Article pubs.acs.org/JAFC
Osthole Enhances Glucose Uptake through Activation of AMP-Activated Protein Kinase in Skeletal Muscle Cells Wei-Hwa Lee,† Ren-Jye Lin,‡ Shyr-Yi Lin,‡,§ Yu-Chien Chen,∥ Hsiu-Ming Lin,∥ and Yu-Chih Liang*,∥,⊥ †
Department of Pathology, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan Department of Primary Care Medicine, Taipei Medical University Hospital, Taipei, Taiwan § Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan ∥ School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan ⊥ Traditional Herbal Medicine Research Center, Taipei Medical University Hospital, Taipei, Taiwan ‡
ABSTRACT: AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole’s glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole’s activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes. KEYWORDS: osthole, AMPK, GLUT4, streptozotocin, skeletal muscle cells
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heat shock, and glucose deprivation.8 In addition, AMPK can also be pharmacologically activated by the antidiabetic drugs metformin9 and troglitazone10 and the cell-permeable activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR),11 as well as the natural polyphenols resveratrol,12 curcumin,13 and epigallocatechin gallate.14 Many natural products are able to increase the rate of glucose uptake and improve hyperglycemia through activation of AMPK in different experimental models. For example, the hot-water extract of Morus alba leaves increased the rates of GLUT4 translocation and glucose uptake in the rat epitrochlearis muscle.15 Increased AMPK activity was regarded as the cause of stimulated glucose uptake in that case. Ganoderma lucidum, a fungus of the family Ganodermataceae, was shown to stimulate uptake of glucose by both PI3K and AMPK in L6 skeletal muscle cells.16 The traditional herbal medicine berberine is used to control blood glucose levels in China. A recent report confirmed that its hypoglycemic activity was associated with increased AMPK activity. 17 Therefore, identifying novel compounds that can activate AMPK and
INTRODUCTION Muscles are the major tissue responsible for insulin-mediated glucose uptake, and type 2 diabetes is characterized by insulin resistance in skeletal muscles.1 Glucose transport is a crucial step in glucose utilization, and this transport is mainly mediated by glucose transporter 4 (GLUT4) translocation during insulin stimulation and muscle contractions.2 Uptake of glucose by skeletal muscles is activated by the phosphatidylinositol 3-kinase (PI3K)/Akt and AMP-activated protein kinase (AMPK) signaling pathways. Activated PI3K and AMPK can both increase the rate of translocation of GLUT4 stored in the cytosol to plasma membranes, resulting in an increased rate of glucose uptake.3,4 Therefore, skeletal muscles are the first candidate tissue for therapy of type 2 diabetes. AMPK is a heterotrimeric protein composed of three subunits, including one catalytic subunit (α) and two noncatalytic subunits (β and γ).5 AMPK functions as a fuel gauge and plays a central role in regulating glucose and lipid metabolism.6 In mammalian cells, AMPK is activated by phosphorylation by at least two upstream kinases, liver kinase (LK) B1 and calmodulin-dependent protein kinase kinases (CaMKKs), as well as allosterically by an increase in the AMP:ATP ratio.7 Activation of AMPK is associated with environmental and nutritional stresses that deplete intracellular ATP levels, such as muscle exercise or contraction, hypoxia, © 2011 American Chemical Society
Received: Revised: Accepted: Published: 12874
September 8, 2011 November 15, 2011 November 20, 2011 November 20, 2011 dx.doi.org/10.1021/jf2036559 | J. Agric.Food Chem. 2011, 59, 12874−12881
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GSKα/β (Cell Signaling Technology, Danvers, MA), anti-GLUT4 (Santa Cruz Biotechnology, Santa Cruz, CA), anti-GAPDH (Abcam, Cambridge, MA), and anti-α-tubulin (Invitrogen Taiwan). The membranes were subsequently incubated with an anti-mouse or antirabbit immunoglobulin G secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and visualized using enhanced chemiluminescence kits (Santa Cruz Biotechnology). Reverse Transcription Polymerase Chain Reaction (RTPCR). Total RNA was isolated from cultured cells, and complementary DNA (cDNA) was prepared as previously described.29 Amplifications of myogenin and β-actin cDNA were performed by incubating 500 ng of total cDNA in 100 mM Tris-HCl buffer (pH 8.3) containing 500 mM KCl, 15 mM MgCl2, 0.1% gelatin, dNTPs (200 μM each), and 50 units/mL Super Taq DNA polymerase with the following oligonucleotide primers: myogenin, 5′-CTGGGGACCCCTGAGCATTG-3′ and 5′-ATCGCGCTCCTCCTGGTTGA-3′; and β-actin, 5′-GTCGACAACGGCTCCGGCATG-3′ and 5′-CTCTTGCTCTGGGCCTCGTCGC-3′. Thermal cycle conditions were as follows: one cycle at 94 °C for 5 min, 30 cycles at 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 30 s, and one cycle at 72 °C for 7 min. PCR products were analyzed on 1.5% agarose gels. Glucose Uptake Assay. Differentiated myotubes were cultured in a 24-well plate. After drug treatment, myotubes were incubated with 50 μM 2-NBDG for 15 min and then washed with phosphate-buffered saline (PBS) three times to remove any remaining 2-NBDG. The fluorescence intensity of cells containing 2-NBDG was measured on a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific, Waltham, MA) with excitation at 485 nm and emission at 535 nm.32 Extraction of Adenine Nucleotides and HPLC analysis. Adenine nucleotides were extracted from cells with perchloric acid as described previously.33 Aliquots of samples were stored at −80 °C until the HPLC analysis. Adenine nucleotide measurements were taken via HPLC analysis using a Waters LC Module I PLUS instrument (Waters, Milford, MA) with a COSMOSIL 5C18-MS-II packed column [4.6 mm (inside diameter) × 250 mm (Nacalai Tesque, Kyoto, Japan)]. The mobile phase contained 25 mM sodium phosphate buffer (pH 5.75), run by an isocratic method at a flow rate of 1.0 mL/min, and adenine nucleotides were detected at 254 nm. Identification of the individual adenine nucleotides was based on comparisons of the retention times of unknown peaks to those of reference standards. Induction of Streptozotocin-Induced Diabetes in Mice and Treatments. Male ICR mice (7 weeks old) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and housed in an air-conditioned animal room. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Mice were starved for 18 h before diabetes was induced with STZ, and then they received a single intraperitoneal (ip) injection of 100 mg of fresh STZ/kg prepared in 0.05 M citrate buffer (pH 4.5).34 The mice were divided into two groups (five or six per group), and osthole was orally administered 5 days per week in the morning for 8 weeks: group 1 was fed a standard diet (Purina rat feed, Ralston Purina, St. Louis, MO) with the vehicle [0.5% sodium carboxymethyl cellulose (CMC)]; group 2 was fed a standard diet with 100 mg osthole/kg in 0.5% CMC. Fasting blood was collected from a tail vein at the end of weeks 3, 5, and 7. At the end of the experiment (week 8), fasting blood was collected via a heart puncture under pentobarbital anesthetization. Determination of the Plasma Glucose Level. The glucose level was measured using a GLUCOSE (GLUC-PAP) kit according the manufacturer’s manual (RANDOX Laboratories, Antrim, U.K.). Statistical Analysis. Cell experimental data were analyzed with a Student’s t test. Animal experimental data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s multiplecomparison test. Data are expressed as the mean ± the standard deviation (SD), and differences were considered significant at p < 0.05.
stimulate muscle glucose uptake could lead to the development of new treatments for type 2 diabetes. Osthole [7-methoxy-8-(3-methylpent-2-enyl)coumarin] (Figure 1) is a derivative of coumarin and is found in the
Figure 1. Chemical structure of osthole.
herb Cnidium monnieri (L.) Cusson, a well-known traditional Chinese medicine used to treat itchy skin, rashes, eczema, and ringworm.18 Recent research found that osthole exhibited several biological functions, including antiosteoporotic, 19 antihepatic,20 antiallergic,21 antiseizure,22 anti-inflammatory,23 antiproliferative,24 and antitumor functions,25,26 and improved lipid metabolism and fatty liver.27,28 Our recent research also found that osthole was able to prevent hyperglycemia in db/db diabetic mice,29 indicating that osthole might be an antidiabetic agent. However, the detailed mechanisms of osthole’s actions remain poorly understood.
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MATERIALS AND METHODS
Materials. Osthole was purchased from Wako Pure Chemical (Osaka, Japan) and kindly provided by C.-C. Wang (Taipei Medical University). The purity was >95% according to high-performance liquid chromatography (HPLC) analysis. Metformin and Compound C were purchased from Tocris Bioscience (Ellisville, MO). Insulin, the adenine nucleotide, and protease inhibitor cocktail were purchased from Sigma Chemical (St. Louis, MO). 2-[N-(7-Nitrobenz-2-oxa-1,3diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was purchased from Invitrogen Taiwan (Taipei, Taiwan). Cell Cultures and Myoblast Differentiation. Mouse C2C12 skeletal myoblasts (BCRC 60083) and human HepG2 hepatoblastoma cells (BCRC 60025) were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heatinactivated fetal bovine serum (FBS) (Invitrogen Taiwan) and modified Eagle’s medium containing 10% FBS, 2 mM L-glutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate, respectively. Rat L6 skeletal myoblasts were kindly provided by J. C. Fong (National Yang-Ming University, Taipei, Taiwan) and were cultured in DMEM containing 10% heat-inactivated FBS. For myoblast differentiation, confluent myoblasts were placed into differentiation medium (DMEM supplemented with 2% heatinactivated horse serum) for 7 days, and the medium was refreshed every 3 days. Extraction of Membrane Proteins and Western Blot Analysis. Membrane proteins were isolated as previously described.30 In brief, harvested cells were homogenized in a Dounce homogenizer with buffer A [20 mM Tris-HC1 (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, and a protease inhibitor cocktail]. After a brief centrifugation, the resulting supernatant was ultracentrifuged at 25000g for 1 h, yielding the supernatant that represented the cytosolic fraction. The membrane pellet was resuspended in buffer A containing 0.5% Triton X-100 and sonicated three times for 30 s each. After ultracentrifugation, the supernatant was collected to represent membrane proteins. Fifty micrograms of protein was resolved by 10 or 12% SDS−PAGE (sodium dodecyl sulfate−polyacrylamide gel electrophoresis) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) as described previously. 31 The PVDF membrane was then incubated with the following primary antibodies: anti-AMPK, anti-phospho-AMPK (Thr172), anti-ACC, anti-phosphoACC (Ser79), anti-phospho-AS160, anti-phospho-Akt, anti-phospho12875
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L6 morphologically changed in terms of the alignment, elongation, and fusion of mononucleated myoblasts into multinucleated myotubes. The expression of myogenin, a specific differentiation marker, was used to evaluate myotube formation in our experiments by RT-PCR. Myogenin began transcription on the first day after cells were cultured in differentiation medium in both cell lines, and expression was sustained for up to 7 days (Figure 2B). We then examined the dose- and time-dependent effects of osthole on AMPK activation in myotubes and hepatocytes. Osthole was used to treat differentiated C2C12 and L6 myotube cells at a concentration of 50 μM, because this concentration was effective in our previous studies29 and did not cause any significant cytotoxicity during a 12 h treatment (data not shown). Osthole treatment significantly induced dose- and time-dependent increases in the level of phosphorylation of AMPK and its downstream mediator, ACC, in differentiated C2C12 and L6 myotube cells (Figure 3). The maximal responses were seen after treatment with osthole for 1 and 2 h in differentiated C2C12 and L6 cells, respectively. In addition, osthole also significantly induced dose- and time-dependent increases in the level of phosphorylation of AMPK and ACC in HepG2 hepatocytes (Figure 4) but was less effective. These results suggest that osthole can activate AMPK in skeletal muscle cells and might have an effect on glucose metabolism. Osthole Stimulated Glucose Uptake That Depended on AMPK Activation in Myotube Cells. To determine whether osthole can stimulate glucose uptake, a fluorescent glucose analogue, 2-NBDG, was used to measure the rate of glucose uptake. Results showed that 12.5 and 50 μM osthole stimulated glucose uptake by ∼1.6- and ∼2.0-fold, respectively, in L6 myotubes (Figure 5A). We next investigated whether osthole stimulates glucose uptake by increasing the extent of translocation of GULT4 to cell membranes. As shown in Figure 5B, a large amount of GLUT4 was present in the cell membrane
RESULTS
Osthole Activated AMPK in Myotube Cells and Hepatocytes. To induce myoblast differentiation, mouse C2C12 skeletal myoblasts and rat L6 skeletal myoblasts were cultured in differentiation medium containing 2% horse serum for different time periods. As shown in Figure 2A, C2C12 and
Figure 2. Differentiation of rat skeletal L6 and mouse skeletal C2C12 myoblasts. (A) Both L6 and C2C12 myoblasts were incubated with 2% horse serum for 4 days to induce myotube differentiation, and representative photographs are shown. (B) Both L6 and C2C12 myoblasts were incubated with 2% horse serum to induce myotube differentiation for different time periods, and total RNA was used to assess the mRNA expression of myogenin and β-actin.
Figure 3. Effects of osthole on the phosphorylation of AMPK and ACC in differentiated myotubes. Differentiated (A and B) L6 or (C and D) C2C12 myotubes were treated with 25 μM osthole for different time periods (A and C) or treated with various concentrations of osthole for 2 h (B and D). Total cell lysates were used to determine the phosphorylation levels of AMPK and ACC by Western blotting. Metformin (Met) was used as a positive control. Abbreviations: p-ACC, phosphorylated ACC; p-AMPK, phosphorylated AMPK; t-ACC, total protein of ACC; t-AMPK, total protein of AMPK. 12876
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To confirm the involvement of AMPK in osthole-stimulated glucose uptake in L6 myotubes, we pretreated cells with Compound C, a selective AMPK inhibitor. As shown in Figure 6A,
Figure 4. Effects of osthole on the phosphorylation of AMPK and ACC in human HepG2 hepatoma cells. Cells were treated with 25 μM osthole for different time periods (A) or treated with various concentrations of osthole for 2 h (B). Total cell lysates were used to determine the phosphorylation levels of AMPK and ACC by Western blotting. Metformin (Met) was used as a positive control. Abbreviations: p-ACC, phosphorylated ACC; p-AMPK, phosphorylated AMPK; t-ACC, total protein of ACC; t-AMPK, total protein of AMPK.
Figure 6. Effects of the AMPK inhibitor, Compound C, on inhibition of the osthole-induced phosphorylation of AMPK and ACC and uptake of glucose by differentiated L6 cells. (A) Differentiated L6 myotubes were treated with 25 μM osthole with or without 10 μM Compound C for 2 h. Total cell lysates were used to determine the phosphorylation levels of AMPK and ACC by Western blotting. (B) Differentiated L6 myotubes were treated with 25 μM osthole with or without 10 μM Compound C for 2 h. 2-NBDG uptake of cells was assessed as described in Materials and Methods. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, compared to the osthole treatment (column 2). Metformin (Met) was used as a positive control. Abbreviations: p-ACC, phosphorylated ACC; p-AMPK, phosphorylated AMPK; t-ACC, total protein of ACC; t-AMPK, total protein of AMPK.
10 μM Compound C indeed decreased the phosphorylation levels of AMPK and its downstream mediator ACC in both osthole- and metformin-treated cells. Osthole-induced glucose uptake was significantly reversed by treatment with Compound C (Figure 6B), suggesting that the osthole-induced increase in the rate of glucose uptake might involve AMPK activation in skeletal muscles. Osthole Increased the Cellular AMP:ATP Ratio. It was reported that 5′-AMP stimulates AMPK phosphorylation by LKB1, and an increase in the AMP:ATP ratio is the leading cause of AMPK activation in skeletal muscles.7 The HPLC profile of adenine nucleotides is shown in Figure 7. The retention times of ATP, ADP, and AMP were ∼6.5, ∼8.9, and ∼18.5 min, respectively. In addition, osthole was found to dosedependently decrease the ATP level but increase the AMP level in myotube cells (Figure 7A). Therefore, osthole significantly increased the cellular AMP:ATP ratio (Figure 7B), which provides evidence of AMPK activation. Osthole Also Activated Akt and AS160 in Myotube Cells. There is a large body of evidence that demonstrates that Akt plays an important role in the translocation of GLUT4 to plasma membranes and thus promotes glucose transport. Further studies revealed that the Akt-mediated phosphorylation
Figure 5. Effects of osthole on the glucose uptake and GLUT4 translocation in differentiated myotubes. (A) Differentiated L6 myotubes were treated with various concentrations of osthole for 2 h or 1 μM insulin for 20 min, and then 2-NBDG uptake was assessed as described in Materials and Methods. Data are presented as the mean ± SD of three independent experiments. *p < 0.05, compared to the control. (B) Differentiated L6 myotubes were treated with 25 or 50 μM osthole for 2 h. Plasma membrane fractions were used to assess GLUT4 expression by Western blotting. Metformin (Met) was used as a positive control.
fraction in osthole-treated cells, the same as with treatment with metformin. 12877
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Figure 7. Effects of osthole on changes in the cellular AMP:ATP ratio in differentiated myotubes. Differentiated L6 myotubes were treated with various concentrations of osthole for 1 h. Total cell extracts were used to determine the concentrations of various kinds of adenine nucleotides by HPLC. (A) Representative HPLC profiles of each point. (B) The AMP:ATP ratios are presented as the mean ± SD of three independent experiments. *p < 0.05, compared to the control.
of AS160 may be involved in GLUT4 translocation,35 and GSK3 may phosphorylate glycogen synthase and then inhibit glycogen synthesis.36 Thus, we investigated phosphorylation levels of Akt, AS160, and GSK3 in L6 myotubes treated with 25 μM osthole for the indicated time periods or at the indicated concentrations for 1 h (Figure 8). The level of osthole-induced phosphorylation of Akt reached a peak at 1 h and then gradually decreased until the 3 h point. Then, a second phase of phosphorylation was observed at 6 h. In addition, the Akt downstream mediators, AS160 and GSK3, were indeed phosphorylated by activated Akt in osthole-treated cells. These results suggest that osthole might increase the rate of translocation of GLUT4 through the Akt/AS160 pathway. Osthole Counteracted Hyperglycemia in Mice with STZ-Induced Diabetes. Mice with STZ-induced diabetes were orally administered a vehicle or osthole for 8 weeks; fasting blood was collected from a tail vein or via heart puncture, and glucose levels were determined by a commercial glucose kit. In diabetic control mice, the fasting blood glucose levels ascended from the third week after STZ administration and reached the highest levels in the fifth week, thereafter descending to a normal glucose level by the seventh week (Figure 9). In osthole-fed diabetic mice, glucose levels were significantly lower than those of vehicle-fed mice at weeks 5, 7,
and 8. The results suggest that osthole reduced blood glucose levels of mice with STZ-induced diabetes.
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DISCUSSION Our previous studies revealed that osthole exhibited hypoglycemia activity in ob/ob and db/db diabetic mice. The underlying mechanisms of osthole’s increase in the rate of glucose uptake are described here. We found that osthole stimulated glucose uptake via the AMPK pathway in skeletal muscle cells and prevented hyperglycemia in mice with STZinduced diabetes. The increase in the AMP:ATP ratio was involved in the activation of AMPK, which subsequently increased the rates of GLUT4 translocation and glucose uptake. Skeletal muscles are the major tissue that uses glucose in the human body. Previous studies found that AMPK is an energy center for controlling glucose and fatty acid metabolism. The commonly used clinical hypoglycemia drug, metformin, and AICAR, an AMPK activator, are thought to increase the rate of uptake of glucose by activating AMPK in skeletal muscles. 37 Muscle contractions also induce glucose uptake in an AMPKdependent manner. On the other hand, the PI3K/Akt signal pathway is also involved in glucose uptake in insulin-stimulated cells. This study demonstrates that osthole stimulated glucose uptake in skeletal muscle cells and prevented hyperglycemia in mice with STZ-induced diabetes. We found that osthole 12878
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Figure 10. Possible mechanisms for the increase in the rate of uptake of glucose by skeletal muscles by osthole. First, osthole may increase the rate of ATP consumption through unknown pathways and results in an increase in the AMP:ATP ratio. Second, a high AMP:ATP ratio leads to activation of AMPK as evidenced by the phosphorylation of AMPK and its downstream target, ACC. Third, activated AMPK phosphorylates and inactivates AS160 and subsequently increases the rates of translocation of GLUT4 to membranes and glucose uptake. On the other hand, osthole may activate PI3K/Akt as evidenced by the phosphorylation of Akt and its downstream target, GSK3. Activated Akt can also phosphorylate and inactivate AS160, which results in an increase in the rate of glucose uptake. However, the PI 3K inhibitor could not decrease the osthole-induced glucose uptake rate. The osthole-induced PI3K/Akt signaling pathway may not be important for glucose uptake in cultured skeletal muscles but may play a role in the increase in the rate of glucose uptake in other tissue types or in vivo.
Figure 8. Effects of osthole on the phosphorylation of Akt, GSK3α/β, and AS160 in differentiated L6 cells. Differentiated L6 myotubes were treated with 25 μM osthole for different time periods (A) or treated with various concentrations of osthole for 1 h (B). Insulin (100 nM, 20 min) was used as the positive control. Total cell lysates were used to determine phosphorylation levels of Akt, GSK3α/β, and AS160 by Western blotting. Abbreviations: p-Akt, phosphorylated Akt; pGSK3α/β, phosphorylated GSK3α/β; p-AS160, total protein of AS160.
phosphorylation of AS160 is involved in the upregulation of GLUT4 translocation and glucose uptake. Glycogen synthase is a key enzyme for converting glucose to glycogen, and it can be phosphorylated and catalytically inactivated by GSK. Activation of Akt also directly phosphorylated GSK3 and resulted in inactivation of GSK3. Therefore, the Akt signal pathway may decrease glucose levels through the ultimate activation of glycogen synthase in vivo. It is possible that osthole might decrease glucose levels in vivo, which is mediated by the Akt/GSK3 pathway. ACC was found to catalyze the rate-limiting step of the fatty acid synthesis pathway in skeletal muscles. Phosphorylation of ACC by AMPK results in inhibition of the enzymatic activity of ACC, which subsequently decreases the rate of fatty acid synthesis. A Western blot analysis revealed an increase in the level of ACC phosphorylation by osthole treatment in skeletal muscle cells and hepatoma cells, indicating that osthole may decrease blood fatty acid levels in vivo. Previous studies found that osthole decreased triglyceride levels in high-fat-fed or alcohol-fed rats.39,40 However, we found no difference in the levels of cholesterol and triglyceride between vehicle-fed and osthole-fed mice with STZ-induced diabetes. p38 MAPK is a member of the MAPK family and is activated by several different kinases, including AMPK.41 Previous studies also demonstrated that activation of p38 MAPK is essential for the maximal increase in the rate of glucose uptake by insulin treatment and contractions of skeletal muscle cells.42,43 We found that treatment of L6 skeletal muscle cells with osthole increased the phosphorylation levels of both AMPK (Figure 3) and p38 MAPK (data not shown). p38 MAPK phosphorylation had two peaks, 10 min and 4 h after osthole treatment. On the other hand, one peak of AMPK phosphorylation seemed to be induced during a 6 h osthole treatment. These results suggest that p38 MAPK phosphorylation might be mediated by both AMPK-dependent and -independent pathways, and activation
Figure 9. Effects of osthole on blood glucose levels in mice with STZinduced diabetes. Mice with STZ-induced diabetes were orally administered osthole for 8 weeks. Fasting blood was collected via a tail vein or heart puncture under pentobarbital anesthetization, and glucose levels were determined as described in Materials and Methods. Data are presented as the mean ± SD. *p < 0.05, compared to the control.
increased the level of phosphorylation of AMPK and its downstream mediator, ACC, and the increase in the rate of glucose uptake could be reversed by the AMPK inhibitor, Compound C. However, the PI3K inhibitor, wortmannin, did not inhibit osthole-induced glucose uptake (data not shown). These results indicate that at the cellular level, the increase in the rate of glucose uptake by osthole seemed to mainly be dependent on activation of AMPK but did not involve PI3K/ Akt. It has been demonstrated that Rab-GTP allows GLUT4 storage vesicles (GSV) to move to and fuse with the plasma membrane. AS160 is a Rab-GTPase-activating protein that can promote hydrolysis of Rab-GTP to Rab-GDP on the GSV. When AS160 is phosphorylated by AMPK and Akt under the conditions of muscle contraction and insulin stimulation, respectively, inactivation of the GTPase activity of AS160 and an increase in the level of Rab-GTP result.38 Therefore, 12879
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B.; Goldman, M. H. Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo role of mitochondrial reactive nitrogen species. J. Biol. Chem. 2004, 279, 43940−43951. (10) Konrad, D.; Rudich, A.; Bilan, P. J.; Patel, N.; Richardson, C.; Witters, L. A.; Klip, A. Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells. Diabetologia 2005, 48, 954−966. (11) Merrill, G. F.; Kurth, E. J.; Hardie, D. G.; Winder, W. W. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am. J. Physiol. 1997, 273, E1107− E1112. (12) Park, C. E.; Kim, M. J.; Lee, J. H.; Min, B. I.; Bae, H.; Choe, W.; Kim, S. S.; Ha, J. Resveratrol stimulates glucose transport in C2C12 myotubes by activating AMP-activated protein kinase. Exp. Mol. Med. 2007, 39, 222−229. (13) Kim, J. H.; Park, J. M.; Kim, E. K.; Lee, J. O.; Lee, S. K.; Jung, J. H.; You, G. Y.; Park, S. H.; Suh, P. G.; Kim, H. S. Curcumin stimulates glucose uptake through AMPK-p38 MAPK pathways in L6 myotube cells. J. Cell. Physiol. 2010, 223, 771−778. (14) Cai, E. P.; Lin, J. K. Epigallocatechin gallate (EGCG) and rutin suppress the glucotoxicity through activating IRS2 and AMPK signaling in rat pancreatic β cells. J. Agric. Food Chem. 2009, 57, 9817− 9827. (15) Ma, X.; Iwanaka, N.; Masuda, S.; Karaike, K.; Egawa, T.; Hamada, T.; Toyoda, T.; Miyamoto, L.; Nakao, K.; Hayashi, T. Morus alba leaf extract stimulates 5′-AMP-activated protein kinase in isolated rat skeletal muscle. J. Ethnopharmacol. 2009, 122, 54−59. (16) Jung, K. H.; Ha, E.; Kim, M. J.; Uhm, Y. K.; Kim, H. K.; Hong, S. J.; Chung, J. H.; Yim, S. V. Ganoderma lucidum extract stimulates glucose uptake in L6 rat skeletal muscle cells. Acta Biochim. Pol. 2006, 53, 597−601. (17) Yin, J.; Gao, Z.; Liu, D.; Liu, Z.; Ye, J. Berberine improves glucose metabolism through induction of glycolysis. Am. J. Physiol. 2008, 294, E148−E156. (18) The Pharmacopoeia Commission of PRC. The Pharmacopoeia of the People’s Republic of China. Part I; Chemical Industry Publishing House: Beijing, 2000; p 256. (19) Li, X. X.; Hara, I.; Matsumiya, T. Effects of osthole on postmenopausal osteoporosis using ovariectomized rats, comparison to the effects of estradiol. Biol. Pharm. Bull. 2002, 25, 738−742. (20) Okamoto, T.; Kawasaki, T.; Hino, O. Osthole prevents anti-Fas antibody-induced hepatitis in mice by affecting the caspase-3-mediated apoptotic pathway. Biochem. Pharmacol. 2003, 65, 677−681. (21) Matsuda, H.; Tomohiro, N.; Ido, Y.; Kubo, M. Anti-allergic effects of cnidii monnieri fructus (dried fruits of Cnidium monnieri) and its major component, osthol. Biol. Pharm. Bull. 2002, 25, 809−812. (22) Luszczki, J. J.; Andres-Mach, M.; Cisowski, W.; Mazol, I.; Glowniak, K.; Czuczwar, S. J. Osthole suppresses seizures in the mouse maximal electroshock seizure model. Eur. J. Pharmacol. 2009, 607, 107−109. (23) Lin, V. C.; Chou, C. H.; Lin, Y. C.; Lin, J. N.; Yu, C. C.; Tang, C. H.; Lin, H. Y.; Way, T. D. Osthole suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt/mTOR pathway. J. Agric. Food Chem. 2010, 58, 4786−4793. (24) Guh, J. H.; Yu, S. M.; Ko, F. N.; Wu, T. S.; Teng, C. M. Antiproliferative effect in rat vascular smooth muscle cells by osthole, isolated from Angelica pubescens. Eur. J. Pharmacol. 1996, 298, 191− 197. (25) Liao, P. C.; Chien, S. C.; Ho, C. L.; Wang, E. I.; Lee, S. C.; Kuo, Y. H.; Jeyashoke, N.; Chen, J.; Dong, W. C.; Chao, L. K.; Hua, K. F. Osthole regulates inflammatory mediator expression through modulating NF-κB, mitogen-activated protein kinases, protein kinase C, and reactive oxygen species. J. Agric. Food Chem. 2010, 58, 10445− 10451. (26) Hung, C. M.; Kuo, D. H.; Chou, C. H.; Su, Y. C.; Ho, C. T.; Way, T. D. Osthole suppresses hepatocyte growth factor (HGF)induced epithelial-mesenchymal transition via repression of the c-Met/
of p38 MAPK contributes to glucose uptake despite the activation pathway of p38 MAPK in osthole-treated cells. In conclusion, our study is the first to report that osthole had an effect on the increase in the rate of uptake of glucose by skeletal muscle cells and the prevention of hyperglycemia in mice with STZ-induced diabetes. Osthole-stimulated glucose uptake was mainly mediated by AMPK activation in skeletal muscles (Figure 10). However, the possibility that activation of the PI3K/Akt signaling pathway by osthole was involved in the increase in the rate of glucose uptake in other tissue types or in vivo cannot be ruled out. Therefore, we suggest that osthole has great potential for treating hyperglycemia disorders.
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AUTHOR INFORMATION Corresponding Author *School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, 250 Wu-Hsing St., Taipei 11014, Taiwan, ROC. Telephone: +886-2-27361661, ext. 3318. Fax: +886-2-27393447. E-mail:
[email protected]. Author Contributions W.-H.L. and R.-J.L. contributed equally to this work. Funding This work was supported by grants (NSC98-2320-B-038-005MY3) from the National Science Council (Taiwan, ROC) and Shuang Ho Hospital (99TMU-SHH-04-1).
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ABBREVIATIONS ACC, acetyl-CoA carboxylase; AICAR, 5-aminoimidazole-4carboxamide ribonucleoside; AMPK, AMP-activated protein kinase; CaMKKs, calmodulin-dependent protein kinase kinase; DMEM, Dulbecco’s modified Eagle’s medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT4, glucose transporter 4; GSK, glycogen synthase kinase; 2-NBDG, 2-[N(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose; PI3K, phosphatidylinositol 3-kinase; PVDF, polymer of vinylidene fluoride; SDS, sodium dodecyl sulfate; RT-PCR, reversetranscription polymerase chain reaction; STZ, streptozotocin
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REFERENCES
(1) Musi, N.; Goodyear, L. J. AMP-activated protein kinase and muscle glucose uptake. Acta Physiol. Scand. 2003, 178, 337−345. (2) Smith, A. G.; Muscat, G. E. Skeletal muscle and nuclear hormone receptors: Implications for cardiovascular and metabolic disease. Int. J. Biochem. Cell Biol. 2005, 37, 2047−2063. (3) Krook, A.; Wallberg-Henriksson, H.; Zierath, J. R. Sending the signal: Molecular mechanisms regulating glucose uptake. Med. Sci. Sports Exercise 2004, 36, 1212−1217. (4) Kurth-Kraczek, E. J.; Hirshman, M. F.; Goodyear, L. J.; Winder, W. W. 5′ AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle. Diabetes 1999, 48, 1667−1671. (5) Kemp, B. E.; Mitchelhill, K. I.; Stapleton, D.; Michell, B. J.; Chen, Z. P.; Witters, L. A. Dealing with energy demand: The AMP-activated protein kinase. Trends Biochem. Sci. 1999, 24, 22−25. (6) Towler, M. C.; Hardie, D. G. AMP-activated protein kinase in metabolic control and insulin signaling. Circ. Res. 2007, 100, 328−341. (7) Hardie, D. G. The AMP-activated protein kinase pathway: New players upstream and downstream. J. Cell Sci. 2004, 117, 5479−5487. (8) Viollet, B.; Mounier, R.; Leclerc, J.; Yazigi, A.; Foretz, M.; Andreelli, F. Targeting AMP-activated protein kinase as a novel therapeutic approach for the treatment of metabolic disorders. Diabetes Metab. 2007, 33, 395−402. (9) Zou, M. H.; Kirkpatrick, S. S.; Davis, B. J.; Nelson, J. S.; Wiles, W. G. IV; Schlattner, U.; Neumann, D.; Brownlee, M.; Freeman, M. 12880
dx.doi.org/10.1021/jf2036559 | J. Agric.Food Chem. 2011, 59, 12874−12881
Journal of Agricultural and Food Chemistry
Article
Akt/mTOR pathway in human breast cancer cells. J. Agric. Food Chem. 2011, 59, 9683−9690. (27) Zhang, Y.; Xie, M.; Xue, J.; Gu, Z. Osthole improves fat milkinduced fatty liver in rats: Modulation of hepatic PPAR-α/γ-mediated lipogenic gene expression. Planta Med. 2007, 73, 718−724. (28) Sun, F.; Xie, M. L.; Zhu, L. J.; Xue, J.; Gu, Z. L. Inhibitory effect of osthole on alcohol-induced fatty liver in mice. Dig. Liver Dis. 2009, 41, 127−133. (29) Liang, H. J.; Suk, F. M.; Wang, C. K.; Hung, L. F.; Liu, D. Z.; Chen, N. Q.; Chen, Y. C.; Chang, C. C.; Liang, Y. C. Osthole, a potential antidiabetic agent, alleviates hyperglycemia in db/db mice. Chem.-Biol. Interact. 2009, 181, 309−315. (30) Liang, Y. C.; Lin-Shiau, S. Y.; Chen, C. F.; Lin, J. K. Suppression of extracellular signals and cell proliferation through EGF receptor binding by (−)-epigallocatechin-3-gallate in human A 431 epidermoid carcinoma cells. J. Cell. Biochem. 1997, 67, 55−65. (31) Liu, D. Z.; Liang, H. J.; Chen, C. H.; Su, C. H.; Lee, T. H.; Huang, C. T.; Hou, W. C.; Lin, S. Y.; Zhong, W. B.; Lin, P. J.; Hung, L. F.; Liang, Y. C. Comparative anti-inflammatory characterization of wild fruiting body, liquid-state fermentation, and solid-state culture of Taiwanofungus camphoratus in microglia and the mechanism of its action. J. Ethnopharmacol. 2007, 113, 45−53. (32) Fukushima, M.; Hattori, Y.; Tsukada, H.; Koga, K.; Kajiwara, E.; Kawano, K.; Kobayashi, T.; Kamata, K.; Maitani, Y. Adiponectin gene therapy of streptozotocin-induced diabetic mice using hydrodynamic injection. J. Gene Med. 2007, 9, 976−985. (33) Hahn-Windgassen, A.; Nogueira, V.; Chen, C. C.; Skeen, J. E.; Sonenberg, N.; Hay, N. Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity. J. Biol. Chem. 2005, 280, 3208−3209. (34) Hayashi, K.; Kojima, R.; Ito, M. Strain differences in the diabetogenic activity of streptozotocin in mice. Biol. Pharm. Bull. 2006, 29, 1110−1119. (35) Giacca, A.; Xiao, C.; Oprescu, A. I.; Carpentier, A. C.; Lewis, G. F. Lipid-induced pancreatic β-cell dysfunction: Focus on in vivo studies. Am. J. Physiol. 2011, 300, E255−E262. (36) Nikoulina, S. E.; Ciaraldi, T. P.; Mudaliar, S.; Carter, L.; Johnson, K.; Henry, R. R. Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle. Diabetes 2002, 51, 2190−2198. (37) Musi, N.; Hirschman, M. F.; Nygren, J.; Svanfeldt, M.; Bavenholm, P.; Rooyackers, O.; Zhou, G.; Williamson, J. M.; Ljunqvist, O.; Efendic, S.; Moller, D. E.; Thorell, A.; Goodyear, L. J. Metformin increases AMP-activated protein kinase activity in skeletal muscle of subjects with type 2 diabetes. Diabetes 2002, 51, 2074−2081. (38) Kei Sakamoto, K.; Holman, G. D. Emerging role for AS160/ TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic. Am. J. Physiol. 2008, 295, E29−E37. (39) Zhang, Y.; Xie, M.; Xue, J.; Gu, Z. Osthole improves fat milkinduced fatty liver in rats: Modulation of hepatic PPAR-α/γ-mediated lipogenic gene expression. Planta Med. 2007, 73, 718−724. (40) Song, F.; Xie, M. L.; Zhu, L. J.; Zhang, K. P.; Xue, J.; Gu, Z. L. Experimental study of osthole on treatment of hyperlipidemic and alcoholic fatty liver in animals. World J. Gastroenterol. 2006, 12, 4359− 4363. (41) Xi, X.; Han, J.; Zhang, J. Z. Stimulation of glucose transport by AMP-activated protein kinase via activation of p38 mitogen-activated protein kinase. J. Biol. Chem. 2001, 276, 41029−41034. (42) Somwar, R.; Koterski, S.; Sweeney, G.; Sciotti, R.; Djuric, S.; Berg, C.; Trevillyan, J.; Scherer, P. E.; Rondinone, C. M.; Klip, A. A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes without affecting GLUT4 translocation. J. Biol. Chem. 2002, 277, 50386−50395. (43) Somwar, R.; Perreault, M.; Kapur, S.; Taha, C.; Sweeney, G.; Ramlal, T.; Kim, D. Y.; Keen, J.; Cote, C. H.; Klip, A.; Marette, A. Activation of p38 mitogen-activated protein kinase A and B by insulin and contraction in rat skeletal muscle: Potential role in the stimulation of glucose transport. Diabetes 2000, 49, 1794−1800. 12881
dx.doi.org/10.1021/jf2036559 | J. Agric.Food Chem. 2011, 59, 12874−12881
