Regulation of Skeletal Muscle Growth and Gene Expression by Insulin-Like Growth Factors and Myostatin
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REGULATION OF SKELETAL MUSCLE GROWTH AND GENE EXPRESSION BY INSULIN-LIKE GROWTH FACTORS AND MYOSTATIN BY DANIEL LEE CLARK DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Animal Sciences in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Assistant Professor Anna C. Dilger, Chair Professor Jonathon E. Beever Professor Emeritus Floyd K. McKeith Associate Professor Clifford F. Shipley ABSTRACT Insulin-like growth factor 2 (IGF2) is a maternally imprinted growth factor that is thought to primarily promote prenatal skeletal muscle growth. A SNP within intron 3 of IGF2 identified as IGF2-intron3-G3072A has a considerable impact on lean yield as a result of increased postnatal IGF2 expression. Our first objective was to characterize carcass cutting yields of pigs with paternal A alleles (APat) and paternal G alleles (GPat) and determine how the mutation affects fresh meat quality and bacon processing. A single heterozygote (AG) boar was bred to homozygous (AA) commercial Yorkshire-cross sows producing F1 barrows and gilts with either GPat or APat alleles. Loin eye area was 10% greater (P = 0.01) and back fat was reduced (P = 0.01) 15% in APat pigs compared with GPat pigs. Consequently, boneless carcass cutting yield was increased (P < 0.01) 2.34 percentage units in APat pigs compared with GPat pigs. Mutations that improve carcass lean yield are often associated with reduced meat quality; however, fresh LM quality was minimally affected by the genotype, as 24-hour pH, drip loss and shear force was not different (P ≥ 0.27) between APat pigs and GPat pigs. Despite the reduced subcutaneous fat accumulation, LM intramuscular extractable lipid was 0.64 percentage units greater (P = 0.02) in APat pigs compared with GPat pigs. While not statistically different (P = 0.30), the magnitude of difference in slicing yield as a percentage of green weight was 1.57 percentage units between bellies from APat pigs (85.83%) and bellies from GPat pigs (87.40%). Therefore, while APat pigs yielded a greater amount of lean product, this may have been at the expense of reduced bacon slicing yields. Our next objectives were to determine the contributions of hyperplasia and hypertrophy to increased muscle mass and delineate the effect of the IGF2 mutation on the expression of myogenic genes during prenatal and postnatal growth. While postnatal IGF2 expression is known to be greater in APat pigs compared with GPat pigs there is ii little data that characterize how the mutation alters prenatal myogenesis. In addition to the expected postnatal increase (P ≤ 0.02) in IGF2 expression, IGF2 expression was also increased (P ≤ 0.06) 1.4-1.5 fold at d90 of gestation and at birth in APat pigs compared with GPat pigs. Additionally, skeletal muscle fiber number within the semitendinosus (ST) tended to be greater (P = 0.10) in APat pigs compared with GPat pigs, possibly as a result of the elevated prenatal IGF2 expression. Together, these data suggest that APat pigs are later maturing than GPat pigs and therefore, may be marketed at heavier weights but with similar subcutaneous fat depth. Furthermore, the elevated IGF2 expression did not affect (P ≥ 0.12) expression of other growth factors such as IGF1 and myostatin. The final objective was to determine how IGF1 and IGF2 cooperate with myostatin to regulate skeletal muscle growth. Therefore, we quantified IGF family member expression in myostatin null (MN) and wild type mice. Skeletal muscle IGF1 expression was surprisingly less (P ≤ 0.04) in skeletal muscle of 7d and 21d old MN mice compared with WT mice. Additionally, IGF2 expression was 1.9 to 2.9-fold greater (P < 0.01) in 21d, 42d, and 70d old MN mice compared with WT mice. Likewise, a similar increase (P < 0.01) was observed in three known Igf2 transcript variants at 21d and 70d in skeletal muscle of MN mice compared with WT mice. Hepatic Igf1 and Igf2 levels were minimally affected by genotype; with an exception at 21d when IGF1 was reduced 1.4-fold (P = 0.04) and IGF2 tended to be 1.3-fold greater (P = 0.06) in MN mice compared with WT mice. Together this implies that during postnatal development, myostatin may repress IGF2 expression in WT mice reducing skeletal muscle growth. Overall, these studies provide evidence that IGF2 is a key factor in regulating both prenatal and postnatal skeletal muscle growth. iii ACKNOWLEDGMENTS First, I want to say thank you to Jesus Christ my Lord and Savior. I can do all things through Christ who strengthens me. -Philippians 4:13 Next, I would like to thank my beautiful wife Diana Clark. Not only has Diana been incredibly instrumental in my teaching and research endeavors, but she has also been unbelievably supportive in everything that I do. Likewise, I would like to thank my parents Rhonda Clark, Steve Clark and Jill Kirts for always being supportive of everything I do. Additionally, I want to thank Bob and Mary Lou Clark, Robert and Thelma Tinsky, Dennis and Irene Doyle, Randy Pezza, and my brother Dustin, all of whom have been supportive and I am confident I would not be where I am today without their support. Professionally, I would like to thank my doctoral research committee. First I would like to thank my adviser Dr. Anna Dilger. Dr. Dilger has provided me with the opportunity to be part of a stellar research program and guidance throughout my time here at the University of Illinois. I truly appreciate everything that she has done for me. I have been very fortunate to have the opportunity to work with Dr. Floyd McKeith. His research and more importantly life advice have been very influential in my life. Dr. Beever has provided me with much technical guidance, taught me many techniques, and provided professional advice all of which greatly enriched my research program, and for that I am very thankful. I also would like to thank Dr. Shipley for serving on my committee and for his expertise that he has provided with embryo transfers. Additionally, I would like to thank Dr. Tom Carr and Dr. John Killefer for their advice during the early stages of my graduate research program. I would like to thank Dr. Dustin Boler iv for his advice and guidance while both a graduate student and now as a faculty member. Additionally, thank you to Brandy Marron as she provided much assistance with the genomic and genotyping procedures. I also very much appreciate my fellow colleagues both past and present, this research would have not been completed without their help. Especially, I would like to thanks Marcos Tavarez, Kellie Kroscher, Ben Bohrer, Elizabeth Hogan and again Diana Clark for all of your help with these projects. And whatever you do, whether in word or deed, do it all in the name of the Lord Jesus, giving thanks to God the Father through him. –Colossians 3:17 v TABLE OF CONTENTS CHAPTER 1 .................................................................................................................................. 1 REVIEW OF LITERATURE ...................................................................................................... 1 INTRODUCTION ...................................................................................................................... 1 INSULIN-LIKE GROWTH FACTORS..................................................................................... 1 MYOSTATIN ............................................................................................................................. 9 MYOSTATIN AND IGF INTERACTION .............................................................................. 11 IGF2 QTL ................................................................................................................................. 15 OBJECTIVES ........................................................................................................................... 19 LITERATURE CITED ............................................................................................................. 21 TABLES ................................................................................................................................... 28 FIGURES .................................................................................................................................. 34 CHAPTER 2 ................................................................................................................................ 37 EFFECTS OF THE PORCINE IGF2 INTRON3-G3072A MUTATION ON CARCASS CUTABILITY, MEAT QUALITY AND BACON PROCESSING ....................................... 37 ABSTRACT .............................................................................................................................. 37 INTRODUCTION .................................................................................................................... 38 MATERIALS AND METHODS .............................................................................................. 39 RESULTS AND DISCUSSION ............................................................................................... 47 IMPLICATIONS ...................................................................................................................... 55 CONCLUSIONS....................................................................................................................... 55 ACKNOWLEDGMENTS ........................................................................................................ 56 LITERATURE