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Cellular and Animal Metabolic Responses to Indispensable Amino Acid Limitation

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Cellular and Animal Metabolic Responses to Indispensable Amino Acid Limitation CELLULAR AND ANIMAL METABOLIC RESPONSES TO INDISPENSABLE AMINO ACID LIMITATION A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Angelos Sikalidis January 2011 © 2011 Angelos Sikalidis CELLULAR AND ANIMAL METABOLIC RESPONSES TO INDISPENSABLE AMINO ACID LIMITATION Angelos Sikalidis, Ph.D. Cornell University 2011 The current thesis was guided by three principal research objectives. The first objective was to identify target genes responsive to indispensable amino acid deprivation with the goal of identifying potential components of the Integrated Stress Response as well as possibly identifying some genes that are differentially expressed in response to amino acid deprivation but not other stress conditions. The second objective was to determine whether the previously identified targets and eIF2α-kinase- mediated responses would be induced in animals fed a diet that was marginal in essential amino acid composition. The third objective was to elucidate the role of elevated 4E-BP1 expression in regulating translation initiation during nutrient deficiency. To assess the role of amino acid deprivation on eIF2α phosphorylation and downstream transcriptional responses, we conducted additional microarray studies for leucine-depleted HepG2/C3A cells and compared these with results for cysteine- depleted cells. The comparison of cells exposed to deficiencies of two different amino acids was done to facilitate selection of genes whose expression is more likely to be altered specifically in response to GCN2-induced eIF2α phosphorylation. In addition, we assessed the effects of both cysteine and leucine deprivation on the phosphorylation of eIF2α and on the protein expression patterns of ATF4 and other ISR-related proteins. We have compiled a list of 120 genes whose expression was differentially expressed in HepG2/C3A cells cultured in either cysteine- or leucine- deficient medium, and this list contains many of the genes known to respond to eIF2α kinase activation and to be components of the ISR. Clearly, amino acid deficiency, including cysteine deficiency in the absence of oxidative stress, induces eIF2α phosphorylation, presumably by activation of GCN2, leading to changes in expression of ATF4 and stress-related target genes. To answer whether eIF2α-kinase–mediated responses would be induced in animals fed a sulfur amino acid (SAA) deficient diet not as imbalanced as one in which a single essential amino acid is totally absent, we fed rats soy protein-based diets that were either adequate or limiting in SAA. Rats fed a SAA-deficient diet grew more slowly than rats fed the control diet. Analysis of liver from rats fed these diets for 7 days showed that the SAA-deficient rats had higher levels of eIF2α phosphorylation and higher levels of activating transcription factor (ATF) 4, ATF3, asparagine synthetase, solute carrier 7A11, cysteinyl-tRNA synthetase, and cystathionine γ-lyase. On the other hand, components of the integrated stress response (ISR) known to promote apoptosis or translational recovery were not induced. These results indicate that rats fed the SAA-deficient diet had a prolonged activation of an eIF2α kinase that leads to upregulation of adaptive components of the ISR. To assess the role of elevated 4E-BP1 expression in regulating translation initiation during nutrient deficiency, we fed rats various protein/amino acid-deficient diets with a varying degree of deficiency and achieved amino acid deficiency and reduction of growth. In these experiments we consistently observed a significant induction of total 4E-BP1 protein levels that did not parallel a reduction in 4E-BP1 phosphorylation and was independent of energy restriction or feed intake. Further, the induction of total 4E-BP1 appeared independent of eIF2α phosphorylation. BIOGRAPHICAL SKETCH Angelos was born on December 13th, 1979 in Thessaloniki, Greece, to Konstantinos a chemist and Eirini a chemist and a medical doctor. A son of two educators, he spent a significant amount of time reading books on a variety of non- science topics, mostly from the fields of literature, ethics, history, politics and humanities in general. He initially planned to become a lawyer but, since in life things take longer than expected and rarely go as planned, he chose to enter the physical sciences track while a lyceum student as he felt that this kind of training fitted his personality and way of thinking better. As an undergraduate, Angelos joined the School of Agriculture of the Aristotle University of Thessaloniki and graduated after attending a 5-year program with a degree in Food Science and Technology in 2003. During his undergraduate tenure Angelos became increasingly more interested in Nutrition and decided to pursue graduate studies in this field thinking that “…if the societal request is for food industries to contribute to public health in addition to the elimination of hunger, food scientists should become nutritionists and vice-versa”, a view thought by many to be rather romantic and naive. At the University of California, Berkeley as a Master’s student, Angelos worked on the mechanisms by which obesity contributes to the risk of colonic cancer in mouse models and graduated with a Master of Science in 2006. He decided to continue with a Ph.D. as a means to broaden his knowledge and understanding in metabolism and with the aspiration of becoming able to contribute to the growth of the Nutritional Sciences academic field back in his homeland where this field is in a more preliminary state. However, as things in life take longer than expected and rarely go as planned whether this path will be walked or not remains to be seen. Regardless of what the future holds, he is glad to have had his Ph.D. experience as it has been a remarkably educational one at various levels, as well as very self-revealing. iii I would like to dedicate this piece of work to my family and all my teachers, mentors and friends who have helped me develop as a person and a scientist. Αγαπητοί μου γονείς, ελπίζω και εύχομαι να μπορέσω να δώσω στα παιδιά μου ό,τι εσείς δώσατε σε μένα... “My beloved parents, I hope and wish to be able to provide to my children what you have given to me…” iv ACKNOWLEDGMENTS I would like to express my appreciation and thanks to several people whose support has been so important and made my “voyage” towards earning my Ph.D. a successful one. In different ways and catering to different needs during this long and demanding yet so educational, maturing and fascinating period of my life, many people have contributed to my achievements in a variety of ways. First of all I would like to warmly, appreciatively and most kindly thank my mentor, Professor Dr. Martha H. Stipanuk for being such a valuable resource of knowledge to me as well as for her guidance and patience throughout my time in her laboratory as a Ph.D. student. My outmost appreciation to my committee members Professor Dr. Patrick J. Stover, Professor Dr. Dennis D. Miller and Professor Dr. Yves R. Boisclair for their useful comments, input and constructive criticism regarding my research projects. I would also like to thank all the members in the Stipanuk Laboratory (Lawrence Hirshberger, Heather Roman, Dr. Jeong-In Lee, Dr. Iori Ueki, Dr. John Dominy) for their support and for enlightening me with their experience while teaching and passing on to me techniques and skills that not only made the completion of my research projects possible, but will follow me as useful research tools for life. In addition, I would like to thank everyone in the Stipanuk Laboratory for fostering such a nice, pleasant and conducive to learning working environment. A big “thank you” to all the administrative staff in the Division of Nutritional Sciences for all their help with the various administrative things I had to deal with as a graduate student in the Division. I would also like to thank my parents Konstantinos and Eirini and my brother Alexandros for their continued support and faith in me throughout my efforts in graduate school all these years that I have been studying in the USA. v Other individuals who have helped me maintain perspective of things and a healthy and positive outlook of life even at difficult times during my graduate school experience, whom I’d like to thank from the bottom of my heart, are: The “older brother” I never had, Prof. Dr. Peter Diamessis (to friends, “Pete”). His wise and thoughtful observations and experience sharing regarding life and grad school in particular, always constituted a beacon of optimism even in the darkest seas and skies of my grad school years. The legendary Olin Café manager Mr. Gregory Halkiopoulos (to friends, “Mr. Greg”), who with his amazing and unparalleled sense of humor, with the necessary dose of sarcasm, kept reminding me that there was/is a whole wide other world waiting for me outside of the “Grad school bubble”. Ailsa Roddie, a special chapter in my life for her support and understanding and the faith she has always exhibited in me as well as for her marvelous quote “the world is your oyster hon”. My friend and colleague Aleksandra Kristo, a Ph.D. student in the University of Maine, for all the conversations, the great, smart humor and the clever Ph.D. comics commentaries when experiments weren’t working and life didn’t seem all that great in the world of research. Geraldine Dupois, for her great sense of humor and unconventional yet great ideas when assays weren’t working, as well as for the history of science lessons which I would always receive when I would talk to her, in an adorable strong French accent. All my other friends in the US who have helped me keep my spirits high and who have been great listeners or talkers, depending on the occasion, but always a source of realistic optimism and positive energy, Dr.
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