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The Microrna Signature in Response to Insulin Reveals Their Implication In

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The Microrna Signature in Response to Insulin Reveals Their Implication In Diabetes Publish Ahead of Print, published online August 31, 2009 Insulin regulates microRNA in skeletal muscle The microRNA signature in response to insulin reveals their implication in the transcriptional action of insulin in human skeletal muscle and the role of a SREBP-1c/MEF2C pathway Running title: Insulin regulates microRNA in skeletal muscle Aurélie Granjon1, Marie-Paule Gustin2, Jennifer Rieusset1, Etienne Lefai1, Emmanuelle Meugnier1, Isabelle Güller1, Catherine Cerutti2, Christian Paultre2, Emmanuel Disse3,4, Rémi Rabasa-Lhoret5, Martine Laville1,3,4, Hubert Vidal1,3,4 and Sophie Rome1* 1 INRA 1235 ; INSERM 870 ; INSA-Lyon ; Régulations Métaboliques Nutrition et Diabète ; Université Lyon; Oullins 69600-FRANCE. 2INSERM ERI22 / EA 4173, Biostatistiques ; Université Lyon; Lyon, 69008- FRANCE 3Hospices Civils de Lyon ; Service de Diabétologie et Nutrition, Hôpital Edouard-Herriot, Lyon, 69008- FRANCE. 4Centre de Recherche en Nutrition Humaine Rhône-Alpes, Oullins 69600-FRANCE. 5Chaire de recherche J-A DeSève, Institut de recherches cliniques de Montréal (IRCM), Montréal (Québec) H2W 1R7. *Correspondence: Sophie Rome Email : [email protected] Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org. Submitted 5 February 2009 and accepted 5 August 2009. This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org Copyright American Diabetes Association, Inc., 2009 Insulin regulates microRNA in skeletal muscle Objective Factors governing microRNA expressions in response to changes of cellular environment are still largely unknown. Our aim was to determine whether insulin, the major hormone controlling whole-body energy homeostasis, is involved in the regulation of microRNA expressions in human skeletal muscle. Research Design and Methods We carried out comparative miRNA expression profiles in human skeletal muscle biopsies before and after a 3-h euglycemic-hyperinsulinemic clamp, with TaqMan low density arrays. Then, using DNA microarrays we determined the response to insulin of the miRNA putative target genes in order to determine their role in the transcriptional action of insulin. We further characterized the mechanism of action of insulin on two representative miRNAs, miR-1 and miR-133a in human muscle cells. Results Insulin down-regulated the expressions of 39 distinct miRNAs in human skeletal muscle. Their potential target mRNAs coded for proteins that were mainly involved in insulin signaling and ubiquitination-mediated proteolysis. Bioinformatic analysis suggested that combinations of different down-regulated miRNAs worked in concert to regulate gene expressions in response to insulin. We further demonstrated that SREBP-1c and MEF2C were involved in the effect of insulin on miR-1 and miR-133a expression. Interestingly, we found an impaired-regulation of miRNAs by insulin in the skeletal muscle of type 2 diabetic patients, likely as consequences of altered SREBP-1c activation. Conclusions This work demonstrates a new role of insulin in the regulation of miRNAs in human skeletal muscleand suggests a possible implication of these new modulators in insulin-resistance. 2 Insulin regulates microRNA in skeletal muscle nsulin is one of the major hormones miRNAs in the regulation of fat metabolism, involved in the control of energy adipocyte differentiation, energy homeostasis I expenditure, carbohydrate, lipid and and glucose-stimulated insulin secretion (9). protein metabolism. It also regulates a variety Skeletal muscle is one of the largest of biological processes such as protein turn- tissues in the human body and is the major over, cell growth and differentiation, and site for insulin-dependent glucose disposal. DNA synthesis. To perform these actions in a Changes in mRNA and protein abundance in concerted manner, insulin coordinates a this tissue are central to a large number of complex program of transcriptional changes metabolic and other disorders including (1; 2) in the human skeletal muscle. insulin-resistance. The discovery that some Recently, our understanding of miRNAs are expressed specifically in skeletal complex gene-regulatory networks governing muscle raises the question of their potential cell physiology has rapidly evolved with the involvement in the transcriptional action of discovery of microRNAs (miRNAs). They insulin in this tissue (10). Here, we report that represent a large class of evolutionary insulin regulates miRNA expressions in the conserved RNAs of 21 to 22 nt which act as human skeletal muscle in vivo which in return negative regulators of gene expression either participate in insulin transcriptional action in by inhibiting mRNA translation or promoting this tissue. We also demonstrated that miR-1 mRNA degradation through base paring to the and miR-133a, specifically expressed in 3’untranslated region (UTR) of target mRNAs muscle tissues (11-13) are regulated by (3). These small noncoding RNAs are insulin via SREBP-1c and MEF2C. transcribed by RNA polymerase II producing Furthermore we found that miR-1 and miR- long primary transcripts (pri-miRNAs) which 133a have impaired insulin response in the are then processed by Drosha and Pasha (also skeletal muscle of type 2 diabetic patients. DGCR8) which result in a stem-loop precursor called pre-miRNA. The pre-miRNA RESEARCH DESIGN AND METHODS is subsequently transported from the nucleus Euglycemic-hyperinsulinemic to the cytoplasm by Exportin-5 (XPO5). Then clamps. Volunteers provided written consent DICER1 (RNase III endonuclease) processes and protocols were approved by ethics the stem-loop to produce a 21-bp RNA committee (Hospices Civils Lyon) . 15 duplex. One strand of the duplex, the miRNA, subjects without history of diabetes were enters the RNA-induced silencing complex submitted to a 3-h euglycemic- (RISC) and directs the complex to target hyperinsulinemic clamp to achieve supra- mRNA (4). In agreement with their important physiological plasma insulin concentrations regulatory functions, global effects of (insulin infusion rate of 2 mU.min-1.kg-1). 5 miRNAs upon the transcriptional profile and subjects among the 15 were selected on the tissue specificity of mRNA expression have basis of their ages (51 + 2 years). Their been reported in response to experimental response to insulin infusion was compared to manipulations of miRNA levels (5-7) and the the response of 5 insulin-resistant type 2 latest estimate indicates that they may diabetic patients (50 + 3 years) who had regulate up to one-third of the mammalian interrupted their usual treatment with oral genome (8). anti-diabetic agents at least 5 days before Important roles for these miRNAs investigations. Metabolic parameters are have emerged in the control of metabolic presented in the online appendix in Table S1. pathways as suggested by studies implicating Percutaneous biopsies of the vastus lateralis 3 Insulin regulates microRNA in skeletal muscle muscle were performed under basal data. qRT-PCR data were computed as fold conditions and after the clamp study. using geometric means and normalized to the Hyperglycemic-euinsulinemic mean value of the control sample in each clamp. Seven healthy men were submitted to paradigm, defined as one. All experiments a 3-h hyperglycemic-euinsulinemic clamp were performed in triplicate and comparisons with infusion of somatostatin to inhibit were analyzed using Student's t-test. Data are endogenous insulin release. Muscle biopsies expressed as mean ± SEM. Significance was of vastus lateralis muscles were performed defined as P < 0.05. under basal conditions and after the clamp Microarray analysis. cDNA study as described previously (14). microarrays are from The Stanford Functional STZ mice. Three groups of 10-week- Genomics Facility old C57Bl/6 males were used (n=6). Control (http://www.microarray.org/sfgf/). The data group was given daily i.p. injections of set is available from GEO database (GSE sodium citrate buffer for 3 days. The 11868). The 6 insulin-sensitive subjects used remaining two groups were made diabetic by for microarray analysis were included in the daily i.p. injection of streptozotocin (STZ) for group of 15 (Table S1). Signal intensities 3 consecutive days. Glucose levels were were log-transformed and normalization was monitored daily and mice were studied when performed by Lowess method. Only spots they achieved fed-blood sugars >500 mg/dl with recorded data on the 6 slides were for at least 3 days. One group was further selected for analysis. With these criteria, treated with 2 injections of insulin 26,108 spots were retrieved. Among them (Insulatard, Novo Nordisk, 3mU). 24 hours 11,864 had a gene symbol and were after the first injection of insulin all animals considered in this study as the list of genes were sacrified and gastrocnemius muscles expressed in human muscle. Genes with fold were removed. changes ≥ |1.19| were considered as miRNA expression profiles in significantly regulated (i.e.; corresponding to human muscle cells or skeletal muscle 95th percentile of genes based on the biospsies. Differentiated myotubes were magnitude of the fold changes) which prepared from 3 different
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