Effects of Paternal Obesity on the Metabolic Profile of Offspring: Alterations In
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Effects of Paternal Obesity on the Metabolic Profile of Offspring: Alterations in Gastrocnemius Muscle GLUT4 Trafficking and Mesenteric Adipose Tissue Transcriptome A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Xinhao Liu August 2018 © 2018 Xinhao Liu. All Rights Reserved 2 This dissertation titled Effects of Paternal Obesity on the Metabolic Profile of Offspring: Alterations in Gastrocnemius Muscle GLUT4 Trafficking and Mesenteric Adipose Tissue Transcriptome by XINHAO LIU has been approved for the Department of Biological Science and the College of Arts and Sciences by Felicia V. Nowak Associate Professor of Biomedical Science Joseph Shields Interim Dean, College of Arts and Sciences 3 ABSTRACT LIU, XINHAO, Ph.D., August 2018, Molecular and Cellular Biology Effects of Paternal Obesity on the Metabolic Profile of Offspring: Alterations in Gastrocnemius Muscle GLUT4 Trafficking and Mesenteric Adipose Tissue Transcriptome Director of Dissertation: Felicia V. Nowak Parental obesity increases the risk of obesity in offspring. The overall hypothesis of this work is that offspring inherit altered epigenetic marks from parents that are obese due to consumption of high fat diet (HFD). The first specific hypothesis was that decreased GLUT4 synthesis or impaired trafficking to plasma membrane occurs in insulin resistant HFDO. This study measured expression of total glucose transporter 4, (GLUT4), the major insulin-stimulated transporter of glucose into muscle and adipose cells, as well as its subcellular distribution in the gastrocnemius muscle. The data showed that the level of total GLUT-4, as indicated by a 46kd protein band, exhibits no difference between LFDO and HFDO at 1.5, 6 or 12 months. However, when data from animals of all ages are combined, total GLUT4 expression is significantly higher in female than male HFDO, suggesting that females are protected from IR at least initially by increased GLUT4 expression facilitating increased glucose transport into skeletal muscle. In contrast, male HFDO have higher plasma membrane GLUT4 expression when compared to male LFDO, 4 suggesting a mechanism in skeletal muscle in males to alter GLUT4 subcellular distribution and prevent worsening of IR. It was next hypothesized that adipose tissue, as an endocrine organ that secretes multiple adipokines, also contributes to IR. Male HFDO also have increased body fat and increased weight of white adipose tissue (WAT), including mesenteric, subcutaneous, gonadal and retroperitoneal depots. Thus, the second specific hypothesis was that altered production of certain adipokines occurs in insulin resistant HFDO. Data showed that cytokines that increase the risk of IR such as IL1b and Fam3b were increased in male HFDO. Ingenuity Pathway Analysis (IPA) analysis showed that differentially expressed genes (DEGs) were enriched in pathways related to lipid metabolism, endocrine function and multiple organ injuries, such as liver and kidney injuries. This study also characterized the transcriptome of adipose tissue and explored the DNA methylation status of CpG islands located in the promoter regions of several DEGs. However, no DNA methylation differences were detected among these genes, suggesting that the observed differential expression is associated with other epigenetic modifications such as modification of non-coding RNAs and microRNAs. The sex-specificity of strategies in offspring to maintain metabolic balance and resist development of IR strongly suggests that epigenetic mechanisms have been set into play by the metabolic status of their obese HFD-fed sires. 5 In conclusion, sex-specific strategies in offspring were identified to maintain metabolic balance and resist development of IR. GLUT4 expression was increased in female offspring and translocation to the active site in plasma membrane was increased in HFDO males. RNA sequence analysis revealed altered expression of specific genes in mesenteric adipose tissue in HFDO males that may contribute to the observed IR. Epigenetic analysis showed that DNA methylation was not the mechanism affecting the DEGs studied. 6 ACKNOWLEDGMENTS The PhD education at Ohio University is an important milestone of my life. I learn precious knowledge that I will benefit from in my future career, and more importantly I gain a better mindset of research. I received great help and guidance from the people around me to whom I am genuinely grateful. First of all, I would like to give my most sincere appreciation to my advisor Dr. Nowak whom I have been working with for five years. I was able to choose the research project and design the study freely. And I received great help from her in developing a challenging method for the study. She gave me the intellectual guidance in research with great patience and cares about me in life. With her tremendous help, I was able to get financial support from University to collaborate with colleagues at the University of Massachusetts Medical School where I got to know great scientists and learn new techniques which allowed me to complete my project. Also Dr. Nowak has made every effort to be supportive of my physician scientist career plan. I would like also to express my sincere appreciation to Dr. Leslie Consitt, Dr. Xiaozhuo Chen and Dr. Allan Showalter. Dr. Consitt has similar research field with me, so she provided me great research insight. I would like to thank Dr. Chen who gave me precious guidance in solving technical issues. Dr. Allan Showalter is considerate to join my committee. 7 I would like to thank Dr. Yuriy Slyvka for editing my manuscript with insightful comments and Jie Xiang for assisting me in completion of the most laborious part of my research project. Last but not the least, I would like to express my genuine gratitude to my family. My mother, Lan Liu, my grandmother, Shufang Duan, and my grandfather, Qikui Liu, granted me the genuine support during my Ph.D. study. Their understanding and support are priceless. 8 TABLE OF CONTENTS Page Abstract ........................................................................................................................... 3 Acknowledgments .......................................................................................................... 6 List of Tables................................................................................................................. 10 List of Figures ............................................................................................................... 11 List of Abbreviation ...................................................................................................... 12 Chapter 1: Background ................................................................................................. 14 1.1 Introduction to Obesity ...................................................................................... 14 1.1.1 Genetic Regulation of Obesity .................................................................... 15 1.1.2 Epigenetic Regulation of Obesity ............................................................... 20 1.2 Introduction to the Effects of Parental Obesity .................................................. 23 1.2.1 Effects of Maternal Obesity ........................................................................ 25 1.2.2 Effects of Paternal Obesity ......................................................................... 26 1.3 Development of Insulin Resistance in Offspring of Obese Sires ....................... 28 1.3.1 Relationship of Insulin Resistance and Adipose Tissue .............................. 28 1.3.2 Epigenetic Mechanisms of Adipokine Regulation ...................................... 29 1.3.3 Relationship of Insulin Resistance and Muscle .......................................... 30 1.3.4 Epigenetic Mechanisms of Muscle Insulin Resistance ............................... 33 1.4 Project Summary ................................................................................................ 33 Chapter 2: Materials And Methods ............................................................................... 36 2.1 Chemicals and Solutions .................................................................................... 36 2.2 Animal Care And Use ........................................................................................ 36 2.3 Glucose Tolerance Test (GTT) ........................................................................... 37 2.4 Insulin Resistance Index .................................................................................... 37 2.5 Total GLUT4 Sample Preparation ...................................................................... 37 2.6 Fractionated GLUT4 Sample Preparation.......................................................... 38 2.7 Analysis of Proteins by Immunoblot .................................................................. 39 9 2.8 RNA Extraction, Library Construction and RNA Sequencing .......................... 40 2.9 RNA Sequencing Analysis ................................................................................. 42 2.9.1 Sequence Data Quality Control .................................................................. 42 2.9.2 Read Alignment and Gene Abundance Estimation ..................................... 42 2.9.3 Differential Gene Expression Analysis ....................................................... 43 2.9.4 Ingenuity Pathway Analysis