Irisin, a Novel Myokine, Regulates Glucose Uptake in Skeletal Muscle Cells via AMPK

Abstract

Irisin is a novel myokine produced by skeletal muscle. However, its metabolic role is poorly understood. In the present study, irisin induced glucose uptake in differentiated skeletal muscle cells. It increased AMP-activated protein kinase (AMPK) phosphorylation and the inhibition of AMPK blocked glucose uptake. It also increased reactive oxygen species (ROS) generation. N-acetyl cysteine, a ROS scavenger, blocked irisin-induced AMPK phosphorylation. Moreover, irisin activated p38 MAPK in an AMPK-dependent manner. The inhibition and knockdown of p38 MAPK blocked irisin-induced glucose uptake. A colorimetric absorbance assay showed that irisin stimulated the translocation of glucose transporter type 4 to the plasma membrane and that this effect was suppressed in cells pretreated with a p38 MAPK inhibitor or p38 MAPK small interfering RNA. In primary cultured myoblast cells, irisin increased the concentration of intracellular calcium. STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, blocked irisin-induced AMPK phosphorylation, implying that calcium is involved in irisin-mediated signaling. Our results suggest that irisin plays an important role in glucose metabolism via the ROS-mediated AMPK pathway in skeletal muscle cells.

Figure 1.. Irisin stimulates glucose uptake in differentiated muscle cells.

A, L6 myotube cells were incubated with irisin (62 ng/mL) for the indicated times and then assayed for glucose uptake. B, Differentiated L6 myotube cells were incubated with different concentrations of irisin for 1 hour and then assayed for glucose uptake. Insulin (100 ng/mL) was treated for 15 minutes and used as a positive control. *, P < .05 vs basal condition.

Figure 2.. Irisin activates AMPK in L6 cells.

A, L6 cells were incubated with irisin (62 ng/mL) for the indicated times. Cell lysates were analyzed by Western blotting, using antibodies against phospho-AMPK (Thr¹⁷²) and ACC (Ser⁷⁹), whereas AMPK and ACC served as controls. B, L6 cells were stimulated for 1 hour at several concentrations of irisin. The cell lysates were analyzed by Western blotting using antibody against phospho-AMPK (Thr¹⁷²) and ACC (Ser⁷⁹), whereas AMPK and ACC served as controls. C, Differentiated L6 myotube cells were incubated for 1 hour with irisin (62 ng/mL) in the presence of the AMPK inhibitor compound C (2μM) and then assayed for glucose uptake. *, P < .05 vs basal condition; **, P < .05 vs irisin-treated condition. D, L6 cells were pretreated with compound C (2μM), then incubated with irisin (62 ng/mL) for 60 minutes. Cell lysates were analyzed by Western blotting using an antibody against phospho-AMPK (Thr¹⁷²), whereas AMPK served as a control. E, L6 cells were transiently transfected with AMPKα2 siRNA for 48 hours. The cell lysates were analyzed by Western blotting using antibody against phosphorylated AMPK2 and ACC, whereas AMPKα2 and β-actin served as controls. F, L6 cells were transiently transfected with AMPKα2 siRNA for 48 hours. Differentiated L6 myotube cells were incubated for 1 hour with irisin (62 ng/mL) and then assayed for glucose uptake. *, P < .05 vs basal condition.

Figure 3.. Irisin activates the AMPK signaling pathway through ROS in myoblasts.

A, Increase of ROS after irisin treatment. L6 cells were treated with irisin (62 ng/mL) for 1 hour. ROS were stained with the marker DCF-DA. Images were captured using a confocal microscope. B, L6 cells were pretreated with the ROS scavenger NAC for 15 minutes and then incubated with irisin (62 ng/mL) for 1 hour. The cell lysates were analyzed by Western blotting with antibodies against phospho-AMPK (Thr¹⁷²) and AMPK. C, Differentiated L6 myotube cells were pretreated with the ROS scavenger NAC for 15 minutes, then incubated with irisin (62 ng/mL) for 1 hour, and then assayed for glucose uptake. *, P < .05 vs basal condition.

Figure 4.. The p38 MAPK pathway is involved in irisin-mediated glucose uptake.

A, L6 cells were incubated with irisin (62 ng/mL) for the indicated times. Lysates were analyzed by Western blotting using an antibody against phospho-p38 MAPK, whereas p38 MAPK served as a control. B, L6 cells were pretreated with compound C (2μM), then incubated with irisin (62 ng/mL) for 60 minutes. Cell lysates were analyzed by Western blotting using an antibody against phospho-p38 MAPK, whereas p38 MAPK served as a control. C, L6 cells were pretreated with the ROS scavenger NAC for 15 minutes and then incubated with irisin (62 ng/mL) for 1 hour. The cell lysates were analyzed by Western blotting with antibodies against phospho-p38 MAPK and p38 MAPK. D, Differentiated L6 myotube cells were incubated with irisin (62 ng/mL) for 1 hour in the presence of the p38 MAPK inhibitor SB202190 (5μM), before assaying for glucose uptake. E, L6 cells were transiently transfected with p38 MAPK siRNA for 48 hours. The cell lysates were analyzed by Western blotting using antibody against p38 MAPK, whereas β-actin served as controls. F, Differentiated L6 myotube cells were transiently transfected with p38 MAPK siRNA (50nM) for 48 hours and then incubated with irisin (62 ng/mL) for 1 hour before assaying for glucose uptake. *, P < .05 vs basal condition; **, P < .05 vs irisin-treated condition.

Figure 5.. Irisin stimulates GLUT4 translocation.

A, Confluent monolayers of myotube cells were incubated with irisin (62 ng/mL) for 60 minutes or insulin for 15 minutes. Cell surface expression of GLUT4myc was detected using an antibody-coupled colorimetric absorbance assay. B, L6 myotube cells were incubated with irisin (62 ng/mL) for 1 hour in the presence of the p38 MAPK inhibitor SB202190 (5 μM), before assaying for GLUT4 translocation. C, L6 myotube cells were transiently transfected with p38 MAPK siRNA (50nM) for 48 hours and then incubated with irisin (62 ng/mL) for 1 hour before assaying for GLUT4 translocation. D, L6 myotube cells were pretreated with NAC for 15 minutes and then incubated with irisin (62 ng/mL) for 1 hour before assaying for GLUT4 translocation. *, P < .05 vs basal condition; **, P < .05 vs irisin-treated condition.

Figure 6.. Irisin activates AMPK and stimulates glucose uptake in primary-cultured myoblasts.

A, Morphology of differentiated primary-cultured myoblasts. Myoblasts were prepared from quadriceps muscle of 3-day mouse. B, Cell lysates were prepared from myoblasts of 0 and 24 hours of differentiation. The cell lysates were analyzed by Western blotting using antibody against myogenin, whereas β-actin served as controls. C, Differentiated primary-cultured myoblasts cells were stimulated for the indicated times with irisin. Cell lysates were analyzed by Western blotting using antibodies against phospho-AMPK (Thr¹⁷²), ACC (Ser⁷⁹), and p38 MAPK, whereas AMPK, ACC, and p38 MAPK served as controls. D, Differentiated primary-cultured myoblast cells were incubated for 1 hour with irisin (62 ng/mL) in the presence of the AMPK inhibitor compound C (2μM), then assayed for glucose uptake. E, Differentiated primary-cultured myoblast cells were incubated for 1 hour with irisin (62 ng/mL) in the presence of the p38 MAPK inhibitor SB202190 (2μM), then assayed for glucose uptake. F, For Ca²⁺ detection, cells were preincubated in Fluo-3 AM (10μM) for 45 minutes. The Ca²⁺ response was measured after treatment of irisin with confocal microscope. Ca²⁺ concentration is correlated with fluorescence intensity. G, Myoblasts were pretreated with the CaMKK inhibitor STO-609 (2μM), then incubated with irisin for 60 minutes. Cell lysates were analyzed by Western blotting using an antibody against phospho-AMPK, whereas AMPK served as a control. H, For Ca²⁺ detection, cells were preincubated in Fluo-3 AM (10μM) for 45 minutes. The Ca²⁺ response was measured after treatment of irisin in the presence of NAC with confocal microscope. I, L6 cells were treated with irisin (62 ng/mL) for 1 hour in the presence of BAPTA. ROS were stained with DCF-DA. Images were captured using a confocal microscope. J, Differentiated primary-cultured myoblast cells were incubated for 1 hour with irisin (62 ng/mL) in the presence of the NAC, then assayed for glucose uptake. K, L6 cells were treated with irisin (62 ng/mL) for 1 hour. ROS were stained with DCF-DA. Images were captured using a confocal microscope. *, P < .05 vs basal condition; **, P < .05 vs irisin-treated condition.