Amino acid-induced impairment of insulin sensitivity in healthy and obese rats is reversible

Abstract

High-protein diets (HPDs) promote weight loss but other studies implicate these diets and their constituent amino acids (AAs) in insulin resistance. We hypothesized that AA-induced insulin resistance is a temporal and reversible metabolic event. L6 myotubes were serum deprived for 4 h and then incubated in AA and/or insulin (100 nmol/L). Another group of cells was incubated overnight in AA + insulin, starved again, and then reincubated with AA and insulin. Mammalian (mechanistic) target of rapamycin complex 1 (mTORC1) signaling and glucose uptake were then measured. Healthy or insulin-resistant rats were gavaged with leucine (0.48 g/kg) and insulin sensitivity was examined. In myotubes, incubation with AA and insulin significantly (P < 0.05) increased the phosphorylation of the mTORC1 substrate ribosomal protein S6 kinase 1 (S6K1, T389) and of insulin receptor substrate 1 (IRS-1, serine residues), but suppressed insulin-stimulated glucose uptake by 40% (P < 0.01). These modifications were mTORC1-dependent and were reversible. In vivo, leucine gavage reversibly increased S6K1 phosphorylation and IRS-1 serine phosphorylation 5- to 12-fold in skeletal muscle and impaired insulin tolerance of glucose (P < 0.05) in lean rats. In insulin-resistant rats, the impairment of whole blood glucose and AA metabolism induced by leucine gavage (0.001 < P < 0.05) was more severe than that observed in lean rats; however, the impairment was reversible within 24 h of treatment. If these data are confirmed in long-term studies, it would imply that the use of leucine/HPD in treating metabolic diseases is unlikely to have lasting negative effects on insulin sensitivity.

Keywords: Glucose metabolism; insulin resistance; leucine; skeletal muscle.

Figure 1.

In L6 myotubes, amino acid and insulin induce S6K1 (T389) and IRS‐1 (serine residues) phosphorylation in a reversible manner. (A) Experimental design for myotube (top) and rat (bottom) studies. Studies conducted on day 2 are in dotted boxes. (B, C) Myotubes were starved for 4 h and then incubated for 30 min in either an amino acid‐free solution or AMEM with added leucine to a final concentration of 800 μmol/L (AA). Incubation with AA continued for up to 2 h in the presence or absence of insulin. Cells were harvested and lysates probed for phosphorylated (B) S6K1 (T389) and (C) IRS‐1 (S 307/612/636/639). Data are mean ± SD. In (B), *P <0.05 versus time 0, AA at 60 and 120 min; ^†P <0.01 versus time 0, AA at 60 and 120 min; ^‡P <0.05 versus AA at 5 min. In (C), *P <0.05 versus time 0; ^†P <0.01 versus AA at all time points. (D, E) Phosphorylated and total S6K1 and IRS‐1 in myotubes incubated with insulin for different lengths of time. Data are mean ± SD. Bars with different symbols differ from one another (P <0.05). (F, G) Total S6K1 and IRS‐1 in myotubes incubated with leucine‐enriched amino acid (AA) with or without insulin for different lengths of time. Data are mean ± SD. In (F), bars with different symbols differ from one another (P <0.05). In (G), *P <0.05 compared to time 0. (H) Myotubes treated as in (B) were incubated in AA and insulin overnight. They were then starved for 4 h (D2 control) and subjected to AA and insulin stimulation (D2 AA+ins). Cell lysates were probed for phosphorylated S6K1 and IRS‐1. Data are mean ± SD, *P ≤0.05 versus D2 control.

Figure 2.

Impairment of insulin‐stimulated glucose transport in myotubes treated with AA + insulin is reversible. (A) Myotubes were starved for 4 h and then incubated for 30 min in either an amino acid‐free solution (Control) or leucine‐enriched AA mixture (AA). Following this, insulin‐stimulated glucose transport was measured. In another group, incubation in AA and insulin continued overnight. Cells were then starved (day 2 control) and insulin‐stimulated glucose transport was then measured. Data are mean ± SE of 5–6 independent experiments; ^†P <0.01 versus Control; *P <0.05 versus Day 2 control. (B) Treatments as in (A) except that incubation with AA was carried out with coincubation with DMSO (AA) or 50 nmol/L rapamycin (AA + Rap). ^†P <0.01 versus Control; *P <0.05 versus AA + Rap. (C, D). Myotubes were starved for 4 h and then incubated with or without AA. Cells were then incubated with insulin in the absence (ins) or presence of AA (aa + ins). Control (ctl) group was not treated with insulin or AA. A fourth group was treated with AA + insulin overnight. Cells were then starved for 4 h and stimulated with insulin (d2). AKT phosphorylation was then analyzed. In the bottom panels, data are expressed as a % of the ins group. Mean ± SE of three experiments (AKT [S473]) or mean ± SD of two experiments (AKT [T308]); ^#P <0.05 versus ins.

Figure 3.

In lean rats, leucine gavage reversibly increases soleus muscle phosphorylation of S6K1 and IRS‐1 (serine residues) and whole body insulin sensitivity. Rats were starved for 18 h and then gavaged with either water (stv) or leucine. They were sacrificed at different times following gavage. Soleus muscles homogenates were analyzed for (A) phospho S6K1 and (B) serine phosphorylated IRS‐1. Another group of rats was gavaged with leucine and 2 h later returned to food. Rats were then starved overnight and, the following day, were gavaged with either water (d2 water) or leucine (the 2 bars to the right in A and B). They were sacrificed at the times indicated and soleus muscles were analyzed for phospho S6K1 and IRS‐1. Mean ± SE, n =5–7, *P <0.05 versus stv. (C) Plasma glucose concentrations, as fractions of time zero value, in rats that were gavaged with water or leucine, and then subjected to ITT 30 min after gavage. A third group was treated as described in (B) and the following day was gavaged with water, followed by ITT (day 2 water). Mean ± SD, n =4–7, *P <0.05 versus water (i.e., rats gavaged with water on day 1). (D) Plasma BCAA during the ITT described in (C). Mean ± SD, n =4–7, *P <0.05, versus T0 in day 2 water group.

Figure 4.

In obese rats, leucine gavage has no effect on soleus muscle phosphorylation of S6K1 and IRS‐1. Rats made obese by being fed high‐fat diet were gavaged with water or with leucine as described in Figure 3. Soleus muscle homogenates were analyzed for (A) phospho S6K1 and (B) serine phosphorylated IRS‐1. Another group of rats was gavaged with leucine and 2 h later returned to food. The rats were then starved overnight and the following day regavaged with either water or leucine (last three columns to the right in A and B). Mean ± SE; n =6. (C) Plasma insulin in lean or obese rats gavaged with leucine. Mean ± SE; n =6, *P <0.001 versus lean.

Figure 5.

Leucine gavage reversibly modifies whole body insulin sensitivity of glucose and amino acid metabolism in obese rats. (A) Plasma glucose concentrations, as fractions of time zero value, in obese rats that were gavaged with water or leucine, and then subjected to ITT 30 min after gavage, as described in Figure 3. A third group was treated as described in Figure 3C and the following day was gavaged with water, followed by ITT (day 2 water). (B) Plasma BCAA during the ITT. Mean ± SE, n =6–7. *P <0.05, ^†P <0.01, ^‡P <0.001 versus corresponding water at the indicated times.