Branched-chain amino acid supplementation restores reduced insulinotropic activity of a low-protein diet through the vagus nerve in rats

Fig. 1. Plasma insulin and glucose concentrations and food consumption during feeding amino acid-deprived diets in rats. Three animal experiments were performed by feeding a control diet with 20% amino acids (20AA) or a diet deprived of single amino acid for 8 h; Asp, Lys, Pro, Ser, Thr, or Val (A, D, G), Gly, Leu, Met, Arg, or Tyr (B, E, H), and Ala, Cys, Phe, His, Ile, or Trp (C, F, I). Plasma insulin and glucose concentrations from 0 to 2 h after feeding are shown as AUC (a–c and d–f, respectively), and food consumptions for 0–1, 1–2 h after feeding are shown as a cumulative bar chart (g–i). Values are expressed as means ± S.E.M. (n = 5 in each group). Results of statistical analysis are given above the graphs (*P < 0.05; **P < 0.01 vs. 20AA). For the food consumption, cumulative value of 0–2 h after feeding was analyzed statistically

Fig. 2. Plasma insulin, glucose, and amino acid concentrations and food consumption during feeding BCAA- restricted diets in rats. Rats were fed a control diet with 20% amino acid (20AA), a low-protein diet with 5% amino acid (5AA), or diets with reduced amount of Val, Leu, Ile, or three BCAAs (BCAA) for 8 h. Plasma insulin and glucose concentrations from 0 to 2 h after feeding are shown as AUC (a and b, respectively), food consumptions for 0–1, 1–2 h after feeding are shown as a cumulative bar chart (c), and plasma BCAA concentrations 30 min after feeding are also shown (d). Values are expressed as means ± S.E.M. (n = 5 in each group). Results of statistical analysis are given above the graphs (*P < 0.05; **P < 0.01 vs. 20AA)

Fig. 3. Plasma insulin, glucose, and amino acid concentrations and food consumption during feeding BCAA-supplemented diets in rats. Rats were fed a control diet with 20% casein (20P), a low-protein diet with 5% casein (5P), or diets supplemented with Val, Leu, Ile, or three BCAAs (BCAA) to 5P for 8 h. Plasma insulin and glucose concentrations from 0 to 2 h after feeding are shown as AUC (a and b, respectively), food consumptions for 0–1, 1–2 h after feeding are shown as a cumulative bar chart (c), and plasma BCAA concentrations 30 min after feeding are also shown (d). Values are expressed as means ± S.E.M. (n = 5 in each group). Results of statistical analysis are given above the graphs (+ P < 0.05, ++ P < 0.01 vs. 20P; *P < 0.05, **P < 0.01 vs. 5P)

Fig. 4. Effect of amino acids on insulin secretion from MIN6 cells. Subconfluent cells were preincubated in Hepes–Krebs buffer containing 3 mM glucose without or with amino acids for 1 h and incubated in the same medium containing 3 mM or 25 mM glucose for another 1 h. In addition to the medium without amino acids (0EAA in a and 0AA in b), amino acid-supplemented media were used; (a) 0AA supplemented with essential amino acids at half, equal or twice the concentration of minimum essential medium (MEM) (1/2EAA, 1EAA, or 2EAA, respectively), (b) 0AA supplemented with Val, Leu, Ile, or three BCAAs twice the concentration of MEM. Insulin concentration in the medium was normalized by protein content of the cell lysates. Values are expressed as means ± S.E.M. (n = 6 in each group). Results of the statistical analysis are given above the graphs (*P < 0.05, **P < 0.01 vs. 0EAA of the same glucose concentration or 0AA)

Fig. 5. Effect of vagotomy and food restriction on plasma insulin, glucose, and amino acid concentrations in rats. a–c Vagotomized or sham-operated rats were fed a control diet with 20% casein (20P), a low-protein diet with 5% casein (5P), 5P supplemented with three BCAAs (5P + BCAA), or 5P supplemented with Leu (5P + Leu) for 1 h. Plasma insulin and glucose concentrations from 0 to 1 h after feeding are shown as AUC (a and b, respectively), and food consumption for 1 h after feeding is also shown (c). Values were expressed as means ± S.E.M. (sham 20P, n = 4; sham 5P, n = 5; sham 5P + BCAA, n = 5; sham 5P + Leu, n = 4; vagotomy 20P, n = 7; vagotomy 5P, n = 3; vagotomy 5P + BCAA, n = 4; vagotomy 5P + Leu, n = 3). Differences between 20P vs. 5P, 5P vs. 5P + BCAA, and 5P vs. 5P + Leu of the same operation group were statistically analyzed, and the results are given above the graphs (*P < 0.05). In addition, P-value of two-way ANOVA is shown below each graph. d–f Rats were fed a control diet with 20% amino acid (20AA) ad libitum (AL) or restricted to 2.5 g (RF) for 1 h. Plasma insulin and glucose concentrations from 0 to 1 h after feeding are shown as AUC (d and e, respectively), and food…