Methionine Restriction Partly Recapitulates the Sympathetically Mediated Enhanced Energy Expenditure Induced by Total Amino Acid Restriction in Rats

Abstract

Total amino acid (AA) restriction promotes hyperphagia and energy expenditure. We determined whether (i) methionine restriction mimics the effects of total AA restriction, (ii) methionine supplementation attenuates these responses, and iii) sympathetic signaling mediates such effects. Rats were injected with either vehicle (V) or 6-hydroxydopamine (S) to induce chemical sympathectomy, and then randomized to four diets: 16% AA (16AA), 5% AA (5AA), 16% AA-methionine (16AA-Met), and 5% AA+methionine (5AA+Met). Propranolol or ondansetron were injected to examine the role of sympathetic and serotonergic signaling, respectively. 5AA, 5AA+Met, and 16AA-Met increased the food conversion rate for 1⁻3 weeks in the V and S groups, and increased mean energy expenditure in V group,; the magnitude of these changes was attenuated in the S group. Propranolol decreased the energy expenditure of V16AA, V5AA, and V5AA+Met and of S5AA, S5AA+Met, and S16AA-Met, whereas ondansetron decreased the energy expenditure in only the S groups. Compared to 16AA, the other V groups had reduced body weights from days 7⁻11 onwards and decreased lean masses throughout the study and the other S groups had decreased body weights and lean masses from day 14 onwards. Total AA restriction enhanced the energy expenditure and reduced the weight and lean mass; these effects were partly recapitulated by methionine restriction and were sympathetically mediated.

Keywords: 6-hydroxydopamine; amino acid restriction; energy balance; energy expenditure; methionine restriction; obesity; obesity-prone rats; sympathetic system.

Figure 1

The experimental timeline: Male obesity-prone rats (6 weeks old) were acclimated to the environmental conditions for 8 days in shoebox cages and 2 days in CLAMS^® (Comprehensive Lab Animal Monitoring System) upon arrival with continuous access to a high-fat control diet (4.4 kcal/g; 33% fat calories; 16% AA). After arrival (day −10), the animals (n = 60) were weight-matched and randomized to two groups to receive injections of either vehicle (V, n = 28) or 6-hydroxydopamine to induce sympathectomy (S, n = 32) on days −9, −8, and −7 and continued on a high-fat diet for 7 days (i.e., until day 0). At 2 days prior to the initiation of the experimental diet (day −2), the rats were then transferred to CLAMS^®. The rats within the S and V groups were then randomized to receive four (n = 8/diet for S and n = 7/diet for V rats) high-fat diets containing 16% AA (control; S16AA and V16AA; 0.46% D,L-methionine w/w as fed), 5% AA (S5AA and V5AA; 0.15% D,L-methionine), 16% AA-methionine (S16AA-Met and V16AA-Met; 0.15% D,L-methionine), or 5% AA+methionine (S5AA+Met and V5AA+Met; 0.46% D,L-methionine) for 21 days. All rats received injections of propranolol, ondansetron, or saline on days 9, 12, 14, and 16 to study the role of sympathetic and serotonergic signaling on energy balance. Metabolic measurements including daily food intake and energy expenditure and weekly body weight and body composition were recorded for 21 days while the rats were individually housed in CLAMS^®.

Figure 2

The effect of dietary methionine restriction or supplementation on energy intake (A,B), body weight (C,D), body fat mass (E,F), and body lean mass (G,H) of vehicle- (A,C,E,G) and 6-hydroxydopamine- (6-OHDA) (B,D,F,H) treated obesity-prone rats during the 3-week study: The data for the antagonists’ administration days (i.e., days 9, 12, 14, and 16) were not included in the graphs or data analysis. The p-values for the overall model effects of treatment, diet, day, diet × day, and treatment × day for energy intake were 0.35, <0.01, <0.01, <0.01, and 0.01, respectively; for body weight were 0.43, <0.01, <0.01, and 0.04, respectively; for body fat were 0.78, 0.04, <0.01, <0.01, and <0.01; and for body lean were 0.77, <0.01, <0.01, <0.01, and <0.01, respectively. The nonsignificant (p > 0.05) interactions are not shown. V16AA, vehicle-treated 16% amino acid diet; V5AA, vehicle-treated 5% amino acid diet; V5AA+Met, vehicle-treated 5% amino acid and methionine-supplemented diet; V16AA-Met, vehicle-treated 16% amino acids and methionine-restricted diet; S16AA, sympathectomized 16% amino acid diet; S5AA, sympathectomized 5% amino acid diet; S5AA+Met, sympathectomized 5% amino acids and methionine-supplemented diet; S16AA-Met, sympathectomized 16% amino acids and methionine-restricted diet. For each variable within each treatment and day, ^a p ≤ 0.05 16AA vs. 5AA, ^b p ≤ 0.05 16AA vs. 5AA+Met, ^c p ≤ 0.05 16AA vs. 16AA-Met, and ^e p ≤ 0.05 5AA vs. 16AA-Met. The values are the mean ± SEM, n = 7–8.

Figure 3

The effect of dietary methionine restriction or supplementation on the energy intake of vehicle- (A,C,E,G) and 6-hydroxydopamine- (6-OHDA) (B,D,F,H) treated obesity-prone rats on day 3 (A,B), 7 (C,D), 15 (E,F), and 20 (G,H): The data for the antagonists’ administration days (i.e., days 9, 12, 14, and 16) were not included in the graphs or data analysis. The first 10 h are the dark period. The p-values for the overall model effects for treatment, diet, hour, and treatment × diet for energy intake on day 3 were <0.01, <0.01, <0.01, and 0.06, respectively; on day 7 were 0.89, 0.26, <0.01, and <0.01, respectively; on day 15 were 0.81, 0.11, <0.01, and <0.01, respectively; and on day 20 were 0.19, <0.01, <0.01, and <0.01, respectively. The nonsignificant (p > 0.05) interactions (i.e., diet × hour, treatment × hour, and treatment × diet × hour) for some days are not shown. V16AA, vehicle-treated 16% amino acid diet; V5AA, vehicle-treated 5% amino acid diet; V5AA+Met, vehicle-treated 5% amino acid and methionine-supplemented diet; V16AA-Met, vehicle-treated 16% amino acids and methionine-restricted diet; S16AA, sympathectomized 16% amino acid diet; S5AA, sympathectomized 5% amino acid diet; S5AA+Met, sympathectomized 5% amino acids and methionine-supplemented diet; and S16AA-Met, sympathectomized 16% amino acids and methionine-restricted diet. For each treatment and day at each hour, ^b p ≤ 0.05 16AA vs. 5AA+Met, ^c p ≤ 0.05 16AA vs. 16AA-Met, and ^e p ≤ 0.05 5AA vs. 16AA-Met. Values are mean ± SEM, n = 7–8.

Figure 4

The effect of dietary methionine restriction or supplementation on the energy expenditure of vehicle- (A,C,E,G,I) and 6-hydroxydopamine- (6-OHDA) (B,D,F,H,J) treated obesity-prone rats on days 3 (A,B), 7 (C,D), 15 (E,F), 17 (G,H), and 18 (I,J): The data for the antagonists’ administration days (i.e., days 9, 12, 14, and 16) were not included in the graphs or data analysis. The first 10 hours are the dark period. The p-values for the overall model effects for treatment, diet, hour, diet × hour, diet × treatment, and treatment × hour for energy expenditure on day 3 were <0.01, <0.01, <0.01, 0.01, 0.47, and 0.55, respectively; for day 7 were 0.92, <0.01, <0.01, 0.05, <0.01, and 0.81, respectively; for day 15 were 0.22, <0.01, <0.01, 0.02, <0.01, and <0.01, respectively; for day 17 were 0.55, <0.01, <0.01, 0.61, 0.04, and 0.46, respectively; and for day 18 were 0.72, <0.01, <0.01, 0.05, <0.01, and <0.01, respectively. The nonsignificant (p > 0.05) interactions are not shown. V16AA, vehicle-treated 16% amino acid diet; V5AA, vehicle-treated 5% amino acid diet; V5AA+Met, vehicle-treated 5% amino acid and methionine-supplemented diet; V16AA-Met, vehicle-treated 16% amino acid and methionine-restricted diet; S16AA, sympathectomized 16% amino acid diet; S-5AA, sympathectomized 5% amino acid diet; S5AA+Met, sympathectomized 5% amino acids and methionine-supplemented diet; S16AA-Met, sympathectomized 16% amino acid and methionine-restricted diet. For each treatment and day at each hour, ^a p ≤ 0.05 16AA vs. 5AA, ^b p ≤ 0.05 16AA vs. 5AA+Met, ^c p ≤ 0.05 16AA vs. 16AA-Met, ^e p ≤ 0.05 5AA vs. 16AA-Met, ^α p ≤ 0.10 16AA vs. 5AA, ^β p ≤ 0.10 16AA vs. 5AA+Met, and ^# p ≤ 0.10 16AA-Met vs. 16AA. The values are mean ± SEM, n = 7–8.

Figure 5

The effect of dietary methionine restriction or supplementation on mean weekly energy intake (A,B) and mean weekly energy expenditure (C,D) of the vehicle- (A,C) and 6-hydroxydopamine- (B,D) treated obesity-prone rats: The data for the antagonists’ administration days (i.e., days 9, 12, 14, and 16) were not included in the graphs or data analysis. The p-values for the overall model effects for treatment, diet, day, and diet × day for energy intake were 0.49, <0.01, 0.44, and 0.01, respectively, and for energy expenditure were 0.10, <0.01, 0.35, and 0.04, respectively. The nonsignificant (p > 0.05) interactions are not shown. V16AA, vehicle-treated 16% amino acid diet; V5AA, vehicle-treated 5% amino acid diet; V5AA+Met, vehicle-treated 5% amino acid and methionine-supplemented diet; V16AA-Met, vehicle-treated 16% amino acid and methionine-restricted diet; S16AA, sympathectomized 16% amino acid diet; S-5AA, sympathectomized 5% amino acid diet; S5AA+Met, sympathectomized 5% amino acids and methionine-supplemented diet; S16AA-Met, sympathectomized 16% amino acid and methionine-restricted diet. For each variable within each treatment and day, ^a p ≤ 0.05 16AA vs. 5AA, ^b p ≤ 0.05 16AA vs. 5AA+Met, ^c p ≤ 0.05 16AA vs. 16AA-Met, ^e p ≤ 0.05 5AA vs. 16AA-Met, ^α p ≤ 0.10 16AA vs. 5AA, ^β p ≤ 0.10 5AA+Met vs. 16AA, ^# p ≤ 0.10 16AA-Met vs. 16AA, and ^δ p ≤ 0.10 5AA vs. 16AA-Met. The values are the mean ± SEM, n = 7–8.