dietary NR.
From these results it is still not possible to conclude what is the minimum level at which NR supplementation impacts the body as there are no intermediate dose test results between 5mg and 30mg / kg but it seems reasonable to conclude that NR supplementation does impact at a dosing level of 30mg / kg in mice.
Using FDA specified guidelines we can calculate the Human Equivalent Dose (HED) for the NR diet given to the mice.Using this guideline 30mg/kg dosing in mice translates into a HED of approx. 2.4mg/kg. Or into approximately 170mg daily nicotinamide riboside dosing for a person weighing 70kg. This is below the limit wrt to side effects and safety and less than the typical serving sizes of 250mg of supplements on the market. The result supports the view that NR supplementation may help maintain health as we age.
Increasing evidence about the importance NAD+ levels in the body continuous to emerge.
This week reseachers in China released a report indicating a link between the onset of nonalcoholic fatty liver disease (NAFLD) and the aging associated decline in NAD+ level.
Further more they found that supplementation with nicotinamide riboside in a NAD+ deficient mice model completely corrected these NAFLD onset symptoms.
Aging is known to be an important risk factor of nonalcoholic fatty liver disease (NAFLD). The researchers wanted to know whether the whether the deficiency of nicotinamide adenine dinucleotide (NAD+) that develops with aging may be the cause or part of the cause.
They setup an experiment that firstly compared NAD+ concentration, together with the protein levels of nicotinamide phosphoribosyltransferase (NAMPT) and several other critical enzymes regulating NAD+ biosynthesis, between middle-aged and aged mice or human patients.
In addition the influences of NAD+ decline on the steatosis (infiltration of liver cells with fat) and steatohepatitis (type of liver disease, characterized by inflammation of the liver with fat accumulation in liver) was evaluated in wild-type (WT) and H247A dominant-negative enzymatic-dead NAMPT transgenic mice (DN-NAMPT) under normal and high-fat diet (HFD).
The found that hepatic NAD+ level (NAD+ level in the liver) decreased in aged mice and also people. NAMPT-controlled NAD+ salvage, but not de novo biosynthesis pathway, was compromised in the liver of elderly mice and human.
The experiment also revealed that under normal diet, middle-age DN-NAMPT mice displayed systemic NAD+ reduction, and had moderate NAFLD phenotypes, like lipid accumulation, enhanced oxidative stress, triggered inflammation and impaired insulin sensitivity in liver.
A high fat diet worsened these NAFLD sympthoms.
A diet that contained nicotinamide riboside however, a natural NAD+ precursor, completely corrected these NAFLD onset sympthoms that were induced by NAD+ deficiency alone or the high fat diet.
From an experiment to evaluate whereas an adenovirus-mediated SIRT1 overexpression would reverse NAFLD phenotypes also it was found that this only partially corrected the disease sympthoms.
Overall this contributes further to the evidence that aging-associated NAD+ deficiency is a critical risk factor for aging related diseases.
In this study it was shown that supplementation of nicotinamide riboside may be a promising therapeutic strategy to prevent and treat NAFLD.
This article will in the future be updated with dosing related information once that comes available so we can calculate the human equivalent dose.
In a study that perhaps more serves as proof that NAD+ deficiency plays a role in diabetes researchers used high levels of nicotinamide riboside supplementation to demonstrate it protects mice from diabetes complications.
There is an association between prediabetic polyneuropathy (PDPN)1 and obesity, about half of individuals with diabetes will also suffer from diabetic peripheral neuropathy (DPN)2, which makes them insensitive to heat and touch.
This condition can progress to foot ulcers and amputations. Unfortunately there are few treatments that are effective for obesity while there are no known treatments for DPN. Best available care is tight glycemic control, lifestyle changes centered on dietary improvement and exercise, and pain medication when DPN is painful.
The researchers noted that in prior research there are indications that supplementation with nicotinamide riboside (NR), a NAD+ precursor vitamin can improve metabolic health in overfed mice. They also noted that research indicates that NR has an ability to protect damaged neurons.
Because of this dual potential the researchers hypothized that NR may improve prediabetic (PD) and diabetic glucose and lipid metabolism while simulatanuously treating the neuropathic complications that come with DPN. The researchers tested the potential positive effect of NR in mouse models.
A large cohort of mice was used in the experiment and several comparison groups created, increasing its relevance.
Out of a group of 60 mice 40 were transferred to a HFD and 20 mice stayed on normal diet. Of the 40 mice 20 were given 20 STZ, a chemical that induces type 2 diabetes (T2D). Finally of each group 10 mice were given NR supplementation at a dosing level of 3g/Kg/day. The groups were then studied for various disease indicators.
The study found that NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy.
In type 2 diabetes mice the results showed that NR strongly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The researchers concluded that the neuroprotective effect of NR could not be explained by glycemic control alone.
They therefore used corneal confocal microscopy to analyse deeper. This revealed a protective effect of NR on small nerve structures in living mice. It was established that hepatic NADP+ and NADPH levels were significantly degraded in prediabetes and type 2 diabetes but were largely protected when mice were supplemented with NR.
The dosing, as mentioned earlier, was very high at 3g/Kg/day. Using FDA specified guidelines we can calculate the Human Equivalent Dose (HED) for the NR diet given to the mice.Using this guideline 3g/kg dosing in mice translates into a HED of approx. 240mg/kg.
Or into approximately 17 gram daily nicotinamide riboside dose for a person weighing 70kg. Typical supplements on the market have serving sizes of 250mg. It should be noted that this dose is well over the limit wrt to side effects and safety.
Nevertheless the data calls for further investigation e.g. repetition at lower dosing and possibly testing of NR in human models of obesity, type 2 diabetes and associated neuropathies.
Given the current dosing ranges it appears likely that a therapeutic approach will need to be done under medical supervision. It should be noted that there are also alternative supplements that hold promise for treatment of diabetes.
NAD+ is required not only for life but for a long life. In this issue, Camacho-Pereira et al. (2016) implicate CD38 in the decline of NAD+ during aging, with implications for combating age-related diseases.
In this issue, Eduardo Chini and col- leagues address an open question in bio- gerontology: why do NAD+ levels fall as we age? They show that the major culprit is an NADase called CD38 whose levels rise during aging. Their results also add to the body of evidence indicating that loss of SIRT3 activity in mitochondria is a cause of age-related metabolic decline (Camacho-Pereira et al., 2016) (Figure 1).
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 nicotin- amide mononucleotide (NMN).
Interest- ingly, 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 metab- olites 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.
These findings suggest that the efficacy of NAD+ precursors may be enhanced by co-supplementation with CD38 inhibitors, which have been recently identified (Escande et al., 2013; Haffner et al., 2015).