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Mechanism by which a High Protein Diet Worsens Heart Disease

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I found this new mouse study [1] to be fascinating. It found feeding mutant mice (genetically prone to forming artherosclerotic plaques like humans) a diet high in fat and protein, especially high in the amino acid leucine (found most abundantly in meat, eggs and dairy) resulted in 30% more unstable plaque formation in the mice's arteries than a diet with the same (large) amount of fat but normal in protein (i.e. with more simple carbs in place of protein).

What was most fascinating was the study's level of detail and specificity regarding the causal pathway.

Here is the visual abstract and summary of the mechanism:

Ingestion and digestion of dietary protein first lead to an acute rise in blood amino acid levels and in turn tissue amino acid levels (including the atherosclerotic plaque). On exposure to rising amino acid levels, mTORC1 is activated in plaque macrophages. A critical downstream effect of activated mTORC1 is inhibition of mitophagy. The resultant build-up of dysfunctional mitochondria triggers the intrinsic apoptosis pathway. Enhanced apoptosis of plaque macrophages contributes to necrotic core formation and a rise in plaque complexity (a surrogate of the vulnerable plaques).

Screenshot_20200204-165014_Foxit PDF.jpg

In other words and in a little more detail, the high fat in the diet triggers macrophage cells in the bloodstream to try to absorb droplets of fat, especially oxidized fat. This leads to "toxic lipid intermediates" inside the macrophage cells that end up damaging the macrophage's mitochondria. Normally, these damaged mitochondria would be detected and cleared out through the process called mitophagy. But in the presence of a high concentration of amino acids (esp. leucine), part of the MTOR complex (MTORC1) inside the macrophage cell is activated in such a way as to promote cell growth while at the same time inhibiting mitophagy.  As a result, damaged mitochondria build up inside the macrophage cell. A high concentration of dysfunctional mitochondria in turn triggers apoptosis of the macrophage cell, effectively killing it off.

Dead macrophage cells overloaded with lipids are a fundamental component of unstable artherosclerotic plaques embedded in artery walls that ultimately are what typically triggers a heart attack. So this study helps elucidate exactly how a diet high in fat and protein increases heart attack risk.



[1] Nature Metabolism 2, 110–125 (2020). https://doi.org/10.1038/s42255-019-0162-4

High-protein diets increase cardiovascular risk by activating macrophage mTOR to suppress mitophagy

Xiangyu Zhang1,2, Ismail Sergin1, Trent D. Evans1, Se-Jin Jeong1,2, Astrid Rodriguez-Velez1,
Divya Kapoor1,2, Sunny Chen1, Eric Song1, Karyn B. Holloway1,2, Jan R. Crowley3, Slava Epelman   4,
Conrad C. Weihl5, Abhinav Diwan   1,2, Daping Fan6, Bettina Mittendorfer7, Nathan O. Stitziel1,
Joel D. Schilling1,8, Irfan J. Lodhi3 and Babak Razani   1,7,8*

High-protein diets are commonly utilized for weight loss, yet they have been reported to raise cardiovascular risk. The mechanisms underlying this risk are unknown. Here, we show that dietary protein drives atherosclerosis and lesion complexity.
Protein ingestion acutely elevates amino acid levels in blood and atherosclerotic plaques, stimulating macrophage mammalian target of rapamycin (mTOR) signalling. This is causal in plaque progression, because the effects of dietary protein are
abrogated in macrophage-specific Raptor-null mice. Mechanistically, we find amino acids exacerbate macrophage apoptosis
induced by atherogenic lipids, a process that involves mammalian target of rapamycin complex 1 (mTORC1)-dependent inhibition of mitochondrial autophagy (mitophagy), accumulation of dysfunctional mitochondria and mitochondrial apoptosis. Using
macrophage-specific mTORC1- and autophagy-deficient mice, we confirm this amino acid–mTORC1–autophagy signalling axis
in vivo. Our data provide insights into the deleterious impact of excessive protein ingestion on macrophages and atherosclerotic
progression. Incorporation of these concepts in clinical studies is important to define the vascular effects of protein-based
weight loss regimens.

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17 hours ago, Dean Pomerleau said:

But in the presence of a high concentration of amino acids (esp. leucine), part of the MTOR complex (MTORC1) inside the macrophage cell is activated in such a way as to promote cell growth while at the same time inhibiting mitophagy. 

Hi Dean!

Very nice find.  

I'm a little confused though:  Why does the article single out leucine?  ("esp. leucine").   I understand the special significance of methionine (it is the first amino acid used in anabolic processes) -- but what's special about leucine?

  --  Saul

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Good article. Here is something else which supports its conclusions (I am not sure if it has been posted elsewhere here):

"we have now found that altered dietary quality – the precise amino acid composition of the diet – regulates metabolic health. Specifically reducing the three branched chain amino acids (leucine, isoleucine, and valine) to the same level as found in a low protein diet is sufficient to improve many aspects of metabolic health, including glucose tolerance and body composition, as effectively as a 2/3rds reduction in total consumption of dietary amino acids."


At first glance, I am not sure how to reconcile this with the methionine restriction studies.

Also, it seems as if the definition of "low" needs to be clarified. With methionine I see studies which consider intake of anything lower than 1.3g per kg to be "low." I am not sure what "low" means for leucine.

Edited by Ron Put
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Thanks for the Sabatini videos. Fascinating stuff. 

Saul, as Mechanism indicates, Sabatini's lab has found that the level of several amino acids are involved in regulating mTORC1. Here is a screen capture from the first Sabatini video that indicates three amino acids, leucine, arginine and lysine are involved in turning on mTORC1:


In addition, it's been found that the other amino acid you mention, methionine (met) activates mTORC1, as shown in the following diagram. Notice that binding sites by which leucine (leu) and arginine (arg) influence mTORC1 activity are also shown in this diagram:


You'll also notice that the sequence of steps by which these amino acids have their influence on mTORC1 are incredibly long and complicated.

But all of these amino acids seem to upregulate mTORC1, putting the cell into an anabolic state. Unfortunately, this anabolic state includes suppressing the breakdown of damaged mitochondria (mitophagy) in macrophages. The accumulation of these damaged mitochondria ultimately appears to cause suicide (apoptosis) of lipid-filled macrophages embedded in artery walls, contributing to the buildup of unstable plaques that reduce blood flow and which can eventually rupture to cause an artery blockage which starves the heart of oxygen (i.e. a heart attack).

I've heard the analogy that macrophages are like firefighters who rush to the scene of a fire, get incapacitated by smoke inhalation, thereby becoming part of the problem rather than part of the solution.

The new paper that started this thread seems to show that the reason the macrophages become incapacitated is that the normal catabolic processes that perform cleanup to keep the macrophages healthy are inhibited by mTORC1 activation, triggered by excess circulation amino acids from a high protein diet. 


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5 hours ago, Saul said:

...Animal protein is almost always higher in methionine than vegetable. Is the same true for the branched amino acids?

Yes, animal protein appears to generally beat plant protein in BCAAs, unless one eats pumpkin seeds by the pound:

"Amount of BCAAs in prepared foods:

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2 hours ago, mccoy said:

LOL, but I think you can use it post-workout without big detriment (Most of its leucine will be sequestered by muscle cells)

I agree and as with anything - the key is 'moderation' - using a couple Tablespoons per day of whey (although whey is a high concentration of protein and BCAAs) when added to a plant-based diet or lacto-ovo vegetarian diet I do not believe falls into the category of a 'high protein' diet; especially when muscles are starving for some AA's from resistance training.

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I am still curious though, has anyone come across some sort of a standard definition of what constitutes a diet "low in methionine" or "low in BCAAs" as it applies to humans?

For methionine, I remember seeing a human study which deemed "low" to be anything 1.3g per kg or less.

But I haven't seen anything defining what "low" means for BCAAs. I can't even find what is the average intake based on SAD, or any other population.

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Mechanism, thank you for the great post! It really helps sort it all out in one place and make better sense of it all.

I am not sure I have any hope of getting my protein intake down to the levels discussed, while still getting a balanced diet. Here is a snapshot of my protein intake over the last three months:


Virtually all of my protein is from plant sources (I eat cheese very rarely) and mostly from flax, nuts and seeds, legumes and cacao nibs.

But, I average close to 70g of fiber daily, so I wonder if having a diet high in fiber reduces the absorption of plant protein, which I believe is a little less bioavailable than animal protein anyway?

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Thanks, Mechanism! Macadamia nuts are on order, next to my walnuts, almonds and Brazil nuts (one a day)

I like MyFoodData, the top item for vegan foods lowest in methionine is Beer! :)

I just ran across this, which among other things discusses median protein and specifically, leucine, intake:

Knowledge Gained from Studies of Leucine Consumption in Animals and Humans
"Food intake surveys underestimate nutrient intakes because of underreporting and my analysis of the UK adult National Diet and Nutrition Survey (8), trimmed of under-reporters (i.e., energy intakes <1.35 × predicted basal metabolic rate), indicates median and 90th percentile intake values for protein of 1.25 and 1.61 g · kg−1 · d−1(14.2 and 17.3% energy) and 108 and 138 mg/kg leucine at 8.3% (9) of the protein intake. Modeling the more recent UK National Diet and Nutrition Survey protein intakes of 17.6% food energy (10) for a physically active 70-kg young man (physical activity level = 2.2) indicates a protein intake of 2.41 g · kg−1 · d−1 or 200 mg · kg−1 · d−1of leucine. Our studies of 90-kg body builders (11) indicated protein intakes up to 3.05 g · kg−1 · d−1(28% protein calories) of a mainly animal protein diet [8.6% leucine (9)]; i.e., a food leucine intake of 262 mg · kg−1 · d−1(∼7 × the recommended dietary allowance). Although little is known about the long-term health impact of these high-protein intakes, it can be assumed that such intakes are widespread."

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  • 4 months later...
On 2/8/2020 at 8:29 PM, Mechanism said:

A potential counter-argument regarding attainability of SAAR in humans is that at least one study found that mere 40% MetR - something more realistically attained -"decreases heart mitochondrial ROS production at complex I during forward electron flow, lowers oxidative damage to mitochondrial DNA and proteins, and decreases the degree of methylation of genomic DNA." ( that's from Gusavo Barja's work:  https://link.springer.com/article/10.1007/s10863-011-9389-9 ). 

However it should be kept in minds that these are merely biomarkers for health and longevity and thus do not necessarily demonstrate life extension per se.

If anyone is interested,  I found the full text of the 2011 Barja article on 40% MetR (in rats) here:

Forty percent methionine restriction lowers DNA methylation, complex I ROS generation, and oxidative damage to mtDNA and mitochondrial proteins in rat heart

A few excerpts:



Methionine dietary restriction (MetR), like dietary restriction (DR), increases rodent maximum longevity. However, the mechanism responsible for the retardation of aging with MetR is still not entirely known. As DR decreases oxidative damage and mitochondrial free radical production, it is plausible to hypothesize that a decrease in oxidative stress is the mechanism for longevity extension with MetR.

 In the present investigation male Wistar rats were subjected to isocaloric 40% MetR during 7 weeks. It was found that 40% MetR decreases heart mitochondrial ROS production at complex I during forward electron flow, lowers oxidative damage to mitochondrial DNA and proteins, and decreases the degree of methylation of genomic DNA. No significant changes occurred for mitochondrial oxygen consumption, the amounts of the four respiratory complexes (I to IV), and the mitochondrial protein apoptosis-inducing factor (AIF).

These results indicate that methionine can be the dietary factor responsible for the decrease in mitochondrial ROS generation and oxidative stress, and likely for part of the increase in longevity, that takes place during DR. They also highlight some of the mechanisms involved in the generation of these beneficial effects.



The magnitude of the longevity extension effect of protein restriction in rodents is up to 50% that of DR. Concerning particular amino acids, methionine restriction (MetR) without restriction of calories increases maximum longevity in rats and mice (Richie et al. 1994; Miller et al. 2005; Sun et al. 2009).

The beneficial effects of MetR in rodents also include decreases in visceral fat, triglycerides, cholesterol, glucose, insulin and IGF1 (Malloy et al. 2006), slowing of cataract development, protection against age-related changes in immunity (Miller et al. 2005), improvements in colon tight junction barrier function (Ramaligan et al. 2010), enhanced metabolic flexibility (Hasek et al. 2010), and lowering of the incidence of cancer (Komninou et al. 2006).

It has been also shown that overexpression of methionine sulfoxide reductase increases animal longevity (Chung et al. 2010).  On the other hand, excessive intake of dietary methionine has toxic effects including increases in oxidative stress (Park et al. 2008; Yalcinkaya et al. 2009; Song et al. 2009; Gomez et al. 2009).Concerning the possible mechanisms involved, previous studies have shown that DR in rodents invariably decreases mitochondrial reactive oxygen species (mitROS) generation and oxidative damage to mitochondrial DNA (mtDNA) and proteins (reviewed in Gredilla and Barja 2005).

This is interesting since long-lived mammals have lower mitROS production and fatty acid unsaturation than short-lived ones (see Pamplona and Barja 2007 for review). Previous studies have shown that protein but not carbohydrate or lipid restriction also decrease mitROS production and oxidative stress (Lopez-Torres and Barja 2008) and the same occurs in the heart after restriction of a single amino acid, methionine (Sanz et al. 2006). Furthermore, when all the other dietary aminoacids except methionine are restricted the benefits in oxidative stress are no longer present (Caro et al. 2009a).

All this suggests that methionine is the dietary factor responsible for the decrease in mitROS generation and oxidative stress in DR. However, the previous studies performed in rat heart (Sanz et al. 2006) were performed at 80% MetR whereas classic DR studies are performed at a level of 40% food restriction.

To be able to attribute to dietary methionine the beneficial effects of standard DR (40%) on oxidative stress, the level of implementation of MetR must be also 40%, not 80%.

Furthermore, the use of 40% MetR also avoids the decrease in growth rate, maturation, and final body size that occurs in 80% MetR and in 40% DR. Those decreases can complicate the interpretation of the results obtained.



In this investigation it is shown for the first time that 7 weeks of 40% MetR significantly lowers mitROS production at complexI during forward electron flow, oxidative damage to mitochondrial DNA, oxidation, glycoxidation and lipoxidation of mitochondrial proteins as well as the methylation level of genomic DNA in the rat heart.  Previous studies have shown that the rate of mitROS generation is lowered at complex I both in long-lived species and in rodents subjected to 40% DR (Barja 2004; Gredilla and Barja 2005) as well as in 80% MtR in rat heart (Sanz et al. 2006).

In this investigation it is shown that 40% MetR is enough to decrease mitROS production in rat heart. Therefore, MetR can be responsible for the decrease in mitROS production and ensuing oxidative damage that occurs in the heart of rats subjected to (40%) DR which can be involved in their longevity extension.

The decrease in mitROS generation after 40% MetR observed in our study took place exclusively at complex I since it occurred only with pyruvate/malate + rotenone, a condition in which the free radical source is confined to complex I. This conclusion is also consistent with the lack of significant changes in mitROS generation in the different assays performed with succinate (a complex II-linked substrate).

The lack of changes with succinate alone indicates that the decrease in mitROS production does not occur during reverse electron flow from complex II to complex I. Instead, it must occur at a free radical generation site working during forward electron flow and located within complex I between the one receiving electrons from NADH and the rotenone inhibition site. This kind of result is consistent with a previous study in other rat tissues (Caro et al. 2009b).



Concerning the cellular mechanism responsible for the qualitative change leading to mitochondria with a lower rate of mitROS generation in MetR, a logical possibility is that changes in DNA methylation are involved.

 Many microarray studies have shown that the longevity extension induced by dietary restriction in rodents is dependent on medium- or long-term changes in gene expression (Park and Prolla 2005). This has not been extensively studied in MetR, although a recent study found changes in various messenger RNAs and some protein signaling molecules in the liver of mice subjected to this dietary manipulation (Sun et al. 2009). Dietary methionine is essential for synthesis of

S-adenosylmethionine that provides methyl groups required for DNA methylation, an important mechanism of gene expression modification. In fact, we have recently showed that lower levels of methionine supplementation in the diet lead to a lower concentration of S-adenosylmethionine (SAM) in the heart of Wistar rats (Gomez et al. 2009). Therefore, a lower level of global methylation of heart genomic DNA was expected in MetR as it was found indeed in this investigation. Thus, it is possible that the qualitative change leading to a lower mitROS production in DR is secondary to changes in gene expression induced by the decrease in DNA methylation observed in the heart genomic DNA, although this does not eliminate the possible additional involvement of other molecular mechanisms.




[…]qualitative rather than quantitative changes in complex I seem to be involved in the decrease in mitROS generation in 40% MetR. As previously suggested for DR models (Gredilla et al. 2001) this qualitative change can be due to a decrease in the steadystate degree of electronic reduction of the complex I generator which would decrease its tendency to univalently reduce oxygen to the superoxide radical.

On the other hand, we observed that the amount of the superoxide dismutase mitochondrial form (MnSOD), the enzyme that scavenges superoxide radical, is not changed by 40% MetR in the rat heart. This is consistent with the idea that the decrease in superoxide radical release from the mitochondria observed in this investigation is due to a true decrease in superoxide radical production and not to an increase in its elimination by MnSOD.



All these results, together with previous ones (Sanz et al. 2004; Caro et al. 2009a) are consistent with the hypothesis that the decreased ingestion of methionine is responsible for the decrease in mitROS generation and oxidative damage to mtDNA that occurs during (40%) DR and protein restriction.

Since ROS can produce DNA single- and double-strand breaks in addition to oxidizing the bases, MetR could contribute to extend lifespan by helping to decrease the formation of mtDNA mutations that occur during rodent and human aging (Barja 2004; Khrapko and Vijg 2007).

In addition to lowering 8- oxodG, in our study a global decrease in oxidative, lipoxidative, and glycoxidative damage to heart mitochondrial proteins was also found. The stronger decrement observed was that of the lipoxidation marker MDAL (58% decrease) whereas the other protein modification markers showed 19%–35% decreases.

This higher sensitivity of MDAL compared to the other four protein modification markers has been also observed in various previous studies of DR and MetR, possibly indicating the prominent role of the decrease in MDA-related lipid peroxidation pathways in these anti-aging models.

On the other hand, the decrease in the two lipoperoxidation-related markers CML and MDAL was not due to decreases in the degree of unsaturation and thus in the sensitivity to oxidative damage of heart mitochondrial fatty acids, because neither the total number of double bonds (DBI) nor the PI were changed by 40% MetR.

Instead, the lower mitROS production of MetR animals seems to directly decrease protein oxidation (lower GSA and AASA) and to also lower glycoxidation and lipoxidation processes finally contributing to a decrease glycoxidative (lower CEL and CML) and lipoxidative (lower CML and MDAL) modification of rat heart mitochondrial proteins.

 Finally, it is known that two traits correlate with maximum longevity across animal species. Long-lived mammals and birds have a low rate of mitROS production and a low degree of fatty acid unsaturation (low DBI), which lower their oxidative stress (Pamplona and Barja 2007). In many previous investigations (40%) DR, a longevity-extending manipulation, lowered mitROS production without changing tissue DBI. We have found that 80% MetR decreases rat heart DBI (Sanz et al. 2006), but when we applied MetR at 40% in the present investigation no changes in this parameter were observed. This kind of result has been also observed in other rat organs (Caro et al. 2008, 2009b).

Therefore, the lack of change in DBI at 40%MetR fits well with the lack of effect of (40%) DR on the global degree of fatty acid unsaturation. Decreases in DBI in MetR are limited to 80% MetR, a dietary modification not occurring in (40%) DR. Therefore, among the two main oxidative stress-related traits of long-lived animals, only a low rate of mitROS production (and not a low degree of fatty acid unsaturation) seems to be involved in the life extension effect of MetR and DR.

Michael Rae's critique (2017) can be found here.


...hopes were raised a few years back by studies by mitochondrial biologist Gustavo Barja and colleagues, who reported that a much more modest 40% MetR lowered the production of ROS from mitochondria extracted from the kidneys and brains of rats, similar to 40% CR.(9) But of course, while reducing mtROS is likely one important mediator of CR, it's not the whole story, and tweaking one mechanism of aging alone doesn't ultimately impact the overall degenerative aging process (as discussed in more detail here). The way you most reliably test to see if an intervention is actually impacting the whole-organism degenerative aging process is by seeing if it impacts the one thing that integrates the effects of all aging processes: maximum lifespan, which is limited precisely by the aging process as opposed to selective vulnerability to individual causes of death. As far as I can see, no proper study of moderate MetR has yet been done. However, what evidence is available does suggest that it doesn't work.  [ETC.]

See also:

Towards a Unified Mechanistic Theory of Aging (Barja, 2019)



A large amount of the longevity-modulating genes discovered during the last two decades are highly conserved during evolution from yeast and invertebrates to mammals. Many different kinds of evidence converge in the concept that life extending manipulations like the dietary restrictions or rapamycin signal the nucleus specifically changing gene expression to increase longevity. The response of the cell aging regulation system is to change the level of activity of many different aging effectors to modulate longevity.

Aging effectors include mitROS production, lipid unsaturation, autophagy, mitochondrial DNA repair and possibly others like apoptosis, proteostasis, or telomere shortening, corresponding to different classic theories of aging. The constitutive spontaneous activity of this aging regulating system, likely including epigenetics, can also explain species longevity.

The aging regulating system reconciles the previously considered independent theories of aging bringing them together into a single unified theory of aging.


The integrated CARS [ Cell Aging Regulation System] is composed of three main parts (Fig. 2):
A) Cytoplasmic Pre-nuclear Signaling (mostly signaling proteins)
B) The nuclear genetic Aging Program (AP)
C) Post-nuclear Aging Effectors (executors of aging).


Fig. 2. Cell Aging Regulation System (CARS). The CARS model broadly integrates known mechanisms of cellular longevity control.

Different kinds of dietary restrictions (DR, MetR and protein restriction) and rapamycin, signals coming from the environment, alter humoral, endocrine, and finally cytosolic signalingproteins like mTOR, AMPK, PI3K, AKT, and many others (part A, left of figure) whose effects are mediated in many cases through TFs like FOXOs, TEFB and othersthat regulate expression of nuclear AP genes (part B center).

The AP output (solid arrows leaving the nucleus on the right of the figure), in turn, modifies the activity  of the multiple Aging Effectors (Part C, right) including: (a) ROSp in mitochondria, b) fatty acid double bonds (measured as double bond index, DBI) that stimulate lipid peroxidation, c) Autophagy, and d) likely others like apoptosis, inflammaging, proteostasis or telomere attrition. There is emerging evidence that that epigeneticsis also involved in CARS action.

The integrated response of the CARS to environmental signals modulates the intra-specific aging rate. An overlapping AP with additional components and wider output activity, composed only of parts B+C in the scheme, could also determine inter-species longevity. In some cases environmental signals can also directly reach aging effectors bypassing the nucleus, as shown for DR and mitochondrial NADH/NAD+. TFs=Transcription factors.  *=telomere shortening will mainly occur in mitotic tissues.

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Dietary sulfur amino acid restriction upregulates DICER to confer beneficial effects

Mol Metab. 2019 Nov; 29: 124–135. Published online 2019 Aug 28. doi: 10.1016/j.molmet.2019.08.017 PMCID: PMC6745493 PMID: 31668384





Dietary restriction (DR) improves health and prolongs lifespan in part by upregulating type III endoribonuclease DICER in adipose tissue. In this study, we aimed to specifically test which missing dietary component was responsible for DICER upregulation.


We performed a nutrient screen in mouse preadipocytes and validated the results in vivo using different kinds of dietary interventions in wild type or genetically modified mice and worms, also testing the requirement of DICER on the effects of the diets.


We found that sulfur amino acid restriction (i.e., methionine or cysteine) is sufficient to increase Dicer mRNA expression in preadipocytes. Consistently, while DR increases DICER expression in adipose tissue of mice, this effect is blunted by supplementation of the diet with methionine, cysteine, or casein, but not with a lipid or carbohydrate source. Accordingly, dietary methionine or protein restriction mirrors the effects of DR. These changes are associated with alterations in serum adiponectin.

We also found that DICER controls and is controlled by adiponectin. In mice, DICER plays a role in methionine restriction-induced upregulation of Ucp1 in adipose tissue. In C. elegans, DR and a model of methionine restriction also promote DICER expression in the intestine (an analog of the adipose tissue) and prolong lifespan in a DICER-dependent manner.


We propose an evolutionary conserved mechanism in which dietary sulfur amino acid restriction upregulates DICER levels in adipose tissue leading to beneficial health effects.



4. Discussion

DICER is a key enzyme required for the expression and function of several small RNA species, among them miRNAs [41]. Over the past few years, our group and others have demonstrated that DICER abundance dynamically changes in adipose tissue in response to metabolic stimuli of different sorts. For example, DICER is downregulated in fat of lipodystrophic patients [12] and in animal models of aging [10], progeria [42], and obesity [42].

These changes in DICER levels reflect the pool of miRNAs in adipocytes [10] and, importantly, markedly affect the levels of miRNAs in circulation [43].

Consistent with the conditions where DICER appear downregulated, adipocyte-specific Dicer knockout (ADicerKO) mice exhibit “whitening” of the brown adipose tissue [12] and a higher risk of premature mortality [12].

On the other hand, interventions that are metabolically beneficial and lead to increased lifespan, such as DR, upregulate DICER in adipose tissue [10]. Interestingly, this upregulation occurs rapidly (within half a week of a 10% DR protocol), anticipates the metabolic effects of DR, and is sustained for as long as DR is maintained [13].

This led us to hypothesize that these dynamic changes in adipose tissue DICER abundance are required for the beneficial effects of DR, in part by ensuring the capacity of adipocytes to express miRNAs under conditions of metabolic stress.

Indeed, ADicerKO mice are unable to increase mitochondrial content in fat and improve insulin sensitivity at the whole-body level in response to DR [13]. Similarly, our studies in C. elegans demonstrate that upregulation of dcr-1 in the intestine of worms is sufficient to improve oxidative stress response [10] and necessary for longevity induced by DR and MR (this study).

While the benefits of maintaining increased DICER levels in adipose tissue have been described, the mechanisms through which DR elicits this upregulation remained unknown. We hypothesized that nutrient restriction could affect DICER levels. Indeed, we found that adipose tissue DICER is sensitive to the levels of sulfur amino acids. Under DR, PR, or MR, DICER levels are upregulated.

On the other hand, when methionine or cysteine are supplemented to DR, DICER abundance is partially reversed toward the levels of ad libitum fed mice, suggesting that dietary sulfur amino acid restriction is both necessary and sufficient to upregulate DICER in adipose tissue.

Importantly, sulfur amino acid restriction prolongs lifespan [6], [34], [44], improves insulin sensitivity [45], [46], promotes adipose tissue browning [39], protects against oxidative stress [47], [48], among other features that have been also directly or indirectly associated with adipose tissue DICER upregulation.

Our data also demonstrate the close relationship between the levels of sulfur amino acids in the diet and various parameters, some of which also relate to DICER levels, like the levels of adiponectin in the blood and 8OHDG in adipose tissue. Importantly, we observed that adiponectin controls Dicer expression in adipose tissue. On the other hand, the absence of Dicer in adipocytes leads to downregulation of blood adiponectin. Together, these results indicate that adiponectin and DICER co-regulate each other in adipocytes, and this mechanism is promoted by sulfur amino acid restriction.

Several studies (including this one) have shown upregulation of adiponectin in response to MR ([49] and Figure 4I). These effects are usually accompanied by upregulation of Ucp1 in inguinal adipose tissue [39] – a bona fide marker of beige adipocyte recruitment [50].

Adiponectin promotes browning by directly binding to anti-inflammatory M2 macrophages, inducing their proliferation and consequent stimulation of beige adipocytes [51]. This suggests that browning induced by MR may involve at least in part M2 macrophages.

MR-induced browning also requires the presence of DICER in adipocytes, which, in turn, is a necessary condition for proper adiponectin production.

We thus propose a model in which adiponectin leads to DICER upregulation, favoring more adiponectin production and promoting induction of browning. Hence, in the absence of adiponectin or DICER, browning is not sustained.

DR and MR also upregulate DCR-1 in C. elegans intestine, and they require DCR-1 to prolong lifespan in nematodes, revealing hundreds of millions of years of evolutionary conservation. C. elegans does not have an obvious adiponectin ortholog but has three orthologs of the adiponectin receptors that play a role in lipid metabolism, stress response, and aging [52], [53]. It is yet to be investigated whether they participate in the effects of sulfur amino acid restriction or if they control DCR-1 expression in C. elegans.

More importantly, this degree of evolutionary conservation gives us confidence that humans may also respond to MR in a similar manner. Indeed, protein and methionine restriction diets have been associated with beneficial health effects in humans [5], [54] Finding the mechanisms through which this association takes place is important to delineate specific interventions that can mimic DR without its heavy demands.

Here we found a sulfur amino acid/adiponectin/DICER axis in adipose tissue that plays an important role in the way DR promotes browning and increases lifespan. This axis represents a promising target for interventions designed to mimic DR and promote beneficial metabolic effects.



Centenarians, but not octogenarians, up-regulate the expression of microRNAs



Centenarians exhibit extreme longevity and a remarkable compression of morbidity. They have a unique capacity to maintain homeostatic mechanisms. Since small non-coding RNAs (including microRNAs) are implicated in the regulation of gene expression, we hypothesised that longevity of centenarians may reflect alterations in small non-coding RNA expression.

We report the first comparison of microRNAs expression profiles in mononuclear cells from centenarians, octogenarians and young individuals resident near Valencia, Spain. Principal Component Analysis of the expression of 15,644 mature microRNAs and, 2,334 snoRNAs and scaRNAs in centenarians revealed a significant overlap with profiles in young individuals but not with octogenarians and a significant up-regulation of 7 small non-coding RNAs in centenarians compared to young persons and notably 102 small non-coding RNAs when compared with octogenarians.

We suggest that the small non-coding RNAs signature in centenarians may provide insights into the underlying molecular mechanisms endowing centenarians with extreme longevity.

Centenarians Maintain miRNA Biogenesis Pathway While It Is Impaired in Octogenarians



Centenarians but not octogenarians up regulate the expression of miRNAs, as we previously reported. We have looked into miRNA biogenesis. We show that RNA POL II, DROSHA, EXPORTIN 5 and DICER, are up-regulated in centenarians compared with octogenarians.

Furthermore, factors involved in the control of these miRNAs biogenesis genes are also up-regulated in centenarians.

Therefore, the up-regulation of miRNA expression in centenarians can be explained in part because miRNA biogenesis pathway is depressed in octogenarians (ordinary aging) while it is maintained in centenarians (extraordinary aging).


DICER is the final enzyme involved inmiRNA maturation. Figure 1d shows that DICER is down-regulatedin octogenarians when compared with young persons orwith centenarians. This is in keeping with the fact that aging is associated to a dysregulation of DICER (Mori et al., 2012; Ungvari et al., 2013).

Our results also show an increased expression of miR-21in centenarians compared with octogenarians (see Figure 1d). This is consistentwith the fact that DICER can regulate miR-21processing. (Ha and Kim, 2014).

We conclude that in centenarians there is an attenuation of the decrease that occurs in miRNome during ordinary aging,because they maintain the miRNA processing machinery activity similar to that found in young individuals.





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All very nice, especially when seen in humans. I'm less enthused about studies in mice/rats, but I guess for certain purposes extrapolating from those is the only realistic hope, as we certainly are not going to have controlled longitudinal studies in humans (i.e. full lifespan). 

What I wonder about, when feeding protein to these mice, is what else comes along for the ride. I think we all understand the importance of a food matrix, and a ton of effort has been wasted in studying a single component in isolation, when that is not what happens in a diet. 

What do we do with studies that feed single amino-acids? Obviously, outside of supplementation, this doesn't happen in a diet - might this not distort things significantly. And on the other side, when feeding something like eggs, how do we make sure to isolate the effect of amino-acids from say, for example, choline - because when we speak of atherosclerosis as in the some of these studies cited, we now know that a large part of it is down to gut bacteria generated choline derived TMAO. Leaping to conclusions that increased atherosclerosis is down to an aminoacid rather than a whole slew of other factors seems incautious. Perhaps food sources with animal amino acid proiles happen to have higher choline levels and it's down to that, rather than the amino acid profile of vegetable sourced protein. And then of course it's all about how cholne is processed and TMAO derived - maybe choline in an environment where other vegetable components in the matrix modify either the gut-bacteria profile or their processing, so you don't get as much TMAO out of the choline that is present (we do know that brassicas can modify this) - again, nothing to do with amino-acid profiles, but all about the matrix and the totality of the diet and perhaps even modified by other factors such as exercise. 

All in all, I think it's dangerous to hold too firmly to some conclusion about cause and effect based on studies that do not sufficiently disentangle all these factors.

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I've spent the last week or so mulling over conflicting goals of optimal metabolic health & reduction in all-cause mortality vs. maximizing muscle strength and size.

I am ... once again, going to attempt return to a wfpb diet.  I will try to maintain as much strength and muscle size as my nutrition naturally allows; with hopes that I don't lose all that much.  I am more concerned with gaining body fat than actually losing muscle.

Much of the above information has motivated me wrt health, along with a quick review of some of the results that I've read from vegan/vegetarian bodybuilders and athletes  ... 


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