Emodin Regulates Glucose Utilization by ActivatingAMPactivated Protein Kinase2.docx

1、Emodin Regulates Glucose Utilization by ActivatingAMPactivated Protein Kinase2Emodin Regulates Glucose Utilization by Activating AMP-activated Protein Kinase*Parkyong Song, Jong Hyun Kim, Jaewang Ghim, Jong Hyuk Yoon, Areum Lee, Yonghoon Kwon, Hyunjung Hyun, Hyo-Youl Moon, Hueng-Sik Choi,* Per-Olof

2、Berggren, Pann-Ghill Suh, and Sung Ho Ryu,1From the Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Republic of Korea, NovaCell Technology Inc., Pohang, Kyungbuk 790-784, Republic of Korea, the Division of Integrative Biosciences and Bi

3、otechnology, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea, the School of Nano-Biotechnology and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan 689-805, Republic of Korea, the *Hormone Research Center, School of Biological Sciences an

4、d Technology, Chonnam National University, Gwangju, Republic of Korea, and the Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, SE-171 77 Stockholm, Sweden1 To whom correspondence should be addressed: POSTECH Biotech Center, San 31 Hyojadong, Pohang 790-784, Republic

5、of Korea., Tel.: Phone: 82-54-279-2292; Fax: 82-54-279-0645; E-mail: rk.ca.hcetsopohgnus.Author information Article notes Copyright and License information Received December 1, 2012; Revised January 5, 2013Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.This articl

6、e has been cited by other articles in PMC.Go to:AbstractAMP-activated protein kinase has been described as a key signaling protein that can regulate energy homeostasis. Here, we aimed to characterize novel AMP-activated kinase (AMPK)-activating compounds that have a much lower effective concentratio

7、n than metformin. As a result, emodin, a natural anthraquinone derivative, was shown to stimulate AMPK activity in skeletal muscle and liver cells. Emodin enhanced GLUT4 translocation and 14Cglucose uptake into the myotube in an AMPK-dependent manner. Also, emodin inhibited glucose production by sup

8、pressing the expression of key gluconeogenic genes, such as phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, in hepatocytes. Furthermore, we found that emodin can activate AMPK by inhibiting mitochondrial respiratory complex I activity, leading to increased reactive oxygen species and Ca

9、2+/calmodulin-dependent protein kinase kinase activity. Finally, we confirmed that a single dose administration of emodin significantly decreased the fasting plasma glucose levels and improved glucose tolerance in C57Bl/6J mice. Increased insulin sensitivity was also confirmed after daily injection

10、of emodin for 8 days using an insulin tolerance test and insulin-stimulated PI3K phosphorylation in wild type and high fat diet-induced diabetic mouse models. Our study suggests that emodin regulates glucose homeostasis in vivo by AMPK activation and that this may represent a novel therapeutic princ

11、iple in the treatment of type 2 diabetic models.Keywords: AMP-activated Kinase (AMPK), Calcium, Glucose Metabolism, Insulin Resistance, Metabolic SyndromeGo to:IntroductionAMPK2 is a highly conserved mammalian serine/threonine protein kinase and acts as a master energy sensor that is responsible for

12、 regulating energy homeostasis (1, 2). These heterotrimeric enzymes are activated under cellular stress conditions such as starvation, exercise, oxidative damage, and hypoxia. Liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) are two regulatory upstream kinases that

13、stimulate AMPK phosphorylation (3). AMPK controls whole body energy homeostasis by regulating glucose and lipid metabolism in multiple peripheral tissues. For example, AMPK activation by 5-aminoimidazole-4-carboxamide-1-ribofuranoside stimulates glucose uptake via PI3K-independent GLUT4 translocatio

14、n in skeletal muscle (4, 5) and inhibits hepatic glucose production (6).Recently, many types of AMPK activators, including cytokines, drugs, and natural compounds, have been identified (3). Among them, metformin (1,1-dimethylbiguanide hydrochloride) and thiazolidinediones are widely used for the tre

15、atment of type 2 diabetes. The main function of metformin is to decrease blood glucose levels by inhibiting hepatic gluconeogenesis and increasing glucose uptake in skeletal muscle (7). Interestingly, the mitochondrial respiratory complex is the common target of metformin, which indirectly activates

16、 AMPK (8). Inhibition of complex I activity can cause changes in the cellular nucleotide ratio (9) and increase reactive oxygen species (10), which are important intermediates to increase AMPK activity. Despite their beneficial effects to glucose utilization, metformin must be administered at high c

17、oncentrations, and they have adverse effects, such as gastrointestinal symptoms and relatively rare lactic acidosis in vivo (11). Furthermore, some human studies have shown that the glucose-lowering effects of metformin are secondary to reduction in hepatic gluconeogenesis (1113). Thus, there is a g

18、rowing demand to identify more effective AMPK activators, which have low dosage and affect various metabolic organs.In this study, we screened a drug library to identify novel AMPK activators. As a result of this screening, emodin (1,3,8-trihydroxy-6-methylanthraquinone), a natural active compound i

19、n the herb Rheum palmatum L. was shown to stimulate the AMPK pathway. Some previous studies have suggested that emodin has an beneficial effect on energy metabolism, including adipocyte differentiation (14), anti-fibrotic effect on pancreatic fibrosis (15), and liver (16). However, validation of the

20、 molecular mechanism and the anti-diabetic effects of this compound has not been fully examined. Here, we show that emodin can increase glucose uptake in skeletal muscle via inhibition of mitochondrial complex I, which then leads to activation of AMPK. In addition, emodin inhibited hepatic gluconeog

21、enesis through AMPK. Importantly, acute intravenous administration of emodin in both wild type and high fat diet-induced diabetic mice significantly decreased blood glucose levels associated with increased glucose utilization in skeletal muscle and liver. Based on the improved glucose metabolism and

22、 insulin sensitivity, this compound holds great promise for eventual use a therapeutic agent to treat type 2 diabetes.Go to:EXPERIMENTAL PROCEDURESMaterials 5-Aminoimidazole-4-carboxamide-1-ribofuranoside was purchased from Toronto Research Chemical Inc. (Toronto, Ontario, Canada). Emodin, dichlorod

23、ihydrofluorescein diacetate, o-phenylenediamine, resveratrol, and metformin were purchased from Sigma, and 2-deoxy14Cglucose was from American Radiolabeled Chemicals Inc. Compound c, an AMPK inhibitor, and SB203580, a p38 MAPK inhibitor, were provided by Merck (RY 70-100).Cell Culture L6 myoblasts w

24、ere grown in -minimal essential medium, and mouse hepatoma Hepa1c1c7 cells were maintained in DMEM containing 10% fetal bovine serum (FBS), 50 units of penicillin/ml, and 50 g of streptomycin/ml at 37 C under humidified air atmosphere containing 5% CO2. Differentiation of myoblast was induced in med

25、ium supplemented with 2% FBS within 7 days.Immunoblotting To prepare total cell lysates, myotube was washed with cold PBS and then lysed in cold lysis buffer (in mmol/liter: 40 HEPES, pH 7.5, 120 NaCl, 1 EDTA, 10 pyrophosphate, 10 glycerophosphate, 50 NaF, 1.5 Na3VO4, 1 PMSF, 5 MgCl2, 0.5% Triton X-

26、100, and protease inhibitor mixture). Transferred membrane was incubated with primary antibodies (1:1000) overnight at 4 C. The following were used: p-AMPK(Thr-172), ACC, p-Akt(Ser-473) (Cell Signaling Technology), AMPK (Upstate), p-ACC(Ser-79) (Millipore), phosphoenolpyruvate carboxykinase (Cayman)

27、, PGC1 (Abcam, UK), and monoclonal anti-Myc antibody (Millipore Corp., Billerica, MA).AMPK Activity Assay Cells were serum-fasted for 3 h and then treated with emodin for 5 min. After precipitation, AMPK activity was evaluated as incorporation of 32P into a synthetic SAMS peptide in kinase reaction

28、buffer (in mmol/liter: 40 HEPES, pH 7.5, 80 NaCl, 1 DTT, 0.2 AMP, 0.2 ATP, 5 MgCl2, 0.1 SAMS, 0.25 -32PATP) at 30 C for 20 min.Determination of 2-Deoxy14Cglucose Uptake in Cells 2-Deoxyglucose uptake was determined as described previously (17). Briefly, differentiated L6 cells were serum-starved for

29、 2 h prior to the assay. Cells were then washed twice with Krebs-Henseleit buffer and were stimulated with or without the indicated agents for 60 min. Glucose uptake was measured by incubation with 0.1 mCi/ml 2-deoxy14Cglucose at room temperature for 10 min.Mitochondrial Complex I Activity Cells wer

30、e disrupted with nondenaturing detergent lauryl maltoside to maintain enzymatic activity and equilibrated with the indicated concentration of metformin and emodin for 10 min. Reaction was initiated by adding the reaction substrate NADH (500 mol/liter) and ubiquinone-1 (50 mol/liter). A decrease in 3

31、40 nm absorbance was recorded over 10 min using a UV spectrophotometer.Fatty Acid Oxidation Serum-fasted myotube was incubated with emodin for 2 h in oxidation media (0.1% lipid-free BSA, 100 m 3Hpalmitate, vaporized for 30 min with N2 gas to saturate palmitate). After oxidation, the rest of the fre

32、e fatty acid was removed with 10% TCA precipitation. Supernatant in a capless microtube was vaporized in a scintillation tube with distilled water added at 50 C for 12 h.Mitochondrial Complex I Activity Cells were disrupted with nondenaturing detergent lauryl maltoside to maintain enzymatic activity and equilibrated with the indicated concentration of metformin and emodin for 10 min. Reaction was initiated by adding the reaction substrate NADH (500 mol/liter) and ubiquinone-1 (50 mol/liter). A decrease in 340 nm absorbance was recorded over 10 min using