Fig 1.
Neither leucine nor KIC gavage affects whole-body insulin tolerance.
Rats were gavaged 0.75 mL/100 g body weight, twice with water, leucine (0.170 mM), or KIC (0.197 mM) 10 minutes apart. They were euthanized at different timepoints (30–180 min) after the gavage. Then HPLC was performed to measure plasma leucine (A) and KIC concentrations (B). Another group of rats was gavaged as described above. One hour after gavage, basal blood samples were taken. Animals were then administered insulin (1 U/kg of body weight). Blood samples were taken at different times to assess glucose levels (C). These blood glucose values at each timepoint were adjusted to basal values of the respective rat. Area under the curve of the blood glucose curve was analyzed (D). Data are means ± SEM, N = 8 each group; * p<0.05, *** p<0.001, **** p<0.0001.
Fig 2.
Leucine, but not KIC, activates S6K1-IRS-1 signalling in the gastrocnemius muscle.
Rats were treated as explained in Fig 1. HPLC was then performed to measure intracellular leucine (A) and KIC (B) concentrations in the gastrocnemius muscle. Proteins were immunoblotted against phosphorylated (ph)-S6K1 (Thr389) (C-D), ph-S6 (Ser235/6) C, E), ph-IRS-1 (Ser612) (C, F), ph-Akt (Ser473) (C, G), and ph-BCKD-E1 α (Ser293) (C, H). BCKD activity assay was performed (I). Data are means ± SEM. N = 8 each group; ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 3.
Leucine, but not KIC, activates S6K1-IRS-1 signalling in the soleus muscle.
Rats were treated as explained in Fig 1. HPLC was then performed to measure intracellular leucine (A) and KIC (B) concentrations in the soleus muscle. Proteins were immunoblotted against ph-S6K1 (Thr389) (C-D), ph-S6 (Ser235/6) C, E), ph-IRS-1 (Ser612) (C, F), ph-Akt (Ser473) (C, G), and ph-BCKD-E1α (Ser293) (C, H). BCKD activity assay was performed (I). Data are means ± SEM. N = 8 each group; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 4.
Leucine, but not KIC, activates S6K1-IRS-1 signalling in the EDL muscle.
Rats were treated as explained in Fig 1. HPLC was then performed to measure intracellular leucine (A) and KIC (B) concentrations in the EDL muscle. Proteins were immunoblotted against ph-S6K1 (Thr389) (C-D), ph-S6 (Ser235/6) C, E), ph-IRS-1 (Ser612) (C, F), ph-Akt (Ser473) (C, G), and ph-BCKD-E1α (Ser293) (C, H). BCKD activity assay was performed (I). Data are means ± SEM. N = 8 each group; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 5.
Leucine and KIC increase liver BCKD activity and phosphorylation of S6.
Rats were treated as explained in Fig 1. HPLC was then performed to measure intracellular leucine (A) and KIC (B) concentrations in the liver. Liver proteins were immunoblotted against ph-S6 (Ser235/6) (C-D), ph-IRS-1 (Ser612) (C, E), and ph-BCKD-E1 α (Ser293) (C, F). BCKD activity assay was performed (G). Data are means ± SEM. N = 7–8 each group; * p<0.05, ** p <0.01, **** p<0.0001.
Fig 6.
Leucine gavage upregulates S6 phosphorylation in the heart.
Rats were treated as explained in Fig 1. HPLC was then performed to measure intracellular A) leucine and B) KIC concentrations in the heart. Heart proteins were immunoblotted against ph-S6 (Ser235/6) (C-D), ph-IRS-1 (Ser612) (C, E), ph-Akt (Ser473) (C, F), and ph-BCKD-E1α (Ser293) (C, G). BCKD activity assay was performed (H). Data are means ± SEM. N = 7–8 each group; * p<0.05, **** p<0.0001.
Fig 7.
Tissues/muscle fiber types have varying levels of BCAA catabolic enzyme abundance.
Gastrocnemius, soleus, EDL, liver, and heart were isolated from rats euthanized 30 min post water gavage. Proteins were immunoblotted against total BCKD-E1α (A-B), ph-BCKD-E1α (Ser293) (A, C), BCAT2 (A, D), BDK (A, E) and pp2Cm (A, F). Data are means ± SEM. N = 7–8 each group. ** p<0.01, *** p<0.001, **** p<0.0001.