Suppression of mTORC1 activation in acid-α-glucosidase-deficient cells and mice is ameliorated by leucine supplementation

Abstract

Pompe disease is due to a deficiency in acid-α-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.

Keywords: Pompe disease; leucine; lysosome; mTORC1; muscle; α-glucosidase.

Fig. 1.

Acid-α-glucosidase (GAA) knockdown in C2C12 cells and GAA deficiency in human infantile Pompe patient fibroblasts. A: C2C12 myoblasts were infected with a control empty lentivirus or lentiviruses encoding two independent GAA shRNAs. The relative expression of GAA mRNA was determined by quantitative RT-PCR. B: lysates from the shRNA knockdown C2C12 cell line (KD#1) and infantile Pompe disease patient fibroblast 1 cell line were immunoblotted for the GAA protein. Expression levels were compared with control C2C12 myoblasts and fibroblasts from a healthy individual. These are representative immunoblots independently performed three times. *P < 0.05 by Student's t-test.

Fig. 2.

Insulin-stimulated mTORC1 activation is reduced in GAA knockdown C2C12 myoblasts. Control empty lentivirus-infected cells (WT) and two independent C2C12 myoblast GAA knockdown cell lines (GAA-KD) were placed in serum and amino acid-free medium for 1 h. The cells were either left untreated or incubated with DMEM containing amino acids with various doses of insulin for 10 min. A: cell lysates were immunoblotted with a pT389-S6K1, S6K1, and GAPDH antibodies. B: quantification of pT389-S6K1/total S6K1 (WT vs. GAA-KD1). GAA-KD2 showed the same results as GAA-KD1 (data not shown). These are representative immunoblots independently performed three times. *P < 0.05 by Student's t-test.

Fig. 3.

Leucine activation of mTORC1 is reduced in GAA knockdown C2C12 myoblasts. Control empty lentivirus-infected cells (WT) and GAA-KD C2C12 myoblasts were placed in serum and amino acid-free medium for 1 h. The cells were either left untreated or incubated with leucine (10 mM) and/or insulin (100 nM) in the absence or presence of an amino acid mixture (DMEM) for 10 min. A: cell lysates were immunoblotted with a pT389S6K1, S6K1, and GAPDH antibodies. B: quantification of pT389S6K1/total S6K1. These are representative immunoblots independently performed three times. *P < 0.05 by Student's t-test. C: WT and GAA-KD cells were either left untreated or incubated with an amino acid mixture (DMEM) and insulin (100 nM) in the absence or presence of leucine (10 mM) or LL-OMe (100 μM) for 10 min. Cell lysates were immunoblotted with pT389S6K1, S6K1, and GAPDH antibodies. D: quantification of pT389-S6K1/total S6K1. These are representative immunoblots independently performed three times. *P < 0.05 by Student's t-test.

Fig. 4.

mTORC1 activation is reduced in human skin fibroblasts of infantile Pompe disease patients. A and B: control human fibroblasts and two fibroblast cell lines from infantile Pompe disease patients were either left untreated or incubated with an amino acid mixture (DMEM) and insulin (10 nM) in the absence or presence of leucine (10 mM) or LL-OMe (100 μM) for 10 min. Cell lysates were immunoblotted with pT389-S6K1, S6K1, and GAPDH antibodies. C: quantification of pT389S6K1/total S6K1. These are representative immunoblots independently performed three times. *P < 0.05 by Student's t-test.

Fig. 5.

mTORC1 activation is reduced in skeletal muscle of the (6^neo/6^neo) GAA knockout Pompe mouse model. A: control wild-type and Pompe mice at 16 wk of age were maintained on a normal chow diet. The mice were then fasted for 12 h and either infused with saline or leucine (30 mg·kg⁻¹·min⁻¹) for 60 min and then injected with either saline or insulin 1 U/kg. The mice were euthanized, and the EDL muscle was isolated, extracted, and immunoblotted for pT389-S6K1, total S6K1, and GAPDH. A representative immunoblot is shown. B: quantification of pT389S6K1/total S6K1 from a total of 6–10 independent mice. Normalization for the WT control mice immunoblotting intensity was used to compare samples from multiple independent immunoblots. *P < 0.05, **P < 0.02 by Student's t-test.

Fig. 6.

Basal and leucine-stimulated mTORC1 activation is reduced in skeletal muscle of Pompe mice at 2 to 10 mo of age. Control wild-type and Pompe mice at 2, 4, and 10 mo (MO) of age were maintained on a normal chow diet. The mice were then fasted for 12 h and either infused with saline or leucine (30 mg·kg⁻¹·min⁻¹) for 60 min. The mice were euthanized and the extensor digitorum longus (EDL) muscle was isolated, extracted, and immunoblotted for pT389S6K1, total S6K1, and GAPDH. The bar graph represents the quantification of pT389S6K1/total S6K1 of 2 mo (n = 5–8), 4 mo (n = 8–12), and 10 mo (n = 5–8) old independent mice. Nonidentical letters indicate measurements that were statistically different from each other at P < 0.05 determined by two-way ANOVA followed by the Tukey's multiple comparisons.

Fig. 7.

Dietary leucine supplementation restores basal and leucine-stimulated mTORC1 activation. A: Control wild-type and Pompe mice at 4 wk of age were either maintained on a normal chow diet with water or leucine-supplemented water (1.5%) for 12 wk. The mice were then fasted for 12 h and either infused with saline or leucine (30 mg·kg⁻¹·min⁻¹) for 60 min, then injected with either saline or insulin at 1 U/kg. The mice were euthanized, and the EDL muscle was isolated, extracted, and immunoblotted for T389-S6K1 phosphorylation, total S6K1, and GAPDH. A representative immunoblot is shown. B: quantification of pT389-S6K1/total S6K1 from a total of 6–10 mice.

Fig. 8.

Dietary leucine supplementation protects Pompe disease mice from changes in body composition, skeletal muscle glycogen, and spinal cord kyphosis. Control wild-type and Pompe mice at 4 wk of age were either maintained on a normal chow diet with water or leucine-supplemented water (1.5%) for 12 wk. A: mice were weighed and analyzed by NMR for fat and lean mass (n = 6–16). B: mice were subject to microCT to determine the degree of spinal cord kyphosis (n = 13–24). C: mice were euthanized, and the gastrocnemius white skeletal muscle was extracted and assayed for total glycogen content (n = 5–7). Nonidentical letters (a, b, c) indicate measurements that were statistically different from each other at P < 0.05 as determined by two-way ANOVA followed by the Tukey multiple-comparison test.

Fig. 9.

Dietary leucine supplementation prevents skeletal muscle protein breakdown and muscle mass loss in Pompe disease mice. Control WT and Pompe mice at 4 wk of age were either maintained on a normal chow diet with water or leucine-supplemented water (1.5%) for 12 wk. A: 3-methylhistidine (3-MH) levels were normalized for urine creatinine (n = 5–8). B: amount of 3-MH per milliliter urine collected over a 24-h period (n = 5–8). C: serum creatine kinase levels (n = 6–7). D: muscle weights of the gastrocnemius (GAS) and EDL muscles (n = 4–6). Nonidentical letters indicate measurements that were statistically different from each other at P < 0.05 determined by two-way ANOVA followed by the Tukey multiple-comparison test.

Fig. 10.

Dietary leucine supplementation improves spontaneous locomotor and running capacity in Pompe disease mice. Control wild type and Pompe mice at 4 wk of age were either maintained on a normal chow diet with water or leucine-supplemented water (1.5%) for 12 wk. A: spontaneous locomotor activity determined over a 72-h period (n = 4). B: after a 3-day training period, mice were placed on a treadmill at 10 m/min with a 15 grade (degree) incline, progressing to 15 m/min. The total distance run to exhaustion was determined in 11–17 mice per group. Nonidentical letters (a, b, c) indicate measurements that were statistically different from each other at P < 0.05 determined by two-way ANOVA followed by the Tukey multiple-comparison test.