**3. Results**

The average survival time was markedly reduced in animals fed with the two diets where EAA were absent or deficient. Indeed, NEAA-100% diet allowed a lifespan of 44.56 ± 3.85 days, while the lifespan of EAA-30%-fed animals was slightly prolonged at 53 ± 1.97 days, on average <20%. Animals fed with Casein-Prot and Casein-AA diets survived for maximum 14 and 16 months, respectively. StD-fed animals survived for maximum 22 months. The longest survival time (25 months) was observed for the EAA-100%-fed animals (Figure 1). The percentage of EAA in the diet is correlated with survival (*r* = 0.901, *p* < 0.001).

**Figure 1.** Percentage of mice's survival according to diet. Animals fed with NEAA-100% and EAA-30% diets (orange and brown lines) had shorter lifespans when compared to StD (blue line). It is interesting to observe the different survival curves between the animals fed with the diet containing casein whole protein (light-green line) and those fed with free AAs of casein (dark-green line). In addition, note the longest survival of the mice fed with EAA-100% diet (red line). Mantel–Cox test: Casein-AA vs. Casein-Prot, z = 3.95, p < 0.001; Casein-AA vs. StD, z = 5.17, *p* < 0.001; EAA-100% vs. StD, z = 2.28, *p* = 0.0226; EAA-30% vs. NEAA-100%, z = 0.21, *p* = 0.83.

#### *3.1. Parameters Evaluation after 2 Months*

#### 3.1.1. Body Weight (BW) and Length (BL)

BW and BL are determined by the quality of nitrogen intake. For the diets poorest in EAA, NEAA-100% and EAA-30% diets, there is a rapid mortality correlating with BW loss (r = 0.92, *p* < 0.000) and also BL (r = 0.99, *p* < 0.000) when compared to all diets. A relatively modest increase in EAA provided by Casein-AA or Casein-Prot diet (which each contain about 19% more EAA than EAA-30% diet), drives a BL increase comparable to those observed in EAA-100% diet and StD. On the contrary, BW is most increased by StD and either Casein-Prot or Casein-AA diet. Instead, EAA-100% diet allows a growth in BL comparable to StD, Casein-Prot and Casein-AA.

EAA-100% diet induced the smallest BW increase among animals fed all diets compatible with prolonged lifespan. That is, the BW of EAA-100%-fed animals, while increasing if compared with the diets poorest in EAA (NEAA-100% and EAA-30%), is significantly less increased (p < 0.001) when compared to both StD and either Casein-Prot or Casein-AA diets.

The BW and BL of mice fed with Casein-Prot and Casein-AA diets were comparable to those of animals fed StD (see Table 2).



WAT, white adipose tissue. BAT, brown adipose tissue.

#### 3.1.2. Food and Water Consumption

The amounts of food and water consumed daily were influenced markedly by di fferent diets. NEAA-100%-fed groups rapidly stopped growing in BL and showed a dramatically rapid BW loss similar to that of EAA-30% (Figure 2A), although the food consumption and therefore caloric intake of NEAA-100% during the first two weeks was significantly higher than any other diet except StD. With the proceeding of BW loss, and particularly in the 80% animals surviving after the sixth week, food consumption also declined, and all animals died in the following two weeks (Figure 2A,B). In NEAA-100%-fed animals, a striking di fference between the grams of daily food intake, and thus calories, and BW was evident. Indeed, NEAA-100% and EAA-30%-fed animals progressively decreased in BW although their caloric intake was similar to that of EAA-100%-fed animals. Indeed, the correlation (*r*) between NEAA-100% or EAA-30% food intake and BW was –0.3 or –0.5, respectively. On the contrary, there was a higher correlation between EAA-100% food intake and BW (r = 0.9, *p* < 0.001) (Figure 2A,B). Only in the case of the NEAA-100% group, water consumption increased significantly from the first week of treatment (p < 0.001, about six-fold), and then decreased progressively to around the average water intake in StD-fed animals, before dying (Figure 2C).

**Figure 2.** Comparison between BW ( **A**), food (g/day) (**B**), and water (mL/day) consumption ( **C**) (mean ± sd) of animals fed with NEAA-100% and EAA-30% diets, StD and EAA-100% diet after 6 weeks. NEAA-100% and EAA-30% diets drive rapid BW decrease, whereas EAA-100% diet slowly increases BW compared to StD ( **A**). Note that EAA-100% diet was consumed in the same amount as NEAA-100% and EAA-30% diets (**B**). NEAA-100%-fed animals showed a higher water intake compared to StD, whereas EAA-30% and EAA-100%-fed animals had a lower water consumption than StD (**C**). Black square, EAA-100%; gray rhombus, NEAA-100%; gray triangle, EAA-30%; gray circle, StD. ANOVA and Bonferroni t-test, \* *p* < 0.05 vs. all diets.

#### 3.1.3. Organ Weights

The weights of specific organs (organ weight, OW) in animals fed with NEAA-100%, EAA-30% and EAA-100% diets were significantly lower than those in animals fed with StD. The weights of the kidneys, livers, BAT and *triceps surae* of the animals fed with NEAA-100% and EAA-30% diets were significantly less than those of the EAA-100%-fed animals. The OW of animals was similar in both NEAA-100% and EAA-30% diets but, interestingly, the rpWAT was near absent in those groups (Table 2).

#### 3.1.4. Blood and Urinary Parameters

The two groups fed with NEAA-100% and EAA-30% diets had altered blood and urine parameters when compared to StD and EAA-100%-fed mice. This is especially evident in the reduction in blood concentration of hemoglobin and albumin, in the increase in neutrophils to lymphocytes ratio (NLR), and in the reduction of albumin and increase of creatinine in the urine. Blood and urine parameters from the EAA-100%-fed group did not di ffer from those of the StD-fed group, except for the Hpg value which was found to be lower (Table 3).


#### *3.2. Parameter Evaluation after 12 Months.*

#### 3.2.1. Body Weight and Length

After one year of follow-up, BW and length were still determined by the quality of nitrogen intake. The growth of mice on Casein-Prot-based diets did not differ from that of StD-fed mice. On the contrary, the EAA-100%-fed animals showed a more modest increase in BW, which remained significantly lower than the BW of the StD, Casein-Prot and EAA-100%-fed groups (Figure 3A, Table 2).

**Figure 3.** BW (**A**), food (**B**) and water (**C**) consumption (mean ± sd) according to StD, EAA-100%, Casein-Prot and Casein-AA diets during the whole survival period of mice. After about eight months, EAA-100%-fed mice significantly increase their water consumption (**C**), although food consumption (**B**) and BW (**A**) remain unchanged. See text for description. Black square, EAA-100%; gray rhombus, StD; gray triangle, Casein-AA; gray circle, Casein-Prot. ANOVA and Bonferroni t-test: \* *p* < 0.05 vs. all diets.

#### 3.2.2. Food and Water Consumption

The Casein-AA, Casein-Prot and EAA-100%-fed animals showed a substantially comparable daily food intake (g/day). All those groups registered a daily food consumption that was significantly reduced compared to that of the StD-fed animals, from the 4th month onwards. Water consumption (ml/day) was similar among StD, Casein-AA and Casein-Prot, whereas EAA-100%-fed mice showed a significantly increased water consumption starting from the eighth month and continuing all along the remaining follow-up (Figure 3C).

## 3.2.3. Organ Weights

Comparable to BW, mice from the EAA-100% group showed the lowest OW except for spleen. In particular, kidney and *triceps surae* weights were significantly lower than those recorded in all other groups. The heart weights of the EAA-100%-fed animals were significantly lower than in the StD and Casein-AA groups, whereas the rpWAT weights were lower in EAA-100% than in both StD and Casein-Prot groups. Liver weight was significantly higher in the StD and Casein-Prot and Casein-AA groups than in EAA-100%. BAT weight in EAA-100% was significantly lower than the Casein-Prot group, while animals fed with Casein-AA and Casein-Prot diets had a significantly increased *triceps surae* weight, when compared to the StD group. Interestingly, the Casein-AA diet induced a significant weight decrease for rpWAT, similar to that observed in EAA-100%-fed animals. On the other hand, rpWAT weights of mice fed Casein-Prot did not differ from those of mice fed with StD (Table 1).

#### 3.2.4. Blood and Urinary Parameters

Serum concentrations of Hpg were markedly increased (about fifty times, p < 0.001) in animals fed with the Casein-AA and Casein-Prot diets as compared to StD-fed animals. In addition, mice fed with the Casein-AA and Casein-Prot diets had increased levels of serum and lowered levels of urinary albumin and creatinine. On the other hand, EAA-100%-fed animals had normal blood parameters and urinary concentrations of albumin and creatinine, and also showed a significantly lower Hpg level even compared to the StD-fed mice (Table 3).

#### *3.3. Parameter Evaluation after 18 Months.*

#### 3.3.1. Body Weight and Length.

After 18 months, only animals fed with the StD and EAA-100% diets survived (Figure 1). EAA-100%-fed animals had lower BW than the StD-fed animals, whereas BL did not vary (Table 2). The fur appearance and spontaneous motor activity of EAA-100%-fed animals seemed to be preserved far better than in StD-fed ones. In fact, the animals fed with EAA-100% showed greater vitality than the others and often clung to the cage, keeping themselves suspended without difficulty.

#### 3.3.2. Food and Water Consumption

The food consumption of EAA-fed animals was consistently lower than in StD-fed ones (Figure 3B). On the contrary, EAA-100%-fed animals showed a progressively and significantly increased water consumption (Figure 3C).
