74 replies to this topic

[Turnbuckle]

 

Interestingly, in waiting 3 hours before exercise, with the following variation on protocol, I find my output capacity is nearly doubled.

 

- 0h

- - 3g niacinimide

- - 5g ribose

- - 2g pyruvate

- - 3g lactate

- - 2g TMG (account for niacinimte metabolism)

- - AMPK activators (mitophagy)

- - - jiaogulan/gynostemma 1g

- - SIRT1 activators (mitophagy)

- - - Fisetin 100mg

- 1.5 hr

- - apigenin (said to have faster metabolism (1hr)) (AMPK activators (mitophagy)) 100mg

- 3hr

- - exercise

 

Apart from the potential balancing component of lactate to pyruvate decreasing NAD/NADH ratio, I'm inclined to think this is a result of having hyper fused or fission-deficient mitochondria (my NAD levels were previously nearly always low)

 

https://www.ncbi.nlm...59/#!po=64.2857

"Here we observed further clustering of nucleoids in mitochondrial fission-deficient cells via mitochondrial hyperfusion. "

"It was recently reported that mitochondrial hyperfusion with highly developed cristae occurs during nutrient starvation to protect mitochondria from autophagic degradation and sustained cell viability "

"However, even though mito-bulbs have highly stacked cristae with abundant mtDNA, they do not enhance respiration, and the physiological roles of mito-bulbs in vivo and in healthy cells remain to be conclusively analyzed."

 

Interestingly, also from that paper:

"In contrast, live imaging of mitochondrial nucleoids in mammals, higher plants, and yeast has revealed that mitochondrial fission occurs independently of nucleoid fission "

 

A few of notes and questions.

 

1. Fission doesn't necessarily decrease ATP production - (hyperfused mitochondria is inefficient)

2. How long does fission take.  Could the reduction in output capacity you see be a result of working out during the process of fission, in which there would indeed be an ATP production diminishment?

3. Wouldn't it be more sensible to work out after fission occurs instead of during?  IE, isn't the idea to destroy faulty mitochondria, not to make them by inducing stress during fission?

 

 

I think the hyperfusion explanation is a stretch. Your first thought is more likely the correct one. Fission results from a high NAD+/NADH ratio, and lactate knocks that down below baseline. See Fig. 2 (B) of this paper. 

 

My purpose in this thread was to drive mitochondria into fission to get more burn for a given amount of work. If you combine increased NAD with fusion, you'll go in the other direction. Normally, the process of fission and fusion is in balance, and occurs very rapidly-- 

 

When mitochondria are viewed in live cells, it becomes immediately apparent that their morphologies are far from static. Their shapes change continually through the combined actions of fission, fusion, and motility. Rapid fission and fusion of mitochondria in cultured fibroblasts allows for the complete redistribution of mitochondrial green fluorescent protein (GFP) from one mitochondrion to all the other mitochondria of a cell within an hour.

https://www.ncbi.nlm...les/PMC4762028/

 

[capob]

https://www.ncbi.nlm...les/PMC3365962/ : Quite an interesting read (I suppose I should have started with it).  In addition to an extraction about lactate

 

"Surprisingly, lactate treatment resulted in an increase in the mitochondrial contents of both the control and NAM-treated cells in a dose-dependent manner (Fig. 2C). This indicates that lactate itself or the lactate-induced decrease in the [NAD+]/[NADH] ratio increases the mitochondrial content."

 

there's also a dynamic of too much NAM which I may have encountered:

 

"... suggest an activation of SIRT1 upon the treatment of 5 mm NAM (Fig. 4C). Meanwhile, NAM at a 10 or 20 mm dose caused an increase of [NAD+] to the levels almost identical to that in the cells treated with 5 mm NAM (Fig. 1B) but resulted in an inhibition of SIRT1 activity during the early time period (data not shown). Importantly, the mitochondrial content was not down-regulated but rather increased in the cells treated with 20 mm NAM"

 

[QuestforLife]

https://www.ncbi.nlm...les/PMC3365962/ : Quite an interesting read (I suppose I should have started with it). In addition to an extraction about lactate

"Surprisingly, lactate treatment resulted in an increase in the mitochondrial contents of both the control and NAM-treated cells in a dose-dependent manner (Fig. 2C). This indicates that lactate itself or the lactate-induced decrease in the [NAD+]/[NADH] ratio increases the mitochondrial content."

there's also a dynamic of too much NAM which I may have encountered:

"... suggest an activation of SIRT1 upon the treatment of 5 mm NAM (Fig. 4C). Meanwhile, NAM at a 10 or 20 mm dose caused an increase of [NAD+] to the levels almost identical to that in the cells treated with 5 mm NAM (Fig. 1B) but resulted in an inhibition of SIRT1 activity during the early time period (data not shown). Importantly, the mitochondrial content was not down-regulated but rather increased in the cells treated with 20 mm NAM"

Yes this is the paper that started this all off. Higher amounts of NAM inhibit SIRT, so cut off mitophagy. But if 3g gives you 5mM (100% absorption), then you need 12g to reach the levels of inhibition. You might be able to achieve the same with lactate.

[capob]

Yes this is the paper that started this all off. Higher amounts of NAM inhibit SIRT, so cut off mitophagy. But if 3g gives you 5mM (100% absorption), then you need 12g to reach the levels of inhibition. You might be able to achieve the same with lactate.

 

 

Where are you getting the 100% absorption.  In my novice calculation:

200lbs * 75% muscle * 75% water * 1 gallon water/10lb = 11.2 gallons of water muscle

= 42.3 liters

NAM: 3grams * 1mole / 122.127g = .0246 moles

.0246 moles / 42.3 liters = .00058 moles per liter = .58mM NAM

 

But, you probably have muscle absorption preferences, potentially even related to the type of exercise.

 

 

A note on teh 20mM NAM application:

Figure 1b (https://www.ncbi.nlm...es/PMC3365962/#!po=45.0000)  : 20mM application of NAM, NAD level remained nearly identical for the extent of the time measured 40 hours.  If this were a rate constraint of NAMPT, then we should expect either that the 20mM would continue to have increasing NAD levels past the 5mM, or that both the 5mM case and 20mM presented adequate NAM for constant NAMPT activity for the entirely, but this would mean the 5mM presented a period of excessive NAM early on, which we might expect to reflect in SIRT1 inhibition (which 5mM does not), and so NAMPT being otherwise regulated apart from constant rate limitation is expectable.  And, I would expect NAMPT is inhibited by NAD (ie high NAD levels (a build up of NMN)).  

 

As such, might a generally increased level of NAD result in a smaller requirement of NAM to block SIRT1 (via less NAM being converted)?  Could that explain the observation of protocol tolerance (ie, not only are there less faulty mitochondria to QC, there is less QC from less SIRT1 activation due to higher NAD levels to start).

 

Considering this dynamic, lactate supplementation would increase NAD+ -> NADH, which would potentially make room for more NAMPT activity, and when combined with NAM, would result in a normalized NAD+/NADH ratio, but a greater quantity of both, which might account for why my exercise output was so high.  Perhaps a useful tool for anyone competing in something athletic.

 

 

I suppose you might want to do something like: quality control, repopulate, strengthen:

 

1. QC fission via increased NAD+/NADH along with SIRT1 activation

2. non QC fission via increased NAD+/NADH with SIRT1 inhibition

 - "In our previous study, knocking down SIRT1 substantially increased the level of the mitochondrial content both in the control and the NAM-treated cells, which rather demonstrates the importance of SIRT1 in the basal level autophagy" (https://www.ncbi.nlm...es/PMC3365962/#!po=45.0000)

3. fusion ("Manipulating mitochondrial dynamics" Turnbuckle thread)

[QuestforLife]

Where are you getting the 100% absorption. In my novice calculation:
200lbs * 75% muscle * 75% water * 1 gallon water/10lb = 11.2 gallons of water muscle
= 42.3 liters
NAM: 3grams * 1mole / 122.127g = .0246 moles
.0246 moles / 42.3 liters = .00058 moles per liter = .58mM NAM

But, you probably have muscle absorption preferences, potentially even related to the type of exercise.


A note on teh 20mM NAM application:
Figure 1b (https://www.ncbi.nlm...es/PMC3365962/#!po=45.0000) : 20mM application of NAM, NAD level remained nearly identical for the extent of the time measured 40 hours. If this were a rate constraint of NAMPT, then we should expect either that the 20mM would continue to have increasing NAD levels past the 5mM, or that both the 5mM case and 20mM presented adequate NAM for constant NAMPT activity for the entirely, but this would mean the 5mM presented a period of excessive NAM early on, which we might expect to reflect in SIRT1 inhibition (which 5mM does not), and so NAMPT being otherwise regulated apart from constant rate limitation is expectable. And, I would expect NAMPT is inhibited by NAD (ie high NAD levels (a build up of NMN)).

As such, might a generally increased level of NAD result in a smaller requirement of NAM to block SIRT1 (via less NAM being converted)? Could that explain the observation of protocol tolerance (ie, not only are there less faulty mitochondria to QC, there is less QC from less SIRT1 activation due to higher NAD levels to start).

Considering this dynamic, lactate supplementation would increase NAD+ -> NADH, which would potentially make room for more NAMPT activity, and when combined with NAM, would result in a normalized NAD+/NADH ratio, but a greater quantity of both, which might account for why my exercise output was so high. Perhaps a useful tool for anyone competing in something athletic.



I suppose you might want to do something like: quality control, repopulate, strengthen:

1. QC fission via increased NAD+/NADH along with SIRT1 activation
2. non QC fission via increased NAD+/NADH with SIRT1 inhibition
- "In our previous study, knocking down SIRT1 substantially increased the level of the mitochondrial content both in the control and the NAM-treated cells, which rather demonstrates the importance of SIRT1 in the basal level autophagy" (https://www.ncbi.nlm...es/PMC3365962/#!po=45.0000)
3. fusion ("Manipulating mitochondrial dynamics" Turnbuckle thread)

You've come up with a far higher figure for NAM dosing because you've worked it out for total water in muscle, whereas I just did serum level (for 5L of blood) assuming 100% bioavailability (as an upper limit, really it wouldn't be this well absorbed). You'd have to take 30g of nicotinamide based on your calculation, which is possibly unsafe even if it is technically correct to replicate the in vitro result.

I'm not sure about your reasoning for the 5mM vs. 20mM dose and NAMPT - I believe the in vitro serum would be maintained at the stated concentration of NAM with more added as required, so there would be a constant rate of conversion via NAMPT to NMN and NAD and then back to NAM via SIRT. Only if the NAM got over a certain level (which it never would in the 5mM case), would SIRT be inhibited and mitochondrial mass start to accumulate again.

Having said all that if you constantly dosed NAM and had no faulty mito loops, you'd still have lower ATP because of the reduced mito mass, which would be tiring. If this isn't happening then maybe NAMPT does reach a point when it can't keep up and NAM does end up inhibiting SIRT and stopping mitophagy.

[capob]

You've come up with a far higher figure for NAM dosing because you've worked it out for total water in muscle, whereas I just did serum level (for 5L of blood) assuming 100% bioavailability (as an upper limit, really it wouldn't be this well absorbed). You'd have to take 30g of nicotinamide based on your calculation, which is possibly unsafe even if it is technically correct to replicate the in vitro result.

I'm not sure about your reasoning for the 5mM vs. 20mM dose and NAMPT - I believe the in vitro serum would be maintained at the stated concentration of NAM with more added as required, so there would be a constant rate of conversion via NAMPT to NMN and NAD and then back to NAM via SIRT. Only if the NAM got over a certain level (which it never would in the 5mM case), would SIRT be inhibited and mitochondrial mass start to accumulate again.

Having said all that if you constantly dosed NAM and had no faulty mito loops, you'd still have lower ATP because of the reduced mito mass, which would be tiring. If this isn't happening then maybe NAMPT does reach a point when it can't keep up and NAM does end up inhibiting SIRT and stopping mitophagy.

 

 

This presents a number of important unknowns.

 

First, from the study, we have "For long term treatment, the medium supplemented with the chemicals was replaced every 2 days."

 

But, even if the 5mM medium was not replenished in the {40hr period illustrating the similarity of NAD levels between 5mM and 20mM}, whether the NAM content of the medium was exhausted depends on the cell content relative to the medium size (unknowns).

 

There are a couple of points of dynamics to consider

 

1. Cell influx rate (whatever the NAM transporter is (does Slc12a8 also transport NAM?))

 

2. bidirectional reaction of NAM salvaging by enzyme NAMPT to NMN

nicotinamide + 5-phosphoribosyl-1-pyrophosphate (PRPP) <=>  nicotinamide mononucleotide (NMN) + pyrophosphate (PPi)

 

3. bidrectional reaction of NMN turned to NAD by NMNAT1, NMNAT2, NMNAT3  

NMN + ATP <=> Nicotinamide adenine dinucleotide (NAD) + PPi

 

4. rate of NAD+ conversion to NADH depending on NAD+/NADH ratio

 

Figure 2A shows a drop in NAD/NADH after day one.  I'm inclined to think this reflects influx rate decrease from NAM exhaustion prior to the day 2 medium replacement, which leads to NAD/NADH auto normalization by the cell.  With the addition of more NAM from medium replacement, the NAM conversion goes up.  The NAM to NAD conversion is upregulated by the amount of NAM, but downregulated by the amount of NAD.  And, after the first medium replacement, there is sufficent NAM to continue the reaction, but it is slowed by the increasing amount of NAD, which is reflective in the declining increase of NAD/NADH.  And, this also is indicated by the fact the mito content actually starts increasing after day 3 - the idea being that there now exists plenty of excess NAM in the cell (inhibiting SIRT1), and that excess NAM is not converting to NAD as quickly due to the presence of increasing NAD).

 

And, I'm inclined to think this might be the reason for the observed issue with regular NAM supplementation.

[QuestforLife]

 

4. rate of NAD+ conversion to NADH depending on NAD+/NADH ratio

 

Figure 2A shows a drop in NAD/NADH after day one.  I'm inclined to think this reflects influx rate decrease from NAM exhaustion prior to the day 2 medium replacement, which leads to NAD/NADH auto normalization by the cell.  With the addition of more NAM from medium replacement, the NAM conversion goes up.  The NAM to NAD conversion is upregulated by the amount of NAM, but downregulated by the amount of NAD.  And, after the first medium replacement, there is sufficent NAM to continue the reaction, but it is slowed by the increasing amount of NAD, which is reflective in the declining increase of NAD/NADH.  And, this also is indicated by the fact the mito content actually starts increasing after day 3 - the idea being that there now exists plenty of excess NAM in the cell (inhibiting SIRT1), and that excess NAM is not converting to NAD as quickly due to the presence of increasing NAD).

 

And, I'm inclined to think this might be the reason for the observed issue with regular NAM supplementation.

 

 

I guess also with lower mitochondrial content NADH will be oxidised to NAD+ at a reduced rate by the electron transport chain, so NAD+ will remain higher of its own accord until such time as the mitochondria increase in number.

 

Just for reference, this in vivo study in humans using a 6 g dose produced a peak concentration in the blood of around 1mM.

 

https://www.ncbi.nlm.../pubmed/1410588

[capob]

I guess also with lower mitochondrial content NADH will be oxidised to NAD+ at a reduced rate by the electron transport chain, so NAD+ will remain higher of its own accord until such time as the mitochondria increase in number.

 

Just for reference, this in vivo study in humans using a 6 g dose produced a peak concentration in the blood of around 1mM.

 

https://www.ncbi.nlm.../pubmed/1410588

 

 

It appears CAC and ETC are balancing, such that, even if mito content is reduced, higher NAD+ will eventually come down (although slower) from higher CAC output relative to ETC output (auto normalization).  It would be interesting to find the dynamic of balancing ETC to CAC activity based on mitochondrial size (and perhaps this is in part what is being balanced with fission/fusion on NAD/NADH imbalances).

 

6g of NAM with 10g ribose (without lactate) did result in an endurance weakening, although not a strength weakening.  Perhaps best to separate occurrences by 48 hours to allow preferenced NAD uptake cells (intestinal cells) to clear excess NAM.

[Ken Mark]

Fascinating discussion.

I have couple of questions.

1. Why take Ribose at all? 4 g seems negligible amount, and it's anyways converted into glucose by body. Ribose is made by cells themselves anyways. I don't see any ribose taken reaching to cells where it's supposed to recombine with Niacinamide.

2. Has there been any gray hair reversal with this protocol. I think this would be most direct and less subjective observation proving age reversal.

[Mr Matsubayashi]

I wonder if low intensity exercise combined with the fission protocol would have further synergy with blood flow restricted training, which also requires low 20-30% effort workouts.

 

Practical Blood Flow Restriction Training Increases Acute Determinants of Hypertrophy Without Increasing Indices of Muscle Damage

https://journals.lww...raining.20.aspx

https://www.physio-p...iction_Training

 

Cheap elastomer straps for this can be purchased from ebay, cheap seat belt covers to cover the straps can also be purchased from ebay. 

[Turnbuckle]

I wonder if low intensity exercise combined with the fission protocol would have further synergy with blood flow restricted training, which also requires low 20-30% effort workouts.

 

 

 

 

I've tried that and found it both horrible and ineffective. Results may vary, of course.

Edited by Turnbuckle, 06 February 2020 - 12:59 PM.

[longcity90]

I understood that this protocol is aimed at fat loss ... but if a person wants to increase lean muscle mass with a caloric deficit is it humanly possible or is there always a need for a surplus? obviously we would have a higher NADH and no longevity benefit by keeping the calories high.

[Amira L.]

Thought this was interesting:https://www.globenew...pertension.html. 

[DJSwarm]

Definite strength and muscle gains, but I can't yet say for sure about the pyruvate.

 

You are hedging.

 

During your "off cycle" time (you have an off cycle to get your new normal, yes?), do you subjectively feel like you can now lift more than when you started? 

 

If there isn't a subjective difference in your current "normal" state then the answer is "no."

 

Personally, I'm back to taking the stairs two at a time, so my answer would be subjectively  "yes."

 

Or if you are a measurer, then when not taking anything, have your measurements changed in good ways? By how much?

Are you on average lifting more weight or more often?

 

I know you track your epigenetic age but if that is a valid measure and you are what 22 years epigenetically younger... well I don't know about you, but I was pretty peppy 22 years ago. If I was really 22 years younger, I'd notice. 

[adamjr]

I followed the protocol as described, but instead of experiencing a weakening effect, I had quite the opposite! I was able to lift the same weights I normally use with much more ease, and for a longer period of time. I had a significant burst in energy, as well. I will say that the burn described was apparent, but I am unsute as to what would cause my experience to be opposite as described. Any thoughts?