2195 replies to this topic

[Andey]

hypnos said:

. That's some of my tools and I'd like to hear from others so I can steal your ideas! 

 

I use a polar HR chest strap and free Elite HRV  heart rate variability app from the Google play-store. I check my HRV most mornings..

 

HRV declines with age and has been suggested as a biomarker for biological age.  A low HRV also indicates a sick heart.  HRV is affected by how you breath so one should use a breathing guide and breath the same during each measurement (5 breaths/minute is standard).

 

HRV varies from day to day. Once a baseline is established, HRV programs use the daily variation to suggest when to exercise hard and when to take it easy.

 

 

 

Hmm..same thoughts )

Ive got extremely inconsistent result from same setup - Polar H7 and Elite HRV. HRV Index changed randomly within one minute laying in bed. As a result Ive get rid of  my H7. 

Probably this problem is specific to me though as my PNS compromised after prolonged illness.

 

BTW you can hyperlink posts directly if you want if copy the link at the top right corner of it (that with #XXX) 

Edited by Andey, 12 July 2017 - 04:46 PM.

[Advocatus Diaboli]

Thanks, #240 RWhigham and #241 Andey for the suggestions. HRV certainly would be interesting to follow during a regimen of Turnbuckle protocol. However, from the mixed results you guys posted, it looks as if I'll need to do some searches for documentations of the experiences of others using the same, or similar, setups in order to get an idea of how reliable they view the results that they get in comparison with your results before I make a final decision to purchase.

 

A few other of my "tools" for assessing potential Turnbuckle-protocol progress-monitoring, that I didn't mention in my previous posts include: a 3-station weight machine on which I do light weight (100 lbs, a tad more than 45 kg) pull-downs, horizontal presses, leg extensions, and "butterfly presses". A couple of 30 lb (about 13.6 kg) dumbbells for curls and overhead presses, and a chin-up structure.

 

OFF TOPIC--It's interesting to note that Andey may be from, and/or lives in Ukraine (as per location data) and asserts a compromised PNS. Given the proximity of Kiev to Chernobyl I wonder if there might be a connection between symptoms and possible ingestion of radioactive decay products from the Chernobyl reactors disaster

 

Results of any nerve-conduction-velocity study (via electromyography) available could be useful to view as a baseline to assess any changes after some period on a Turnbuckle protocol. In addition, self-tests of simple reaction time would be interesting to follow in order to note any possible improvement that might be partially attributed to the protocol.

 

Iodine-131 which would have been released from Chernobyl could also play a role in certain PNS pathologies as well as perhaps affect metabolic rate, and so forth. Compromised thyroid function, as might be expected in partial/total destruction of the thyroid, via I-131, might also adversely affect the PNS by interfering with peripheral nerve regeneration as aided by thyroid hormones.

 

 

Edited by hypnos, 12 July 2017 - 08:08 PM.

[RWhigham]

 

The protocols in this thread are based on the premise that increasing NAD+ "increases fusion". I've been thinking about what it means to "increase fusion" and arrived at the following possibility:

 

A mitochondria typically spends 7 sec fused and 21 sec fissioned, and completes a housekeeping cycle every 28 sec. Once things have equilibrated, a snapshot at any given point in time will show 75% of the mitochondria fissioned and 25% fused.

 

 

Now if a mitochondria spends less time fissioned, say 7 sec, a complete cycle will take just 14 sec. Once things have equilibrated, a snapshot at any given point in time will show 50% of the mitochondria fissioned and 50% fused. An observer would say "fusion has increased".  post234

 

 

By speeding  up housekeeping cycles with NAD+ or some other means, the rate at which defective daughters are produced by housekeeping cycles would also increase. This could upset the balance between producing and clearing defective daughters and cause the number of defective daughters to continually increase. 

 

A short time after raising NAD+ in the above scenario an observer would say "fusion increased". Later with a growing population of non-fusing defective daughters the observer would say "fission increased".

 

When NAD+ is kept elevated enough, mitophagy will cause a decline in the total mitochondrial mass of up to 50% in 5-10 days. ref

 

Hypothesis: Increasing NAD+ increases the rate of housekeeping cycles which can fill cells with defective daughters and make one feel wiped out. Reducing NAD+ afterward reduces the housekeeping rate so biogenesis can catch up and restock the mito population, hopefully with better quality mitos from the surviving population.

 

Anecdotal reports indicate that reducing your mitochondrial mass and then letting it rebuild does increase health.

 

Note on exercise:  Exercise increase NAMPT which is the rate-limiting enzyme in the conversion of nicotinamide into NAD+. So exercise after taking a dose of nicotinamide will increase NAD+ more effectively. 

Edited by RWhigham, 12 July 2017 - 09:22 PM.

[Advocatus Diaboli]

RWhigham, a lot of discussion here, about mito fission-fusion, seems to center on the effects on muscles. Do you have any thoughts about how purposefully-induced mito fission and fusion, Turnbuckle protocol, e.g., might affect neurons?

 

OFF TOPIC--Please tell me to shut up if you consider me to be too much of a quidnunc.  You mention in post #240 "an unusual beat pattern". If the nature of the unusual pattern is runs of ectopic beats such as bigeminy,or trigeminy, among others, then the ectopic beats may be an indication of an intracellular magnesium deficiency. Thyroid problems also may manifest in irregular heart beats as well.

The "double hump" in your heartbeat frequency distribution could be an indication of atrial fibrillation, or afib. With afib, some people will complain of a "trout thrashing around in my chest" and others will not have any overt symptoms at all, and are only made aware of the condition after a doctor listens to their heart and/or runs an EKG test. Some afib sufferers may complain of a minor "lack of wind" climbing stairs or jogging.

A pulse reading of 180 is not inconsistent with afib, where atrial beats can reach into several hundreds of beats per minute but may be registered on a Fitbit, for example, as a significantly lower value (180 for example) depending upon the sampling rate of the measuring device, which might not be able to process input data quickly enough to reflect an actual heart rate.

In addition, BNP is released during a paroxysmal (see below) afib attack and its release can result in an urge to urinate.

A good quality fingertip pulse oximeter will show an irregular waveform when in afib--it will be quite noticeably different from the waveform seen in normal sinus rhythm, and the indicated pulse rate displayed on the device will jump around erratically--e.g., 104, 188, 132, 160, 112, all within a few seconds time.

Afib is generally categorized into vagal, adrenergic and mixed types. each of which can be further categorized as paroxysmal, persistent, long-standing persistent, and permanent. Manifestation of an adrenergic afib attack can occur during, as well as after engaging in, strenuous exercise, for example. Whereas a vagal-mediated afib attack may occur after drinking a glass of ice water or after a heavy meal, or bending down.

[RWhigham]

RWhigham, a lot of discussion here, about mito fission-fusion, seems to center on the effects on muscles. Do you have any thoughts about how purposefully-induced mito fission and fusion, Turnbuckle protocol, e.g., might affect neurons?

 

I don't know. It's a worry.  Some of us may be susceptible.

 

The "double hump" in your heartbeat frequency distribution could be an indication of atrial fibrillation

There is no afib and no ectopic beats. EKG is fine. 24 hr Holter monitor was fine. There is nothing noticeably wrong with the EKG in the time domain. I don't think the cardiologist was interested in frequency domain analysis on a perfectly normal looking EKG. 

Edited by RWhigham, 13 July 2017 - 04:50 AM.

[Turnbuckle]

 

 

The protocols in this thread are based on the premise that increasing NAD+ "increases fusion". I've been thinking about what it means to "increase fusion" and arrived at the following possibility:

 

 

 

 

A short time after raising NAD+ in the above scenario an observer would say "fusion increased". Later with a growing population of non-fusing defective daughters the observer would say "fission increased".

 

 

 

 

Can you provide evidence for this surprising assertion that NAD+ increases fusion?

Edited by Turnbuckle, 14 July 2017 - 11:21 PM.

[Richard McGee]

Active autophagy coupled with rapid mitochondrial fusion and fission constitutes an important mitochondrial quality control mechanism and is critical to cellular health. In our previous studies, we found that exposure of cells to nicotinamide causes a decrease in mitochondrial content and an increase in mitochondrial membrane potential (MMP) by activating autophagy and inducing mitochondrial fragmentation. Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD]/ [NADH] ratio and the activation of SIRT1, an NAD-dependent deacetylase that plays a role in autophagy flux. The [NAD]/[NADH] ratio was inversely correlated with the mitochondrial content.Autophagy is the major cellular mechanism removing organelles, including mitochondria, and along with coordinated mitochondrial fission and fusion is believed to selectively remove damaged (depolarized) mitochondria (7). Therefore, the persistence of a high level of autophagy flux and mitochondria, structural dynamics may be the key to the maintenance of mitochondrial quality. In fact, the longevity of model organisms has been linked to the efficient maintenance of autophagy, a cellular process that is down-regulated during aging (8). The role of autophagy in the maintenance of health and longevity was recently highlighted in studies that showed that calorie restriction exerts its effect by enhancing mitochondrial autophagy (9). However, the mitochondrial status in the cells in which autophagy is activated has rarely been examined in detail. Limited information is available as to whether the content of mitochondria decreases or their quality increases in the cells in which autophagy has been induced.
 

http://www.jbc.org/c...inamide-inducedMitophagy EVENT MEDIATED BY HIGH NAD/NADH RATIO AND SIRT1 PROTEIN ACTIVATION*

 

There seems little doubt that raising NAD+ levels contribute to improved autophagy flux. A still open question (at least in my mind) is how effectively the fusion side of the process works during long-term elevation of NAD+ levels. Anecdotal evidence in this thread (increasing tiredness) suggests that fission stimulation should be limited in duration.

[Turnbuckle]

It seems that people are forgetting what the protocol is and posting and debating based on assertions that are 180 degrees out of whack. That is unfortunate, as these assertions will be here for years and people will quote them as if they are true. To recap the protocol:

 

1. Mitochondria are fissioned to the smallest possible size and mitophagy is set in motion, removing defective mtDNA. (This can be set in motion by increasing the NAD+/NADH ratio.)

2. Fusion and bio-genesis replace the mtDNA mass lost in step 1, producing a mito population that is healthier than  before. 

 

A healthier mito population translates to more ATP and improved cellular operations, which translates to healthier organs, which translates to a healthier organism.

 

The immediate effect of step 1 is to decrease ATP production and decrease performance, which can be used to enhance the effectiveness of exercise. See Exercise Like a Girl.

The somewhat more gradual effect of step 2 is to increase ATP production and increase performance. 

 

These steps have to be used together. Step 1 without step 2 can result in damage to tissues such as tendons that have few mitochondria, while step 2 without step 1 will result in a long term decline in mito quality as mito QC will not be functioning.

[Richard McGee]

Some of the misunderstanding people have is due to the complexity of the fission/fusion process and the fact that, in a young and healthy person this process is ongoing. My understanding is that the fission protocol tips the balance in favor of fission, without completely eliminating fusion (and vice versa for the fusion protocol). So the ongoing fission/fusion process is not a binary either-or state of affairs. (Note I'm not accusing anyone of promoting such a view, but it's a complex subject and easily misunderstood.) If I'm understanding correctly, this protocol pushes the balance in favor of one or the other but does not completely eliminate its complementary function. The "stronger" the fission half of the protocol, the more likely it is you experience some negative reaction sooner rather than later. It is a matter of degree. It is probable that, over time, continuing the enhanced cycles becomes less useful after most of the damaged mitochondria are cleaned up.

 

I suggest that after one undergoes an optimum number of cycles of enhanced fission/fusion, perhaps a switch to a milder "housekeeping" mode might be in order (each individual will obviously respond differently). An example of a housekeeping protocol might be NAM + R in the morning and ascorbic acid and PQQ in the evening. Since my reaction to the fission side of the protocol has diminished significantly, I may be at this point already. 

[Turnbuckle]

Advocatus Diaboli--"Do you have any thoughts about how purposefully-induced mito fission and fusion, Turnbuckle protocol, e.g., might affect neurons?"

 

 

Continuously feeding mice with high levels of NR is useful for Alzheimer's--

 

we found (1) dietary treatment of Tg2576 mice [an AD mouse model] with 250 mg/kg/day of NR for 3 months significantly attenuates cognitive deterioration in Tg2576 mice and coincides with an increase in the steady-state levels of NAD+ in the cerebral cortex; (2) application of NR to hippocampal slices (10 µM) for 4 hours abolishes the deficits in long-term potentiation recorded in the CA1 region of Tg2576 mice; (3) NR treatment promotes PGC-1α expression in the brain coinciding with enhanced degradation of BACE1 and the reduction of Aβ production in Tg2576 mice. 

 

https://www.ncbi.nlm...les/PMC3632303/

 

 

So far as I know, no one has looked at using NR (or N+R) intermittently in brain studies, but it should be superior for the same reasons as in the protocol. Another NAD+ precursor, NMN, has been shown to have benefits to neurons--

 

We have previously found that aging significantly reduces levels of the essential cofactor nicotinamide adenine dinucleotide (NAD+) in multiple peripheral tissues (Yoshino et al, 2011). This age-associated decrease in NAD+ levels is due to a decline in protein levels of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the biosynthetic pathway of NAD+ from nicotinamide (Fig​(Fig1A)1A) (Revollo et al, 2004; Yoshino et al, 2011). Nampt converts nicotinamide, a major precursor in mammalian NAD+ biosynthesis, and 5′-phosphoribosyl-1-pyrophosphate to nicotinamide mononucleotide (NMN), a key NAD+ intermediate. NMN is then converted to NAD+ by nicotinamide/nicotinic acid mononucleotide adenylyltransferase (Nampt) (Revollo et al, 2004, 2007). We and others have previously reported that the expression of Nampt in the brain is extremely low compared to peripheral tissues (Revollo et al, 2007; Friebe et al, 2011). However, Nampt has uniquely strong expression in the hippocampus (Zhang et al, 2010; Wang et al, 2011a). Because recent studies show that the energetic demands of stem cell proliferation and lineage specification require distinct metabolic programs (Folmes et al, 2012), we hypothesized that NSPCs would be particularly sensitive to changes in NAD+ levels and accordingly alter their proliferation, self-renewal, and differentiation.

 

https://www.ncbi.nlm...les/PMC4194122/

 

 

NMN is nicotinamide mononucleotide, and does more than just convert to NAD+ in the brain--

 

 

Nicotinamide mononucleotide could restore cognition in Alzheimer's disease rat model.

 

Treatment of intracerebroventricular Aβ oligomer infusion AD model rats with NMN (500mg/kg, intraperitoneally) sustained improvement in cognitive function as assessed by the Morris water maze. In OHCs, Aβ oligomer- treated culture media with NMN attenuated neuronal cell death.NMN treatment also significantly prevented the Aβ oligomer-induced inhibition of LTP. Furthermore, NMN restored levels of NAD(+) and ATP, eliminated accumulation of reactive oxygen species (ROS) in the Aβ oligomer - treated hippocampal slices. 

 

http://www.greenmedi...ers-disease-rat

 

 

How it might work intermittently I don't know, but it should be easy to try as it appears to require only the addition of a phosphate source, at least in vitro--

 

Conditions Necessary for NMN Synthesis-The failure of previous workers

to obtain results comparable with those reported here may be attributed

to two factors. In order to obtain maximum synthesis, the cells

must be washed prior to incubation and must be incubated in a medium

containing inorganic phosphate in excess of that found in plasma. 

http://www.jbc.org/c.../2/889.full.pdf

 

 

 

Or perhaps IP6 instead of inorganic phosphate to produce NMN. In any case, I would be surprised if these various intermediates to NAD didn't have other functions in the cell, and might act differently in neurons. Nicotinamide certainly does-- https://www.ncbi.nlm.../pubmed/2952896

 

 

 

[Shai Hulud]

Don't think it has been especially mentioned, but integrity and quality of the inner mitochondrial membrane is important for the ETC;

Needed are cardiolipin (phosphatidylglycerol), phosphatidylcholine, DHA/EPA.

Supplementation should be helpful, though when would be the best time?

 

 

[Turnbuckle]

Don't think it has been especially mentioned, but integrity and quality of the inner mitochondrial membrane is important for the ETC;

Needed are cardiolipin (phosphatidylglycerol), phosphatidylcholine, DHA/EPA.

Supplementation should be helpful, though when would be the best time?

 

 

Likely during biogenesis.

[zorba990]

Don't think it has been especially mentioned, but integrity and quality of the inner mitochondrial membrane is important for the ETC;
Needed are cardiolipin (phosphatidylglycerol), phosphatidylcholine, DHA/EPA.
Supplementation should be helpful, though when would be the best time?

Interesting! Cardiolipin seems to be anti viral as well..haven't found a decent priced source yet

https://www.ncbi.nlm...les/PMC3359948/
Phosphatidylglycerol Suppresses Influenza A Virus Infection
"We examined the potency of POPG as an anti-IAV agent using a mouse model of viral infection. Female BALB/c mice (6 wk old) were inoculated intranasally, with the mouse-adapted influenza strain, H1N1-PR8-IAV (80 pfu/mouse), in either the absence or presence of 3 mg POPG. At 5 days after infection, the animals were killed, and the lungs were lavaged, harvested, and analyzed for the effects of viral infection (26). The results presented in Figure 7A demonstrate that the POPG treatment clearly suppressed viral propagation in the lung by a factor of 10 "


Perhaps we want Distearoyl phosphatidylglycerol which is the stearic acid version. (DSPG) http://www.nipponsei...hospholipid.htm


Maybe taking purified phoschol , the defatted version and stearic acid simultaneously would work. https://shop.nutrasa...ode=Cerebra_2OZ

Edited by zorba990, 16 July 2017 - 03:13 AM.

[RWhigham]

It seems that people are forgetting what the protocol is and posting and debating based on assertions that are 180 degrees out of whack. That is unfortunate, as these assertions will be here for years and people will quote them as if they are true. To recap the protocol:

 

1. Mitochondria are fissioned to the smallest possible size and mitophagy is set in motion, removing defective mtDNA. (This can be set in motion by increasing the NAD+/NADH ratio.)

2. Fusion and bio-genesis replace the mtDNA mass lost in step 1, producing a mito population that is healthier than  before. 

 

Mea culpa. I misspoke. This thread is clearly based on NAD+ increasing fission. I reported my posts #234 & #243 and asked that they be flagged with warnings.

 

 

Edited by RWhigham, 16 July 2017 - 05:14 PM.

[RWhigham]

A red herring theory:  no more after this I promise.

 

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy  explains "housekeeping" cycles

 

Synopsis:

 

Mitos were examined in culture.

The average time a mito spent in aggregation was 7 sec and the average time spent fragmented was 21 sec.

So the average fusion/fission "housekeeping" cycle took 28 sec.

When fusion and fission reached balance 25% (with 7sec dwell) of the mitos were aggregated and 75% (with 21sec dwell) were fragmented.

 

After fission, healthy and defective daughters were created. The defective daughters would thereafter remain fragmented until they were taken by autophagy (except for a few that would recover several hours later). They showed that autophagy targeted the defective daughters.

 

The authors concluded that during aggregation mito parts were segregated based on quality to opposite ends of the aggregate.

 

Take away:

 

Quality control cycles normally happen very fast, several times per minute.

Quality control requires fusion because defects cannot be segregated while fragmented. 

 

Table:     Hypothetical dwell times in fusion & fission

 

    Avg time     Avg time    Housekeeping     Resulting

    in fusion     in fission       cycle time           Quality

 

1)  7 sec         21 sec         28 sec          normal - 25% of the mitos are aggregated and engaged in defective daughter creation

 

2)  7 sec         7 sec          14 sec           better - cycling in half the time doubles the defective daughter creation rate 

 

3)  21 sec        7 sec          28 sec          best - 75% of the mitos are aggregated, tripling the defective daughter creation rate

     

4)  long           long           slowest           poor - the number of cycles is very reduced, although defects are still being segregated

 

5)  7 sec         long             slow              worst - the number of cycles is low, and the % of the mitos segregating defects is very low

 

Red Herring Conclusion: The quality control cycles are severely impaired if most of the mitos are fragmented (scenario 5).  

If mitophagy is simultaneously increased, it has to be relatively non-selective, not just taking the now rare defective daughters.

Perhaps mitos that were fission-resistant and mitophagy-resistant would be eliminated, but the absence of normal quality control cycles

would result in deteriorating quality in the remaining mitos.

 

Reality:

In Experiment #5 Inducing Mitophagy with 3750 mg NR per Day  Fafner55 kept NAD+ raised for 6 days. Afterward his energy and skin were improved. His mito quality improved. If his 28 sec mito quality control cycles were largely missing for 6 days it did not matter.

 

Edited by RWhigham, 16 July 2017 - 05:46 PM.

[Turnbuckle]

 

RWhigham: Red Herring Conclusion: The quality control cycles are severely impaired if most of the mitos are fragmented (scenario 5).  

If mitophagy is simultaneously increased, it has to be relatively non-selective, not just taking the now rare defective daughters.

Perhaps mitos that were fission-resistant and mitophagy-resistant would be eliminated, but the absence of normal quality control cycles

would result in deteriorating quality in the remaining mitos.

 

 

 

 

Mitochondria are not chomped up without a label, it seems. Cellular mito QC marks defective mitochondria with a protein that says, "okay to eat."

 

We have shown that Parkin is recruited to depolarized mitochondria and that Parkin promotes their autophagic degradation. 

https://www.ncbi.nlm...les/PMC2592826/

 

 

How rare these depolarized mitochondria are is questionable as young and old cells are different. The natural process of QC keeps cells young for decades, but then mitochondria appear that are defective but cannot be removed. They take up space but do not produce ATP, or may produce oxidants instead. Thus the QC process declines, more such mitochondria appear, and the cell goes into a death spiral unless a spring cleaning is forced upon it.

 

Cells from aging tissues and late passage cultures exhibit abnormalities in mitochondrial electron transport chain (ETC)3 such as cytochrome c oxidase negativity and mtDNA mutations (1). Defective ETCs produce large amounts of reactive oxygen species (ROS) and thereby play a major role in the induction of cellular senescence and possibly tissue aging and are strongly associated with various aging-associated degenerative diseases and cancers as well (2, 3). For this reason, maintenance of mitochondria quality is of utmost importance to body health and longevity (4). Mitochondrial quality control is mediated largely by the removal of dysfunctional parts of mitochondria and biogenesis of new parts of mitochondria. However, during senescence and aging, the removal of damaged mitochondria is attenuated, resulting in an increase of mitochondrial mass and cellular mitochondrial content (5, 6). Autophagy is the major cellular mechanism removing organelles, including mitochondria, and along with coordinated mitochondrial fission and fusion is believed to selectively remove damaged (depolarized) mitochondria (7). Therefore, the persistence of a high level of autophagy flux and mitochondria, structural dynamics may be the key to the maintenance of mitochondrial quality. In fact, the longevity of model organisms has been linked to the efficient maintenance of autophagy, a cellular process that is down-regulated during aging (8).

 

http://www.jbc.org/c...7/23/19304.long

 

 

 

While fission/fusion may proceed quickly for normal mitochondria, fission may not occur at all for what one paper calls "zombie mitochondria." These things are the organelle equivalent of a virulent cancer, growing and reproducing without producing ATP, and infecting other mitochondria by fusion.

 

When normal mitophagic organelle elimination (Figure 7A) is suppressed by Parkin insufficiency, abnormal undead or zombie mitochondria accumulate and (as zombies will do) contaminate the normal mitochondrial population by fusing with normal organelles (Figure 7B). Mitochondrial fusion that is ordinarily protective, therefore, becomes the mechanism for a general contagion of mitochondrial dysfunction. Interrupting mitochondrial fusion prevents contamination of functionally normal mitochondria by virulent zombie mitochondria (Figure 7C), sequestering abnormal mitochondria that can then be removed by alternate, albeit almost certainly less efficient, elimination pathways.

 

https://www.ncbi.nlm...les/PMC4392818/

 

 

To fission these zombies one will have to resort to heroic means, such as forcing all mitochondria to minimal size by increasing the NAD+/NADH ratio. This has been tried in vitro over a period of days, and it's found that the mito mass eventually levels off after 3 days, suggesting that healthy mitochondria (ie, those with functional mtDNA) are resistant to further elimination.

 

 

In our previous studies, we found that exposure of cells to nicotinamide causes a decrease in mitochondrial content and an increase in mitochondrial membrane potential (MMP) by activating autophagy and inducing mitochondrial fragmentation. Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD+]/[NADH] ratio and the activation of SIRT1, an NAD+-dependent deacetylase that plays a role in autophagy flux. The [NAD+]/[NADH] ratio was inversely correlated with the mitochondrial content, and an increase in the ratio by the mobilization of the malate-aspartate shuttle resulted in autophagy activation and mitochondrial transformation from lengthy filaments to short dots. Furthermore, treatment of cells with SIRT1 activators, fisetin or SRT1720, induced similar changes in the mitochondrial content.

 

Fig. 1A shows an example of the change in the mitochondrial content that is commonly observed in various tested human cells (including normal fibroblasts and MCF-7, H460, and HCT116 cancer cell lines) after supplementation of 5 mm NAM in culture media. The mitochondrial content as determined by flow cytometry using two different mitochondrion-specific dyes was substantially decreased for the first 3 days and thereafter remained at a level about 70% that in the untreated cells, as reported previously

 

https://www.ncbi.nlm...les/PMC3365962/

 

 

Edited by Turnbuckle, 16 July 2017 - 08:53 PM.

[stephen_b]

1. Salidroside (an active component of rhodiola) stimulates mitochondrial biogenesis (PMID 24868319).
2. Astaxanthin seems to promote mitophagy.

[Shai Hulud]

 

1. Astaxanthin seems to promote mitophagy.

 

 

 

If I get the paper right, astaxanthin is degraded by the same enzymatic system as beta carotin. Even though they used knockout mice, it's seems important to mention that there are several known clinically relevant SNPs, at least in BCO1 and these are relatively prevalent (at least in Caucasian populations, don't know about others). 

As astaxanthin is pretty potent and too much antioxidants will at some increase oxidative stress, I'd recommend either researching this further or start rather low with astaxanthin, to avoid it building up in one's system.

I just found this in examine's article on it:

 

 

According to one study, Astaxanthin had a plasma elimination half-life of 52 hours with a standard deviation of 40.[13] That being said, there appears to be large differences between individuals and non-linear kinetics of astaxanthin. Doses as small as 10mg can persist in the body for upwards of a day whereas superdoses of 100mg can persist for 72 hours.[30]

 

This seems to fit well with the idea of interindividual differences in carotenoid metabolism. 

I think it could get pretty tricky to fit it into Turnbuckle's protocol, as it's long half-life could interfere with the fission phase (depending on how long the chosen phases are). 

See complete article with sources here

[hotbit]

 

A red herring theory:  no more after this I promise.

 

Fission and selective fusion govern mitochondrial segregation and elimination by autophagy  explains "housekeeping" cycles

 

Synopsis:

[......]

Table:     Hypothetical dwell times in fusion & fission

    Avg time     Avg time    Housekeeping     Resulting

    in fusion     in fission       cycle time           Quality

1)  7 sec         21 sec         28 sec          normal - 25% of the mitos are aggregated and engaged in defective daughter creation

[......]

 

Thanks for linking this interesting article.

 

What do you exactly mean by 'Avg time in fusion'? Do you mean time mitochondria stay in coupled, fused state or time of transition between solitary and coupled states?  As for the times mitochondria stay in solitary or coupled states from Fig. 2 description of the linked article times are respectively around ~80 sec and ~1300s. These time are quite in range of times used in an article I linked in one of my previous posts where mitochondrial dynamics was simulated. Thus I'm a bit puzzled. Average housekeeping cycle would rather take  ~28 min instead of 28 sec... Well, not exactly, as under Fig. 9 they write:

 

After being depolarized and solitary for a few hours, the mitochondrion is removed by autophagy.

 

Membrane potential recovery of a defective mitochondrion and fusion with another one needs to be prevented for a couple of hours for mitophagy to occur. Thus 'house keeping cycle' or QC cycle requires several hours.

 

Skin test.

Skin cells renew / live for about 30 days only. Therefore skin seems to be a good candidate for testing and results should be largely visible within 1-3  months.

 

I've mixed some NAM and R into a cream base and applied every 2-3 days for a month to half of my face. During this month I've also done twice 1 day of general 'fission'. I'm almost 50 and during the last 2-3 years some wrinkles and silver hair quickly emerged.

The only 'tests' of the skin I could perform are visual inspection and finger tip feeling. I doesn't seem to be an easy task, though. Since I was a teenager I didn't stare into my mirrored face to much and took very few photos recently. To my surprise, left and right sides of my face are more asymmetric than I thought. When I grin,  wrinkles around my left and right eye have very different patterns and sizes. This makes comparisons difficult. Eye on the treated side seems larger, cheek less saggy (I'm thin so they are not that saggy anyway), no difference in wrinkles spotted. Under the eye I found discoloured patch of skin along the wrinkle. No idea whether it was there before the treatment or NAM + R caused it. Due to apparent asymmetry of my face it is difficult to spot small changes or attribute some spotted features to the treatment. Under the finger tip* both sides feel same. No hair de-greying observed.

*finger tip has incredibly high resolution

Edited by hotbit, 17 July 2017 - 02:28 PM.

[Turnbuckle]

 

Skin test.

Skin cells renew / live for about 30 days only. Therefore skin seems to be a good candidate for testing and results should be largely visible within 1-3  months.

 

I've mixed some NAM and R into a cream base and applied every 2-3 days for a month to half of my face. During this month I've also done twice 1 day of general 'fission'. I'm almost 50 and during the last 2-3 years some wrinkles and silver hair quickly emerged.

The only 'tests' of the skin I could perform are visual inspection and finger tip feeling. I doesn't seem to be an easy task, though. Since I was a teenager I didn't stare into my mirrored face to much and took very few photos recently. To my surprise, left and right sides of my face are more asymmetric than I thought. When I grin,  wrinkles around my left and right eye have very different patterns and sizes. This makes comparisons difficult. Eye on the treated side seems larger, cheek less saggy (I'm thin so they are not that saggy anyway), no difference in wrinkles spotted. Under the eye I found discoloured patch of skin along the wrinkle. No idea whether it was there before the treatment or NAM + R caused it. Due to apparent asymmetry of my face it is difficult to spot small changes or attribute some spotted features to the treatment. Under the finger tip* both sides feel same. No hair de-greying observed.

*finger tip has incredibly high resolution

 

 

 

I've not noticed any de-graying for N+R, or for any product whatsoever except for hair dye.

 

As for skin, there are creams featuring either nicotinamide or ribose, and on Amazon the nicotinamide creams get good reviews. I have not tried these, but I've found that a dermaroller with a home-made lysine/water solution works very well for eliminating wrinkles and tightening skin. And while that worked, I don't recommend N+R combined with dermarolling, as it seemed to irritate skin more than anything.

Edited by Turnbuckle, 17 July 2017 - 03:00 PM.

[ceridwen]

Feeling much better now. Tinnitus much better. Taken 1 Niacin tablet today.

[RWhigham]

I'm going to respond to hotbit, but I think this is beating a dead horse.

 

hotbit: What do you exactly mean by 'Avg time in fusion'? Do you mean time mitochondria stay in coupled, fused state or time of transition between solitary and coupled states?  As for the times mitochondria stay in solitary or coupled states from Fig. 2 description of the linked article times are respectively around ~80 sec and ~1300s. These time are quite in range of times used in an article I linked in one of my previous posts where mitochondrial dynamics was simulated. Thus I'm a bit puzzled. Average housekeeping cycle would rather take  ~28 min instead of 28 sec... Well, not exactly, as under Fig. 9 they write  

 

Good catch -- a cycle was 28 min instead of 28 sec.

 

A cycle is about 10 min herea: Mitochondrial Fusion, Fission and Autophagy as a Quality Control Axis: The Bioenergetic View : 

Remarkably, studies that track individual mitochondria through fusion and fission found that the two events are paired and that fusion triggers fission. On average each mitochondrion would go though ~5 fusion:fission cycles every hour.

 

Measurement of Δψm during single fusion and fission events demonstrate that fission may yield uneven daughter mitochondria where the depolarized daughter less likely to become involved in a subsequent fusion and is more likely to be targeted by autophagy. According to this hypothesis pairs of fusion and fission allow for the reorganization and sequestration of damaged mitochondrial components into daughter mitochondria that are segregated from the networking pool and then becoming eliminated by autophagy.

 

I call perusing the above theories "beating a dead horse" because to me it has been convincingly established that the Turnbuckle protocol alters the normal fusion/fission balance in favor fission, and provides a net benefit.

 

Indeed, the Turnbuckle protocol may be the single most important way to extend life and healthspan.

I note in passing that rapamycin stimulates autophagy, and could also be doing mito cleanup.

Edited by RWhigham, 17 July 2017 - 11:17 PM.

[Fafner55]

... the Turnbuckle protocol may be the single most important way to extend life and healthspan.

 

 

I agree that cleaning out dysfunctional mitochondria ranks at or near the top of interventions that may extend life and healthspan, and will add that this intervention should be viewed in a larger context for those readers that might interpret it as a silver bullet. 

 

It is self evident that the exponential accumulation of dysfunctional mitochondria implies a self reinforcing mechanism. Clearing dysfunctional mitochondria offers a reset for that vicious cycle, but without addressing the underlying cause dysfunctional mitochondria might quickly re-accumulate. 

 

Interventions that could further help break the cycle are

• Reducing the number of senescent cells (which cause inflammation leading to elevated CD38 and NAD+ depletion)
• Repleting NAD+ 
• Inducing mitophagy
• Reducing the rate of oxidative damage (likely with C60)
• Repairing single and double strand DNA breaks (with NR)

[Turnbuckle]

Of all the theories of aging, I would put the decline of mitochondria first, and the decline of stem cell pools a close second. 

[RWhigham]

Here is a list I made for myself:

 

To Induce fission: 

   + NAD+:   Nicotinamide-&-Ribose-&-exercise, Niagen NR, Pomegranate, Fisetin, Dynveo_GSE

   + SIRT1:   Cocoa, Hydroxytyrosol, Pterostilbene, Resveratrol, Myricetin, Luteolin, Silymarin

 

To Induce autophagy:

  - TORC1: Rapamycin, EGCG, Fisetin, Metformin, Epimedium, Vit-D, Spermidine, Garcinol

           Fisetin both increases NAD+ and inhibits TORC1

  + Tor-independent-autophagy:  EGCG, Spermidine, Carbamazepine, Valporic_acid

         EGCG has TOR_dependant and TOR_independent autophagy pathways

         Carbamazepine & Valporic_acid are anti-seizure drugs

 + PINK/Parkin (tag mitos for elimination):  Cinnamon, Rosemary_extract

 + Other: Fasting, Coffee/tea, Ursolic Ac, Ketones

 

Avoid:  These interfere with fission + autophagy: 

 - autophagy: Leucine, Insulin, (perhaps other bodybuilding supplements could go here)

 - fission:  Sulforaphane (-Drp1)

 + fusion: Stearic acid, PQQ, AMPK_activators

       AMPK_activators: AICAR, Actv-AMP (jiaogulan), Lithium, Naringin, Berberine, Quercetin

 + Membrane potential (MP):  Creatine, anti-oxidants  (higher MP protects mitos)

     Antioxidants: CoQ10, C60, vit-C, Astaxanthin

 Astaxanthin, Trehalose:   +autophagy but also +fusion 

    Trehalose is theoretically destroyed by digestion, but seems to have effects

 

Advice:

Watch out for the avoids.  Sulforaphane for example will prevent fission & prevent everything else from having any effect.

At first, don't try to over do it. You can get too "wiped out"  (follow the Turnbuckle examples).

 

It should be possible to abort with sulforaphane (Jarrow BroccoMax)  and  something from the +fusion items

[aribadabar]

Biogenesis (used with fusion)

Resveratrol (200mg)

PQQ (20mg)

BCAA (10g)

Hydroxytyrosol (25mg)

Epicatechin (500mg)

 

1. Astaxanthin seems to promote mitophagy.

 

Here is a list I made for myself:

 

To Induce fission: 

   + NAD+:   Nicotinamide-&-Ribose-&-exercise, Niagen NR, Pomegranate, Fisetin, Dynveo_GSE

   + SIRT1:   Cocoa, Hydroxytyrosol, Pterostilbene, Resveratrol, Myricetin, Luteolin, Silymarin

 

...

 

Avoid:  These interfere with fission + autophagy: 

 - autophagy: Leucine, Insulin, (perhaps other bodybuilding supplements could go here)

 - fission:  Sulforaphane (-Drp1)

 + fusion: Stearic acid, PQQ, AMPK_activators

       AMPK_activators: AICAR, Actv-AMP (jiaogulan), Lithium, Naringin, Berberine, Quercetin

 + Membrane potential (MP):  Creatine, anti-oxidants  (higher MP protects mitos)

     Antioxidants: CoQ10, C60, vit-C, Astaxanthin

 Astaxanthin, Trehalose:   +autophagy but also +fusion 

    

 

I was looking at these three posts and I was wondering:

 

How do you reconcile inducing auto-/mitophagy (supposedly occurring during the fission phase) and the contradictory statements that the items in red whereTurbuckle suggests they are to be used during fusion while RWhigham states to use to induce fission?

 

As to Astaxanthin, how it could both boost mito/autophagy (a fission state process) and fusion at the same time? When would be best to take it?

 

Thanks!

Edited by aribadabar, 18 July 2017 - 04:10 PM.

[RWhigham]

aribadabar: As to Astaxanthin, how it could both boost mito/autophagy (a fission state process) and fusion at the same time? When would be best to take it?  See Astaxanthin Mediates Mitochondrial Dynamics in Mice : "elevated expression of phosphorylation of unc-51 like autophagy activating kinase 1(ULK1) on Ser757, and uncoupling protein 2, mitofusin 2, and decreased protein levels of p62 and ATG13 indicating potentiated mitophagy and fusion in liver of WT fed the astaxanthin diet". (emphasis mine)

 

aribadabar: How do you reconcile inducing auto-/mitophagy (supposedly occurring during the fission phase) and the contradictory statements that the items in red whereTurbuckle suggests they are to be used during fusion.  It's not contradictory, because SIRT1 is required in both  mitophagy and biogenesis.

 

SIRT1 required for mitophagy

Nicotinamide-induced Mitophagy EVENT MEDIATED BY HIGH NAD+/NADH RATIO AND SIRT1 PROTEIN ACTIVATION: Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD+]/[NADH] ratio and the activation of SIRT1.    Importantly, the activators induced mitochondrial fragmentation only when SIRT1 expression was intact.    Furthermore, treatment of cells with SIRT1 activators, fisetin or SRT1720, induced similar changes in the mitochondrial content.

Sirt1 and the Mitochondria: Sirt1 also appears to be important for the turnover of defective mitochondria by mitophagy.

 

SIRT1 required for biogenesis

Sirt1 and the Mitochondria: I discuss here evidences for Sirt1’s regulation of mitochondrial biogenesis

 

Caveat:

I don't know if boosting SIRT1 will actually intensify fission and mitophagy. Initially, nicotinamide-ribose-exercise suffices to produce fission & mitophagy. As you become more tolerant, you might want to try adding other things for a more complete cleanup (mito rejuvenation). Endogenous SIRT1 decreases with age and obesity, so supplements for SIRT1 could help some more than others.

Edited by RWhigham, 19 July 2017 - 03:34 AM.

[RWhigham]

 

Caveat:

I don't know if boosting SIRT1 will actually intensify fission and mitophagy. Initially, nicotinamide-ribose-exercise suffices to produce fission & mitophagy. As you become more tolerant, you might want to try adding other things for a more complete cleanup (mito rejuvenation). Endogenous SIRT1 decreases with age and obesity, so supplements for SIRT1 could help some more than others.

 

SIRT1 is shorthand for SIRT1 activation 

Edited by RWhigham, 19 July 2017 - 04:36 AM.

[APBT]

Sulforaphane, a phytochemical in broccoli sprouts, ameliorates obesity

https://www.scienced...70307100402.htm

http://diabetes.diab...g/content/66/5/1222


 

Sulforaphane causes a major epigenetic repression of myostatin in porcine satellite cells

https://www.ncbi.nlm...les/PMC3528693/

[RWhigham]

 

To Induce fission: 

   + NAD+:   Nicotinamide-&-Ribose-&-exercise, Niagen NR, Pomegranate, Fisetin, Dynveo_GSE

   + SIRT1:   Cocoa, Hydroxytyrosol, Pterostilbene, Resveratrol, Myricetin, Luteolin, Silymarin

 

To Induce autophagy:

  - TORC1: Rapamycin, EGCG, Fisetin, Metformin, Epimedium, Vit-D, Spermidine, Garcinol

           Fisetin both increases NAD+ and inhibits TORC1

  + Tor-independent-autophagy:  EGCG, Spermidine, Carbamazepine, Valporic_acid

         EGCG has TOR_dependant and TOR_independent autophagy pathways

         Carbamazepine & Valporic_acid are anti-seizure drugs

 + PINK/Parkin (tag mitos for elimination):  Cinnamon, Rosemary_extract

 + Other: Fasting, Coffee/tea, Ursolic Ac, Ketones

 

Avoid:  These interfere with fission + autophagy: 

 - autophagy: Leucine, Insulin, (perhaps other bodybuilding supplements could go here)

 - fission:  Sulforaphane (-Drp1)

 + fusion: Stearic acid, PQQ, AMPK_activators

       AMPK_activators: AICAR, Actv-AMP (jiaogulan), Lithium, Naringin, Berberine, Quercetin

 + Membrane potential (MP):  Creatine, anti-oxidants  (higher MP protects mitos)

     Antioxidants: CoQ10, C60, vit-C, Astaxanthin

 Astaxanthin, Trehalose:   +autophagy but also +fusion 

    Trehalose is theoretically destroyed by digestion, but seems to have effects

The above is for fission & autophagy.   For fusion & biogenesis stearic acid seems to be great for a fast restoration. Other things to "avoid" during fission may also enhance fusion.  SIRT1 activation is required for both fission and fusion, so the SIRT1 enhancers could be useful both times.

 

The following are my results so far:

    Experiment #1 - very strong effects

    Experiment #2 - recommended protocol for beginners, worked great

    Experiment #3 - tried Turnbuckle's  atomic protocol

    Experiment #4 - added a fission inhibitor and an autophagy inhibitor. Totally prevented any effect 

 

Details:

Experiment #1  June 5, 2017

Day 1 promoting fission & autophagy, at 8 AM I took

  NAD+ promoter   Nicotinamide 2 g  & ribose 5 g

  NAD+ promoter   Niagen NR 200 mg

  NAD+ promoter   Pomegranate (1g  Ellagic acid)

  NAD+ promoter   Dynveo_GSC 200 mg (grape seed extract)

  SIRT1 promoter   Pterostilbene 100 mg

  SIRT1 promoter   Resveratrol 500 mg  (and DHEA 75mg, to prevent resveratrol side-effects per Turnbuckle)

  At 9 AM I tried to exercise but couldn't do much

Day 2 and 3  just rested  - day 2 protocols were still new

Result  I was too weak to exercise much.  I felt sick and slept a lot. Wife said I looked sick. It took 48 hrs to recover. I believer the added SIRT1 promoters, or the extra NAD+ promoters (or both) caused a lot of fission and autophagy,  I can't prove it. 

 

Experiment #2  June 11, 2017

Day 1 promoting fission & autophagy, at 8 AM I took

   NAD+ promoters     nicotinamide 2g and ribose 5g

   NAD+ promoter      At 9 AM I exercised hard for 1 hr

   NAD+ promoter      At 11 AM I took enough Pomegranate extract to have 1g ellagic acid

Day 2 promoting fusion & biogenesis, at 8 AM I took

   Fusion promoter    Stearic acid 10g 

   Antioxidant  (autophagy inhibitor)    Vit-C crystals 2g 

Result - went for a very fast walk with frequent sprinting and sustained HR of 170-180 bpm for 30 min. Felt great.

 

Experiment #3  Jun 14, 2017 

Day 1 promoting fission & autophagy, at 8 AM I took

   NAD+ promoter   Nicotinamide 2g & Ribose 5g (8AM),

   TMG 1 g  & lysine 2 g  at 8:30 AM       Atomic protocol

   NAD+ promoter   Exercise 9AM-10AM,

   NAD+ promoter   Pomegranate extract 1g at 11AM    

   PINK/Parkin promoter   Cinnamon 1/2 tsp  at  11AM 

Effect I experienced low blood pressure all morning after the TMG & lysine 

3 PM  I feel completely recovered already, expecting to feel great again after stearic acid tomorrow

Day 2 promoting fusion and autophagy,  

    + Stemcell replication   Curmumin (Ultra-Cur) 2.4 g  7 AM 

    Antioxidant (inhibit autophagy)  Vit-C crystals 2 g  8 AM

    Fusion promoter   stearic acid 10g  8:30 AM

    Treadmill walk 9 AM to 10 AM

    Biogenesis promoter   PQQ  20 mg   11 AM

    Biogenesis promoter  Sulforaphane 60 mg   11 AM

    After lunch  Very sleepy.  Slept for 2 hours

    4 PM     Arms and shoulders unusually sore 

    5 PM     Ran an errand. Felt like I was walking on "spring" shoes. Felt good to walk very fast.

 

Experiment #4  Jun 25, 2017

Day 1 promoting fission & autophagy, at 8 AM I took

   NAD+ promoter   Nicotinamide 2 g & Ribose 5 g

   NAD+ promoter   Sulforaphane 6 0mg (BroccoMax)   [Mistake--although Sulforaphane is likely a NAD+ promoter, it is also a very strong fission inhibitor]

   At 8:30 AM   TMG 1 g  & lysine 2 g   (my 2nd attempt at Turnbuckle atomic protocol)

   NAD+ promoter  At 9 AM  exercised hard, and drank my sport drink containing leucine [2nd mistake--leucine is a strong autophagy inhibitor}

Result: Felt no effects. The TMG & lysine did not lower my BP like in experiment #3.  Inhibiting fission (sulforaphane) and autophagy (leucine) prevented any noticeable result--demonstrating that fission and autophagy are likely responsible for feeling wiped out.

Edited by RWhigham, 19 July 2017 - 10:49 PM.