MAPK ERK signaling regulates insulin sensitivity to control glucose metabolism in drosophila

MAPK/ERK SIGNALING REGULATES INSULIN SENSITIVITY TO CONTROL GLUCOSE METABOLISM IN DROSOPHILA ZHANG WEI INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2012 MAPK/ERK SIGNALING REGULATES INSULIN SENSITIVITY TO CONTROL GLUCOSE METABOLISM IN DROSOPHILA ZHANG WEI (B.Sc., Fudan University, China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2012 Acknowledgments First of all, I would like to thank my supervisor Dr Stephen Cohen, for giving me the opportunity and freedom to work on whatever I like in his lab It was great luck for me to start my last PhD rotation upon his lab moving from EMBL Heidelberg, Germany to Temasek Life Sciences Laboratory (TLL), Singapore in August 2007 Since then, I have been impressed by his talent and enthusiasm towards science, as well as the open lab atmosphere under his leadership It is an honor to work with so many wonderful lab members, former and present, after settling down in Dr Cohen’s lab at the end of 2007 Among them, I am deeply indebted to Ville Hietakangas, who now is leading an independent lab in Finland Ville did the FoxO gain-of-function screen in the Drosophila eye and initiated this project I am very grateful for his guidance and help during the long battle to get this work published I would also like to thank our lab managers Lim Singfee and Chen Ya-wen for their invaluable support in the lab I have enjoyed working with them so much I am also very happy to have had the chance of working with Eva Loser, Thomas Sandmann and Sebastien Szuplewski, from whom I learned enormous knowledge about cell culture, Q-PCR and fly genetics I am very thankful, as well, to Jishy Varghese for discussing science with me and sharing his experimental experience on metabolism Out of the Cohen’s group, I would like to express my appreciation to Sung HsinHo for conversations about my work, science and the future Hsinho shares lab space with I us since we moved to Institute of Molecular and Cell Biology (IMCB) in July 2010 He also helped me with some experiments when I was traveling around Of course, I have to thank my friends made in Singapore: Liu Fangfang, Luo Hang, Qian Neng and Li Jingping It’s the best to have you around me during the four-year PhD study Finally, I would like to thank the most important people in my life, my parents Although they are in China, far away from Singapore, I always have the feeling that they are just next to me supporting me I owe all my achievements to them To express my gratitude to them, I specially wrote a Chinese version of this ‘Acknowledgements’ for my father and mother Zhang Wei IMCB Dec 2011 II 致谢 首先，我要感谢我的导师 Stephen Cohen 博士，谢谢他给我机会和自由在他的实 验室从事我想做的工作。 在 2007 年的 月，当他的实验室从德国海德堡 EMBL 搬来新加坡淡马锡生命科学实验室(TLL)的时候, 我很幸运能够在他的实 验室做我的第三个实验室试用项目。从那时起，他对科学的天赋和热忱，以及 在他领导下实验室开放的科学氛围，给我留下了深刻的映像。 因此在 2007 年 12 月，我正式加入 Cohen 博士的实验室，并有幸与一群杰出的 实验室同僚共事。在他们中间，我深深地受惠于 Ville Hietakangas，他现在在芬 兰已经有了自己独立的实验室。Ville 用果蝇的复眼做了 FoxO 获得性功能的遗 传筛选，并为我打开了这个项目的大门。从那时起一直到现在我的工作发表， 我十分感激他这一路对我的指导和帮助。 另外我也要感谢我们的实验室主管林声慧和陈雅雯，谢谢她们在工作上莫大的 支持。我很高兴能够和她们一起共事到现在。同时我也很高兴曾经和 Eva Loser, Thomas Sandmann，还有 Sebastien Szuplewski 在同一个实验室共事，他们教会 了我很多细胞培养，定量 PCR 和果蝇遗传学的知识。再有，我要感谢 Jishy Varghese，谢谢他和我讨论科学，教授我关于新陈代谢方面的实验技巧。 在 Cohen 小组之外，我要向宋欣和表达我的谢意，谢谢他常常和我谈论工作， 科学和对未来的规划。在 2010 年 月，我们搬到了分子细胞生物研究所 (IMCB)。自那时起，欣就成了我们的邻居。他也在我出国时帮忙做了一些实验。 III 当然，我也不能忘记我在新加坡遇到的好友：刘方芳，罗航，钱能和李菁萍。 这四年留学生涯有你们在我身边是最美好的。 最后，我要感谢我人生中最重要的两个人，他们是我的父母。虽然他们远在千 里之外的中国，我却常常感到他们一直在我身边支持我。我今天所取得的一切 成就都归功于他们。为了表达我对他们的感恩，我特地将这篇中文致词献给我 的爸爸妈妈。 张威 IMCB 2011 年 12 月 IV Table of Contents Acknowledgments I Table of Contents V Summary VIII List of Tables X List of Figures .XI List of Abbreviations XIII Publication XV Introduction 1.1 Insulin-like signaling pathway 1.1.1 The phosphatidylinositol-3-kinase (PI3K)/AKT Pathway 1.1.2 Two TOR Complexes 1.1.2.1 TORC1 1.1.2.2 TORC2 1.1.3 The transcription factor FoxO (Forkhead box “O”) 1.1.4 Negative feedback regulation 11 1.1.5 Metabolism 13 1.2 Mitogen-activated protein kinase pathways/ Extracellular signal-regulated kinase pathway 14 1.2.1 The extracellular signal-regulated kinase (ERK) pathway 15 1.2.2 Scaffold protein: Kinase Suppressor of Ras (KSR) 17 V 1.2.3 Ets-1 transcription factor Pointed 19 1.3 Crosstalk between insulin-like signalling and MAPK/ERK pathway 20 Results 23 2.1 kinase suppressor of ras (ksr) was identified as enhancer of FoxO gain-offunction 23 2.2 KSR regulates FoxO activity in an AKT-mediated manner 26 2.3 Roles of other MAPK pathway components 29 2.4 KSR acts upstream of PI3K 32 2.5 MAPK signaling regulates inr expression 34 2.6 inr is regulated at the transcriptional level by the ETS-1 orthologue Pointed 38 2.7 EGFR is upstream of MAPK/ERK-mediated control in inr expression 45 2.8 MAPK/ERK regulates inr gene expression to control glucose metabolism 47 Discussion 52 3.1 Growth and metabolism through crosstalk during development 52 3.2 Short term vs long term mechanisms to modulate insulin responsiveness 53 3.3 Screen of transcription factors downstream of ERK regulating inr 54 3.4 The role of two Pnt splicing isoforms 57 3.5 Transcriptional regulation of inr expression by FoxO and by Pointed 58 3.6 Metabolism of circulating sugars, stored glycogen and triglyceride 61 3.7 Evolutionary conservation of MAPK/ERK-Pnt-InR axis 62 Perspective 64 Materials and Methods 66 VI 5.1 Fly strains 66 5.2 Plasmids 66 5.2.1 pMT-InR-Flag 66 5.2.2 pMT-Pnt-P2-Myc 67 5.2.3 A series of inr promoter luciferase reporter constructs 67 5.2.4 Mutagenesis of Pnt consensus site 70 5.3 Cell culture and treatments 70 5.4 In vitro dsRNA transcription for S2 cell RNAi 70 5.5 Cell imaging 74 5.6 Fat body FoxO immunofluorescent staining 74 5.7 Immunoblotting 75 5.8 RNA extraction and quantitative RT-PCR 76 5.9 Glucose and trehalose assay from hemolymph of Drosophila larvae 79 5.10 Triglyceride and Glycogen assay 80 Reference 82 VII Summary Insulin-like signaling is an important and conserved physiological regulator of growth and metabolism in multicellular animals In humans, disturbance in insulin sensitivity leads to impaired clearance of glucose from the blood stream, due to less glucose uptake by liver and fat and other tissues, which is a hallmark of diabetes While the core components of insulin-like pathway have been well established, the mechanisms that adjust insulin responsiveness are only known to a limited extent A genetic screen in Drosophila that was designed to identify regulators of cellular insulin sensitivity in an in vivo context was done in our lab This screen identified kinase suppressor of ras (ksr), an essential scaffold protein involved in MAPK/ERK signaling, as an enhancer of FoxO overexpression phenotype Based on this screen, surprisingly, I discovered cross-talk between the epidermal growth factor receptor (EGFR)-activated MAPK/ERK and insulin-like signaling pathways Cellular insulin resistance observed was due to downregulation of insulin-like receptor (inr) gene expression following persistent MAPK/ERK inhibition The MAPK/ERK pathway regulates inr expression via the ETS-1 transcription factor Pointed This regulation permits physiological adjustment of insulin sensitivity and subsequent maintenance of circulating glucose at appropriate levels, as failure of this regulation in the fat body leads to elevated circulating glucose levels, likely reflecting impaired clearance of dietary glucose from the circulation by the fat body Overall, I provide evidence for a regulatory feed-forward mechanism through PI3K and InR that allows for dynamic transient responsiveness as well as more stable, long VIII Table Primers for in vitro dsRNA synthesis Target CG Number Primer Name Primer Sequence KSR CG2899 PI3K/Dp110 CG4141 EGFP2 (GFP control) D-MEK/Dsor CG15793 Raf CG2845 GAP1 CG6721 pnt CG17077 PVR CG8222 KSR-F KSR-R CG4141F CG4141R dsEGFPT7-up dsEGFPT7-low Dsor1B(F) Dsor1B(R) Raf1-F Raf1-R Raf2-F Raf2-R dsGAP1-f dsGAP1-r dsGAP2-f dsGAP2-r 17077-f 17077-r pntp1-f pntp1-r pntp2-f pntp2-r dsPVR1-f dsPVR1-r dsPVR2-f ACATTAATACGACTCACTATAGGGAGAGCCTGCATGAACCCACCATA ACATTAATACGACTCACTATAGGGAGAGCTTCATCCCACGGCCAACCTG TTATTAATACGACTCACTATAGGGAGATTCGGCATCCTCAGCCTCGGCGAG CCATTAATACGACTCACTATAGGGAGAAGGAACTGATCAGGGGATAGTC CTAATACGACTCACTATAGGGAGAGGAGCTGTTCACCGGGG CTAATACGACTCACTATAGGGAGTCGCGCTTCTCGTTGGGG CACTTAATACGACTCACTATAGGGAGAGTAAACGAACCGCCGCCCAAG CACTTAATACGACTCACTATAGGGAGAGCCCAGCGTCAACGCTCTATTG ACATTAATACGACTCACTATAGGGAGAAGCCACCATCTTCATCCTCCGGCA ACATTAATACGACTCACTATAGGGAGAACGGCCACGGGACCGTGCCAA ACATTAATACGACTCACTATAGGGAGAGCAACTAACGCCGGATATGTG ACATTAATACGACTCACTATAGGGAGAGCTGCTCATATTGAAGCGGA CACTTAATACGACTCACTATAGGGAGAGGCAAAGAATCTGAGCAGTC CACTTAATACGACTCACTATAGGGAGATCAGTTCACTGTTCTCCTTG CACTTAATACGACTCACTATAGGGAGAGATTGTCTGGACTGCACTAC CACTTAATACGACTCACTATAGGGAGACCGTGCAGAACTTCTTGTAC CACTTAATACGACTCACTATAGGGAGAGTGGAAGCACCGGAAACAGC CACTTAATACGACTCACTATAGGGAGAGAATTCCCCAACGACGAGCCA CACTTAATACGACTCACTATAGGGAGACATAGCTTTGCAGGACCATG CACTTAATACGACTCACTATAGGGAGAATGCATTTACTGACAGTGCA CACTTAATACGACTCACTATAGGGAGAGCAAGGTCAACGAGGTACTG CACTTAATACGACTCACTATAGGGAGACCGACGTCACATAATCCATG CACTTAATACGACTCACTATAGGGAGAATGGAATGGTTCACTCCGTC CACTTAATACGACTCACTATAGGGAGAATCACACTGACGTTGTACTC CACTTAATACGACTCACTATAGGGAGATCGGTTCACATTGAGACCAG yan CG3166 Sin3A CG8815 lilli CG8817 bowl CG10021 NFAT CG11172 spen CG18497 kay CG33956 dsPVR2-r 3166-f 3166-r 8815-f 8815-r 8817-f 8817-r 10021-f 10021-r 11172-f 11172-r 18497-f 18497-r 33956-f 33956-r CACTTAATACGACTCACTATAGGGAGACAGACCGATCAGGATGAGGA CACTTAATACGACTCACTATAGGGAGAGCTCCCAGTACAGTTATCATTG CACTTAATACGACTCACTATAGGGAGACCAGACTGTGAGCAGAACTG CACTTAATACGACTCACTATAGGGAGATTTGGGTGCAACGACTCTTCC CACTTAATACGACTCACTATAGGGAGAAGCGAATTGTCCGAAATGTGC CACTTAATACGACTCACTATAGGGAGACGTCCAGCGTCACAAACAATG CACTTAATACGACTCACTATAGGGAGAATCGTCCCACTTGTTGCTCC CACTTAATACGACTCACTATAGGGAGACGACGAGGAGGATTCGTTGC CACTTAATACGACTCACTATAGGGAGACCCAGGTGGTACAGAATATCG CACTTAATACGACTCACTATAGGGAGACACCAATTCCACAATGAGTG CACTTAATACGACTCACTATAGGGAGAGGTGAATGTTGTTGCTGCTG CACTTAATACGACTCACTATAGGGAGACACCGTTAATTCTGTCACAG CACTTAATACGACTCACTATAGGGAGACGATAAAGAGTGTCCTTGTC CACTTAATACGACTCACTATAGGGAGAGTCCAAGCTGGTCAGCTTATA CACTTAATACGACTCACTATAGGGAGAACGCTCCTGTTTGCTTTCTAC Table Primers for real time PCR reaction using Sybr Green reagent Target CG Number Kinesin rp49 KSR InR pri-InR InR-A InR-B InR-C InR-D Pnt/pntp1 pntp2 PVR CG7765 CG7939 CG2899 CG18402 CG18402 CG18402 CG18402 CG18402 CG18402 CG17077 CG17077 CG8222 Forward Primer CTTTTCATAGCGTCGCTTCC GCTAAGCTGTCGCACAAA AGCCGAGCGAAGATTGTAAA CTGGTGGTGCTGACAGAGAA CAAGAGACAGCAACAAAAGG CAATGCACGACAACAAAACC TCGAAACGTTGAGATCGTTG GTGCCTCGCACTTTGCTTAT CGTTGTTGTTGCTGCTGTTT CGATGCGAATGCCTACTACACG TTTCTGTCCAGCCTAGTTGAGTCG AATGACCACCGTCCCAGAAGAC Reverse Primer GCTAAGCTGTCGCACAAA TCCGGTGGGCAGCATGTG TCCCGATACATGCCTACACA GCAGCTGACAACTGGCATTA GCTTGCATGTGTTGGTGAGC GGCACTCGATGATCATTTCA CGCACTTGTATTTCGTGGAA ATACGCTCACCAACACATGC GTCTCGTGCTCTCCTGCTCT TGCTGGTGTTGTAGCCTGAAC AACTGCACAGATCCTTGCATCC CAGGCGGGTTGTACGGAGTTATTG ... Regulates Insulin Sensitivity to Control Glucose Metabolism in Drosophila PLoS Genet 7(12): e1002429 doi:10.1371/journal.pgen.1002429 XV Introduction 1.1 Insulin- like signaling pathway Insulin- like signaling. .. damage-inducible protein 45 GAP GTPase-activating protein GEF guanine nucleotide exchange factor GTP guanosine triphosphate IGF insulin- like growth factor ILP insulin- like peptides InR insulin- like... insulin- like signaling pathways Cellular insulin resistance observed was due to downregulation of insulin- like receptor (inr) gene expression following persistent MAPK/ ERK inhibition The MAPK/ ERK