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View of Methods for Visceral Adipose Tissue Analysis Evaluating the role of fibroblast activation protein and fibroblast growth factor 21 in growth hormone-induced adipose tissue fibrosis ____________________________________ A Thesis Presented to The Honors Tutorial College Ohio University _______________________________________ In Partial Fulfillment of the Requirements for Graduation from the Honors Tutorial College with the degree of Bachelor of Science in Biological Sciences ______________________________________ by Delaney Kate Geitgey April 2020 TABLE OF CONTENTS TABLE OF CONTENTS .............................................................................................................. 2 ABSTRACT .................................................................................................................................... 4 INTRODUCTION.......................................................................................................................... 6 Characteristics of adipose tissue and adipocytes ........................................................................ 7 Adipocyte function....................................................................................................................... 7 Adipose tissue depots ................................................................................................................... 8 Main classifications ................................................................................................................. 8 Subcutaneous depots ................................................................................................................ 9 Intra-abdominal depots .......................................................................................................... 10 Characteristics of fibrosis ............................................................................................................ 11 Extracellular matrix (ECM) and fibrosis ................................................................................... 11 Fibrotic disease states ................................................................................................................ 12 Development of fibrosis ............................................................................................................. 15 Contributing factors to fibrosis .................................................................................................. 16 Fibroblasts in fibrosis ............................................................................................................ 16 Fibroblast activation protein ................................................................................................. 17 Fibroblast growth factor 21 ................................................................................................... 18 Epithelial-mesenchymal transition ........................................................................................ 19 WAT fibrosis ............................................................................................................................. 21 AT fibrosis mechanisms ......................................................................................................... 23 Characteristics of growth hormone ............................................................................................ 26 Growth hormone mechanisms ................................................................................................... 26 GH/IGF-1 axis ........................................................................................................................... 27 Influence of GH on nutrient metabolism ................................................................................... 28 Clinical conditions associated with altered GH levels ............................................................... 30 Laron Syndrome ..................................................................................................................... 30 GH deficiency......................................................................................................................... 32 GH excess............................................................................................................................... 33 Growth hormone mouse lines .................................................................................................... 34 GH and WAT fibrosis ................................................................................................................ 37 Research question and approach ................................................................................................ 39 Pathway of interest ..................................................................................................................... 39 2 Specific aims .............................................................................................................................. 40 METHODS ................................................................................................................................... 42 Mice ........................................................................................................................................... 42 Body weight and composition analysis ...................................................................................... 42 Plasma collection ....................................................................................................................... 43 Dissection ................................................................................................................................... 43 Enzyme-linked immunosorbent assays (ELISAs) ..................................................................... 44 RNA isolation ............................................................................................................................ 44 cDNA and qPCR ........................................................................................................................ 45 Statistical analysis ...................................................................................................................... 46 RESULTS ..................................................................................................................................... 47 Total body data .......................................................................................................................... 47 Tissue-specific data .................................................................................................................... 48 Fat depot data ........................................................................................................................ 48 Metabolic organ data ............................................................................................................. 50 Serum/plasma protein data ......................................................................................................... 51 RNA isolation ............................................................................................................................ 54 DISCUSSION ............................................................................................................................... 55 Fibroblast activation protein (FAP) ........................................................................................... 56 Fibroblast growth factor 21 (FGF21) ......................................................................................... 57 Beta-klotho (β-klotho) ............................................................................................................... 58 Procollagen peptides .................................................................................................................. 59 Procollagen 1 ......................................................................................................................... 59 Procollagen 3 ......................................................................................................................... 60 Conclusion ................................................................................................................................. 61 Significance of results ............................................................................................................ 61 Limitations of findings ........................................................................................................... 61 Future directions .................................................................................................................... 63 REFERENCES .............................................................................................................................. 66 3 ABSTRACT Adipose tissue (AT) is a unique energy storage tissue able to undergo significant hypertrophy and atrophy, dependent, in part, on nutrient status. When hypertrophy is in excess and sustained, the result is obesity, a common and widespread health problem. However, when AT stores are selectively depleted or abnormally deposited, the resultant condition known as lipodystrophy can lead to comparable health outcomes as obesity. That is, either extreme in AT mass results in similar metabolic outcomes (insulin resistance, increased immune cell infiltration, and increased production of inflammatory cytokines). With either chronic obesity or lipodystrophy, AT can undergo major remodeling, which can ultimately result in unresolved chronic inflammation
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