Chlorogenic acid stimulates glucose transport in skeletal muscle via AMPK activation: a contributor to the beneficial effects of coffee on diabetes

Abstract

Chlorogenic acid (CGA) has been shown to delay intestinal glucose absorption and inhibit gluconeogenesis. Our aim was to investigate the role of CGA in the regulation of glucose transport in skeletal muscle isolated from db/db mice and L6 skeletal muscle cells. Oral glucose tolerance test was performed on db/db mice treated with CGA and soleus muscle was isolated for 2-deoxyglucose transport study. 2DG transport was also examined in L6 myotubes with or without inhibitors such as wortmannin or compound c. AMPK was knocked down with AMPKα1/2 siRNA to study its effect on CGA-stimulated glucose transport. GLUT 4 translocation, phosphorylation of AMPK and Akt, AMPK activity, and association of IRS-1 and PI3K were investigated in the presence of CGA. In db/db mice, a significant decrease in fasting blood sugar was observed 10 minutes after the intraperitoneal administration of 250 mg/kg CGA and the effect persisted for another 30 minutes after the glucose challenge. Besides, CGA stimulated and enhanced both basal and insulin-mediated 2DG transports in soleus muscle. In L6 myotubes, CGA caused a dose- and time-dependent increase in glucose transport. Compound c and AMPKα1/2 siRNA abrogated the CGA-stimulated glucose transport. Consistent with these results, CGA was found to phosphorylate AMPK and ACC, consistent with the result of increased AMPK activities. CGA did not appear to enhance association of IRS-1 with p85. However, we observed activation of Akt by CGA. These parallel activations in turn increased translocation of GLUT 4 to plasma membrane. At 2 mmol/l, CGA did not cause any significant changes in viability or proliferation of L6 myotubes. Our data demonstrated for the first time that CGA stimulates glucose transport in skeletal muscle via the activation of AMPK. It appears that CGA may contribute to the beneficial effects of coffee on Type 2 diabetes mellitus.

Figure 1. CGA lowered fasting blood glucose in db/db mice and stimulated glucose transport into soleus muscle.

A: Oral glucose tolerance test was performed on db/db mice (n = 4) treated with different treatments. 2 g/kg glucose was loaded at 0 minute. Blood samples were collected at −10, 0, 15, 30 60 and 120 minutes for glucose measurement. Results are the mean ± SD of four mice. B: Soleus muscle was isolated from db/db mice and treated with CGA or/and 100 nmol/l insulin or 2 mmol/l metformin for 30 minutes. 2-deoxyglucose uptake was measure over a 30-minute period, using liquid scintillation counter. Results are the mean ± SE of three independent experiments. *P<0.05, **P<0.01 compared with controls. DC = Diabetic Control.

Figure 2. Dose- and time-dependent stimulation of glucose transport in L6 myotubes by CGA.

A: L6 myotubes were incubated with incremental concentrations of CGA for 24 hours. B: L6 myotubes were incubated with 2 mmol/l CGA at different incubation periods up to 24 hours. 2-deoxyglucose uptake was measure over a 30-minute period, using liquid scintillation counter. Readings are expressed as percentage increase over basal uptake of cells incubated with vehicle. Results are the mean ± SE of three independent experiments. *P<0.05, **P<0.01 compared with vehicle-treated control.

Figure 3. Effects of compound c on CGA-stimulated glucose transport.

L6 myotubes were incubated with 2 mmol/l CGA for 24 hours. A: Myotubes were preincubated with 100 nmol/l wormannin for 30 minutes before incubated with CGA or insulin. Myotubes were then incubated with 100 nmol/l insulin 30 minutes before 2-deoyglucose uptake measurement. B: Myotubes were preincubated with 10 µmol/l compound c for 30 minutes before incubated with CGA or metfformin. Myotubes were then incubated with 2 mmol/l metformin 2 hours before 2-deoyglucose uptake measurement. 2-deoxyglucose uptake was measured over a 30-minute period using liquid scintillation counter. Readings are expressed as percentage increase over basal uptake of cells incubated with vehicle. Results are the mean ± SE of three independent experiments. *P<0.05, **P<0.01 compared with controls.

Figure 4. Effects of gene silencing of AMPK on CGA-stimulated glucose transport in L6 myotubes.

L6 myotubes were transfected with vehicle, unrelated siRNA or AMPKα1/2 siRNA as described in Research Design and Methods. A: Expression of AMPKα1/2 after transfection with or without unrelated siRNA or AMPKα1/2 siRNA. B: Transfected or non-transfected myotubes were incubated with 2 mmol/l CGA for 24 hours. 2-deoxyglucose uptake was measure over a 30-minute period using liquid scintillation method. Readings were expressed as percentage increase over basal uptake that was obtained from non-transfeted cells incubated with vehicle. Results are the mean ± SE of three independent experiments. **P<0.01 compared with non-transfected-control treated with CGA.

Figure 5. Dose- and time- dependent phosphorylation of AMPK.

A: Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24 hours. Plasma membranes were isolated and detected for GLUT 4 and GLUT 1 through immunoblotting. B: Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24 hours. C: Myotubes were treated with incremental doses of CGA for 24 hours. Whole cell lysate was used for the detection of p-AMPK, AMPK, p-ACC, ACC and CaMKKβ. Illustrated are the representative images of three independent experiments. Results are the mean ± SE of three independent experiments. *P<0.05, **P<0.01 compared with controls.

Figure 6. Increased AMPK activity and Akt phosphorylation in the absence of PI3K activation by CGA.

A: Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24 hours. B: Myotubes were treated with 2 mmol/l CGA for various incubation periods up to 24 hours. Whole cell lysate was immunoprecipitated with anti-AMPK α1/2. Immunoprecipitate was assayed against SAMS peptide in the presence of [γ-³²P]ATP. Kinase activity was expressed as incorporated ATP/mg protein/minute. C: Myotubes were treated with vehicle, 100 nmol/l insulin or 2 mmol/l CGA. Whole cell lysate was immunoprecipitated with IRS-1 and immunoblotted for IRS-1 and p85 subunit of PI3K. D: Myotubes were treated with vehicle, 100 nmol/l insulin or 2 mmol/l CGA. Whole cell lysate was detected for p-Akt through immunoblotting. Results are the mean ± SE of three independent experiments. *P<0.05, **P<0.01 compared with controls.

Figure 7. Effect of CGA on cell viability and cell proliferation of L6 myotubes.

Myotubes were incubated with incremental concentrations of CGA for 24 hours. A: Viability of myotubes was measured using MTT staining. Readings are expressed as a percentage of non-viable cells compared to vehicle-treated myotubes. B: Numbers of cells in cell-cycle phases were examined using propidium iodide staining and FACS analysis. Readings are expressed as percentage of cells stained by propidium iodide at different phases. Results are the mean ± SE of three independent experiments. *P<0.05 compared with vehicle-treated control.