Weight pulling protects from high protein diet induced weight and fat gain.

(A) Experimental design. (B-C) Food consumption per mouse (B) or normalized to body weight (C) after ∼6 weeks on the indicated diets. n=8/group. (D-F) Body weight (D), lean mass (E) and fat mass (F) over time, and change (Δ) from the beginning to end of study. n=7-8 mice/group. (G-J) Weight of the iWAT (G), eWAT (H), BAT (I), and liver (J) at the conclusion of the study. n=6-7 mice per group. (B-J) Statistics for the overall effects of diet, training, and the interaction represent the p value from a two-way ANOVA; *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

Dietary protein content and resistance training did not significantly impact liver lipid droplet or inguinal white adipocyte size.

(A-C) Representative Oil-Red-O stained liver sections from mice in the indicated groups, with quantification of average lipid droplet size (B) and number (C). n=3-7/group. (D-F) Representative H&E stained iWAT sections from mice in the indicated groups, with quantification of average lipid droplet size (E) and number (F). n = 7-8/group. (B-C, E-F) Statistics for the overall effects of diet, training, and the interaction represent the p value from a two-way ANOVA; *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Scale bar = 100µM. Data represented as mean ± SEM.

Effect of diet and exercise on glycemic control and blood metabolites.

(A-B) Glucose (A) and Insulin (B) tolerance tests were performed after 9-10 weeks on the diet, respectively, and area under the curve (AUC) was calculated. n=7-8 mice/group. (C-E) Blood was collected from animals after a 16-hour overnight fast; fasting blood glucose (C) and insulin (D) were determined and used to calculate HOMA2-IR (E). n=7-8 mice/group. (F-I) Blood was collected from animals after a 16-hour overnight fast after 16 weeks on the diets. Fasting FGF21 (F), ketones (G), triglycerides (H) and cholesterol (I) were determined. n=4-8 mice/group. (A-I) statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA, *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

Low protein fed animals have increased energy expenditure regardless of training regimen.

(A-F) Metabolic chambers were used to examine metabolic parameters after mice were fed the indicated diets for 8-9 weeks. These included food consumption (A), spontaneous activity (B), Respiratory Exchange Ratio (RER) (C-D), and energy expenditure (E) over a 24 hour period. (F) Energy expenditure (EE) as a function of body weight was calculated (data for each individual mouse are plotted, and slopes and intercepts were calculated using ANCOVA). n=7-8 mice/group. (A, B, D) Statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA conducted separately for the light and dark cycles; *p<0.05 from a Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

Strength and muscle growth is maximized by high protein and progressive resistance exercise.

(A-C) Weight pulling was performed three times per week for 12 weeks. The average maximum weight pulled each week with area under the curve (AUC) (A), and the number of sets achieved (B) is shown. Similarly, the number of stimulatory touches (C) by the investigator on all mice was averaged through multiple weeks of training and plotted per set. (A-C) n=8 mice/group (A-B); for (C) values for 8 mice/group were assayed over n=5 weeks. Statistics for the overall effects of diet, time or set, and the interaction represent the p value from a two-way RM ANOVA or REML; *p<0.05 from a Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. (D-G) Physical performance was assessed by an inverted cling test (D-E) or rotarod test (F-G). (E, G) Cling time (E) or rotarod time (G) as a function of body weight was calculated (data for each individual mouse are plotted, and slopes and intercepts were calculated using ANCOVA). n=7-8 mice/group. Statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA, *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

Weight pulling and high protein diet increased FDL mass but not mitochondrial respiration.

(A-C) The muscle mass of the FDL in absolute mass (A), normalized to body weight (B), and (C) normalized to tibia length. n=8/group. (D-H) Mitochondrial respiration parameters as measured in the FDL, including mitochondrial leak (D), and following addition of 0.25 mM (E), 0.5 mM (F), and 5.5 mM ADP (G). (H) 10mM succinate was provided to evaluate complex I and II driven respiration. n=7-8 mice/group. (A-H) Statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA, *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

Weight pulling and high protein diet increase the mass of specific muscles.

(A-L) The mass of muscles was measured in absolute terms (A-D) and normalized to tibia length (E-H). The muscles measured were the quadriceps (A, E), soleus (B, F), plantaris (C, G) and forearm flexor complex (D, H). n=6-8 mice/group. Statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA, *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

Bicep and forearm hypertrophy is maximized by HP diets and WP.

(A-C) Representative images of arm musculature (A) with quantification of biceps (B) and forearm (C) diameter. (B-C) n=7-8 mice per group. Statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA, *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.

FDL fiber type hypertrophy is maximized by high protein diets and weight pulling.

(A-H) Representative images of the FDL and fiber type with quantification of mid-belly CSA (B), fibers per cross section (C), fiber CSA (D), and individual muscle fiber type size: type I (E), type IIA (F), type IIB (G) and type IIX (H).n=5-8 mice per group. Statistics for the overall effects of diet, training and the interaction represent the p value from a two-way ANOVA, *p<0.05, Sidak’s post-test examining the effect of parameters identified as significant in the 2-way ANOVA. Data represented as mean ± SEM.