Late-life isoleucine restriction promotes physiological and molecular signatures of healthy aging

Abstract

In defiance of the paradigm that calories from all sources are equivalent, we and others have shown that dietary protein is a dominant regulator of healthy aging. The restriction of protein or the branched-chain amino acid isoleucine promotes healthspan and extends lifespan when initiated in young or adult mice. However, many interventions are less efficacious or even deleterious when initiated in aged animals. Here, we investigate the physiological, metabolic, and molecular consequences of consuming a diet with a 67% reduction of all amino acids (Low AA), or of isoleucine alone (Low Ile), in male and female C57BL/6J.Nia mice starting at 20 months of age. We find that both diet regimens effectively reduce adiposity and improve glucose tolerance, which were benefits that were not mediated by reduced calorie intake. Both diets improve specific aspects of frailty, slow multiple molecular indicators of aging rate, and rejuvenate the aging heart and liver at the molecular level. These results demonstrate that Low AA and Low Ile diets can drive youthful physiological and molecular signatures, and support the possibility that these dietary interventions could help to promote healthy aging in older adults.

Keywords: BCAA; aging; dietary protein; dietary restriction; isoleucine; metabolic health; protein restriction.

Figure 1.. Low Ile and Low AA diets promote leanness in aged C57BL/6J.Nia mice.

(A) Experimental scheme. Three different amino acid defined diets were utilized: Control, Low Ile, and Low AA. Aged mice began their respective diets at 20 months of age, while Young mice were fed the Control diet starting at 6 months of age. (B–F) Body weight (B), with change in fat mass (C) and lean mass (D) of male mice was tracked over time. (E) Change in body weight, fat, and lean mass during the course of the experiment. (F) Body composition percentage. (B–F) n=10–13/group; (E–F) ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. (G) Food consumption of male mice throughout the experiment (n=5–6 cages/group, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. (H–L) Body weight (H), with change in fat mass (I) and lean mass (D) of female mice was tracked over time. (J) Change in body weight, fat, and lean mass during the course of the experiment. (K) Body composition percentage. (H–L) n=10–11/group; (K–L) ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. (M) Food consumption of female mice throughout the experiment. n=2–4 cages/group, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data presented as mean ± SEM.

Figure 2.. Late-life feeding of Low Ile and Low AA diets promote aspects of healthspan, particularly in male mice.

(A–C) Frailty score of male mice (A) was tracked throughout the experiment between 20 and 24 months of age (n=10–13/group, p-value represents the result of the indicated 2-way mixed-effects analysis). (B–C) Selected individual frailty categories, presented as the average of scores during the 3^rd and 4^th month of the experiment. (A-C) n=10–13/group at the beginning of the experiments, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. (D–E) Male rotarod (D) and inverted cling (E) performance were assessed between 22–23 months of age (n=8–11/group, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice). (F–G) Male Barnes Maze Test performance at 24 months of age (F). n=7–10/ group, p-value represents the effect of diet in the indicated 2-way ANOVA, acquisition time only. (G) Barnes Maze Test acquisition trial duration with loitering (test on loitering time only, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice). (H–J) Frailty score of female mice (H) was tracked throughout the experiment between 20 and 24 months of age (n=10–11/group, p-value represents the effect of diet in the indicated 2-way ANOVA). (I–J) Selected individual frailty categories, presented as the average of scores during the 3^rd and 4^th month of the experiment. (H–J) n=10–11/group at the beginning of the experiments, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. (K–L) Female rotarod (K) and inverted cling (L) performance were assessed between 22–23 months of age (n=8–11/group, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice). (M–O) Female Barnes Maze Test performance at 24 months of age (M). n=8–10/ group, p-value represents the effect of diet in the indicated 2-way ANOVA, acquisition time only. (O) Barnes Maze Test acquisition trial duration with loitering (test on loitering time only, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data presented as mean ± SEM.

Figure 3.. Late-life feeding of a Low Ile or Low AA diet improves glycemic control and boosts energy expenditure.

(A–D) Glucose tolerance test in male (A) and female (B) mice fed the indicated diets. Insulin tolerance test in male (C) and female (D) mice fed the indicated diets. n=10–13/group, ANOVA followed by Dunnett’s test vs. Aged Control-fed mice. (E–H) Metabolic chambers were used to determine the respiratory exchange ratio (RER) male (E) and females (F) and the energy expenditure normalized to body weight in males (G) and females (H). n=7–10/group, ANOVA conducted separately for the light and dark cycles followed by Dunnett’s vs. Aged Controlfed mice. (I–J) ANCOVA of energy expenditure with lean mass as a covariate in males (I) and females (J). n=7–10/group. (K–L) The serum FGF21 level at the end of the experiment, after 16 hr fasting overnight and 3 hr refeeding. n=5–7/group, ANOVA followed by Dunnett’s test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data presented as mean ± SEM.

Figure 4.. Low Ile and Low AA diet ameliorates multiple molecular indicators of aging rate in the liver.

(A–D) Diet and age alters aging rate indicators related to (A) mTOR signaling, (B) MAPK ERK signaling, (C) capindependent translation (CIT), and (D) lipid oxidation in the liver of male mice. (E–H) Diet and age alters aging rate indicators related to (E) mTOR signaling, (F) MAPK ERK signaling, (G) cap-independent translation (CIT), and (H) lipid oxidation in the liver of female mice. (I) Representative Western blots of the proteins analyzed. n=6/group, ANOVA followed by Dunnet’s test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data presented as mean ± SEM.

Figure 5.. Low Ile diet promotes youthful functional and molecular aspects of the female mice heart.

(A–E) Echocardiogram evaluation of female mice at 25 months of age. (A) Left ventricle posterior wall diameter, (B) left ventricle inner diameter, (C) stroke volume, (D) heart rate, and (E) cardiac output. n=5–10/group, *p<0.05, **p<0.01, ANOVA followed by Dunnett’s test. (F–G) Statistically significant phosphatidylglycerols (F) and all cardiolipins (G) in female mice hearts at 24 months of age after 4 months of dietary intervention. n=5/group, #p<0.05 Aged Control vs. Aged Low Ile, $p<0.05 Aged Control vs. Young Control, t-test. (H) LION lipid ontology analysis of significantly altered lipid species in the female mice heart. Data presented as mean ± SEM.

Figure 6.. Late-life feeding of a Low Ile diet rejuvenates the liver transcriptome.

(A–B) Volcano plots of differentially expressed genes in the liver of male (blue) and female (red) mice with age (A) and diet (B). All DEGs are determined at α = 0.10. (C) A summary of the effect of Low Ile on the age-driven differentially expressed gene sets in males (left) and females (right). Inner ring represents the genes up or downregulated by aging, while the outer ring represents the effect of a Low Ile diet on the DEGs altered with aging. n=5–6/group.