**5. Conclusions**

We investigated the effects of different diets providing the same amount of carbohydrates, lipids, micronutrients and nitrogen, but containing various proteins or free EAA/NEAA ratios, on lifespan in mice. To our knowledge, this is the first report showing that diets providing nitrogen as free EAA are compatible with a prolonged lifespan in mice. The most relevant finding of our study was the inverse relationship between NEAA dietary content and lifespan. On the contrary, the diet with the highest amount of EAA increased lifespan and maintained low BW, while reducing systemic inflammation and preserving a balanced protein metabolism. On the contrary, the diet with a reduction of just less than 20% in EAA content compared to that usually provided by food proteins (about 45/49%) triggered the rapid catabolic processes characterized by rapid muscle mass loss, and led to precocious death of animals. We confirmed that casein, although among the reference proteins for rodents, is a protein that does not allow a lifespan comparable to that allowed by standard laboratory diets and providing adequate amounts of sulphur-containing AAs, which are insufficiently present in casein. However, in casein diets we could observe that a formulation of free AAs reproducing casein AAs content promoted the longest survival, in our opinion probably consequent to a more elevated absorption of methionine and cisteine.

It is commonly claimed that lifespan is affected by the amount of calories consumed [57]. Our data sugges<sup>t</sup> that this is an oversimplified assumption. On the contrary, lifespan is conditioned by EAA and NEAA dietary content, since this ratio modulates phenotypic modifications in different organs, especially adipose and muscle tissues, and induces profound biological modifications. If we evaluate a diet in terms of the extension of lifespan, the total AAs content provided by any dietary protein should not be considered the optimal parameter. Indeed, our study suggests that an elevated intake of NEAA, provided by normal nutritional and food proteins, negatively affects health and correlates with lifespan, at least in mice. So, the concept of "optimal nutrition", being very hard to define anyway because it depends on so many variables (the aims of diet, sex, age, health, environment, species etc.), should be deeply revised on the basis of the present data. We think that the ratio among EAA and NEAA is the most likely factor responsible for the health-promoting effects of proteins in any diet, and eventually for prolonged or reduced survival, at least in rodents.

These data also led us to the question of why some AAs are provided exclusively with diet, whereas others can be synthesized by the organism itself. The origin and evolutionary significance of the relationships between EAA and NEAA is not known. We sugges<sup>t</sup> as an hypothesis worth to be further explored, that the excessive introduction of NEAA may trigger certain natural selection mechanisms connected with the shortening of lifespan.

**Author Contributions:** Conceptualization, F.S.D., G.C. and C.R.; Methodology, G.C. and C.R.; Software, C.R. and G.C.; Formal Analysis, V.F., E.P., G.C.; Investigation, G.C. and C.R.; Data Curation, G.C., C.R. V.F.; Writing—Original Draft Preparation, G.C., C.R., V.F. and E.P.; Writing—Review, E.M., A.P., R.C. and F.S.D.; Writing—Editing. C.R., G.C., V.F.; Supervision, F.S.D.; Project Administration, F.S.D.; Funding Acquisition, F.S.D. and G.C.

**Funding:** This work was supported by grants from Nutriresearch s.r.l. (Milan, Italy) (G.C.). It was also partially supported by a gran<sup>t</sup> provided by Dolomite-Franchi S.p.a. (Marone, Brescia, Italy) to G.C.

**Conflicts of Interest:** All authors have no competing financial interests to declare.

**Compliance with Ethical Standards:** All animal procedures followed the European Communities Council Directive of November 24, 1986 (86/609/EEC), and complied with the Italian Ministry of Health and The National Animal Protection Guidelines.
