Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice

doi:10.1016/j.cmet.2023.10.005

. 2023 Nov 7;35(11):1976-1995.e6.

doi: 10.1016/j.cmet.2023.10.005.

Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice

Cara L Green¹, Michaela E Trautman², Krittisak Chaiyakul³, Raghav Jain⁴, Yasmine H Alam⁵, Reji Babygirija⁶, Heidi H Pak², Michelle M Sonsalla⁷, Mariah F Calubag⁶, Chung-Yang Yeh¹, Anneliese Bleicher¹, Grace Novak¹, Teresa T Liu⁸, Sarah Newman¹, Will A Ricke⁸, Kristina A Matkowskyj⁹, Irene M Ong¹⁰, Cholsoon Jang⁵, Judith Simcox¹¹, Dudley W Lamming¹²

Affiliations

¹ Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA.
² Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Nutrition and Metabolism Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
³ Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁴ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Integrated Program in Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁵ Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA.
⁶ Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁷ Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Comparative Biomedical Sciences Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁸ George M. O'Brien Center of Research Excellence, Department of Urology, University of Wisconsin, Madison, WI 93705, USA.
⁹ William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA; University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA.
¹⁰ Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53705, USA; University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA; Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI 53705, USA.
¹¹ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Integrated Program in Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706, USA.
¹² Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Nutrition and Metabolism Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA; Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA; Comparative Biomedical Sciences Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA; University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA. Electronic address: dlamming@medicine.wisc.edu.

PMID: 37939658
PMCID: PMC10655617
DOI: 10.1016/j.cmet.2023.10.005

Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice

Cara L Green et al. Cell Metab. 2023.

. 2023 Nov 7;35(11):1976-1995.e6.

doi: 10.1016/j.cmet.2023.10.005.

Authors

Affiliations

¹ Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA.
² Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Nutrition and Metabolism Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
³ Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53705, USA.
⁴ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Integrated Program in Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁵ Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA.
⁶ Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁷ Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Comparative Biomedical Sciences Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA.
⁸ George M. O'Brien Center of Research Excellence, Department of Urology, University of Wisconsin, Madison, WI 93705, USA.
⁹ William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Department of Pathology and Laboratory Medicine, University of Wisconsin-Madison, Madison, WI, USA; University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA.
¹⁰ Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, Madison, WI 53705, USA; University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA; Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Madison, WI 53705, USA.
¹¹ Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Integrated Program in Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA; Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, WI 53706, USA.
¹² Department of Medicine, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA; Nutrition and Metabolism Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA; Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI 53706, USA; Comparative Biomedical Sciences Graduate Program, University of Wisconsin-Madison, Madison, WI 53706, USA; University of Wisconsin Carbone Comprehensive Cancer Center, University of Wisconsin, Madison, WI 53705, USA. Electronic address: dlamming@medicine.wisc.edu.

PMID: 37939658
PMCID: PMC10655617
DOI: 10.1016/j.cmet.2023.10.005

Abstract

Low-protein diets promote health and longevity in diverse species. Restriction of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine recapitulates many of these benefits in young C57BL/6J mice. Restriction of dietary isoleucine (IleR) is sufficient to promote metabolic health and is required for many benefits of a low-protein diet in C57BL/6J males. Here, we test the hypothesis that IleR will promote healthy aging in genetically heterogeneous adult UM-HET3 mice. We find that IleR improves metabolic health in young and old HET3 mice, promoting leanness and glycemic control in both sexes, and reprograms hepatic metabolism in a sex-specific manner. IleR reduces frailty and extends the lifespan of male and female mice, but to a greater degree in males. Our results demonstrate that IleR increases healthspan and longevity in genetically diverse mice and suggests that IleR, or pharmaceuticals that mimic this effect, may have potential as a geroprotective intervention.

Keywords: aging; branched-chain amino acids; frailty; isoleucine; lifespan; metabolic health; mice; nutritional interventions; protein restriction.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests D.W.L. has received funding from, and is a scientific advisory board member of, Aeovian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases.

Figures

**Figure 1:. IleR reduces body weight and improves glycemic control in young HET3 mice.**
(A) Experimental scheme. (B-C) Male (B) and female (C) mouse weight. (D-K) Change in weight (D-E), fat mass (F-G), lean mass (H-I) and adiposity (J-K) over 14 weeks. (L-M) Glucose tolerance of male (L) and female (M) mice after 10 weeks on the indicated diets. (B-M) n=11–12 mice/group. (B-C) Tukey test following residual maximum-likelihood (REML) analysis model with Geisser-Greenhouse correction. (D-K) Tukey test following 2-way ANOVA. (L, M) Tukey test following ANOVA. (B-M) *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. P-values for the overall effect of Sex, Diet and the interaction represent the significant p-values from the two-way ANOVA. Data represented as mean ± SEM. See also Figure S1.

**Figure 2:. IleR alters energy balance in young HET3 mice.**
(A-B) Daily food consumption was measured in home cages after 3 weeks on diet in males (A) and females (B). (C-D) Calculated daily isoleucine intake for males (C) and females (D). (A-D) n=11–12 mice/group. (E-J) Energy expenditure measure in males (E-G) and females (H-J); n=8–10 mice/group. (A-F, H-I) Tukey test following 2-way ANOVA, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. P-values for the overall effect of Diet, Sex and the interaction represent the significant p-values from the two-way ANOVA. Data represented as mean ± SEM. See also Figure S2.

**Figure 3:. IleR reduces body weight and improves glycemic control in HET3 mice when started in mid-life.**
(A) Experimental scheme. At 24 months of age, a pre-selected cohort of mice in each group was euthanized for molecular analysis. (B-E) Male (B-C) and female (D-E) body weight and change from 6–24 months of age. (B, D) n varies by month; maximum 47–53 mice/group. (C) n=9–15 mice/group. (E) n=11–19 mice/group. (F-O) Body composition over the course of the lifespan and change from 6–24 months of age. (F, H, J, L) n varies by month; maximum 47–53 mice/group. (G, K, N) n=9–15 mice/group. (I, M, O) n=11–19 mice/group. (P-Q) Glucose tolerance after 16 months (22 months of age) on the indicated diets in male (P) and female (Q) mice; n=14–16 mice/group for males, 15–20 mice/group for females. (R-S) GTT area-under-the-curve (AUC) for male (R) and female (S) mice over the course of the experiment. Initial n=30–33/group in males, n=30–32 mice/group in females. (T-U) Insulin tolerance after 16 months on diet (22 months of age) on the indicated diets in male (T) and female (U) mice; n=19–27 mice/group for males, 19–29 mice/group for females. (B, D, F, H, J, L) Mixed-effects model with Geisser-Greenhouse correction followed by Dunnett’s multiple comparison correction test, (C, E, G, I, K, M-Q, T,U) Tukey test following ANOVA. (R, S) Tukey test following 2-way ANOVA. (B-U) *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. P-values for the overall effect of Diet, Age and the interaction represent the significant p-values from the two-way ANOVA. Data represented as mean ± SEM. See also Figure S3.

**Figure 4:. IleR alters energy balance in HET3 mice when started in mid-life.**
(A-C) Food and isoleucine consumption in male mice after 1 month on diet (A-B) and over the course of the experiment (C). (D-F) Food and isoleucine consumption in female mice after 1 month on diet (D-E) and over the course of the experiment (F). (A, B) n=17–18 mice/group. (C) n varies by month; maximum 47–51 mice/group (D, E) n=20–22 mice/group. (F) n varies by month; maximum 51–53 mice/group. (G-L) Energy expenditure in males and females after 18 months on diet. (G, I, K) n=23–32 mice/group. (H, J, L) n=24–32 mice/group. (M-R) Expression of the indicated genes in the iWAT of 24-month old male (M-O) and female (P-R) mice fed the indicated diets. n=4–8 mice/group. (A,B,D-E,G-J,M-R) Tukey test following ANOVA. (C, F) Tukey test following 2-way ANOVA. (A-R) *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. P-values for the overall effect of Diet, Age and the interaction represent the significant p-values from the two-way ANOVA. Data represented as mean ± SEM. See also Figure S4.

**Figure 5:. Correlation analysis identifies diet and age dependent and independent physiological and metabolic responses to a Low Ile diet.**
(A) Phenotypic measurements correlated with consumption of isoleucine (kcals) in each mouse (Pearson’s correlation) and clustered (hierarchical clustering). Phenotypic measurements that do not cluster as well appear in the middle of the correlation plot surrounded by a black box. (B-D) Phenotypic measurements from PCA of young and old mice of both sexes were visualized; positively correlated variables point to the same side of the plot, negatively correlated variables point to opposite sides of the plot. Length and color of arrows indicate contribution to the principal components. Young n=11–12 mice/group, old n=17–22 mice/group. (F-Q) Selected phenotypic measurements. Circulating FGF21 n=6–7 mice/group, Hepatic *Fgf21* n=6–8 mice/group, FBG n=11–21 mice/group. Tukey test following 2-way ANOVA; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. P-values for the overall effect of Sex, Diet and the interactions represent the significant p-values from the two-way ANOVA. Data represented as mean ± SEM. See also Table S2. See also Figure S5.

**Figure 6:. IleR and Low AA diets have sex- and age-dependent molecular impacts.**
(A-D) Top 50 significantly differentially expressed (SDE) genes for the indicated comparisons, with hierarchical clustering across all groups in each figure. Venn Diagrams below each heatmap indicating significant gene overlap for the comparisons, unadjusted p<0.05. n=4–8 mice/group. (E) Enriched transcriptomic pathways across all male groups (red = upregulated, blue = downregulated, grey = not significant). n=6–8 mice/group. (F-G) Spearman’s rank order correlation matrix of phenotypic, transcriptomic, metabolomic and lipidomic changes across young (F) and old (G) male Control vs Low Ile mice. Mega-clusters identified by hierarchical clustering are outlined in black (Table S5D). N=6–8 mice/group. (H) Genes from KEGG “Longevity regulating pathway” significantly altered in old and young male mice on Low AA or Low Ile diets. n=6–8 mice/group. See also Tables S3A–S5D. See also Figure S6.

**Figure 7:. IleR increases lifespan and improves healthspan in HET3 male mice.**
(A-B) Frailty of male (A) and female (B) mice over time. n varies by month; max n=44–49 mice/group. (C-D) Void spot assay conducted in 27-month-old male (C) and female (D) mice. n=2–14 mice/group. (E-H) Inverted cling test in 20-month-old mice and over time. (E-F) n=5–11 mice/group. (G-H) n varies by month; max n=16–22 mice/group. (I-L) Rotarod test in 20-month-old mice and over time. (I-J) n=5–10 mice/group. (K-L) n varies by month; max n=15–22 mice/group. (M-N) Tumor incidence at necropsy; n=31–38 mice/group. (O-P) Pathology observed at necropsy. n=22–32 mice/group. (Q-R) Kaplan-Meier survival curves by diet in male (Q) and female (R) mice. n=47–53 mice/group. (S-T) Average top 10% of lifespans in male (S) and female (T) mice by diet, n=8 mice/group. (U-V) Spearman’s rank correlation of frailty measured at 28 months with final lifespan across male (U) and female (V) mice. n=13–25 mice/group. (A-B, G-H, K-L) Mixed-effects model (REML) for time and diet with post-hoc Tukey testing for multiple comparisons. #P<0.05 Control vs Low AA, *P<0.05 Control vs Low Ile. (C-F, I-J, S-T) Tukey test following ANOVA; *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. (M-N) Two-sided chi-squared test, *P<0.05. (Q-R) Log-rank test for Control vs Low AA and Control vs Low Ile. Outlier removed from (D) using ROUT outlier test. Data represented as mean ± SEM. See also Tables S6A and S6C. See also Figure S7.

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References

1. Osborne TB, Mendel LB, and Ferry EL (1917). The Effect of Retardation of Growth Upon the Breeding Period and Duration of Life of Rats. Science 45, 294–295. 10.1126/science.45.1160.294. - DOI - PubMed
1. Weindruch R, Walford RL, Fligiel S, and Guthrie D (1986). The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. The Journal of nutrition 116, 641–654. 10.1093/jn/116.4.641. - DOI - PubMed
1. Colman RJ, Beasley TM, Kemnitz JW, Johnson SC, Weindruch R, and Anderson RM (2014). Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys. Nature communications 5, 3557. 10.1038/ncomms4557. - DOI - PMC - PubMed
1. McCay CM, Crowell MF, and Maynard LA (1935). The Effect of Retarded Growth Upon the Length of Life Span and Upon the Ultimate Body Size. The Journal of nutrition 10, 63–79. 10.1093/jn/10.1.63. - DOI - PubMed
1. Green CL, Lamming DW, and Fontana L (2022). Molecular mechanisms of dietary restriction promoting health and longevity. Nat Rev Mol Cell Biol 23, 56–73. 10.1038/s41580-021-00411-4. - DOI - PMC - PubMed

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[1] Osborne TB, Mendel LB, and Ferry EL (1917). The Effect of Retardation of Growth Upon the Breeding Period and Duration of Life of Rats. Science 45, 294–295. 10.1126/science.45.1160.294. - DOI - PubMed

[2] Osborne TB, Mendel LB, and Ferry EL (1917). The Effect of Retardation of Growth Upon the Breeding Period and Duration of Life of Rats. Science 45, 294–295. 10.1126/science.45.1160.294. - DOI - PubMed

[3] Weindruch R, Walford RL, Fligiel S, and Guthrie D (1986). The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. The Journal of nutrition 116, 641–654. 10.1093/jn/116.4.641. - DOI - PubMed

[4] Weindruch R, Walford RL, Fligiel S, and Guthrie D (1986). The retardation of aging in mice by dietary restriction: longevity, cancer, immunity and lifetime energy intake. The Journal of nutrition 116, 641–654. 10.1093/jn/116.4.641. - DOI - PubMed

[5] Colman RJ, Beasley TM, Kemnitz JW, Johnson SC, Weindruch R, and Anderson RM (2014). Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys. Nature communications 5, 3557. 10.1038/ncomms4557. - DOI - PMC - PubMed

[6] Colman RJ, Beasley TM, Kemnitz JW, Johnson SC, Weindruch R, and Anderson RM (2014). Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys. Nature communications 5, 3557. 10.1038/ncomms4557. - DOI - PMC - PubMed

[7] McCay CM, Crowell MF, and Maynard LA (1935). The Effect of Retarded Growth Upon the Length of Life Span and Upon the Ultimate Body Size. The Journal of nutrition 10, 63–79. 10.1093/jn/10.1.63. - DOI - PubMed

[8] McCay CM, Crowell MF, and Maynard LA (1935). The Effect of Retarded Growth Upon the Length of Life Span and Upon the Ultimate Body Size. The Journal of nutrition 10, 63–79. 10.1093/jn/10.1.63. - DOI - PubMed

[9] Green CL, Lamming DW, and Fontana L (2022). Molecular mechanisms of dietary restriction promoting health and longevity. Nat Rev Mol Cell Biol 23, 56–73. 10.1038/s41580-021-00411-4. - DOI - PMC - PubMed

[10] Green CL, Lamming DW, and Fontana L (2022). Molecular mechanisms of dietary restriction promoting health and longevity. Nat Rev Mol Cell Biol 23, 56–73. 10.1038/s41580-021-00411-4. - DOI - PMC - PubMed

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Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice

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Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice

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