Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

doi:10.1038/s41467-020-16568-z

. 2020 Jun 9;11(1):2894.

doi: 10.1038/s41467-020-16568-z.

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Yann W Yap^#¹, Patricia M Rusu^#¹, Andrea Y Chan¹, Barbara C Fam², Andreas Jungmann^{3

4}, Samantha M Solon-Biet⁵, Christopher K Barlow⁶, Darren J Creek^{6

7}, Cheng Huang⁶, Ralf B Schittenhelm⁶, Bruce Morgan⁸, Dieter Schmoll⁹, Bente Kiens¹⁰, Matthew D W Piper¹¹, Mathias Heikenwälder¹², Stephen J Simpson⁵, Stefan Bröer¹³, Sofianos Andrikopoulos², Oliver J Müller^{4

14}, Adam J Rose¹⁵

Affiliations

¹ Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.
² Department of Medicine (Austin Health), University of Melbourne, Heidelberg, VIC, 3084, Australia.
³ Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany.
⁴ German Center for Cardiovascular Research (DZHK), Partner sites Kiel and Heidelberg, Germany.
⁵ Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia.
⁶ Biomedical Proteomics and Metabolomics Facility and the Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.
⁷ Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, 3052, Australia.
⁸ Institute for Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany.
⁹ Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, Frankfurt am Main, 65926, Germany.
¹⁰ Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, 2100, Copenhagen, Denmark.
¹¹ School of Biological Sciences, School of Life and Environmental Sciences, Monash University, Clayton, VIC, 3800, Australia.
¹² Division of Chronic Inflammation and Cancer, German Cancer Research Center, 69120, Heidelberg, Germany.
¹³ Research School of Biology, Australian National University, Canberra, ACT, 0200, Australia.
¹⁴ Department of Internal Medicine III, University of Kiel, Kiel, Germany.
¹⁵ Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia. [email protected].

^# Contributed equally.

PMID: 32518324
PMCID: PMC7283339
DOI: 10.1038/s41467-020-16568-z

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Yann W Yap et al. Nat Commun. 2020.

. 2020 Jun 9;11(1):2894.

doi: 10.1038/s41467-020-16568-z.

Authors

Affiliations

¹ Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.
² Department of Medicine (Austin Health), University of Melbourne, Heidelberg, VIC, 3084, Australia.
³ Department of Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany.
⁴ German Center for Cardiovascular Research (DZHK), Partner sites Kiel and Heidelberg, Germany.
⁵ Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia.
⁶ Biomedical Proteomics and Metabolomics Facility and the Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.
⁷ Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, 3052, Australia.
⁸ Institute for Biochemistry, Centre for Human and Molecular Biology (ZHMB), Saarland University, 66123, Saarbrücken, Germany.
⁹ Sanofi-Aventis Deutschland GmbH, Industriepark Hoechst, Frankfurt am Main, 65926, Germany.
¹⁰ Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, 2100, Copenhagen, Denmark.
¹¹ School of Biological Sciences, School of Life and Environmental Sciences, Monash University, Clayton, VIC, 3800, Australia.
¹² Division of Chronic Inflammation and Cancer, German Cancer Research Center, 69120, Heidelberg, Germany.
¹³ Research School of Biology, Australian National University, Canberra, ACT, 0200, Australia.
¹⁴ Department of Internal Medicine III, University of Kiel, Kiel, Germany.
¹⁵ Department of Biochemistry and Molecular Biology, Metabolism, Diabetes and Obesity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia. [email protected].

^# Contributed equally.

PMID: 32518324
PMCID: PMC7283339
DOI: 10.1038/s41467-020-16568-z

Abstract

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

PubMed Disclaimer

Conflict of interest statement

D.S. is an employee of Sanofi-Aventis Deutschland GmbH, a pharmaceutical company. All other authors declare no competing interests.

Figures

**Fig. 1. Dietary amino acids are required for the systemic metabolic effects of dietary protein dilution.**
a Serum urea levels of mice in response to a 3-week treatment with diets containing 20% energy from protein (20P), 5% energy from protein (5P), and 5% energy from protein and 15% energy from amino acids to match that of 20P. Data are mean and SEM; n = 5 individual mice per group. Data were analysed by one-way ANOVA with Holm–Sidak post-hoc tests. Different than 20P: *P < 0.05, **P < 0.01, ***P < 0.001. Different than 5P: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. b Feed efficiency of mice as in (a). c Energy expenditure over the different day phases of mice as in (a). d The rate of O₂ consumption (VO₂) over the different day phases of mice as in (a). e The rate of CO₂ production (VCO₂) over the different day phases of mice as in (a). f Scatter plot of energy expenditure (EE) versus body weight of mice as in (a). g Physical activity as assessed by laser beam breaks across three physical dimensions (sumXYZ) over the different day phases of mice as in (a). h Serum fibroblast growth factor 21 (FGF21) levels of mice as in (a). i Insulin sensitivity index during fasting (ISI(f)) of mice as in (a).

**Fig. 2. Dietary essential amino acid restriction, independent from non-essential amino acid or carbohydrate supply, dictates the systemic metabolic response to dietary protein dilution.**
a Hepatic portal vein amino acid levels in response to refeeding a low-protein diet following a week of diet adaptation. Data (mean and SEM) are represented as a proportion of the control group fed a normal protein diet (20%P). n = 5/group derived from samples of individual mice. Data were analysed by Student’s t-test. Different than 20%P, *P < 0.05. b Liver tissue amino acid levels in response to refeeding a low-protein diet following a week of diet adaptation. Data are represented as a proportion of the control group fed a normal protein diet (20%P). n = 5/group derived from samples of individual mice. Data were analysed by Student’s t-test. Different than 20%P, *P < 0.05. c Nutrient source breakdown as % contribution to total energy for the experimental groups. EAA: essential amino acids. NEAA: non-essential amino acids. d Urinary urea output of mice in response to a 3-week treatment with diets as per the protocol of SF1A containing nutrient energy sources as in (c). Data are mean and SEM; n = 5 individual mice per group. Data were analysed by one-way ANOVA with Holm–Sidak post-hoc tests. Different than 20P: *P < 0.05, **P < 0.01, ***P < 0.001. Different than 5P: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. e Feed efficiency of mice as in (d). f Energy expenditure of mice as in (d). g Serum fibroblast growth factor 21 (FGF21) levels of mice as in (d). h Blood glucose levels during an intraperitoneal glucose tolerance test (ipGTT) of mice as in (d). i Plasma insulin levels during an intraperitoneal glucose tolerance test (ipGTT) of mice as in (d). j Insulin sensitivity index during fasting (ISI(f)) of mice as in (d).

**Fig. 3. Certain essential amino acids including threonine and tryptophan are sufficient and necessary for the systemic metabolic effects of dietary protein dilution.**
a Feed efficiency of mice in response to a 3-week treatment with diets containing 18% from amino acids (normal amino acid; NAA), 4.5% essential AA (LEAA; as of diet E in Fig. 2c), and LEAA supplemented with either lysine, threonine, and tryptophan (LEAA + KTW), phenylalanine, histidine, and methionine (LEAA + FHM), or isoleucine, leucine, and valine (LEAA + ILV), all with other AA equally adjusted to give 18% AA in total. Data are mean and SEM (n = 6 individual mice per group). Data were analysed by one-way ANOVA with Holm–Sidak post-hoc tests. Different than diet NAA: *P < 0.05, **P < 0.01, ***P < 0.001. Different than diet LEAA: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. b Energy expenditure of mice as in (a). c Serum fibroblast growth factor 21 (FGF21) levels of mice as in (a). d Insulin sensitivity index during fasting (ISI(f)) of mice as in (a). e Feed efficiency of mice in response to a 3-week treatment with diets containing 18% from amino acids (AA; NAA), 4.5% essential AA (LEAA; as of diet E in Fig. 2c), and diet singly with restricted amounts of lysine (LK), threonine (LT), and tryptophan (LW), all with other AA equally adjusted to give 18% AA in total. Data are mean and SEM; n = 6 individual mice per group. Data were analysed by one-way ANOVA with Holm–Sidak post-hoc tests. Different than diet NAA: *P < 0.05, **P < 0.01, ***P < 0.001. Different than diet LEAA: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. f Energy expenditure of mice as in (e). g Serum FGF21 levels of mice as in (e). h ISI(f) of mice as in (e). i Feed efficiency of mice in response to a 3-week treatment with diets containing 18% from amino acids (normal amino acid; NAA), 4.5% AA (LAA; as of diet B in Fig. 2c), and LAA supplemented with threonine and tryptophan while keeping total AA at 4.5% (LAA(TW)). Data are mean and SEM (n = 5 individual mice per group). Data were analysed by one-way ANOVA with Holm–Sidak post-hoc tests. Different than NAA: *P < 0.05, **P < 0.01, ***P < 0.001. Different than LAA: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. j Energy expenditure of mice as in (e). k Serum FGF21 levels of mice as in (e). l ISI(f) of mice as in (e).

**Fig. 4. The systemic metabolic response to dietary AA restriction is conserved in mature male and female mice.**
a The change in body, fat, and lean mass of 6-month-old male (shown left) mice in response to an 8-week treatment with diets containing 18% from amino acids (normal amino acid; NAA), 4.5% AA (LAA; as of diet B in Fig. 2c), 4.5% essential AA (LEAA; as of diet E in Fig. 2c), and a diet low in threonine but with matching total AA to NAA (LT). Data are mean and SEM (n = 5 individual mice per group). Data were analysed by one-way ANOVA with Holm–Sidak post-hoc tests. Different than NAA: ^aP < 0.05, ^aaP < 0.01, ^aaaP < 0.001. Different than LAA: ^bP < 0.05, ^bbP < 0.01, ^bbbP < 0.001. Different than LEAA: ^cP < 0.05, ^ccP < 0.01, ^cccP < 0.001. b The change in body, fat, and lean mass of 6-month-old female (shown left) mice treated as in (a). c Tissue weights of mice at the end of the treatment as in (a). d Tissue weights of mice at the end of the treatment as in (b). e Feed efficiency during the 8-week treatment of mice as in (a). f Feed efficiency during the 8-week treatment of mice as in (b). g Energy expenditure of mice as in (a). h Energy expenditure of mice as in (b). i Serum fibroblast growth factor 21 (FGF21) levels of mice as in (a). j Serum FGF21 of mice as in (b). k Insulin sensitivity index during fasting (ISI(f)) of mice as in (a). l ISI(f) of mice as in (b).

**Fig. 5. Threonine restriction is a common feature of other models of systemic AA restriction and retards obesity-induced metabolic dysfunction in mice.**
a Hepatic portal vein serum amino acid (AA) concentrations in Slc6a19 knockout (−/−) or wildtype (+/+) littermate mice in the refed state on a standard control diet. Data are mean and SEM; n = 5 individual mice per group group. Data were analysed by a Student’s t-test. Different than +/+: *P < 0.05. b Plasma FGF21 levels from Slc6a19 knockout (−/−) or wildtype (+/+) littermate mice in the refed state following intraperitoneal administration of the amino acids threonine and tryptophan (TW), phenylalanine and histidine (FH), or vehicle (VEH: saline solution) on a standard control diet. Data are mean and SEM; n = 5 individual mice per group. Data were analysed by a two-way repeated measures ANOVA. Different than +/+: *P < 0.05, **P < 0.01, ***P < 0.001. Different than VEH: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. c Blood glucose levels during a 4-week treatment of New Zealand Obese mice fed diets containing 18% from amino acids (normal amino acid; NAA) or a diet low in threonine but with matching total AA to NAA (LT). Data are mean and SEM; n = 8 individual mice per group. Data were analysed by one-way repeated measures ANOVA. Different than NAA: *P < 0.05, **P < 0.01, ***P < 0.001. d Serum triglyceride (TG) levels at the end of mice at the end of treatment as in (c). Data were analysed by a Student’s t-test. Different than NAA: *P < 0.05, **P < 0.01, ***P < 0.001. e Serum fibroblast growth factor 21 (FGF21) levels of mice at the end of treatment as in (c).

**Fig. 6. Liver-derived fibroblast growth factor 21 is necessary for the systemic metabolic remodelling with dietary threonine restriction.**
a Serum fibroblast growth factor 21 (FGF21) levels of Fgf21fl/fl mice at the end of an 8-week treatment with diets containing 18% from amino acids (AA; NAA) or low threonine with other AA equally adjusted to give 18% AA in total (LT); with pre-treatment with adeno-associated viruses to express Cre-recombinase (AAV-CRE) or green fluorescent protein (AAV-GFP) in an hepatocyte-selective manner. Data are mean and SEM (n = 6 NAA × AAV-GFP; n = 7 LAA AAV-GFP; n = 6 LT × AAV-CRE; n = 8 LT × AAV-CRE). Data were analysed by two-way ANOVA with Holm–Sidak post-hoc tests. Different than NAA: *P < 0.05, **P < 0.01, ***P < 0.001. Different than AAV-GFP: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. b Feed efficiency during the 8-week treatment of mice as in (a). c Energy expenditure of mice as in (a). d Insulin sensitivity index during fasting (ISI(f)) of mice as in (a).

**Fig. 7. Enforced hepatic threonine biosynthetic capacity reverses the systemic metabolic effects to dietary threonine restriction.**
a Feed efficiency of mice in response to a 3-week treatment with diets containing 18% from amino acids (normal amino acid; NAA; yellow bars) and a diet with restricted amounts of threonine (LT; green bars), following prior treatments with adeno-associated viruses to transduce the liver to express yeast threonine biosynthetic enzymes (AAV-yTHR1 + THR4) or a negative control (AAV-GFP). Data are mean and SEM (N = 6 individual mice per group). Data were analysed by two-way ANOVA with Holm–Sidak post-hoc tests. Different than diet NAA: *P < 0.05, **P < 0.01, ***P < 0.001. Different than AAV-GFP: ^#P < 0.05, ^##P < 0.01, ^###P < 0.001. b Energy expenditure of mice as in (a). c Serum fibroblast growth factor 21 (FGF21) levels of mice as in (a). d Insulin sensitivity index during fasting (ISI(f)) of mice as in (a).

See this image and copyright information in PMC

Cited by

Meta-analysis links dietary branched-chain amino acids to metabolic health in rodents.
Solon-Biet SM, Griffiths L, Fosh S, Le Couteur DG, Simpson SJ, Senior AM. Solon-Biet SM, et al. BMC Biol. 2022 Jan 14;20(1):19. doi: 10.1186/s12915-021-01201-2. BMC Biol. 2022. PMID: 35031039 Free PMC article.
The Role of Reduced Methionine in Mediating the Metabolic Responses to Protein Restriction Using Different Sources of Protein.
Fang H, Stone KP, Ghosh S, Forney LA, Gettys TW. Fang H, et al. Nutrients. 2021 Jul 29;13(8):2609. doi: 10.3390/nu13082609. Nutrients. 2021. PMID: 34444768 Free PMC article.
Dietary restriction of isoleucine increases healthspan and lifespan of genetically heterogeneous mice.
Green CL, Trautman ME, Chaiyakul K, Jain R, Alam YH, Babygirija R, Pak HH, Sonsalla MM, Calubag MF, Yeh CY, Bleicher A, Novak G, Liu TT, Newman S, Ricke WA, Matkowskyj KA, Ong IM, Jang C, Simcox J, Lamming DW. Green CL, et al. Cell Metab. 2023 Nov 7;35(11):1976-1995.e6. doi: 10.1016/j.cmet.2023.10.005. Cell Metab. 2023. PMID: 37939658 Free PMC article.
Macronutrient Determinants of Obesity, Insulin Resistance and Metabolic Health.
Wali JA, Solon-Biet SM, Freire T, Brandon AE. Wali JA, et al. Biology (Basel). 2021 Apr 16;10(4):336. doi: 10.3390/biology10040336. Biology (Basel). 2021. PMID: 33923531 Free PMC article. Review.
Kinetic proteomics identifies targeted changes in liver metabolism and the ribo-interactome by dietary sulfur amino acid restriction.
Jonsson WO, Borowik AK, Pranay A, Kinter MT, Mirek ET, Levy JL, Snyder EM, Miller BF, Anthony TG. Jonsson WO, et al. Geroscience. 2023 Aug;45(4):2425-2441. doi: 10.1007/s11357-023-00758-w. Epub 2023 Mar 28. Geroscience. 2023. PMID: 36976488 Free PMC article.

See all "Cited by" articles

References

1. Rose WC. II. The sequence of events leading to the establishment of the amino acid needs of man. Am. J. Public Health Nation’s Health. 1968;58:2020–2027. - PMC - PubMed
1. Popkin BM, Adair LS, Ng SW. Global nutrition transition and the pandemic of obesity in developing countries. Nutr. Rev. 2012;70:3–21. - PMC - PubMed
1. Mitchell SE, et al. The effects of graded levels of calorie restriction: I. Impact of short term calorie and protein restriction on body composition in the C57BL/6 mouse. Oncotarget. 2015;6:15902–15930. - PMC - PubMed
1. Solon-Biet SM, et al. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab. 2014;19:418–430. - PMC - PubMed
1. Solon-Biet SM, et al. Dietary protein to carbohydrate ratio and caloric restriction: comparing metabolic outcomes in mice. Cell Rep. 2015;11:1529–1534. - PMC - PubMed

Publication types

Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions
Actions
Actions

LinkOut - more resources

Full Text Sources
Molecular Biology Databases
- Mouse Genome Informatics (MGI)
- NIAID Data Ecosystem - Find datasets on Infectious and Immune-mediated Diseases

[1] Rose WC. II. The sequence of events leading to the establishment of the amino acid needs of man. Am. J. Public Health Nation’s Health. 1968;58:2020–2027. - PMC - PubMed

[2] Rose WC. II. The sequence of events leading to the establishment of the amino acid needs of man. Am. J. Public Health Nation’s Health. 1968;58:2020–2027. - PMC - PubMed

[3] Popkin BM, Adair LS, Ng SW. Global nutrition transition and the pandemic of obesity in developing countries. Nutr. Rev. 2012;70:3–21. - PMC - PubMed

[4] Popkin BM, Adair LS, Ng SW. Global nutrition transition and the pandemic of obesity in developing countries. Nutr. Rev. 2012;70:3–21. - PMC - PubMed

[5] Mitchell SE, et al. The effects of graded levels of calorie restriction: I. Impact of short term calorie and protein restriction on body composition in the C57BL/6 mouse. Oncotarget. 2015;6:15902–15930. - PMC - PubMed

[6] Mitchell SE, et al. The effects of graded levels of calorie restriction: I. Impact of short term calorie and protein restriction on body composition in the C57BL/6 mouse. Oncotarget. 2015;6:15902–15930. - PMC - PubMed

[7] Solon-Biet SM, et al. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab. 2014;19:418–430. - PMC - PubMed

[8] Solon-Biet SM, et al. The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell Metab. 2014;19:418–430. - PMC - PubMed

[9] Solon-Biet SM, et al. Dietary protein to carbohydrate ratio and caloric restriction: comparing metabolic outcomes in mice. Cell Rep. 2015;11:1529–1534. - PMC - PubMed

[10] Solon-Biet SM, et al. Dietary protein to carbohydrate ratio and caloric restriction: comparing metabolic outcomes in mice. Cell Rep. 2015;11:1529–1534. - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Affiliations

Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

LinkOut - more resources

Full Text Sources

Molecular Biology Databases

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

LinkOut - more resources

Full Text Sources

Molecular Biology Databases