Conceived and designed the experiments: KWO AH BKHT. Performed the experiments: KWO AH. Analyzed the data: KWO AH. Contributed reagents/materials/analysis tools: BKHT. Wrote the paper: KWO.
The authors have declared that no competing interests exist.
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.
Regular consumption of coffee has been associated with a lower risk of Type 2 diabetes mellitus and it has been replicated across sexes, geographical locations and obesity levels
Besides caffeine, coffee contains numerous compounds like phenols, diterpenes, trigonelline and minerals such as potassium and magnesium. Among them, chlorogenic acid
Van Dijk et al. (2009) showed that CGA ingestion significantly reduced early fasting glucose and insulin responses in overweight men during an OGTT
CGA, DMEM, Krebs-Ringer bicarbonate buffer (KRBB), antibiotic/antimycotic, insulin, wortmannin, cytochalasin B, MTT, AMP, PI/RNase were obtained from Sigma (St. Louis, MO, USA). Rat L6 skeletal muscle myoblasts were obtained from ATCC (Manassas, VA, USA). FBS was from Hyclone (Cramlington, UK). DMSO was purchased from MP Biomedicals (Illkirch, France). Glucose oxidase kits were obtained from Thermo Scientific (Waltham, MA, USA). Compound c and NP 40 were obtained from Merck (Darmstadt, Germany). 2-Deoxy-[3H]D-glucose and [γ-32P]-ATP were purchased from PerkinElmer (Waltham, MA, USA). Protease inhibitor cocktail was purchased from Abcam (Cambridge, UK). AMPK α1/2 and an unrelated siRNA (control siRNA-A) were purchased from Santa Cruz (Santa Cruz, CA). Antibodies like anti-IRS-1, anti-PI3-kinase p85α, anti-GLUT 4, anti-GLUT 1, anti-CAMKKβ, anti-phospho-AMPK α1/2, anti-phospho-Akt1 were obtained from Santa Cruz (Santa Cruz, CA) too. anti-GAPDH and anti-phospho-ACC were from Cell Signaling Technology (Danvers, MA, USA) and Milipore (Billerica, MA, USA) respectively. Oligofectamine and OPTI-MEM were purchased from Invitrogen (Carlsbad, CA, USA). Bradford protein estimation kit was from Bio-Rad (Hercules, CA, USA). G-Sepharose beads and ECL detection kit were obtained from GE Healthcare (Piscataway, NJ, USA). SAMS peptide was purchased from Tocris Bioscience (Minneapolis, MN, USA).
Thirty male
The Principles of Laboratory Animal Care (NIH, 1985) were followed throughout the duration of experiment. The experimental protocol for animal study was approved by NUS Institutional Animal Care and Use Committee (IACUC) (Protocol No: 085/07(A3)10).
Twenty
Soleus muscle was isolated from db/db mice as described previously
The culture was maintained in DMEM containing 10% FBS and 1% antibiotic/antimycotic in a humidified atmosphere of 5% CO2 at 37°C. Differentiation of myoblasts into myotubes was carried out as described by Klip et al.
The cells were grown and differentiated in 96-well plates. After the indicated periods of incubation with different treatments, the cells were rinsed with KRPH (HEPES-buffered Krebs-Ringer phosphate) buffer, consisting of 118 mmol/l NaCl, 5 mmol/l KCl, 1.3 mmol/l CaCl2, 1.2 mmol/l MgSO4, 1.2 mmol/l KH2PO4 and 30 mmol/l HEPES (pH 7.4). CGA was prepared in 5% DMSO and diluted with appropriate amount of DMEM to obtain different concentrations. 5% DMSO was used for all drug preparations. For the study with CGA+insulin, myotubes were treated with 2 mmol/l CGA for 24 hours and stimulated with 100 nmol/l insulin for 30 mins before 2DG uptake measurement. For the study involving inhibitors, myotubes were pre-incubated with 100 nmol/l wortmannin or 10 µmol/l compound c for 30 mins before adding CGA. Washed cells were incubated with 10 µmol/l 2-Deoxy-[3H]D-glucose (1 µCi/ml) in KRPH buffer for 30 mins at 37°C. The 2-DG uptake was terminated immediately by aspirating the radioactive incubation solution and washing three times by ice-cold phosphate-buffered saline (PBS). The cells were then lysed with 0.5 N NaOH, followed by 0.5 N HCL for neutralization. The quantity of 2-DG taken up by the cells was measured with liquid scintillation counter. Non-specific uptake was measured in the presence of 10 µmol/l cytochalasin B and subtracted from the total uptake. 2-DG uptake was expressed as a percentage of the basal uptake of cells incubated with KRPH buffer only.
The subcellular fractionation of myotubes was done as previously described
Transfection of siRNA into myotubes was done as described previously with a minute modification
Treated cells were scraped gently from 6-well plate and pelleted with ice-cold PBS at 3,000 rpm, 4°C for 5 minutes. Cell pellet was then lysed in lysis buffer (50 mmol/l Tris [pH 8], 170 mmol/l NaCl, 1 mmol/l DTT, 0.5% NP40 and protease inhibitor cocktail for 30 minutes at 4°C. Cell lysate was then centrifuged at 13,000 rpm for 10 minutes to remove cell debris. 2 µl of the cell lysate was used for Bradford protein estimation. 1 mg of total cellular protein was immunoprecipitated with 1 µg of anti-IRS-1 antibody coupled to 40 µl of 100 mg/ml protein G-Sepharose beads. Immunoprecipitated proteins, together with the beads, were separated with SDS-PAGE and blotted as mentioned in the western blot analysis below. Blotted proteins were probed with anti-PI3-kinase p85α antibody.
Treated cells were scraped from 6-well plates and pelleted with ice-cold PBS at 3,000 rpm, 4°C for 5 minutes. Cell pellets were then lysed in lysis buffer containing 250 mmol/l sucrose, 150 mmol/l NaCl, 50 mmol/l HEPES (pH 7.4) 10 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1 mmol/l DTT, 0.5 mmol/l EDTA, 1% Triton-X 100 and proteases inhibitors cocktail. Cell lysates were separated via SDS-PAGE and the separated proteins were blotted onto nitrocellulose membrane. Membranes were probed with anti-GAPDH, anti-GLUT 4, anti-GLUT 1, anti-phospho-AMPK α1/2, anti-phospho-Akt1 and anti-phospho-ACC. They were then developed using the ECL detection kit.
Myotube viability and proliferation were assayed with MTT and propidium iodode (PI) staining. For the MTT assay, cells were treated with different concentrations of CGA for 24 hours and then incubated with 5 mg/ml MTT for a fruther 4 hours at 37°C. Medium was then removed and 100 µl of DMSO was added to dissolve the formazan crystal formed. Absorbance was measured at a wavelength of 500 nm with a microplate reader (Tecan Infinite m200, Mannedorf, Switzerland). Similarly, for the PI staining, cells were trypsinized and washed with PBS after incubation with different concentrations of CGA for 24 hours. The cells were then fixed in 70% ethanol at 4°C for at least 2 hours. After fixation, the cells were stained with PI/RNase in PBS supplemented with 1% FBS at room temperature for 20 mins before proceeding to flow cytometry analysis, using cyAn ADP Flow Cytometer (Beckman Coulter, Brea, CA).
Myotubes were treated with CGA of different concentrations for indicated periods of time. Treated cells were scraped and pelleted with ice-cold PBS at 3,000 rpm, 4°C for 5 minutes. Cell pellets were then lysed in lysis buffer containing 250 mmol/l sucrose, 150 mmol/l NaCl, 50 mmol/l HEPES (pH 7.4) 10 mmol/l sodium fluoride, 1 mmol/l sodium pyrophosphate, 1 mmol/l sodium orthovanadate, 1 mmol/l DTT, 0.5 mmol/l EDTA, 1% Triton-X 100 and proteases inhibitors cocktail. 2 µl of the cell lysate was used for Bradford protein estimation. 1 mg of total cellular protein was immunoprecipitated with 1 µg of anti-AMPK α1/2 antibody coupled to 40 µl of 100 mg/ml protein G-Sepharose beads. Kinase reaction was carried out on washed immunoprecipitate in 40 mmol/l HEPES (pH 7.0), 0.2 mmol/l AMP, 80 mmol/l NaCl, 0.8 mmol/l DTT, 5 mmol/l MgCl2, 0.2 mmol/l ATP (2 mCi [γ-ATP]) and 0.1 mmol/l SAMS peptide for 20 minutes at 37°C. Reaction mixture was then spotted on P81 Whatman filter paper and washed extensively using phosphoric acid and acetone. Radioactivity on the filter paper was measured using liquid scintillation. Kinase activity was expressed as incorporated ATP/mg protein/minute.
Experiments were repeated three times, each time in triplicate. Values are expressed as mean ± SE. One-way ANOVA followed by Tukey's
In an oral glucose tolerance test, 250 mg/kg CGA was found to significantly lower fasting blood glucose for 31±9% compared to diabetic control 10 minutes after the intraperitoneal injection (
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.
Soleus muscle isolated from db/db mice showed a significant increase in glucose transport after treated with CGA (
Treatment of myotubes with increasing concentrations of CGA for 24 hours led to a significant dose-dependent increase in glucose transport, which was first observed at 1 mmol/l (55±8%). The highest increase was observed at 2 mmol/l (63±6%) and the stimulation was maintained up to the highest concentration tested, which was 10 mmol/l (
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.
To explain the mechanism underlying the glucose transport stimulated by CGA, we examined the effect of several molecules which are capable of mediating the stimulation of glucose transport. Wortmannin is a well-known selective inhibitor for phosphatidylinositol 3-kinase (PI3K), a key regulator in insulin signaling
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.
Besides using inhibitor compound c, AMPKα content was reduced with RNA silencing. AMPKα 1/2 siRNA nucleotides reduced the total expression of AMPKα 1/2 by 74±7% compared with transfection with equal concentration of unrelated control siRNA sequence (
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.
Glucose transport is mediated by the members of GLUT protein family, which consists of 12 transmembrane transporters
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.
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.
Activation of PI3K requires association of its p85 subunit to IRS-1 as shown by insulin in
To discard the possibility that increases in glucose transport in response to CGA might be due to the changes in myotube numbers, we assessed the effect of increasing doses of CGA on cell viability and cell proliferation. No significant changes were observed up to a concentration of 4 mmol/l (
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.
The estimated prevalence of diabetes among adults in 2001 was 171 million people and was expected to increase to 366 million people by 2030
In the present study, we showed for the first time the effect of CGA on fasting blood glucose in a diabetic animal model. db/db mice with homozygous spontaneous mutation of leptin receptor is a diabetic model showing Type 2 diabetic characteristics such as obese, uncontrolled rise in blood glucose and insulin. Intraperitoneal injection of CGA lowered the fasting blood glucose in db/db mice and this is consistent with the findings from Van Dijk et al. (2009) in overweight patients
To further support the data we found using the db/db mice, we investigated the effect of CGA on glucose transport in L6 myotubes and the possible mechanisms to execute its function in glucose transport. The results of our study showed that CGA stimulated glucose transport in L6 myotubes in a dose- and time-dependent manner. Optimum increase (∼1.5 fold) was observed at the dose of 2 mmol/l after 24-hour incubation period, compared to vehicle-treated control. Using non-radioactive glucose transport assay, Prakhabar and Doble (2009) demonstrated that CGA caused significant increase of glucose transport at micromolar concentration. However, we found that micromolar amounts of CGA only mildly stimulated glucose transport (data not shown). To examine the mechanism by which CGA exerts its effect on glucose transport, we used several selective glucose transport inhibitors, such as wortmannin and compound c, which inhibit PI3K and AMPK respectively. We found that wortmannin did not affect CGA-stimulated glucose transport whereas compound c significantly decreased glucose uptake stimulated by CGA. In addition, AMPK knockdown also abolished the stimulatory effect caused by CGA on glucose transport of myotubes. Taken together, these results suggest that the effect of CGA on glucose transport may possibly be mediated by AMPK.
AMPK is a master sensor and regulator of cellular energy balance
Several studies have reported that AMPK activation by the well-known AMPK activator, AICAR, led to cell cycle arrest which involved accumulation of tumor suppressor protein p53
In summary, we have demonstrated that CGA decreases fasting blood glucose in db/db mice using a glucose tolerance test. Also, CGA stimulated glucose transport in skeletal muscle through the translocation of GLUT 4, mediated by the activation of AMPK. At 2 mmol/l, CGA did not cause any deleterious effect on cell viability and cell proliferation. As a second major component in coffee, these data, together with those shown by other studies
The authors thank Flow Cytometry Lab, Department of Pharmacology for the guidance on operating the flow cytometer. We would like to thank to Ms. Chew Xin Yi, Department of Pharmacology, for her guidance on performing immunoprecipitation.