While there remains room for debate, it certainly now appears that methionine restriction actually works to retard aging (and thus, extend maximum lifespan) independently of CR. However, it is not a viable anti-aging lifestyle for humans.
MetR is not Protein Restriction (PR), and CR does not Involve MetR or PR
It's important to note first that methionine restriction is not protein restriction, and the benefits of CR do not come from protein or methionine restriction: and in the MetR studies, and the best CR studies, they explicitly keep total protein the same. This is noted quite explicitly (and not just observable in the 'Methods') in (7):
These results cannot be attributed to diminished protein intake; MR rats consumed more protein than their CF [control-fed] counterparts when corrected for either body weight (MR: 0.87 g/day vs. CF: 0.58 g/day) or lean body mass (MR: 6.2 g/day vs. CF: 4.9 g/day).
Conventional CR, where carbohydrate and not protein content is sacrificed, is neither protein restriction nor MR. In a preliminary comparison of weight-matched MR and 40% CR rats, CR rats consumed more methionine (CR: 0.16±0.03 g/day vs. MR: 0.02±0.003 g/day) than MR and amounts comparable to CF (0.18 g/day).(7)
There are many, many CR studies (eg, (1-5) in which the percentage of protein in the chow fed to the CR rodents is increased in order to ensure that the absolute intake of protein (and, as a result, Met) is kept exactly the same between the CR and AL groups, and the CR works just fine (better, in fact, in adult-onset organisms). In fact, this is the standard CR protocol of the best labs.
Similarly,(5a) compared one group of animals under 40% total diet CR (including 40% reduction of all macronutrients across the board) with another under 40% CR but with the %protein in the chow boosted up so that they consumed the same absolute amount of protein as the AL animals. The mean and maximum lifespan of the two CR groups was the same. The CR+PR group did fared slightly better on maintaining their renal health with age than the CR-no-PR group (the strain of rat used (F344) is highly prone to nephropathy, which can be ameliorated by PR alone), but only at the very end of life (age 30 months), and only in the form of a modest increase in the number of the lowest grade of lesions.
The non-involvement of PR in CR was also shown in a painstaking meta-analysis of CR studies with varying levels of protein intake.(6)
Protein restriction alone has little or no effect on LS except by introducing "crypto-CR:" studies claiming otherwise either don't track weight and/or Calories rigorously, or are in animals with specific diseases in which a low-pro diet is beneficial, or don't actually measure maximum LS, or uses short-lived controls, the Original Sin of Biogerontology.
Now, with that out of the way:
What Would it Take to Do This?
What about MetR itself? MetR is one of the very few things rigorously documented to retard the biological aging process in humans. Could you avoid the hassle and huger of CR and still get true anti-aging benefits by restricting your methionine (+cysteine) intake?
Many people would like to believe so. In fact, some (including the ignominious Greger and possibly Mark McCarty) take this idea a step further, suggesting that because plant-based proteins tend to have lower % and/or absolute amounts of Met+Cys compared to animal proteins, one could effect MetR and thus reap anti-aging benefits by simply substituting vegetal for animal proteins.
This Will Not Work. The kind of MetR that retards aging in mice and rats is not a mere "moderation" of Met intake, but a radical and dangerous restriction from normal intake: 80% MetR (ie, about 20% of the rat "RDA" for Met, plus no cysteine in the diet). For instance, in (7) above (an important MetR study that gave some of the first evidence that MetR is not merely a form of "crypto-CR"), "The animals were fed on a purified diet containing either 0.86% methionine (CF) or 0.17% methionine (MR) (Orentreich et al., 1993). The diets were devoid of L-cystine; hence methionine was the sole source of sulfur amino acid."(7) As noted in the quote, MetR animals wound up consuming 0.02±0.003 g Met/day, vs. 0.18 g/day in controls, with no Cys in either group.(7) Similarly, in (8) (another important study, as it was the first to show clearly that MetR works in mice and not just rats):
studies of F344 and other rat strains [refs] have shown that maximal lifespan can be extended by diets in which cystine and cysteine are absent and methionine levels are as low as possible. ... [W]e placed a group of CB6F1 female mice on a semi-purified diet containing 0.1% methionine at 6 weeks of age. Control mice were fed a semi-purified diet similar in all respects except that it contained 0.43% methionine by weight. Methionine levels for the experimental group were increased to 0.12% when the mice reached 4 months of age, and again to 0.15% when the mice were 6 months of age, in an effort to reduce the proportion of experimental mice dying as a consequence of rectal prolapse. The experimental diet remained at 0.15% for the duration of the experiment. [Both diets contained 0% cystine].(8)
This is a moderately less severe degree of MetR than is standard in the rat studies (starts off at 75% MetR, but loosens up to 66% MetR by age 6 months), but still at all points significantly lower than is achievable by simply consuming vegetal proteins. In his own work on MetR in mice, MetR pioneer Norm Orentreich used a 0.86% Met control diet, but a 0.12% Met diet for MetR — even lower, in absolute terms, than he used in rats, despite the higher-Met control diet.
The human Met+Cys RDA is 19 mg/kg•d, or 1330 mg for a 70 kg adult; to achieve the kind of MetR that retards aging in rodents, would require consuming somewhere between 266 (rat studies) to 443 (peak in the mouse study (8)) mg Met per day, with no compensatory Cys — which turns out to be important (see below)).
This cannot be achieved on a natural-food diet. Even a non-CR diet of 2500 Calories of potatoes would contain 1000 mg Met + 800 mg Cys, and it's even worse if you throw in some broccoli (2900 and 2100 mg, respectively). Legumes — as the LORD hath said of counting to 5 with the Holy Hand Grenade of Antioch — are right out.
Now, when I've pointed this out in the past, some people have insisted that this can't possibly be right. Some have argued, for instance, that we shouldn't calculate the degree of restriction from the RDA, but from typical human intakes, derived from either typical American diets or worldwide estimates from the WHO. But this isn't what they do with the rodents: the control chow contains the rodent RDA — no more, no less — and Met is cut back from there in the MetR animals, who additioinally consume zero Cys.
And this more moderate interpretation of the MetR protocol clearly isn't accepted in the scientific community, either. In studies trying to translate the salutary effects of MetR in rodents on metabolism in obese people(11) and in enhancing the effects of chemotherapy,(12-17) Met is radically restricted and Cys excluded entirely, through resort to special chemically-defined medical shakes such as Hominex®-2 and XMet Maxamum®, which were originally designed for people with genetic disorders of vitamin B6-nonresponsive homocystinuria or hypermethioninemia), sometimes supplemented with very low levels of potatoes and vegetables.
In (11) (coauthored by MetR pioneer Dr. Norm Orentreich), "Twenty-six obese subjects (six male and 20 female) meeting criteria for metabolic syndrome were randomized to a diet restricted to 2 mg methionine/kg body weight per day [140 mg in a 70 kg adult!] and were provided capsules containing either placebo (n = 12) or 33 mg methionine/kg body weight per day (n = 14)." The same 2 mg/kg•d was used in one of the cancer chemo enhancement studies(16,17), while in the others,(12-15) subjects consumed zero Met+Cys during the brief (1-4 day) window in which they were administered their chemo.
No Cheating with Cysteine
As I've noted earlier, the rodent MetR studies involve diets that are not only extremely low in Met, bu contain no Cys at all. This turns out to be quite important. Some people have wondered if you might be able to get the benefits of MetR while minimizing the side-effects by consuming some Cys. Instead, consumption of Cys clearly impairs the metabolic effects of MetR.
Although cysteine is the precursor of two sulfur compounds with antioxidant properties, taurine and glutathione (ref), plasma total cysteine (tCys) levels are positively associated with body fat mass (ref), obesity (ref), hypercholesterolemia (ref), and metabolic syndrome (ref) in humans. [See also (27) below]. ...
To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats.
MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels.
Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that (24).
In followup work, they showed that many of the gene expression changes in fat, liver, and especially muscle that are induced by MetR are reversed by adding 0.5% Cys.(25) Similarly, "NAC supplementation in MR rats raised tCys and partly or completely reversed MR effects on weight, fat %, Scd1 expression in liver and white adipose tissue, and estimated SCD1 activity. In CF [control-fed] rats, NAC decreased body fat % and lowered SCD1-18 activity index (P<0.001)."(25)
On the other hand, "Taurine supplementation of MR rats did not restore weight gain or hepatic Scd1 expression or indices to CF levels, but further decreased adiposity. Taurine supplementation of CF rats did not affect adiposity, but lowered triglyceridemia."(26)
Pseudo-Studies of Moderate MetR
Now, 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.
As mentioned above, (5a) compared two CR groups: a CR+PR group in which 40% CR was achieved by cutting all macronutrients across the board, and a CR-no-PR group in which the percentage of protein in the chow was increased even as the amount of food was reduced, so that CR animals consumed 40% fewer Calories but the same absolute amount of protein as the AL animals. The strain of rat used (F344) is highly prone to nephropathy, and as summarized above, the CR+PR group did fare slightly better on maintaining their renal health with age than the CR-no-PR group. However, the mean and maximum lifespan of the two CR groups was the same.(5a)
Of course, cutting the protein meant that the CR+PR group was consuming 40% less Met+Cys than the CR-no-PR group as well, just as in Barja's mtROS study. Yet CR worked just as well without cutting protein or Met, aside from the renal outcome that is unlikely to be driven by mtROS specifically.
In a similar study in a different strain of rats, "caloric restricted male rats were fed 18, 30 or 42 percent casein diets that provided two-thirds of the quantity of diet consumed by groups fed 12, 20, or 28 percent casein diets ad libitum, respectively. Hence, caloric restricted groups consumed the same amount of protein as their paired ad libitum fed groups but one-third fewer calories." At one year (youngish adulthood in a rat), the low-protein CR18pro group lived slightly longer than the other two groups — but at two years (seniority), the CR30 and CR40 groups had substantially higher survival (31%, 61%, and 53% survival, respectively).(18) Importantly, in the ad libitum group (in which protein was varied without cutting Calories), the two-year survival was also impaired at 12% protein: 19% survival, vs. 31 and 33% survival on the higher-protein diet.(18) Again, reducing protein intake (and, concomitantly, Met intake) by a moderate amount didn't extend life, and may have shortened it. However, this isn't a very convincing study, because all of these animals were short-lived.
More recently, the matter was put to a more direct test, which seems on its face to give strong evidence that mere methionine moderation yields no life extension benefit. In (10), "three levels of methionine were provided to mice to represent a severe restriction (0.16%) [standard MetR]; approximately 50% reduction (0.43%) compared with standard rodent diets and more achievable in human diets; and an enriched level (1.3%) that remained below toxicity levels." Now, unfortunately, they didn't include a group receiving standard (0.86% Met) chow, but the results initially seem reasonably clear:
--------------0.16% Met -|- 0.43% Met -|- 1.3% Met
Median LS: ---787 days -|- 581 days ---|- 556 days
Maximum†: 1087days ---|-888 days -|--- 799 days
†As tenth-decile survivorship
(Pardon the messy attempt at a table). As you can see, the moderate MetR did not enjoy any substantial life extension. However, there are two problems with this study. First, both the moderate-MetR and moderate-Met-overdose groups were somewhat short-lived, and even the MetR group was actually only normally longevous, not demonstrating any actual life extension (and it seems unlikely that moderate MetR would have shortened life; the lack of a normal-Met group makes that difficult to asssess). So the failure of moderate MetR here can't be taken as strong evidence.
Second, their "moderate MetR" group used what others would consider to be a normal level of Met for mice, not a restriction. The group seems to have based their numbers on classical MetR studies in rats, but standard mouse chows today contain more like the 0.43% Met used as moderate MetR in this study — indeed, Rich Miller's strong MetR mouse study above (8) used a 0.43% Met control chow. So it's not clear that this even was "moderate MetR," or indeed MetR at all.
So, the definitive study to test moderate MetR hasn't yet been done. But from what evidence is available, it certainly appears that it Met Moderation will not work as an anti-aging therapy.
Moderating Met is Still Good for You
That said, there certainly is some evidence that "moderate MetR" is healthy — altho' this may simply amount to saying that plant-based sources of protein is good for you, potentially for reasons not related to Met.
[In a] "prospective cohort study consisting of 1981 coronary disease free men from eastern Finland, aged 42-60 years at baseline" and with "an average follow-up time of 14.0 years," "adjusting for age, examination years, BMI, urinary nicotine metabolites and protein intake (excluding methionine), the relative risks of acute coronary event in the three highest quarters of dietary methionine intake were 1.31 (95% CI: 0.92, 1.86) [for an intake of 1.7-2 g Met/d], 1.31 (95% CI: 0.88, 1.96) [2-2.6 g Met/d], and 2.08 (95% CI: 1.31, 3.29) [> 2.6 g Met/d] as compared with the lowest quarter [<1.7 g Met/d]."(19) The major dietary sources of methionine were meat and meat products (31.8%), milk and milk products (31.7%), cereal (17.7%) [meaning, grains: grains are high-Met as a relative proportion of protein, which is why they are used to "complement" the protein in beans] and fish (9.8%)."(19)
Not the saturated fat: The significance of the specific finding for methionine may possibly be even greater than that reported, as there was a surprising inverse correlation between methionine intake and saturated fat, which "indicates that although the absolute saturated fat intake increases with increasing methionine intake, its relative proportion of total energy intake decreases. That may indicate that the subjects who consumed more methionine also consumed more energy, but chose protein (and thus methionine) sources with low saturated fat content."(19)
This could happen with low-fat dairy, lower-fat cuts of meat, low-fat fish, or conceivably even more grain intake, though it's hard to get a meaningful increase in % dietary protein from grains. The "covariates used in the statistical models included saturated fat" which should in principle have zeroed any confounding effects; still, one wonders if the effect of Met might be even greater than that reported, if it were indeed blunted by their relatively low SFA intake of high Met consumers.
Not the homocysteine: Your initial thought will likely be to chalk this up to homocysteine. Aside from the fact that the causal connection of Hcy to cardiovascular disease is at this point on very shaky ground in general, it just won't wash as an explanation in this specific study. "In our study, however, plasma tHcy was inversely associated with methionine intake. In addition, in a recent randomized controlled trial, a six-month high-protein, high-methionine diet did not raise plasma tHcy concentrations compared with a low-protein, low-methionine diet . Similar results have been observed in other studies. ... One hypothesis [for this disconnect] is that the enzymes in the methionine cycle adapt to the high methionine intake and thus maintain a normal concentration of Hcy in circulation."(19) Another, more likely one is the confounding of meat, methionine, and B12 intakes. As noted in my post on supplementation for vegetarians, elevated Hcy is quite common in veg(etari)ans, as a dose-response curve (Hcy & B12 deficiency omnivores < lacto-ovos < vegans).
More protein is better:
However, ... total protein intake ... tended to decrease the risk."(19) Specifically, "The mean (±SD) intake of protein was 90.7 ± 25.1 g/day (15.5 ± 2.6% of energy)" and "The age and examination year adjusted [relative risks] of acute coronary event in quarters of energy-adjusted protein intake were 1.0 [for 12.9% of Calories from protein, which would average about 75 g protein/day], 0.89 [14.9%/ 87 g protein], 0.86 [16.1%/ 94 g protein], and 0.78 [18.0%/105 g protein].
If energy adjusted methionine intake was added in the models [to disentangle the fact that a higher intake of protein normally entails a higher intake of methionine, already separately shown to be a risk factor], the RRs for quarters of energy adjusted protein intake (excluding methionine) were 1.0, 0.76, 0.63, and 0.43. Further adjustments did not change the result.(19)
That's right: if you had average Met intake (~2 g/d), but were in the highest protein intake group (~94 g protein/d), you were less than half as likely to suffer an acute coronary event over the course of the next 14 years as a man with similar Met intake but only getting 75 g — which is already for most people more than the RDA.
"Interestingly, total protein intake was not associated with increased risk of acute coronary events, but rather tended to modestly decrease the risk, consistent with previous findings in women ." (19) This refers to findings from the Nurses' Health Study, which is amongst the highest-quality epidemiological studies ever produced (large, with prospective analysis of a relatively homogeneous cohort, very well-funded, long-term, repeated measures of dietary and metabolic outcomes along the way, etc etc). They found similar results extending to even higher absolute (as well as relative) protein intake, as is more typical of North American diets — and this in women, who are smaller and weigh less than the men in (19): "After age, smoking, total energy intake, percentages of energy from specific types of fat, and other ischemic heart disease risk factors were controlled for, high protein intakes were associated with a low risk of ischemic heart disease; when extreme quintiles of total protein intake were compared [14.7% vs 24.0% protein], the relative risk was 0.74 (95% CI: 0.59, 0.94). Both animal and vegetable proteins contributed to the lower risk. This inverse association was similar in women with low-or high-fat diets."(20)
In a much longer-term followup including data from both the Nurses’ Health Study (followed up from 1980 to June 1, 2012) and the all-male Health Professionals Follow-up Study (followup 1986 to January 31, 2012):
The median protein intake, as assessed by percentage of energy, was 14%for animal protein (5th-95th percentile, 9%-22%) and 4%for plant protein (5th-95th percentile, 2%-6%). After adjusting for major lifestyle and dietary risk factors, animal protein intake was weakly associated with higher mortality, particularly cardiovascular mortality (HR, 1.08 per 10% energy increment; 95%CI, 1.01-1.16; P for trend = .04), whereas plant protein was associated with lower mortality (HR, 0.90 per 3% energy increment; 95%CI, 0.86-0.95; P for trend < .001). These associations were confined to participants with at least 1 unhealthy lifestyle factor based on smoking, heavy alcohol intake, overweight or obesity, and physical inactivity, but not evident among those without any of these risk factors.(22)
Note that this was for total protein intake from each source, without consideration of what that higher protein intake replaced. Taking displacement effects into account:
Replacing animal protein of various origins with plant protein was associated with lower mortality. In particular, the HRs for all-cause mortality were 0.66 (95% CI, 0.59-0.75) when 3% of energy from plant protein was substituted for an equivalent amount of protein from processed red meat; 0.88 (95%CI, 0.84-0.92) from unprocessed red meat; and 0.81 (95%CI, 0.75-0.88) from egg.(22)
Similarly, in the Iowa Women’s Health Study,"Among women in the highest intake quintile, CHD mortality decreased by 30% from an isoenergetic substitution of vegetable protein for carbohydrate (95% confidence interval (CI): 0.49, 0.99) and of vegetable for animal protein (95% CI: 0.51, 0.98), following multivariable adjustment."(21)
To return to (19):
The risk was further decreased when the intake of methionine was taken into account in the models [of total protein intake vs. acute coronary event risk], which supports our findings of the risk increasing effects of high methionine intake. This could also explain why the increased risk associated with high methionine intake became more apparent only after total protein intake (excluding methionine) had been taken into account in the statistical models. In other words, the risk reduction associated with total protein intake masks the risk associated with its minor component, methionine. ...
in addition to the findings by Hu and colleagues [,] Very low levels of animal protein intake have been associated with increased risk of hemorrhagic stroke in women , and low total intake of protein with hemorrhagic stroke in Japan . Furthermore, a significant inverse association between dietary protein and blood pressure in both sexes has been found in a meta-analysis of cross-sectional studies . In addition, recent studies have found that short-term high protein diets decrease triglyceride and LDL cholesterol and increase HDL cholesterol concentrations or have no significant effect on blood lipid measures, although the concurrent weight loss may explain at least part of the effect . However, it seems that high protein diets may be beneficial for cardiovascular health, although longer-term studies are needed for more definite evidence.(19)
There is also a provocative study (28) in which they showed that protein restriction (per se: 7% protein diet, vs. 21% normal rodent chow) or simple replacement of animal protein (lactalbumin, with or without casein) with the same amount of vegetal protein (wheat and corn gluten plus isolated soy protein) substantially inhibited the growth of a variety of implanted cancers in vivo, ostensibly independently of Calorie intake. However, it doesn't appear from reading the paper that they actually measured how much food the animals ate, or how much the animals weighed: the chow was isocaloric, but without measuring food intake you can't rule out crypto-CR. And, implanted cancer cells aren't as good a model as actual "spontaneous" (age-related) cancer incidence and mortality over a lifetime of feeding one diet vs. another.
In practice, a high-protein, low-methionine diet is one composed of a lot of non-grain vegetarian protein. All legume proteins are good, and lentils and favas are exceptionally noteworthy as being high %protein, low-Calorie, and unusually low-Met and low leucine even for a legume (see here and here on leucine moderation), per gram of protein and per Calorie. Dairy (aside from whey) is moderate in methionine; Quorn, despite being of vegetal source, is not very low — and not just because of its eggwhite content: the vegan version is ev is also pretty acceptable (certainly compared with meat).
Eggwhites, however, are very high-Met.
Additionally, tho' not enamored of vegans, gelatin is a very screwy protein, which contains no Trp and very little Met indeed; it is so messed up that it shouldn't be a major component of the diet (especially not if it isn't otherwise protein-packed), but if you add a small amount of supplemental Trp to gelatin or sugar-free Jell-O, the near absence of Met turns into a bonus.
Reminder on Protein, IGF-1, and CR
Additionally, although plant-sourced protein is in general good for one, and although I emphasize again that MetR ≠ PR, still people on CR should also engage in protein moderation to avoid overriding the reduction of IGF-1 induced by CR — ie, on CR, your protein should be at or a little above the RDA . Here again plant-based proteins are your friends, as vegetal protein is much less IGF-1-inducing than animal protein. In my own case, as I've noted before, even at ≈80 g protein and 16% of energy from mostly vegetal protein, my free and total IGF-1 levels were too low (and I attribute some negative health effects thereto), and I subsequently bumped it up to 20% protein, with the extra protein being either dairy or more highly-absorbable vegetal sources (tho' I have a hard time titrating my protein intake to get a consistent, agreeable IGF-1 profile).
A Plea for Rigor
Now, whatever you think of my review of the evidence and analysis, a plea. The life extension community is suffering from widespread confusion on this point that comes from — and is certainly perpetuated by — overly-loose use of language (and what I suspect is willful ignorance at best on the part of overly-zealous vegan propagandists). I have begged people over and over and over and over again to please, please, please, stop muddying the water by referring to limiting one's intake of some nutrient to RDAish levels as "restriction" of that nutrient. As we've seen, biogerontological studies of protein-, Met + Cys-, Leu-, Trp-, or Calorie restriction involve restricting consumption of these nutrients to levels far below the animals' "RDA" intake; with the exception of the Calories in CR, I don't practice or endorse that, and neither does anyone I know of (including a few folks who do, unfortunately, refer to what they do as "restriction" of that nutrient).
1: Weindruch R, Walford RL. Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence. Science. 1982 Mar 12;215(4538):1415-8. PubMed PMID: 7063854.
2: Lee CK, Pugh TD, Klopp RG, Edwards J, Allison DB, Weindruch R, Prolla TA. The impact of alpha-lipoic acid, coenzyme Q10 and caloric restriction on life span and gene expression patterns in mice. Free Radic Biol Med. 2004 Apr 15;36(8):1043-57. PubMed PMID: 15059645.
"An additional group of mice were 151 subjected to CR at 14 months of age (as detailed in ) to serve as a positive control." Their reference (39) is this study from the same authors, which says "The restricted diet was nearly isocaloric to the normal diet, but enriched in protein, vitamins, and minerals to avoid malnutrition." They cite back to an earlier "Methods" guideline of theirs (PMID 10537025, "Controlling caloric consumption: protocols for rodents and rhesus monkeys"), which says even more explicitly, "The protein (casein), minerals, and vitamins are enriched in the [CR] diet such that nearly identical amounts of these components are fed to both [AL and CR] animals after a reduction in diet."
3: Dhahbi JM, Kim HJ, Mote PL, Beaver RJ, Spindler SR. Temporal linkage between the phenotypic and genomic responses to caloric restriction. Proc Natl Acad Sci U S A. 2004 Apr 13;101(15):5524-9. Epub 2004 Mar 25. PubMed PMID: 15044709; PubMed Central PMCID: PMC397416.
"Control mice were fed 93 kcal per week of chemically defined control diet (AIN-93M, Diet No. F05312, Bioserv, Frenchtown, NJ). ... CR was introduced by reducing calories to 77 kcal per week of chemically defined CR diet for 2 weeks, followed by 52.2 kcal per week of CR diet thereafter (AIN-93M 40% Restricted, Diet No. F05314, Bioserv)." As is spelled out in the main investigators' own patent for "Methods of evaluating caloric restriction and identifying caloric restriction mimetics," "Mice on the control diet were fed 93 kcal per week fo the control diet (AIN-93M). Mice on the CR diets were fed 77 kcal per week of the CR diet or 52 kcal per week Of the CR diet (40% calorie restricted AIN-93M). ... Each mouse in the LT-CR [Long-Term CR] group 106 was subjected to a LT-CR dietary program with feeding of 52.2 kcal per week of a semi-purified CR diet (AIN-93M 40% Restricted, Diet No. F05314, BIO-SERV). The control diet program consist of about 14 gm/100 gm diet casein, about 0.2 gm/100 gm diet L-cysteine ... The CR diet consist of about 23.3 gm 100 gm diet casein, about 0.3 gm/100 gm diet cysteine ... Note that the 40% CR diet composition listed in Table 12 is for both the 52 kcal per week CR diet and the 77 kcal per week CR diet. The dietary composition for the diet in the reduction stage, where the diet includes a 77 kcal per week diet program, can be adjusted accordingly from the CR diet to obtain a 77 kcal per week diet. The CR diet was used for both 52 and 77 kcal per week CR diets." I'm sure you can do the math .
4: Pugh TD, Oberley TD, Weindruch R. Dietary intervention at middle age: caloric restriction but not dehydroepiandrosterone sulfate increases lifespan and lifetime cancer incidence in mice. Cancer Res. 1999 Apr 1;59(7):1642-8. PubMed PMID: 10197641.
5: Davis TA, Bales CW, Beauchene RE. Differential effects of dietary caloric and protein restriction in the aging rat. Exp Gerontol. 1983;18(6):427-35. PubMed PMID: 6673988.
5a: Masoro EJ, Iwasaki K, Gleiser CA, McMahan CA, Seo EJ, Yu BP. Dietary modulation of the progression of nephropathy in aging rats: an evaluation of the importance of protein.
Am J Clin Nutr. 1989 Jun;49(6):1217-27. PMID: 2729159 [PubMed - indexed for MEDLINE]
6: Speakman JR, Mitchell SE, Mazidi M. Calories or protein? The effect of dietary restriction on lifespan in rodents is explained by calories alone. Exp Gerontol.2016 Dec 15;86:28-38. doi: 10.1016/j.exger.2016.03.011. Epub 2016 Mar 19. Review.PubMed PMID: 27006163.
7: Malloy VL, Krajcik RA, Bailey SJ, Hristopoulos G, Plummer JD, Orentreich N. Methionine restriction decreases viseral fat mass and preserves insulin action in aging male Fischer 344 rats independent of energy restriction. Aging Cell. 2006 Aug;5(4):305-14. Epub 2006 Jun 26. PMID: 16800846
8: Miller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M. Methionine-deficient diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell. 2005 Jun;4(3):119-25. PubMed PMID: 15924568.
9: Pilar Caro, Jose Gomez, Ines Sanchez, Alba Naudi, Victoria Ayala, Monica López-Torres, Reinald Pamplona, and Gustavo Barja. Forty percent methionine restriction decreases mitochondrial oxygen radical production and leak at complex I during forward electron flow and lowers oxidative damage to proteins and mitochondrial DNA in rat kidney and brain mitochondria. Rejuvenation Res. 2009 Dec;12(6):421-34. PubMed PMID 20041736.
10: Brown-Borg HM, Rakoczy SG, Wonderlich JA, Rojanathammanee L, Kopchick JJ, Armstrong V, Raasakka D. Growth hormone signaling is necessary for lifespan extension by dietary methionine. Aging Cell. 2014 Dec;13(6):1019-27. doi: 10.1111/acel.12269. Epub 2014 Sep 19. PubMed PMID: 25234161; PubMed Central PMCID: PMC4244257.
11: Plaisance EP, Greenway FL, Boudreau A, Hill KL, Johnson WD, Krajcik RA, Perrone CE, Orentreich N, Cefalu WT, Gettys TW. Dietary methionine restriction increases fat oxidation in obese adults with metabolic syndrome. J Clin Endocrinol Metab. 2011 May;96(5):E836-40. doi: 10.1210/jc.2010-2493. Epub 2011 Feb 23. PubMed PMID: 21346062; PubMed Central PMCID: PMC3085194.
12: Durando X, Farges MC, Buc E, Abrial C, Petorin-Lesens C, Gillet B, Vasson MP, Pezet D, Chollet P, Thivat E. Dietary methionine restriction with FOLFOX regimen as first line therapy of metastatic colorectal cancer: a feasibility study. Oncology. 2010;78(3-4):205-9. doi: 10.1159/000313700. Epub 2010 Apr 26. PubMed PMID: 20424491.
13: Thivat E, Farges MC, Bacin F, D'Incan M, Mouret-Reynier MA, Cellarier E, Madelmont JC, Vasson MP, Chollet P, Durando X. Phase II trial of the association of a methionine-free diet with cystemustine therapy in melanoma and glioma. Anticancer Res. 2009 Dec;29(12):5235-40. PubMed PMID: 20044642.
14: Durando X, Thivat E, Farges MC, Cellarier E, D'Incan M, Demidem A, Vasson MP, Barthomeuf C, Chollet P. Optimal methionine-free diet duration for nitrourea treatment: a Phase I clinical trial. Nutr Cancer. 2008;60(1):23-30. doi: 10.1080/01635580701525877. PubMed PMID: 18444132.
15: Thivat E, Durando X, Demidem A, Farges MC, Rapp M, Cellarier E, Guenin S, D'Incan M, Vasson MP, Chollet P. A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer. Anticancer Res. 2007 Jul-Aug;27(4C):2779-83. PubMed PMID: 17695447.
16: Epner DE, Morrow S, Wilcox M, Houghton JL. Nutrient intake and nutritional indexes in adults with metastatic cancer on a phase I clinical trial of dietary methionine restriction. Nutr Cancer. 2002;42(2):158-66. PubMed PMID: 12416254.
17: Epner DE. Can dietary methionine restriction increase the effectiveness of chemotherapy in treatment of advanced cancer? J Am Coll Nutr. 2001 Oct;20(5 Suppl):443S-449S; discussion 473S-475S. Review. PubMed PMID: 11603655.
18: Davis TA, Bales CW, Beauchene RE. Differential effects of dietary caloric and protein restriction in the aging rat. Exp Gerontol. 1983;18(6):427-35. PubMed PMID: 6673988.
19: Virtanen JK, Voutilainen S, Rissanen TH, Happonen P, Mursu J, Laukkanen JA, Poulsen H, Lakka TA, Salonen JT. High dietary methionine intake increases the risk of acute coronary events in middle-aged men. Nutr Metab Cardiovasc Dis. 2006 Mar;16(2):113-20. Epub 2005 Nov 2. PMID: 16487911
20: Hu FB, Stampfer MJ, Manson JE, Rimm E, Colditz GA, Speizer FE, Hennekens CH, Willett WC. Dietary protein and risk of ischemic heart disease in women. Am J Clin Nutr. 1999 Aug;70(2):221-7. PMID: 10426698 [PubMed - indexed for MEDLINE]
21: Kelemen LE, Kushi LH, Jacobs DR Jr, Cerhan JR. Associations of dietary protein with disease and mortality in a prospective study of postmenopausal women. Am J Epidemiol. 2005 Feb 1;161(3):239-49. PubMed PMID: 15671256.
22: Song M, Fung TT, Hu FB, Willett WC, Longo VD, Chan AT, Giovannucci EL. Association of Animal and Plant Protein Intake With All-Cause and Cause-Specific Mortality. JAMA Intern Med. 2016 Oct 1;176(10):1453-1463. doi: 10.1001/jamainternmed.2016.4182. PubMed PMID: 27479196; PubMed Central PMCID: PMC5048552.
23: Ables GP, Perrone CE, Orentreich D, Orentreich N. Methionine-restricted C57BL/6J mice are resistant to diet-induced obesity and insulin resistance but have low bone density. PLoS One. 2012;7(12):e51357. doi: 10.1371/journal.pone.0051357. Epub 2012 Dec 7. Erratum in: PLoS One. 2014;9(7):e104050. PubMed PMID: 23236485; PubMed Central PMCID: PMC3518083.
24: Elshorbagy AK, Valdivia-Garcia M, Mattocks DA, Plummer JD, Smith AD, Drevon CA, Refsum H, Perrone CE. Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase. J Lipid Res. 2011 Jan;52(1):104-12. doi: 10.1194/jlr.M010215. Epub 2010 Sep 25. PubMed PMID: 20871132; PubMed Central PMCID: PMC2999932.
25: Elshorbagy AK, Valdivia-Garcia M, Mattocks DA, Plummer JD, Orentreich DS, Orentreich N, Refsum H, Perrone CE. Effect of taurine and N-acetylcysteine on methionine restriction-mediated adiposity resistance. Metabolism. 2013 Apr;62(4):509-17. doi: 10.1016/j.metabol.2012.10.005. Epub 2012 Nov 13. PubMed PMID: 23154184.
26: Perrone CE, Mattocks DA, Plummer JD, Chittur SV, Mohney R, Vignola K, Orentreich DS, Orentreich N. Genomic and metabolic responses to methionine-restricted and methionine-restricted, cysteine-supplemented diets in Fischer 344 rat inguinal adipose tissue, liver and quadriceps muscle. J Nutrigenet Nutrigenomics. 2012;5(3):132-57. doi: 10.1159/000339347. Epub 2012 Oct 9. PubMed PMID: 23052097.
27: Elshorbagy AK, Valdivia-Garcia M, Refsum H, Butte N. The association of cysteine with obesity, inflammatory cytokines and insulin resistance in Hispanic children and adolescents. PLoS One. 2012;7(9):e44166. doi: 10.1371/journal.pone.0044166. Epub 2012 Sep 11. PubMed PMID: 22984471; PubMed Central PMCID: PMC3439485.
28: Fontana L, Adelaiye RM, Rastelli AL, Miles KM, Ciamporcero E, Longo VD, Nguyen H, Vessella R, Pili R. Dietary protein restriction inhibits tumor growth in human xenograft models. Oncotarget. 2013 Dec;4(12):2451-61. PubMed PMID: 24353195; PubMed Central PMCID: PMC3926840.
 Iso H, Stampfer MJ, Manson JE, et al. Prospective study of fat and protein intake and risk of intraparenchymal hemorrhage in women. Circulation 2001;103:856-63.
 Shimamoto T, Komachi Y, Inada H, et al. Trends for coronary heart disease and stroke and their risk factors in Japan. Circulation 1989;79:503-15.
 Liu L, Ikeda K, Sullivan DH, Ling W, Yamori Y. Epidemiological evidence of the association between dietary protein intake and blood pressure: a meta-analysis of published data. Hypertens Res 2002;25:689-95.
 Halton TL, Hu FB. The effects of high protein diets on thermogenesis, satiety and weight loss: a critical review. J Am Coll Nutr 2004;23:373-85.