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Erythropoietin Reduces Insulin Resistance Via Regulation of Its Int. J. Biol. Sci. 2017, Vol. 13 1329 Ivyspring International Publisher International Journal of Biological Sciences 2017; 13(10): 1329-1340. doi: 10.7150/ijbs.19752 Research Paper Erythropoietin Reduces Insulin Resistance via Regulation of Its Receptor-Mediated Signaling Pathways in db/db Mice Skeletal Muscle Yu Pan1, Xiu Hong Yang2, Li Li Guo3,Yan Hong Gu2, Qing Yan Qiao2, Hui Min Jin2 1. Division of Nephrology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; 2. Division of Nephrology, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China; 3. Hemodialysis Center, Baoshan Branch of Shanghai No.1 People’s Hospital, Shanghai, China. Corresponding author: Hui Min Jin, MD., Address: Division of Nephrology, Shanghai Pudong Hospital, Fudan University, Pudong Medical Center, 2800 Gongwei Road, Huinan Town, Pudong, Shanghai 201399, China. Fax: +86 21 68036053. E-mail: [email protected] © Ivyspring International Publisher. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions. Received: 2017.02.21; Accepted: 2017.08.08; Published: 2017.10.17 Abstract Erythropoietin (EPO) can reduce insulin resistance (IR) in adipocytes; however, it is unknown whether EPO can decrease IR in skeletal muscle. Here we investigated whether EPO could reduce IR in type 2 diabetic mouse skeletal muscle and its possible signaling mechanisms of action. Twelve-week-old db/db diabetic mice were employed in this study. Systemic use of EPO improved glucose profiles in type 2 diabetic mice after 4 and 8 weeks treatment. EPO up-regulated EPOR protein expression in skeletal muscle, and subsequently activated downstream signaling molecules such as JAK2, IRS-1, PI3K, AKT, and eNOS. We next constructed lentivirally-delivered shRNAs against EPOR and transfected skeletal muscle cells to knockdown EPOR. EPOR knockdown inhibited EPO induced JAK2, IRS-1, PI3K, AKT, eNOS signaling transduction, autophagy and Glut 4 translocation, as well as promoted apoptosis in skeletal muscle. Thus, EPO reduces skeletal muscle IR in type 2 diabetic mice via its specific receptor, EPOR. Possible mechanisms involved in its action may include increased autophagy and reduced apoptosis in type 2 diabetic skeletal muscles, which provides a new strategy for the treatment of IR. Key words: Erythropoietin (EPO), Erythropoietin receptor EPOR, Insulin resistance (IR), Skeletal muscle, type 2 diabetes, Insulin signaling. Introduction Type 2 diabetes mellitus is highly prevalent many studies suggest EPO is also active locally in worldwide, and is characterized by insulin resistance many other tissues, including cardiovascular, renal, (IR). IR occurs most commonly within peripheral endothelium, nerves, and muscle in response to adipocytes and skeletal muscle 1, and recently, these physical and metabolic stress2-7. In non-hematopoietic tissues have been of research interest due to the cells, however, it remains controversial whether EPO increasing importance of strategically targeting IR for exerts its effect by binding EPOR. In our previous the therapeutic prevention of type 2 diabetes. research8, we confirmed that EPOR is expressed in Erythropoietin (EPO) is a glycoprotein produced 3T3-L1 adipocytes, and EPO improved IR in cultured during fetal development within kidney and liver adipocytes via EPOR. Blockade of EPOR with tissue, and has been widely used in the clinic to treat antagonists or siRNA completely reversed IR decrease anemia induced by chronic kidney disease, tumors induced by EPO, indicating EPO reduces IR via EPOR and other diseases. EPO primarily activates and mediated signaling cascades in adipocytes. promotes erythroid cell proliferation and growth via The role and regulatory processes involving EPO its specific cell surface receptor, EPOR. However, and EPOR in skeletal muscle remain to be elucidated. http://www.ijbs.com Int. J. Biol. Sci. 2017, Vol. 13 1330 Part of the difficulties in ascertaining this information µl) was collected from the tail vein (t=0). Serum is due to inconsistent results with respect to prior in glucose levels were determined using the One Touch vitro and in vivo studies. Evidence suggests C2C12 Basic glucose meter (Lifescan Canada, Burnaby, myoblasts show elevated JAK2, STAT5 and AKT British Columbia, Canada). Next, a glucose tolerance phosphorylation when treated with EPO, similar to test was performed after mice were fasted overnight. signaling responses observed in neural cells9, 10. Briefly, mice were injected intraperitoneally with Interestingly, STAT5 activation was not observed in 1g/kg glucose. Serum glucose was quantified in tail rat skeletal muscle tissue in response to EPO blood samples collected at 0 (prior to glucose stimulation11. In human skeletal muscle, acute EPO administration), 30, 60, 90 and 120 min after glucose administration failed to induce phosphorylation and administration. An insulin tolerance tests was activation of STAT5, AKT and MAPK signaling performed 3 days following tests for glucose pathways12. Discrepancies in the measurement of tolerance. All mice with free access to water and food EPOR mRNA and protein levels may be related to were injected intraperitoneally with insulin (1 IU/kg) potential species differences and inherent potential and serum glucose was quantified in tail blood variability in in vitro and in vivo models. Using samples collected at 0 (prior to insulin primary human muscle cells and examining mouse administration), 30, 60, 90 and 120 min after insulin muscle in vivo will aid in clarifying these administration. Plasma insulin was assayed using an inconsistencies. Depending on this background ELISA kit (Alpco, Salem, USA). information, we designed and executed experiments using db/db mice, a type 2 diabetic mouse model, to Muscle NO level measurement explore whether EPO therapy can reduce skeletal Muscle NO levels were measured using a 280i muscle IR, and elucidate potential signaling pathways Nitric Oxide Analyzer (GE Analytical Instruments), that may be involved in the mechanism of EPO-EPOR according to the manufacturer’s instructions. In brief, signaling in type 2 diabetes skeletal muscle. ice-cold ethanol was added into muscle homogenate samples at a ratio of 2:1. The mixture was kept at 0°C Materials and Methods for 30 min and then centrifuged at 14,000 RPM for 5 min. The supernatant was then used for NO analysis Animal model and in vivo experimental design based on a gas-phase chemiluminescent reaction Twelve-week-old db/db mice and background between NO and ozone. control mice (db/m) were purchased from SLAC Laboratories (Shanghai, China). All mice were housed Periodic Acid–Schiff staining and under controlled light (12 h light/12 h dark) and immunofluorescence temperature conditions, and had ad libitum access to Mice were anesthetized and hind legs food (normal rodent chow) and water. All procedures gastrocnemius muscles were immediately dissected. were conducted in accordance with the guidelines of Tissues were then fixed in 4% buffered the Chinese Council on Animal Care and were paraformaldehyde for 2 days, embedded in paraffin approved by Shanghai Jiao Tong University Animal and processed for sectioning. Tissue sections (3µm) Care Committee. were assessed by Periodic Acid–Schiff staining. All experiments involving db/db mice were Immunofluorescence analyses of mouse muscle performed at 12 weeks of age following determination sections were accomplished with DAPI labeling of cell that glucose concentrations in fasting blood samples nuclei, and specific antibody according to standard indicated the mice were diabetic. Mice were divided protocols. Briefly, sections were incubated overnight into the following treatment groups (n=10/each, 5 at 4°C with rabbit anti-EPOR antibody (Santa Cruz male and 5 female): (1) non-treated db/db (treated with Biotechnology, Santa Cruz, CA, USA).After extensive 0.9% saline), (2) EPO (0.4μg/kg/w) (recombinant washes, sections were then incubated with anti-rabbit human Epoetin alpha, 3SBIO, China), and (3) EPO FITC-conjugated secondary antibody (DAKO, (1.2μg/kg/w). Saline and EPO were administered to Carpinteria, CA, USA) for 30 min. Following labeling mice once a weekfor8 week via subcutaneous and coverslip mounting, images were acquired using injection. a Nikon (ECLIPSE-E1000) microscope connected to a video camera equipped with a color monitor and Fasting glycemia, glucose tolerance (IGTT) ImagePlus software. and insulin tolerance (ITT insulin) tests Mice were subjected to glucose tolerance and Preparation of primary muscle cells insulin tolerance tests at days 0, 28 and 56. Briefly, Primary muscle cells were obtained from lower mice were fasted for 15h, and a basal blood sample (20 hind limb muscles of adult db/db animals (10 week old) using a protocol modified from previous http://www.ijbs.com Int. J. Biol. Sci. 2017, Vol. 13 1331 description13. In brief, The muscles were excised from cytosolic proteins were extracted using a Membrane the lower limbs of mice, thoroughly minced and Protein Extraction Reagent (Thermo Fisher, Waltham, digested. And next the cells were collected via a MA, USA). Extracted proteins were used for Western 5-minute centrifugation at 1800g. The collected cells blot analysis. were exposed to trypsin-EDTA (solarbio) (0.25% with 1mM EDTA) at 37°C for 10 minutes. Fibroblasts mRNA Isolation and real-time
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