**1. Introduction**

Proteins are macromolecules serving a vast array of functions within the cell, and a balanced protein synthesis and degradation is crucial for preserving cell homeostasis. Hence, increased proteolysis and reduced protein synthesis have been associated with the severe depletion of body protein reserves, eventually resulting in malnutrition. This may impact the progression of several disease-associated

conditions [1]. For example, muscle wasting in people aged over 65 years old, hospitalized for a variety of chronic disease conditions, has been related with the disarrangement of protein balance [2].

Therefore, an adequate nutritional supply of protein may represent a relevant means for the managemen<sup>t</sup> of patients who are malnourished as a consequence of reduced food intake or increased metabolic demand (i.e., chronic and acute diseases).

Dietary proteins with their various compositions of amino acids (AAs) are nitrogen (N) sources for almost all organisms. From a nutritional point of view, AAs can be classified as non-essential (NEAA) or essential (EAA), depending on their potential to be synthesized endogenously or not [3,4], although the original definitions of the two terms focused on their e fficiency in promoting protein deposition [5,6].

Adequate dietary provision of AAs is essential for the growth, development, health, and survival of animals and humans [7]. It is also established that the administration of an adequate EAA+NEAA mix favors an increase in rat body weight, which is considered an appropriate parameter to evaluate the success of the animal in terms of growth and wellness [3,7–9]. However, this concept should be profoundly reconsidered if associated to lifespan. Indeed, caloric restriction and short-term caloric deficit improve the e fficiency of mitochondria in humans just as in rodents, which might have the potential to increase their longevity [10].

Previous work by our group showed that the supplementation of a laboratory standard diet, containing special EAA formulations, increased rodents' lifespan in older mice [11]. At the molecular level, such a dietary regimen was able to promote mitochondrial biogenesis and to induce organelle ultrastructural changes in the heart, skeletal muscle and adipose tissue [12–14]. In addition, an EAA-rich diet prevented liver damage induced by chronic ethanol consumption [15,16], boosted the e ffects of rosuvastatin on the kidneys [17] and accelerated wound healing in late middle-aged rats, by promoting collagen integrity [18]. Furthermore, in vitro data showed that variations in the EAA/NEAA ratio might be crucial for the fate of cancer cells via the induction of apoptosis [19]. Taken as a whole, these findings indicate that varying dietary EAA/NEAA ratios may a ffect cell metabolism.

While dietary proteins are the major source of AAs, the exact amounts of EAA contained in animal and vegetable proteins, introduced daily with diet, are di fficult to establish. Indeed, the EAA content varies considerably depending on the source. However, any dietary protein has an EAA/NEAA ratio ≤0.9 at best. In other words, we introduce a very large amount of NEAA to meet the need for EAA and the excess of NEAA must be eliminated through complex metabolic pathways [20].

Many individual AAs have been tested by dietary exclusion studies in rodents, in order to demonstrate their influence on metabolism and health. For example, the restriction of methionine increases the expression of FGF21 with fall-out e ffects on insulin-dependent glucose uptake [21]. Other studies have been carried out in rodents with the restriction of branched-chain-AAs [22] or leucine alone [23], assessing their e ffects on various metabolic aspects (e.g., improving glucose tolerance or white and brown adipose tissue remodeling, respectively). The e ffects of caloric, protein and carbohydrate restriction on animal survival and welfare have also been studied [24–26]. While the restriction of individual AAs and groups of AAs has been looked at extensively in terms of metabolism and ageing, no studies have been performed specifically about EAA/NEAA ratio in the context of longevity.

Recent data from our studies on late middle-aged animals, fed for one month with iso-caloric and iso-nitrogenous diets containing di fferent EAA/NEAA ratios, showed significant changes in body mass and blood parameters [27]. We therefore investigated the lifelong e ffects on male mice of iso-caloric and iso-nitrogenous special diets containing five specific EAA/NEAA ratios, compared to a standard laboratory rodent diet.

#### **2. Materials and methods**
