Dietary restriction (DR), a reduction in food intake without starvation, extends lifespan in many organisms: yeast 8
, invertebrates 9
and mammals 1
, including primates 10
. DR in rodents and primates also produces a broad-spectrum improvement in health during ageing 1,10
. Reduced calorie intake has been suggested to underlie extended lifespan in rodents. However, specific amino acids may be as or more important 11-13
. DR lowers fecundity 2
, for instance in the nematode worm Caenorhabditis elegans
, the fruit fly Drosophila melanogaster
and rodents 16
. The prevailing view is that DR induces an evolved response to food shortages 3,17
. If somatic maintenance and reproduction compete for limiting nutrients then, with abundant food, reproduction is prioritized, and somatic maintenance is allocated only the nutrients necessary to ensure survival during the reproductive period, which, due to extrinsic hazards in the wild, falls far short of intrinsic, potential lifespan 4
. With food shortage, reproduction becomes dangerous for the parent and offspring survive poorly, and nutrients are hence reallocated to somatic maintenance, thus increasing survival to reproduce successfully when the food supply returns 3,5
. High survival, associated with DR, and high reproductive rate, associated with full feeding, would thus be mutually exclusive.
We have tested this prediction in Drosophila
. DR is implemented by dilution of the diet, without compensation of food intake rates 18-20
, resulting in increased lifespan and reduced fecundity, measured as egg laying 20
. In nature, Drosophila
eat yeasts 21
and, although many manipulations of dietary balance can alter lifespan 22,23
, enhanced longevity by DR is modulated almost exclusively by dietary yeast, independent of calorie intake 18,22-24
We investigated which nutrients in yeast produce high fecundity in fully-fed flies, and whether these same nutrients also decrease lifespan, as predicted by the reallocation hypothesis. The ratio and type of food components were optimised to maximise both lifespan with DR and fecundity with full feeding 24
, and we examined the effect of adding back nutrients to the DR diet. Since availability of free nutrients will be higher than that in yeast, we first measured fecundity with addition of all nutrients in the ratio present in yeast (see Methods), at several concentrations. We then used the concentration that increased fecundity to the level with full feeding (Table S1
). Adding back vitamins, lipids or carbohydrates did not affect fecundity or lifespan (), indicating that they do not limit fecundity during DR, and that increased intake of calories per se
does not reduce lifespan. In contrast, addition of amino acids increased fecundity and decreased lifespan, as for full feeding ().
Amino acids mediate lifespan and fecundity changes in fly DR
To test for non-nutritional toxicity of amino acids, we measured the osmolarity and pH of each diet. Compared with full feeding, amino acid additions to DR food caused small changes in osmolarity that do not correlate with lifespan (446mOsM for DR increased to 495mOsM with All AA and 1081mOsM for full feeding), and no detectable change in pH, indicating that changes in these factors do not account for the lifespan differences (Fig. S2
). Furthermore, provision of excess water did not abrogate life-shortening by amino acids, but completely rescued that of 0.8% salt addition to DR food (Fig. S2
), demonstrating the efficacy of water provision.
Reallocation of amino acids from reproduction to somatic maintenance could explain the responses of lifespan and fecundity to amino acid add-back. Alternatively, different amino acids could independently produce the two responses. We first investigated the 10 essential and 10 non-essential amino acids, similar in Drosophila
to those in mammals 25
. Adding back non-essential amino acids (N-EAAs) slightly decreased lifespan, with no effect on fecundity (). In contrast, adding back essential amino acids (EAAs) increased fecundity as much as did all 20 amino acids or full feeding (), and also substantially decreased survival, again as much as full feeding (). Adding back N-EAAs increased dietary nitrogen concentration by 9% more than adding back EAAs (Table S1
), suggesting that specific amino acids rather than increased dietary nitrogen were responsible. Further increasing the concentration of EAAs led to further increased fecundity and decreased survival (Fig. S3
). The effects of full feeding can thus be attributed to EAAs in the diet, consistent with reallocation of EAAs from reproduction to somatic maintenance upon DR.
Essential amino acids cause the DR effect
We next determined which EAAs affected fecundity and lifespan. In rodents, lifespan can be extended by restricting either methionine or tryptophan 11-13
. Adding-back EAAs except methionine and tryptophan did not increase fecundity (), indicating that one of these is limiting. Adding back EAAs except methionine also did not increase fecundity from the DR condition (), indicating that methionine is essential, while omission of tryptophan produced the full increase (). Furthermore, adding back methionine (but not tryptophan or any other EAA) alone to a DR diet increased fecundity as much as addition of all 10 EAAs and full feeding (Fig. & S3
). Methionine alone is thus necessary and sufficient for the increase in fecundity. Importantly, egg quality, as indicated by hatching of larvae, was normal upon methionine addition (Fig. S5
). Elevated fecundity with amino acid addition could have resulted from increased food intake. However, direct feeding observations and dye accumulation assays 19
showed that feeding behaviour and rate of food intake were unaltered (Fig. S6
). Adding back a range of methionine concentrations (0.07mM to 13mM) increased female fecundity to a plateau ( and Fig. S7a
) and only addition of other, now limiting, EAAs could increase fecundity further (Fig. S8
). Thus methionine probably does not act as a signal to determine fecundity, because its effects depended upon the ratio of methionine to other EAAs, suggesting instead that it acts through nutritional limitation of reproduction.
Methionine is necessary and sufficient to increase DR egg laying
Surprisingly, adding back methionine did not decrease lifespan (), even when it was added back at much higher concentrations than that limiting for fecundity (Fig. S7b & c
). Hence, reduction in lifespan upon full feeding does not result from reallocation of nutrients from somatic maintenance to reproduction, because the nutrient that increased fecundity, methionine, did not reduce lifespan. Furthermore, the fact that high fecundity and high lifespan can co-occur is inconsistent with the idea that any aspect of reproduction directly inflicts damage on the soma to shorten lifespan 26
. We obtained identical results using a fly diet made with another yeast commonly used for fly DR studies 24
, indicating that these results are not diet-specific (Fig. S9
). Nor can decreased lifespan with full feeding be attributed to unidentified toxins in the food 20,24
. Instead, the responses of lifespan and fecundity to full feeding are independently mediated by different amino acids.
Amino acids, insulin signaling and DR
Adding-back each EAA individually did not decrease lifespan while, again, methionine alone increased fecundity (Fig. S4
). Interestingly, adding-back all EAAs except methionine restored lifespan to the DR level, whereas omission of tryptophan had no effect (). Notably, restriction of methionine alone also increases lifespan in rodents 12,13
. Methionine thus acts in combination with one or more other EAAs to shorten lifespan upon full feeding. Full feeding thus increases fecundity and decreases lifespan through different nutrients in Drosophila
, the former through methionine alone and the latter through a combination of methionine and other EAAs (Fig. S1
). There is thus an imbalance in the ratio of amino acids in yeast relative to what the fly requires for the high fecundity from full feeding, and some consequence of this imbalance decreases lifespan.
Genetic interventions that reduce insulin / insulin-like growth factor signalling (IIS) also extend lifespan of worms, flies and mice 7
. There has been debate on the role of IIS in lifespan-extension by DR 9
has a single IIS receptor, dInR, which mediates both the growth and metabolic functions of IIS 27
. We tested the role of IIS in the responses to DR and EAAs, by over-expressing a dominant-negative form of dInR (InRDN), which extends fly lifespan 28
. InRDN-expressing flies were longer-lived than controls even with DR and, like that of controls, their lifespan was unchanged by the addition of methionine (Fig. & S10
). However, in sharp contrast to controls, lifespan was hardly (trial 1) or not at all (trial 2) reduced by EAA add-back or full feeding (Fig. & S10
). InRDN expression also reduced the responses of egg-laying to methionine and full feeding. Thus, reduced IIS can both extend lifespan beyond the maximal response to DR, showing that mechanisms additional to those of DR are involved, but it can also protect against the lifespan-shortening effects of amino acid imbalance upon full feeding and EAA addition, showing that IIS is required for the lifespan-shortening.
Amino acids that are not used in reproduction in the flies could shorten lifespan through metabolic costs of their removal, through consequent damage, for instance to the excretory malpighian tubules, or through other life-shortening physiological responses. Nutrient imbalance in the diet could also account for the responses of lifespan and fecundity to DR in other organisms, including mammals, if specific nutrients in their diet are also limiting for full physiological function. Indeed, protein quality is implicated in human health, because the ratio of amino acids in the diet can affect traits important for aging, such as glucose homeostasis and bone health 29
. The mechanisms that influence lifespan are conserved over the large evolutionary distances between invertebrates and mammals 7
, and our results hence imply that in mammals too the benefits of DR for health and lifespan may be obtained without impaired fecundity and without DR itself, by a suitable balance of nutrients in the diet.