Decreased Consumption of Branched-Chain Amino Acids Improves Metabolic Health

Abstract

Protein-restricted (PR), high-carbohydrate diets improve metabolic health in rodents, yet the precise dietary components that are responsible for these effects have not been identified. Furthermore, the applicability of these studies to humans is unclear. Here, we demonstrate in a randomized controlled trial that a moderate PR diet also improves markers of metabolic health in humans. Intriguingly, we find that feeding mice a diet specifically reduced in branched-chain amino acids (BCAAs) is sufficient to improve glucose tolerance and body composition equivalently to a PR diet via metabolically distinct pathways. Our results highlight a critical role for dietary quality at the level of amino acids in the maintenance of metabolic health and suggest that diets specifically reduced in BCAAs, or pharmacological interventions in this pathway, may offer a translatable way to achieve many of the metabolic benefits of a PR diet.

Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. A naturally sourced low protein diet improves the metabolic health of mice

Glucose (A) and pyruvate (B) tolerance tests on male C57BL/6J mice fed a naturally sourced 21% or 7% protein diet for 3 or 5 weeks, respectively (a, b: n=6-9/group; Tukey-Kramer test following ANOVA, * = p < 0.05). Mice were fasted overnight and (C) blood glucose and (D) insulin were measured, and (E) the HOMA2-IR was calculated after 6 weeks on the specified diets. (F) Food consumption was measured after 2 weeks on the 21% or 7% protein diets. (G) Weight was determined immediately prior to diet start and after 3 and 8 weeks (c-g: n=6-9/group; two-tailed t-test, * = p < 0.05). Error bars represent standard error.

Figure 2. Reduced consumption of BCAAs improves glycemic control

(A) Glucose tolerance test (GTT) on male C57BL/6J mice fed either an amino-acid defined Control diet, or one of two diets (Low AA and ExLow AA) with reduced amino acid content for three weeks (n=9 mice/group; for GTT, Dunnett’s test following ANOVA, a = p < 0.05 Control vs. Low AA, b = p < 0.05 Control vs. ExLow AA; for AUC, Dunnett’s test following ANOVA, * = p < 0.05). (B) GTT on male C57BL/6J mice fed a Control diet, a Low AA diet, or a diet in which either leucine (Low Leu) or all BCAAs (Low BCAA) is reduced by 2/3rds for three weeks (n=8-12 per group; for GTT, Dunnett’s test following ANOVA, a = p < 0.05 Control vs. Low AA, b = p < 0.05 Control vs. Low BCAA, c = p < 0.05 Control vs. Low Leu; for AUC, Dunnett’s test following ANOVA, * = p < 0.05, ** = p < 0.01). (C) GTT on male C57BL/6J mice fed a Control diet, a Low AA diet, a Low BCAA diet, or a Low FHKMTW diet in which six essential amino acids (F,H,K,M,T, and W) are reduced by 2/3rds, for three weeks (n=8 per group; for GTT, Dunnett’s test following ANOVA, a = p < 0.05 Control vs. Low AA, b = p < 0.05 Control vs. Low BCAA, c = p < 0.05 Control vs. Low FHKMTW; for AUC, Dunnett’s test following ANOVA, ** = p < 0.01). (D) Pyruvate tolerance test (PTT) on male C57BL/6J mice fed the indicated diets for 5 weeks (n=8-12 per group; for PTT, Dunnett’s test following ANOVA, a = p < 0.05 Control vs. Low AA (7%), b = p < 0.05 Control vs. Low BCAA, c = p < 0.05 Control vs. Low Leu; for AUC, Dunnett’s test following ANOVA, * = p < 0.05, ** = p < 0.01). Mice were fasted overnight and (E) blood glucose and (F) insulin were measured, and (G) the HOMA2-IR was calculated after 7 weeks on the specified diets (n=8-12 mice/group; Dunnett’s test following ANOVA, * = p < 0.05). (H-I) Gene expression in the liver of mice fasted overnight after 11 weeks of feeding the indicated diets was determined by quantitative PCR (n= 5-6/group, Dunnett’s test following ANOVA, * = p < 0.05 vs. control; for grouped analysis, n = 6-18/group, two-tailed t-test, * = p < 0.05). Error bars represent standard error.

Figure 3. Ex vivo analysis of pancreatic islet and β cell function

(A) An ex vivo insulin secretion assay was performed to assess (A) insulin secretion per islet and (B) islet insulin content in response to low (1.7 mM) and high (16.7 mM) glucose in mice fed either Control, Low AA, or Low BCAA diets for 11 weeks (n = 6 mice/group, Dunnett’s test following ANOVA, * = p < 0.05 vs. Control). (C) The impact of decreased BCAAs or total AAs on ATP/ADP and Ca2+ oscillations of pancreatic β cells was determined by simultaneous imaging after 17 weeks of feeding the indicated diets, and (D) plateau fraction and (E) amplitude was then calculated (n=149-176 islets; Dunnett’s test following ANOVA). Error bars represent standard error.

Figure 4. Dietary branched chain amino acids regulate food intake, body composition and adipose mass

(A) Food consumption after 3 weeks on diets (n=9 mice/group, means with the same letter are not significantly different from each other (Tukey–Kramer test following ANOVA, p < 0.05)). (B) Weight and body composition were measured immediately prior to diet start and after 3 and 10 weeks on the indicated diets (n= 7-12/group, means with the same letter are not significantly different from each other (Tukey–Kramer test following ANOVA, p < 0.05)). (C) Paraffin-embedded skin samples were collected after feeding mice the indicated diets for 11 weeks, sectioned, H&E stained and the thickness of dermal white adipose tissue (dWAT) was quantified (D) for non-anagen stage skin samples, measuring from muscle to dermis; scale bar = 100μM (n=5-11/group, means with the same letter are not significantly different from each other (Tukey–Kramer test following ANOVA, p < 0.05)). (E) The epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT) was collected at necropsy and weighed (n=6-9/group, means with the same letter are not significantly different from each other (Tukey–Kramer test following ANOVA, p < 0.05)). Error bars represent standard error.

Figure 5. The effects of reduced branched chain amino acids are independent of FGF21

(A) FGF21 was measured in the plasma of mice fed the indicated diets for 17 weeks and sacrificed following an overnight fast (n=8-9/group, Dunnett’s test following ANOVA, * = p < 0.05). (B) Fgf21 expression in the liver, skeletal muscle and adipose tissue of mice fasted overnight after 11 weeks of feeding the indicated diets was determined by quantitative PCR (n= 5-11/group, Dunnett’s test following ANOVA, * = p < 0.05). (C) Energy expenditure was measured after 4-6 weeks of feeding the indicated diets (n= 5-9/group, Dunnett’s test following ANOVA, * = p < 0.05). (D) Adiponectin was measured in the plasma of mice fed the indicated diets following an overnight fast after 17 weeks of diet feeding (n=5-9/group, Dunnett’s test following ANOVA, * = p < 0.05). (E) Ppargc1a expression in the liver of mice fasted overnight after 11 weeks of feeding the indicated diets was determined by quantitative PCR (n= 5-6/group, Dunnett’s test following ANOVA, * = p < 0.05 vs. control). Error bars represent standard error.