Nghiên cứu chiến lược điều trị kháng insulin trong các tình trạng bệnh lý chuyển hóa

Therapeutic Strategies for the Treatment of Insulin Resistance in Various Metabolic Disease States Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By: Michelle Lynn Asp, M. The Ohio State University Nutrition Graduate Program The Ohio State University 2010 Dissertation Committee: Martha A. Copyright by Michelle Lynn Asp 2010 Abstract Insulin resistance is present in multiple metabolism-related diseases. It is an almost universal phenomenon in type 2 diabetes, and many times obesity plays an important role in its development.

In contrast, insulin resistance is also present in cancer cachexia, a syndrome characterized by severe wasting of adipose tissue and skeletal muscle. The overarching goal of this research was to better understand insulin resistance in these two diverse disease states and to test novel therapeutic agents for improving insulin sensitivity and disease outcomes. The first objective was to determine whether dietary oil supplementation with conjugated linoleic acid (CLA) or safflower oil (SAF) improved markers of glycemia, insulin sensitivity, blood lipids, and inflammation in obese, post-menopausal women with type 2 diabetes. In humans, CLA induces modest weight loss, which can subsequently improve insulin sensitivity and glycemia.

SAF was chosen as a comparison oil. Women were supplemented with 8 grams of CLA and SAF in a 16-week double-masked, crossover study. CLA had no effect on any metabolic measurements despite modestly decreasing body weight and fat mass. Unexpectedly, SAF improved HbA1c, fasting glucose, HDL ii cholesterol, C-reactive protein, and the insulin sensitivity index QUICKI.

These improvements may have been in part due to decreased trunk fat mass. All SAF-induced metabolic changes occurred after an increase in serum linoleic acid was detected, suggesting serum fatty acid composition may have a role in inducing the observed metabolic improvements in women with type 2 diabetes. The second objective was to understand the development and consequences of insulin resistance in a mouse model of cancer cachexia. Insulin resistance has been observed in patients with cancer cachexia, but its time course was not known.

Male CD2F1 mice were inoculated with 1x106 colon-26 adenocarcinoma cells. Insulin resistance, measured by an insulin tolerance test, was observed prior to weight loss in tumor-bearing mice, suggesting insulin resistance may be a cause rather than a consequence of weight loss. Additionally, activation of Akt, a central signaling molecule in the insulin signaling pathway, was decreased, and proteolytic gene expression was increased at the end of the study. Treating mice with the insulin sensitizer rosiglitazone (RGZ) prevented tumor- induced insulin resistance and normalized proteolytic gene expression in early-stage cachexia.

Because of the improvements seen with RGZ treatment in mice with early-stage cachexia, the third objective was to determine whether RGZ could improve outcomes in late-stage cachexia. Tumor-bearing mice treated with intraperitoneal injections of RGZ iii (10mg/kg body weight daily) had a two-day delay in weight loss compared to tumor- bearing mice without RGZ. Additionally, RGZ increased adipose mass and adipocyte size. No improvements were seen in muscle mass, strength or proteolytic gene expression.

Delayed weight loss with RGZ was dependent on increased food intake, was associated with a delay in tumor-induced muscle gene expression related to substrate switching from glucose to fatty acids, and was associated with a modest improvement in insulin-induced Akt activation. These data suggest that improved insulin sensitivity and food intake may contribute to the ability of RGZ to delay the onset of overt cachexia. iv Dedicated to my Lord and Savior Jesus Christ “I can do anything through him who gives me strength.” Philippians 4:13 v Acknowledgements The work presented in this dissertation would not have been accomplished without the support and contributions of numerous people. Thank you Dr.

Belury, for encouraging me to pursue my PhD and allowing someone who didn’t know how to use a pipet enter your lab and learn everything from the very beginning. You have provided me so many opportunities throughout the past 6.5 years to grow and develop as a scientist, including presenting and publishing my work. You have prepared me for the next step of my career and I am very appreciative of your support and guidance. Thank you Dr.

Guttridge, for offering your expertise about cachexia to help me develop this new area of research in the Belury Lab. Thank you also for providing opportunities to present my work and dialogue with other muscle researchers through your organization of the OSU and Nationwide Children’s Muscle Group. Finally, thank you for your helpful advice about applying and interviewing for post-doctoral positions. vi Thank you Dr.

McCarthy for offering your expertise to help me learn about the colon-26 cachexia mouse model. I appreciate your generosity of time to teach and assist in the necropsy room during my mouse studies, as well as allowing us to harvest and analyze some tissues from your mouse studies. Thank you Dr. Harrison for offering your expertise and time to serve on my committee.

Your extensive knowledge and experience in the nutrition sciences is inspiring and challenges me to continue to read and study outside my area of research expertise. Thank you Min, Mike, and Kara, for your support, encouragement, and intriguing scientific conversations both related and unrelated to our research. You all have taught me so much and challenged me to be the best I can be. You are the greatest lab-mates one could ever ask for and I feel so lucky that I have been able to work with you all.

Thank you Angie, Aparna, and Li-Fen, for answering at least a million random questions during my first year in the lab, and for not running away when you saw me coming. Thank you Belury Lab members, past and present, who have assisted with animal studies. Animal studies require a large team effort and I appreciate every person’s contribution. Thank you Dr.

Failla and Dr. Your excitement about science is contagious, and your encouraging words have given me confidence in my abilities. vii Thank you Mom, Dad, and Katie for encouraging me to pursue my dreams. Your prayers and your unconditional love mean so much to me.

I am so blessed to have you in my life. Thank you Carol, Barbara, Pam, Britt, Shayna, Jill, Paula, and Julia for your prayers, support, and friendship. Thank you for being there when I have struggled and celebrating with me in my successes. Thank you to the many people and organizations that contributed financially in the form of fellowships, scholarships and research awards – The Ohio State University Presidential Fellowship, Myrtle Wolcott Cram Graduate Research Fellowship, P.

Scholar Award, Russell Klein Award, Virginia M. Vivian Graduate Research Endowment, and the Energy and Macronutrient Metabolism Research Interest Group of the American Society of Nutrition. (Dietetics; Chemistry), Concordia College Moorhead, Minnesota 2001-2002…………………………………………….Dietetic Intern, Medical Dietetics The Ohio State University, Columbus, Ohio 2002………………………….Student Research Associate, Department of Endocrinology The Ohio State University, Columbus, Ohio 2002……………………………….Mead Johnson/Bristol-Myers Squibb Scholarship American Dietetic Association 2002-2004……………………………….Graduate Research Associate, Medical Dietetics The Ohio State University, Columbus, Ohio 2004……………………. (Allied Medical Professions, emphasis Medical Dietetics) The Ohio State University, Columbus, Ohio 2004-2006………………………….Research Associate, Department of Human Nutrition The Ohio State University, Columbus, Ohio 2006-2010…………….…Graduate Research Associate, Department of Human Nutrition The Ohio State University, Columbus, Ohio 2007………………………………….

Vivian Graduate Research Endowment Department of Human Nutrition The Ohio State University, Columbus, Ohio 2008……………………………………………………………………….Travel Award Energy and Macronutrient Metabolism Research Interest Group American Society of Nutrition ix 2008-2009………………………….Myrtle Wolcott Cram Graduate Research Fellowship College of Education and Human Ecology The Ohio State University, Columbus, Ohio 2009-2010………………………………………………………….Presidential Fellowship The Ohio State University, Columbus, Ohio 2009………………………………………….…Russell Klein Memorial Research Award Department of Human Nutrition The Ohio State University, Columbus, Ohio 2009………………………………………………………………….Preparing Future Faculty Fellow The Ohio State University, Columbus, Ohio Publications Tian M, Nishijima Y, Asp ML, Stout MB, Peiser PJ, Belury MA. Cardiac alterations in cancer-induced cachexia in mice. Asp ML, Tian M, Wendel AA, Belury MA. Evidence for the contribution of insulin resistance to the development of cachexia in tumor-bearing mice.

Norris LE, Collene AL, Asp ML, Hsu JC, Liu LF, Richardson JR, Li D, Bell D, Osei K, Jackson RD, Belury MA. Comparison of dietary conjugated linoleic acid with safflower oil on body composition in obese postmenopausal women with type 2 diabetes mellitus. Am J Clin Nutr 2009;90:468-76. Asp ML, Hertzler SR, Chow J, Wolf BW.

Gamma-cyclodextrin lowers postprandial glylcemia and insulinemia without carbohydrate malabsorption in healthy adults. J Am Coll Nutr 2006;25:49-55. Belury MA, Asp ML, Collene AL, Koster CG, Liu LF, Purushotham A, Shrode G, Wendel A. Is there a role for conjugated linoleic acid to aid in the prevention of type 2 diabetes? In: Huang YS, Yanagita T, Knapp H (eds).

Dietary Fats and Risk of Chronic Disease. Champaign, IL: American Oil Chemists Society, 2006:263-73. x Hertzler SR, Kim Y, Khan R, Asp M, Savaiano D. Intestinal disaccharidase depletions.

In: Shils ME, Shike M, Ross AC, Caballero B, Cousins RJ (eds). Modern Nutrition in Health and Disease, 10th ed. Philadelphia:Lippincott Williams & Wilkins, 2006:1189- 1200. Stout MB, Wendel AA, Asp ML, Tian M, Cole RM, Smith JW, Belury MA.

Dietary conjugated linoleic acid alters adipose phenotype in a depot-specific manner in mice. Keystone Symposia - Adipose Tissue Biology, Keystone, Colorado, 2010:#318. Asp ML, Tian M, Stout MB, Belury MA. Effects of the insulin sensitizer rosiglitazone on cachexia outcomes in mice with colon-26 tumors.

5th Cachexia Conference, Barcelona, Spain, 2009:#030. Tian M, Asp ML, Stout MB, Nishijima Y, Reiser PJ, Belury MA. Cardiac alterations in cancer-induced cachexia in mice. 5th Cachexia Conference, Barcelona, Spain, 2009:#086.

Asp ML, Tian M, Wendel AA, Belury MA. Evidence for the early involvement of insulin resistance in the development of cachexia in mice bearing colon-26 tumors. Tian M, Asp ML, Wendel AA, Belury MA. Dysregulation of lipid metabolism in cardiac muscle of mice with cachexia.

Norris L, Collene A, Asp M, Liu L, Hsu D, Li D, Bell D, Osei K, Jackson R, Belury M. Conjugated linoleic acid reduces body weight and body fat in postmenopusal women with type 2 diabetes. Norris L, Collene A, Asp M, Liu L, Hsu D, Li D, Bell D, Osei K, Jackson R, Belury M. Comparative effects of dietary oils on markers of insulin sensitivity in postmenopausal women with type 2 diabetes.

Asp ML, Chow J, Hertzler SR. Effect of gamma-cyclodextrin on postprandial glycemia, insulinemia, and colonic hydrogen production in healthy humans. xi Fields of Study Major Field: The Ohio State University Nutrition xii Table of Contents Abstract……………………………………………………………………………. ix List of Tables………………………………………………………………….

xv List of Figures. xvi List of Abbreviations. 5 Chapter 2: Literature Review 2.2 Role of PPARS in Insulin Sensitivity.3 Type 2 Diabetes Mellitus. 35 Chapter 3: Safflower oil, but not Conjugated Linoleic Acid, Improves Glycemia, Inflammation and Blood Lipids in Obese, Post-Menopausal Women with Type 2 Diabetes 3.3 Research Design and Methods.

82 xiii Chapter 4: Evidence for the Contribution of Insulin Resistance to the Development of Cachexia in Tumor-Bearing Mice 4.3 Materials and Methods. 114 Chapter 5: Effects of Rosiglitazone on Cachexia Pathophysiology in Mice with Colon-26 Tumors 5.3 Materials and Methods.150 List of References. 156 xiv List of Tables 3.1 Markers of Glycemia with CLA and SAF Supplementation.2 Blood Lipids and Inflammatory Markers with CLA and SAF Supplementation.3 Serum levels of Linoleic Acid and Conjugated Linoleic Acid with CLA and SAF Supplementation.1 Study 1 Tissue Weights, Serum Metabolites and Food Intake.2 Study 2 Tissue Weights, Serum Metabolites and Food Intake.1 Body Weight Change, Tissue Mass and Plasma Metabolites for Late-Stage Cachexia Study. 131 xv List of Figures 2.1 Various Componenets of the Insulin Signaling Pathway.2 Causes of Insulin Resistance.3 Overview of Physiological and Pharmacological Roles of the PPARs in Energy Metabolism.4 Mechanism of Adipocyte Differentiation and Role of PPARγ.5 Eicosanoid Synthesis Pathway.6 Molecular Structures of t10c12-CLA, c9t11-CLA and Linoleic Acid.7 Defining Features of Cachexia.8 Structure of the Proteasome.9 Patients with Cancer Cachexia have Insulin Reistance.1 Pre-Determined Steps Analysis for Fasting Glucose, QUICKI, and Adiponectin.2 Pre-Determined Steps Analysis for CRP and HDL Cholesterol.3 Pre-Determined Steps Analysis for Serum Fatty Acids.1 Body Weight and Insulin Tolerance Test.