Figures
Abstract
We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK.
Citation: Shen Y, Honma N, Kobayashi K, Jia LN, Hosono T, Shindo K, et al. (2014) Cinnamon Extract Enhances Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myocytes by Inducing LKB1-AMP-Activated Protein Kinase Signaling. PLoS ONE 9(2): e87894. https://doi.org/10.1371/journal.pone.0087894
Editor: Josep Bassaganya-Riera, Virginia Tech, United States of America
Received: September 26, 2013; Accepted: January 4, 2014; Published: February 14, 2014
Copyright: © 2014 Shen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Nihon University individual research grant program (to T.S.). This work was also supported by grants from the Japan Scienceand Technology Agency (JST) A-STEP program (to T.H.). Y.S. is supported by a Fellowship from the Academic Frontier Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The Cinnamon (Cinnamomum zeylanicum) used in this study was a gift from House Foods Corporation (Tokyo). This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. The authors have declared that no other competing interests exist.
Introduction
Diabetes mellitus is a common chronic disease in which a patient has persistent hyperglycemia. A number of epidemiologic studies have shown that as of year 2000, at least 171 million people worldwide have diabetes; and the number is estimated to be 2.8% in the world of the population [1]. Controlling the blood sugar concentration as close as possible to normal is very important to prevent the complication of diabetes. Thus therapeutic agents that ameliorate insulin resistance or stimulate the secretion of insulin and several kinds of insulin preparations for direct injection have received considerable attention. In recent years, several insulin-sensitizing agents have been developed that improve hyperglycemia through the activation of AMP-activated protein kinase (AMPK) [2], [3].
AMPK is highly conserved from yeast to plants and animals, and it plays a key role in the regulation of energy metabolism [4]. The activation of AMPK induces the translocation of glucose transporter 4 (GLUT4) to the plasma membrane [5], and several studies have demonstrated that AMPK and its signaling pathway are potential molecular targets in the development of drugs for the treatment of type 2 diabetes and obesity [6]–[9]. http://en.wikipedia.org/wiki/Diabetes_mellitus - cite_note-Wild2004-1#cite_note-Wild2004-1.
Cinnamon is an important component in Chinese medicine. Preparations made from the bark of trees of the genus Cinnamomum have been prescribed for more than 2000 years in China, and the first record for their use as Chinese medicine appeared in Shen-Nong’s Herbal [10]. There have been a number of in vitro and in vivo studies showing that cinnamon improves both insulin resistance and glucose metabolism [11]–[27]. However, the detailed mechanism of this anti-diabetic activity has not yet been clarified and is still controversial. Our previous study demonstrated that oral administration of a hot-water extract of cinnamon up-regulates mitochondrial uncoupling protein-1 (UCP-1) and enhances the production and translocation of GLUT4 in muscle and, at least, the translocation of GLUT4 in adipose tissues [27]. In this present study, we investigated the detailed molecular basis underlying the anti-diabetic effect of cinnamon by focusing on the insulin- and/or AMPK-signal pathways mediating glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE.
Materials and Methods
Preparation of Cinnamon Extract
Cinnamon (Cinnamomum zeylanicum) was a gift from House Foods Corporation (Tokyo). The cinnamon sticks (250 g) were soaked into 2,500 ml of water for 24 h at room temperature and then heated for 30 min at 100°C. The cinnamon extract was lyophilized and stored at −20°C until used.
Reagents
The following primary antibodies against Akt, acetyl-CoA carboxylase (ACC), AMPKα, LKB1, p-Akt (Ser473), p-ACC (Ser79), p-AMPKα (Thr172), and p-LKB1 (Ser428) were purchased from Cell Signaling Technology (MA, USA). Anti-GLUT4 was from EMD Chemicals Inc. (Damstadt, Germany); and anti-E-cadherin, from Santa Cruz Biotechnology, Inc. (TX, USA). Anti-β-actin antibody was purchased from Sigma-Aldrich (MO, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (PA, USA). Insulin and compound C were obtained from Sigma-Aldrich and tyrphostin AG 1024 came from Alexis Biochemicals (CA, USA).
Cell Culture
3T3-L1 fibroblasts (RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan) were cultured and induced to differentiate into adipocytes by the method described previously [28]. Lipid droplets that appeared in the mature adipocytes were assayed colorimetrically after fixation with formaldehyde and stained with 0.5% Oil-Red O as described by Ramírez-Zacarías et al. [29]. C2C12 myoblasts (RIKEN BRC) were cultured in growth medium (DMEM with 4.5 g/l glucose, 4 mM glutamine, and 10% vol/vol fetal bovine serum). After 90% confluence had been reached the medium was changed to the differentiation medium (DMEM with 4.5 g/l glucose, 4 mM glutamine and 2% vol/vol fetal bovine serum) to stimulate myotube formation. The myoblasts were cultured in the differentiation medium for 8 days prior to the experiment.
Measurement of Glucose Uptake
Glucose uptake was measured by the method described by Ragolia et al. [30]. Briefly, 3T3-L1 adipocytes and C2C12 myocytes cultured in 60-mm dishes (Becton Dickinson, NJ, USA) were serum-starved in DMEM for 16 h, and the cells were then washed 3 times with PBS (pH 7.4) and subsequently incubated for 30 min in the following medium: DMEM alone, DMEM containing either 100 nM insulin or 30 µg/ml CE, or the combination of both insulin and CE. Then, 0.5 mM 2-deoxy-D-[2,6–3H] glucose (1.5 µCi/well, Moravek Biochemicals, CA, USA) was added to the cells, which were then incubated for 15 min. Finally, the cells in triplicate were washed 4 times with PBS containing 0.3 mM phloretin and thereafter lysed in 1 ml of 1N NaOH for scintillation counting.
Western Blot Analysis
Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore Corporation, MA, USA) for Western blotting. The membrane was incubated at 4°C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, GLUT4 (1∶1000), E-cadherin (1∶200), and β-actin (1∶2000). After the incubation, anti-mouse IgG horseradish peroxidase-conjugate (HRP, 1∶2000, for β-actin) or anti-rabbit IgG HRP (1∶2000, for p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, and GLUT4) was added; and the membrane was incubated for 30 min at room temperature. The antigenic proteins on the membrane were visualized by chemiluminescence by use of a Lumi-LightPLUS Western blotting kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated by using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan).
Isolation of Plasma Membranes from 3T3-L1 Adipocytes
3T3-L1 adipocytes were homogenized by sonication for 5 min at 3 kHz/130 W (UCD-130TM, Cosmo Bio Co., Tokyo, Japan) in ice-cold HES buffer (0.02 M HEPES, 0.25 M sucrose, and 2 mM EGTA; pH 7.4) and centrifuged at 700×g for 7 min to remove unhomogenized cellular debris and nuclei from the homogenate. The harvested supernatant was further centrifuged at 760×g for 10 min to remove mitochondria. It was recentrifuged at 35,000×g for 60 min, and the resulting pellet was used as the plasma membrane fraction of the adipocytes. The supernatant was used as the cytosol fraction [31]. These membrane and cytosol fractions were subjected to Western blotting to detect GLUT4 and E-cadherin, respectively. The amounts of protein in the cytosolic fraction and membrane pellet were quantified by using Protein Assay Dye Reagent (Bio-Rad Laboratories, Inc., PA, USA).
RNA Isolation and Real-time PCR
Total RNA was extracted from 3T3-L1 adipocytes and C2C12 myotubes by use of Isogen reagent (Wako Pure Chemical Industries). Real-time reverse transcription-PCR was performed by the fluorescent dye SYBR Green I method using SYBR Premix Ex Taq and Perfect Real Time (Takara Bio, Shiga, Japan) with a StepOne PCR system (Life Technologies, CA, USA). The primers used in this study were designed based on GenBank information (Stk11, NM_011492) and synthesized by Life Technologies. The PCR primers used for LKB1 were 5′-CAC ACT TTA CAA CAT CAC CA-3′ and 5′-CTC ATA CTC CAA CAT CCC TC-3′ `.
Silencing of LKB1 Gene Expression by RNA Interference
LKB1 siRNA (Stealth RNAi, Life Technologies) and control siRNA (Life Technologies) were used. The 3T3-L1 adipocytes or C2C12 myotubes cultured in 60-mm dishes (Becton Dickinson) were transfected with a 0.5 µM concentration of either siRNA by using the transfection reagent Lipofectamine 2000 (Life Technologies). Prior to transfection, 12.5 µl of siRNA and 5 µl of transfection reagent were diluted with 500 µl of serum-free medium in separate tubes at first, and then the diluted siRNA and Lipofectamine were mixed together. The mixtures were incubated for 10 min at room temperature and then added dropwise to each culture well. Four hours after transfection, the medium was exchanged for fresh medium. The cells were cultured for 24 h prior to the experiment. The efficiency of LKB1 silencing was monitored by both quantitative reverse transcription-PCR and Western blotting.
Animal Experiment
Four-week-old male Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type 2 diabetes mellitus, and Long-Evans Tokushima Otsuka (LETO), a non-diabetic normal control, were provided by Otsuka Pharmaceutical Co., Ltd. (Tokushima Research Institute, Tokushima, Japan) and housed individually in a stainless steel wire-bottomed cage in a temperature-controlled room at 22–23°C with a 12 h photoperiod. OLETF and LETO rats were kept on AIN-93G diet (Clea Japan Inc., Tokyo, Japan) and water ad libitum. After 1 week of acclimation, rats were divided into 2 groups with matched body weight; i.e., 1) Control group without cinnamon (LETO, OLETF); 2) cinnamon treated group (LETO+CE, OLETF+CE). All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Nihon University Animal Care and Use Committee.
Oral Glucose Tolerance Test and Insulin Tolerance Test
OLETF and LETO rats were orally administered CE (100 mg/kg. b.w./day) for 15 weeks. For the glucose tolerance test, rats were fasted for 16 h followed by oral administration of glucose (1.2 g/kg b.w.). The blood-glucose concentration was measured by a DEXTER-Z II (Bayer Medical Co., Ltd., Leverkusen, Germany) using a blood collected from a tail vein. For insulin tolerance test, rats were fasted for 4 h, then the insulin (0.75 U/kg b.w.) was administered intraperitoneally followed by the measurement of blood glucose concentration as described above. Plasma insulin concentration was assayed by using an ELISA kit (Shibayagi Co., Ltd., Gunma, Japan).
Statistical Analysis
The Student’s t test was used in comparing two sets of data. Statistical differences among more than three groups were determined using one-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni multiple comparison tests. Statistical analysis was performed by using GraphPad Prism software v6.0 (GraphPad Software, CA, USA). A value of p<0.05 was considered statistically significant. All data are expressed as mean ± S.E.
Results
CE Stimulated Glucose Uptake and GLUT4 Translocation in 3T3-L1 Adipocytes
To clarify the anti-diabetic effect of CE, we first examined the effect of CE on glucose uptake and translocation of GLUT4 to the plasma membrane in differentiated 3T3-L1 adipocytes. Oil red O-stained 3T3-L1 adipocytes that differentiated from the 3T3 fibroblasts are shown in Fig. 1A. As shown in Fig. 1B, 30 µg/ml CE significantly increased the 2-deoxyglucose uptake into the 3T3-L1 adipocytes in a treatment time-dependent manner, with peak uptake at 30 min. Insulin (100 nM), which we employed as a positive control, also potently stimulated the uptake of 2-deoxyglucose. Consistent with the glucose uptake, significant translocation of GLUT4 was also observed in both insulin- and CE-treated adipocytes (Fig. 1C). The concentration of CE we used in this study did not show any cytotoxicity toward 3T3-L1 adipocytes, as assessed by the MTT assay (data not shown).
A: Appearance of oil red O-stained 3T3-L1 adipocytes differentiated from mouse 3T3-L1 fibroblasts. The photomicrographs show typical adipocytes found in different fields of observation. B: 3T3-L1 adipocytes were serum-starved for 16 h in DMEM, and then challenged with CE (30 µg/ml) for 15–240 min or with insulin (100 nM) for 30 min, and thereafter assayed for the uptake of 2-deoxyglucose by the cells, as described in the Materials and Methods section (n = 3). C: GLUT4 expression in the adipocytes stimulated with either insulin or CE for 30 min. The adipocytes were retrieved after the stimulation, and the plasma membrane fraction prepared was analyzed by Western blotting as described in the text (n = 4). Values are the means ± S.E. *, p<0.05 between insulin and control; †, p<0.05 between CE and control (Bonferroni test). PM, plasma membrane fraction; lys, lysate of the 3T3-L1 adipocytes.
CE Caused Phosphorylation of AMPK in 3T3-L1 Adipocytes
To investigate the molecular mechanism underlying CE-stimulated GLUT4 translocation and glucose uptake, we studied key kinases involved in the insulin signaling pathway. Insulin-induced glucose uptake was completely cancelled by pretreatment of the adipocytes with the insulin receptor inhibitor AG1024; however, AG1024 could not block the effect of CE (Fig. 2A). Intriguingly, insulin or CE increased the phosphorylation of Akt at its Ser473, and this increase was significantly blocked by AG1024 (Fig. 2B). These results suggest that insulin receptor-Akt signaling was not a major pathway involved in the CE-stimulated glucose uptake. Then we evaluated the contribution of AMPK to the CE-stimulated glucose uptake. CE potently increased the level of phosphorylated AMPK (Fig. 2C). AG1024 did not show any effect on this CE-induced AMPK phosphorylation, but it significantly increased the insulin-mediated phosphorylation of AMPK. Insulin seemed to have little direct effect on the phosphorylation of AMPK in the adipocytes and this inefficacy was abrogated by pre-incubation of the cells with AG1024. Taken together, these results indicate that CE up-regulated glucose uptake through the phosphorylation of AMPK pathway.
3T3-L1 adipocytes were serum-starved for 16 h. For the inhibitor treatment, the cells were pretreated with 60 µM AG1024 for 1 h. The medium was then changed to fresh medium containing 100 nM insulin or 30 µg/ml CE and thereafter stimulated for another 30 min. A: Glucose uptake was measured as described in Materials and Methods section. Each value represents the mean ± S.E. of 3 different experiments. B: Cell lysates were analyzed by Western blotting using anti-phospho-Akt and anti-Akt antibodies. C: Cell lysates were analyzed by Western blotting using anti-phospho-AMPK anti-AMPK antibodies. The data represent the mean ± S.E. of 5 different experiments. *, p<0.05 vs control; †, p<0.05 vs insulin; ‡, p<0.05 vs CE; §, p<0.05 vs insulin+CE; ns: indicates no significant difference (Bonferroni test).
LKB1, AMPK, and ACC are Activated by CE
To corroborate the role of AMPK in CE-mediated glucose uptake, we examined the effect of an AMPK inhibitor. First, 3T3-L1 adipocytes were pre-treated with compound C (200 µM, Com.C), an AMPK inhibitor, to test the effect of it on the phosphorylation of AMPK and uptake of 2-deoxyglucose by the adipocytes stimulated with CE (30 µg/ml). The CE-induced phosphorylation of AMPK was significantly decreased by pre-incubation of the adipocytes with Com.C (Fig. 3A). The CE-stimulated glucose uptake was also attenuated by Com.C (Fig. 3B), indicating the involvement of the AMPK pathway in the CE-induced glucose uptake. Next, we examined the phosphorylation of factors located downstream and upstream of AMPK. As shown in Fig. 3C, the increase in phosphorylated AMPK was accompanied by that of phosphorylated ACC (Ser79), which enzyme is located downstream of AMPK. The phosphorylation of LKB1, which acts upstream of AMPK, was also increased by CE treatment (Fig. 3D). Therefore, these findings show that CE activated the LKB1-AMPK-ACC signaling cascade, eventually leading to increased glucose uptake.
3T3-L1 adipocytes were serum-starved for 16 h. For the inhibitor treatment, cells were pretreated with 200 µM Compound C (Com.C), an inhibitor of AMPK, for 20 min, after which the medium was changed to fresh medium containing 100 nM insulin or 30 µg/ml CE. The cells were then incubated for another 30 min. A: Cell lysates were analyzed by Western blotting using anti-phospho-AMPK and anti-AMPK antibodies. The data represent the mean ± S.E. of 5 different experiment *: p<0.05 vs control; ‡: p<0.05 vs CE; ns: indicates no significant difference (Bonferroni test). B: Glucose uptake was measured as described in the Materials and Methods section. Each value represents the mean ± S.E. of 3 different experiments. *: p<0.05 vs control; †: p<0.05 vs CE; ns: indicates no significant difference (Bonferroni test). C, D: Cell lysates were analyzed by Western blotting using anti-phospho-ACC, anti-phospho- LKB1, anti-ACC, and anti-LKB1 antibodies. The data represent the mean ± S.E. of 5 different experiments. *: p<0.05 vs control; ns: indicates no significant difference (Bonferroni test).
CE Stimulated AMPK through the LKB1 Pathway in C2C12 Myotubes
In addition to the white adipose tissue, muscle plays physiologically important roles in the glucose metabolism. To confirm further the function of LKB1 in the muscle cells, LKB1 siRNA was used to transiently transfect C2C12 muscle cells. The appearance of the differentiated C2C12 cells used in this study is shown in Fig. 4A. The 2 photomicrographs, taken at different fields of a culture, show the typical myotube phenotype. The LKB1 mRNA level and the CE-induced phosphorylation of LKB1 at its Ser428 were decreased by approximately 60% and 40%, respectively, with siRNA pools specific for the LKB1 gene (siLKB1) in comparison with the respective values for the cells transfected with the RNAi negative control (Fig. 4B and C).
Differentiated C2C12 cells (A) were transfected with LKB1 siRNA for 24 h. Two different fields of observation are shown. B: Quantitative real-time PCR was used to determine the mRNA expression levels of LKB1 by the C2C12 cells that had been treated with LKB1 siRNA. The data represent the mean ± S.E. of 3 different experiments. **: p<0.01 (Student’s t test). C: Effect of siRNA duplexes designed to reduce LKB1 expression on the LKB1 protein levels and on the phosphorylation of LKB1 in C2C12 myotube cells examined by Western blotting. These silencing effects were observed even in the culture at 24 h after the transfection. The data represent the mean ± S.E. of 4 different experiments. *: p<0.05 vs control; †: p<0.05 vs CE; ns: indicates no significant difference (Bonferroni test). D: CE (30 µg/ml) was added or not to LKB1 siRNA-transfected cells or negative control siRNA-transfected cells, which were then incubated for 30 min and thereafter harvested and analyzed by Western blotting using anti-phospho-AMPK, and anti-AMPK antibodies. The data represent the mean ± S.E. of 3 different experiments. *: p<0.05 vs control; †: p<0.05 vs CE; ns: indicates no significant difference (Bonferroni test). E: Differentiated C2C12 cells were transfected or not with LKB1 siRNA or negative control siRNA for 24 h and then treated or not with CE (30 µg/ml) for 30 min. The glucose uptake was measured by the method as described in the Materials and Methods section. The data represent the mean ± S.E. of 3 different experiments. *: p<0.05 vs control; †: p<0.05 vs CE; ns: indicates no significant difference (Bonferroni test).
Knockdown of LKB1 attenuated the CE-induced phosphorylation of AMPK (Fig. 4D). The knockdown of LKB1 by siRNA also suppressed CE-stimulated glucose uptake (Fig. 4E). These results suggest that AMPK mediated the effect of CE on signaling pathway in an LKB1-AMPK-dependent manner.
CE-administration Improved Glucose Tolerance in Type 2 Diabetes Model Rats
To determine the effect of CE on type 2 diabetes, we performed the oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) in type 2 diabetes model rats in vivo (Fig. 5). CE administration did not show any effect on body weight (Table 1). In OGTT, CE-treated OLETF rats had lower blood glucose concentrations than that of vehicle-treated OLETF rats (Fig. 5A, 5B) indicating that the improved glucose tolerance in type 2 diabetes model rats. On the other hand, CE did not show any effect on the blood glucose concentration of LETO rats.
A: For oral glucose tolerance test (OGTT), the rats were fasted for 6 h prior to performing the test. Glucose (1.2 g/kg b.w.) was orally administered and the blood-glucose concentration was measured by using the blood obtained from a tail vein at the indicated time points after the oral glucose loading. B: The area under the curve (AUC) of the OGTT for A. C: For insulin tolerance test (ITT), rats were fasted for 4 h. Then insulin (0.75 U/kg b.w.) was intraperitoneally injected and the blood-glucose concentration was measured as described above. D: The area under the curve (AUC) of the ITT for C. Data were analyzed by Bonferroni test. (n = 5–10 for each group, mean ± S.E.). *: p<0.05 vs LETO; †: p<0.05 vs OLETF; ns: indicates no significant difference.
ITT revealed that insulin sensitivity was not influenced by CE treatment in both LETO and OLETF rats (Fig. 5C, 5D).
Discussion
The principal finding of this study is that a hot-water extract of cinnamon (CE) had a metabolic effect on adipocytes and skeletal muscles. Specifically, we demonstrated for the first time that the LKB1-AMPK signaling pathway is involved in CE-stimulated glucose metabolism and that CE improves glucose tolerance in vivo.
AMPK is a heterotrimeric complex composed of a catalytic subunit α and regulatory β and γ subunits [32]. AMPK is activated by phosphorylation of its Thr172 residue located in the activation-loop of the α subunit [33]–[35]. It has been reported that AMPK is activated synergistically by AMP and the phosphorylation of this Thr172 for insulin-independent glucose transport [36]. Activation of AMPK induces the recruitment of GLUT4 to the plasma membrane, resulting in up-regulated glucose uptake [5], [37]. In this study, we demonstrated that CE stimulated the translocation of GLUT4 to the plasma membrane and increased glucose uptake in a concentration and time-dependent manner (Fig. 1B and C). To clarify the relationship between CE-activated glucose metabolism and insulin-independent glucose transport, we used an insulin receptor inhibitor, tyrphostin AG 1024 (AG1024) and an AMPK inhibitor, compound C, in addition to transiently LKB1 knock-downed cells, which have been proven to lack insulin or AMPK-signaling. In fact, we confirmed that CE stimulated glucose uptake was cancelled by inhibition of AMPK and its upstream kinase, the LKB1 (Figs. 3B and 4E). These results strongly suggest that activation of LKB1-AMPK signaling by CE in both adipocyte and muscle cell up-regulates glucose uptake into the cells, but not insulin signaling.
CE caused the phosphorylation of Akt, a kinase acting downstream of the insulin receptor, but little is known about the cross talk between AMPK and insulin signaling. In this study, insulin did not show any additive or synergistic effect with CE in respect to glucose uptake. Actually, insulin signaling was very weakly influenced by CE treatment in comparison with the effect of CE on AMPK signaling. Intriguingly, inhibition of insulin receptors by AG1024 caused an increase in the AMPK activity in the presence of insulin (Fig. 2C), thus suggesting negative regulation of AMPK activity by insulin signaling. It has been reported that insulin inhibits AMPK activity via the PI3K pathway in ischemic cardiac muscle [38]–[39]. To the best of our knowledge, this is the first report to demonstrate the antagonistic effect between insulin and AMPK signaling in adipocytes.
Berberine, an effective compound of Chinese traditional herbal medicine, and S-allyl cysteine in garlic have been reported to ameliorate diabetes and obesity via stimulation of AMPK activity; however, the mechanism for AMPK phosphorylation or its upstream signaling remains unclear [40]–[42]. The tumor suppressor LKB1, a protein ubiquitously expressed by mammalian cells, has been demonstrated to activate AMPK [35], [43]–[47]. It has been reported that LKB1 is activated by two putative kinases; i.e., STE20-related adaptor protein (STRAD) and mouse protein 25 (MO25) [48]. STRAD possesses a domain with high sequence homology to protein kinases but it lacks the key catalytic residues required for catalysis and has therefore been classified a ‘pseudokinase’. MO25 has no sequence homology with other proteins in the database [49]. LKB1 has been shown to phosphorylate and activates AMPK in response to metformin and energy depletion in cell lines [35], [43] as well as in mouse liver and muscle [50], [51]. Although there are many papers reporting the activation of AMPK by natural compounds, the detailed mechanism(s) for the activation still remains unclear. In this study, we speculate that CE promotes the formation of LKB1 complex with STRAD and MO25 to activate AMPK by the phosphorylation of Thr172.
Finally, we observed that CE improved glucose intolerance but not insulin resistance in type 2 diabetic rats (Fig. 5). This observation is consistent with the anti-diabetic effect of CE on insulin-uncontrolled type 1 diabetic rats which we reported previously [27]. In our studies, we identified two compounds in the CE by high-performance liquid chromatography analyses; the CE preparation contained cinnamaldehyde (8.5 µg/mg CE) and cinnamyl alcohol (3.6 µg/mg CE) [27]. Since both cinnamaldehyde and cinnamyl alcohol potently stimulated glucose uptake in 3T3-L1 adipocytes in our studies (data not shown), these compounds are thought to be, at least in partly, responsible for the effect of CE.
In conclusion, the current study demonstrated for the first time that CE stimulates LKB1-mediated phosphorylation of AMPK to increase glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes. Since insulin resistance has been linked with the progressive development of type 2 diabetes mellitus, natural compounds that stimulate AMPK activity should have a significant clinical impact on type 2 diabetes mellitus.
Acknowledgments
We thank House Foods Co. (Tokyo, Japan) for providing the sticks of Cinnamon. We also thank Dr. Etsuko Muraki for helpful discussions and advice.
Author Contributions
Conceived and designed the experiments: YS. Performed the experiments: YS NH KK. Analyzed the data: YS NH KK. Wrote the paper: YS. Contribution to the discussion section of the manuscript: LNJ KS TH TS. Conducted some of the research: TH. Reviewed/edited the manuscript: TA TS.
References
- 1. Wild S, Roglic G, Green A, Sicree R, King H (2004) Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 27: 1047–1053.
- 2. Sewter C, Vidal-Puig A (2002) PPARγ and the thiazolidinediones: molecular basis for a treatment of ‘Syndrome X’? Diabetes Obes Metab 4: 239–248.
- 3. Leverve XM, Guigas B, Detaille D, Batandier C, Koceir EA, et al. (2003) Mitochondrial metabolism and type-2 diabetes: a specific target of metformin. Diabetes Metab 29: 6S88–94.
- 4. Kahn BB, Alquier T, Carling D, Hardie DG (2005) AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 1: 15–25.
- 5. Russell 3rd RR, Bergeron R, Shulman GI, Young LH (1999) Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR. Am J Physiol 277: H643–649.
- 6. Fryer LG, Parbu-Patel A, Carling D (2002) The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways. J Biol Chem 277: 25226–25232.
- 7. Riddle MC (1999) Oral pharmacologic management of type 2 diabetes. Am Fam Physician 60: 2613–2620.
- 8. Nawrocki AR, Rajala MW, Tomas E, Pajvani UB, Saha AK, et al. (2006) Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists. J Biol Chem 281: 2654–2660.
- 9. Hawley SA, Gadalla AE, Olsen GS, Hardie DG (2002) The antidiabetic drug metformin activates the AMP-activated protein kinase cascade via an adenine nucleotide-independent mechanism. Diabetes 51: 2420–2425.
- 10. Cheng BC (1983) A review of cinnamon. Zhong Cao Yao 14: 134.
- 11. Berrio LF, Polansky MM, Anderson RA (1992) Insulin activity: stimulatory effects of cinnamon and brewer’s yeast as influenced by albumin. Horm Res 37: 225–229.
- 12. Onderoglu S, Sozer S, Erbil KM, Ortac R, Lermioglu F (1999) The evaluation of long-term effects of cinnamon bark and olive leaf on toxicity induced by streptozotocin administration to rats. J Pharm Pharmacol 51: 1305–1312.
- 13. Qin B, Nagasaki M, Ren M, Bajotto G, Oshida Y, et al. (2003) Cinnamon extract (traditional herb) potentiates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats. Diabetes Res Clin Pract 62: 139–148.
- 14. Anderson RA, Broadhurst CL, Polansky MM, Schmidt WF, Khan A, et al. (2004) Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity. J Agric Food Chem 52: 65–70.
- 15. Qin B, Nagasaki M, Ren M, Bajotto G, Oshida Y, et al. (2004) Cinnamon extract prevents the insulin resistance induced by a high-fructose diet. Horm Metab Res 36: 119–125.
- 16. Verspohl EJ, Bauer K, Neddermann E (2005) Antidiabetic effect of Cinnamomum cassia and Cinnamomum zeylanicum in vivo and in vitro. Phytother Res 19: 203–206.
- 17. Kannappan S, Jayaraman T, Rajasekar P, Ravichandran MK, Anuradha CV (2006) Cinnamon bark extract improves glucose metabolism and lipid profile in the fructose-fed rat. Singapore Med J 47: 858–863.
- 18. Kim SH, Hyun SH, Choung SY (2006) Anti-diabetic effect of cinnamon extract on blood glucose in db/db mice. J Ethnopharmacol 104: 119–123.
- 19. Mang B, Wolters M, Schmitt B, Kelb K, Lichtinghagen R, et al. (2006) Effects of a cinnamon extract on plasma glucose, HbA, and serum lipids in diabetes mellitus type 2. Eur J Clin Invest 36: 340–344.
- 20. Suppapitiporn S, Kanpaksi N, Suppapitiporn S (2006) The effect of cinnamon cassia powder in type 2 diabetes mellitus. J Med Assoc Thai 89 Suppl 3S200–205.
- 21. Vanschoonbeek K, Thomassen BJ, Senden JM, Wodzig WK, van Loon LJ (2006) Cinnamon supplementation does not improve glycemic control in postmenopausal type 2 diabetes patients. J Nutr 136: 977–980.
- 22. Blevins SM, Leyva MJ, Brown J, Wright J, Scofield RH, et al. (2007) Effect of cinnamon on glucose and lipid levels in non insulin-dependent type 2 diabetes. Diabetes Care 30: 2236–2237.
- 23. Cao H, Polansky MM, Anderson RA (2007) Cinnamon extract and polyphenols affect the expression of tristetraprolin, insulin receptor, and glucose transporter 4 in mouse 3T3-L1 adipocytes. Arch Biochem Biophys 459: 214–222.
- 24. Pham AQ, Kourlas H, Pham DQ (2007) Cinnamon supplementation in patients with type 2 diabetes mellitus. Pharmacotherapy 27: 595–599.
- 25. Subash Babu P, Prabuseenivasan S, Ignacimuthu S (2007) Cinnamaldehyde-a potential antidiabetic agent. Phytomedicine 14: 15–22.
- 26. Wang JG, Anderson RA, Graham 3rd GM, Chu MC, Sauer MV, et al. (2007) The effect of cinnamon extract on insulin resistance parameters in polycystic ovary syndrome: a pilot study. Fertil Steril 88: 240–243.
- 27. Shen Y, Fukushima M, Ito Y, Muraki E, Hosono T, et al. (2010) Verification of the antidiabetic effects of cinnamon (Cinnamomum zeylanicum) using insulin-uncontrolled type 1 diabetic rats and cultured adipocytes. Biosci Biotechnol Biochem 74: 2418–2425.
- 28. Seki T, Miyasu T, Noguchi T, Hamasaki A, Sasaki R, et al. (2001) Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes. J Cell Physiol 189: 72–78.
- 29. Ramirez-Zacarias JL, Castro-Munozledo F, Kuri-Harcuch W (1992) Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with Oil red O. Histochemistry. 97: 493–497.
- 30. Ragolia L, Begum N (1997) The effect of modulating the glycogen-associated regulatory subunit of protein phosphatase-1 on insulin action in rat skeletal muscle cells. Endocrinology 138: 2398–2404.
- 31. Nishiumi S, Ashida H (2007) Rapid preparation of a plasma membrane fraction from adipocytes and muscle cells: application to detection of translocated glucose transporter 4 on the plasma membrane. Biosci Biotechnol Biochem 71: 2343–2346.
- 32. Carling D (2004) The AMP-activated protein kinase cascade-a unifying system for energy control. Trends Biochem Sci 29: 18–24.
- 33. Hawley SA, Davison M, Woods A, Davies SP, Beri RK, et al. (1996) Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol Chem 271: 27879–27887.
- 34. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, et al. (2004) LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23: 833–843.
- 35. Shaw RJ, Kosmatka M, Bardeesy N, Hurley RL, Witters LA, et al. (2004) The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 101: 3329–3335.
- 36. Goodyear LJ, Kahn BB (1998) Exercise, glucose transport, and insulin sensitivity. Annu Rev Med 49: 235–261.
- 37. Amitani H, Asakawa A, Cheng K, Amitani M, Kaimoto K, et al. (2013) Hydrogen improves glycemic control in type1 diabetic animal model by promoting glucose uptake into skeletal muscle. PLoS One 8: e53913.
- 38. Gamble J, Lopaschuk GD (1997) Insulin inhibition of 5′ adenosine monophosphate-activated protein kinase in the heart results in activation of acetyl coenzyme A carboxylase and inhibition of fatty acid oxidation. Metabolism 46: 1270–1274.
- 39. Beauloye C, Marsin AS, Bertrand L, Krause U, Hardie DG, et al. (2001) Insulin antagonizes AMP-activated protein kinase activation by ischemia or anoxia in rat hearts, without affecting total adenine nucleotides. FEBS Lett 505: 348–352.
- 40. Cheng Z, Pang T, Gu M, Gao AH, Xie CM, et al. (2006) Berberine-stimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK. Biochim Biophys Acta 1760: 1682–1689.
- 41. Lee YS, Kim WS, Kim KH, Yoon MJ, Cho HJ, et al. (2006) Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes 55: 2256–2264.
- 42. Hwang YP, Kim HG, Choi JH, Do MT, Chung YC, et al. (2013) S-allyl cysteine attenuates free fatty acid-induced lipogenesis in human HepG2 cells through activation of the AMP-activated protein kinase-dependent pathway. J Nutr Biochem 24: 1469–1478.
- 43. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, et al. (2003) Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2: 28.
- 44. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, et al. (2005) Calmodulin-dependent protein kinase kinase-β is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2: 9–19.
- 45. Hurley RL, Anderson KA, Franzone JM, Kemp BE, Means AR, et al. (2005) The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases. J Biol Chem 280: 29060–29066.
- 46. Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, et al. (2005) Ca2+/calmodulin-dependent protein kinase kinase-β acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2: 21–33.
- 47. Pil Hwang Y, Gyun Kim H, Choi JH, Truong Do M, Tran TP, et al. (2013) 3-Caffeoyl, 4-dihydrocaffeoylquinic acid from Salicornia herbacea attenuates high glucose-induced hepatic lipogenesis in human HepG2 cells through activation of the liver kinase B1 and silent information regulator T1/AMPK-dependent pathway. Mol Nutr Food Res 57: 471–482.
- 48. Alessi DR, Sakamoto K, Bayascas JR (2006) LKB1-dependent signaling pathways. Annu Rev Biochem 75: 137–163.
- 49. Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, et al. (2004) LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J 23: 833–843.
- 50. Sakamoto K, McCarthy A, Smith D, Green KA, Grahame Hardie D, et al. (2005) Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction. EMBO J 24: 1810–1820.
- 51. Shaw RJ, Lamia KA, Vasquez D, Koo SH, Bardeesy N, et al. (2005) The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310: 1642–1646.