NAD+NAD+ Research (Part 1)

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NAD+ Research (Part 1)

Post by jocko6889 » Wed Jan 15, 2020 6:11 pm

NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis(Braidy, 2020)

https://sci-hub.se/downloads-ii/2020-01 ... 110831.pdf

Human studies documenting the beneficial effects of raising NAD+ levelsin the CNS are nascent in the current literatire. At present,oral or i.v. NADH have been reported to reduce anxiety (Alegre et al., 2010), attenuatesleep disturbances (Santaella et al., 2004;Forsyth et al., 1999), improvecognitive performance(Birkmayer, 1996), lower the number and duration ofheadaches (Forsyth et al., 1999), and amelioratesymptoms of jet lag (Birkmayer et al., 2002). Treatment with NADH has also been shown to slow downthe progression of dementia, and improve outcomes inverbal fluency and visual-constructional ability (Demarin et al., 2004). Treatment with i.v NAD+ and NADH has also been shown toimprove motor symptoms inParkinson's disease (Birkmayer, 1993; Grant et al 2019)


Nicotinamide adenine dinucleotide (NADH) in patients with chronic fatigue syndrome(De Sevilla, 2010)

https://www.sciencedirect.com/science/a ... 6510001591

Administration of oral NADH was associated to a decrease in anxiety and maximum heart rate, after a stress test in patients with CFS. On the contrary, this treatment did not modify other clinical variables and the global functional performance.

Comparison of oral nicotinamide adenine dinucleotide (NADH) versus conventional therapy for chronic fatigue syndrome
(Santealla, 2004)


https://www.ncbi.nlm.nih.gov/pubmed/15377055

The twelve patients who received NADH had a dramatic and statistically significant reduction of the mean symptom score in the first trimester (p < 0.001). However, symptom scores in the subsequent trimesters of therapy were similar in both treatment groups. Elevated IgG and Ig E antibody levels were found in a significant number of patients.

Nicotinamide adenine dinucleotide (NADH)--a new therapeutic approach to Parkinson's disease. Comparison of oral and parenteral application (Birkmayer, 1994)

https://www.ncbi.nlm.nih.gov/pubmed/8101414

The reduced coenzyme nicotinamide adenine dinucleotide (NADH) has been used as medication in 885 parkinsonian patients in an open label trial. About half of the patients received NADH by intravenous infusion, the other part orally by capsules. In about 80% of the patients a beneficial clinical effect was observed: 19.3% of the patients showed a very good (30-50%) improvement of disability, 58.8% a moderate (10-30%) improvement. 21.8% did not respond to NADH. Statistical analysis of the improvement in correlation with the disability prior to treatment, the duration of the disease and the age of the patients revealed the following results: All these 3 parameters have a significant although weak influence on the improvement. The disability before the treatment has a positive regression coefficient (t value < 0.01). The duration of the disease has a negative regression coefficient (< 0.01) and so has the age a negative regression coefficient (t value < 0.05). In other words younger patients and patients with a shorter duration of disease have a better chance to gain a marked improvement than older patients and patients with longer duration of the disease. The orally applied form of NADH yielded an overall improvement in the disability which was comparable to that of the parenterally applied form

Treatment of Alzheimer’s disease with stabilised oral nicotinamide adenine dinucleotide: A randomised double-blind study (Demarin, 2004)

https://www.ncbi.nlm.nih.gov/pubmed/15134388

The present trial was a randomized, placebo-controlled, matched-pairs, double-blind, 6-month clinical study. Patients with probable AD (n = 26) were randomized to receive either stabilized oral NADH (10 mg/day) or placebo. Twelve pairs of subjects were matched for age and baseline total score on the Mattis Dementia Rating Scale (MDRS) and the Mini Mental State Examination. After 6 months of treatment, subjects treated with NADH showed no evidence of progressive cognitive deterioration and had significantly higher total scores on the MDRS compared with subjects treated with placebo (p < 0.05). Analysis of MDRS subscales revealed significantly better performance by NADH subjects on measures of verbal fluency (p = 0.019), visual-constructional ability (p = 0.038) and a trend (p = 0.08) to better performance on a measure of abstract verbal reasoning. There were no differences between groups in measures of attention, memory, or in clinician ratings of dementia severity (Clinical Dementia Rating). Consistent with earlier studies, the present findings support NADH as a treatment for AD.


Combination of NAD+ and NADPH Offers Greater Neuroprotection in Ischemic Stroke Models by Relieving Metabolic Stress (Huang, 2018)

10.1007/s12035-017-0809-7

Both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and β-nicotinamide adenine dinucleotide hydrate (NAD+) have been reported to have potent neuroprotective effects against ischemic neuronal injury. Both NADPH and NAD+ are essential cofactors for anti-oxidation and cellular energy metabolism. We investigated if combined NADPH and NAD+ could offer better neuroprotective effects on cellular and animal models of ischemic stroke. In vitro studies with primary cultured neurons demonstrated that NAD+ was effective in protecting neurons against oxygen-glucose deprivation/reoxygenation (OGD/R) injury when given during the early time period of reoxygenation. In vivo studies in mice also suggested that NAD+ was effective for ameliorating ischemic brain damage when administered within 2 h after reperfusion. The combination of NADPH and NAD+ provided not only greater beneficial effects but also larger therapeutic window in both cellular and animal models of stroke. The combination of NADPH and NAD+ significantly increased the levels of adenosine triphosphate (ATP) and reduced the levels of reactive oxygen species (ROS) and oxidative damage of macromolecules. Furthermore, the combined medication significantly reduced long-term mortality, improved the functional recovery, and inhibited signaling pathways involved in apoptosis and necroptosis after ischemic stroke. The present study indicates that the combination of NAD+ and NADPH can produce greater therapeutic effects with smaller dose of NADPH; on the other hand, NADPH can significantly prolong the therapeutic window of NAD+. The current results suggest that the combination of NADPH and NAD+ may provide a novel effective therapy for ischemic stroke.

In summary, NAD+replenishment exhibited neuroprotectionagainst ischemia/reperfusion-induced neuronal injury bothin vivo and in vitro. The combination of NAD+andNADPH provided greater neuroprotective effects in both cel-lular and animal models of ischemic stroke. NADPH couldprolong the therapeutic window of NAD+, whereas NAD+could reduce the dosage of NADPH to produce the desirabletherapeutic effects. The current study suggests that the com-bination of NAD+and NADPH may provide a novel effectivetherapy for ischemic stroke.


Nicotinamide phosphoribosyltransferase regulates cocaine reward through Sirtuin 1(Kong, 2018)

https://sci-hub.se/10.1016/j.expneurol.2018.05.010

Our results suggest that NAMPT-mediated NAD biosynthesis may modify cocaine behavioral effects through SIRT1. Moreover, our findings reveal that the interplay between NAD biosynthesis and SIRT1 regulation may comprise a novel regulatory pathway that responds to chronic cocaine stimuli.


Fasting- and ghrelin-induced food intake is regulated by NAMPT in the hypothalamus (Treebak, jan 2020)

https://www.ncbi.nlm.nih.gov/pubmed/31900990

Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signaling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied.
METHODS:
We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration.
RESULTS:
NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+ , increased levels of ROS, and affected the expression of Agrp, Pomc, and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake.
CONCLUSION:
Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.


(NAD+) Diphosphopyridine Nucleotide in the Prevention, Diagnosis and Treatment of Drug Addiction (Ohollaren, 1961)

https://daks2k3a4ib2z.cloudfront.net/5a ... n-1961.pdf

DPN = old name for NAD+
The author has previously reported the successful use of DPN in the treatment of acute and chronic alcoholism. In the administration of nearly 1000 Gm. to more than 100 patients there has been no toxic effect whatsoever; from the coenzyme DPN in its oxidized form, when administered at a speed tolerated by the patient.


Intravenous NAD+ effectively increased the NAD metabolome, reduced oxidative stress and inflammation, and increased expression of longevity genes safely in elderly humans(Brady, 2019-pending)

https://dergipark.org.tr/jcnos/issue/48187/610084

NAD+ injection HAS been tested in humans and is far more effective than NR capsules. Some research by Naidy Brady presented at a conference shows 1 week of NAD+ IV in humans . They haven’t published this yet, so don’t have much detail, but we know they found NAD+ injections for 1 week had significantly more more effect than 3 weeks of NR. Markers such as CRP improved with NAD+, not with NR.

"Nicotinamide adenine dinucleotide (NAD+) serves important roles in hydrogen transfer and as the cosubstrate for poly(ADP-ribose) polymerase (PARPs), the sirtuin (SIRT1-7) family of enzymes, and CD38 glycohydrolases. Recently, intravenous (IV) NAD+ therapy has been used as a holistic approach to treat withdrawal from addiction, overcome anxiety and depression, and improve overall quality of life with minimal symptoms between 3-7 days of treatment. We evaluated repeat dose IV NAD+ (1000 mg) for 6 days in a population of 8 healthy adults between the ages of 70 and 80 years.

Our data is the first to show that IV NAD+ increases the blood NAD+ metabolome in elderly humans. We found increased concentrations of glutathione peroxidase -3 and paraoxonase-1, and decreased concentrations of 8-iso-prostaglandin F2α, advanced oxidative protein products, protein carbonyl, C-reactive protein and interleukin 6. We report significant increases in mRNA expression and activity of SIRT1, and Forkhead box O1, and reduced acetylated p53 in peripheral blood mononuclear cells isolated from these subjects. No major adverse effects were reported in this study. The study shows that repeat IV dose of NAD+ is a safe and efficient way to slow down age-related decline in NAD+."


A pilot study investigating changes in the human plasma and urine NAD+ metabolome during a 6 hour intravenous infusion of NAD+(Brady, 2019)

https://www.frontiersin.org/articles/10 ... 7/abstract

This study therefore documented changes in plasma and urine levels of NAD+ and its metabolites during and after a 6 hour 3 μmol/min NAD+ intravenous infusion.
Surprisingly, no change in plasma [NAD+] or metabolites (nicotinamide, methylnicotinamide, ADP ribose and nicotinamide mononucleotide) were observed until after 2 hours. Increased urinary excretion of methylnicotinamide and NAD+ were detected at 6 hours, however no significant rise in urinary nicotinamide was observed.
This study revealed for the first time that i) at an infusion rate of 3 μmol/min NAD+ is rapidly and completely removed from the plasma for at least the first 2 hours, ii) the profile of metabolites is consistent with NAD+ glycohydrolase and NAD+ pyrophosphatase activity and iii) urinary excretion products arising from an NAD+ infusion include NAD+ itself and meNAM but not NAM.


Treatment of Alzheimer's disease with stabilized oral nicotinamide adenine dinucleotide: a randomized, double-blind study (Demarin, 2004)

https://www.ncbi.nlm.nih.gov/pubmed/15134388/

This study was designed to evaluate the effect of stabilized oral reduced nicotinamide adenine dinucleotide (NADH) on cognitive functioning in patients with Alzheimer's disease (AD). NADH is a coenzyme that plays a key role in cellular energy production and stimulates dopamine production. In previous trials NADH has been shown to improve cognitive functioning in patients with Parkinson's disease, depression and AD. The present trial was a randomized, placebo-controlled, matched-pairs, double-blind, 6-month clinical study. Patients with probable AD (n = 26) were randomized to receive either stabilized oral NADH (10 mg/day) or placebo. Twelve pairs of subjects were matched for age and baseline total score on the Mattis Dementia Rating Scale (MDRS) and the Mini Mental State Examination. After 6 months of treatment, subjects treated with NADH showed no evidence of progressive cognitive deterioration and had significantly higher total scores on the MDRS compared with subjects treated with placebo (p < 0.05). Analysis of MDRS subscales revealed significantly better performance by NADH subjects on measures of verbal fluency (p = 0.019), visual-constructional ability (p = 0.038) and a trend (p = 0.08) to better performance on a measure of abstract verbal reasoning. There were no differences between groups in measures of attention, memory, or in clinician ratings of dementia severity (Clinical Dementia Rating). Consistent with earlier studies, the present findings support NADH as a treatment for AD.


Insight into Molecular and Functional Properties of NMNAT3 Reveals New Hints of NAD Homeostasis within Human Mitochondria (Chiarugi, 2013)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796565/

we show that extracellular NAD, but not its metabolic precursors, sustains mitochondrial NAD pool in an ATP-independent manner

Intriguingly, we found that cellular NAD depletion could be completely prevented by adding 1 mM NAD to the culture media (Fig. 6A). However, identical concentrations of the NAD precursors Nam, NMN, nicotinamide riboside and nicotinic acid were not effective (Fig. 6A).

A widely appreciated dogma of cell biology is that mitochondria are impermeable to NAD [16], [33]. Several findings, however, indicate that this tenet should be revisited. First, both yeast and plant mitochondria have NAD transporters [34], [35]. Second, transporters for NAD precursors in the plasmamembrane or different cell organelles including mitochondria have not been identified so far. Third, inability of mitochondria to transport NAD has been only demonstrated in vitro, and it might be due to the fact that, under these experimental settings, transport systems are impaired or lack a co- or counter-molecule necessary for their functioning. Consistently, both yeast and plant mitochondrial NAD transporters work as nucleotide exchangers [34], [35]. Fourth, increase in the extracellular concentrations of NAD in cultured cells raises the dinucleotide content within their mitochondria and boosts cellular respiration [8]. Finally, prior work found evidence for metabolic state-dependent NAD fluxes through the inner mitochondrial membrane [36], [37]. These findings taken together, plus data of the present study indicating that exogenous NAD but not NMN, NR, NA or Nam prevents FKSG76-dependent mitochondrial NAD depletion, suggest that, akin to yeast and plants, yet-to-be defined mitochondrial NAD transporters are present in mammalian cells.


SIRT1-mediated eNAMPT secretion from adipose tissue regulates hypothalamic NAD+ and function in mice

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426056/

lowering glucose dramatically enhanced eNAMPT secretion 3.5–5.5-fold

Adipose tissue-specific Nampt knockout mice exhibit reduced plasma eNAMPT levels and defects in NAD+ biosynthesis not only in adipose tissue but also in the hypothalamus

while other tissues such as the liver and skeletal muscle did not show any change in NAD+ levels (Figure 5B), the hypothalamus, but not the hippocampus, showed significant decreases in NAD+ levels

confirming the direct effect of eNAMPT on hypothalamic NAD+ levels.

Furthermore, the defect appears to be specific at least to the hypothalamus because other tissues and organs, including the liver, skeletal muscle, and the hippocampus, do not show any reduction in NAD+ levels

female ANKO mice showed significantly reduced physical activity during the dark time (Figure 6A), consistent with reduced hypothalamic NAD+

it is conceivable that eNAMPT secreted from adipose tissue plays a critical role in supplying NAD+ to the hypothalamus

FASCINATING!


Effects of Chronic NAD Supplementation on Energy Metabolism and Diurnal Rhythm in Obese Mice (Roh, 2018)

https://www.ncbi.nlm.nih.gov/pubmed/30230244
http://sci-hub.se/https://www.ncbi.nlm. ... d/30230244

C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks and received an intraperitoneal injection of either saline or NAD (1 mg/kg/day) for the last 4 weeks.

The control mice were fed a chow diet and injected with saline for the same period. Body weights were monitored daily. Daily rhythms of food intake, energy expenditure, and locomotor activity were measured at the end of NAD treatment. The effect of NAD treatment on the clock gene Period 1 (PER1) transcription was also studied.
RESULTS:
Chronic NAD supplementation significantly attenuated weight gain in HFD-fed obese mice. Furthermore, NAD treatment recovered the suppressed rhythms in the diurnal locomotor activity patterns in obese mice. In addition, exogenous NAD supply rescued cellular NAD depletion-induced suppression of PER1 transcriptional activity in hypothalamic neuron cells as well as blunted daily fluctuations of hypothalamic arcuate nucleus PER1 expression in obese mice.
CONCLUSIONS:
NAD supplementation showed therapeutic effects in obese mice with altered diurnal behaviors.

Previous studies have reported the benefi- cial metabolic effects of supplementation with NAD precursors NR and NMN in mice and humans (11,14‒17). Similar to our findings, 12-week supplementation with NR (400 mg/kg/d in drinking water) in HFD-fed mice reduced body weight gain and fat mass and increased EE (14). Twelve-month NMN administration in mice (100-300 mg/kg in drinking water) attenuated aging-associated body weight gain (16). Moreover, intraperitoneal administration of NMN (500 mg/kg/d) for 1 week improved glucose metabolism in diabetic mice (11) and restored mitochondrial functions in the muscles of aged mice (15). It is notable that supplementation with NAD by itself, at a 100-times lower dose compared with those of its precursors NMN and NR (25,26), caused a beneficial metabolic effect;

Mice that received NAD+ weighed less, were more active, and had better glucose control than mice that received placebo

Nicotinamide adenine dinucleotide suppresses epileptogenesis at an early stage

To confirm whether NAD+ penetrates the BBB, we harvested the hippocampus and measured the NAD+ level at 30 min and 60 min after NAD+ injection in normal male C57BL/6 mice. As shown in Fig. 1a, compared with control mice (1.00 ± 0.10, N = 5), the NAD+ level in the hippocampus was significantly high at 30 min after the i.p. injection of NAD+ (100 mg/kg, i.p.; 1.43 ± 0.05, N = 6) and was still higher at 60 min after the injection (1.24 ± 0.11, N = 5)

NAD+ in blood crossed to brain and hippocampus

NAD+ Injections Reversed NAD+ Depletion in SE Model Mice at Early-Stage Epileptogenesis

Early-Stage Injection of NAD+ Attenuated the Incidence of Seizures in SE Model Mice at SRS Stage

Early-Stage Intervention With NAD+ Reversed Abnormal EEG Activity in SE Model Mice at SRS Stage

early-stage intervention with NAD+ improved contextual fear memory impairment

intraperitoneal injection of 50 mg/kg NAD+ decreases ischemic brain damage in ischemic model mice

intraperitoneal injection of 100 mg/kg NAD+ alleviates doxorubicin-induced liver damage in mice


Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway (Pillai, 2009)

https://www.ncbi.nlm.nih.gov/pubmed/19940131


http://sci-hub.tw/10.1074/jbc.M109.077271

Mice were simultaneously treated with NAD at 1 mg/kg/day for 2 weeks
these data demonstrated that NAD treatment was capable of maintaining cellular NAD levels
NAD treatment restored the cellular NAD levels

In summary, we demonstrated that exogenous NAD can block cardiac hypertrophy via activation of SIRT3.


Exogenous NAD+ administration significantly protects against myocardial ischemia/reperfusion
injury in rat model (Zhang, 2016)

https://www.ncbi.nlm.nih.gov/pmc/articl ... 8-3342.pdf

Our observations have suggested that exogenous NAD+ administration can pro- foundly decrease I/R hearts injury
Saline or NAD+ (5 mg/kg, 10 mg/kg, 20 mg/kg, dissovled in saline) were injected intra- venously right before ischemia
Rats were sacrificed 6 hours or 24 hours after reperfusion
Compared with other drugs that have shown protective effects on myocardial I/R injury, the NAD+ produced 85% decrease in the infarct size has suggested that NAD+ is one of the drugs that have greatest capacity to decrease myocardial
We observed obvious protective effects of 10 mg/ kg and 20 mg/kg NAD+ on the infarct formation (Figure 1A).
Quantifications of the myocar- dial infarct volume indicate that NAD+ dose dependently decreased infarct formation

Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia. (Ying, 2007)

https://www.bioscience.org/2007/v12/af/ ... lltext.htm

10 and 20 mg / kg NAD+

Also tested 10 mg/kg NAM - not effective

Six ml of NAD+ or nicotinamide, which was dissolved in phosphate-buffered saline (PBS), was applied in one side of the nose of rats each time, with the nose at another side blocked for 5 seconds to enhance the influx of the solutions through the nostrial tract. This procedure was repeated every 2 minutes alternatively on each side of the nose, totally for 10 times

The profound protective effects of the intranasal NAD+ administration were also observed at 72 hrs after ischemia.

intranasal administration with 10 mg / kg NAD+, but not with 5 mg / kg NAD+, significantly attenuated the ischemia / reperfusion-produced neurological deficits

In contrast to the profound protective effects of 10 mg / kg NAD+, intranasal administration with 10 mg / kg nicotinamide did not decrease infarct formation

Our results provide the first in vivo evidence that NAD+ administration can profoundly decrease brain damage under certain pathological conditions

It is noteworthy that NAD+ can reduce infarct formation by up to 86 % even when administered at 2 hrs after ischemic onset. Compared with other studies that apply drugs during post-ischemia phases in order for decreasing ischemic brain injury, the protective effect of NAD+ could be one of the most profound effects ever reported

Intranasal drug delivery approach could have multiple merits over traditional drug delivery approaches: First, it may deliver drugs into the brains by bypassing the blood-brain barriers

Cumulative evidence has suggested that NAD+ may mediate cell death via multiple mechanisms (5). For examples, NADH / NAD+ ratio is a major index of cellular reducing potential, which can modulate MPT --- a mediator of both apoptosis and necrosis under many conditions; and both NAD+ and NADH mediate energy metabolism that could determine cell death modes


Pharmacological Effects of Exogenous NAD on Mitochondrial Bioenergetics, DNA Repair and Apoptosis (Pittelli, 2011)

http://molpharm.aspetjournals.org/conte ... 6.full.pdf

Although the canonical view considers NAD unable to permeates lipid bilayers (Di Lisa and Ziegler, 2001), several studies report evidence for exogenous NAD (eNAD) uptake by different cells
we further investigated cellular uptake of eNAD, and also focused on its possible functional effects as indirect evidence of eNAD entrance
Together, our findings on the one hand strengthen the hypothesis that eNAD crosses intact the plasmamembrane, and on the other provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

The ability of eNAD to increase the mitochondrial NAD content and energy production is of particular relevance
it is worth noting that the present study indicates that mitochondria sense cytoplasmic concentrations of NAD and/or its precursors, resetting their pyridine nucleotide pool accordingly
mitochondrial fluctuation of NAD contents according to those present in the cytoplasm does not seem bidirectional
It seems, therefore, that these organelles are able to maintain their NAD content when that of cytosol decreases, but readily increase the pyridine nucleotide pool when the cytoplasmic availability of NAD and/or its precursors increases

A key finding of our study is that apoptosis is reduced by increasing the extracellular concentrations of NAD
Our data are consistent with previous reports showing that eNAD affords protection from various stresses such as beta-amyloid (Qin et al., 2006), ischemia (Wang et al., 2008), NMNAT inactivation (Wang et al., 2005) or PARP-1-dependent cell death
These findings are at odds with the hypothesis that eNAD increase iNAD contents because of extracellularly-formed NAD precursors

eNAD is transported intact across the plasmamembrane


Prevention of Traumatic Brain Injury-Induced Neuron Death by Intranasal Delivery of Nicotinamide Adenine Dinucleotide (won, 2012)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972775/

The present study demonstrates that intranasally-administered NAD+ increases hippocampal NAD+ levels and reduces TBI-induced neuronal death in the hippocampus.

Intraperitoneal injection of the same NAD+ concentration that we used for intranasal administration showed no neuroprotective effects on TBI-induced neuronal death.

Compared with NAD+-treated rats, nicotinamide treatment showed no protective effects against TBI-induced neuronal death

Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia (ying, 2007)

https://www.bioscience.org/2007/v12/af/ ... lltext.htm

Increasing evidence has supported the hypothesis that PARP-1 induces cell death by depleting intracellular NAD+. We observed that intranasal NAD+ delivery significantly increased NAD+ contents in the brains.

Intranasal delivery with 10 mg / kg NAD+ at 2 hours after ischemic onset profoundly decreased infarct formation when assessed either at 24 or 72 hours after ischemia. The NAD+ administration also significantly attenuated ischemia-induced neurological deficits. In contrast, intranasal administration with 10 mg / kg nicotinamide did not decrease ischemic brain damage. These results provide the first in vivo evidence that NAD+ metabolism is a new target for treating brain ischemia, and that NAD+ administration may be a novel strategy for decreasing brain damage in cerebral ischemia and possibly other PARP-1-associated neurological diseases.

Our results provide the first in vivo evidence that NAD+ administration can profoundly decrease brain damage under certain pathological conditions. It is noteworthy that NAD+ can reduce infarct formation by up to 86 % even when administered at 2 hrs after ischemic onset. Compared with other studies that apply drugs during post-ischemia phases in order for decreasing ischemic brain injury, the protective effect of NAD+ could be one of the most profound effects ever reported. In this study we also provided evidence that by the intranasal delivery approach NAD+ can be delivered into the brains.

Multiple studies have suggested that nicotinamide can decease ischemic brain injury, but at doses higher than 125 mg / kg (19). Our study shows that intranasal administration with nicotinamide at 10 mg / kg can not affect ischemic brain damage, in contrast to the profound protective effects of 10 mg / kg NAD+.


Exogenous nicotinamide adenine dinucleotide administration alleviates ischemia/reperfusion-induced oxidative injury in isolated rat hearts via Sirt5-SDH-succinate pathway( lui, jul 2019)

http://sci-hub.tw/https://www.ncbi.nlm. ... d/31278893

We first found that myocardial total NAD level was remarkably increased with NAD treatment (10 mg/kg) for 14 days.

NAD administration significantly decreased the lactate dehydrogenase (LDH) level in coronary leakage, decreased the malondialdehyde (MDA) level and increased the reduced glutathione/oxidized glutathione disulfide ratio (GSH/GSSG) in myocardial tissue.

In addition, NAD treatment effectively attenuated the depression of cardiac function in the isolated rat hearts after ischemia-reperfusion. Furthermore, we found that exogenous NAD attenuated the succinate accumulation during ischemia and decreased its depleting rate during reperfusion.

we found that NAD administration promoted the Sirt5 and SDH-a interaction and decreased the succinylation level of SDH-a.

These results implied that exogenous NAD administration promoted Sirt5-mediated SDH-a desuccinylation and decreased the activity of SDH-a, which attenuated the succinate accumulation during ischemia and its depleting rate during reperfusion and finally alleviated reactive oxygen species generation.


SIRT2, ERK and Nrf2 Mediate NAD+ Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497790/

We found that NAD+ treatment can increase the GSH/GSSG ratios in the cells under basal conditions. These findings have suggested not only the great nutritional potential of NAD+, but also a novel mechanism underlying the protective effects of the NAD+ administration in the disease models: the NAD+ administration can enhance the resistance of the normal cells to oxidative insults by increasing the antioxidant capacity of the cells.

The major findings of our current study include: first, NAD+ treatment can increase the GSH/GSSG ratio of PC12 cells under basal conditions, suggesting that NAD+ treatment can increase directly the antioxidant capacity of the cells; Collectively, our study has indicated that NAD+ can enhance directly the antioxidant capacity of the cells

Our recent study reported that NAD+ induced increases in intracellular ATP levels under basal conditions through its degradation into adenosine (Zhang et al., 2018).

NAD+ can pass through BBB to enter the brain under normal conditions: Roh et al. (2018) reported that exogenous NAD+ crossed the BBB through the Connexin 43 gap junction and entered the hypothalamus in its intact form; and Huang et al. (2018) reported that intravenous injection of NAD+ significantly increased the NAD+ level in the brain under physiological conditions, which has further suggested that NAD+ can cross the BBB under normal conditions.


Extracellular Degradation Into Adenosine and the Activities of Adenosine Kinase and AMPK Mediate Extracellular NAD+-Produced Increases in the Adenylate Pool of BV2 Microglia Under Basal Conditions (Zhang, Oct2018)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200843/

intranasal administration of NAD+, but not nicotinamide, can profoundly decrease ischemic brain injury (Ying et al., 2007) and traumatic brain injury (Won et al., 2012), which argues against the possibility that extracellular NAD+ produces its protective effects via nicotinamide – one of its degradation products. However, extracellular NAD+ can also be degraded into adenosine by multiple potential mechanisms

treatment of 10, 100, and 500 μM NAD+ can significantly increase the intracellular levels of ATP, ADP, and AMP of the cells (Figures Figures1A1A–C). NAD+ treatment can also increase the intracellular levels of NAD+

Our time-course study showed that extracellular adenosine levels increased by threefold after only 10 min of the NAD+ treatment

The major findings of our current study includes: first, NAD+ treatment can significantly increase the intracellular levels of ATP, ADP, and AMP of BV2 microglia under basal conditions.

Third, NAD+ treatment can significantly increase the extracellular adenosine levels,

Fourth, NAD+ treatment can significantly increase the intracellular adenosine levels of the cells.

Previous studies have indicated that extracellular NAD+ significantly decreases the damage of the cells exposed to various pathological insults. Therefore, our study has indicated that the mechanisms underlying the NAD+ treatment-produced effects on the cells under pathological insults are not applicable to the cells under basal conditions. These observations are not surprising for the following reasons: the cytosolic NAD+ concentrations are normally in the ranges between 1 and 10 mM (Ying, 2008). Because NAD+ enters cells by gradient-driven transport (Alano et al., 2010), the NAD+ at the concentrations between 0.01 and 0.5 mM, which was used in our study, should not be able to enter the cells to influence the adenylate pools of BV2 microglia under basal conditions.

extracellular NAD+ is degraded into adenosine extracellularly, that enters the cells through ENTs, which is converted to AMP by adenosine kinase. Increased AMP can lead to both increased AMPK activity and increased intracellular ADP levels, which jointly produce the increased intracellular ATP levels of BV2 cells under basal conditions.

Although the NAD+ at the concentrations between 0.01 and 0.5 mM cannot directly enter cells to increase intracellular NAD+ levels, we still found that the NAD+ can significantly increase the intracellular levels of NAD+ and adenylate of BV2 microglia through extracellular degradation into adenosine.

Since intracellular adenosine levels are normally in the nanomolar range (Latini and Pedata, 2001), the extracellular NAD+-generated adenosine can enter cells to increase intracellular adenosine levels thus increasing the intracellular adenylate levels through the activities of adenosine kinase and AMPK. In other words, the exceedingly low intracellular adenosine levels under normal physiological conditions is an unique ‘attractor’ and base for the relatively low concentrations of extracellular NAD+ to produce its significant biological effects on cells.

our current study has provided the first direct evidence showing that extracellular adenosine generated from extracellular NAD+ degradation mediates the biological effects of exogenous NAD+ by adenosine kinase- and AMPK-mediated pathways.

Our study has shown that relatively low concentrations of NAD+ can increase both extracellular and intracellular adenosine levels, the AMPK activity and intracellular adenylate pools, all of these factors have been shown to enhance defensive potential of both normal cells and stressed cells against toxic insults

Collectively, our current study has suggested a novel mechanism to account for the profound protective effects of NAD+ administration in the animal models of a number of diseases and aging

It is noteworthy that the intravenous NAD+ administration in all of the animal studies should lead to the NAD+ concentrations that are well below milimolar range (Ying et al., 2007; Wang et al., 2014; Zhang et al., 2016; Xie et al., 2017). Since the cytosolic concentration of NAD+ is normally in the range between 1 and 10 mM, it is usually assumed that the NAD+ administration-produced NAD+ concentrations in the blood should not be able to enter cells to produce biological effects. However, our study has shown that as low as 10 μM NAD+ can lead to significant increases in the ATP levels of all of the cell types we have studied on this topic, including BV2 microglia, PC12 cells, and C6 glioma cells (Zhang et al., 2018). Therefore, our findings have general value for understanding the mechanisms underlying the biological effects of NAD+ administration in models of diseases, aging or healthy controls: the relatively low concentrations of extracellular NAD+ can still produce its profound effects on cells through its extracellular degradation into adenosine, which may lead to increased intracellular levels of adenosine, AMP, ADP, and ATP on the basis of the activities of ENTs, adenosine kinase and AMPK.


NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864043/

These results establish NAD+ depletion as causal event in PARP-1 mediated cell death

Prior studies provide indirect evidence for uptake of extracellular NAD+ by neurons (T. Araki et al., 2004; J. Wang et al., 2005), and direct evidence for NAD+ uptake by other cell types (S. Bruzzone et al., 2001; C. C. Alano et al., 2004; R. A. Billington et al., 2008).

PARP-1 activation is a major cause of neuronal death in brain ischemia, trauma, and other settings, but the role of NAD+ depletion in this cell death pathway has been unresolved. The present findings show that PARP-1-mediated neuronal death is blocked when NAD+ depletion is prevented with exogenous NAD+. NAD+ repletion also prevents the intermediary steps in this cell death pathway that otherwise result from PARP-1 activation: glycolytic inhibition, mitochondrial depolarization, and mitochondrial AIF release. Conversely, depletion of cytosolic NAD+ with NAD+ glycohydrolase causes glycolytic inhibition and mitochondrial AIF release, independent of PARP-1 activation. These findings establish cytosolic NAD+ depletion as a necessary and sufficient event in the PARP-1 cell death pathway.


Contribution of P2X7 receptors to adenosine uptake by cultured mouse astrocytes (Okuda, 2010)

https://www.ncbi.nlm.nih.gov/pubmed/20645413/

Extracellularly applied NAD(+) prevents astrocyte death caused by excessive activation of poly(ADP-ribose) polymerase-1, In this study, we examined whether the intact form of NAD(+) is incorporated into astrocytes. A large portion of extracellularly added NAD(+) was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring-labeled [(14)C]NAD(+), but not nicotinamide moiety-labeled [(3)H]NAD(+), Taken together, these results indicate that exogenous NAD(+) is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R-associated channel/pore.


Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment (Lasmezas, 2015)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4840455/

Mice were treated daily with 30 mg/kg NAD+ by the intranasal route as a means to circumvent the blood–brain barrier

Four days after the onset of treatment, we observed a substantial difference in the level of activity of NAD+- versus PBS-treated mice.

The former started moving and exploring almost immediately after being placed in the cage, while the majority of the latter remained prostrated (Supplementary Video 1).
Rotarod testing starting 1 week after the onset of treatment showed better performance in NAD+- versus PBS-treated mice (Supplementary Fig. 8). Quantification of motor activity in the open field showed a significant difference between the two groups (Fig. 7C).

Addition of NAD+ 3 days after TPrP exposure completely and dose-dependently rescued cells from TPrP-induced death (Fig. 2A). The rescuing effect of NAD+ was independent of the reduced or oxidized status of the metabolite (Fig. 2B). Blockage of the salvage synthesis pathway (that uses nicotinamide as a precursor for NAD+) using the compound FK866 abolished the protective effect of nicotinamide in a dose-dependent manner (but not that of NAD+ itself)

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.

Prion diseases are fatal brain diseases of animals and humans and belong to the group of protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, frontotemporal dementia or amyotrophic lateral sclerosis.

we discovered that TPrP induces neuronal death via a profound depletion of intracellular nicotinamide adenine dinucleotide (NAD+) levels causing metabolic failure. Neuronal death can be rescued in vitro and in vivo by NAD+ replenishment.

our data demonstrate for the first time that a failure of NAD+ metabolism is the cause of neuronal ailing

Moreover, our TPrP toxicity model reveals a new mechanism of NAD+ depletion independent of PARP1.

misfolded amyloidogenic protein can induce neuronal death by genuine NAD+ starvation and that ailing neurons can be completely rescued by NAD+ treatment

our study shows that neuronal death induced by NAD+ depletion is reversible and that NAD+ replenishment mitigates neurodegeneration

We propose the development of NAD+-replenishment strategies for the treatment of prion diseases.


Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells (Nikiforov, 2019)

https://www.mdpi.com/2218-1989/9/12/293/htm

Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam.

Interestingly, exogenous nucleotides including NMN, NAMN, NAD+, and NAAD can support the maintenance of intracellular NAD pools as well as the nucleoside NR [12,13,14,15].

Moreover, the human ecto-enzyme CD73 has been described to catalyze both the cleavage of NAD+ to NMN and AMP as well as the subsequent dephosphorylation of the mononucleotides to the corresponding nucleosides, NR and adenosine [19,20].

In addition, extracellular NAD+ is used as a substrate of the glycohydrolases CD38 and CD157 which generate the second messengers ADP-ribose and cyclic ADP-ribose with the release of Nam [21] (Figure 1).

there are several studies supporting the direct uptake of NMN or NAD+ into human cells [13,23,24].

………………………

The quantitative analysis revealed that after 12 h of incubation in the presence of cells, less than 40% of the originally added amount of NAD+ remained. Moreover, after 48 h, NAD+ was undetectable in the medium (Figure 3B).

At the same time, a considerable amount of NMN was detectable, but also the formation of Nam was observed.

Surprisingly, NAD+ was efficiently degraded to NMN and Nam in the culture medium, even in the absence of cells. Within 24 h of incubation of NAD+ in the standard medium, DMEM supplemented with 10% heat-inactivated FBS; at 37 °C more than half of the added dinucleotide was degraded, whereas after 48 h less than 20% of the originally added amount remained (Figure 3B).

The mononucleotide NMN exhibited a higher stability, even though it was also significantly degraded to NR and Nam in the absence of cells. In the presence of cells, NMN was degraded faster, but even under these conditions, after 24 and 48 h still 80% and 60%, respectively, of the added mononucleotide were present in the medium (Figure 3C).

Likewise, we observed cleavage of NR to Nam in the medium without cells.

Strikingly, in the control sample that contained water instead of FBS, the extent of NR cleavage was similar (Figure 4, right lower panel).

These data indicate that NR efficiently hydrolyses to Nam (and ribose) in aqueous solutions

As shown here, any intermediate of NAD metabolism could potentially contribute to the maintenance of intracellular NAD. However, it is unlikely that they are equally relevant for this function. Physiologically, the intermediates are differently available.

NMN exhibits a relatively high chemical stability but is partly dephosphorylated to NR by the cells.


Extracellular ATP and β-NAD alter electrical properties and cholinergic effects in the rat heart in age-specific manner (kuzmin, mar 2019)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439013/


Nicotinamide phosphoribosyltransferase-related signaling pathway in early Alzheimer's disease mouse models(Chen, dec2019)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854586/

NAD+ group were intraperitoneally injected with NAD+ (30 mg/kg) at 20 weeks of age and once every other day for 4 weeks.

The administration of NAD+ alleviated the spatial learning and memory of APP/PS1 mice and reduced senile plaques. Administration of NAD+ may also increase the expression of the key protein NAMPT and its related protein sirtuin 1 as well as the synthesis of NAD+. Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.

NAD+ group were intraperitoneally injected with NAD+ (30 mg/kg) at 20 weeks of age and once every other day for 4 weeks.

NAMPT expression levels in the hippocampus in the NAD+ group were also significantly increased after intraperitoneal injection of NAD+

Intraperitoneally injecting NAD+ increased the expression of NAMPT in the cortex in the NAD+ mice (P<0.05).

Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.


Age-Associated Changes In Oxidative Stress and NAD+ Metabolism In Human Tissue

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407129/

“Age associated increases in oxidative nuclear damage have been associated with PARP-mediated NAD+ depletion and loss of SIRT1 activity”
“This study provides quantitative evidence in support of the hypothesis that hyperactivation of PARP due to an accumulation of oxidative damage to DNA during aging may be responsible for increased NAD+ catabolism in human tissue.
The resulting NAD+ depletion may play a major role in the aging process, by limiting energy production, DNA repair and genomic signalling.”

“There is a growing awareness that oxidative stress (OS) plays a key role not only in the aging process, but also in various degenerative diseases including Alzheimer's disease, cancer, diabetes, and chronic inflammation”

“However, at high concentrations, they are capable of damaging proteins, lipids and DNA [6].”

“Studies showing an increase in intracellular ROS through exogenous hydrogen peroxide treatment in vitro, or inhibition of endogenous ROS scavenging enzymes such as superoxide dismutase (SOD) and catalase, have been shown to promote premature aging and significantly lower lifespan”

“the mitochondria (the site of oxidative phosphorylation and ATP generation) is the major source of ROS production”

“The human body has a number of physiological protection and repair systems, including activation of the DNA nick sensor poly(ADP-ribose) polymerase-1 (PARP)”

“excessive DNA damage leads to over-activation of PARP, and increased NAD+ catabolism [23], [24], resulting in suppression of NAD+-dependent ATP generation and possible energy crisis”

“PARP activity was significantly increased in adults, older adults and elderly subjects compared to newborns”

“A significant decrease in total NAD+ content was observed in adults (p<0.05), older adult (p<0.05) and elderly (p<0.05) subjects compared to newborns”

“we found no significant difference in SIRT1 activity between any of the four age categories”
Lipid Peroxidation Increases with Age
Oxidative DNA Damage Accumulates with Age
PARP Activity increases with age leading to NAD+ Depletion

“report for the first time that PARP activity increases with age in human skin and correlates with both age and NAD+ depletion . “

“we report for the first time a significant decline in SIRT1 activity with age in post pubescent males (Fig. 5A, line b), though surprisingly, not in females (Fig. 5B).

We also did not find a correlation between NAD+ levels and SIRT1 activity in males”

“the lack of correlation between NAD+ levels and SIRT1 activity suggests that NAD+ availability, though required, is not the most sensitive modulator of SIRT1 activity in humans.”

“This study reports for the first time a link between oxidative stress and PARP activity, aging and a decline in NAD+ levels, in human tissue”


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Re: NAD+ Research (Part 2)

Post by jocko6889 » Wed Jan 15, 2020 6:12 pm

NAD+ metabolism: Bioenergetics, signaling and manipulation for therapy

https://www.ncbi.nlm.nih.gov/pubmed/27374990

We survey the historical development of scientific knowledge surrounding Vitamin B3, and describe the active metabolite forms of Vitamin B3, the pyridine dinucleotides NAD+ and NADP+ which are essential to cellular processes of energy metabolism, cell protection and biosynthesis.

NICOTNAMIDE

In humans and mammals, nicotinamide and nicotinic acid are routed in non-overlapping pathways to NAD+

For mammalian cells the central challenge in NAD+ homeostasis is successful recycling of nicotinamide, released from NAD+ consuming processes, back to NAD+

Published data for NAD+ turnover in vivo indicate halflives of as little as 4-10 hours

3 g of nicotinamide is required to be resynthesized to NAD+ up to several times per day

These facts implicate efficient nicotinamide recycling as the basis for effective NAD+ maintenance in humans

the role of Nampt in setting the NAD+ level in cells confirms that the level of the enzyme, and not nicotinamide concentrations themselves, have the largest effect on setting NAD+ level

Interestingly, Nampt levels appear to be upregulated by dietary intake and exercise

the entire NAD+ pool is being replaced 2-4 times per day

Figure 6 provides a comprehensive view of NAD+ metabolic pathways as found in humans and how these pathways intersect and converge on the central metabolite NAD+. This figure also conveys the fact that different NAD+ precursors can enter NAD+ biosynthetic pathways and can converge to NAD+ via independent and also overlapping pathways.

Nicotinamide had ability to increase NAD+ level in liver (47%), but was weaker in kidney (2%), heart (20%), blood (43%) or lungs (17%). Nicotinic acid raised NAD+ in liver (47%), and impressively raised kidney (88%), heart (62%), blood (43%) and lungs (11%)


The NAD World 2.0: the importance of the inter-tissue communication mediated by NAMPT/NAD+/SIRT1 in mammalian aging and longevity control

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516857/

hypothalamus may be the “biological clock”

organs with low inampt, like pancreas, brain, hypothalamus, depend on circulating nmn or nampt to create needed NAD+

They are most susceptible to decreasing NAD+

NAMPT-mediated NAD+ biosynthesis declines with age in multiple organs and tissues, such as pancreas, adipose tissue, skeletal muscle, liver, and brain

These findings strongly suggest that the hypothalamus functions as a high-order control center of aging in mammals, controlling the process of aging and limiting lifespan

the secretion of eNAMPT is regulated by adipose NAD+ and SIRT1,36 which positions adipose tissue to be a sensor of NAD+ in the system.

the feedback mechanism between the hypothalamus and adipose tissue predicts the importance of NMN as a systemic signaling molecule that maintains biological robustness

boosting NAD+ biosynthesis with NMN or nicotinamide riboside, another NAD+ intermediate that is converted to NMN, in key system-controlling organs and tissues could maintain biological robustness and delay the aging process in mammals

NAMPT-mediated NAD+ biosynthesis also independently declines over age in adipose tissue, likely due to chronic inflammation developed within adipose tissue

Thus, the system would eventually reach a point where the robustness of this feedback loop between the hypothalamus and adipose tissue can no longer be maintained


Nampt Expression Decreases Age-Related Senescence in Rat Bone Marrow Mesenchymal Stem Cells by Targeting Sirt1

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5268649/

(previous studies show that ) Caloric restriction (CR) and exercise significantly upregulated Nampt expression, which contributes to prolonging the life expectancy of individuals and reducing the incidence of age-related diseases

expression levels of Nampt were both lower in the MSCs obtained from the old group than in the young group

Lower Nampt levels in MSCs obtained from old rats were associated with lower levels of intracellular NAD+ synthesis, which attenuated Sirt1 expression and activity

Sirt1 protein expression was 2.34-fold lower (Fig 6A) and its mRNA expression was 1.54-fold lower (Fig 6B) than the levels in their young counterparts

Sirt1 activity was 50% lower in old MSCs than in young MSCs

MSCs derived from old rats displayed significantly lower intracellular NAD+ concentrations than were observed in the young rats


Loss of NAD homeostasis leads to progressive and reversible degeneration of skeletal muscle (AUG 9 2016)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985182/

(NAMPT) Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content

accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance.

Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass, despite having only a modest effect on the intramuscular NAD pool.

lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice

Such restrictions have been reported during states of genotoxic stress that accompany a growing list of diseases, including cancer and neurodegeneration, as well as the course of natural aging

(Nampt), as well as its product, nicotinamide mononucleotide (NMN), are found in both intracellular and extracellular compartments

However, the specific sites to which intact NAD precursors are distributed and utilized in vivo have not been demonstrated experimentally.

decreases in the NAD content of brain, liver, and muscle, coincident with declines in the function of these tissues

Our findings establish a lower threshold of tolerability for NAD loss in muscle and shed light on potential mechanisms driving the age-related declines in muscle function and metabolic capacity

Muscles from mNKO mice are deficient in Nampt and contain <15% of the normal intramuscular concentrations of oxidized and reduced NAD by 3 months of age in both sexes

Muscle from mNKO mice exhibited a >60% decline in ATP content, clearly indicating energetic stress

Surprisingly, mNKO mice aged 3 months appeared to fatigue at the same rate as littermate controls

By 7 months of age, the difference in muscle mass was apparent in the hindlimbs

we found that 7-month-old mNKO mice could no longer maintain the treadmill performance of littermate controls

We were surprised to find fully 33% of detectable genes to be significantly altered in mNKO muscle

Nicotinamide riboside functionally and morphologically restores NAD-deficient muscle

Thus, our data are inconsistent with the hypothesis that a block in glycolysis is the primary metabolic defect in vivo

We also noted that Nampt deletion did not result in the accumulation of its NAM substrate, but rather in a higher steady state concentration of methyl-NAM, an alternative fate for NAM equivalents

we dissolved (NR) in the drinking water to deliver an effective daily dose of ~400 mg/kg

Mice beginning at age 5.5 months received the treatment continuously for 6 weeks

this intervention appeared to completely prevent the development of exercise intolerance observed in 7-month-old mNKO mice and reversed lactic acidosis at the point of exhaustion

experienced a complete restoration of exercise capacity after only one week, which persisted for the duration of the treatment

near complete restoration of force generated by isolated mNKO muscles, as well as normalization of mass in all major hindlimb muscles

we again found that NAD content of whole NR-treated mNKO muscle remained severely depleted, with only a trend toward improvement when compared to the untreated knockouts

we designed an isotope-labeled NR tracer, with a single 13C and a single deuterium

Accordingly, labeled NR was readily detectable in liver, but not in skeletal muscle

suggesting the existence of a naturally occurring pool capable of interconverting with NMN

oral NR dosing increased circulating NAM ~40-fold while NMN remained unchanged and NR was detected only at trace levels in the blood

Thus, the majority of the orally administered NR that reaches the muscle appears to enter in the form of liberated NAM or as NMN

Nampt overexpression prevents age-related decline in muscle function through a direct metabolic mechanism

Neither the natural abundance of circulating NMN, nor extracellular Nampt (eNampt) (Revollo et al., 2007b) appear sufficient to alleviate the resulting pathology

we were surprised to find that NR exerts only a subtle influence on the steady state concentration of NAD in muscles

this is largely attributable to breakdown of orally delivered NR into NAM prior to reaching the muscle

Nonetheless, our results indicate that NR is more effective than NAM for reversing mNKO phenotypes

subtle changes in NAD can disproportionately modulate aerobic metabolism

It is important to note that NAD turnover may vary independently from NAD concentration and that small changes in average tissue concentration might reflect larger changes in specific cells or subcellular compartments.

Such indirect activities may help to explain how oral NR administration clearly mitigates the severity of insults to a growing list of tissues in which robust NAD decrements were not observed before treatment

while mNKO mice clearly exhibited progressive weakness and loss of both endurance and bone structure, the decline in NAD was more severe than has been observed for normal aging

the modest age-related decline in NAD can have functional consequences is in stark contrast to the ability of young mice to initially tolerate a much more severe depletion

suggests that one or more additional factors may aggregate over time to exacerbate the dependence of muscle function on internal NAD stores


NAD AND THE AGING PROCESS: ROLE IN LIFE, DEATH AND EVERYTHING IN BETWEEN

CD38 is clearly the main NADase in mammalian tissues and contributes to the degradation of NAD both in cultured cells and in tissues. CD38 will consume NAD during its normal catalysis, generating the products NAM and ADPR
CD38 is mostly an ectoenzyme highly expressed in immune cells
it has been shown that CD38 and its homolog BST-1/CD157 degrade both NMN and NR
Another group of enzymes that uses NAD as a substrate and can regulate cellular NAD levels are the PARP enzymes
mitochondria are regulated by nuclear NAD
levels of NAMPT decline during replicative senescence of human smooth muscle cells, and in peripheral tissue
whereas exercise training has the opposite effect, at least in skeletal muscle
CD38 is the main enzyme involved in the degradation of the NAD precursor nicotinamide mononucleotide (NMN) in vivo (Camacho-Pereira et al., 2016),
indicating that CD38 has a key role in age-related NAD decline and as a modulator of NAD-replacement therapy for aging and metabolic diseases.

It has been hypothesized that CR, by increasing the levels of intracellular NAD, could stimulate SIRTUIN activity, and then extend the lifespan of organisms, (Lin et al., 2000; Longo et al., 2015; Lu and Lin, 2010).
Recently, it was reported that a CR mimetic, 2-deoxyglucose (2-DG), can extend the replicative lifespan of Hs68 cells by increasing intracellular NAD and SIRT1 activity (Yang et al., 2011).
However, several other mechanisms, independent of SIRTUINS, have also been implicated in the biological effects of CR
including inhibition of the IGF-1 pathway and modulation of the AMPK-mTOR pathway

Dissecting Systemic Control of Metabolism and Aging in the NAD World: The Importance of SIRT1 and NAMPT-mediated NAD Biosynthesis

significant amounts of eNAMPT exist in mouse and human blood circulation,

Based on these findings, it has been proposed that eNAMPT contributes to extracellular biosynthesis of NMN that is distributed to all tissues and organs to promote NAD biosynthesis at a systemic level [12,15].

Indeed, major metabolic tissues can rapidly incorporate NMN from blood circulation and utilize it for NAD biosynthesis (our unpublished results)


The dynamic regulation of NAD metabolism in mitochondria (Roberts, 2012)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3683958/

Indeed, in certain circumstances where glucose is the predominant exogenous substrate, depletion of cytoplasmic NAD by 50% or more can block glycolysis and mitochondrial substrate flux and eventually lead to loss of cellular viability

Interestingly, in the liver, NR also shows a mild increase (~5-fold) compared to a ~15-fold increase of NMN after NMN administration

suggesting that some NMN may be converted to NR before uptake

The mitochondrial NAD pool is relatively distinct from that of the rest of the cell [15, 16]. While cytoplasmic NAD/NADH ratios range between 60 and 700 in a typical eukaryotic cell, mitochondrial NAD/NADH ratios are maintained at 7 to 8

Aging also has a significant impact on NAD biosynthesis at the cellular and organismal levels, resulting in reduced NAD levels in human cells [93, 94] and rodents [52, 94, 95].

Consistent with the decreases in NAD levels, aged mice have significantly decreased NAMPT levels in peripheral tissues [52]

Firstly, ATP can be used to phosphorylate NR, generating NMN,

NAD precursors and intermediates are converted to NMN in the cytoplasm, and NMN is transported into mitochondria for NAD biosynthesis

Both intermediates can also be systemically delivered to cells in different tissues and organs.

Recently, it has been shown that NMN and NR can be efficiently transported into cells and multiple tissues and utilized for NAD biosynthesis [52, 53].

For instance, within fifteen minutes after administration of a single dose of NMN to mice, NMN levels in the liver, pancreas, and white adipose tissue (WAT) increase significantly, and NMN levels in the pancreas and WAT reach 10-fold higher levels than NMN levels in the liver

These changes are accompanied by a concomitant significant increase in NAD levels (2.0 fold) in the liver, and a more modest one in the pancreas and WAT (1.4 fold and 1.2–1.5 fold, respectively)


Sublingual and Oral delivery

http://www.ijplsjournal.com/issues%20PD ... 011/10.pdf

any drug diffusing across the oral mucosa membranes has direct access to the systemic circulation via capillaries and venous drainage and will bypass hepatic metabolism

the oral cavity offers relatively consistent and friendly physiological conditions for drug delivery

Enzyme degradation in the GI tract is a major concern for oral drug delivery. In comparison, the buccal and sublingual regions have less enzymes and lower enzyme activity, which is especially favorable to protein and peptide delivery

The intra-oral method of absorption i.e. used in oral spray vitamins - has been shown to be up to 90% effective, whereas in (fig.3) The Physician's Desk Reference shows that vitamins and minerals in a pill form are only 10-20% absorbed by the body


Quantitative Analysis of NAD Synthesis-Breakdown Fluxes(Liu, Rabinowitz, 2018)
Ling liu dissertation


https://dataspace.princeton.edu/jspui/b ... _12390.pdf
Later published:
https://sci-hub.tw/https://doi.org/10.1 ... 018.03.018

NR and NMN were administered by intravenous bolus or oral gavage at a relatively low dose (50 mg/kg)

Readily detectable concentrations of intact NR were observed in the blood following IV injection, but not after oral administration, indicating nearly complete first-pass metabolism (Figure 2.7d)

Irrespective of the route of delivery, the main circulating product of the administered NR or NMN was NAM, which rose ~20x within 5 min of IV NR or NMN; oral NR or NMN administration led to a more modest rise in circulating NAM

Examination of tissue NAD labeling indicated some direct assimilation of oral NR and NMN into liver NAD, based on M+2 labeling that made up a minority of the signal, but was nonetheless readily detectable.

The active formation of liver NAD from NR and NMN is consistent with both compounds being subject to substantial hepatic first pass metabolism. In contrast, extrahepatic tissues displayed minimal M+2 NAD (Figure 2.7e), suggesting that orally delivered NR and NMN are converted into NAM before reaching the systemic circulation.

IV injection of NR or NMN, on the other hand, resulted in substantial M+2 NAD in both liver and kidney.

In the brain, we detected only M+1 NAD, indicating a reliance on circulating NAM and suggesting that intact NR and NMN may not cross the blood-brain barrier.

Other tissues, in contrast, relied almost exclusively on circulating NAM made by the liver.

Liver synthesis of NAD and excretion of NAM occurred even when serum NAM was elevated by co-infusion of tryptophan and NAM; thus, liver constitutively produces NAM to support NAD synthesis throughout the rest of the body

We also explored the metabolism of two NAD precursors that have recently received attention for their ability to elevate tissue NAD levels, NR and NMN. Interestingly, we found that neither compound was able to enter the circulation intact in substantial quantities when delivered orally.


Nicotinamide adenine dinucleotide is transported into mammalian mitochondria

https://elifesciences.org/articles/33246

http://sci-hub.tw/https://elifesciences ... SyoymS8%3D

mitochondria do not synthesize NAD at all, but rather take it up intact from the cytosol, which in turn, can take up NAD from the extracellular space

Here we present evidence that mitochondria directly import NAD.

also results in labeling of mitochondrial NAD, suggesting that fully formed NAD, rather than NMN, is transported

Taken together, our experiments confirm that despite the lack of any recognized transporter, mammalian mitochondria, like their yeast and plant counterparts, are capable of importing NAD(H)

While mammalian mitochondria are generally considered to be impermeable to pyridine nucleotides (32,33), at least two studies have previously reported evidence for uptake of NAD

leading the authors to propose that intact NAD crosses the plasma membrane and subsequently enters the mitochondria directly

unequivocally demonstrate that the mitochondrial NAD pool can be established through direct import of NAD

the finding of Felici et al. that extracellular NAD but not nicotinamide riboside is able to restore mitochondrial NAD in cells overexpressing FKSG76

implying that both NMN and NAD import contribute to the mitochondrial NAD pool

This observation suggests that a mitochondrial transporter for NMN may also await discovery

In summary, we show that mammalian mitochondria are capable of directly importing NAD (or NADH). This finding strongly suggests the existence of an undiscovered transporter in mammalian mitochondria


Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis

http://sci-hub.tw/https://www.ncbi.nlm. ... /21917911/

“Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.”

“as expected, nicotinamide, nicotinamide riboside, and NMN exposure augmented iNAD, although to an extent several fold lower than that prompted by NAD”

“In the present study we report that exposure to eNAD substantially increases the dinucleotide cellular pool, suggesting plasma membrane permeability”

“It is noteworthy that these increments take place in different cellular compartments and alter specific NAD-dependent reactions”

“present study indicates that mitochondria sense cytoplasmic concentrations of NAD and/or its precursors, resetting their pyridine nucleotide pool accordingly.”

“A key finding of our study is that apoptosis is reduced by increasing the extracellular concentrations of NAD”

It is known, however, that almost complete (98% at 8 min) single-pass transformation of NAD infused into the portal vein or hepatic artery takes place in the perfused rat liver (Broetto-Biazon et al., 2008), indicating rapid degrada- tion by hepatocytes.


AMPK activation protects cells from oxidative stress‐induced senescence via autophagic flux restoration and intracellular NAD + elevation

https://www.ncbi.nlm.nih.gov/pmc/articl ... 6-bib-0006

AMPK activation restored the H2O2‐impaired autophagic flux in senescent cells

AMPK restored NAD+ synthesis in cells with H2O2‐induced senescence

AMPK activation restored the NAD + levels in the senescent cells

“AMPK activation raises the intracellular NAD+ concentrations and activates SIRT1”

“The involvements of NAD+ synthesis assessed next. The known pathways of NAD+ synthesis were diagrammed in Fig. 5E. Our results showed that mRNA and protein abundance of nicotinamide phosphoribosyl transferase (NAMPT), a rate‐limiting enzyme of NAD+ synthesis in salvage pathway, significantly decreased in senescent cells (Fig. 5F); however, the mRNA level of quinolinic acid phosphoribosyl transferase (QPRT), a rate‐limiting enzyme of NAD+ synthesis in de novo pathway, was increased (Fig. S8A), while no expressional alteration in nicotinamide mononucleotide adenylyltransferase (NMNAT) (Fig. S8A). In addition, the situation of NAD+ consumption was examined via measuring the activity of a major NAD+‐consuming enzyme poly‐ADP‐ribose polymerase (PARP‐1). Resultantly, PARP‐1 activity significantly increased in senescent cells (Fig. S9). These results demonstrate that the NAD+ decline found in senescent cells is relevant to both its synthetic decline and consumptive elevation, and for the synthesis, the involvement of salvage pathway seems dominating.”

“These results demonstrate the connection of NAD+ synthesis with the AMPK activity in our system and particularly emphasize the involvement of the salvage NAD+ synthesis pathway.”

senescent cells less NAMPT, less NAD salvage. MORE de novo NAD. MORE PARP-1. Less NAD overall.

NAMPT required for AMPK to increase NAD+ thru salvage pathway

“the activation of AMPK can suppress this type of cellular senescence by restoring both autophagy flux and NAD+ synthesis”

“Moreover, the result from Burkewitz et al., 2014 is also interesting. It suggests that sustained stimulation of AMPK lead to irreversible senescence, while acute activation of AMPK catabolic pathway permitted a rapid adaptation or resistance to external and internal stresses”

“inhibition of NAD+ synthesis in normal cells could obviously suppress the autophagic flux, suggesting that NAD+ homeostasis is required for the maintenance of the autophagic flux.”

“Although it is currently unclear why adding NMN cannot restore the autophagic flux”

“antisenescence effect of AMPK rely on both the activation of autophagy and the restoration of NAD+ synthesis”

“we found that BBR treatment significantly suppressed mTOR phosphorylation in senescent cells, similar and even stronger than that induced by rapamycin and insulin as an mTOR activation control”

“Met and BBR upregulated the cellular NAD+ level”

“autophagic dysfunction and a decline in NAD+ are two features of senescent cells induced by oxidative stress, and the activation of AMPK can suppress this type of cellular senescence by restoring both autophagy flux and NAD+ synthesis.”


NAD+ metabolism: Bioenergetics, signaling and manipulation for therapy

mitochondrial NAD+ pool is likely more stable compared to the cytosolic pool

“Evidence indicates the entire NAD+ pool is consumed and resynthesized in mammals several times a day [72]. Under normal cellular and tissue conditions, synthesis of NAD+ is affected by the availability of possible precursors, so that availabilities of nicotinic acid, nicotinamide riboside, nicotinamide and tryptophan can alter NAD+ synthetic rates, thus affecting NAD+ level. “

“For mammalian cells the central challenge in NAD+ homeostasis is successful recycling of nicotinamide,
released from NAD+ consuming processes, back to NAD+ “

“Assuming comparable numbers for a 75 kg human, 3 g of nicotinamide is required to be resynthesized to NAD+ up to several times per day “
“The nicotinamide recycling reaction is catalyzed by an enzyme called nicotinamide phosphoribosyltransferase (nampt). Nampt couples nicotinamide with PRPP to form nicotinamide mononucleotide (NMN) “
“Experiments to assess the role of Nampt in setting the NAD+ level in cells confirms that the level of the enzyme, and not nicotinamide concentrations themselves, have the largest effect on setting NAD+ level “
Evidence indicates the entire NAD+ pool is consumed and resynthesized in mammals several times a day [72]. Under normal cellular and tissue conditions, synthesis of NAD+ is affected by the availability of possible precursors, so that availabilities of nicotinic acid, nicotinamide riboside, nicotinamide and tryptophan can alter NAD+ synthetic rates, thus affecting NAD+ level. Not each of these precursors is bioequivalent in this respect. Strikingly, nicotinamide is not limiting in many tissues, except possibly in liver, so availability of nicotinamide is not crucially tied to NAD+ formation rate, and even when administered at fairly substantial doses via diet [73]

This is remarkable, if one considers the possibility that the entire NAD+ pool is being replaced 2-4 times per day, suggesting that only 0.1-0.2 % nicotinamide is lost per turnover cycle. This result implicates a highly efficient NAD+ resynthesis capacity in humans

“Interestingly, Nampt levels appear to be upregulated by dietary intake and exercise “

“NR and NaR raise NAD+ levels dose-dependently and up to 2.7 fold in mammalian cells [110] in a manner unlike the behavior of nicotinamide or nicotinic acid “

“Kirkland and co-workers explored effects of high doses of nicotinamide or nicotinic acid on tissue NAD+ levels in rats, and concluded that these precursors had different abilities to raise NAD+ levels in different tissues [73]

Nicotinamide had ability to increase NAD+ level in liver (47%), but was weaker in kidney (2%), heart (20%), blood (43%) or lungs (17%). Nicotinic acid raised NAD+ in liver (47%), and impressively raised kidney (88%), heart (62%), blood (43%) and lungs (11%) [73] [both nicotinamide and nicotinic acid were administered at 1000 mg/kg diet] “

“CD38-/animals were protected from weight gain also showed impressive mitochondrial biogenesis in skeletal muscle [66] “


NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences

Nampt/PBEF/visfatin in plasma raises an intriguing possibility that this protein might generate NMN extracellularly using the nicotinamide in plasma as a substrate, which may be subsequently transported into cells for NMNATcatalyzed NAD synthesis (238). While many future studies are needed to demonstrate this hypothesis, our understanding regarding NAD synthesis could be significantly revised if this hypothesis were demonstrated: The processes of NAD synthesis may not only occur intracellularly in the nucleus and other subcellular organelles, but also occur extracellularly. It has also been proposed that the cytokine-like functions of PBEF and the insulin-mimetic functions of visfatin may be accounted for by the NAD -synthesizing functions of Nampt (238). Demonstration of this hypothesis would further deepen our understanding about the biological functions of the extracellular metabolic intermediates in NAD synthesis
Fasting and CR induce NAMPT expression 2–3 fold in the liver and skeletal muscle

On the other hand, HFD feeding significantly reduces NAMPT protein levels in the liver and WAT

Like HFD feeding, aging significantly reduces NAMPT protein levels.

Consistent with the decreases in NAD levels, aged mice have significantly decreased NAMPT levels in peripheral tissues

The brain, sirtuins, and ageing

plasma levels of NMN decrease with age

NMN also ameliorates disease conditions, including type 2 diabetes induced by high-fat diet or age140, brain damage in a cerebral ischaemia–reperfusion mouse model141, and cognitive impairment and amyloid deposition in Alzheimer disease

It takes two to tango: NAD+ and sirtuins in aging/longevity control

NMN is rapidly incorporated to major metabolic tissues and converted to NAD

NAMPT and NAD+ levels decline with age in multiple organs, such as pancreas, adipose tissue, skeletal muscle, liver, and brain


NAMPT regulates mitochondria biogenesis via NAD metabolism and calcium binding proteins during skeletal muscle contraction

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241898/

“The purpose of this study was to investigate the effect that muscle contraction induced NAD metabolism via NAMPT has on mitochondrial biogenesis.”

“the AMPK-NAMPT signal is a key player for muscle contraction induced SIRT1 expression and PGC-1α deacetylation, which influences mitochondrial biogenesis”

“It is known that muscle contraction elevates NAD/NADH levels and regulates various signal proteins including SIRT 1”

“In skeletal muscle, NAMPT is increased by exercise or calorie restriction and is dependent on AMPK activation [13]”

“AMPK is not only a key regulatory factor for energy metabolism but also for mitochondria biogenesis [4].”

“Nampt-dependent recycling of nicotinamide to NAD (see Fig. 1) may represent a physiologically important homeostatic mechanism to avoid depletion of the intracellular NAD pool during its active use as a substrate.”

“Uncontrolled PARP-1 activity in response to high levels of DNA damage can, however, lead to a severe depletion of intracellular NAD pools, decreased resistance to stress, and cell death”

“it is tempting to conclude that elevated levels of Nampt may in fact represent a physiological adaptive response increasing cell resistance to environmental stress.”

“Because of its central role in the recycling pathway allowing NAD biosynthesis from nicotinamide, Nampt occupies a pivotal position in controlling the activity of several NAD-dependent enzymes”

“although NAD can in principle be synthesized by alternative sources, virtually all cells of the organism rely on nicotinamide to maintain adequate intracellular NAD levels “

“available data strongly suggest that cells adjust systemic NAD biosynthesis to regulate the activity of several NAD-dependent enzymes such as sirtuins.”

“The recent finding that rhythmic oscillations in protein levels of Nampt lead to circadian oscillation on NAD strongly support the central role of Nampt in the regulation of NAD homeostasis”


NAD+ metabolism and the control of energy homeostasis a balancing act between mitochondria and the nucleus

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4487780/

“NAD+ has emerged as a vital cofactor that can rewire metabolism, activate sirtuins and maintain mitochondrial fitness”

“As a first step, NAD+ is cleaved to NMN and 5’-AMP. Next NMN is rapidly hydrolyzed to NR, which in turn is more slowly converted into NAM “

“blood concentrations of NA are relatively low (~100 nM), yet when pharmacologically primed , can increase and be rapidly converted to NAM by the liver.
Strikingly, NAM levels in fasted human plasma are also too low to support NAD+ biosynthesis in cells.
All of these results suggest that these NAD+ precursors are metabolized very quickly in mammalian blood and tissues”
“sirtuin activity is inhibited by NAM,which could explain why NAM failed to provide the benefits of NA,
however, in some situations NAM treatments can have beneficial effects”
NMN might be present in the bloodstream at concentrations around 50 μM (Revollo et al., 2007).

In contrast, NAD+ increases in mammalian cells and tissues in response to exercise or calorie restriction (CR) (, both of which are interventions associated with metabolic and age-related health benefits.
Therefore, despite the classical misconception that intracellular NAD+ levels rarely change , the evidence above unequivocally demonstrates the ability of NAD+ to respond dynamically to physiological stimuli. So, how do changes in NAD+ levels take place innately?”

“NAM can also be an NAD+ precursor through its metabolism into NAM mononucleotide (NMN) by the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT)”

“NA is initially metabolized by the NA phosphoribosyltransferase (NAPRT) into NAMN, converging with the de novo pathway”
“NR is transported into cells by nucleoside transporters and is then phosphorylated by the NR kinases 1 and 2 (NRKs) , generating NMN.
After the generation of NMN, NMNAT enzymes can then catalyze the formation of NAD+”
NR is not salvage or de novo a unique pathway?
“different lines of evidence suggest that NR is the primary metabolite transported into the cell and metabolized into NAD”
“The presence of a circulating extracellular form of the NAMPT enzyme (eNAMPT), to convert NAM to NMN, supports this possibility.
Recent evidence indicates that eNAMPT activity in the plasma is required to safeguard hypothalamic NAD+ levels”
“All the above suggest that plasma levels of most NAD+ precursors are probably unable to systematically sustain high NAD+ production rates.”
“This, however, does not rule out a limited contribution of circulating NR, NMN, NAM or Trp to NAD+ biosynthesis under basal conditions.”
“further technical improvements will be needed, especially for NR, NMN and NAM determination, to precisely evaluate whether circulating precursors contribute to tissular NAD+ homeostasis”
“intracellular NAD+ levels are maintained between 0.2 and 0.5 mM, depending on the cell type or tissue. However, NAD+ levels can change, up to ~2-fold, in response to diverse physiological stimuli”
“It was recently shown that exogenous NAD+ can elevate mitochondrial NAD+ levels more than cytoplasmic levels, indicating that NAD+ precursors or intermediates traverse the mitochondrial membrane”
“NR is likely converted to NMN in the cytosol and NMN may traverse the mitochondrial membrane to produce NAD+ via NMNATs” conflicting with other views…

“This way, both NMN and NAM might act as the main intracellular forms for regulating NAD+ levels between compartments”

“Excessive DNA damage dramatically reduces NAD+ levels (Berger, 1985), even down to 20-30% of their normal levels”

“PARP activity leads to SIRT1 inactivation, by limiting NAD+ levels, in the case of PARP1 ”
however:
“SIRT1 was shown to directly inhibit PARP1”

“NAD+ levels can become so low following cell stress or senescence that SIRT1 no longer has the activity to keep PARP1 in check.

This is supported by the fact that NAD+-repletion by expression of NAMPT can protect against PARP1 overexpression in a SIRT1-mediated manner”

“Thus, it is likely that diverse cellular fates and metabolic decisions are closely regulated by the balance of the reciprocal regulation of SIRT1 and PARP1 activities, under the guidance of NAD+ levels”

“Recent work has further strengthened the hypothesis that PARP1 and SIRT1 have counterbalancing roles in metabolism and aging.

For instance, PARP1 activity is enhanced with aging and high-caloric intake , yet reduced upon nutrient scarcity “

CD38
“Mice deficient in Cd38 show significantly elevated levels of NAD+ (10-30-fold) in tissues such as liver, muscle, brain, and heart, with corresponding SIRT1 activation, confirming the role of CD38 as a major NAD+ consumer”
“increase in NAD+ observed in Cd38-deficient mice is ~30 fold, while most other strategies described to date lead to a ~2-fold increase in NAD+ at best. The massive effect of CD38 on NAD+ levels could therefore be indicative for major alterations in additional NAD+-utilizing metabolic pathways.

Niacin
“the effects of niacin relied on the ability of NA or NAM to elevate NAD+ levels and activate the sirtuins”

NAM
“NAM, but not NA, can recover this drop in NAD+ levels (Ho and Hashim, 1972). Later it was demonstrated that the NAD+ reduction induced by STZ was due to increased DNA damage, stimulating PARP1 activity”

“NAM has the capacity to exert end-product inhibition on SIRT1 deacetylase activity. However, long-term NAM treatment increases NAD+ levels via the NAD+ salvage pathway, which likely tips the balance of the NAD+/NAM ratio such that SIRT1 is activated”

“OLETF rats, a rodent model of obesity and type 2 diabetes, exhibited profound metabolic improvements following NAM treatment (100mg/kg for 4 weeks). This treatment induced liver NAD+ levels, which were complimented by enhanced glucose control”

“exposing neuronal cells to toxic prion proteins to model protein misfolding in Alzheimer’s and Parkinson’s disease induced NAD+ depletion that was improved with exogenous NAD+ or NAM”

“NR might have a privileged position among different NAD+ precursors in the prevention of neurodegeneration, as the effect of NR may be enhanced by the increase in NRK2 during axonal damage”

CANCER
“niacin supplementation can decrease the development of skin cancer
NR can both reduce the incidence of cancer and have a therapeutic effect on fully formed tumors”
“some evidence suggests that the protective effects of niacin against the development of skin cancer are due to elevation in both PARPs and SIRT1 activity”
AGING
“most data agree that sirtuin activation in mammals delays the onset of age-related degenerative processes”

“The association between metabolism, health and lifespan have long been proposed based on similarities, between metabolic dysfunction and disease (e.g. obesity, diabetes, neurodegeneration, cancer) and the aging process. Only recently have these processes been linked so tightly by multiple proteins, including the sirtuins and PARPs, all of which are tightly controlled by the regulation and subcellular balance of the metabolite NAD+”

“As such, we have never been so close to solving the ancient question of how we age and what we can do to slow this process, while simultaneously not compromising on our quality of life.”
drkris69
Posts: 304
Joined: Tue Aug 20, 2019 2:41 pm

Re: NAD+ Research (Part 1)

Post by drkris69 » Thu Jan 16, 2020 8:07 am

Wow thank you Jocko for this thread. I will be studying this and it will be great using it as a reference. ;)
Pigeon
Posts: 10
Joined: Sat Sep 21, 2019 6:20 am

Re: NAD+ Research (Part 1)

Post by Pigeon » Thu Jan 16, 2020 7:58 pm

A fantastic resource Jocko, thank you. I do wish we had a pocket of this forum where we could stash all of the relevant published papers that have passed through the forum since its inception.
Drdavid
Posts: 488
Joined: Thu Aug 15, 2019 9:59 am

Re: NAD+ Research (Part 1)

Post by Drdavid » Fri Jan 17, 2020 7:15 am

jocko6889 wrote:
Wed Jan 15, 2020 6:11 pm
NAD+ therapy in age-related degenerative disorders: A benefit/risk analysis(Braidy, 2020)

https://sci-hub.se/downloads-ii/2020-01 ... 110831.pdf

Human studies documenting the beneficial effects of raising NAD+ levelsin the CNS are nascent in the current literatire. At present,oral or i.v. NADH have been reported to reduce anxiety (Alegre et al., 2010), attenuatesleep disturbances (Santaella et al., 2004;Forsyth et al., 1999), improvecognitive performance(Birkmayer, 1996), lower the number and duration ofheadaches (Forsyth et al., 1999), and amelioratesymptoms of jet lag (Birkmayer et al., 2002). Treatment with NADH has also been shown to slow downthe progression of dementia, and improve outcomes inverbal fluency and visual-constructional ability (Demarin et al., 2004). Treatment with i.v NAD+ and NADH has also been shown toimprove motor symptoms inParkinson's disease (Birkmayer, 1993; Grant et al 2019)


Nicotinamide adenine dinucleotide (NADH) in patients with chronic fatigue syndrome(De Sevilla, 2010)

https://www.sciencedirect.com/science/a ... 6510001591

Administration of oral NADH was associated to a decrease in anxiety and maximum heart rate, after a stress test in patients with CFS. On the contrary, this treatment did not modify other clinical variables and the global functional performance.

Comparison of oral nicotinamide adenine dinucleotide (NADH) versus conventional therapy for chronic fatigue syndrome
(Santealla, 2004)


https://www.ncbi.nlm.nih.gov/pubmed/15377055

The twelve patients who received NADH had a dramatic and statistically significant reduction of the mean symptom score in the first trimester (p < 0.001). However, symptom scores in the subsequent trimesters of therapy were similar in both treatment groups. Elevated IgG and Ig E antibody levels were found in a significant number of patients.

Nicotinamide adenine dinucleotide (NADH)--a new therapeutic approach to Parkinson's disease. Comparison of oral and parenteral application (Birkmayer, 1994)

https://www.ncbi.nlm.nih.gov/pubmed/8101414

The reduced coenzyme nicotinamide adenine dinucleotide (NADH) has been used as medication in 885 parkinsonian patients in an open label trial. About half of the patients received NADH by intravenous infusion, the other part orally by capsules. In about 80% of the patients a beneficial clinical effect was observed: 19.3% of the patients showed a very good (30-50%) improvement of disability, 58.8% a moderate (10-30%) improvement. 21.8% did not respond to NADH. Statistical analysis of the improvement in correlation with the disability prior to treatment, the duration of the disease and the age of the patients revealed the following results: All these 3 parameters have a significant although weak influence on the improvement. The disability before the treatment has a positive regression coefficient (t value < 0.01). The duration of the disease has a negative regression coefficient (< 0.01) and so has the age a negative regression coefficient (t value < 0.05). In other words younger patients and patients with a shorter duration of disease have a better chance to gain a marked improvement than older patients and patients with longer duration of the disease. The orally applied form of NADH yielded an overall improvement in the disability which was comparable to that of the parenterally applied form

Treatment of Alzheimer’s disease with stabilised oral nicotinamide adenine dinucleotide: A randomised double-blind study (Demarin, 2004)

https://www.ncbi.nlm.nih.gov/pubmed/15134388

The present trial was a randomized, placebo-controlled, matched-pairs, double-blind, 6-month clinical study. Patients with probable AD (n = 26) were randomized to receive either stabilized oral NADH (10 mg/day) or placebo. Twelve pairs of subjects were matched for age and baseline total score on the Mattis Dementia Rating Scale (MDRS) and the Mini Mental State Examination. After 6 months of treatment, subjects treated with NADH showed no evidence of progressive cognitive deterioration and had significantly higher total scores on the MDRS compared with subjects treated with placebo (p < 0.05). Analysis of MDRS subscales revealed significantly better performance by NADH subjects on measures of verbal fluency (p = 0.019), visual-constructional ability (p = 0.038) and a trend (p = 0.08) to better performance on a measure of abstract verbal reasoning. There were no differences between groups in measures of attention, memory, or in clinician ratings of dementia severity (Clinical Dementia Rating). Consistent with earlier studies, the present findings support NADH as a treatment for AD.


Combination of NAD+ and NADPH Offers Greater Neuroprotection in Ischemic Stroke Models by Relieving Metabolic Stress (Huang, 2018)

10.1007/s12035-017-0809-7

Both reduced nicotinamide adenine dinucleotide phosphate (NADPH) and β-nicotinamide adenine dinucleotide hydrate (NAD+) have been reported to have potent neuroprotective effects against ischemic neuronal injury. Both NADPH and NAD+ are essential cofactors for anti-oxidation and cellular energy metabolism. We investigated if combined NADPH and NAD+ could offer better neuroprotective effects on cellular and animal models of ischemic stroke. In vitro studies with primary cultured neurons demonstrated that NAD+ was effective in protecting neurons against oxygen-glucose deprivation/reoxygenation (OGD/R) injury when given during the early time period of reoxygenation. In vivo studies in mice also suggested that NAD+ was effective for ameliorating ischemic brain damage when administered within 2 h after reperfusion. The combination of NADPH and NAD+ provided not only greater beneficial effects but also larger therapeutic window in both cellular and animal models of stroke. The combination of NADPH and NAD+ significantly increased the levels of adenosine triphosphate (ATP) and reduced the levels of reactive oxygen species (ROS) and oxidative damage of macromolecules. Furthermore, the combined medication significantly reduced long-term mortality, improved the functional recovery, and inhibited signaling pathways involved in apoptosis and necroptosis after ischemic stroke. The present study indicates that the combination of NAD+ and NADPH can produce greater therapeutic effects with smaller dose of NADPH; on the other hand, NADPH can significantly prolong the therapeutic window of NAD+. The current results suggest that the combination of NADPH and NAD+ may provide a novel effective therapy for ischemic stroke.

In summary, NAD+replenishment exhibited neuroprotectionagainst ischemia/reperfusion-induced neuronal injury bothin vivo and in vitro. The combination of NAD+andNADPH provided greater neuroprotective effects in both cel-lular and animal models of ischemic stroke. NADPH couldprolong the therapeutic window of NAD+, whereas NAD+could reduce the dosage of NADPH to produce the desirabletherapeutic effects. The current study suggests that the com-bination of NAD+and NADPH may provide a novel effectivetherapy for ischemic stroke.


Nicotinamide phosphoribosyltransferase regulates cocaine reward through Sirtuin 1(Kong, 2018)

https://sci-hub.se/10.1016/j.expneurol.2018.05.010

Our results suggest that NAMPT-mediated NAD biosynthesis may modify cocaine behavioral effects through SIRT1. Moreover, our findings reveal that the interplay between NAD biosynthesis and SIRT1 regulation may comprise a novel regulatory pathway that responds to chronic cocaine stimuli.


Fasting- and ghrelin-induced food intake is regulated by NAMPT in the hypothalamus (Treebak, jan 2020)

https://www.ncbi.nlm.nih.gov/pubmed/31900990

Neurons in the arcuate nucleus of the hypothalamus are involved in regulation of food intake and energy expenditure, and dysregulation of signaling in these neurons promotes development of obesity. The role of the rate-limiting enzyme in the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT), for regulation energy homeostasis by the hypothalamus has not been extensively studied.
METHODS:
We determined whether Nampt mRNA or protein levels in the hypothalamus of mice were affected by diet-induced obesity, by fasting and re-feeding, and by leptin and ghrelin treatment. Primary hypothalamic neurons were treated with FK866, a selective inhibitor of NAMPT, or rAAV carrying shRNA directed against Nampt, and levels of reactive oxygen species (ROS) and mitochondrial respiration were assessed. Fasting and ghrelin-induced food intake was measured in mice in metabolic cages after intracerebroventricular (ICV)-mediated FK866 administration.
RESULTS:
NAMPT levels in the hypothalamus were elevated by administration of ghrelin and leptin. In diet-induced obese mice, both protein and mRNA levels of NAMPT decreased in the hypothalamus. NAMPT inhibition in primary hypothalamic neurons significantly reduced levels of NAD+ , increased levels of ROS, and affected the expression of Agrp, Pomc, and genes related to mitochondrial function. Finally, ICV-induced NAMPT inhibition by FK866 did not cause malaise or anhedonia, but completely ablated fasting- and ghrelin-induced increases in food intake.
CONCLUSION:
Our findings indicate that regulation of NAMPT levels in hypothalamic neurons is important for the control of fasting- and ghrelin-induced food intake.


(NAD+) Diphosphopyridine Nucleotide in the Prevention, Diagnosis and Treatment of Drug Addiction (Ohollaren, 1961)

https://daks2k3a4ib2z.cloudfront.net/5a ... n-1961.pdf

DPN = old name for NAD+
The author has previously reported the successful use of DPN in the treatment of acute and chronic alcoholism. In the administration of nearly 1000 Gm. to more than 100 patients there has been no toxic effect whatsoever; from the coenzyme DPN in its oxidized form, when administered at a speed tolerated by the patient.


Intravenous NAD+ effectively increased the NAD metabolome, reduced oxidative stress and inflammation, and increased expression of longevity genes safely in elderly humans(Brady, 2019-pending)

https://dergipark.org.tr/jcnos/issue/48187/610084

NAD+ injection HAS been tested in humans and is far more effective than NR capsules. Some research by Naidy Brady presented at a conference shows 1 week of NAD+ IV in humans . They haven’t published this yet, so don’t have much detail, but we know they found NAD+ injections for 1 week had significantly more more effect than 3 weeks of NR. Markers such as CRP improved with NAD+, not with NR.

"Nicotinamide adenine dinucleotide (NAD+) serves important roles in hydrogen transfer and as the cosubstrate for poly(ADP-ribose) polymerase (PARPs), the sirtuin (SIRT1-7) family of enzymes, and CD38 glycohydrolases. Recently, intravenous (IV) NAD+ therapy has been used as a holistic approach to treat withdrawal from addiction, overcome anxiety and depression, and improve overall quality of life with minimal symptoms between 3-7 days of treatment. We evaluated repeat dose IV NAD+ (1000 mg) for 6 days in a population of 8 healthy adults between the ages of 70 and 80 years.

Our data is the first to show that IV NAD+ increases the blood NAD+ metabolome in elderly humans. We found increased concentrations of glutathione peroxidase -3 and paraoxonase-1, and decreased concentrations of 8-iso-prostaglandin F2α, advanced oxidative protein products, protein carbonyl, C-reactive protein and interleukin 6. We report significant increases in mRNA expression and activity of SIRT1, and Forkhead box O1, and reduced acetylated p53 in peripheral blood mononuclear cells isolated from these subjects. No major adverse effects were reported in this study. The study shows that repeat IV dose of NAD+ is a safe and efficient way to slow down age-related decline in NAD+."


A pilot study investigating changes in the human plasma and urine NAD+ metabolome during a 6 hour intravenous infusion of NAD+(Brady, 2019)

https://www.frontiersin.org/articles/10 ... 7/abstract

This study therefore documented changes in plasma and urine levels of NAD+ and its metabolites during and after a 6 hour 3 μmol/min NAD+ intravenous infusion.
Surprisingly, no change in plasma [NAD+] or metabolites (nicotinamide, methylnicotinamide, ADP ribose and nicotinamide mononucleotide) were observed until after 2 hours. Increased urinary excretion of methylnicotinamide and NAD+ were detected at 6 hours, however no significant rise in urinary nicotinamide was observed.
This study revealed for the first time that i) at an infusion rate of 3 μmol/min NAD+ is rapidly and completely removed from the plasma for at least the first 2 hours, ii) the profile of metabolites is consistent with NAD+ glycohydrolase and NAD+ pyrophosphatase activity and iii) urinary excretion products arising from an NAD+ infusion include NAD+ itself and meNAM but not NAM.


Treatment of Alzheimer's disease with stabilized oral nicotinamide adenine dinucleotide: a randomized, double-blind study (Demarin, 2004)

https://www.ncbi.nlm.nih.gov/pubmed/15134388/

This study was designed to evaluate the effect of stabilized oral reduced nicotinamide adenine dinucleotide (NADH) on cognitive functioning in patients with Alzheimer's disease (AD). NADH is a coenzyme that plays a key role in cellular energy production and stimulates dopamine production. In previous trials NADH has been shown to improve cognitive functioning in patients with Parkinson's disease, depression and AD. The present trial was a randomized, placebo-controlled, matched-pairs, double-blind, 6-month clinical study. Patients with probable AD (n = 26) were randomized to receive either stabilized oral NADH (10 mg/day) or placebo. Twelve pairs of subjects were matched for age and baseline total score on the Mattis Dementia Rating Scale (MDRS) and the Mini Mental State Examination. After 6 months of treatment, subjects treated with NADH showed no evidence of progressive cognitive deterioration and had significantly higher total scores on the MDRS compared with subjects treated with placebo (p < 0.05). Analysis of MDRS subscales revealed significantly better performance by NADH subjects on measures of verbal fluency (p = 0.019), visual-constructional ability (p = 0.038) and a trend (p = 0.08) to better performance on a measure of abstract verbal reasoning. There were no differences between groups in measures of attention, memory, or in clinician ratings of dementia severity (Clinical Dementia Rating). Consistent with earlier studies, the present findings support NADH as a treatment for AD.


Insight into Molecular and Functional Properties of NMNAT3 Reveals New Hints of NAD Homeostasis within Human Mitochondria (Chiarugi, 2013)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796565/

we show that extracellular NAD, but not its metabolic precursors, sustains mitochondrial NAD pool in an ATP-independent manner

Intriguingly, we found that cellular NAD depletion could be completely prevented by adding 1 mM NAD to the culture media (Fig. 6A). However, identical concentrations of the NAD precursors Nam, NMN, nicotinamide riboside and nicotinic acid were not effective (Fig. 6A).

A widely appreciated dogma of cell biology is that mitochondria are impermeable to NAD [16], [33]. Several findings, however, indicate that this tenet should be revisited. First, both yeast and plant mitochondria have NAD transporters [34], [35]. Second, transporters for NAD precursors in the plasmamembrane or different cell organelles including mitochondria have not been identified so far. Third, inability of mitochondria to transport NAD has been only demonstrated in vitro, and it might be due to the fact that, under these experimental settings, transport systems are impaired or lack a co- or counter-molecule necessary for their functioning. Consistently, both yeast and plant mitochondrial NAD transporters work as nucleotide exchangers [34], [35]. Fourth, increase in the extracellular concentrations of NAD in cultured cells raises the dinucleotide content within their mitochondria and boosts cellular respiration [8]. Finally, prior work found evidence for metabolic state-dependent NAD fluxes through the inner mitochondrial membrane [36], [37]. These findings taken together, plus data of the present study indicating that exogenous NAD but not NMN, NR, NA or Nam prevents FKSG76-dependent mitochondrial NAD depletion, suggest that, akin to yeast and plants, yet-to-be defined mitochondrial NAD transporters are present in mammalian cells.


SIRT1-mediated eNAMPT secretion from adipose tissue regulates hypothalamic NAD+ and function in mice

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426056/

lowering glucose dramatically enhanced eNAMPT secretion 3.5–5.5-fold

Adipose tissue-specific Nampt knockout mice exhibit reduced plasma eNAMPT levels and defects in NAD+ biosynthesis not only in adipose tissue but also in the hypothalamus

while other tissues such as the liver and skeletal muscle did not show any change in NAD+ levels (Figure 5B), the hypothalamus, but not the hippocampus, showed significant decreases in NAD+ levels

confirming the direct effect of eNAMPT on hypothalamic NAD+ levels.

Furthermore, the defect appears to be specific at least to the hypothalamus because other tissues and organs, including the liver, skeletal muscle, and the hippocampus, do not show any reduction in NAD+ levels

female ANKO mice showed significantly reduced physical activity during the dark time (Figure 6A), consistent with reduced hypothalamic NAD+

it is conceivable that eNAMPT secreted from adipose tissue plays a critical role in supplying NAD+ to the hypothalamus

FASCINATING!


Effects of Chronic NAD Supplementation on Energy Metabolism and Diurnal Rhythm in Obese Mice (Roh, 2018)

https://www.ncbi.nlm.nih.gov/pubmed/30230244
http://sci-hub.se/https://www.ncbi.nlm. ... d/30230244

C57BL/6 mice were fed a high-fat diet (HFD) for 12 weeks and received an intraperitoneal injection of either saline or NAD (1 mg/kg/day) for the last 4 weeks.

The control mice were fed a chow diet and injected with saline for the same period. Body weights were monitored daily. Daily rhythms of food intake, energy expenditure, and locomotor activity were measured at the end of NAD treatment. The effect of NAD treatment on the clock gene Period 1 (PER1) transcription was also studied.
RESULTS:
Chronic NAD supplementation significantly attenuated weight gain in HFD-fed obese mice. Furthermore, NAD treatment recovered the suppressed rhythms in the diurnal locomotor activity patterns in obese mice. In addition, exogenous NAD supply rescued cellular NAD depletion-induced suppression of PER1 transcriptional activity in hypothalamic neuron cells as well as blunted daily fluctuations of hypothalamic arcuate nucleus PER1 expression in obese mice.
CONCLUSIONS:
NAD supplementation showed therapeutic effects in obese mice with altered diurnal behaviors.

Previous studies have reported the benefi- cial metabolic effects of supplementation with NAD precursors NR and NMN in mice and humans (11,14‒17). Similar to our findings, 12-week supplementation with NR (400 mg/kg/d in drinking water) in HFD-fed mice reduced body weight gain and fat mass and increased EE (14). Twelve-month NMN administration in mice (100-300 mg/kg in drinking water) attenuated aging-associated body weight gain (16). Moreover, intraperitoneal administration of NMN (500 mg/kg/d) for 1 week improved glucose metabolism in diabetic mice (11) and restored mitochondrial functions in the muscles of aged mice (15). It is notable that supplementation with NAD by itself, at a 100-times lower dose compared with those of its precursors NMN and NR (25,26), caused a beneficial metabolic effect;

Mice that received NAD+ weighed less, were more active, and had better glucose control than mice that received placebo

Nicotinamide adenine dinucleotide suppresses epileptogenesis at an early stage

To confirm whether NAD+ penetrates the BBB, we harvested the hippocampus and measured the NAD+ level at 30 min and 60 min after NAD+ injection in normal male C57BL/6 mice. As shown in Fig. 1a, compared with control mice (1.00 ± 0.10, N = 5), the NAD+ level in the hippocampus was significantly high at 30 min after the i.p. injection of NAD+ (100 mg/kg, i.p.; 1.43 ± 0.05, N = 6) and was still higher at 60 min after the injection (1.24 ± 0.11, N = 5)

NAD+ in blood crossed to brain and hippocampus

NAD+ Injections Reversed NAD+ Depletion in SE Model Mice at Early-Stage Epileptogenesis

Early-Stage Injection of NAD+ Attenuated the Incidence of Seizures in SE Model Mice at SRS Stage

Early-Stage Intervention With NAD+ Reversed Abnormal EEG Activity in SE Model Mice at SRS Stage

early-stage intervention with NAD+ improved contextual fear memory impairment

intraperitoneal injection of 50 mg/kg NAD+ decreases ischemic brain damage in ischemic model mice

intraperitoneal injection of 100 mg/kg NAD+ alleviates doxorubicin-induced liver damage in mice


Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway (Pillai, 2009)

https://www.ncbi.nlm.nih.gov/pubmed/19940131


http://sci-hub.tw/10.1074/jbc.M109.077271

Mice were simultaneously treated with NAD at 1 mg/kg/day for 2 weeks
these data demonstrated that NAD treatment was capable of maintaining cellular NAD levels
NAD treatment restored the cellular NAD levels

In summary, we demonstrated that exogenous NAD can block cardiac hypertrophy via activation of SIRT3.


Exogenous NAD+ administration significantly protects against myocardial ischemia/reperfusion
injury in rat model (Zhang, 2016)

https://www.ncbi.nlm.nih.gov/pmc/articl ... 8-3342.pdf

Our observations have suggested that exogenous NAD+ administration can pro- foundly decrease I/R hearts injury
Saline or NAD+ (5 mg/kg, 10 mg/kg, 20 mg/kg, dissovled in saline) were injected intra- venously right before ischemia
Rats were sacrificed 6 hours or 24 hours after reperfusion
Compared with other drugs that have shown protective effects on myocardial I/R injury, the NAD+ produced 85% decrease in the infarct size has suggested that NAD+ is one of the drugs that have greatest capacity to decrease myocardial
We observed obvious protective effects of 10 mg/ kg and 20 mg/kg NAD+ on the infarct formation (Figure 1A).
Quantifications of the myocar- dial infarct volume indicate that NAD+ dose dependently decreased infarct formation

Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia. (Ying, 2007)

https://www.bioscience.org/2007/v12/af/ ... lltext.htm

10 and 20 mg / kg NAD+

Also tested 10 mg/kg NAM - not effective

Six ml of NAD+ or nicotinamide, which was dissolved in phosphate-buffered saline (PBS), was applied in one side of the nose of rats each time, with the nose at another side blocked for 5 seconds to enhance the influx of the solutions through the nostrial tract. This procedure was repeated every 2 minutes alternatively on each side of the nose, totally for 10 times

The profound protective effects of the intranasal NAD+ administration were also observed at 72 hrs after ischemia.

intranasal administration with 10 mg / kg NAD+, but not with 5 mg / kg NAD+, significantly attenuated the ischemia / reperfusion-produced neurological deficits

In contrast to the profound protective effects of 10 mg / kg NAD+, intranasal administration with 10 mg / kg nicotinamide did not decrease infarct formation

Our results provide the first in vivo evidence that NAD+ administration can profoundly decrease brain damage under certain pathological conditions

It is noteworthy that NAD+ can reduce infarct formation by up to 86 % even when administered at 2 hrs after ischemic onset. Compared with other studies that apply drugs during post-ischemia phases in order for decreasing ischemic brain injury, the protective effect of NAD+ could be one of the most profound effects ever reported

Intranasal drug delivery approach could have multiple merits over traditional drug delivery approaches: First, it may deliver drugs into the brains by bypassing the blood-brain barriers

Cumulative evidence has suggested that NAD+ may mediate cell death via multiple mechanisms (5). For examples, NADH / NAD+ ratio is a major index of cellular reducing potential, which can modulate MPT --- a mediator of both apoptosis and necrosis under many conditions; and both NAD+ and NADH mediate energy metabolism that could determine cell death modes


Pharmacological Effects of Exogenous NAD on Mitochondrial Bioenergetics, DNA Repair and Apoptosis (Pittelli, 2011)

http://molpharm.aspetjournals.org/conte ... 6.full.pdf

Although the canonical view considers NAD unable to permeates lipid bilayers (Di Lisa and Ziegler, 2001), several studies report evidence for exogenous NAD (eNAD) uptake by different cells
we further investigated cellular uptake of eNAD, and also focused on its possible functional effects as indirect evidence of eNAD entrance
Together, our findings on the one hand strengthen the hypothesis that eNAD crosses intact the plasmamembrane, and on the other provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

The ability of eNAD to increase the mitochondrial NAD content and energy production is of particular relevance
it is worth noting that the present study indicates that mitochondria sense cytoplasmic concentrations of NAD and/or its precursors, resetting their pyridine nucleotide pool accordingly
mitochondrial fluctuation of NAD contents according to those present in the cytoplasm does not seem bidirectional
It seems, therefore, that these organelles are able to maintain their NAD content when that of cytosol decreases, but readily increase the pyridine nucleotide pool when the cytoplasmic availability of NAD and/or its precursors increases

A key finding of our study is that apoptosis is reduced by increasing the extracellular concentrations of NAD
Our data are consistent with previous reports showing that eNAD affords protection from various stresses such as beta-amyloid (Qin et al., 2006), ischemia (Wang et al., 2008), NMNAT inactivation (Wang et al., 2005) or PARP-1-dependent cell death
These findings are at odds with the hypothesis that eNAD increase iNAD contents because of extracellularly-formed NAD precursors

eNAD is transported intact across the plasmamembrane


Prevention of Traumatic Brain Injury-Induced Neuron Death by Intranasal Delivery of Nicotinamide Adenine Dinucleotide (won, 2012)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972775/

The present study demonstrates that intranasally-administered NAD+ increases hippocampal NAD+ levels and reduces TBI-induced neuronal death in the hippocampus.

Intraperitoneal injection of the same NAD+ concentration that we used for intranasal administration showed no neuroprotective effects on TBI-induced neuronal death.

Compared with NAD+-treated rats, nicotinamide treatment showed no protective effects against TBI-induced neuronal death

Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia (ying, 2007)

https://www.bioscience.org/2007/v12/af/ ... lltext.htm

Increasing evidence has supported the hypothesis that PARP-1 induces cell death by depleting intracellular NAD+. We observed that intranasal NAD+ delivery significantly increased NAD+ contents in the brains.

Intranasal delivery with 10 mg / kg NAD+ at 2 hours after ischemic onset profoundly decreased infarct formation when assessed either at 24 or 72 hours after ischemia. The NAD+ administration also significantly attenuated ischemia-induced neurological deficits. In contrast, intranasal administration with 10 mg / kg nicotinamide did not decrease ischemic brain damage. These results provide the first in vivo evidence that NAD+ metabolism is a new target for treating brain ischemia, and that NAD+ administration may be a novel strategy for decreasing brain damage in cerebral ischemia and possibly other PARP-1-associated neurological diseases.

Our results provide the first in vivo evidence that NAD+ administration can profoundly decrease brain damage under certain pathological conditions. It is noteworthy that NAD+ can reduce infarct formation by up to 86 % even when administered at 2 hrs after ischemic onset. Compared with other studies that apply drugs during post-ischemia phases in order for decreasing ischemic brain injury, the protective effect of NAD+ could be one of the most profound effects ever reported. In this study we also provided evidence that by the intranasal delivery approach NAD+ can be delivered into the brains.

Multiple studies have suggested that nicotinamide can decease ischemic brain injury, but at doses higher than 125 mg / kg (19). Our study shows that intranasal administration with nicotinamide at 10 mg / kg can not affect ischemic brain damage, in contrast to the profound protective effects of 10 mg / kg NAD+.


Exogenous nicotinamide adenine dinucleotide administration alleviates ischemia/reperfusion-induced oxidative injury in isolated rat hearts via Sirt5-SDH-succinate pathway( lui, jul 2019)

http://sci-hub.tw/https://www.ncbi.nlm. ... d/31278893

We first found that myocardial total NAD level was remarkably increased with NAD treatment (10 mg/kg) for 14 days.

NAD administration significantly decreased the lactate dehydrogenase (LDH) level in coronary leakage, decreased the malondialdehyde (MDA) level and increased the reduced glutathione/oxidized glutathione disulfide ratio (GSH/GSSG) in myocardial tissue.

In addition, NAD treatment effectively attenuated the depression of cardiac function in the isolated rat hearts after ischemia-reperfusion. Furthermore, we found that exogenous NAD attenuated the succinate accumulation during ischemia and decreased its depleting rate during reperfusion.

we found that NAD administration promoted the Sirt5 and SDH-a interaction and decreased the succinylation level of SDH-a.

These results implied that exogenous NAD administration promoted Sirt5-mediated SDH-a desuccinylation and decreased the activity of SDH-a, which attenuated the succinate accumulation during ischemia and its depleting rate during reperfusion and finally alleviated reactive oxygen species generation.


SIRT2, ERK and Nrf2 Mediate NAD+ Treatment-Induced Increase in the Antioxidant Capacity of PC12 Cells Under Basal Conditions

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497790/

We found that NAD+ treatment can increase the GSH/GSSG ratios in the cells under basal conditions. These findings have suggested not only the great nutritional potential of NAD+, but also a novel mechanism underlying the protective effects of the NAD+ administration in the disease models: the NAD+ administration can enhance the resistance of the normal cells to oxidative insults by increasing the antioxidant capacity of the cells.

The major findings of our current study include: first, NAD+ treatment can increase the GSH/GSSG ratio of PC12 cells under basal conditions, suggesting that NAD+ treatment can increase directly the antioxidant capacity of the cells; Collectively, our study has indicated that NAD+ can enhance directly the antioxidant capacity of the cells

Our recent study reported that NAD+ induced increases in intracellular ATP levels under basal conditions through its degradation into adenosine (Zhang et al., 2018).

NAD+ can pass through BBB to enter the brain under normal conditions: Roh et al. (2018) reported that exogenous NAD+ crossed the BBB through the Connexin 43 gap junction and entered the hypothalamus in its intact form; and Huang et al. (2018) reported that intravenous injection of NAD+ significantly increased the NAD+ level in the brain under physiological conditions, which has further suggested that NAD+ can cross the BBB under normal conditions.


Extracellular Degradation Into Adenosine and the Activities of Adenosine Kinase and AMPK Mediate Extracellular NAD+-Produced Increases in the Adenylate Pool of BV2 Microglia Under Basal Conditions (Zhang, Oct2018)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200843/

intranasal administration of NAD+, but not nicotinamide, can profoundly decrease ischemic brain injury (Ying et al., 2007) and traumatic brain injury (Won et al., 2012), which argues against the possibility that extracellular NAD+ produces its protective effects via nicotinamide – one of its degradation products. However, extracellular NAD+ can also be degraded into adenosine by multiple potential mechanisms

treatment of 10, 100, and 500 μM NAD+ can significantly increase the intracellular levels of ATP, ADP, and AMP of the cells (Figures Figures1A1A–C). NAD+ treatment can also increase the intracellular levels of NAD+

Our time-course study showed that extracellular adenosine levels increased by threefold after only 10 min of the NAD+ treatment

The major findings of our current study includes: first, NAD+ treatment can significantly increase the intracellular levels of ATP, ADP, and AMP of BV2 microglia under basal conditions.

Third, NAD+ treatment can significantly increase the extracellular adenosine levels,

Fourth, NAD+ treatment can significantly increase the intracellular adenosine levels of the cells.

Previous studies have indicated that extracellular NAD+ significantly decreases the damage of the cells exposed to various pathological insults. Therefore, our study has indicated that the mechanisms underlying the NAD+ treatment-produced effects on the cells under pathological insults are not applicable to the cells under basal conditions. These observations are not surprising for the following reasons: the cytosolic NAD+ concentrations are normally in the ranges between 1 and 10 mM (Ying, 2008). Because NAD+ enters cells by gradient-driven transport (Alano et al., 2010), the NAD+ at the concentrations between 0.01 and 0.5 mM, which was used in our study, should not be able to enter the cells to influence the adenylate pools of BV2 microglia under basal conditions.

extracellular NAD+ is degraded into adenosine extracellularly, that enters the cells through ENTs, which is converted to AMP by adenosine kinase. Increased AMP can lead to both increased AMPK activity and increased intracellular ADP levels, which jointly produce the increased intracellular ATP levels of BV2 cells under basal conditions.

Although the NAD+ at the concentrations between 0.01 and 0.5 mM cannot directly enter cells to increase intracellular NAD+ levels, we still found that the NAD+ can significantly increase the intracellular levels of NAD+ and adenylate of BV2 microglia through extracellular degradation into adenosine.

Since intracellular adenosine levels are normally in the nanomolar range (Latini and Pedata, 2001), the extracellular NAD+-generated adenosine can enter cells to increase intracellular adenosine levels thus increasing the intracellular adenylate levels through the activities of adenosine kinase and AMPK. In other words, the exceedingly low intracellular adenosine levels under normal physiological conditions is an unique ‘attractor’ and base for the relatively low concentrations of extracellular NAD+ to produce its significant biological effects on cells.

our current study has provided the first direct evidence showing that extracellular adenosine generated from extracellular NAD+ degradation mediates the biological effects of exogenous NAD+ by adenosine kinase- and AMPK-mediated pathways.

Our study has shown that relatively low concentrations of NAD+ can increase both extracellular and intracellular adenosine levels, the AMPK activity and intracellular adenylate pools, all of these factors have been shown to enhance defensive potential of both normal cells and stressed cells against toxic insults

Collectively, our current study has suggested a novel mechanism to account for the profound protective effects of NAD+ administration in the animal models of a number of diseases and aging

It is noteworthy that the intravenous NAD+ administration in all of the animal studies should lead to the NAD+ concentrations that are well below milimolar range (Ying et al., 2007; Wang et al., 2014; Zhang et al., 2016; Xie et al., 2017). Since the cytosolic concentration of NAD+ is normally in the range between 1 and 10 mM, it is usually assumed that the NAD+ administration-produced NAD+ concentrations in the blood should not be able to enter cells to produce biological effects. However, our study has shown that as low as 10 μM NAD+ can lead to significant increases in the ATP levels of all of the cell types we have studied on this topic, including BV2 microglia, PC12 cells, and C6 glioma cells (Zhang et al., 2018). Therefore, our findings have general value for understanding the mechanisms underlying the biological effects of NAD+ administration in models of diseases, aging or healthy controls: the relatively low concentrations of extracellular NAD+ can still produce its profound effects on cells through its extracellular degradation into adenosine, which may lead to increased intracellular levels of adenosine, AMP, ADP, and ATP on the basis of the activities of ENTs, adenosine kinase and AMPK.


NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864043/

These results establish NAD+ depletion as causal event in PARP-1 mediated cell death

Prior studies provide indirect evidence for uptake of extracellular NAD+ by neurons (T. Araki et al., 2004; J. Wang et al., 2005), and direct evidence for NAD+ uptake by other cell types (S. Bruzzone et al., 2001; C. C. Alano et al., 2004; R. A. Billington et al., 2008).

PARP-1 activation is a major cause of neuronal death in brain ischemia, trauma, and other settings, but the role of NAD+ depletion in this cell death pathway has been unresolved. The present findings show that PARP-1-mediated neuronal death is blocked when NAD+ depletion is prevented with exogenous NAD+. NAD+ repletion also prevents the intermediary steps in this cell death pathway that otherwise result from PARP-1 activation: glycolytic inhibition, mitochondrial depolarization, and mitochondrial AIF release. Conversely, depletion of cytosolic NAD+ with NAD+ glycohydrolase causes glycolytic inhibition and mitochondrial AIF release, independent of PARP-1 activation. These findings establish cytosolic NAD+ depletion as a necessary and sufficient event in the PARP-1 cell death pathway.


Contribution of P2X7 receptors to adenosine uptake by cultured mouse astrocytes (Okuda, 2010)

https://www.ncbi.nlm.nih.gov/pubmed/20645413/

Extracellularly applied NAD(+) prevents astrocyte death caused by excessive activation of poly(ADP-ribose) polymerase-1, In this study, we examined whether the intact form of NAD(+) is incorporated into astrocytes. A large portion of extracellularly added NAD(+) was degraded into metabolites such as AMP and adenosine in the extracellular space. The uptake of adenine ring-labeled [(14)C]NAD(+), but not nicotinamide moiety-labeled [(3)H]NAD(+), Taken together, these results indicate that exogenous NAD(+) is degraded by ectonucleotidases and that adenosine, as its metabolite, is taken up into astrocytes via the P2X7R-associated channel/pore.


Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment (Lasmezas, 2015)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4840455/

Mice were treated daily with 30 mg/kg NAD+ by the intranasal route as a means to circumvent the blood–brain barrier

Four days after the onset of treatment, we observed a substantial difference in the level of activity of NAD+- versus PBS-treated mice.

The former started moving and exploring almost immediately after being placed in the cage, while the majority of the latter remained prostrated (Supplementary Video 1).
Rotarod testing starting 1 week after the onset of treatment showed better performance in NAD+- versus PBS-treated mice (Supplementary Fig. 8). Quantification of motor activity in the open field showed a significant difference between the two groups (Fig. 7C).

Addition of NAD+ 3 days after TPrP exposure completely and dose-dependently rescued cells from TPrP-induced death (Fig. 2A). The rescuing effect of NAD+ was independent of the reduced or oxidized status of the metabolite (Fig. 2B). Blockage of the salvage synthesis pathway (that uses nicotinamide as a precursor for NAD+) using the compound FK866 abolished the protective effect of nicotinamide in a dose-dependent manner (but not that of NAD+ itself)

The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.

Prion diseases are fatal brain diseases of animals and humans and belong to the group of protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, frontotemporal dementia or amyotrophic lateral sclerosis.

we discovered that TPrP induces neuronal death via a profound depletion of intracellular nicotinamide adenine dinucleotide (NAD+) levels causing metabolic failure. Neuronal death can be rescued in vitro and in vivo by NAD+ replenishment.

our data demonstrate for the first time that a failure of NAD+ metabolism is the cause of neuronal ailing

Moreover, our TPrP toxicity model reveals a new mechanism of NAD+ depletion independent of PARP1.

misfolded amyloidogenic protein can induce neuronal death by genuine NAD+ starvation and that ailing neurons can be completely rescued by NAD+ treatment

our study shows that neuronal death induced by NAD+ depletion is reversible and that NAD+ replenishment mitigates neurodegeneration

We propose the development of NAD+-replenishment strategies for the treatment of prion diseases.


Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells (Nikiforov, 2019)

https://www.mdpi.com/2218-1989/9/12/293/htm

Here, we studied the metabolism of extracellular NAD+ and its derivatives in human HEK293 cells using normal and serum-free culture medium. Using medium containing 10% fetal bovine serum (FBS), mono- and dinucleotides were degraded to the corresponding nucleosides. In turn, the nucleosides were cleaved to their corresponding bases. Degradation was also observed in culture medium alone, in the absence of cells, indicating that FBS contains enzymatic activities which degrade NAD+ intermediates. Surprisingly, NR was also rather efficiently hydrolyzed to Nam in the absence of FBS. When cultivated in serum-free medium, HEK293 cells efficiently cleaved NAD+ and NAAD to NMN and NAMN. NMN exhibited rather high stability in cell culture, but was partially metabolized to NR. Using pharmacological inhibitors of plasma membrane transporters, we also showed that extracellular cleavage of NAD+ and NMN to NR is a prerequisite for using these nucleotides to maintain intracellular NAD contents. We also present evidence that, besides spontaneous hydrolysis, NR is intensively metabolized in cell culture by intracellular conversion to Nam.

Interestingly, exogenous nucleotides including NMN, NAMN, NAD+, and NAAD can support the maintenance of intracellular NAD pools as well as the nucleoside NR [12,13,14,15].

Moreover, the human ecto-enzyme CD73 has been described to catalyze both the cleavage of NAD+ to NMN and AMP as well as the subsequent dephosphorylation of the mononucleotides to the corresponding nucleosides, NR and adenosine [19,20].

In addition, extracellular NAD+ is used as a substrate of the glycohydrolases CD38 and CD157 which generate the second messengers ADP-ribose and cyclic ADP-ribose with the release of Nam [21] (Figure 1).

there are several studies supporting the direct uptake of NMN or NAD+ into human cells [13,23,24].

………………………

The quantitative analysis revealed that after 12 h of incubation in the presence of cells, less than 40% of the originally added amount of NAD+ remained. Moreover, after 48 h, NAD+ was undetectable in the medium (Figure 3B).

At the same time, a considerable amount of NMN was detectable, but also the formation of Nam was observed.

Surprisingly, NAD+ was efficiently degraded to NMN and Nam in the culture medium, even in the absence of cells. Within 24 h of incubation of NAD+ in the standard medium, DMEM supplemented with 10% heat-inactivated FBS; at 37 °C more than half of the added dinucleotide was degraded, whereas after 48 h less than 20% of the originally added amount remained (Figure 3B).

The mononucleotide NMN exhibited a higher stability, even though it was also significantly degraded to NR and Nam in the absence of cells. In the presence of cells, NMN was degraded faster, but even under these conditions, after 24 and 48 h still 80% and 60%, respectively, of the added mononucleotide were present in the medium (Figure 3C).

Likewise, we observed cleavage of NR to Nam in the medium without cells.

Strikingly, in the control sample that contained water instead of FBS, the extent of NR cleavage was similar (Figure 4, right lower panel).

These data indicate that NR efficiently hydrolyses to Nam (and ribose) in aqueous solutions

As shown here, any intermediate of NAD metabolism could potentially contribute to the maintenance of intracellular NAD. However, it is unlikely that they are equally relevant for this function. Physiologically, the intermediates are differently available.

NMN exhibits a relatively high chemical stability but is partly dephosphorylated to NR by the cells.


Extracellular ATP and β-NAD alter electrical properties and cholinergic effects in the rat heart in age-specific manner (kuzmin, mar 2019)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439013/


Nicotinamide phosphoribosyltransferase-related signaling pathway in early Alzheimer's disease mouse models(Chen, dec2019)

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854586/

NAD+ group were intraperitoneally injected with NAD+ (30 mg/kg) at 20 weeks of age and once every other day for 4 weeks.

The administration of NAD+ alleviated the spatial learning and memory of APP/PS1 mice and reduced senile plaques. Administration of NAD+ may also increase the expression of the key protein NAMPT and its related protein sirtuin 1 as well as the synthesis of NAD+. Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.

NAD+ group were intraperitoneally injected with NAD+ (30 mg/kg) at 20 weeks of age and once every other day for 4 weeks.

NAMPT expression levels in the hippocampus in the NAD+ group were also significantly increased after intraperitoneal injection of NAD+

Intraperitoneally injecting NAD+ increased the expression of NAMPT in the cortex in the NAD+ mice (P<0.05).

Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.


Age-Associated Changes In Oxidative Stress and NAD+ Metabolism In Human Tissue

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3407129/

“Age associated increases in oxidative nuclear damage have been associated with PARP-mediated NAD+ depletion and loss of SIRT1 activity”
“This study provides quantitative evidence in support of the hypothesis that hyperactivation of PARP due to an accumulation of oxidative damage to DNA during aging may be responsible for increased NAD+ catabolism in human tissue.
The resulting NAD+ depletion may play a major role in the aging process, by limiting energy production, DNA repair and genomic signalling.”

“There is a growing awareness that oxidative stress (OS) plays a key role not only in the aging process, but also in various degenerative diseases including Alzheimer's disease, cancer, diabetes, and chronic inflammation”

“However, at high concentrations, they are capable of damaging proteins, lipids and DNA [6].”

“Studies showing an increase in intracellular ROS through exogenous hydrogen peroxide treatment in vitro, or inhibition of endogenous ROS scavenging enzymes such as superoxide dismutase (SOD) and catalase, have been shown to promote premature aging and significantly lower lifespan”

“the mitochondria (the site of oxidative phosphorylation and ATP generation) is the major source of ROS production”

“The human body has a number of physiological protection and repair systems, including activation of the DNA nick sensor poly(ADP-ribose) polymerase-1 (PARP)”

“excessive DNA damage leads to over-activation of PARP, and increased NAD+ catabolism [23], [24], resulting in suppression of NAD+-dependent ATP generation and possible energy crisis”

“PARP activity was significantly increased in adults, older adults and elderly subjects compared to newborns”

“A significant decrease in total NAD+ content was observed in adults (p<0.05), older adult (p<0.05) and elderly (p<0.05) subjects compared to newborns”

“we found no significant difference in SIRT1 activity between any of the four age categories”
Lipid Peroxidation Increases with Age
Oxidative DNA Damage Accumulates with Age
PARP Activity increases with age leading to NAD+ Depletion

“report for the first time that PARP activity increases with age in human skin and correlates with both age and NAD+ depletion . “

“we report for the first time a significant decline in SIRT1 activity with age in post pubescent males (Fig. 5A, line b), though surprisingly, not in females (Fig. 5B).

We also did not find a correlation between NAD+ levels and SIRT1 activity in males”

“the lack of correlation between NAD+ levels and SIRT1 activity suggests that NAD+ availability, though required, is not the most sensitive modulator of SIRT1 activity in humans.”

“This study reports for the first time a link between oxidative stress and PARP activity, aging and a decline in NAD+ levels, in human tissue”
Great reports! Would you mind posting this in the NAD research archive thread?
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Re: NAD+ Research (Part 1)

Post by jocko6889 » Fri Jan 17, 2020 11:17 am

DrDavid, that was sort of my plan, to merge what you started with this. See my response to you on the other thread you started.
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Re: NAD+ Research (Part 1)

Post by smokinjoe » Fri Jan 17, 2020 6:20 pm

Wow, this is a treasure trove! Thank you for this!
Male, 41
no i don't smoke :)

taking:
NAD+ nasal since August 15, 2019
NMN powder, 125 mg per day; 625 per day since Oct 1, 2019
Resveratrol 600mg, since September 15, 2019
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