179 replies to this topic

[Turnbuckle]

Some thoughts on tweaking the protocol for removing zombie mitochondria--

 

Research in rats dating back to the sixties shows that NAD+ takes a long time to build up from a single large dose of nicotinamide. NAD reaches a maximum at around six hours--about an hour after to dose is gone. Thus this should be dosed at t=0.

 

See Fig. 1 in this paper: On the Relative Efficacy of Nicotinamide and Nicotinic Acid as Precursors of Nicotinamide Adenine Dinucleotide.

 

PQQ: Since PQQ converts NADH to NAD+ (taking about 2 hrs in vitro) and according to one source, "PQQ peaked in serum at 2–3 h after each dose with a half-life of 3–5 h." Thus this could be taken at t=3 hours so that its peak coincides with the nicotinamide peak.

 

NaB: As I've seen widely varying figures for the biological half life of sodium benzoate and it's not clear how long it actually takes to activate PINK1 and Parkin, I'm including 1 g at t=0 and t=3.

 

So that's it. I'm leaving off everything else previously mentioned to keep this simple--

 

Zombie Protocol 2

--------------------------------------------------------

t=0

 

2 g of nicotinamide

1 g of NaB

 

t=3 hours

 

20 mg of PQQ

1 g of NaB

--------------------------------------------------------

 

I haven't tried this yet, just putting it up for comments.

[Adam1]

I really like this way of designing a protocol with optimized times for peak levels. My only question is re dose of sodium benzoate, as this is not something I am familiar with. It seems possibly high, and I would prefer to start with a low dose and titrate up carefully, due to its potential to damage mitochondria. (Also as you know, avoid combining it with vitamin C and citric acid so that it does not generate benzene.)

 

Generally antibiotics at high doses can damage mitochondria due to these organelles having had evolutionary origins as bacteria (according to endosymbiosis theory). Sodium benzoate is an anti-microbial, and its unusual targeting of mitochondria leads one to ask what the therapeutic window actually is.

[Turnbuckle]

I really like this way of designing a protocol with optimized times for peak levels. My only question is re dose of sodium benzoate, as this is not something I am familiar with. It seems possibly high, and I would prefer to start with a low dose and titrate up carefully, due to its potential to damage mitochondria. (Also as you know, avoid combining it with vitamin C and citric acid so that it does not generate benzene.)

 

Generally antibiotics at high doses can damage mitochondria due to these organelles having had evolutionary origins as bacteria (according to endosymbiosis theory). Sodium benzoate is an anti-microbial, and its unusual targeting of mitochondria leads one to ask what the therapeutic window actually is.

 

 

Thank you for expressing these concerns. NaB has been used for over a hundred years and does indeed have anti-microbial properties, but these seem to be associated with low pH--4.5 and below. It has been taken at the multi-gram level for days or weeks with no apparent harm, but nevertheless, Professor Peter W Piper caused a stink in the UK press roughly ten years ago, claiming that it could harm mitochondria. His concerns are echoed in his paper from that time, but seem to be mainly speculative, as effects were once again were found at a low pH environment.

 

The above-described research into yeast preservative resistance has been mainly prompted by the desire to achieve substantial reductions in large-scale preservative use. There is considerable consumer demand for less preserved foods, as these are perceived as more natural. Although the compounds in Figure 1a all occur in natural materials, recent evidence raises some concerns over whether their widespread, large-scale use is a completely safe practice. Benzoic acid can react with the ascorbic acid in soft drinks in order to produce benzene, a potentially carcinogenic compound. In addition, both sorbic and benzoic acids are mutagenic towards the yeast mitochondrial genome, due to their capacity to generate severe oxidative stress. Whether they are also capable of exerting a similar damage to the mitochondrial DNA in man is uncertain, but it represents an aspect that was not properly addressed in the earlier safety testing of these compounds.

 

http://onlinelibrary...2/yea.1576/full

 

 

He obviously has an ax to grind against all preservatives, but still, I looked further into it and found one paper that gave me pause about the logic of using NaB, but for a different reason. In this case it was given at the half gram level, and it was discovered that the blood level peaked at 30 minutes and quickly disappeared, and in some cases disappeared before 30 minutes--

 

Based on subject weights, the administered dose of 480 mg sodium benzoate corresponded to a per kg dose of 5.65 mg/kg/dose (range 4.5–7 mg/kg/dose), substantially lower than the doses used clinically for treatment of hyperammonemia and non-ketotic hyperglycinemia. The process measures for the study are displayed in Figure 3, demonstrating a substantial rise in the mean value of benzoate and hippurate following ingestion of each of the two sodium benzoate-containing beverages. Benzoate and hippurate peak at 30 minutes, and fall dramatically by 120 minutes, indicating rapid metabolism and clearance. Interestingly, although hippurate showed a rise in all subjects, benzoate itself was not detectable in two out of the fourteen subjects following the water with benzoate beverage, suggesting that it had been completely metabolized by 30 minutes. 

 

http://www.ncbi.nlm..../PMC4289147/#R9

 

 

 

One problem is, I don't know if the autophagic genes respond like a light switch or if they require a continuous stimulus. In any case, I'm going back to cinnamon, which produces NaB as a metabolite and thus should give a longer residence time. (Though I might throw in some extra NaB at the five hour mark.)

Edited by Turnbuckle, 16 May 2016 - 12:57 PM.

[maxwatt]

Two more arrows in our quiver for apoptosis and mitochondrial biogenesis:  dihydromyricetin for autophagy and baicalin for neogenesis

 

http://www.ncbi.nlm....bmed/25896550 

Dihydromyricetin improves skeletal muscle insulin sensitivity by inducing autophagy via the AMPK-PGC-1α-Sirt3 signaling pathway.

Insulin resistance in skeletal muscle is a key feature in the pathogenesis of type 2 diabetes (T2D) that often manifests early in its development. Pharmaceutical and dietary strategies have targeted insulin resistance to control T2D, and many natural products with excellent pharmacological properties are good candidates for the control or prevention of T2D. Dihydromyricetin (DHM) is a natural flavonol which provides a wide range of health benefits including anti-inflammatory and anti-tumor effects. However, little information is available regarding the effects of DHM on skeletal muscle insulin sensitivity as well as the underlying mechanisms. In the present study, we found that DHM activated insulin signaling and increased glucose uptake in skeletal muscle in vitro and in vivo. The expression of light chain 3, the degradation of sequestosome 1, and the formation of autophagosomes were also upregulated by DHM. DHM-induced insulin sensitivity improvement was significantly abolished in the presence of 3-methyladenine, bafilomycin A1, or Atg5 siRNA in C2C12 myotubes. Furthermore, DHM increased the levels of phosphorylated AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor coactivator-1α (PGC-1α), and Sirt3 in skeletal muscle in vitro and in vivo. Autophagy was inhibited in the presence of Sirt3 siRNA in C2C12 myotubes and in skeletal muscles from Sirt3-/- mice. Additionally, PGC-1α or AMPK siRNA transfection attenuated DHM-induced Sirt3 expression, thereby abrogating DHM-induced autophagy in C2C12 myotubes. In conclusion, DHM improved skeletal muscle insulin sensitivity by partially inducing autophagy via activation of the AMPK-PGC-1α-Sirt3 signaling pathway.

 

http://www.ncbi.nlm....MC4146053/     Baicalin influences the dendritic morphology of newborn neurons in the hippocampus of chronically stressed rats

....Baicalin treatment increased the number of both class I and class II doublecortin-positive neurons. In addition, doublecortin-positive neurons showed less complexity in dendritic morphology after corticosterone injection, and this change was totally reversed by baicalin treatment. These findings suggest that baicalin exhibits a beneficial effect on adult neurogenesis.

 

http://www.ncbi.nlm....lin islet cells Baicalin reduces mitochondrial damage in streptozotocin-induced diabetic Wistar rats.

....Baicalin had also increased the plasma leptin content (p < 0.05) versus the diabetic control, which in turn had effected the total expression of hepatic mitochondria per cell indicating its effects in SIRT1 activity. The increase in mitochondrial number was further complemented with similar trends in the hepatic citrate synthase activity.

 

 

[Adam1]

baicalin for neogenesis…

 

I like the additional arrows. One comment on the opposite functions involved in quality control.

 

Baicalin and baicalein do whatever is needed under a number of conditions. These related compounds not only span both biogenesis and autophagy, but can induce extensive autophagy to destroy cancer cells (Aryal, Pramod, et al. "Baicalein induces autophagic cell death through AMPK/ULK1 activation and downregulation of mTORC1 complex components in human cancer cells." Febs Journal 281.20 (2014): 4644-4658.) or suppress autophagy when a virus takes over a host’s autophagy (Zhu, Hai-yan, et al. "Baicalin inhibits autophagy induced by influenza A virus H3N2." Antiviral research 113 (2015): 62-70.)

 

Other familiar phytochemicals can match baicalin’s offer of one-stop shopping. From a recent review: “Curcumin improves mitochondrial dynamics regarding mitochondrial biogenesis and mitophagy" (de Oliveira, Marcos Roberto, et al. "Curcumin, mitochondrial biogenesis, and mitophagy: Exploring recent data and indicating future needs." Biotechnology Advances 2016). Resveratrol is another compound that is an excellent inducer of both mitophagy and mitogenesis (de Oliveira, Marcos Roberto, et al. "Resveratrol and the mitochondria: From triggering the intrinsic apoptotic pathway to inducing mitochondrial biogenesis, a mechanistic view." Biochimica et Biophysica Acta (BBA)-General Subjects 2016).

 

This might be explained in part by the compounds being mediators of our bidirectional gateways for mitochondrial entrances and exits (Hang, Liting, John Thundyil, and Kah‐Leong Lim. "Mitochondrial dysfunction and Parkinson disease: a Parkin–AMPK alliance in neuroprotection." Annals of the New York Academy of Sciences 1350.1 (2015): 37-47.)

 

Interestingly, the role of Parkin in the biogenesis and clearance of mitochondria is akin to that performed by the energy sensor AMP-activated protein kinase (AMPK), suggesting that the two proteins might act in a functionally converging manner to maintain the quality of cellular mitochondria.

 

[Turnbuckle]

Trial of Zombie Protocol 2 (modified)

--------------------------------------------------------

t=0

2 g nicotinamide

4 g cinnamon extract

 

t=3.5 hours

20 mg PQQ

4 g cinnamon extract

 

t=5 hours

1/4 g NaB

--------------------------------------------------------

 

The only notable side effect was at around t=3 hours, when I felt antsy with various small pains all over that dissipated after ten or twenty minutes. The next day I felt normal, with no noticeable change in energy level. At the gym (t=20 hours), however, I did a double take at the weight stack on the first machine, as it seemed much easier than normal. Most of the machines were that way, although I'd expected it to go the other way.

[Adam1]

Encouraging results for the refined protocol! I am learning from you Turnbuckle.

 

When using cinnamon it’s possible its compounds may be helping mitochondrial function in other ways too. As mentioned above, mitochondrial dysfunction can result from antibiotics. While I neglected to reference this above, see e.g.  Moullan, Norman, et al. "Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research." Cell reports 10.10 (2015): 1681-1691. Cinnamon has a lower likelihood of doing this, while still protecting mitochondria from bacteria.

 

Mitochondria are “a target of choice for bacterial pathogens” (Lobet, Elodie, Jean-Jacques Letesson, and Thierry Arnould. "Mitochondria: a target for bacteria." Biochemical pharmacology 94.3 (2015): 173-185.)

 

Also, enteric bacteria such as Clostridia spp., “produce short-chain fatty acid metabolites that are potentially toxic to the mitochondria” (Frye, Richard E., et al. "Gastrointestinal dysfunction in autism spectrum disorder: the role of the mitochondria and the enteric microbiome." Microbial ecology in health and disease 26 (2015).)

 

There may prove to be compounds in cinnamon and other herbs such as rosemary that target bacterial pathogens while preserving and improving mitochondrial function and dynamics.

[Nate-2004]

Turnbuckle in reference to the first post why nicotinamide (NAM) as opposed to Nicotinamide Riboside (NR)?

[Turnbuckle]

Turnbuckle in reference to the first post why nicotinamide (NAM) as opposed to Nicotinamide Riboside (NR)?

 

Or why NR as opposed to NAM? Or niacin, for that matter? 

 

I've tried niacin, btw, and in some ways it's more convenient because its effect on NAD is so fast. But the flushing is tremendous, so if you're not used to it, don't try this--

 

Trial of Zombie Protocol 3

--------------------------------------------------------

t=0

20 mg PQQ

 

t=1 hr

2 g niacin

2 g cinnamon extract

 

t=1.5 hrs

1/4 g NaB

--------------------------------------------------------

 

Initially I felt less energy, but by the time I went running at 3 hours, I felt fine. Did it do anything? I don't know. Nothing obvious, anyway.

[Nate-2004]

Or why NR as opposed to NAM? Or niacin, for that matter? 

 

Main reason is the considerable number of references and fairly good arguments in the NR thread in addition to the fact that it's a better precursor because it jumps ahead of the process that can otherwise inhibit SIRT1/3 when it comes to increased nicotinamide. Granted it's expensive, but then again it'd be less expensive if A: There were no patent and B: There were a reason not to take it all the time (context).

Edited by Nate-2004, 30 May 2016 - 05:17 PM.

[to age or not to age]

Three weeks ago, two chemists I have been working with for almost a year successfully synthesized beta lapachone.  I had a proton NMR done on the sample and it is indeed pure beta lapachone.  Now we are making 10 grams which will be finished and tested hopefully by June 15th.  I am taking samples with me to the Glenn Symposium.  Several scientists I know are very interested in this compound.  We are going to make perhaps 5 grams available for scientific research purposes.  The price will be 1,500 to 2,000 per gram. 

[Turnbuckle]

 

Or why NR as opposed to NAM? Or niacin, for that matter? 

 

Main reason is the considerable number of references and fairly good arguments in the NR thread in addition to the fact that it's a better precursor because it jumps ahead of the process that can otherwise inhibit SIRT1/3 when it comes to increased nicotinamide. Granted it's expensive, but then again it'd be less expensive if A: There were no patent and B: There were a reason not to take it all the time (context).

 

 

That is indeed my impression of NR--that it is being marketed on the basis of providing a shorter path to NAD. Earlier I tried it at a 500 mg dose but didn't see anything. Eventually I will get around to upping the dose to the NAM level, but I haven't seen a NAD response curve as exists for NAM and niacin, so I don't know how to time the dose.

[gamesguru]

supplements that block fusion might not selectively shrink bad mitochondria. it's quite possible, as suggested, some antioxidants actually help to flush out the baddies, because they interact with mitophagy-related pathways.

Possible antioxidant mechanism of melanoidins extract from Shanxi aged vinegar in mitophagy-dependent and mitophagy-independent pathways.
Yang L1, Wang X, Yang X.

Melanoidins are widely reported to have antioxidant activity; however, their mechanism has not been frequently studied. In this study, we found that melanoidins from Shanxi aged vinegar induced mitopahgy, the specific autophagic elimination of mitochondria, as assessed by up-regulation of the autophagy markers LC3-II and Beclin1 as well as degradation of the autophagy substrate p62 and mitochondrial proteins. Melanoidins reduced reactive oxygen species (ROS) in normal human liver cells and mouse livers through a mitophagy-dependent pathway, by the observation that the reducing ROS effect of melanoidins was partially lost when mitophagy was inhibited by chloroquine. Impaired Akt signaling was found in cells treated with melanoidins, which might explain the activation of autophagy induced by melanoidins. These results suggest that in addition to direct free radical scavenging activity, melanoidins decreased ROS levels through mitophagy in which damaged mitochondria, the source of ROS, were degraded.

 

Inducing mitophagy in diabetic platelets protects against severe oxidative stress.
Lee SH1, Du J1, Stitham J1, Atteya G1, Lee S2, Xiang Y1, Wang D1, Jin Y1, Leslie KL1, Spollett G3, Srivastava A4, Mannam P4, Ostriker A1, Martin KA1, Tang WH5, Hwa J6.

Diabetes mellitus (DM) is a growing international concern. Considerable mortality and morbidity associated with diabetes mellitus arise predominantly from thrombotic cardiovascular events. Oxidative stress-mediated mitochondrial damage contributes significantly to enhanced thrombosis in DM A basal autophagy process has recently been described as playing an important role in normal platelet activation. We now report a substantial mitophagy induction (above basal autophagy levels) in diabetic platelets, suggesting alternative roles for autophagy in platelet pathology. Using a combination of molecular, biochemical, and imaging studies on human DM platelets, we report that platelet mitophagy induction serves as a platelet protective mechanism that responds to oxidative stress through JNK activation. By removing damaged mitochondria (mitophagy), phosphorylated p53 is reduced, preventing progression to apoptosis, and preserving platelet function. The absence of mitophagy in DM platelets results in failure to protect against oxidative stress, leading to increased thrombosis. Surprisingly, this removal of damaged mitochondria does not require contributions from transcription, as platelets lack a nucleus. The considerable energy and resources expended in "prepackaging" the complex mitophagy machinery in a short-lived normal platelet support a critical role, in anticipation of exposure to oxidative stress.

 

Ceramide targets autophagosomes to mitochondria and induces lethal mitophagy
R David Sentelle,    Can E Senkal,    Wenhui Jiang (2012)

Mechanisms by which autophagy promotes cell survival or death are unclear. We provide evidence that C18-pyridinium ceramide treatment or endogenous C18-ceramide generation by ceramide synthase 1 (CerS1) expression mediates autophagic cell death, independent of apoptosis in human cancer cells. C18-ceramide–induced lethal autophagy was regulated via microtubule-associated protein 1 light chain 3 β-lipidation, forming LC3B-II, and selective targeting of mitochondria by LC3B-II–containing autophagolysosomes (mitophagy) through direct interaction between ceramide and LC3B-II upon Drp1-dependent mitochondrial fission, leading to inhibition of mitochondrial function and oxygen consumption. Accordingly, expression of mutant LC3B with impaired ceramide binding, as predicted by molecular modeling, prevented CerS1-mediated mitochondrial targeting, recovering oxygen consumption. Moreover, knockdown of CerS1 abrogated sodium selenite–induced mitophagy, and stable LC3B knockdown protected against CerS1- and C18-ceramide–dependent mitophagy and blocked tumor suppression in vivo. Thus, these data suggest a new receptor function of ceramide for anchoring LC3B-II autophagolysosomes to mitochondrial membranes, defining a key mechanism for the induction of lethal mitophagy.

From wikipedia: Several studies have attempted to define further the specific role of ceramide in the events of cell death and some evidence suggests ceramide functions upstream of the mitochondria in inducing apoptosis. However, owing to the conflicting and variable nature of studies into the role of ceramide in apoptosis, the mechanism by which this lipid regulates apoptosis remains elusive.^[9]

 

Rotenone-induced mitophagy. (but prolonged high doses may cause Parkinson's!!)
Charleen T. Chu,    Jing Ji,    Ruben K. Dagda,    Jian Fei Jiang, et al.

(a–d) Rotenone (Rot) increased the number of GFP–LC3 puncta and the level of co-localization with mitochondria (arrows) in SH-SY5Y cells (a–c; 1 μM) and primary cortical neurons (d; 250 nM), quantified in Fig. 3f and Supplementary Fig. S1b,c. Veh, vehicle. (e,f) Rotenone increased delivery of MitoTracker Green-stained mitochondria (mt) to LysoTracker Red (LTR)-stained lysosomes, inhibited by siRNA knockdown of ATG7 or LC3 in SH-SY5Y cells. Ctrl, control. Right: RNAi knockdown. (g) Rotenone decreased IMM [inner mitochondrial membrane] (COXIV), OMM (TOM40) and matrix (MnSOD) protein expression levels in primary neurons, reversed by bafilomycin (Baf) inhibition of autolysosomal degradation, quantified in Supplementary Fig. S1e. Mean ± s.d. of n = 7 independent experiments for b,c, and n = 3 independent experiments for f,g (see Supplementary Table S4 for statistics source data); *P<0.05 versus vehicle control; †P<0.05 versus Rot/control siRNA. Scale bars, 10 μm. See Supplementary Fig. S7 for uncropped blots.

 

and something as simple as caffeine can make a good addition to this stack?

Effects of Caffeine on Metabolism and Mitochondria Biogenesis in Rhabdomyosarcoma Cells Compared with 2,4-Dinitrophenol
Roger A. Vaughan,1–3 Randi Garcia-Smith,2 Marco Bisoffi,2 Kristina A. Trujillo,2 and Carole A. Conn3

Purpose:
This work investigated if treatment with caffeine or 2,4-dinitrophenol (DNP) induce expression of peroxisome proliferator-activated receptor coactivator 1 alpha (PGC-1α) and increase both mitochondrial biosynthesis and metabolism in skeletal muscle.
Methods:
Human rhabdomyosarcoma cells were treated with either ethanol control (0.1% final concentration) caffeine, or DNP at 250 or 500 μM for 16 or 24 hours. PGC-1α RNA levels were determined using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). PGC-1α protein and mitochondrial content was determined using flow cytometry and immunohistochemistry. Metabolism was determined by quantification of extracellular acidification rate and oxygen consumption rate.
Results:
Treatment with either caffeine or DNP induced PGC-1α RNA and protein as well as mitochondrial content compared with control. Treatment with caffeine and DNP also significantly increased oxidative metabolism and total metabolic rate compared with control. Caffeine similarly increased metabolism and mitochondrial content compared with DNP.
Conclusion:
This work identified that both caffeine and DNP significantly induce PGC-1α, and increase both metabolism and mitochondrial content in skeletal muscle.

Caffeine increases mitochondrial function and blocks melatonin? signaling to mitochondria in Alzheimer's mice and cells.
Dragicevic N1, Delic V, Cao C, Copes N, Lin X, Mamcarz M, Wang L, Arendash GW, Bradshaw PC.

Caffeine and melatonin have been shown to protect the Swedish mutant amyloid precursor protein (APP(sw)) transgenic mouse model of Alzheimer's disease from cognitive dysfunction. But their mechanisms of action remain incompletely understood. These Alzheimer's mice have extensive mitochondrial dysfunction, which likely contributes to their cognitive decline. To further explore the mechanism through which caffeine and melatonin protect cognitive function in these mice, we monitored the function of isolated mitochondria from APP(sw) mice treated with caffeine, melatonin, or both in their drinking water for one month. Melatonin treatment yielded a near complete restoration of mitochondrial function in assays of respiratory rate, membrane potential, reactive oxygen species production, and ATP levels. Caffeine treatment by itself yielded a small increase in mitochondrial function. However, caffeine largely blocked the large enhancement of mitochondrial function provided by melatonin. Studies with N2a neuroblastoma cells stably expressing APP(sw) showed that specific inhibition of cAMP-dependent phosphodiesterase (PDE) 4 or cGMP-dependent PDE5 also blocked melatonin protection of mitochondrial function, but A(2a) and A₁ adenosine receptor antagonists were without effect. Melatonin or caffeine at the concentrations used to modulate mitochondrial function in the cells had no effect on cAMP-dependent PDE activity or cellular cAMP or cGMP levels. Therefore, caffeine and increased cyclic nucleotide levels likely block melatonin signaling to mitochondria by independent mechanisms that do not involve adenosine receptor antagonism. The results of this study indicate that melatonin restores mitochondrial function much more potently than caffeine in APP(sw) transgenic mouse and cell models of Alzheimer's disease.

 

while reading about quercetin and autophagy, i found this reference. again like with antioxidants, many flavonoids are antiglycative, so it remains to see if they selectively target healthy mitochondria or if you really have to avoid them in the early phase.

Lo MC, Lu CI, Chen MH, Chen CD, Lee HM, Kao SH (2010) Glycoxidative stress-induced mitophagy modulates mitochondrial fates. Ann N Y Acad Sci 1201:1-7

Edited by gamesguru, 10 June 2016 - 01:56 PM.

[Adam1]

Gamesguru, it is fascinating that bioactives can send ambivalent signals. UV light, for example, is both cytotoxic (or induces autophagy through JNK) and a promoter of mitogenesis (Blagosklonny, M. V. "Carcinogenesis, cancer therapy and chemoprevention." Cell Death & Differentiation 12.6 (2005): 592-602.) Perhaps the above-mentioned cancer review provides an analogy that is applicable:

 

 

The term carcinoagents emphasizes the dual nature of cytotoxic agents. Carcinoagents include not only carcinogens and drugs but also physiological restrictive signaling molecules such as TGF-beta and immunological suppressors, for instance. All could be used to control cancer.

 

The paradigm we live in labels compounds as good or bad, because at this point we cannot study the vastly complicated network of tradeoffs. In real life, the compounds have a predominant tendency but enter a matrix where they may be translated anywhere along a continuum of diametric opposites.

[Fafner55]

Turnbuckle, I am curious about your opinion on how honokiol might contribute to improving the population of mitochondria.

 

Honokiol has been shown to increase Sirt3.  It was found that in the heart muscle cells from mice honokiol nearly doubled Sirt3 levels within 24 hours.  “Honokiol blocks and reverses cardiac hypertrophy in mice by activating mitochondrial Sirt3” (2015) http://www.nature.com/ncomms/2015/150414/ncomms7656/abs/ncomms7656.html

The authors write, “To the best of our knowledge, this is the first report to describe a pharmacologic activator of Sirt3. Until now, caloric restriction combined with endurance exercise has been the only way to boost Sirt3 levels.” http://www.sciencedaily.com/releases/2015/04/150414125815.htm

[Turnbuckle]

Honokiol is interesting. I've taken it in magnolia extract, both with and without NR, but haven't seen in effect at all with either supplement or together. At least no immediate effect, anyway.

[LeeYa]

An excellent thread, thanks turnbuckle!

Do you consider to include Alpha lipoic acid and acetyl-l-carnitine?

The Effects and Mechanisms of Mitochondrial Nutrient α-Lipoic Acid on Improving Age-Associated Mitochondrial and Cognitive Dysfunction: An Overview
http://link.springer...1064-007-9403-0

Quercetin could be considered as well:

Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance
http://ajpregu.physi...6/4/R1071.short
 
What about NAC?
http://www.lmreview....ne-up-part-III/




What about NO-Boosters? http://science.scien...nt/299/5608/896

Edited by LeeYa, 30 June 2016 - 03:16 PM.

[Turnbuckle]

An excellent thread, thanks turnbuckle!

Do you consider to include Alpha lipoic acid and acetyl-l-carnitine?

The Effects and Mechanisms of Mitochondrial Nutrient α-Lipoic Acid on Improving Age-Associated Mitochondrial and Cognitive Dysfunction: An Overview
http://link.springer...1064-007-9403-0

Quercetin could be considered as well:

Quercetin increases brain and muscle mitochondrial biogenesis and exercise tolerance
http://ajpregu.physi...6/4/R1071.short
 
What about NAC?
http://www.lmreview....ne-up-part-III/




What about NO-Boosters? http://science.scien...nt/299/5608/896

 

I presently take a near daily mix of powers that contain ALCAR and NAC, but not ALA. The combo of ALA and ACAR I don't like, so I take ALA separately, a couple of times a week.

[maxwatt]

Apparently Urolithin A is a one-stop molecule for eliminating unhealthy mitochondria and generating healthy ones.  Only tested in worms and mice so far.  See this thread: http://www.longecity...geing/?p=782588

 

Above only references the New Scientist write-up of the work from Auwerx' lab, but the original paper in Nature can be found below:

 

http://www.nature.co...ll/nm.4132.html

 

There is no ready source of urolithin A, but if one is lucky enough to have the right gut bacteria (Gordonibacter species) ellagic acid in pomegranate will be metabolized to urolithin A. 

 

 

 

[Nate-2004]

Apparently Urolithin A is a one-stop molecule for eliminating unhealthy mitochondria and generating healthy ones.  Only tested in worms and mice so far.  See this thread: http://www.longecity...geing/?p=782588

 

Above only references the New Scientist write-up of the work from Auwerx' lab, but the original paper in Nature can be found below:

 

http://www.nature.co...ll/nm.4132.html

 

There is no ready source of urolithin A, but if one is lucky enough to have the right gut bacteria (Gordonibacter species) ellagic acid in pomegranate will be metabolized to urolithin A. 

 

Interesting where did you find that it was Gordonibacter? I assumed it was Clostridium leptum based on Wikipedia as to what produces Urolithins.

[gamesguru]

It's not produce urolithin A?  But it may be produced indirectly, after the fact.  So both bacteria may work the same?

This allowed the detection of catabolic intermediates [urolithins M-5, M-6, M-7, C, and 2,3,8,10-tetrahydroxy urolithin (urolithin E)].

 

The study on Gordonibacter sheds some light.

The HPLC-MS analyses showed the sequential production of pentahydroxy-urolithin (urolithin M-5), tetrahydroxy-urolithin (urolithin M-6) and trihydroxy-urolithin (urolithin C), while dihydroxy-urolithins (urolithin A and isourolithin A), and monohydroxy-urolithin (urolithin B) were not produced in pure cultures. Consequently, either other bacteria from the gut or the physiological conditions found in vivo are necessary for completing metabolism until the final urolithins (dihydroxy and monohydroxy urolithins) are produced. This is the first time that the urolithin production capacity of pure strains has been demonstrated.

Edited by gamesguru, 14 July 2016 - 04:24 PM.

[maxwatt]

As far as I could find, Clostridium leptum is associated with higher Urolithin levels in fecal samples, but has not been observed actually producing Urolithin.  It may be only an association, not a cause.  Gordonibacter, on the other hand, has been shown to actually convert ellagic acid to Urolithin A*.  I googled bacteria ellagic acid urolithin to find ghe papers.

 

Now where an one find a probiotic containing either of these species?  Combine that with pomegranate extract and voila.  I'd like to try it for my bed-ridden mother.

 

http://www.ncbi.nlm....pubmed/24909569

 

 

Apparently this bacterium is a necessary but not a sufficient condition, as gamesguru pointed out quoting this papr, above.

 

 

Edited by maxwatt, 14 July 2016 - 06:18 PM.

[Flex]

That was helpful and interesting. Indeed PINK1 and Parkin are critical in mediating fission and mitophagy and are worth enhancing. Further mix ‘n match candidates for the autophagy portion of the protocol (mostly from Nixon, Ralph A. "The role of autophagy in neurodegenerative disease." Nature medicine 19.8 (2013): 983-997.):

 

TORC1 inhibition – curcumin

AMPK activation – lithium, trehalose, quercetin, catechins, naringen, berberine, curcumin

PP2A activation – palm oil for tocopherols, melatonin, forskolin, epigallocatechine gallate

Other induction targets: parthenolide in feverfew, fisetin, luteolin

 

Many phytochemicals can have oxidant or antioxidant effects depending on factors such as dose and what they encounter. Some compounds may both induce and inhibit autophagy. For example, berberine is also an inhibitor (Hundeshagen, Phillip, et al. "Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy." BMC biology 9.1 (2011): 38.) It appears specific antioxidants can have a direct role in mitophagy (Yang, Lei, Xuping Wang, and Xiaolan Yang. "Possible antioxidant mechanism of melanoidins extract from Shanxi aged vinegar in mitophagy-dependent and mitophagy-independent pathways." Journal of agricultural and food chemistry 62.34 (2014): 8616-8622.)

 

Ideally the protocol would be pared down and individualized to contain just the rate-limiting steps of mitochondrial turnover through mitophagy, biogenesis, dynamics of fusion/fission, and could also address selective protein quality control. I am interested in learning about ways of implementing this.

 

Rescue of GABAB and GIRK function in the lateral habenula by protein phosphatase 2A inhibition ameliorates depression-like phenotypes in mice.
http://www.ncbi.nlm....pubmed/26808347

 

Seems to have pro-depressant properties on the other hand. Do You know accidentally a PP2A inhibitor ?
 

[Nate-2004]

This seems contradictory:

 

Quercetin inhibits AMPK/TXNIP activation and reduces inflammatory lesions to improve insulin signaling defect in the hypothalamus of high fructose-fed rats.

 

Abstract

Fructose is a nutritional composition of fruits and honey. Its excess consumption induces insulin resistance-associated metabolic diseases. Hypothalamic insulin signaling plays a pivotal role in controlling whole-body insulin sensitivity and energy homeostasis. Quercetin, a natural flavonoid, has been reported to ameliorate high fructose-induced rat insulin resistance and hyperlipidemia. In this study, we investigated its regulatory effects on the hypothalamus of high fructose-fed rats. Rats were fed 10% fructose in drinking water for 10 weeks. After 4 weeks, these animals were orally treated with quercetin (50 and 100 mg/kg), allopurinol (5 mg/kg) and water daily for the next 6 weeks, respectively. Quercetin effectively restored high fructose-induced hypothalamic insulin signaling defect by up-regulating the phosphorylation of insulin receptor and protein kinase B. Furthermore, quercetin was found to reduce metabolic nutrient sensors adenosine monophosphate-activated protein kinase (AMPK) activation and thioredoxin-interacting protein (TXNIP) overexpression, as well as the glutamine-glutamate cycle dysfunction in the hypothalamus of high fructose-fed rats. Subsequently, it ameliorated high fructose-caused hypothalamic inflammatory lesions in rats by suppressing the activation of hypothalamic nuclear factor κB (NF-κB) pathway and NOD-like receptor 3 (NLRP3) inflammasome with interleukin 1β maturation. Allopurinol had similar effects. These results provide in vivo evidence that quercetin-mediated down-regulation of AMPK/TXNIP and subsequent inhibition of NF-κB pathway/NLRP3 inflammasome activation in the hypothalamus of rats may be associated with the reduction of hypothalamic inflammatory lesions, contributing to the improvement of hypothalamic insulin signaling defect in this model. Thus, quercetin with the central activity may be a therapeutic for high fructose-induced insulin resistance and hyperlipidemia in humans.

 

Yet:

 

Quercetin activates AMP-activated protein kinase by reducing PP2C expression protecting old mouse brain against high cholesterol-induced neurotoxicity.

Abstract

It is known that a high-cholesterol diet induces oxidative stress, inflammatory response, and beta-amyloid (Abeta) accumulation in mouse brain, resulting in neurodegenerative changes. Quercetin, a naturally occurring flavonoid, has been reported to possess numerous biological activities beneficial to health. Our previous studies have demonstrated that quercetin protects mouse brain against D-galactose-induced oxidative damage. Against this background, we evaluated the effect of quercetin on high-cholesterol-induced neurotoxicity in old mice and explored its potential mechanism. Our results showed that oral administration of quercetin significantly improved the behavioural performance of high-cholesterol-fed old mice in both a step-through test and the Morris water maze task. This is at least in part caused by decreasing ROS and protein carbonyl levels and restoring Cu--Zn superoxide dismutase (Cu, Zn-SOD) activity. Furthermore, quercetin also significantly activated the AMP-activated protein kinase (AMPK) via down-regulation of protein phosphatase 2C (PP2C), which reduced the integral optical density (IOD) of activated microglia cells and CD11b expression, down-regulated iNOS and cyclooxygenase-2 (COX-2) expression, and decreased IL-1beta, IL-6, and TNF-alpha expression in the brains of high-cholesterol-fed old mice through the suppression of NF-kappaB p65 nuclear translocation. Moreover, AMPK activation significantly increased 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase (ACC) phosphorylation and reduced fatty acid synthase (FAS) expression in the brains of high-cholesterol-fed old mice, which reduced cholesterol levels, down-regulated cholesterol 24-hydroxylase (CYP46A1) and beta-amyloid converting enzyme 1 (BACE1) expression, decreased eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation, and lowered Abeta deposits. However, the neuroprotective effect of quercetin was weakened by intraperitoneal injection of compound C, an AMPK inhibitor. These results suggest that AMPK activated by quercetin may be a potential target to enhance the resistance of neurons to age-related diseases.

 

What's the deal?

Edited by Nate-2004, 17 August 2016 - 04:30 AM.

[Adaptogen]

Preservation of Cognitive Function by Lepidium meyenii (Maca) Is Associated with Improvement of Mitochondrial Activity and Upregulation of Autophagy-Related Proteins in Middle-Aged Mouse Cortex

[gamesguru]

happy sunday, all

 Acute upregulation of neuronal mitochondrial type-1 cannabinoid receptor and its role in metabolic defects and neuronal apoptosis after TBI

Zhen Xu,corresponding author#1 Xiao-Ai Lv,#2 Qun Dai,3 Yu-Qing Ge,3 and Jie Xu4 (2016)

 

Similar results (1.19 folds higher than vehicle to pyruvate, 1.17 folds higher than vehicle to lactate, 86.2 % of vehicle to oxygen consumption, 87.3 % of vehicle to ATP production, 1.60 folds higher than vehicle to cyt c and 1.44 folds higher than vehicle to AIF in mitochondria of neurons/1.17 folds higher than vehicle to pyruvate, 1.15 folds higher than vehicle to lactate, 88.2 % of vehicle to oxygen consumption, 89.3 % of vehicle to ATP production, 1.58 folds higher than vehicle to cyt c and 1.40 folds higher than vehicle to AIF in mitochondria of wild types) were also seen in rotenone treatment groups (Fig. 4A2, A3, B2 and ​andB3).B3). The forskolin treatment showed opposite effects in mitochondria separated from cultured neurons and wild type mice (Fig. 4 A4, B4). Same dose of HU-210 and AM251 were also administrated to mitochondria separated from CB1^−/− mice. Results showed no changes were found in mitochondrial cAMP/PKA/complex activity suggesting CB1 receptors were the specific targets (Fig. 4c).

 

Statins, fibrates and retinoic acid upregulate mitochondrial acylcarnitine carrier gene expression.
Iacobazzi V1, Convertini P, Infantino V, Scarcia P, Todisco S, Palmieri F. (2009)

In this study, we investigated the effects of statins, fibrates, 9-cis-retinoic acid and forskolin on the transcription of the mitochondrial carnitine/acylcarnitine carrier (CAC) gene. Statins, fibrates, retinoic acid and forskolin activate luciferase gene reporter activity driven by the -334/+3 bp region of the human CAC promoter containing wild-type (but not mutated) PPRE. These four agents also increase CAC transcript and protein levels. The combinations of statins and fibrates, retinoic acid and fibrates and fibrates and forskolin act synergistically. Mevalonate abolishes the activation of CAC gene expression by statins; the inhibitor of the PKA pathway H89 suppresses the stimulation of CAC gene expression by forskolin. Because CAC is essential for fatty acid beta-oxidation, the above results on the regulation of CAC gene expression provide a novel contribution to the understanding of the hypolipidemic action of statins, fibrates and retinoic acid.

PQQ stimulates mitochondrial biogenesis through cAMP response element-binding protein phosphorylation and increased PGC-1alpha expression.
Chowanadisai W1, Bauerly KA, Tchaparian E, Wong A, Cortopassi GA, Rucker RB. (2010)

(couldn't find anything much on CoQ10)
Bioactive compounds reported to stimulate mitochondrial biogenesis are linked to many health benefits such increased longevity, improved energy utilization, and protection from reactive oxygen species. Previously studies have shown that mice and rats fed diets lacking in pyrroloquinoline quinone (PQQ) have reduced mitochondrial content. Therefore, we hypothesized that PQQ can induce mitochondrial biogenesis in mouse hepatocytes. Exposure of mouse Hepa1-6 cells to 10-30 microm PQQ for 24-48 h resulted in increased citrate synthase and cytochrome c oxidase activity, Mitotracker staining, mitochondrial DNA content, and cellular oxygen respiration. The induction of this process occurred through the activation of cAMP response element-binding protein (CREB) and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a pathway known to regulate mitochondrial biogenesis. PQQ exposure stimulated phosphorylation of CREB at serine 133, activated the promoter of PGC-1alpha, and increased PGC-1alpha mRNA and protein expression. PQQ did not stimulate mitochondrial biogenesis after small interfering RNA-mediated reduction in either PGC-1alpha or CREB expression. Consistent with activation of the PGC-1alpha pathway, PQQ increased nuclear respiratory factor activation (NRF-1 and NRF-2) and Tfam, TFB1M, and TFB2M mRNA expression. Moreover, PQQ protected cells from mitochondrial inhibition by rotenone, 3-nitropropionic acid, antimycin A, and sodium azide. The ability of PQQ to stimulate mitochondrial biogenesis accounts in part for action of this compound and suggests that PQQ may be beneficial in diseases associated with mitochondrial dysfunction.

Melatonin Improves mitochondrial function by promoting MT1/SIRT1/PGC-1 alpha-dependent mitochondrial biogenesis in cadmium-induced hepatotoxicity in vitro.
Guo P1, Pi H1, Xu S1, Zhang L1, Li Y2, Li M1, Cao Z1, Tian L1, Xie J1, Li R1, He M1, Lu Y1, Liu C1, Duan W1, Yu Z1, Zhou Z3. (2014)

Melatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, and it has been under clinical investigation for expanded applications. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. This study investigates the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. Consistent with this finding, Cd exposure was associated with decreased Sirtuin 1 (SIRT1) protein expression and activity, thus promoted acetylation of PGC-1 alpha, a key enzyme involved in mitochondrial biogenesis and function, although Cd did not disrupt the interaction between SIRT1 and PGC-1 alpha. However, all cadmium-induced mitochondrial oxidative injuries were efficiently attenuated by melatonin pretreatment. Moreover, Sirtinol and SIRT1 siRNA each blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT1/ PGC-1 alpha signaling. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to promote SIRT1/ PGC-1 alpha signaling. In summary, our results indicate that SIRT1 plays an essential role in the ability of moderate melatonin to stimulate PGC-1 alpha and improve mitochondrial biogenesis and function at least partially through melatonin receptors in cadmium-induced hepatotoxicity.

Melatonin promotes adipogenesis and mitochondrial biogenesis in 3T3-L1 preadipocytes.
Kato H1, Tanaka G1, Masuda S2, Ogasawara J3, Sakurai T3, Kizaki T3, Ohno H3, Izawa T1,4. (2015)

Melatonin is synthesized in the pineal gland, but elicits a wide range of physiological responses in peripheral target tissues. Recent advances suggest that melatonin controls adiposity, resulting in changes in body weight. The aim of this study was to investigate the effect of melatonin on adipogenesis and mitochondrial biogenesis in 3T3-L1 mouse embryo fibroblasts. Melatonin significantly increased the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a master regulator of adipogenesis, and promoted differentiation into adipocytes. Melatonin-treated cells also formed smaller lipid droplets and abundantly expressed several molecules associated with lipolysis, including adipose triglyceride lipase, perilipin, and comparative gene identification-58. Moreover, the hormone promoted biogenesis of mitochondria, as indicated by fluorescent staining, elevated the citrate synthase activity, and upregulated the expression of PPAR-γ coactivator 1 α, nuclear respiratory factor-1, and transcription factor A. The expression of uncoupling protein 1 was also observable both at mRNA and at protein level in melatonin-treated cells. Finally, adiponectin secretion and the expression of adiponectin receptors were enhanced. These results suggest that melatonin promotes adipogenesis, lipolysis, mitochondrial biogenesis, and adiponectin secretion. Thus, melatonin has potential as an anti-obesity agent that may reverse obesity-related disorders.

Korean red ginseng (Panax ginseng) improves insulin sensitivity and attenuates the development of diabetes in Otsuka Long-Evans Tokushima fatty rats.
Lee HJ1, Lee YH, Park SK, Kang ES, Kim HJ, Lee YC, Choi CS, Park SE, Ahn CW, Cha BS, Lee KW, Kim KS, Lim SK, Lee HC. (2009)

Ginseng has been reported to ameliorate hyperglycemia in experimental and clinical studies; however, its mechanism of action remains unclear. In this study, we investigated the metabolic effects and putative molecular mechanisms of Korean red ginseng (KRG, Panax ginseng) in animal models for type 2 diabetes mellitus (T2DM) and peripheral insulin-responsive cell lines. Korean red ginseng was administered orally at a dose of 200 mg/(kg d) to Otsuka Long-Evans Tokushima fatty rats for 40 weeks. Initially, chronic administration of KRG reduced weight gain and visceral fat mass in the early period without altering food intake. The KRG-treated Otsuka Long-Evans Tokushima fatty rats showed improved insulin sensitivity and significantly preserved glucose tolerance compared with untreated control animals up to 50 weeks of age, implying that KRG attenuated the development of overt diabetes. KRG promoted fatty acid oxidation by the activation of adenosine monophosphate-activated protein kinase (AMPK) and phosphorylation of acetyl-coenzyme A carboxylase in skeletal muscle and cultured C2C12 muscle cells. Increased expression of peroxisome proliferator-activated receptor-gamma coactivator-1alpha, nuclear respiratory factor-1, cytochrome c, cytochrome c oxidase-4, and glucose transporter 4 by KRG treatment indicates that activated AMPK also enhanced mitochondrial biogenesis and glucose utilization in skeletal muscle. Although these findings suggest that KRG is likely to have beneficial effects on the amelioration of insulin resistance and the prevention of T2DM through the activation of AMPK, further clinical studies are required to evaluate the use of KRG as a supplementary agent for T2DM.

Estrogenic control of mitochondrial function and biogenesis.
Klinge CM1. (2008)

Estrogens have cell-specific effects on a variety of physiological endpoints including regulation of mitochondrial biogenesis and activity. Estrogens regulate gene transcription by the classical genomic mechanism of binding to estrogen receptors alpha and beta (ERalpha and ERbeta) as well as the more recently described nongenomic pathways involving plasma membrane-associated ERs that activate intracellular protein kinase-mediated phosphorylation signaling cascades. Here I will review the rapid and longer-term effects of estrogen on mitochondrial function. The identification of ERalpha and ERbeta within mitochondria of various cells and tissues is discussed with a model of estrogen regulation of the transcription of nuclear respiratory factor-1 (NRF-1, NRF1). NRF-1 subsequently promotes transcription of mitochondrial transcription factor Tfam (mtDNA maintenance factor, also called mtTFA) and then Tfam targets mtDNA-encoded genes. The nuclear effects of estrogens on gene expression directly controlling mitochondrial biogenesis, oxygen consumption, mtDNA transcription, and apoptosis are reviewed. Overall, we are just beginning to evaluate the many direct and indirect effects of estrogens on mitochondrial activities.

Apigenin and quercetin ameliorate mitochondrial alterations by tunicamycin-induced ER stress in 3T3-L1 adipocytes.
Nisha VM1, Anusree SS, Priyanka A, Raghu KG. (2014)

Endoplasmic reticulum (ER) is an important organelle with functions like protein synthesis, folding, and calcium homeostasis. ER stress, a condition that dramatically affects protein folding homeostasis in cells, has been associated with a number of metabolic disorders. Emerging clinical and preclinical evidence support the notion that pharmacological modulators of ER stress have therapeutic potential as a novel target for treating metabolic diseases. ER is in physical contact with mitochondria, and there is a strong cross talk between these organelles at functional level. The present investigation was aimed to check the mitochondrial alterations in adipocytes with tunicamycin-induced ER stress and modulation by apigenin and quercetin. For this, differentiated adipocytes were incubated with tunicamycin (2 μg/ml) for 18 h, and changes in mitochondrial membrane potential, biogenesis, reactive oxygen species production, and adiponectin secretion were seen. Tunicamycin-induced ER stress altered reactive oxygen species (ROS) (6.34-fold↑), membrane potential (4.1-fold↑), mitochondrial biogenesis (2.4-fold↓), and adiponectin secretion (3.5-fold↓). Apigenin and quercetin ameliorated alterations in mitochondria. From results, we conclude that ER stress significantly alters mitochondrial functions and both the bioactives significantly protected mitochondrial alterations during ER stressand reestablished adiponectin secretion.

 

Defect of mitochondrial respiratory chain is a mechanism of ROS overproduction in a rat model of alcoholic liver disease: role of zinc deficiency.
Sun Q1, Zhong W2, Zhang W2, Zhou Z3. (2016)

Morphological and functional alterations of hepatic mitochondria have been documented in patients with alcoholic liver disease (ALD). Our recent study demonstrated that zinc level was decreased in whole liver and mitochondria by chronic alcohol feeding. The present study was undertaken to determine whether zinc deficiency mediates alcohol-induced mitochondrial electron transport chain (ETC) defect and whether defective ETC function may lead to generation of reactive oxygen species (ROS). Male Wistar rats were pair fed with the Lieber-DeCarli control or ethanol diet for 5 mo. Chronic alcohol exposure increased hepatic triglyceride, free fatty acid, and 4-hydroxynonenal (4HNE) levels; meanwhile hepatic mitochondrial 4HNE level was also increased. Moreover, hepatic mitochondrial respiratory complexes I, III, IV, and V and hepatic ATP production were decreased by chronic alcohol exposure. Chronic alcohol feeding decreased peroxisome proliferator-activated receptor gamma coactivator-1-alpha (PGC1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), and mitochondrial DNA. HepG2 cells were treated with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) for 6 h. Zinc deficiency significantly decreased mitochondrial respiratory complexes I, III, and IV. In addition, PGC1α, NRF1, and TFAM levels as well as mitochondrial DNA were significantly decreased by TPEN treatment. Knockdown of mitochondrial respiratory complexes I, III, or IV by shRNA caused a decrease in mitochondrial membrane potential and an increase in ROS production. These results suggest that alcohol-induced hepatic zinc deficiency could inactivate mitochondrial biogenesis pathway and decrease mitochondrial DNA replication, which, in turn, decreases mitochondrial complex protein expression. The defect of mitochondrial respiratory complexes may worsen alcohol-induced ROS production.

Lithium increases PGC-1alpha expression and mitochondrial biogenesis in primary bovine aortic endothelial cells.
Struewing IT1, Barnett CD, Tang T, Mao CD. (2007)

Lithium is a therapeutic agent commonly used to treat bipolar disorder and its beneficial effects are thought to be due to a combination of activation of the Wnt/beta-catenin pathway via inhibition of glycogen synthase kinase-3beta and depletion of the inositol pool via inhibition of the inositol monophosphatase-1. We demonstrated that lithium in primary endothelial cells induced an increase in mitochondrial mass leading to an increase in ATP production without any significant change in mitochondrial efficiency. This increase in mitochondrial mass was associated with an increase in the mRNA levels of mitochondrial biogenesis transcription factors: nuclear respiratory factor-1 and -2beta, as well as mitochondrial transcription factors A and B2, which lead to the coordinated upregulation of oxidative phosphorylation components encoded by either the nuclear or mitochondrial genome. These effects of lithium on mitochondrial biogenesis were independent of the inhibition of glycogen synthase kinase-3beta and independent of inositol depletion. Also, expression of the coactivator PGC-1alpha was increased, whereas expression of the coactivator PRC was not affected. Lithium treatment rapidly induced a decrease in activating Akt-Ser473 phosphorylation and inhibitory Forkhead box class O (FOXO1)-Thr24 phosphorylation, as well as an increase in activating c-AMP responsive element binding (CREB)-Ser133 phosphorylation, two mechanisms known to control PGC-1alpha expression. Together, our results show that lithium induces mitochondrial biogenesis via CREB/PGC-1alpha and FOXO1/PGC-1alpha cascades, which highlight the pleiotropic effects of lithium and reveal also novel beneficial effects via preservation of mitochondrial functions.

OM2, a Novel Oligomannuronate-Chromium(III) Complex, Promotes Mitochondrial Biogenesis and Lipid Metabolism in 3T3-L1 Adipocytes via the AMPK-PGC1α Pathway
Jiejie Hao (2016)

Background
In our previous studies, we prepared novel oligomannuronate-chromium(III) complexes (OM2, OM4) from marine alginate, and found that these compounds sensitize insulin action better than oligomannuronate(OM), chromium, and metformin in C2C12 skeletal muscle cells. In the present study, we studied their effects on mitochondrial biogenesis, lipid metabolism, and the underlying molecular mechanisms in differentiated 3T3-L1 adipocytes.

Methodology/Principal Findings
We firstly used the pGL3-PGC1α and pGL3-ATGL promoter plasmids to compare their effects on PGC1α and ATGL transcription activities. Then mitochondrial biogenesis was quantified by transmission electron microscopy and MitoTracker staining. Mitochondrial oxygen consumption and fatty acid oxidation were measured by an oxygen biosensor system and ³H-labelled water scintillation. The mitochondrial DNA and mRNA involved in mitochondrial biogenesis and lipid oxidation were evaluated by real-time PCR. AMPK together with other protein expression levels were measured by western blotting. The inhibitor compound C and siRNA of PGC1α were used to inhibit the OM2-induced AMPK-PGC1α signaling pathway. And we found that  OM2 stimulated  AMPK-PGC1α pathway in the 3T3-L1 adipocytes, which were correlated with induced mitochondrial biogenesis, improved mitochondrial function, and reduced lipid accumulation by enhanced fatty acid β-oxidation and augmented ATGL protein expression. 

Conclusions/Significance
Our data indicated that the marine oligosaccharide-derived OM2 might represent a novel class of molecules that could be useful for type 2 diabetes prevention and treatment by up-regulating AMPK-PGC1α signaling pathway.

[Adam1]

 

That was helpful and interesting. Indeed PINK1 and Parkin are critical in mediating fission and mitophagy and are worth enhancing. Further mix ‘n match candidates for the autophagy portion of the protocol (mostly from Nixon, Ralph A. "The role of autophagy in neurodegenerative disease." Nature medicine 19.8 (2013): 983-997.):

 

TORC1 inhibition – curcumin

AMPK activation – lithium, trehalose, quercetin, catechins, naringen, berberine, curcumin

PP2A activation – palm oil for tocopherols, melatonin, forskolin, epigallocatechine gallate

Other induction targets: parthenolide in feverfew, fisetin, luteolin

 

Many phytochemicals can have oxidant or antioxidant effects depending on factors such as dose and what they encounter. Some compounds may both induce and inhibit autophagy. For example, berberine is also an inhibitor (Hundeshagen, Phillip, et al. "Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy." BMC biology 9.1 (2011): 38.) It appears specific antioxidants can have a direct role in mitophagy (Yang, Lei, Xuping Wang, and Xiaolan Yang. "Possible antioxidant mechanism of melanoidins extract from Shanxi aged vinegar in mitophagy-dependent and mitophagy-independent pathways." Journal of agricultural and food chemistry 62.34 (2014): 8616-8622.)

 

Ideally the protocol would be pared down and individualized to contain just the rate-limiting steps of mitochondrial turnover through mitophagy, biogenesis, dynamics of fusion/fission, and could also address selective protein quality control. I am interested in learning about ways of implementing this.

 

Rescue of GABAB and GIRK function in the lateral habenula by protein phosphatase 2A inhibition ameliorates depression-like phenotypes in mice.
http://www.ncbi.nlm....pubmed/26808347

 

Seems to have pro-depressant properties on the other hand. Do You know accidentally a PP2A inhibitor ?

 

 

 

Flex, you asked re a PP2A inhibitor. There seem to be some caveats, e.g. with an extract used in TCM:

 

Inhibition of PP2A is considered to promote cancer development through the induction of phosphorylation and activation of several substrate kinases, including IκB kinase, c-Jun N-terminal kinase (JNK), extracellular signal-related kinase, p38, Akt and protein kinase C (PKC), the majority of which accelerate growth

It appears astragalus may have some PP2A inhibitory activity, at least with regards to the MIN6 cell line. However, I personally feel safer with a wide-spectrum multi-target approach.

[Fafner55]

Apparently Urolithin A is a one-stop molecule for eliminating unhealthy mitochondria and generating healthy ones.  Only tested in worms and mice so far.  See this thread: http://www.longecity...geing/?p=782588

 

Above only references the New Scientist write-up of the work from Auwerx' lab, but the original paper in Nature can be found below:

 

http://www.nature.co...ll/nm.4132.html

 

There is no ready source of urolithin A, but if one is lucky enough to have the right gut bacteria (Gordonibacter species) ellagic acid in pomegranate will be metabolized to urolithin A. 

 

The impressive results obtained with Urolithin A are a reminder of the importance of reducing the accumulation of dysfunctional mitochondria.

In vivo experiments with UA showed that induction of mitophagy resulted in

• A lifespan increase 45% in C. elegans (a nematode used as a model organism in labs)

• An average 42% greater running endurance in aged mice (24 months of age) and Wistar rats.

 

On the surface it might seem possible that ingesting a sufficient quantity of pomegranate extract could achieve the same purpose, but I found that to be impossible.

 

First, the two in vivo studies I found showed that 70 to 80% of the subjects had the right gut bacteria to metabolize pomegranate ellagitannins into UA.

• "Pomegranate Juice and Extracts Provide Similar Levels of Plasma and Urinary Ellagitannin Metabolites in Human Subjects" (2008) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3196216/
• “Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers” (2015) http://www.ncbi.nlm.nih.gov/pubmed/26189645 , http://www.vitafoods.eu.com/visiting/poster-presentations/poster-presentations/poster-pomx-pomegranate-extract-stimulates-growth-of-gut-bacteria-implications-for-prebiotic-and-metabolic-effects

While the types of gut bacteria responsible for production of UA are not definitively known, the species that show promise are not those found in common probiotics.

• “Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers” (2015) http://www.ncbi.nlm.nih.gov/pubmed/26189645 http://www.vitafoods.eu.com/visiting/poster-presentations/poster-presentations/poster-pomx-pomegranate-extract-stimulates-growth-of-gut-bacteria-implications-for-prebiotic-and-metabolic-effects
• “Description of urolithin production capacity from ellagic acid of two human intestinal Gordonibacter species” (2014) http://www.ncbi.nlm.nih.gov/pubmed/24909569
• “Gut Microbiota Conversion of Dietary Ellagic Acid into Bioactive Phytoceutical Urolithin-A Inhibits Heme Peroxidases” (2016) http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0156811

 

Then, to estimate the dose of pomegranate extracted needed, I started with

• For a 70 kg human, 4 mg/kg/day in mice converts to 280 mg UA
• Standardized pomegranate extract is said to contain 40% punicalagins
• Assumed pomegranate extract  metabolizes its punicalagin content into ellagic acid at an average rate of 25%, based on the wide range colonic fermentation https://examine.com/supplements/punicalagins/
• Guessed that 10 to 20% of the ellagic acid might be metabolized into UA

These assumptions give an estimate of pomegranate extract needed = 5.6  to 11.2 gm.

 

I started self experiments with 2 gm pomegranate extract twice per day dissolved in water.  It tasted awful. Coffee worked better.  After 4 or 5 days I had a fever and a painful case of uncomplicated diverticulitis, probably caused by small particles of pomegranate skin, etc in the extract. Antibiotic treatment and a week's rest was needed to recover.

 

This experiment was a failure.  The dose of pomegranate extract that I consumed does not appear sufficient to induce mitophagy but can cause diverticulitis.  It does not seem possible to consume enough pomegranate extract to be effective without causing severe gastric distress.

[Fafner55]

Estimated Dose of NR Needed to Induce Mitophagy

• "Nicotinamide enhances mitochondria quality through autophagy activation in human cells" (2009) http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2009.00487.x/full

• “Novel NAD+ metabolomic technologies and their applications to Nicotinamide Riboside interventions” (2016)  http://ir.uiowa.edu/etd/3203/  (Ph.D. thesis)

• “GENERALLY RECOGNIZED AS SAFE (GRAS) DETERMINATION FOR NIAGEN ™ (NICOTINAMIDE RIBOSIDE CHLORIDE)“ (2015-12) http://www.fda.gov/downloads/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm505226.pdf

 Decrease of mitochondrial mass in the NAM-treated human fibroblasts.jpg   36.56KB   11 downloads

 

The following data points and facts are known.

• Keeping NAD+ elevated by 50% for 5 to 7 days significantly decreases mtDNA mass by 50% (in vitro)  "Nicotinamide enhances mitochondria quality through autophagy activation in human cells" (2009) http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2009.00487.x/full

• 1000 mg/day (15 mg/kg) in a 52 year old (n=1) increased (peak, after 8 hours) plasma concentrations of

  • NAD+ by 317%

  • NAM by 75% (from 0.8 to 1.4 µM)  

• Dose responses of NR chloride (in vivo, n=12) increased

  • NAM by ≈ 62% for 300 mg NR

  • NAM by ≈ 140% for 1000 mg NR

• Predose plasma concentration of NAM in the n=12 experiments ranged from 0.2 to 0.55 µM, and from 0.7 to 0.8 µM  in the 52 year old, indicating a wide range.

• NAM levels rose to a range of 0.6 to 0.85 µM for 1000 mg NR in the n=12 experiment.

• NR increases NAD+ in a dose-dependent manner.

• NR has a serum half life of about 4 hours.

 

From these data points and other facts I can surmise that

• My normal dose of NR at 250 mg twice per day with 50 mg pterostilbene should be sufficient to maintain a generally healthy population of mitochondria.

• While NR induces mitophagy, it has not shown in vivo results as impressive as Urolithin-A, which implies that additional mitophagy might be possible beyond those shown with 300 to 1000 mg doses of NR.

• 500 mg every 6 hours for 5 days (2 gm/day) might further increase mitophagy and cause a substantial clearance of defective mitochondria.

 

Experiment #1

Hypothesis: 500 mg (about 7.5 mg/kg) every 6 hours (2 gm/day) for 6 days might induce mitophagy and cause substantial clearance of defective mitochondria.

 

Test: NR is well tolerated at 1 gm/day (clinical trial), and at 2.5 gm/day (anecdotal) and showed no adverse conditions in mice at 400 mg/kg/day (≈32 gm/day HED, FDA submission).  Since it appears safe, and since there are no in vivo experiments on inducing mitophagy with NR, taking about 4 gm/day should give clear results if this treatment works.

 

NR is packaged in 125 mg capsules.  Taking 10 capsules (1250 mg) three times per day is a total daily dose of 3750 mg.  This amount is nearly twice the 2000 mg/day does I estimate from in vitro data that is needed to induce mitophagy.  In the absence of in vivo data, along with general safety of NR, this higher dose appears warranted, particularly since treatment with Urolithin A showed a significant 42% increase in running endurance in aged rats, which is a better performance increase than I have seen reported for NR and suggests that greater levels of mitophagy are possible.

 

2016-09-28  Day 1. Begin taking 10 capsules (1250 mg) three times per day. I have not taken C60-OO for a month or, for 10 days, any supplements or medications .

2016-09-29  Day 2 AM: My skin is definitely smoother and I have more energy, the kind of energy and sense of health and well being one feels in youth.  Otherwise there are no apparent side effects.  Smoother skin is not something I anticipated, but it is consistent with research showing cells returning to normal shape when (aged or dysfunctional) mitochondria is removed.  

“Mitochondria shown to trigger cell ageing” (2016) http://www.ncl.ac.uk/press/news/2016/02/mitochondriashowntotriggercellageing/, http://emboj.embopress.org/content/35/7/724

Late afternoon - my face has the feeling of being slightly flushed, as if I were taking a niacin.

2016-09-30   Day 3. Woke up with a headache, otherwise OK.  The headache could have been caused by dehydration.  It went away after drinking water and a cup of coffee. My muscles feel firmer and stronger: I am slouching less when sitting.  This sensation is probably related to increased ATP production and other benefits of NR, and not related to mitophagy.

2016-10-01  Day 4. The ambient temperature is 76° and I feel slightly cold.  Less body heat generation is consistent with reduced mitochondrial mass.  I feel no other symptoms.

2016-10-02  Day 5. Today is the fifth of this treatment.  I am unusually clear minded.  As for other symptoms, there is a slight feeling of niacin-like flushing when I take NR, otherwise nothing.  Since in vitro experiments should additional decrease in mitochondrial mass through 7 days of treatment, I plan to continue taking NR for one or two more days.  

“Nicotinamide enhances mitochondria quality through autophagy activation in human cells" (2009) http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2009.00487.x/full  

2016-10-03  Day 6 and final day of this treatment.  My muscles feel small, inadequate and ache, which is consistent with significant decrease in mitochondria.  Otherwise I feel OK. General autophagy does not seem to be affected, as lipofuscin deposits on my hands remain the same.  Another observation is that my sense of smell has been unusually acute for the last several days.  My mitochondrial mass should be reduced by about 30% by the end of today if in vitro measurement results are an accurate indication.

2016-10-04  Day 7.  Took 10 mg PQQ about 7 AM, which should stimulate mitogenesis and restore my mass of mitochondria back to normal within hours.  Noontime: Other than muscle aches, I feel good, energetic and healthy.

2016-10-04  Day 8.  I feel  completely normal.

2016-10-05  Day 9.  I feel energetic.

2016-10-06  Day 10.   I feel even more energetic and my skin is noticeably smoother on my arms and face.  This treatment is unequivocally beneficial.  

 

 

Other thoughts

1. mtDNA 4977 bp deletion has been shown to increase at and average rate (in years) t ≈ 0.0002 exp(0.042 t)   “Mitochondrial DNA 4977 bp deletion is a common phenomenon in hair and increases with age” (2012)  http://www.ncbi.nlm....les/PMC4362429/

2. Measurements of averaged accumulation of dysfunctional mitochondria bely much higher concentrations in aged and postmitotic cells.

3. If in vitro tests are any indication, the experiment I described above should have reduced my mitochondria mass by an estimated 30%

4. Since dysfunctional mitochondria are preferentially recycled, as 30% reduction in mass might correspond to a 60% reduction in the mass of dysfunctional mitochondria (just a guess, assuming twice rate).

5. Then 3 treatments should reduced dysfunctional mitochondria by 65%, or by about 20 years average accumulation.  6 treatments would reduce dysfunctional mitochondria by about 89%, equal to about 50 years average accumulation.  

I am 61 years old.

[niner]

Fafner55, thanks for the pomegranate extract report.  Although I'm not certain that a Human Equivalent Dose calculation is called for in this particular case, it probably is.  If that's the case, then the HED for a mouse getting 4mg/kg/d in a 70kg human is 280/12 = 23 mg UA.  Using the same assumptions as you have above, that would translate to about one half to one gram per day, which is a lot easier to stomach.   I think it's unlikely that the pomegranate extract caused your diverticulitis.  I think it's more likely that it was coincidental, although you did take a lot of extract, so it's not like we can rule it in or out as a cause. 

 

If you did have a higher level of mitophagy, how would you know?  A sudden change in apparent cardiovascular fitness?  What sort of time course would one expect in such an improvement?

 

ps:  Just saw the NR post.  Very interesting!  We need better ways to quantify the effects.

Edited by niner, 12 October 2016 - 12:49 AM.