e v. f‘ «a .W f: {5 .5 . . . . 8.. ”.5 .n . i. .- fim... m}, u... M. .35 . x 3... :1 $33. .333. s. .2 .. . P 1.5.3:) .. . . . , . . . , . . , , ..fa& , , ,. , . . ,... ”.wa ._ . :1 V1.3... , , . . 3 . , , . . .gAgnglfl . 0%.Qh I... .1\ .t . x , s . .2» mitt-9 LIBRARIES MICHIGAN STATE UNIVERSITY EAST LANSING, MICH 48824-1048 <2. j) (2519‘ I? 27 I This is to certify that the dissertation entitled DIFFERENTIATION OF BOVINE INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES presented by Aaron Christopher Grant has been accepted towards fulfillment of the requirements for the Doctoral degree in Animal Science QMQ mm Major Professor’s Signature 2/23/05 Date MSU is an Affin'native Action/Equal Opportunity Institution PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 C‘fiHC/DatoDueJWJS DIFFERENTIATION OF BOVINE INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES By Aaron Christopher Grant A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 2005 ABSTRACT DIFFERENTIATION OF BOVINE INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES ' By Aaron Christopher Grant Intramuscular (i.m.) and subcutaneous (s.c.) adipose tissue substantially influence beef carcass value, and are of economic importance to the beef industry. Adipose development is at least partially controlled by the differentiation of preadipocytes into mature adipocytes. We hypothesized that bovine i.m. and so. preadipocyte differentiation would be enhanced by a peroxisome proliferator-activated receptor (PPARH agonist, troglitazone (TRO), and a glucocorticoid, dexamethasone (DEX), with the relative response being greater in i.m. than s.c. cells. In the first set of experiments, preadipocytes from i.m. and so. stromal- vascular (S-V) cells of an Angus steer were cloned and used to optimize culture conditions supporting adipose differentiation, as well as compare the adipogenic responses of i.m. and so. preadipocytes to TRO. A higher percentage of isolated s.c. clones (47%) were identified as adipogenic than i.m. (12.5%) (P < 0.001). A s.c. preadipocyte clone was used to optimize differentiation culture conditions. Addition of 10 and 20 uUmL serum lipid (8L) to serum-free media containing 280 nM insulin increased glycerol-3-phosphate dehydrogenase (GPDH) activity. Inclusion of 1.25 to 10 uM TRO to media containing insulin and SL also increased GPDH activity (P < 0.001). No GPDH activity was detected when medium included insulin, octanoate, and acetic acid, following 48 h exposure to DEX. However, insulin, SL, TRO, and DEX stimulated GPDH activity (P < 0.001). Omission of TRO or insulin from this media lowered GPDH activity (P < 0.001), while removal of DEX tended to reduce activity (P = 0.06). When i.m. (n = 3) and so. (n = 2) clones were compared, all clones responded to addition of 20 to 60 uM TRO (P < 0.02), with no depot differences (P = 0.47). In the second set of experiments, i.m. and so S-V cells isolated from three Angus steers were used to determine the adipogenic effects of DEX and TRO. Forty and 60 pM TRO increased GPDH activity 2.9- and 3.4-fold compared to non-treated cells (P < 0.03), with no depot differences (P = 0.32). When DEX and TRO were tested in combination, DEX stimulated a 1.8-fold increase in GPDH activity (P = 0.006), while TRO induced a 2.5-fold increase in GPDH activity (P = 0.02). No DEX x TRO interaction (P = 0.53) or depot effects (P = 0.41) were found. In clonal analysis experiments, DEX increased the percentage of adipogenic colonies by 1.2-fold (P = 0.02), while TRO increased the proportion of differentiated colonies by 2.3-fold (P = 0.01). No DEX x TRO interaction (P = 0.65) or depot differences (P = 0.10) were found. However, the percentage of differentiated cells within adipogenic colonies was 6.4-fold greater in so. isolates than i.m. (P < 0.001). Dexamethasone had no significant effect (P = 0.10) while TRO increased the proportion of differentiated cells by 10-fold (P < 0.001). In summary, differentiation of bovine i.m. and so. preadipocytes was enhanced in response to SL, DEX, and TRO. Under identical media conditions, so. preadipocytes have a greater capacity to differentiate compared to i.m. This thesis is dedicated to my parents who have always encouraged me to pursue my dreams and have been there for me with their love and guidance throughout my life. And to the rest of my family, all of whom have shown endless love and support towards me during my years abroad. ACKNOWLEDGEMENTS I am indebted to the support, assistance, and encouragement of many, to which without, the completion of this thesis would not have been possible. I would first like to thank my major professor, Dr. Daniel Buskirk for allowing me the opportunity to learn and work under his tutelage. His guidance and friendship have enabled me to broaden my knowledge in many different aspects of beef cattle science in an environment that nurtured to both basic and applied research. Dr. Buskirk’s constant enthusiasm as well as personal involvement in all aspects of my research program will not be forgotten. In addition, a special thanks is due to Dr. Matthew Doumit, who was instrumental in the development of the research methods applied within this dissertation, and for the countless hours of help, advisement, and friendship. Your commitment to helping students is unsurpassed. I would also like to thank the other members of my guidance committee, Drs. Harlan Ritchie, Torn Herdt and Dale Romsos for their insight in developing my graduate and research programs as well as the completion of this dissertation. I would also like to acknowledge the efforts of Dr. Rob Tempelman for his assistance with the statistical analysis included in this dissertation. I would like to thank my fellow graduate students and lab mates, Guillermo Ortiz-Coldn, Matt McCurdy, Jason Scheffler and Chuck Allison for their help, guidance, and support during the course of my research program. I would also like to thank Emily Helman for assistance with laboratory work as well as Brigitte Grobbel and Nick Ehrke who have aided in lab material preparation and data collection. This thesis would not have been completed had it not been for the cumulative efforts of all of you. I am also indebted to Tom Forton, Jennifer Dominguez and the Meat Lab crew for harvesting the cattle used in these studies, as well as aiding in the sample isolation procedures. Lastly, I am indebted for the time spent with the faculty, students, and staff within the Department of Animal Science. My tenure at Michigan State has been truly enjoyable, and was a direct result of the quality and caring people of which I had the opportunity to interact with on a daily basis. vi TABLE OF CONTENTS LIST OF TABLES .................................................................................................. x LIST OF FIGURES .............................................................................................. xi LIST OF ABBREVIATIONS ................................................................................ xv INTRODUCTION .................................................................................................. 1 CHAPTER I Review of Literature Marbling and palatability ................................................................................... 4 Flavor ..................................................................................................... 4 Juiciness ................................................................................................ 5 Tenderness ............................................................................................ 6 Adipose tissue development ............................................................................. 8 Heterogeneity of adipose depots ............................................................ 8 Preadipocyte proliferation and differentiation .................................................. 13 In vitro preadipocyte experiments ........................................................ 13 Preadipocyte proliferation (h yperplasia) ............................................... 14 Preadipocyte differentiation .................................................................. 15 Factors influencing preadipocyte proliferation and differentiation ................... 18 Growth factors ...................................................................................... 18 Adipogenic signalling agents ................................................................ 20 Depot differences in preadipocyte development ............................................. 29 Proliferation capabilities ....................................................................... 29 Differentiation capabilities .................................................................... 30 Summary ........................................................................................................ 32 Literature cited ................................................................................................ 34 CHAPTER II Isolation, cloning, and optimization of differentiation of bovine preadipocytes Abstract ........................................................................................................... 48 Introduction ..................................................................................................... 49 Materials and Methods .................................................................................... 50 Results and Discussion ................................................................................... 60 Implications ..................................................................................................... 70 Literature Cited ............................................................................................... 77 vii CHAPTER III Effects of dexamethasone and troglitazone on differentiation of stromal- vascular cells isolated from bovine intramuscular and subcutaneous adipose tissues Abstract ........................................................................................................... 81 Introduction ..................................................................................................... 82 Materials and Methods .................................................................................... 83 Results and Discussion ................................................................................... 92 Implications ................................................................................................... 100 Literature Cited ............................................................................................. 106 CHAPTER IV Interpretive Summary ..................................................................................... 109 APPENDICES APPENDIX A Adipogenic cloning efficiency and clone inventory ........................................ 124 APPENDIX B Effects of adipogenic stimuli on the differentiation of preadipocyte clones 125 APPENDIX C Effects of adipogenic stimuli on the differentiation of stromal-vascular cells and clonaIIy-derived adipogenic colonies ............................................. 128 APPENDIX D Characteristics of steers used for stromaI-vascular cell isolations ................ 132 APPENDIX E StromaI-vascular cell extraction protocol ...................................................... 133 APPENDIX F Glycerol-3-phosphate dehydrogenase cell solubilization and assay protocol142 APPENDIX G Cell inventory identification system ............................................................... 149 APPENDIX H Media components ........................................................................................ 151 APPENDIX | Ex-Cyte media supplement components ...................................................... 153 viii LIST OF TABLES CHAPTER I Table 1-1. Depot differences in adipocyte characteristics .................................... 9 CHAPTER III Table 3-1. Effect of dexamethasone or troglitazone on the percentage estimates and 95% confidence intervals (CL), of clonaIIy-derived, stromaI-vascular cells isolated from intramuscular and subcutaneous adipose tissues of three steers ................................ 105 APPENDIX D Table D-1. Identification, live, and carcass characteristics of steers used for stromaI-vascular cell isolations ......................................... 132 APPENDIX I Table I-1. Fatty acid composition of Ex-Cyte lipid supplement ......................... 153 LIST OF FIGURES Images in this dissertation are presented in color CHAPTER I Figure 1-1. Possible mechanisms of preadipocyte differentiation ...................... 16 CHAPTER II Figure 2-1. Effect of serum lipid supplementation on glycerol-3-phosphate dehydrogenase (GPDH) activity and protein concentration of subcutaneous bovine clonalIy-derived preadipocytes. Values are least squares means and SEM of three independent experiments. Means without a common superscript letter differ (P < 0.05). ....................................................................................... 71 Figure 2-2. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH) activity of subcutaneous bovine clonalIy-derived preadipocytes. Values are least squares means and SEM of three independent experiments. Means without a common superscript letter differ (P < 0.05). ..................... 72 Figure 2-3. Photomicrographs of a subcutaneous bovine preadipocyte clone exposed for 12—d to serum-free differentiation medium supplemented with 280 nM insulin, 0.25 uM dexamethasone, 1 mM octanoate, and 10 mM acetate (T RT 1) (A), or to serum- free differentiation medium supplemented with 280 nM insulin, 20 lemL serum lipids, and 40 pM troglitazone (TRT 4) (B). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 um. ................................................................... 74 Figure 2-4. Effect of adipogenic agents on glycerol-3-phosphate dehydrogenase (GPDH), activity and protein concentration of subcutaneous bovine clonally.derived preadipocytes. Components included (+) or excluded (-) from the media were troglitazone (TRO) (40 pM), dexamethasone (DEX) (0.25 nM), serum lipids (SL) (20 uUmL), insulin (280 nM), octanoate (1 mM), and acetate (10 mM). Values are least squares means and SEM of three independent experiments. Means without a common superscript letter differ (P < 0.05). .................................... 75 Figure 2-5. Effect of troglitazone supplementation on glycerol-3- phosphate dehydrogenase (GPDH) activity. Values are least xi squares means and SEM for two independent experiments of three subcutaneous (s.c.), and two intramuscular (i.m.), bovine clonally derived preadipocyte cell lines. Means without a common superscript letter differ (P < 0.05). .................................. 76 CHAPTER III Figure 3-1. Effect of troglitazone supplementation on glycerol-3- phosphate dehydrogenase (GPDH) activity. Values are least squares means and SEM of subcutaneous (s.c.), and intramuscular (i.m.), stromal-vascular cells from three steers. Least squares means without a common superscript letter differ (P < 0.05). ............................................................................ 101 Figure 3-2. Effect of 0.25 uM dexamethasone (DEX) (upper panel), and 40 uM troglitazone (TRO) (bottom panel) on glycerol—3-phosphate dehydrogenase (GPDH) activity. Values are least squares means and SEM of subcutaneous, and intramuscular, stromal- vascular cells from three steers. Least squares means without a common superscript letter differ (P < 0.05). ............................... 102 Figure 3-3. Photomicrographs of subcutaneous clonally—derived adipogenic colonies exposed for 10-d to serum-free differentiation medium (A), or serum-free differentiation medium supplemented with 0.25 uM dexamethasone and 40 uM troglitazone (B). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. ....................... 104 APPENDIX B Figure 8-1. Photomicrographs of a bovine subcutaneous preadipocyte clone (SC3-C31) exposed for 12-d to differentiation media containing: 1 mM octanoate, 10 mM acetic acid, and 0.25 uM dexamethasone (DEX) (A); 20 lemL serum lipids (SL) and 0.25 uM DEX (B); 20 uUmL SL and 40 uM troglitazone (TRO) (C); 20 lemL SL, 0.25 (M DEX, and 40 [M TRO (D). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 um. ................................................................. 125 Figure 8-2. Effect of troglitazone supplementation on glycerol-3- phosphate dehydrogenase (GPDH), activity. Values are least squares means and SEM for two independent experiments of three subcutaneous (s.c.), and two intramuscular (i.m.), bovine preadipocyte clonal cell lines. ........... 126 Figure B-3. Effect of troglitazone supplementation on glycerol-3- xii phosphate dehydrogenase (GPDH), activity. Values are pooled least squares means and SEM for two independent experiments of three subcutaneous (s.c.), and two intramuscular (i.m.), bovine preadipocyte clonal cell lines. ........... 127 APPENDIX C Figure C-1. Figure 02 Figure C-3. Figure C-4. Effect of troglitazone supplementation on glycerol-3- phosphate dehydrogenase (GPDH), activity. Values are pooled least squares means and SEM of subcutaneous (s.c.), and intramuscular (i.m.), stromal-vascular cells from three steers. ........................................................................................... 128 Effect of 0.25 pM dexamethasone (DEX) (upper panel), and 40 uM troglitazone (TRO) (lower panel), on glycerol-3- phosphate dehydrogenase (GPDH) activity. Values are pooled least squares means and SEM of subcutaneous (s.c.), and intramuscular (i.m.), stromal-vascular cells from three steers. ........................................................................................... 129 Photomicrographs of subcutaneous clonally—derived adipogenic colonies exposed for 10-d to differentiation media containing: 20 pL/mL serum lipids (SL) (A); 20 pL/mL SL and 0.25 pM DEX (B); 20 pL/mL SL and 40 uM troglitazone (TRO) (C); 20 pUmL SL, 0.25 uM DEX, and 40 uM TRO (D). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 um. .................................................................. 130 Photomicrographs of intramuscular clonally-derived adipogenic colonies exposed for 10-d to differentiation media containing: 20 pUmL serum lipids (SL) (A); 20 pUmL SL and 0.25 uM DEX (B); 20 uleL SL and 40 uM troglitazone (TRO) (C); 20 uUmL SL, 0.25 uM DEX, and 40 uM TRO (D). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. .................................................................. 131 APPENDIX E Figure E-1. Figure E-2. Photos of 1000 pm screen taped in funnel (A); 500 and 53 um screens secured in modified conical tube tops (B); filtration technique using 1000 um screen (C); and filtration technique using 500 and 53 pm screens (D). ................................................ 139 Extraction process of longissimus muscle, subcutaneous (A), and perirenal adipose tissue samples (B). Carcass following extraction (C) ................................................................................ 140 xiii Figure E-3. Excision of i.m. adipose tissue from muscle (A); mincing of so. adipose tissue (B); incubation of 50 mL tubes in Lab Line incubator (C); and addition of final cell suspension to cryogenic vials (D) ......................................................................... 141 xiv ACC BSA cAMP CIEBP DEX DMEM EDTA FA FABP FAS FBS FGF GLUT GPDH HCL i.t. i.m. IGF IGFBP IGF R LPL LM IMX ME mRNA NADH ont LIST OF ABBREVIATIONS Acetyl-CoA Carboxylase Bovine Serum Albumin Cyclic AMP CCAAT/Enhancer Binding Protein Dexamethasone Dulbecco’s Modified Eagle’s Medium Ethylenediaminetetraacetic Acid Fatty Acid Fatty Acid Binding Protein Fatty Acid Synthase Fetal Bovine Serum Fibroblast Growth Factor Glucose Transport Protein Glycerol-3-Phosphate Dehydrogenase Hydrochloric Acid lnterrnuscular Intramuscular Insulin-like Growth Factor Insulin-like Growth Factor Binding Protein Insulin-like Growth Factor Receptor Insulin Receptor Lipoprotein Lipase Longissimus Muscle Methylisobutylxanthine Malic Enzyme Messenger Ribonucleic Acid Nicotinamide Adenine Dinucleotide, Reduced Form Omental ORO PBS PGan PGlz PGJz 15d-PGJ2 PPAR PPRE p.r. PREF1 PUFA RA RAR RXR s.c. SL S-V TGF TNF TRO TZD USDA Oil Red 0 Phosphate-Buffered Saline Prostaglandin F20 Prostacyclin Prostaglandin J2 15-deoxy-A‘2'14-Prostaglandin J2 Peroxisome Proliferator-Activated Receptor Peroxisome Proliferator-Activated Receptor Response Element Perirenal Preadipocyte Factor-1 Polyunsaturated Fatty Acid Retinoic Acid Retinoic Acid Receptor Retinoid X Receptor Subcutaneous Bovine Serum Lipids Stromal-vascular Transforming Growth Factor Tumor Necrosis Factor Troglitazone Thiazolidinedione United States Department of Agriculture xvi INTRODUCTION The production of high quality beef that is tender, juicy, and flavorful, while minimizing fat trim from excessive subcutaneous fat, provides a continual challenge for the beef industry. Adipose tissue development is of critical economic importance to beef cattle production, as intramuscular fat (marbling) contributes to palatability characteristics, while subcutaneous fat is wasteful to the industry. Results from the 2000 National Beef Quality Audit estimated the total value lost to the beef industry due to insufficient marbling was $21 per steer and heifer harvested, while $43 per head, or $1.3 billion total, was being lost due to excess external fat annually. Many researchers have attempted to increase marbling in beef cattle through genetic selection and nutritional management practices. However, knowledge of the events involved in adipose cell development may result in a better understanding of fat accretion in cattle. Cellular characteristics of bovine adipose tissues have been shown to differ by depot. Notably, adipocytes from subcutaneous depots have been reported to be larger, and have greater lipogenic capabilities than those from intramuscular tissues. In addition, subcutaneous and intramuscular adipocytes may utilize different substrates for adipogenesis, further suggesting that cells from these depots are inherently different. Adipose tissue development is partially controlled by adipocyte precursor cell (preadipocyte) proliferation, and differentiation into mature adipocytes. Preadipocyte differentiation has been characterized as a morphological and biochemical change from a fibroblast-like cell, to a mature adipocyte capable of lipid storage. The differentiation process is regulated by the expression and activation of adipogenic genes, many of which are influenced by endogenous and(or) exogenous factors. One such gene that has been described as a key regulator of adipogenesis is peroxisome proliferator-activated receptor (PPAR)-y. Peroxisome proliferator-activated receptoray is a transcription factor that is upregulated and activated in response to adipogenic stimuli. Upon activation, PPARy is responsible for the upregulation of several adipogenic genes characteristic of mature adipocytes. Evidence suggests that preadipocytes derived from different depots may vary in the expression of, or ability to respond to PPARy agonists, thus influencing differentiation. Although much research has been conducted using established clonally derived murine preadipocyte cell lines, and murine and human stromal-vascular cells, few studies have used bovine cells. In addition, few if any studies have directly focused on determining adipogenic factors that may differentially influence the differentiation of bovine intramuscular and subcutaneous preadipocytes. Given the importance of these adipose tissue depots in beef cattle, it seems logical to investigate the differential development of preadipocytes derived from these adipose tissues. Therefore, the goals of the present study were to: 1) refine the methodology for bovine preadipocyte isolation, culture, and cloning; 2) optimize culture conditions that induce differentiation of bovine preadipocytes; and 3) determine the differential responses of intramuscular and subcutaneous preadipocytes and stromal- vascular cells to adipogenic stimuli. A better understanding of adipogenic factors responsible for differences in bovine adipose tissue development could lead to the manipulation of cellular events that enhance intramuscular and(or) reduce subcutaneous fat accretion. This may ultimately allow for the production of cattle possessing carcasses with superior palatability characteristics and minimal waste. CHAPTER I REVIEW OF LITERATURE Marbling and palatability Marbling has been defined as intramuscular fat distributed amongst perimysial connective tissue and near circulatory vessels (Aberle et al., 2001 ). Marbling makes a positive contribution to beef palatability characteristics, namely flavor, juiciness, and tenderness (Blumer, 1963). Flavor refers to beef's overall taste and odor, while juiciness is defined as liquid detected during chewing. Although tenderness cannot be defined by a single term, it is associated with the amount of force required to chew, along with the fragmentation characteristics of muscle fibers, and residues after chewing. More specifically, tenderness relates to the amount of connective tissue present within a muscle (Blumer, 1963) and its solubility due to heating, post-mortem proteolytic enzyme activity of the calpain system, and muscle fiber shortening during cooking (Wulf et al., 1996). Because marbling has long been associated with the overall eating experience (Blumer, 1963; Smith et al., 1984; Smith et al., 1987; Wheeler et al., 1999), and carcass value of beef (Harper and Pethick, 2004), developing methods that increase intramuscular fat accretion in cattle could improve consumer satisfaction with beef and the profitability of the industry. Flavor. Studies have found a positive relationship between beef marbling and flavor desirability and(or) intensity (McBee and Wiles, 1967; Tatum et al., 1980; Dolezal et al., 1982; Smith et al., 1984; Smith et al., 1987; Wheeler et al., 1999). Steaks from carcasses of similar maturity that possess higher marbling scores and USDA Quality Grades have been found to be more flavorful than steaks from lower grading carcasses (McBee and Wiles, 1967; Tatum et al., 1980; Smith et al., 1984; Wheeler et al., 1999). However, some experiments have shown no positive association between intramuscular fat and beef flavor (Parrish et al., 1973; Dikeman and Crouse, 1975; Garcia-de-Siles et al., 1977; Wheeler et al., 1994). Inconsistencies between studies may be partially attributed to variation in diet, aging time, breed-type, sample size, and degree of doneness. For example, differences in aging time could account for variation in results between studies, as it has been shown that aging steaks for a longer period of time improves beef flavor ratings (Wheeler et al., 1999). In addition, cattle breed-type could also differentially influence experimental results, as some studies used cattle of primarily British origin while others obtained steaks from a more representative cross section of the current industry. Despite the inconsistencies in results among studies, marbling is commonly regarded to positively influence beef flavor characteristics. Juiciness. Marbling may enhance juiciness through increasing the water- holding capacity of meat, and by lubricating muscle fibers during cooking. A positive correlation between marbling score or quality grade and juiciness has been shown by many researchers (Cover et al., 1956; McBee and Wiles, 1967; Jennings et al., 1978; Tatum et al., 1980; Dolezal et al., 1982; Smith et al., 1987; Wheeler et al., 1994; Wheeler et al., 1999). Fatty acids (FA) present in muscle and intramuscular (i.m.) fat that melt at or below the temperature at which meat is cooked, have been hypothesized to make a significant contribution to beef juiciness (Blumer, 1963). Melted fat may maintain juiciness and(or) tenderness in cooked steaks by coating perimysial connective tissue, thereby reducing moisture loss (Aberle et al., 2001). Steaks from carcasses with higher marbling scores have been found to maintain juiciness to a greater extent when cooked well done when compared to those with less marbling (Wheeler et al., 1999). Approximately 15% of the variation in juiciness of cooked steaks may be accounted for by differences in the edible portion of fat (Cover et al., 1956). McBee and Wiles (1967) showed that juiciness increased in a linear manner with increasing levels of marbling. Other studies (Jennings et al., 1978; Tatum et al., 1980; Dolezal et al., 1982; Smith et al., 1984; Smith et al., 1987; Wheeler et al., 1994; Wheeler et al., 1999) have demonstrated similar positive relationships between juiciness and marbling scores and(or) quality grades. Although, a few experiments have found no apparent association between marbling and juiciness (Parrish et al., 1973; Dikeman and Crouse, 1975; Garcia-de-Siles et al., 1977), a small positive association between the two is generally accepted. Tendemess. Marbling may influence tenderness by decreasing the bulk density of a portion of meat, as well as lubricating muscle fibers during cooking. However, no consistent relationship between marbling and beef tenderness has been demonstrated. Some have reported positive correlations between marbling and tenderness (McBee and Wiles, 1967; Jennings et al., 1978; Tatum et al., 1980; Dolezal et al., 1982; Jones and Tatum, 1994; Wheeler et al., 1994; Wheeler et al., 1999), while others have found no significant association between the two (Carpenter et al., 1972; Parrish et al., 1973; Dikeman and Crouse, 1975; Garcia-de-Siles et al., 1977; Davis et al., 1979). Taste panel ratings suggest that marbling score can account for between 0.01% and 36% of the variation in tenderness (Blumer, 1963; Wheeler et al., 1999), and that marbling score was 30 to 38% accurate in predicting the tenderness of loin steaks from youthful carcasses (< 30 mo.) (Smith et al., 1987). Data from Wheeler et al. (1999) suggest that steaks from carcasses grading USDA High Choice were more likely to remain tender when cooked well done compared to those of Low Select. Marbling score, among many different estimates of composition recorded on fresh carcasses, has been found to be the best single predictor of beef tenderness. However, when used as the sole predictor of tenderness, marbling only accounted for between 5 and 9% of the variation in tenderness (Jones and Tatum, 1994; Wheeler et al., 1994). Likewise, Carpenter et al. (1972) reported that 23% of carcasses grading USDA Choice were considered undesirable in tenderness, and Davis et al. (1979) suggested that variation in tenderness exists, even among carcasses of the same quality grade. Differences in tenderness findings between studies could be a result of many factors including breed type and animal-to-animal variation, post-mortem aging and degree of doneness of the cooked product. Although results vary between studies, marbling appears to have a positive relationship with all three palatability contributors (flavor, juiciness, and tenderness). Adipose tissue development Lipid deposition within adipose tissue is controlled by hyperplasia of adipocyte precursor cells (preadipocytes), preadipocyte differentiation into adipocytes, and adipocyte hypertrophy (lipid filling) (Hood, 1982). Adipocyte hypertrophy is influenced by de novo FA synthesis, FA uptake from blood, FA oxidation, and lipolysis (Hood, 1983). Ailhaud et al. (1992) suggested that while E preadipocytes are capable of proliferation, fully differentiated adipocytes lose their capacity for mitosis. Therefore, preadipocytes are responsible for adipose tissue accumulation through hyperplasia, while differentiated adipocytes E contribute to adipose tissue accumulation through hypertrophy. Although white adipose tissue formation begins before birth in most species, fat accretion increases rapidly after birth as a result of increased fat cell number and size (Gregoire et al., 1998) with different depots showing varying degrees of potential for development. As animals continue to grow and(or) fatten, hyperplasia and hypertrophy continue at varying rates within different adipose tissues (Vernon, 1986). Heterogeneity of adipose depots. Several studies have demonstrated differences between adipose depots in rats (Broad et al., 1983; DiGirolamo et al., 1998), humans (Adams et al., 1997; Hutley et al., 2003), sheep (Broad et al., 1983; Soret et al., 1999), and cattle (Hood and Allen, 1973; Broad et al., 1983; Cianzio et al., 1985; Dubeski et al., 1997; Brethour, 2000) (Table 1-1). In humans, adipocytes of subcutaneous (s.c.) origin were capable of higher rates of both triglyceride synthesis and lipolysis than cells from the intra-abdominal depot Table 1-1. Depot differences in adipocyte characteristicsa DepotD Item Species p.r. o.m. so. it i.m. Author TG° synthesis Human i.d Hd Edens, (1993) Lipolysis Human L H Edens, (1993) Bovine L L H L Rule, (1992) Leptin secretion Human L H Montague, (1997) Leptin expression Human L H Montague, (1997) E Glucose uptake Human H L Stolic, (2002) , Ovine H L Broad, (1983) 5 Rat H L Broad, (1983) i Insulin receptor, no. Human H L Lefebvre, (1998) Bovine H H L McGrattan, (2000) I“ FGF° expression Human H L Gabrielsson, (2002) Accretion rate Bovine H H Md L L Hood, (1983) GPDHf activity Bovine H M M L Mendizabal, (1999) Bovineg L L H H Eguinoa, (2003) Cell diameter Bovine B" B M sd s Cianzio, (1985) aCalculated on a per cell basis bp.r. = perirenal, o.m. = omental, so. = subcutaneous, i.t. = interrnuscular, i.m. = intramuscular ‘Triacylglycerol dL = low, H = high, M = medium, B = big, S = small: compared to depots within the same row °Fibroblast growth factor fGlycerol-3-phosphate dehydrogenase 9Values in this row calculated taking differences in adipocyte size into account (Edens et al., 1993). Montague et al. (1997) also showed that leptin (a protein secreted by adipocytes to aid in energy storage regulation) production in cm. adipose tissue is less than that found in so. regions. It was suggested that leptin may have less of an inhibitory effect on omental (o.m.) adipose tissue growth, which could contribute to problems with central obesity in humans. In addition, cellular glucose uptake has been shown to differ between adipose regions. Greater insulin stimulated glucose uptake (Stolic et al., 2002; Virtanen et al., 2002), along with a greater number of insulin receptors (Lefebvre et al., 1998) have been found in cm. versus s.c. adipocytes in the human. These studies indicate that hormonal response may differ among various adipose tissues. Differences in gene expression between cells of different adipose depots also appear to exist, as in human adipose cells it was found that leptin messenger ribonucleic acid (mRNA) levels were higher in so than o.m. adipocytes (Montague et al., 1997). In addition to this, it was found that mRNA of certain members of the fibroblast growth factor (FGF) family were more highly expressed in o.m. than s.c. cells (Gabrielsson et al., 2002). Depot differences in bovine adipose tissue development appear to exist. Reports have shown that adipocyte size and number vary between adipose depots in the bovine (Hood and Allen, 1973; Smith and Crouse, 1984.; Cianzio et al., 1985; Miller et al., 1991; May et al., 1994). In vivo adipose tissue depot accretion rates in cattle have been shown to occur in the following order: perirenal (p.r.) and cm. > so. > intermuscular (i.t.) > i.m. (Hood, 1983). Hood and Allen (1973) also showed that most bovine adipocyte hyperplasia was 10 completed by 8 months of age in p.r. and so. fat depots, while cell hyperplasia was still evident in i.m. tissue up to 14 months of age. After these respective points in time, the authors suggested that cell hypertrophy was primarily responsible for adipose tissue accumulation. In addition, Cianzio et al. (1985) reported that cell hyperplasia occurred within the i.m. fat depot up to 15 months of age, while the brisket fat depot maintained hyperplasic growth past 15 months F of age in cattle. These data, combined with their measurements of adipocyte cell diameter, (kidney fat > mesenteric > so > i.t. > i.m. > brisket fat) led Cianzio et ,3, al. (1985) to conclude that i.m. and brisket fat depots develop at slower rates ,. than the other depots. Additionally, Allen et al. (1976), suggested that because bovine i.m. adipocytes were smaller in size than those from other depots, total cell number was critical in determining the quantity of i.m. lipid. It was also postulated that larger adipocytes have greater lipogenic capabilities than smaller cells (Hood, 1983). When adipocytes from o.m., p.r., so, and i.t. depots of growing steers were compared, p.r. adipocytes were found to be largest in size and had the highest lipogenic enzyme activities (glycerol-3-phosphate dehydrogenase (GPDH) and fatty acid synthase (FAS)) when based on a per cell basis (Mendizabal et al., 1999). The same authors found cells from the i.t. depot to be the smallest in size with the lowest enzyme activities. Additionally, lipolytic rates were found to be highest within s.c. adipose tissue in relation to o.m., p.r., and i.t. fat depots (Rule et al., 1992). These data collectively suggest that differences in metabolic activity, as well as, rates of lipogenesis and lipolysis exist between bovine adipose tissues. 11 Not only are there apparent differences in size and enzyme activity between ruminant adipose tissues, substrate utilization may also vary between depots. While glucose is known to be the primary precursor of FA synthesis in monogastrics, acetate (the primary volatile FA produced in the rumen) has been implicated as the major precursor of FA synthesis in ruminants, whereas glucose contributes little (Hanson and Ballard, 1967; Hood et al., 1972). However, Smith and Crouse (1984) determined that glucose provided 50 to 75% of the acetyl units utilized for lipogenesis in i.m. tissue explants compared to 1 to 10% in so. tissue explants. To support this, Miller et al. (1991) found the activity of two glycolytic enzymes, hexokinase and phosphofructokinase, to be greater in i.m. than s.c. adipocytes when expressed on a per cell basis. These data suggest that i.m. cells may have an enhanced ability to utilize glucose for lipogenesis when compared with so. cells. Nevertheless, due to differences in relative tissue size between so (large) and i.m. (small) adipose depots in cattle, absolute glucose consumption may still be higher in so. tissue (Schoonmaker et al., 2004). Smith and Crouse (1984) also determined that acetate provided 70 to 80% of the acetyl units for lipogenesis within s.c. tissue, while only contributing 1 to 10% in i.m. tissue. The previous studies suggest that variations in substrate utilization exist between ruminant adipose tissues, thus leading to speculation that these differences may be manipulated to influence adipose depot development. 12 Preadipocyte proliferation and differentiation Adipose tissue accretion is partially influenced by the proliferation and differentiation of preadipocytes. The rate and extent of preadipocyte proliferation and differentiation has been described as a multifaceted process that may be influenced by a number of factors such as age, species, and adipose depot (Djian et al., 1983, 1985; Kirkland et al., 1990; Soret et al., 1999; Hausman et al., 2001). Preadipocytes represent a cell population that are capable of influencing adipose tissue development through replication both pre and post-natally (Hausman et al., 1980; Ailhaud et al., 1992). It has been postulated that preadipocytes originate from multipotent stem cells, which first undergo determination into unipotential preadipocytes (Ailhaud et al., 1992). Preadipocytes generally display a fibroblast-like morphology, and during hyperplastic growth do not typically express measurable adipogenic enzyme activity or accumulate lipid until the post confluent stage of in vitro cell culture (Plaas and Cryer, 1980). In vitro preadipocyte experiments. Although preadipocyte cell culture experiments generally attempt to relate in vitro findings to physiological events taking place in vivo, the cells used within these experiments can differ significantly. Many researchers choose to utilize immortal cell lines (originally derived from fat pads of rats and mice) for research purposes due to their homogeneity and availability for use (Gharbi-Chichi et al., 1984). However, these cells may lack many of the intrinsic characteristics present within cells of the species of interest. Because of this, many researchers choose to extract and 13 isolate preadipocytes from the specific animals they intend to study. Cells may be further classified as “primary” (cells isolated from adipose depots of interest, which may contain non-adipose cells) or “clonal” (cells derived from one adipogenic precursor cell) preadipocytes. Primary cell cultures of isolated stromal-vascular (S-V) cells may include other cell types such as fibroblasts, myoblasts, and endothelial cells that may influence adipose tissue development E both in vitro and in vivo (Sato et al., 1996). Other researchers have chosen to develop clonal cell cultures to study the intrinsic cellular properties of i preadipocytes (Djian et al., 1983; Aso et al., 1995; Tchkonia et al., 2002). The I. patterns of preadipocyte development between primary and clonal cell cultures appear to be similar. Tchkonia et al. (2002) found that variations in adipogenesis between human adipose depots were similar in primary and clonally-derived cell cultures. Using both culture methods, differentiation capacity was greatest in subcutaneous, intermediate in mesenteric, and least in omental cells. This indicates that the characteristics defining regional differences in preadipocyte development were inherent within the cells themselves. Although differences exist between the preadipocyte cell culture methods mentioned, each has the potential to provide valuable information regarding adipose tissue development within and across species of interest. Preadipocyte proliferation (hyperplasia). Many different factors such as hormones, growth factors and cytokines are known to be involved in regulating preadipocyte proliferation. Wright and Hausman (1995) showed that insulin-like growth factor (IGF)-1 served as an effective mitogenic and adipogenic stimulant 14 of porcine preadipocytes. Other factors that are capable of increasing preadipocyte proliferation include: transforming growth factor-[3 (TGFIl), tumor necrosis factor-a (TNFa), and FGF (Ailhaud et al., 1992; Butterwith, 1994; Wright and Hausman, 1995; Gregoire et al., 1998; Friihbeck et al., 2001; Hausman et al., 2001). These studies suggest that the regulation of preadipocyte proliferation is complex and can be influenced by multiple factors. However, more research has focused on factors that determine preadipocyte differentiation. Preadipocyte differentiation. Preadipocyte differentiation has been described as the morphological transformation from an undifferentiated fibroblast-like cell into a rounded, lipid-filled cell concomitant with the expression of transcription factors, genes, cell surface receptors, binding proteins and lipogenic enzymes associated with mature fat cells (Butterwith, 1994; Gregoire et al., 1998). Differentiation appears to be a multi-step process that can vary greatly between preadipocytes of different origins (Adams et al., 1997; Wu et al., 2000; Tchkonia et al., 2002). A model of possible mechanisms and pathways involved in preadipocyte differentiation previously reported in the literature are shown (Figure 1-1). This model has not been fully elucidated using bovine cells. Generally, before undergoing terminal differentiation, preadipocytes must first exit the cell cycle (Ailhaud et al., 1989). Following growth arrest, a cascade of gene regulation events appear to proceed, which ultimately lead to the development of a mature adipocyte. Initially, the expression of two transcription factors, CCAAT/enhancer binding protein (CIEBPHI and CIEBP6, are transiently upregulated. In clonal cell lines such as 3T3-L1, this upregulation may be 15 LInoIeIc Acld Glucocortlcolds Extracellular Arachldonlc Acld PUFAJ'ZJ TNFa Insulin Intracellular FABP, LPL, GPDH, ME, FAS, GLUT4, etc. Figure 1-1. Possible mechanisms of preadipocyte differentiation. cAMP = Cyclic AMP; ClEBP(a,B,6) = CCAAT/enhancer binding protein isoforms (a,B,6); FABP = Fatty acid binding protein; FAS = Fatty acid synthase; Glut4 = Glucose transport protein-4; GPDH = Glycerol-3-phosphate dehydrogenase; LPL = Lipoprotein lipase; ME = Malic enzyme; PG02 = Prostaglandin Dz; PGI2 = Prostacyclin; PGJz = Prostaglandin J2; PPARy = Peroxisome proliferator-activated receptorq; PPRE = Peroxisome proliferator—activated receptor response element; PREF1 = Preadipocyte factor-1; PUFA= Polyunsaturated fatty acid; RA= Retinoic Acid; RXR= Retinoid X receptor; TZD= Thiazolidinedione; TNFa= Tumor necrosis factor. += Stimulate; - = Inhibit. 16 induced by hormonal stimuli such as glucocorticoids and methylisobutylxanthine (MIX) (Yeh et al., 1995). CCAATIenhancer binding protein-B has been associated with the subsequent upregulation of two other transcription factors, CIEBPa and peroxisome proliferator-activated receptor (PPAR)-’y (Yeh et al., 1995; Wu et al., 1996), which are known to transactivate many adipocyte specific genes later in differentiation (Grégoire et al., 1998). Although C/EBP5 role in the differentiation cascade is less clear, it is thought that it may act in unison with CIEBPB in upregulating PPARy (Wu et al., 1996). CCAATIenhancer binding protein-a expression occurs later in the differentiation cascade and may be upregulated by PPARy in addition to CIEBPB (Wu et al., 1999). Both ClEBPa and PPARy are also capable of inducing the transcription of one another in a positive feedback loop, which ensures continued expression of both transcription factors in mature adipocytes (Mandrup and Lane, 1997; Wu et al., 1999). Upon upregulation and activation, both PPARy and(or) ClEBPa may induce the expression of numerous adipogenic genes such as: fatty acid-binding protein (Tontonoz et al., 1994a), lipoprotein lipase (LPL) (Schoonjans et al., 1996), stearoyI-CoA desaturase—1 (Wilson Miller and Ntambi, 1996), glucose transporter (GLUT)-4 (Kaestner et al., 1990), malic enzyme (ME) (Castelein et al., 1994), and acetyl-CoA carboxylase (ACC) (T ae et al., 1995). Also during differentiation, other enzymes involved in lipogenesis and triacylglycerol synthesis are expressed including: ATP citrate lyase (Benjamin et al., 1994), FAS (Paulauskis and Sul, 1988), GPDH, and glyceraldehyde-3-phosphate dehydrogenase (Spiegelman et al., 1983). These cellular changes allow for enhanced FA or 17 triacylglycerol synthesis within the cells, thus taking on characteristics representative of mature adipocytes. Factors influencing preadipocyte proliferation and differentiation There are many factors involved in the regulation of preadipocyte proliferation and differentiation. Several agents capable of influencing these cellular events include: fetal bovine serum (FBS), IGF-1, insulin, glucocorticoids, FA, prostaglandins, and thiazolidinediones (TZD), and are discussed below. Growth factors. Within most in vitro cell culture systems, some type of ( serum or growth factor supplement is required for preadipocyte survival and optimal proliferation rates. Fetal bovine serum, which is commonly used in preadipocyte cell culture, contains various growth factors, binding proteins, along with nutritional, hormonal, and other poorly defined components capable of sustaining and promoting cell growth. Therefore, when used as a media supplement, serum serves to mimic the environmental conditions of cells in vivo (Barnes and Sato, 1980). Although its components are relatively undefined, FBS has been utilized extensively as a growth promoting additive within cell cultures of various cell lines and species (Djian et al., 1983; Hauner, 1989; Kliewer et al., 1995; Soret et al., 1999; Tchoukalova et al., 2000; Wu et al., 2000). Although Broad and Ham (1983) have described serum-free media that supports ovine S- V cell growth, proliferation rates were still sub-optimal when compared to media containing FBS. In addition, due to the complexity of factors required to sustain preadipocyte growth in serum-free media, most researchers still rely on sera to provide these nutrients. 18 Insulin-like growth factor-1 is regarded as an important regulator of adipogenesis, although the processes by which it exerts its effects are not well understood. Chen et al. (1996) reported that porcine S-V cells secrete IGF-1 and IGF binding proteins (IGFBP). As well, preadipocytes have also been found to express IGF-1 receptors (IGFR) (Entingh-Pearsall and Kahn, 2004). This suggests that IGF-1 may play an autocrine and(or) paracrine role in adipogenesis. Insulin-like growth factor-1 has been shown to be mitogenic for S- V cells and clonally derived preadipocytes (Deslex et al., 1987; Ramsay et al., 1989b; Buttenivith and Goddard, 1991; Wright and Hausman, 1995) and has .1. been termed a potent stimulator of proliferation (Hausman et al., 2001). It has been suggested that IGF-1 exerts its mitogenic effects via the IGFR (Boney et al., 2000). Once activated, this receptor stimulates a mitogen-activated protein kinase cascade, which results in cell proliferation. Insulin-like growth factor-1 effects are subject to regulation by IGFBP (Hausman et al., 2001). These binding proteins may influence IGF-1 through regulating its transport and bioavailability (Butterwith, 1994). Along with its mitogenic effects, IGF-1 is known to promote preadipocyte differentiation. In vitro studies have shown that IGF-1 stimulates differentiation in various S-V cell types and preadipocyte cell lines (Deslex et al., 1987; Smith et al., 1988; Hausman, 1989; Ramsay et al., 1989b; Wright and Hausman, 1995; Gregoire et al., 1998). Insulin-like growth factor-1 and insulin are both capable of stimulating similar receptors and signalling pathways that enhance differentiation (Entingh-Pearsall and Kahn, 2004). However, IGF-1 may be a more potent 19 stimulator of differentiation than insulin, as undifferentiated preadipocytes express a greater number of IGFR than insulin receptors (Entingh et al., 2003; Entingh-Pearsall and Kahn, 2004). Insulin-like growth factor-1 appears to enhance differentiation through the activation of its receptor (Boney et al., 2000), which induces a signal transduction cascade capable of upregulating genes associated with mature adipocytes (Smith et al., 1988; Steele-Perkins et al., 1988). Although these data support the role of IGF-1 in modulating adipogenesis, deletion of the IGFR did not inhibit differentiation of immortalized brown preadipocyte clones (Entingh-Pearsall and Kahn, 2004). Adipogenic signalling agents. Due to its multiple anabolic actions, insulin is known to be a contributing factor in adipose tissue accretion and appears to have varying effects on adipocyte development. Numerous studies have shown that insulin stimulates adipogenesis within adipose tissues and cells of various species (Etherton and Evock, 1986; Guller et al., 1988; Hausman and Jewell, 1988; Hausman, 1989; Miller et al., 1991; Faulconnier et al., 1994). Using 3T3- F442A cells, Guller et al. (1988) demonstrated that insulin was a critical factor in adipogenesis, as its removal from media reduced differentiation by 97%. In addition, Sztalryd et al. (1991) showed that insulin stimulated glucose uptake in differentiating rat S-V cells. More specifically, in studies using 3T3-L1 cells, insulin was shown to enhance the transcription of genes such as fatty acid synthetase, acyl-CoA synthetase, stearoyl CoA desaturase-1, and glucose transporter proteins (Paulauskis and Sul, 1988; Garcia de Herreros and Birnbaum, 1989; Weineret al., 1991). Also, physiological levels of insulin 20 stimulate lipogenic enzyme activity (GPDH and LPL) in rat and porcine S-V cells, thereby enhancing differentiation (Deslex et al., 1987; Hausman and Jewell, 1988; Hausman, 1989). Insulin promotes adipogenesis through binding and activation of the insulin receptor (IR) (Entingh-Pearsall and Kahn, 2004). Although the quantity of IR were found to be lower than IGF R in undifferentiated murine preadipocytes, IR numbers increased significantly during differentiation and were greater than IGFR in mature adipocytes (Entingh et al., 2003). It was also shown that differentiation was dependant on IR in murine preadipocytes, as IR deficiency inhibited differentiation (Entingh-Pearsall and Kahn, 2004). Pharmacological concentrations of insulin have also been shown to bind both insulin and IGF-1 receptors, which may increase its effects on adipogenesis (Smith et al., 1988). Many cell culture experiments use high concentrations of insulin in their differentiation media, potentially for this reason (Kirkland et al., 1996; Wu et al., 2000; Tchkonia et al., 2002; Ding et al., 2003). In general, it has been found that ruminant adipose tissues are less responsive to the effects of insulin than fat depots of other species (Bauman, 1976). To support this, Broad and Ham (1983) found that increasing insulin concentrations in a defined differentiation media did not affect differentiation of ovine S-V cells. Broad et al. (1983) and Sasaki (1990) also found that while insulin stimulated lipogenesis in rat adipocytes, ovine adipocytes showed little adipogenic response to increased insulin concentrations. An explanation for these findings may be that ruminant adipocytes are less sensitive to insulin, possibly due to a reduced ability to specifically bind the hormone (Vasilatos et al., 21 1983). More recently, Aso et al. (1995) found that, while GLUT1 protein levels increased during differentiation, no evidence of GLUT4 transporters were found in clonally derived bovine intramuscular preadipocytes. Therefore, the reduced response to insulin observed in ovine and bovine adipose tissues could also arise from a lower number, or lack, of insulin responsive glucose transporters. However, insulin is normally included as a differentiation media component in E ruminant S-V cell culture studies (Plaas and Cryer, 1980; Broad and Ham, 1983; Aso et al., 1995; Torii et al., 1998; Soret et al., 1999; Wu et al., 2000), and has i been reported to be effective at concentrations as low as 0.9 nM (Soret et al., 1.- 1999). In addition, Adams et al. (1996) found that ovine S-V cells failed to differentiate when insulin was removed from differentiation media. Cumulatively, these data support insulin’s involvement in potentiating differentiation in numerous cell models. Glucocorticoids or their analogs, specifically dexamethasone (DEX), have been used as adipogenic components of differentiation media in preadipocyte cultures from many species and cell lines (Hauner et al., 1987; Gaillard et al., 1991; Smas et al., 1999; Tchoukalova et al., 2000; Wu et al., 2000). Dexamethasone has been shown to influence differentiation through the down regulation of preadipocyte factor-1 (PREF 1), an epidermal growth factor-like protein (Wolf, 1999). This protein is highly expressed in certain preadipocyte cell lines and S-V cells but is absent in mature fat cells (Smas et al., 1998). Dexamethasone has also been shown to induce a cascade that leads to the successive expression of several transcription factors, CIEBP, (a, B, and 6) and 22 PPAR, ((1, y, and 8), resulting in preadipocyte differentiation (Yeh et al., 1995; Wu et al., 1996). The CCAAT enhancer binding proteins [3, 5, and or appear to act sequentially, with CIEBPB and CIEBP6 expression occurring early in the differentiation process while ClEBPa is considered a terminal differentiation factor. Cao et al. (1991) also determined that DEX was capable of directly upregulating CIEBP?) in 3T3-L1 cells. In addition, Wu et al. (1996) showed that glucocorticoids enhanced the expression of CIEBPS and C/EBPB, which in turn induced PPARy expression and initiated adipogenesis in 3T3-L1 cells. Peroxisome proliferator—activated receptor-y is primarily expressed in adipose .‘ tissue as compared to PPARa and PPARS, and plays an important role in regulating adipogenesis (Brun et al., 1996). Many in vitro studies only expose cells to DEX for the initial 48 h to initiate differentiation, as CIEBPS and CIEBPB are involved in the early events of adipose conversion and are diminished during the later stages (Yeh et al., 1995; Wu et al., 1996). Although CIEBP and PPAR appear to be critical to the development of adipose tissue, not all adipogenic cell types respond similarly to DEX and(or) these transcription factors. Hausman (1999) found no in vivo association between glucocorticoid treatment and CIEBP expression in adipose tissue of late gestation fetal pigs. As well, Yeh et al. (1995) found that while CIEBPB can induce expression of C/EBPa in 3T3-L1 cells, this does not occur in NIH-3T3 cell lines. Glucocorticoids may also be involved in the regulation of insulin, as they have been found to increase insulin receptor expression in other cell types such as lymphocytes (McDonald and Goldfine, 1988). Reciprocally, insulin has also been shown to increase the 23 number and binding affinity of glucocorticoid receptors in porcine S-V cells (Chen et al., 1995). These interactions between glucocorticoids and insulin could potentially enhance preadipocyte differentiation. In addition, Soret et al. (1999) also observed an additive effect on differentiation when DEX and TZD were added to serum-free media in S-V cells derived from fattened sheep. This effect was attributed to the ability of DEX to increase PPARy concentrations, while TZD ’ E activated the transcription factor, in differentiating preadipocytes. In addition to their apparent influence on adipogenic transcription factors, glucocorticoids may also stimulate differentiation by influencing arachidonic acid metabolism, thereby , enhancing prostaglandin synthesis (Ailhaud et al., 1992). In addition to their role as an energy source, FA and their derivatives are capable of affecting preadipocyte differentiation by influencing processes such as gene expression, FA uptake, and de novo FA synthesis. A wide variety of FA, ranging in chain length and degree of saturation, have been used to study adipose differentiation in preadipocyte and S-V cell cultures (De la Llera et al., 1981; Broad and Ham, 1983; Gaillard et al., 1989; Tontonoz et al., 1994b; Aso et al., 1995; Adams et al., 1996; Sato et al., 1996; Ohyama et al., 1998; Soret et al., 1999; Wu et al., 2000; Hutley et al., 2003). In bovine S-V cells, Sato et al. (1996) found that octanoate increased lipid filling and GPDH activity compared to octanoate deprived media. In the same study, cholesterol increased the adipogenic effects of octanoate, although cholesterol alone was not beneficial. Acetate has also been used successfully as a source for FA synthesis in the differentiation of bovine s.c. S-V cells (Plaas and Cryer, 1980) and i.m. 24 preadipocytes (Aso et al., 1995). Aso et al. (1995) observed an increase in acetic acid incorporation compared to cellular glucose uptake during bovine preadipocyte differentiation, suggesting that the FA produced were derived primarily from acetate in these cells. As well, addition of lipid supplements, composed mostly of various triacylglycerol, to culture media significantly increased differentiation and GPDH activity of ovine (Adams et al., 1996) and bovine S-V cells (Ohyama et al., 1998; Soret et al., 1999; Wu et al., 2000). Arachidonic acid and its metabolites, specifically prostaglandins, are capable of both stimulating and inhibiting differentiation in preadipocytes. Preadipocytes are capable of producing prostaglandins from arachidonic acid, which could in turn influence preadipocyte differentiation (Hyman et al., 1982; Négrel et al., 1989). Three members of the prostaglandin family found to modulate preadipocyte differentiation are prostaglandin-F20, (PGan), prostacyclin (PGIz), and 15-deoxy-A‘2'14-prostaglandin J2 (15d-PGJ2). Contrasting reports document the effects of these prostaglandins on preadipocyte differentiation. Gaillard et al. (1989) found that PGan enhanced the ability of arachidonic acid to induce adipose conversion in Ob1771 cells but was ineffective when used alone. However, Serrero et al. (1992) found that PGFZQ inhibited the expression of adipocyte specific genes, as well as triacylglycerol accumulation in primary cultures of rat preadipocytes. Prostaglandin-F2.ll inhibited differentiation of 3T3- L1 cells through the activation of calcium/calmodulin dependant protein kinase which, in turn, led to an increase in DNA synthesis and cell proliferation (Wilson Miller et al., 1996). Prostacyclin has been found to stimulate differentiation 25 through the upregulation of adipogenic transcription factors via its ability to increase the concentration of intracellular cAMP (Négrel et al., 1989; Ailhaud et al., 1992; Gregoire et al., 1998). Négrel et al. (1989) found that PGI2 triggered the differentiation of Ob1771 cells by binding to its cell surface receptor which then stimulated cAMP production. As a cAMP response element has been found in the promoter region of the CIEBP8 gene (Cantwell et al., 1998), PGlz may :- increase differentiation via this pathway. In addition, Brun et al. (1996) and Hertz l et al. (1996) stated that PGIz was capable of activating PPAR. Although the mechanisms of PGlz action were somewhat unclear, Hertz et al. (1996) E suggested that PGI2 may have acted as a direct intracellular activator of PPAR, indirectly as a proligand for PPAR activation through a PGIz derivative, or through influencing PPAR-associated proteins. Fifteen-deoxy-A‘z'“- prostaglandin J2 is an arachidonic acid metabolite that has been identified as a direct endogenous PPARy ligand in NIH 3T3 and C3H10T1l2 adipogenic cell lines respectively (Forrnan et al., 1995; Kliewer et al., 1995). Fifteen-deoxy- A‘z'“-prostaglandin J2 is a derivative of prostaglandin J2 (PGJz), which has been found to be incorporated into cell nuclei (Narumiya et al., 1987), thus indicating its ability to potentially bind PPARy within the nuclei. These results suggest that arachidonic acid and its metabolites may be important regulators of adipose tissue development. However, Torii et al. (1998) found that addition of 15d-PGJ2 to differentiation media had no effect on the differentiation of bovine i.m. S-V cells. Therefore, the role of arachidonic acid metabolites on the differentiation of bovine preadipocytes is yet to be clearly defined. 26 Vitamin A derivatives (retinoids) and their receptors appear to be involved in cellular differentiation. Vitamin A main metabolite, retinoic acid (RA) is capable of influencing gene transcription through its binding and activation of retinoid receptors (Villarroya et al., 1999). Retinoid receptors are transcription factors that belong to the same receptor family as PPAR, and have been shown to be highly involved in adipose tissue metabolism through heterodimerization with other receptors (Keller et al., 1993). In order to bind DNA and activate transcription, PPAR isoforms must first heterodimerize with retinoid X receptors (RXR) (Keller et al., 1993). As PPAR are inactive as homodimers, RXR must be available for transcription to occur (Lemberger et al., 1996). Retinoic acid has been shown to both positively and negatively influence preadipocyte differentiation. Safonova et al. (1994) found that RA enhanced the rate of differentiation in Ob1771 cells when included at physiological concentrations through its activation of the retinoid acid receptor (RAR). However, when tested at supraphysiological concentrations, RA negatively affected differentiation. Additionally, RA was shown to decrease the differentiation of bovine (Ohyama et al., 1998) and porcine (Suryawan and Hu, 1997; Brandebourg and Hu, 2005) S-V cells. Brandebourg and Hu (2005) suggested that RA inhibited differentiation through the down regulation of PPARy and RXR expression with simultaneous activation of RAR. Others have found that RA blocked adipogenesis by inhibiting CIEBPB (Schwarz et al., 1997). Adipose differentiation may also depend on the intracellular pr0portion of RAR and RXR within the cell. Villarroya et al. (1999) suggested that decreased expression of RAR may allow greater 27 “L I l' 1.. heterodimerization of RXR/PPAR dimers instead of RAR/RXR, thus enhancing differentiation. In agreement with this, Xue et al. (1996) found that RAR was downregulated during the differentiation of 3T3-L1 cells. Although it is unclear wether they are stimulatory or inhibitory towards differentiation, these studies suggest that retinoids may play an important role in regulating adipose tissue development. Thiazolidinediones are known antidiabetic agents that enhance adipose conversion through increasing cellular insulin sensitivity, as well as, directly activating PPARy through high affinity ligand binding (Fon'nan et al., 1995; Lehmann et al., 1995). As stated previously, PPAR combines with RXR to form heterodimers, which upon ligand binding, become transcriptional activators (Kliewer et al., 1992). These heterodimers are capable of binding to PPAR response elements within the promoter segments of various genes, causing their upregulation (Kliewer et al., 1992). It appears that TZD may also effect CIEBPa expression through the stimulation of PPARy, which upregulates ClEBPa and aids in maintaining the differentiated state (Wu et al., 1999). Indeed, TZD enhanced CIEBPa expression in porcine S-V cells (Tchoukalova et al., 2000). Exposure of cultured S-V cells from various species to TZD consistently results in stimulation of adipogenesis (Adams et al., 1997; Ohyama et al., 1998; Torii et al., 1998; Hutley et al., 2003). For example, less than 5% of human s.c. preadipocytes accumulated lipid in non TZD supplemented differentiation media, while 15 to 20% of preadipocytes accumulated lipid following addition of a TZD (Adams et al., 1997). Torii et al. (1998) also showed that TZD stimulated 28 differentiation of bovine i.m. S-V cells in a dose-dependent manner. Cumulatively, these studies indicate the importance of TZD and(or) PPARy ligands in stimulating preadipocyte differentiation. Depot differences in preadipocyte development Regional differences in preadipocyte development appear to exist between adipose tissues of various species. Preadipocytes from different depots E may differ in response to endogenous/exogenous stimuli, proliferation and(or) differentiation rate, and lipogenic enzyme activities. Proliferation capabilities. Although most preadipocyte cell culture studies I that compare regional differences focus on differentiation between depots, differences also exist in proliferation rates. Rat p.r. preadipocytes replicated more rapidly than those of epididymal origin in all ages of rats studied (Djian et al., 1983; Kirkland et al., 1990). Although neither study provided any substantial evidence to explain the observed differences in proliferation between depots, both suggested that cellular differences in response to hormones or growth factors may have been causative. Djian et al. (1983) also suggested that differences in growth rate may be due to physiological age (previous in vivo doublings) of the tissues, stating that rat or cells may be from a “younger" adipose depot than those of epididymal origin, and therefore have higher replicative rates. More recent studies using rat (Dieudonne et al., 2000) and human (Anderson et al., 2001) S-V cells have shown that an estrogen derivative stimulated a greater relative increase in proliferation of so. compared to cm. cells, indicating that differential depot responses to hormones do exist. 29 Differentiation capabilities. The capacity for preadipocyte differentiation appears to differ between depots and may be influenced by numerous factors. In vitro studies using preadipocytes or S-V cells of several species have shown that differentiation capabilities can vary among adipose depots within a species when cells are cultured under identical conditions (Djian et al., 1985; Deslex et al., 1987; Wiederer and Loffler, 1987; Kirkland et al., 1990; Sztalryd et al., 1991; Adams et al., 1996; Adams et al., 1997; Soret et al., 1999; Wu et al., 2000; Hutley et al., 2003). Primary cultures of rat p.r. preadipocytes contain a higher percentage of cells capable of extensive differentiation than those from the epididymal region (Djian et al., 1983, 1985; Wiederer and Loffler, 1987). Similar experiments found higher LPL and GPDH activities of rat S-V cells from retroperitoneal and pr. regions when compared to epididymal depots (Sztalryd and Faust, 1990; Kirkland et al., 1996). Depot differences in the capacity of differentiating S-V cells to accumulate FA have also been found. Castera et al. (2001) determined that, during differentiation, FA uptake progressed more rapidly in rat p.r. than epididymal cells. Along with these results, FA binding protein levels were higher in p.r. than epididymal cells. In the same study, it was also found that the incorporation of FA into human o.m. S-V cells were greater than those from the so. region. These data indicate that heterogeneity among adipose tissues is partially accounted for by inherent differences in preadipocytes. Both intracellular and extracellular factors have been shown to induce differential responses in preadipocyte differentiation among depots. 30 Glucocorticoids may exert depot specific effects on preadipocyte differentiation. Bujalska et al. (1999) observed that cortisol enhanced lipogenic enzyme activity in cm. compared to so. human S-V cells. In addition, Ramsay et al. (1989a) found that porcine s.c. S-V cells differentiated in response to hydrocortisone addition while p.r. cells showed no response. In contrast to these results, Hausman and Poulos (2004) reported that DEX equally increased the relative proportion of differentiated porcine so. and i.m. S-V cells, even though, regardless of treatment, a higher percentage of so. cells differentiated compared to i.m. Thiazolidinediones may induce depot specific effects on adipogenesis through their effects on PPARy. Thiazolidinediones have been found to promote increased differentiation in human s.c. S-V cells, while the same effect was not observed in cm. cells (Adams et al., 1997; Hutley et al., 2003). To compliment this, Sewter et al. (2002) showed that PPARy isoforms are more highly expressed in so. S-V cells than o.m., thus allowing them to be more responsive to TZD treatment. In addition, Tchkonia et al. (2002) attributed reduced PPARy expression to the low responsiveness of cm. preadipocytes to TZD compared to so. cells. These studies lend to evidence suggesting that preadipocytes from different regions of the body are unique to one another. Although less information is available regarding differences in preadipocyte differentiation between adipose depots in ruminants, reports note regional variations similar to those found in other species. When ovine S-V cells were cultured under identical conditions, it was found that i.t. and so S-V cells 31 displayed greater differentiation capabilities in the presence of insulin, triiodothyronine, and a mixture of lipids, compared to 0m. cells (Adams et al., 1996). Although no explanations for the variations in differentiation were given, differential cellular responses to the media components may have been causative. Soret et al. (1999) also demonstrated that ovine s.c. S-V cells were capable of greater differentiation than o.m. cells. These depot specific differences were reduced however, when TZD were added to the media. The authors thus suggested that a cell’s ability to produce endogenous PPARy ligands may account for an increased capacity for differentiation. Wu et al. (2000) reported similar findings in bovine S-V cells, as a PPARy agonist stimulated a greater relative increase in differentiation of 0m. than s.c. cells. The authors stated that differences in a cells’ ability to utilize triacylglycerol and(or) FA as natural PPAR ligands may influence their ability to differentiate. Summary An understanding of the cellular mechanisms responsible for differential rates of adipose tissue development in beef cattle could lead to an increase in the efficiency of current beef production practices, and product quality. Although adipose precursor cells isolated from other species have been extensively studied, experiments using bovine S-V cells and(or) preadipocytes are limited. In addition, most of these have not focused on cellular differences among adipose depots. Although S-V cell isolation and culture techniques have been documented, significant differences in the methodology exist among laboratories and species. 32 In vitro cell culture experiments provide a direct model with which to study the mechanisms involved in adipose tissue development, thus it seems appropriate to apply this model to the bovine. Preadipocyte differentiation characteristics of different adipose depots have been shown to be unique. Therefore, we hypothesize that differentiation of bovine i.m. and so. S-V cells would be enhanced by adipogenic stimuli, and that the relative response to these stimuli would be greater in i.m. than s.c. cells. 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Cascade regulation of terminal adipocyte differentiation by three members of the CIEBP family of Ieucine zipper proteins. Genes Dev. 9:168-181. 47 CHAPTER II ISOLATION, CLONING, AND OPTIMIZATION OF DIFFERENTIATION OF BOVINE PREADIPOCYTES Abstract The objectives of these experiments were to: 1) optimize differentiation of - clonally derived bovine preadipocytes in serum-free media; and 2) determine if a peroxisome proliferator-activated receptor agonist troglitazone (T RO) preferentially induces differentiation of i.m. compared to so. clonally-derived preadipocytes. StromaI-vascular cells from i.m. and so. fat depots of an Angus steer (558 kg, 13.5 mo. old) were cloned using dilution or cloning ring techniques. A higher percentage of isolated s.c. clones (47%; n = 30 of 64) were deemed adipogenic compared to i.m. (12.5%; n = 6 of 48) (P < 0.001), as determined by oil red O staining. A s.c. preadipocyte clone was used to determine the adipogenic effects of insulin, bovine serum lipids (SL), octanoate, acetic acid, dexamethazone (DEX), and TRO when added to serum-free media. Addition of 10 and 20 uUmL SL to low glucose DMEM media containing 280 nM insulin increased glycerol-3-phosphate dehydrogenase (GPDH) activity (P < 0.01). Inclusion of 1.25 to 10 pM TRO to media containing 280 nM insulin and 20 pUmL SL also increased GPDH activity (P < 0.001). The combination of 280 nM insulin, 1 mM octanoate, and 10 mM acetic acid, with 48 h exposure to 0.25 uM DEX caused morphological differentiation in a small number of cells but did not stimulate GPDH activity (P = 1.0). When used together, 280 nM insulin, 20 uUmL SL, 40 uM TRO, and 0.25 (M DEX stimulated differentiation compared to 48 the aforementioned treatment (P < 0.001). Omission of TRO or insulin from this media lowered GPDH activity by 68% (P < 0.001), while removal of DEX tended to reduce activity (P = 0.06). Preadipocyte clones from so. (n = 3) and i.m. (n = 2) adipose tissues were used to determine if the effects of TRO on differentiation were depot-specific. Twenty to 60 uM TRO enhanced differentiation compared to control (P < 0.02). No depot differences in response to TRO were detected (P = 0.47). These data demonstrate that clonally derived s.c. preadipocytes isolated from an Angus steer are capable of differentiation in response to combinations of insulin, octanoate, acetate, SL, DEX, and TRO. In addition, no depot differences in differentiation were observed between so. and i.m. preadipocytes in response to TRO. Introduction Excessive external and insufficient intramuscular fat were reported as two of the top six quality challenges for the beef industry (Smith et al., 2000). Adipose tissue development is partially controlled by adipocyte precursor cell (preadipocyte) proliferation and differentiation. Preadipocyte differentiation is a transformation from a fibroblast-like cell to a lipid-filled cell, with the expression of transcription factors, genes, and enzymes indicative of a mature fat cell (Butterwith, 1994; Grégoire et al., 1998). Bovine stromal-vascular (S-V) cells (Sato et al., 1996; Torii et al., 1998) and clonally- derived preadipocytes (Aso et al., 1995) are capable of differentiation in response to adipogenic stimuli. Wu et al. (2000) demonstrated that bovine 49 omental (o.m.) S-V cells exhibit a greater relative increase in differentiation when exposed to media including a peroxisome proliferator-activated receptor (PPAR) ~y agonist than subcutaneous (s.c.) cells. Thus, the potential may exist to differentially affect adipose depot development in beef cattle. Direct comparisons of preadipocyte differentiation from the two most economically important bovine adipose depots, intramuscular (i.m.) and so, are lacking. We hypothesized that a PPARy agonist would enhance the differentiation of bovine i.m. and so. preadipocytes, with the relative response being greater in i.m. than s.c. cells. Our initial studies found that previously described conditions for culture of bovine preadipocytes yielded insufficient differentiation to allow accurate comparisons between i.m. and so. preadipocytes. Thus, given the paucity of information regarding manipulation of culture conditions to optimize bovine preadipocyte differentiation, we conducted experiments described herein to: 1) optimize differentiation using insulin, fatty acids, serum lipids, glucocorticoids, and a PPARy agonist; and 2) determine differential responses of i.m. and so. preadipocytes to a PPARy agonist. Materials and Methods Clonally derived bovine preadipocyte cell lines, isolated from i.m. and so. adipose depots, were used to determine the effects of adipogenic stimuli on adipose differentiation. 50 Animal description Subcutaneous and i.m. adipose tissues were collected from an Angus steer (age, 13.5 mo; hot carcass weight, 345 kg; fat thickness, 20.3 mm; marbling score, Moderate”). Before harvest the steer was fed a high concentrate, corn-based diet for 209 d. An anabolic implant (200 mg progesterone, 20 mg estradiol benzoate) was administered on d 18 and 87 of the feeding period. Animal care was conducted according to procedures approved by the Michigan State University Committee on Animal Use and Care (AUF No. 1 0/03-1 30-00). Reagents The bovine serum lipid (SL) culture media supplement was an aqueous lipoprotein concentrate containing a mixture of fatty acids, cholesterol, and phospholipids, derived from bovine serum (Ex-Cyte; Serologicals Corp.,Norcross, GA). Troglitazone (TRO), a thiazolidinedione PPARy agonist, was obtained from Calbiochem, La Jolla, CA. Unless otherwise stated, all other reagents were of tissue culture grade and were purchased from Sigma (St. Louis, MO). Stromal-vascular cell isolation Adipose tissue samples were collected immediately after exsanguination from the left side of the carcass. lncisions were made between the 12th and 13th rib and a sample containing a portion of both so. adipose tissue and longissimus muscle (LM) was extracted. Upon collection, samples were immediately placed in a sterile ice-cold solution of 171.1 mM sodium chloride, 3.4 mM potassium chloride, 10.1 mM sodium phosphate-dibasic, and 1.8 mM potassium phosphate- 51 monobasic, (pH 7.4) (phosphate-buffered saline; PBS), and transported to the laboratory. Stromal-vascular cells from so. and i.m. adipose tissues were isolated under sterile conditions using a modification of the method described by Forest et al. (1987). Briefly, s.c. adipose tissue was separated from the LM and visible collagenous connective tissue. Visible connective and muscle tissues were carefully dissected away from i.m. adipose tissue. All excised adipose samples were then cut into approximately 2 mm sections. For each depot, samples of approximately 3 g were aliquoted into 50 mL conical tubes and digested in 6 mL Dulbecco’s modified Eagle’s medium (DMEM; 5.5 mM glucose) (lnvitrogen Corp., Carlsbad, CA) containing 2 mglmL collagenase (06885; > 125 collagenase digestion units/mg solid) and 2% bovine serum albumin (BSA). Initially, samples were incubated in a 37°C water bath, with inversion of vials at 0, 5, 10, and 15 min. Samples were then transferred to an incubator (Lab-Line Instruments Inc., Melrose Park, IL) and further digested with shaking for 45 min at 37°C and 230 rpm. The digested material was then sequentially filtered through 1000, 500, and 53 um nylon mesh, after which the filtrates were centrifuged twice for 10 min at 800 x g. Resulting cell pellets were resuspended in growth medium comprised of base media (DMEM, 1% antibiotic-antimycotic, 0.1% gentamicin, 33 pM biotin, 17 pM pantothenate, and 100 uM ascorbate), supplemented with 10% fetal bovine serum (FBS) subsequent for cloning. Preadipocyte cloning One gram tissue-equivalent of cells from each depot was suspended in 4 mL growth media and seeded equally into 2 wells of a 6-well tissue culture plate 52 (35 mm diameter; Corning Inc., Corning, NY). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. Cells were grown to approximately 70% confluence, washed twice with PBS, and then removed from the wells by trypsinization (0.5 glL trypsin and 0.02 g/L ethylenediaminetetraacetic acid (EDTA) in PBS (pH 7.2)). One of two cloning procedures were used to isolate preadipocytes. In one procedure, cells were counted, diluted, and plated at a density of 400 cells/10-cm culture plate and incubated in growth medium for 10 to 14 d. Individual colonies were isolated using 0.5 to 1.0 cm diameter sterile glass cloning-rings dipped in silicone grease. After placement of cloning rings, cells of a colony were washed twice with PBS, trypsinized, and transferred into one well of a 24-well culture plate containing growth media. In a second cloning procedure, cells were counted and diluted to one cell per 200 uL in growth media. Cells were seeded at 200 uLlwell into 96- well culture plates. Wells observed to contain colonies derived from a single cell were then propagated in growth media for 8 to 13 d. Wells were washed twice with PBS, trypsinized, and transferred into one well of a 24-well culture plate containing growth media. All isolated clones were then grown to approximately 70% confluence in 24-well and 6-well culture plates (2 to 5 d each). Clones were then seeded at 1800 cells/cm2 into 10-cm diameter culture plates and again grown to approximately 70% confluence (5 to 7 d each). Clones were then trypsinized, counted using a hemocytometer, and resuspended in freezing media (base media supplemented with 20% FBS and 10% dimethyl sulfoxide). Cells were 53 aliquoted into 1.8 mL cryogenic vials and placed in a styrofoam rack overnight at -80°C, with subsequent storage in liquid nitrogen. Identification of preadipocyte clones Sub-samples of cells from each isolated clone were suspended in 10 mL growth media and seeded at clonal densities (400 cells/well) in 10-cm plates to determine if clones were adipogenic. In initial clonal characterizations, cells were grown for 8 to 10 d, then exposed to differentiation media consisting of base media supplemented with: 8.7 nM bovine insulin, 20 mM glucose, 1 mM octanoate, and 10 mM acetic acid (EMD Chemicals Inc., Gibbstown, NJ) with addition of 0.25 (M dexamethasone (DEX) for the first 48 h of the differentiation period. Differentiation media similar to that stated above stimulated differentiation in an i.m. preadipocyte cell line derived from Japanese Black cattle (Aso et al., 1995). After discovery of improved conditions for bovine preadipocyte differentiation in our laboratory, clones were grown for 8 to 10 d and induced to differentiate in base media containing 280 nM bovine insulin, 20 mM glucose, and 10 uleL SL with 48 h exposure to 0.25 uM DEX. After 10 to 12 d exposure to differentiation media, all plates were stained with oil red O (ORO) and visualized for the percentage of colonies containing triacylglycerol. Clones having at least 1 cell/colony stained with ORO in 2 85% of their colonies were characterized as preadipocytes. Clones identified as preadipocytes were seeded at 1800 cells/cm2 into 10-cm diameter culture plates and sequentially propagated to approximately 70% confluence (5 to 7 d each) until reaching the 8‘" or 9th passage. All experiments using preadipocyte clones were performed using cells 54 at the 9th or 10th passage. To optimize differentiation conditions, experiments 1, 2, and 3 were conducted using one so clone. General Procedures Cell culture. Clonally derived preadipocytes were seeded in 6-well plates at a density of 5200 cells/cm2 and incubated in growth medium at 37°C in a humidified atmosphere of 95% air and 5% CO2. After 24 h, plates were washed twice with PBS and fresh growth medium was added. Growth medium was replaced every 2 d until cells reached confluence (4 to 6 d). After reaching confluence, plates were washed twice with PBS and experimental differentiation treatments applied. For all experiments, differentiation media were replaced with fresh media every 2 d for 10 to 12 d. Media additions were present for the entire differentiation period except DEX, which was supplemented only for the initial 48 h. All wells were washed twice with PBS before media replacement on d-2 of the differentiation period. Gcherol-3-phosphate dehydrogenase activity assay. Cell differentiation was quantified biochemically by measuring glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity using a modification of the method described by Adams et al. (1997). Cells were washed twice with ice-cold PBS. Contents of two wells per treatment were combined and harvested in a total volume of 200 uL of ice- cold Tris (pH 7.4) containing 1 mM EDTA and 50 uM dithiothreitol (extraction buffer), then transferred into prechilled 1.5 mL microcentrifuge tubes. Each sample was then disrupted by sonification 3 times at 40 W (3 s bursts with 1 min cooling on ice between bursts) using a Sonifier—Cell Disrupter 350 (Branson 55 Sonic Power Co., Danbury, CT). Samples were then centrifuged at 16,000 x g for 15 min at 2°C. Solutions containing 50 pL of the resulting supernatant and 150 uL of assay buffer were assayed in duplicate within 30 min for GPDH activity. The final concentration of the supernatant and assay buffer solution was: 100 mM triethanolamine-HCL (pH 7.4), 2.5 mM EDTA, 50 uM dithiothreitol, 0.8 mM dihydroxyacetone phosphate, and 0.317 mM NADH. Each reaction was initiated by the addition of assay buffer to the supernatant in one well of a 96-well plate (lmmulon 1B; Fisher Scientific, Hampton, NH). Fifty uL of extraction buffer served as the reagent blank. To obtain the reaction rate, the AA340 was recorded at 15 5 intervals for 6 min at 30°C using a spectrophotometer (Versamax Tunable Microplate Reader, Molecular Devices, Sunnyvale, CA). All reactions measured were linear for at least 220 s and the Vmax used to calculate GPDH activity was obtained from the linear range. Enzyme activity was expressed as nanomoles of NADH oxidized - min'1 - mg protein“. Protein quantification. Aliquots (20 pL) of supernatant were assayed in duplicate to determine protein concentration using the bicinchoninic acid assay (BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). Samples were mixed with 200 pL of assay reagent, incubated for 30 min at 37°C, and absorbance at 562 nm determined. Protein concentration was determined using BSA as a standard. Morphological differentiation assay. Cell differentiation was morphologically assessed by observation of cells containing lipid droplets stained with ORO. The ORO solution was prepared using a protocol described by 56 Humason (1972). Briefly, a solution of 0.35 g ORO and 100 mL isopropanol was incubated at room temperature overnight without stirring, then filtered through Whatman #40 filter paper. Seventy-five mL of distilled water was added to the solution, which was incubated overnight at 4°C, filtered twice using Whatman #40 filter paper, and stored at room temperature. A solution of nuclear stain was made by dissolving 1 g of giemsa into 66 mL glycerol and 66 mL of methanol. This solution was filtered using Whatman #1 filter paper. Wells were initially aspirated of media and washed twice with PBS. Cells were then fixed by addition of 3.7% formaldehyde (diluted from a stock of 37% formaldehyde solution containing 10% methanol; Mallinckrodt Baker Inc., Phillipsburg, NJ) in PBS for 4 min. which was then aspirated, and the wells washed twice with PBS. After fixation, cells were washed twice with PBS and incubated at room temperature with ORO solution for 1 h. Residual ORO solution was aspirated and the cells were washed twice with distilled water (15 min incubation/wash). Cell nuclei were stained by adding 1 mL giemsa solution to each well for 1 h, after which the cells were washed twice in distilled water. Wells were aspirated of water and stored at 4°C. Cells were visualized within 24 h of staining. Digital photographs were taken using a Nikon CoolPix 5000 digital camera (Nikon lnc., Melville, NY) fitted to a Zeiss inverted microscope (Carl Zeiss Inc., Thornwood, NY). Experiment 1 This experiment was conducted to determine the effect of increasing concentrations of SL on preadipocyte differentiation using a 5.0. clonal cell line. 57 Im. fl-—'_ _. - Three independent experiments were performed using differentiation media consisting of base medium supplemented with 280 nM bovine insulin, 20 mM glucose, and 5, 10, 20, or 40 uleL SL.' Protein concentrations and GPDH activities were measured after a 12-d differentiation period. Experiment 2 This experiment was designed to evaluate the adipogenic effects of increasing concentrations of TRO. Three independent experiments were performed using differentiation media consisting of base medium supplemented with 280 nM bovine insulin, 20 mM glucose, 20 lemL SL, and 1.25, 2.5, 5, or 10 uM TRO. Troglitazone was solubilized in dimethyl sulfoxide at a stock concentration of 22.7 mM before addition to media. Addition of dimethyl sulfoxide to differentiation media alone did not significantly affect differentiation (data not shown). Protein concentrations and GPDH activities were measured after a 10-d differentiation period. Experiment 3 The purpose of this experiment was to investigate the effects of FA source and(or) removal of individual components from serum-free media on preadipocyte differentiation. Three independent experiments were performed to compare different differentiation media additives. Differentiation media treatments consisted of base medium with 20 mM glucose, supplemented with: 280 nM bovine insulin, 0.25 uM DEX, 1 mM octanoate, and 10 mM acetic acid (TRT 1); 280 nM bovine insulin, 20 pUmL SL, and 0.25 pM DEX (TRT 2); 280 nM b0Vine insulin, 20 pL/mL SL, 0.25 lJM DEX, and 40 pM TRO (TRT 3); 280 nM 58 min- ”Ah-P - ' I ‘ bovine insulin, 20 uUmL SL, and 40 uM TRO (TRT 4); 20 uL/mL SL, 0.25 uM DEX, and 40 uM TRO (TRT 5). Protein concentrations and GPDH activities were measured after a 12-d differentiation period. Experiment 4 This experiment was conducted to determine if so and i.m. preadipocytes respond differentially to the lipogenic effects of TRO. Two independent experiments were performed using three so. and two i.m. clonal cell lines. Differentiation media consisted of base medium supplemented with 280 nM bovine insulin, 20 mM glucose, 20 uUmL SL, and 5, 10, 20, 40, or 60 pM TRO. Protein concentrations and GPDH activities were measured after a 12-d differentiation period. Statistical analysis Data were analyzed using the Mixed Model procedure of SAS (SAS, Cary, NC) appropriate for completely randomized designs. In determining the percentage of adipogenic clones isolated from i.m. and so. depots, means were generated using depot as the fixed effect with clone defined as the experimental unit. In all experiments, pooled cells from 2 wells of a 6-well plate were considered the experimental unit. In Exp.1, 2, and 3, means were calculated using the fixed effects of SL, TRO, and treatment, respectively. Regression models for dose responses were determined using the Proc REG procedure of SAS. In Exp. 4, to satisfy the conditions of normality and homogeneity of variance, GPDH data were loge transformed. Least squares means were then calculated using the fixed effects of TRO, depot, and TRO x depot. Clone within 59 depot and TRO x clone within depot were included as random variables. In all experiments, when main effects were considered significant (P < 0.05), differences between means were investigated using Tukey’s multiple comparison test. Results and Discussion Results from these studies document the isolation and cloning of bovine preadipocytes and characterize the response of these cells to adipogenic stimuli. Isolation and culture of S-V cells from Japanese Black cattle have been reported for so, o.m. (Wu et al., 2000), perirenal (Ohyama et al., 1998), and i.m. (Aso et al., 1995; Sato et al., 1996; Torii et al., 1998) adipose tissue depots. Isolation of i.m. S-V cells from an Angus steer proved more tedious and yielded fewer cells compared to the so. depot. However, careful dissection and isolation resulted in the successful culture and cloning of preadipocytes capable of differentiation from both so and i.m. adipose tissues. A higher percentage of clones isolated from the so. depot (47%; n = 30 of 64) were identified as adipogenic based on ORO staining compared with adipogenic clones (12.5%; n = 6 of 48) from the i.m. depot (P < 0.001). The lower percentage of identified preadipocytes from the i.m. depot suggests that a greater proportion of non-adipogenic cell types may exist in these S-V isolates. Alternatively, the apparent non-adipogenic clones may have been preadipocytes that were unresponsive to our differentiation conditions. Depot differences in the percentage of non-adipogenic cells in S-V isolates may confound results of depot 60 comparison studies (Tchkonia et al., 2002). Therefore, to study and compare cells identified as preadipocytes from i.m. and so. depots, our experiments were conducted using adipogenic clones. Experiment 1 Addition of SL to serum-free differentiation media for 12 d increased GDPH activity (Figure 2-1). Preadipocyte differentiation was not detected using control media without SL. However, cells containing lipid droplets were observed with the addition of 5 uleL or greater SL, and concentrations of 10 (P < 0.01) and 20 uL/mL (P < 0.001) increased GPDH activity when compared to the control. Gcherol-3-phosphate dehydrogenase activities did not differ (P = 0.37) between 10 and 20 pUmL SL. Inclusion of 40 uUmL SL showed no difference in GPDH activity (P = 0.99) when compared to the control. Visually, cell morphology and viability appeared to be negatively affected with addition of 40 pUmL SL. Previous studies using bovine (Wu et al., 2000) and ovine (Soret et al., 1999) S-V cells have shown that lipid supplementation enhances differentiation. Wu et al. (2000) demonstrated that addition of a lipid emulsion (containing phosphatidylcholine and triacylglycerols) to differentiation media stimulated bovine S-V cell differentiation. Also, the inclusion of very low-, low-, or high- density lipoproteins, derived from human plasma, enhanced adipose conversion. Using the same SL used in our experiments, Soret et al. (1999) showed that 10 uUmL SL stimulated lipid accumulation in ovine S-V cells. Fatty acids, especially long chain polyunsaturated FA, serve as precursors of other FA in triacylglycerol 61 synthesis, and(or) stimulate preadipocyte differentiation through ligand binding to PPAR (Kliewer et al., 1997; Krey et al., 1997). Of note, linoleic acid has been found to increase lipid accumulation in human (Hutley et al., 2003) and porcine (Ding et al., 2003) S-V cells. Likewise, arachidonic acid, a linoleic acid derivative, has also been found to enhance differentiation in Ob1771 preadipocytes (Massiera et al., 2003), and may exert its effects via its eicosanoid E products (Krey et al., 1997), including the prostaglandins (Kliewer et al., 1997; , Massiera et al., 2003). Peroxisome proliferator-activated receptor-y protein expression has been documented in undifferentiated bovine (Torii et al., 1998) and porcine (Kim et al., 2000) S-V cells derived from adipose tissue. Ding et al. (2003) suggested that addition of PPARy ligands, such as FA, stimulate differentiation in porcine S-V cells through ligand binding to PPARy, as well as potentially serving as substrates for triacylglycerol synthesis. Although we did not measure PPARy protein expression, we speculate that these mechanisms are responsible for the SL-induced increase in differentiation observed in this experiment. Experiment 2 Increasing concentrations of TRO, previously shown to stimulate differentiation in S-V cells isolated from Japanese Black cattle (Ohyama et al., 1998; Torii et al., 1998), were added to differentiation media in an attempt to increase the adipose conversion of the so. clone used in Exp. 1. A quadratic increase in GPDH activity (P < 0.001) was observed with increasing 62 concentrations of TRO (Figure 2-2). Treatment with 10 (M TRO resulted in a 6.3-fold increase in GPDH activity above the control (P < 0.001). Soret et al. (1999) reported a 1.9- to 2.5-fold increase in GPDH activity of S-V cells derived from 6- to 8-mo-old wether lambs when 0.1 uM TZD was added to media containing 10 uUmL SL. In preliminary studies, we did not observe increased GPDH activity above that of SL addition using the same TZD concentration as Soret (1999). In our experiments, 20 uUmL SL were used, which may have contributed to the lack of response observed at that TZD concentration. In addition, differences in the specific culture media and TZD used between studies, as well as possible species effects, may have influenced results. However, the concentrations of TRO found to stimulate differentiation in our experiment were within the range of TRO (1 to 100 (M) previously shown to enhance adipose conversion of pr. (Ohyama et al., 1998) and i.m. (Torii et al., 1998) S-V cells isolated from Japanese Black cattle. Thiazolidinediones such as TRO, are known to be high affinity PPARy ligands (Lehmann et al., 1995). Peroxisome proliferator-activated receptorq is primarily expressed in adipose tissues of most species investigated (Chawla et al., 1994; Braissant et al., 1996; Sundvold et al., 1997), including Japanese Black cattle (T orii et al., 1998) and has been implicated in the differentiation process through its activation by endogenous (Kliewer et al., 1997; Krey et al., 1997) and synthetic (Lehmann et al., 1995; Spiegelman, 1998) ligands. Ligand-bound PPARy forms heterodimers with retinoid X receptors, which are then capable of binding to PPAR response elements in the promoters of adipogenic genes, thus stimulating differentiation 63 (Kliewer et al., 1992). Experiment 1 showed that inclusion of SL to our differentiation media stimulated differentiation in a so. bovine preadipocyte clonal line. Furthermore, Exp. 2 determined that a PPARy agonist, in addition to SL, increased adipose conversion. This increase in differentiation was likely a result of TRO being a more potent activator of PPARy than the FA in SL. Enhanced ligand binding to PPARy may increase adipogenic gene transcription, thus stimulating a greater number of cells to differentiate and(or) increase the adipogenic capacity of the differentiated cells. Therefore, while TRO likely acted as the more potent PPARy agonist, SL may have contributed to differentiation as a substrate source for triacylglycerol synthesis in this study. Experiment 3 Using media components shown to increase differentiation in our previous experiments, and those reported in other studies, various adipogenic media were tested to further investigate factors that induce differentiation in bovine preadipocytes. The combination of 1 mM octanoate and 10 mM acetic acid with 0.25 (M DEX, and 280 nM insulin (T RT 1) resulted in the morphological differentiation of a small number of cells (Figure 2-3). However, GPDH activity was below the detection limit of our assay (Figure 2-4). Although this treatment did not stimulate biochemical differentiation using the so. clone, octanoate (Aso et al., 1995; Sato et al., 1996; Wu et al., 2000) and acetate (Aso et al., 1995) have previously been incorporated into differentiation media of bovine S-V cells and preadipocytes, respectively. When used in combination with DEX and 1- methyl-3-isobutylxanthine, Sato et al. (1996) found that octanoate stimulated a 5- 64 fold increase in GPDH activity using bovine i.m. S-V cells, while Wu et al. (2000) observed no significant response in cm. cells. Han et al. (2002) suggested that octanoate may serve as a PPARy ligand and possibly activate differentiation in 3T3-L1 cells. Acetate is considered the major precursor for FA synthesis in ruminant adipose tissues (Vernon, 1980), and Aso et al. (1995) showed increased acetate incorporation into bovine preadipocytes during adipose conversion. Therefore, acetate was incorporated into our differentiation medium to serve as a substrate for FA synthesis. Lipid emulsions have also been shown to enhance S-V cell differentiation (Soret et al., 1999; Wu et al., 2000). Therefore, we replaced the relatively ineffective FA combination used in TRT 1 with the lipid supplement used in Exp. 1. The combination of insulin, SL and DEX (TRT 2) was sufficient to generate GPDH activity as well as visually increase the number of cells containing lipid droplets when compared with the combination of octanoate and acetate. The concentration of FA in SL (0.19 mglmL) used in this study was within the concentration range of octanoate (0.14 mglmL) and acetate (0.60 mglmL) provided to the cells. Therefore, the observed increase in differentiation observed with SL is not merely explained by an increase in FA concentration in relation to octanoate or acetate. In addition, a 1.7-fold increase in protein concentration was observed when SL replaced octanoate and acetate, indicating that the supplement was more favorable for cell viability and differentiation. The relative effects of TRO, DEX, SL, and insulin on preadipocyte differentiation were tested (Fig. 2-4). The combination of all four components 65 (TRT 3) resulted in the greatest GPDH activity. Omission of DEX (TRT 4) tended to reduce GPDH activity (P = 0.06), whereas the removal of TRO (TRT 2) decreased GPDH activity by 68% (P < 0.001) and also lowered protein concentrations (P = 0.03). It has been suggested that glucocorticoids are capable of increasing differentiation through the upregulation of COAT/enhancer- binding proteins, which in turn, influence the expression of PPAR) in 3T3-L1 cells (Wu et al., 1996). Also, glucocorticoids may increase differentiation through the enhancement of arachidonic acid metabolism (Gaillard et al., 1991). An increase in arachidonic acid metabolism may result in the production of prostacyclin, which has been found to activate PPAR (Hertz et al., 1996). Thus, the potential for synergism between DEX and TRO in enhancing differentiation exists. Soret et al. (1999) found the combination of DEX and a thiazolidinedione to be additive in stimulating the differentiation of S-V cells from 6- to 8-mo-old wether lambs. However, results using S-V cells from suckling lambs (Soret et al., 1999), and ' those from 5 to 7 day old pigs (Tchoukalova et al., 2000) demonstrated that the combination of DEX and a thiazolidinedione did not stimulate differentiation above that of either compound when included individually. Soret et al. (1999) therefore suggested that the effects of these compounds on S-V cell differentiation are dependant on species, depot, and animal age. In addition, Soret et al. (1999) also found that DEX and a thiazolidinedione increased protein concentrations individually, and in combination with SL. Based on the reduced activity observed upon their removal from media, our study suggests that TRO is a more potent stimulator of differentiation than DEX. lf DEX and TRO primarily 66 function via the pathways mentioned previously, a lack of selective endogenous PPARy agonists, rather than PPARy protein expression, may be limiting adipose conversion of the so. clone. Insulin appeared to enhance adipose differentiation and(or) viability of the so. clone, since a 74% reduction in GPDH activity (P < 0.001) was recorded upon ommision of insulin (TRT 5), from differentiation media (TRT 3). Insulin is known to stimulate adipogenesis of swine (Suryawan et al., 1997) and ovine (Adams et al., 1996) S-V cells, and has been included in differentiation media of bovine S-V cells (Aso et al., 1995; Torii et al., 1998; Wu et al., 2000). Although ruminant adipose tissues are generally considered less responsive to the effects of insulin than fat depots of most non-ruminant species (Vernon, 1980), Adams et al. (1996) documented that exclusion of insulin from differentiation media prevented the adipose conversion of ovine S-V cells. Although the specific effects of insulin on preadipocyte differentiation have yet to be fully detailed, insulin has been shown to influence the expression of adipogenic genes. Insulin enhanced the transcription of genes such as fatty acid synthase (Paulauskis and Sul, 1988; Moustaid et al., 1994), glyceraldehyde-3-phosphate dehydrogenase (Nasrin et al., 1990), acyl-CoA synthetase, and stearoyl CoA desaturase-1 (Weiner et al., 1991), in studies using 3T3-L1 cells. Insulin is also known to stimulate glucose uptake into 3T3-L1 adipocytes through increased translocation of the glucose transporter-4 protein to the plasma membrane (Calderhead et al., 1990). In addition, Stahl et al. (2002) showed that insulin increased cellular uptake of long chain FA through stimulating FA transport protein translocation 67 from intracellular pools to the plasma membrane in 3T3-L1 adipocytes. Therefore, insulin may act as a modulator of cellular differentiation, as well as serve to increase substrate availability for adipogenesis in adipocytes. In our study, the omission of insulin visually appeared to reduce lipid accumulation within cells. However, the percentage of cells visually estimated to contain lipid appeared to be similar in both insulin treated and non-treated cultures. This suggests that the lack of insulin may have reduced FA and(or) glucose entry into the cells, thus reducing GPDH activity. The exclusion of insulin also caused a reduction in protein concentration (P < 0.001) compared to TRT 2, 3, or 4. This again suggests a critical role for insulin in anabolic processes and(or) viability of bovine preadipocytes. We also tested the removal of SL from TRT 3, which resulted in substantial cell detachment from the plates 2 d after exposure to differentiation media, thus, the treatment was discontinued. The SL appear to contain components that aid in maintaining cell viability and(or) attachment during differentiation. The latter appears to be dependant upon the presence of DEX and(or) TRO, because medium containing no SL was capable of supporting cell viability in the absence of DEX and TRO (Figure 2-1). Experiment 4 To examine if the adipogenic effect of TRO was depot-specific, we exposed clonal cell lines derived from both i.m. and so. adipose tissues to increasing concentrations of TRO. No significant depot x treatment (P = 0.51) interaction or depot (P = 0.47) effects were apparent in this experiment, 68 therefore, only the main effects of TRO are shown (Figure 2-5). Increasing concentrations of TRO caused a dose-dependent increase in GPDH activity. Addition of 20 to 60 uM TRO increased enzyme activity in relation to control values (P < 0.02). Previous studies using S-V cells isolated from 6- to 8-mo-old wether lambs (Soret et al., 1999) and 4- to 5-yr-old Holstein cows (Wu et al., 2000) have shown a greater relative increase in GPDH activity in cm. compared to so. cells with addition of a PPARy agonist to differentiation media. The authors suggested that o.m. cells might be limited in their ability to produce natural PPARy ligands when compared to so. cells. In contrast, studies using human S-V cells have shown that thiazolidinedione addition stimulated differentiation in s.c., while o.m. cells were less responsive to treatment (Adams et al., 1997; Sewter et al., 2002; Tchkonia et al., 2002). Adams et al. (1997) observed no differences in PPARy protein abundance between depots and offered no definitive explanation for the depot-specific responses to thiazolidinedione. However, more current studies showed that PPARy messenger RNA (Sewter et al., 2002; Tchkonia et al., 2002) and protein expression (Tchkonia et al.. 2002) were greater in human s.c. S-V cells than o.m., and thus, may account for depot differences in adipogenesis. When taken together, the aforementioned studies suggest that regional variation in adipogenesis exists in S-V cells isolated from different depots. Variations in the extent of differentiation among clones within and between depot were apparent in this experiment (Figure 2-5). However, this was not unexpected, as both Tchkonia et al. (2002) and Djian et al. (1985) found variations in the 69 percentage of differentiated cells among clonally derived colonies originating from the same depot. Although bovine i.m. and so. S-V cells have been shown to respond to lipid supplements (Sato et al., 1996; Wu et al., 2000) and thiazolidinediones (Torii et al., 1998), this study represents the first direct depot comparison of bovine i.m. and so. preadipocytes to a PPARy ligand. Implications Clonally-derived subcutaneous preadipocytes isolated from an Angus steer are capable of increased differentiation in response to serum lipids, dexamethasone, and troglitazone. As well, the relative increase in differentiation of both subcutaneous and intramuscular preadipocytes was similar when exposed to troglitazone. This suggests that proliferator-activated receptor-y is likely involved in the differentiation of both depots. Thus, the potential to influence adipose tissue development via manipulation of pathways leading to peroxisome proliferator-activated receptoray expression and(or) activation exist. The use of clonally-derived preadipocytes coupled with the refinement of cell culture conditions allowed for direct comparison between bovine i.m. and so. preadipocytes. This culture system will permit future investigations on the cellular mechanisms involved in bovine adipose tissue development, and elucidation of depot differences in preadipocyte differentiation. 70 Figures 12 Y _._Protein ' 10 C 79. XI . 0.5 3 o I... 8 . o. .1 a, h 0.4 g g 2’ TE 6 - - g . 0.3 .5 T: '6 E I- : 4 . “- I . 0.2 D 8 2 d I 001 0 L 0.0 0 5 10 20 40 Serum lipids, pLImL Figure 2-1. Effect of serum lipid supplementation on glycerol-3-phosphate dehydrogenase (GPDH) activity and protein concentration of subcutaneous bovine clonally-derived preadipocytes. Values are least squares means and SEM of three independent experiments. Means without a common superscript letter differ (P < 0.05). 71 200 160 . 120 .. 80. 8 GPDH nmoI-min'1-mg protein" Troglitazone, pllll Figure 2-2. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH) activity of subcutaneous bovine clonally—derived preadipocytes. Values are least squares means and SEM of three independent experiments. Means without a common superscript letter differ (P < 0.05). 72 Figure 2-3. Photomicrographs of a subcutaneous bovine preadipocyte clone exposed for 12-d to serum-free differentiation medium supplemented with 280 nM insulin, 0.25 uM dexamethasone, 1 mM octanoate, and 10 mM acetate (T RT 1) (A), or to serum-free differentiation medium supplemented with 280 nM insulin, 20 pUmL serum lipids, and 40 pM troglitazone (TRT 4) (B). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. 73 80 .m. u 5 .mm 74 80 .. . 0.7 - GPDH z ,7 2 '§ +Protein - 0-5 ‘5 so . h .. 0.5 o. ..| g E . . 0.4 is 40 - E: .E .. 0.3 '5 '2' § . 0.2 o. C I 20 . E . 0.1 0 0 - 0.0 1 2 3 4 5 Treatment TRO - - + + + DEX + + + - + s|_ - + + + + Insulin + + + + - Octanoate + - - - - Acetate + - - - - Figure 2-4. Effect of adipogenic agents on glycerol-3-phosphate dehydrogenase (GPDH), activity and protein concentration of subcutaneous bovine clonally- derived preadipocytes. Components included (+) or excluded (-) from the media were troglitazone (TRO) (40 pM), dexamethasone (DEX) (0.25 (M), serum lipids (SL) (20 pL/mL), insulin (280 nM), octanoate (1 mM), and acetate (10 mM). Values are least squares means and SEM of three independent experiments. Means without a common superscript letter differ (P < 0.05). 75 .. ‘ d Is 5 - d o c E d O. 4 bed a I 5 abc 'i: ab .5 3 I: '2 ' a r: 2 . I a .. n. (D 1 . a“: O u .1 o 1 I r T I I I T I I I I 1 0 10 20 30 40 50 60 Troglitazone, pM Figure 2-5. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH) activity. 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Induction of peroxisome proliferator-activated receptor y during the conversion of 3T3 fibroblasts into adipocytes is mediated by C/EBPB, C/EBP8, and glucocorticoids. Mol. Cell. Biol. 16:4128-4136. 80 CHAPTER III EFFECTS OF DEXAMETHASONE AND TROGLITAZONE ON DIFFERENTIATION OF STROMAL-VASCULAR CELLS ISOLATED FROM BOVINE INTRAMUSCULAR AND SUBCUTANEOUS ADIPOSE TISSUES Abstract The objectives of these experiments were to compare differentiation of bovine stromal-vascular (S-V) cells isolated from intramuscular (i.m.) and subcutaneous (s.c.) adipose tissues in response to a PPARy agonist and a glucocorticoid. Stromal-vascular cells were isolated from i.m. and so. fat depots of three Angus steers (age, 13.5 mo; hot carcass weight, 345 to 350 kg; 12th rib fat thickness, 12.5 to 22.0 mm; marbling score, Small80 to Moderate”). Cells were used to determine the adipogenic effects of troglitazone (TRO) and dexamethasone (DEX) when added to differentiation medium containing 280 nM insulin and 20 pL/mL bovine serum lipids. Addition of 40 and 60 (M TRO increased glycerol-3-phosphate dehydrogenase (GPDH) activity 2.9- and 3.4-fold respectively, compared to control medium (P < 0.02). No interaction between TRO and depot (P = 0.70) or depot differences (P = 0.32), were observed. In a second study, DEX stimulated a 1.8-fold increase in GPDH activity (P = 0.01), while TRO induced a 2.5-fold increase in GPDH activity (P = 0.02) compared to cells not treated with DEX or TRO, respectively. No interactions between DEX, TRO, and depot (P > 0.59), or depot differences (P = 0.41) were found. Morphological assessment of adipogenic colonies showed that DEX induced a 1.2-fold increase in the percentage of adipogenic colonies (P = 0.02), while TRO 81 increased the proportion of adipogenic colonies by 2.3-fold (P = 0.01) compared to those not treated with DEX or TRO, respectively. No depot differences in the percentage of adipogenic colonies were found (P = 0.10). However, the percentage of differentiated cells within adipogenic colonies was found to be 6.4- fold greater in so. isolates compared to i.m. (P < 0.001). Addition of TRO increased the proportion of differentiated cells by 10-fold when compared to those of non-treated colonies (P < 0.001). The percentage of differentiated cells within adipogenic colonies was not increased by DEX (P = 0.10). These data indicate that bovine i.m. and so. S-V cells are capable of enhanced enzymatic differentiation in response to DEX and TRO, while morphological differentiation of adipogenic cells within colonies was increased by TRO but not DEX. Most importantly, inherent differences in the capacity to differentiate appear to exist between adipogenic bovine i.m. and so. S-V cells. Introduction Adipose tissue development is partially controlled by adipocyte precursor cell (preadipocyte) proliferation and differentiation into mature adipocytes. Preadipocyte differentiation may be transcriptionally influenced by glucocorticoids (Wu et al., 1996) and the peroxisome-proliferator activated receptor (PPARH, which is highly expressed in bovine adipose tissue (Sundvold et al., 1997). Glucocorticoids have been implicated in preadipocyte differentiation via pathways that lead to the upregulation or activation of PPARy (Wu et al., 1996; Kliewer et al., 1997; Krey et al., 1997), which is known to increase the 82 transcription of adipogenic genes (Tontonoz et al., 1994; Schoonjans et al., 1996). Glucocorticoids increase adipogenic differentiation of bovine (Aso et al., 1995; Wu et al., 2000), ovine (Soret et al., 1999), and porcine (Ramsay et al., 1989; Tchoukalova et al., 2000) stromal-vascular (S-V) cells. Biochemical differentiation was induced in porcine subcutaneous (s.c.) S-V cells, while perirenal cells did not respond, to glucocorticoids (Ramsay et al., 1989). r Ligands of PPARy have been shown to increase differentiation of perirenal (p.r.) and intramuscular (i.m.) S-V cells isolated from Japanese Black cattle (Ohyama et al., 1998; Torii et al., 1998). Wu et al. (2000) found that a PPARy agonist induced a greater relative response in differentiation of bovine omental (o.m.) compared to s.c. S-V cells. To our knowledge, studies comparing bovine i.m. and s.c. S-V cells, or their responses to a PPARy agonist, are lacking. We hypothesized that differentiation of bovine i.m. and s.c. S-V cells would be enhanced by a PPARy agonist and(or) glucocorticoid, and that the relative response to these stimuli would be greater in i.m. than s.c. cells. Knowledge of factors that differentially influence bovine S-V cells derived from i.m. and s.c. depots may lead to methods that improve marbling and(or) reduce subcutaneous fat. Materials and Methods Animal descriptions Subcutaneous and i.m. adipose tissues were collected from three Angus steers (age, 13.5 mo; hot carcass weight, 345 to 350 kg; 12th rib fat thickness, 83 12.7 to 21.6 mm; marbling score, Small80 to Moderate”). Before harvest the steers were fed a high concentrate, corn-based diet for 209 to 249 d. Anabolic implants (200 mg progesterone, 20 mg estradiol benzoate) were administered on approximately d 30 and 100 of the feeding period. All experiments were performed using i.m. and s.c. S-V cells isolated from 3 steers. Animal care was conducted according to procedures approved by the Michigan State University E Committee on Animal Use and Care (AUF No. 10/03-130-00). Reagents The bovine serum lipid (SL) culture media supplement was an aqueous E lipoprotein concentrate containing a mixture of fatty acids, cholesterol, and phospholipids, derived from bovine serum (Ex-Cyte; Serologicals Corp., Norcross, GA). Troglitazone (TRO), a thiazolidinedione PPARy agonist, was. obtained from Calbiochem, La Jolla, CA. Unless otherwise stated, all other reagents were of tissue culture grade and were purchased from Sigma (St. Louis, MO). Stromal-vascular cell isolation Adipose tissue samples were collected immediately after exsanguination from the left side of the carcass. lncisions were made between the 12th and 13th rib and a sample from each steer containing a portion of both s.c. adipose tissue and longissimus muscle (LM) was extracted. Upon collection, samples were immediately placed in a sterile ice-cold solution of 171.1 mM sodium chloride, 3.4 mM potassium chloride, 10.1 mM sodium phosphate-dibasic, and 1.8 mM potassium phosphate-monobasic, (pH 7.4) (phosphate-buffered saline; PBS), 84 and transported to the laboratory. Stromal-vascular cells from s.c. and i.m. adipose tissues were isolated under sterile conditions using a modification of the method described by Forest et al. (1987). Briefly, s.c. adipose tissue was separated from the LM and visible collagenous connective tissue. Visible connective and muscle tissues were carefully dissected away from i.m. adipose tissue. All excised adipose samples were then cut into approximately 2 mm F sections. For each depot, samples of approximately 3 g were aliquoted into 50 mL conical tubes and digested in 6 mL Dulbecco’s modified Eagle’s medium (DMEM; 5.5 mM glucose) (lnvitrogen Corp., Carlsbad, CA) containing 2 mglmL b collagenase (C6885; > 125 collagenase digestion units/mg solid) and 2% bovine serum albumin (BSA). Initially, samples were incubated in a 37°C water bath, with inversion of vials at 0, 5, 10, and 15 min. Samples were then transferred to an incubator (Lab-Line Instruments Inc., Melrose Park, IL) and further digested with shaking for 45 min at 37°C and 230 rpm. The digested material was then sequentially filtered through 1000, 500, and 53 um nylon mesh, after which the filtrates were centrifuged twice for 10 min at 800 x g. Resulting cell pellets were resuspended in growth medium comprised of base media (DMEM, 1% antibiotic- antimycotic, 0.1% gentamicin, 33 pM biotin, 17 pM pantothenate, and 100 pM ascorbate) supplemented with 10% fetal bovine serum (FBS) for proliferation, or resuspended in freezing media (base medium supplemented with 20% FBS and 10% dimethyl sulfoxide). Cells suspended in freezing medium were aliquoted into 1.8 mL cryogenic vials and placed in a styrofoam rack overnight at -80°C, with subsequent storage in liquid nitrogen. 85 General Procedures Cell culture. One gram tissue-equivalent of cells from each depot was suspended in 4 mL growth medium and seeded equally into 2 wells of a 6-well tissue culture plate (35 mm diameter; Corning Inc., Corning, NY). Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% C02. After reaching approximately 70% confluence, cells were washed twice with PBS and removed from the wells by trypsinization (0.5 g/L trypsin and 0.02 g/L L' flikbl‘ rh- I ethylenediaminetetraacetic acid (EDTA) in PBS (pH 7.2)). Cells were then resuspended in growth medium and seeded at 1800 cells/cm2 into 10-cm diameter culture plates. Cells were grown to approximately 70% confluence in 10-cm plates. At this time, cells were washed twice with PBS, trypsinized, resuspended in freezing medium, and frozen for future experiments. Cells frozen at this stage were considered to be in the second passage, and all experiments were performed on tertiary cultures. Differentiation media in all experiments consisted of base medium including 280 nM bovine insulin, 20 mM glucose, and 20 pL/mL SL with additions of TRO, and(or) a glucocorticoid analog, dexamethasone (DEX) as indicated. Differentiation media were replaced with fresh media every 2 d. Media additions were present for the entire differentiation period except DEX, which was supplemented only for the initial 48 h. Troglitazone was solubilized in dimethyl sulfoxide at a stock concentration of 22.7 mM before addition to media. Addition of dimethyl sulfoxide to differentiation media alone did not significantly affect 86 differentiation (data not shown). All plates were washed twice with PBS before media replacement on d-2 of the differentiation period. Gcherol-3-phosphate dehydrogenase activity assay. Cell differentiation was quantified biochemically by measuring glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity using a modification of the method described by Adams et al. (1997). Cells were washed twice with ice-cold PBS. Contents of two wells F per treatment were combined and harvested in a total volume of 200 pL of ice- cold Tris (pH 7.4) containing 1 mM EDTA and 50 pM dithiothreitol (extraction buffer), then transferred into prechilled 1.5 mL microcentrifuge tubes. Each E sample was then disrupted by sonification three times at 40 W (3 s bursts with 1 min cooling on ice between bursts) using a Sonifier—Cell Disrupter 350 (Branson Sonic Power Co., Danbury, CT). Samples were then centrifuged at 16,000 x g for 15 min at 2°C. Solutions containing 50 (IL of the resulting supernatant and 150 uL of assay buffer were assayed in duplicate within 30 min for GPDH activity. The final concentration of the supernatant and assay buffer solution was: 100 mM triethanolamine—HCL (pH 7.4), 2.5 mM EDTA, 50 (M dithiothreitol, 0.8 mM dihydroxyacetone phosphate, and 0.317 mM NADH. Each reaction was initiated by the addition of assay buffer to the supernatant in one well of a 96-well plate (lmmulon 13; Fisher Scientific, Hampton, NH). Fifty uL of extraction buffer served as the reagent blank. To obtain the reaction rate, the AA340 was recorded at 15 s intervals for 6 min at 30°C using a spectrophotometer (Versamax Tunable Microplate Reader, Molecular Devices, Sunnyvale, CA). All reactions measured were linear for at least 180 s and the Vmax used to calculate GPDH activity was 87 obtained from the linear range. Enzyme activity was expressed as nanomoles of NADH oxidized - min‘1 - mg protein". Protein determination. Aliquots (20 (IL) of supernatant were assayed in duplicate to determine protein concentration using the bicinchoninic acid assay (BCA Protein Assay Reagent Kit, Pierce, Rockford, IL). Samples were mixed with 200 pL of assay reagent, incubated for 30 min at 37°C, and absorbance at 562 nm determined. Protein concentration was determined using BSA as a standard. Morphological differentiation assay. Cell differentiation was morphologically assessed by observation of cells containing lipid droplets stained with oil red O (ORO). The ORO solution was prepared using the protocol described by Humason (1972). Briefly, a solution of 0.35 g ORO and 100 mL isopropanol was incubated at room temperature overnight without stirring, then filtered through Whatman #40 filter paper. Seventy-five mL of distilled water was added to the solution, which was incubated overnight at 4°C, filtered twice using Whatman #40 filter paper, and stored at room temperature. A solution of nuclear stain was made by dissolving 1 g of giemsa into 66 mL glycerol and 66 mL of methanol. This solution was filtered using Whatman #1 filter paper. Wells were initially aspirated of media and washed twice with PBS. Cells were then fixed by addition of 3.7% formaldehyde (diluted from a stock 37% formaldehyde solution containing 10% methanol; Mallinckrodt Baker lnc., Phillipsburg, NJ) in PBS for 4 min. After fixation, cells were washed twice with PBS and incubated at room temperature with ORO solution for 1 h. Residual ORO solution was aspirated 88 and the cells were washed twice with distilled water (15 min incubation/wash). Cell nuclei were stained by adding 1 mL giemsa solution to each well for 1 h, after which the cells were washed twice in distilled water. Wells were aspirated of water and stored at 4°C. Cells were visualized within 24 h of staining. Digital photographs were taken using a Nikon CoolPix 5000 digital camera (Nikon |nc., Melville, NY) fitted to a Zeiss inverted microscope (Carl Zeiss lnc., Thornwood, NY). Experiment 1 This experiment was designed to evaluate the effects of increasing concentrations of TRO on the adipogenic differentiation of S-V cells isolated from bovine i.m. and s.c. adipose depots. Cells were seeded in six-well tissue culture plates at a density of 5200 cells/cmz and incubated in growth medium. After 24 h, wells were washed twice with PBS and fresh growth medium was added. After reaching confluence (3 d), cells were washed twice with PBS and differentiation media were added. Differentiation treatments included 0, 5, 10, 20, 40, or 60 pM TRO. Protein concentrations and GPDH activities were measured after a 12-d differentiation period. Experiment 2 The objective of this experiment was to investigate the effects of DEX and TRO on enzymatic and morphological adipogenic differentiation of bovine i.m. and s.c. S-V cells. Biochemical differentiation was determined on S-V cells grown as described for Exp. 1, while morphological analysis was conducted on 89 individual colonies, each presumably derived from a single cell. Differentiation media in this experiment included additions of 40 uM TRO and(or) 0.25 uM DEX. Cells used for morphological analysis were seeded at clonal densities (400 cellsl10-cm plate) and incubated in growth media undisturbed for 8 (I, when colonies reached appropriate sizes (approximately 250 to 500 cells) for enumeration. Plates were washed twice with PBS and differentiation treatments were applied. After a 10-d differentiation period, cells were fixed and stained with ORO and giemsa for morphological analysis. The percentage of colonies with differentiated cells were determined by microscopic analysis and the proportion of differentiated cells within colonies were determined by analysis of photomicrographs. A differentiated cell was defined as a cell having one or more lipid droplets 2 10 pm in diameter. A colony with at least one differentiated cell was defined as adipogenic. Any colony considered to be derived from more than one cell was omitted from analysis. Adipogenic colonies were counted and compared to the total number of colonies to determine the percentage of adipogenic colonies on each of 24 plates. Photomicrographs of 10 randomly selected adipogenic colonies on each plate were taken to determine the proportion of differentiated cells within adipogenic colonies. Three to seven photomicrographs of each colony were taken across the diameter of the colony to capture a representative cross-section of cells at 200X magnification. All enumerations were conducted by an evaluator blinded to depot and treatment. 90 Statistical analysis Data were analyzed using the Mixed Model procedure of SAS (SAS, Cary, NC). In Exp. 1, pooled cells from two wells of a six-well plate were considered the experimental unit. To satisfy the conditions of normality and homogeneity of variance, GPDH data from both experiments were loge transformed. Means were calculated using the fixed effects of TRO, depot, and TRO x depot, with steer, steer x depot, and steer x TRO included as random variables. In Exp. 2, GPDH data were generated using two wells of a six-Well plate as the experimental unit. Data were loge transformed as in Exp. 1. Means were calculated using the fixed effects of TRO, DEX, depot, and their two- and three- way interactions, with steer, steer x depot, steer x DEX, and steer x TRO included as random variables. For morphological analysis, plate served as the experimental unit in the determination of the percentage of adipogenic colonies, as well as for data collected on differentiated cells within colony. The logit transformation, as described by Ramsey and Schafer (2002), of each value was then calculated to satisfy conditions of normality and homogeneity of variance. Least squares means for the percentage of differentiated colonies, and differentiated cells within colony, were calculated using the fixed effects of depot, DEX, TRO, and their two- and three-way interactions. When analyzing colony data, steer, steer x depot, steer x DEX, and steer x TRO were included as random variables. Steer, and plate within steer, DEX, TRO, and depot were included as random variables when differentiated cells within colony were 91 analyzed. No interactions were determined to be significant in the experiments described herein (P > 0.27). Main effects were considered significant at P < 0.05. When main effects were determined to be significant, differences between means were investigated using Tukey’s multiple comparison test. Results and Discussion Experiment 1 To our knowledge, direct comparisons of differentiation between bovine i.m. and s.c. S-V cells in response to a PPARy agonist have not been reported. Therefore, we compared the effects of 5 to 60 uM TRO on the differentiation of i.m. and s.c. S-V cells. Addition of 40 or 60 (M TRO increased GPDH activity 2.9- and 3.4-fold, respectively, when compared to control (P < 0.02) (Figure 3-1). However, no significant depot effects were evident (P = 0.32). Protein concentrations were similar between S-V cells from each depot (P = 0.35), and TRO had no effect on protein concentration of S-V cells (P = 0.17) (data not shown). Peroxisome proliferator-activated receptor-y has been described as a critical transcriptional regulator in preadipocyte differentiation (Spiegelman, 1998) and is primarily expressed in adipose tissues of most species (Chawla et al., 1994; Braissant et al., 1996) including cattle (Sundvold et al., 1997). Peroxisome proliferator-activated receptor-y is activated by endogenous (Kliewer et al., 1997; Krey et al., 1997) and synthetic (Lehmann et al., 1995; Spiegelman, 1998) ligands. Once ligand-bound, PPARy dimerizes with a retinoid X receptor. This 92 dimer is then capable of binding to PPAR response elements in the promoters of adipogenic genes, which stimulate differentiation (Kliewer et al., 1992). Undifferentiated bovine (Torii et al., 1998) and porcine (Kim et al., 2000) S-V cells express PPARy protein. Therefore, differentiation in S-V cells may be stimulated through agonist binding to PPARy (Ding et al., 2003). Thiazolidinediones, such as TRO, are known to be high affinity PPARy ligands (Lehmann et al., 1995) and have been used to enhance differentiation of bovine (Torii et al., 1998), ovine (Soret et al., 1999), and porcine (Tchoukalova et al., 2000) S-V cells. Depot differences in response to PPARy ligands have been reported between cm. and s.c. S-V cells isolated from 6- to 8-mo-old wether lambs (Soret et al., 1999) and 4- to 5-yr-old Holstein cows (Wu et al., 2000). Both studies found that s.c. S-V cells differentiated to a greater extent than o.m. cells in media containing lipid supplements. However, when differentiated in media containing a selective PPARy ligand, a greater relative increase in differentiation was observed in o.m. compared to s.c. cells. The authors suggested that o.m. cells may have a lower capacity to produce endogenous PPARy ligands than s.c. cells. However, thiazolidinedione addition to human S-V cells stimulated greater differentiation in s.c. when compared to 0m. cells (Adams et al., 1997; Sewter et al., 2002; Tchkonia et al., 2002). Adams et al. (1997) found no differences in PPARy protein expression between depots. Alternatively, Sewter et al. (2002) and Tchkonia et al. (2002) showed that greater PPARy mRNA and protein expression in s.c. cells may account for the enhanced differentiation observed. Both i.m. and s.c. S-V cells responded similarly to the 93 concentrations of TRO used in our experiment, which are within the range of those used in a previous bovine study (T orii et al., 1998). Although we did not directly measure PPARy protein expression in this experiment, it does not appear that PPARy signalling in the presence of a PPARy ligand differed between i.m. and s.c. depots. Experiment 2 Glucocorticoids have been shown to increase differentiation in porcine s.c. S-V cells while having no effect on p.r. cells (Ramsay et al., 1989). In addition, while bovine s.c. S-V cells showed a greater ability to differentiate than o.m. cells in media containing a lipid supplement, these depot differences were reduced when cells were differentiated in media containing a PPARy agonist (Wu et al., 2000). Experiments using purified bovine preadipocytes are lacking, as, to our knowledge, a marker capable of identifying bovine preadipocytes prior to differentiation has not been reported. Therefore, we tested the adipogenic effects of DEX, TRO, and their combination on the differentiation of bovine i.m. and s.c. S-V cells in mass culture and using clonal analysis. No significant depot differences in the enzymatic differentiation of i.m. and s.c. cells were observed in response to 40 uM TRO and 0.25 (M DEX (P = 0.41). Therefore, the main effects of DEX and TRO were investigated (Figure 3-2). Dexamethasone initiated a 1.8-fold increase in GPDH activity over cells not treated with DEX (P = 0.01), while a 2.5-fold increase in GPDH activity was observed with TRO addition (P = 0.02) compared to cells not treated with TRO (Figure 3-2). As no interaction between DEX and TRO was found (P = 0.53), the 94 combination of both compounds was considered additive. Protein concentrations were found to be similar between depots regardless of treatment (P = 0.46). Clonally-derived colonies were analyzed to determine treatment effects on the percentage of adipogenic colonies, and the proportion of differentiated cells within adipogenic colonies. The percentage of adipogenic i.m. and s.c. colonies present in each S-V cell population were found to be similar (P = 0.10) (Table 3- 1), therefore, the main effects of TRO and DEX were investigated. Addition of TRO to differentiation media enhanced the percentage of adipogenic colonies by 2.3-fold (P = 0.01), while DEX induced a 1.2-fold increase over the control (P = 0.02) (Table 3-1). Due to the lack of an apparent interaction (P = 0.65), the effects of DEX and TRO were considered additive. To further investigate preadipocytes isolated from i.m. and s.c. depots, the effects of TRO and DEX on the proportion of differentiated cells within adipogenic colonies were determined (Figure 3-3). Regardless of depot, addition of TRO increased the proportion of differentiated cells by 10-fold when compared to non- treated colonies (P < 0.001) (Table 3-1), suggesting the capacity for enhanced differentiation in response to an exogenous PPARy agonist exists in both i.m. and s.c. depots. Addition of DEX tended (P = 0.10) to increase the percentage of differentiated cells within colony in both depots when compared to those of non- treated colonies (Table 3-1). Although the percentage of differentiated cells within colony varied in both depots, a 6.4-fold greater percentage of s.c. cells differentiated when compared to i.m. cells (P < 0.001) (Table 3-1). Thus, despite 95 similar responses to TRO or DEX, s.c. preadipocytes generally appear to be more adipogenic than i.m. Previous studies have tested the adipogenic effects of TRO and DEX using S-V cells isolated from adipose tissues. However, comparisons of these compounds on S-V cells derived from different bovine adipose tissue depots are lacking. Although their specific effects are not well understood, glucocorticoids have been used to enhance differentiation in i.m. (Aso et al., 1995; Sato et al., 1996), s.c., and cm. (Wu et al., 2000) S-V cells isolated from Japanese Black (Aso et al., 1995; Sato et al., 1996; Wu et al., 2000) and Holstein (Wu et al., 2000) cattle. They have also been shown to increase adipose conversion in ovine (Soret et al., 1999) and porcine (Ramsay et al., 1989; Tchoukalova et al., 2000) S-V cells. Glucocorticoids may influence adipocyte differentiation through the upregulation of CCAAT/enhancer binding protein expression in 3T3-L1 preadipocytes (Yeh et al., 1995) and fibroblasts (Wu et al., 1996). In addition, glucocorticoids have been found to increase arachidonic acid metabolism, with subsequent prostaglandin production, in Ob1771 preadipocytes (Gaillard et al., 1991). These pathways may lead to the upregulation of PPARy protein, or increased ligand binding to PPARy, respectively (Wu et al., 1996; Kliewer et al., 1997; Krey et al., 1997), which may result in the transcription of adipogenic genes (Tontonoz et al., 1994; Schoonjans et al., 1996). Soret et al. (1999) found that DEX stimulated a 3.6- and 4.3-fold increase in GPDH activity compared to control in s.c. and cm. ovine S-V cells, respectively. In addition, Ramsay et al. (1989) found that while porcine s.c. S-V cells increased GPDH activity in 96 response to glucocorticoid administration, p.r. cells did not respond. Our results demonstrate that the addition of DEX to differentiation media increased GPDH activity in bovine i.m. and s.c. preadipocytes, possibly through one or more of the aforementioned pathways. Given that DEX may increase the expression of PPARy, and that both DEX and TRO appear capable of activating PPARy, the potential for an interaction between these compounds exists. However, to our knowledge, studies investigating possible interactions between the two compounds, and(or) their potential depot specific effects on bovine S-V cell differentiation, have not been conducted. Tchoukalova et al. (2000) found that DEX and a thiazolidinedione enhanced GPDH activity in porcine S-V cells when added individually to differentiation media. However the addition of both compounds was not additive. Using S-V cells isolated from 6- to 8-mo-old wether lambs, Soret et al. (1999) found that addition of DEX in combination with a PPARy agonist had additive effects on GPDH activity of both cm. and s.c. cells, with no depot differences reported. While no depot specific responses in GPDH activity were found in our experiment, we showed that TRO and DEX individually stimulated GPDH activity in bovine S-V cells. As well, due to the lack of a treatment interaction, the combination of both compounds was assumed to be additive. The differentiation characteristics of clonally-derived adipogenic colonies were investigated using morphological analysis. We found no significant differences in the percentage of adipogenic colonies between i.m. and s.c. 97 depots. This suggests that our isolation procedures yielded a similar proportion of adipogenic cells from both adipose tissues. In addition, both DEX and TRO were capable of increasing the proportion of colonies classified as adipogenic within i.m. and s.c. S-V isolates in this study. The relative percentage of preadipocytes within adipogenic colonies from both i.m. and s.c. depots increased in response to addition of TRO. This suggests that TRO was able to trigger preadipocyte differentiation through PPARy activation. Although DEX also tended to increase the percentage of differentiated cells within adipogenic colonies, it had a reduced effect when compared to TRO. Djian et al. (1985) reported that, although clonally-derived colonies of rat preadipocytes showed varying capacities for differentiation within depot, a greater proportion of cells differentiated in colonies derived from p.r. when compared to cells of epididymal origin. Tchkonia et al. (2002) found that the percentage of differentiated cells was greatest in human s.c., intermediate in mesenteric, and lowest in cm. adipogenic colonies arising from single cells. These results were partially attributed to greater PPARy expression found in s.c. cells (Tchkonia et-al., 2002). lmportantly in this experiment, we found that clonally-derived s.c. cells exhibited a greater inherent ability to differentiate than those from i.m. when exposed to our culture conditions. The reason(s) for the difference in the adipogenic capacity of s.c. and i.m. cells in this experiment are unclear. The potential for differential expression of PPARy between depots exists, although PPARy expression was not tested in this study. Preliminary western immunoblot analysis results from our laboratory showed no apparent 98 difference in PPARy protein expression between i.m. and s.c. S-V cells (unpublished data). Therefore, depot specific responses to medium components other than DEX or TRO, such as SL and(or) insulin may be involved. As well, differential expression of other transcription factors not studied in these experiments may exist, and thus, contribute to the observed differences in differentiation. For example, Tchkonia et al. (2002) determined the expression of CCAAT/enhancer binding protein-a, another key adipogenic transcription factor, to be greater in human s.c. compared to cm. S-V cells. Certainly, preadipocyte differentiation is a complex process that involves the coordination of many factors and events that lead to cellular lipid accumulation. We conclude that bovine S-V cells isolated from i.m. and s.c. depots are capable of enhanced differentiation in response to DEX and TRO. Although no depot specific responses due to treatment were found, we are the first to report that bovine s.c. preadipocytes differentiate more extensively than i.m. preadipocytes when cultured in similar environments. This suggests that inherent differences in the capacity for adipose differentiation between bovine i.m. and s.c. preadipocytes exist. Studies further characterizing these depot differences, along with identifying factors that differentially influence differentiation of preadipocytes derived from i.m. and s.c. adipose tissues, will aid in improving marbling and(or) reducing excess external fat on beef carcasses. 99 Implications Differentiation of bovine intramuscular and subcutaneous stromal-vascular cells increase in response to dexamethasone and(or) troglitazone, possibly through involvement with the adipogenic transcription factor, peroxisome proliferator-activated receptorey. Interestingly, the capacity for differentiation appears to be greater in adipogenic subcutaneous than intramuscular stromal- vascular cells. While the mechanisms responsible for these observed differences remain to be elucidated, our study supports previous work noting the importance of peroxisome proliferator-activated receptor-y in bovine adipose differentiation. Thus, the potential to manipulate bovine adipose tissue development through factors that influence peroxisome proliferator-activated receptor-y appear to exist. Determining factors that favor bovine intramuscular adipose development, when compared to subcutaneous fat accretion, may lead to more highly marbled beef with less trimmable subcutaneous fat. 100 Table and Figures I b 75 3- b 3 2 Q - ab a g b 75 2- ab 3 .E 3 E ‘ a C E a 1. 0 0°) 0 d _l o T I I I I I I I I j I I I 0 1o 20 30 4o 50 so Troglitazone, uM Figure 3-1. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH) activity. Values are least squares means and SEM of subcutaneous (s.c.), and intramuscular (i.m.), stromal-vascular cells from three steers. Least squares means without a common superscript letter differ (P < 0.05). 101 Log. GPDH nmoI-min"-mg protein" N 1. 0' I NODEX 5. 4. 3'1 a Log. GPDH nmoI-min"-mg protein" N No TRO TRO Figure 3-2. Effect of 0.25 pM dexamethasone (DEX) (upper panel), and 40 uM troglitazone (T RO) (bottom panel) on glycerol-3-phosphate dehydrogenase (GPDH) activity. Values are least squares means and SEM of subcutaneous, and intramuscular, stromal-vascular cells from three steers. Least squares means without a common superscript letter differ (P < 0.05). 102 Figure 3-3. Photomicrographs of subcutaneous clonally-derived adipogenic colonies exposed for 10-d to serum-free differentiation medium (A), or serum-free differentiation medium supplemented with 0.25 (M dexamethasone and 40 pM troglitazone (B). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. 103 104 Table 3-1. Effect of dexamethasone or troglitazone on the percentage estimates and 95% confidence intervals (CL), of clonally-derived, stromal- vascular cells isolated from intramuscular and subcutaneous adipose tissues of three steers Lower Upper GI. C.l. P-value Adipogenic, %a Colonies” No Dexamethasone 41.6 18.0 69.7 Dexamethasone 51 .2 24.5 77.2 0.02 No Troglitazone 28.1 10.6 56.3 Troglitazone 65.6 36.8 86.2 0.001 Intramuscular 37.4 14.2 68.4 Subcutaneous 55.5 25.8 81.7 0.10 Differentiated, %3 Cells within adipogenic colonies° No Dexamethasone 5.1 1.7 14.1 Dexamethasone 10.4 3.7 26.3 0.10 No Troglitazone 2.2 0.7 6.3 Troglitazone 21.9 8.3 46.4 <0.001 Intramuscular 2.8 0.9 8.1 Subcutaneous 17.9 6.7 40.1 <0.001 aAdipogenic colonies were defined as colonies having at least one differentiated cell. 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Thiazolidinedione induces the adipose differentiation of fibroblast-like cells resident within bovine skeletal muscle. Cell Biol. Int. 22:421-427. Wu, P., K. Sato, F. Suzuta, Y. Hikasa, and K. Kagota. 2000. Effects of lipid- related factors on adipocyte differentiation of bovine stromal-vascular cells in primary culture. J. Vet. Med. Sci. 62:933-939. Wu, Z., N. L. R. Bucher, and S. F. Farmer. 1996. Induction of peroxisome proliferator-activated receptor y during the conversion of 3T3 fibroblasts into adipocytes is mediated by CIEBPB, CIEBPS, and glucocorticoids. Mol. Cell. Biol. 16:4128-4136. Yeh, W. C., 2. Cao, M. Classon, and S. L. McKnight. 1995. Cascade regulation of terminal adipocyte differentiation by three members of the CIEBP family of leucine zipper proteins. Genes Dev. 92168-181. 108 CHAPTER IV INTERPRETIVE SUMMARY Adipose tissue development in beef cattle provides a constant challenge for the beef industry. Marbling, or intramuscular (i.m.) fat, is required to enhance palatability characteristics of beef, and is the primary indicator of carcass quality, while excess subcutaneous (s.c.) fat is generally considered as waste. Although fat accretion in general is associated with high caloric intake, indicative of the finishing phase of production in the US, rates of adipose tissue accretion are not well correlated between depots. Generally, a greater rate of accretion occurs within subcutaneous when compared to intramuscular adipose depots in cattle fed high-energy diets. Depending on feeding strategy, this may result in carcasses that possess excessive subcutaneous fat and(or) insufficient marbling, both of which contribute to reduced profitability for the industry. Numerous in vivo studies have attempted to increase intramuscular fat accretion without simultaneous increases in subcutaneous adipose tissue, but most have proven inconsistent or ineffective in stimulating marbling above that of current production practices. The differences in rate of development between intramuscular and subcutaneous adipose tissues suggest that these two depots may inherently differ. In vitro experiments provide a method with which to study the cellular features of adipose tissues, and may prove useful in determining specific factors that contribute to regional variations in adipose tissue development. Adipose 109 tissue accretion is partially controlled by adipocyte precursor cell (preadipocyte) proliferation, and differentiation into mature adipocytes. Few experiments have utilized cell culture techniques to document the differentiation characteristics of bovine preadipocytes. Therefore, the objectives of the following studies were to: 1) refine the methodology for bovine preadipocyte isolation, culture, and cloning; 2) optimize culture conditions that induce preadipocyte differentiation; and 3) determine the differential responses of intramuscular and subcutaneous preadipocytes and stromal-vascular cells to a PPARy agonist and a glucocorticoid analog. These experiments were conducted to explore the factors that influence bovine preadipocyte differentiation. Initially, S-V cells were isolated using a modified procedure from those previously used for the isolation of rat preadipocytes. The isolation of S-V cells from s.c. tissue proved a relatively trouble-free process. The greater quantity of readily available tissue enabled more total grams of s.c. tissue to be isolated than i.m. Excision of i.m. adipose tissue from muscle was more labor intensive and required great care for successful isolation. In addition, fewer total grams of i.m. adipose tissue were able to be recovered than s.c. in the time permitted to maintain cell viability. Therefore, to improve cell yields in future isolations, efforts should be concentrated towards extraction of i.m. cells. This may be accomplished by assigning a minimum of three people to extract i.m. adipose tissue from muscle, while two would suffice for s.c. cell isolation. In addition, freezing cells immediately after isolation may reduce cell viability and survivability, as, upon thawing and plating, a lower percentage of these cells 110 appeared capable of plate attachment and proliferation compared to those seeded directly after isolation. Therefore, an attempt should be made to seed and propagate freshly isolated cells prior to freezing. This may allow for greater cell survival and increased overall cell yield. One of two cloning procedures was used to isolate preadipocytes. In one procedure, cells were counted, diluted, and plated at clonal densities in 10-cm plates. The second cloning procedure involved the dilution and plating of cells at a concentration of one cell per well in 96-well plates. Both cloning methods proved effective in isolating adipogenic clones. However, a direct comparison between cloning procedures was not conducted. Preadipocyte isolation using 96-well plates required greater effort and time spent replacing media, in addition to visually analyzing wells to determine which colonies were derived from single cells. Alternatively, the cloning ring procedure required more skill in placing the rings over isolated colonies, as well as ensuring that a proper seal between the ring and culture plate was made in order to extract cells. Therefore, researcher preference and cloning efficiency (Appendix A) will likely determine which method is used in future isolations. A greater percentage of clones isolated from the s.c. depot were identified as preadipocytes compared to the i.m. depot. This suggests that a greater proportion of non-adipose cell types may exist in i.m. S-V isolates. However, the apparent “non-adipogenic” clones may also be preadipocytes that were unresponsive to our differentiation conditions. Differentiation media used to identify a majority of the preadipocytes included insulin, dexamethasone, 111 octanoate, and acetate. Reevaluation of clones using optimized differentiation media including serum lipids, troglitazone, and dexamethasone may stimulate differentiation in a portion of clones previously determined to be “non- adipogenic”. Lipids resident in bovine serum may act as physiological activators of preadipocyte differentiation in vivo. Therefore, a bovine-derived, serum lipid (SL) supplement was initially titrated to test its effects on the differentiation of an s.c. clone. The admixture of fatty acids (FA), cholesterol, and phospholipids present in SL stimulated morphological (visual observation of cells containing lipid droplets) and biochemical, glycerol-3-phosphate dehydrogenase (GPDH), preadipocyte differentiation. Linoleic and arachidonic acid are two polyunsaturated FA (PUFA) included in the SL supplement. Metabolites of both FA have been implicated in the upregulation and(or) activation of peroxisome proliferator-activated receptor-y (PPARy), which is an important adipogenic transcription factor. Although the specific activator($) of differentiation in SL were not determined, it is possible that one, or a combination of FA present in SL stimulated differentiation through the activation of PPARy. Studies using porcine preadipocytes have suggested that individual PUFA are stimulatory towards differentiation by providing PPARy ligands. Therefore, future studies should investigate the effects of individual PUFA on bovine preadipocyte differentiation. We next examined the adipogenic effects of a specific PPARy agonist on the differentiation of a s.c. clone when added to differentiation medium containing insulin and SL. Increasing concentrations of troglitazone (T RO) enhanced 112 preadipocyte differentiation. The potential mechanism of action for the enhanced differentiation was likely increased ligand binding and activation of PPARy. As TRO is a highly selective PPARy ligand, these results may indirectly indicate that PPARy is expressed in the undifferentiated cells of the s.c. clone. In addition, if TRO and the components of SL both stimulate differentiation through activation of PPARy, it would appear that TRO is a more potent activator of PPARy. However, SL may provide a dual purpose in supporting adipogenesis, as the FA present in the supplement may serve as substrate for FA and(or) triacylglycerol synthesis. Studies measuring the expression pattern of PPARy during bovine preadipocyte differentiation would further characterize its involvement in the differentiation process. In further attempts to optimize differentiation, we tested the effects of FA source and(or) removal of adipogenic compounds from serum-free medium using the following treatments: 1) insulin, octanoate, acetate, and dexamethasone (DEX); 2) insulin, SL, and DEX; 3) insulin, SL, DEX, and TRO; 4) insulin, SL, and TRO; and 5) SL, DEX, and TRO. Treatment 1 stimulated lipid accumulation in a small portion of cells, but did not induce measurable GPDH activity of the s.c. clone. No differentiated cells were observed when a supraphysiological concentration of insulin was included as the only differentiation media additive (Figure 2-1). This suggests that octanoate, acetate, and DEX, in combination with insulin, were capable of inducing morphological differentiation compared to their omission. However, octanoate, acetate, and DEX were not tested individually. Thus, in this study, it cannot be determined which agent(s) were 113 responsible for the appearance of lipid containing cells. One cannot discount the potential effects of octanoate and acetate on preadipocyte differentiation. However, preliminary studies, not included in this document, found that addition of DEX to medium containing insulin was capable of stimulating differentiation in a small portion of cells. An increase in differentiation was observed when SL were substituted for octanoate and acetate (Trt 2 vs. Trt 1). The enhanced adipogenic effect of SL did not appear to be a result of increased FA concentration, as the concentration of FA in SL (0.19 mglmL) used in this study was within the concentration range of octanoate (0.14 mglmL) and acetate (0.60 mglmL) provided to the cells. Therefore, if the FA were responsible for the differentiation observed in both treatments, the FA in SL were more potent activators of PPARy and(or) provided a more readily available substrate for FAltriacnglycerol synthesis than the combination of octanoate and acetate. Again, this leads to speculation that long chain PUFA, such as linoleic and arachidonic acid or other SL components may be responsible for the increased differentiation observed. The combination of insulin, SL, DEX, and TRO (Trt 3) caused the most extensive differentiation of any treatment. Removal of DEX from this media tended to reduce differentiation. The specific effects of DEX on differentiation were not studied in these experiments. However, glucocorticoids may be involved in the upregulation of CCAAT/enhancer binding proteins, as well as, increase the metabolism of arachidonic acid, which stimulates prostaglandin production. Both of these potential modes of action may lead to increased 114 PPARy protein expression and(or) activation, respectively, thus enhancing differentiation. However, expression of PPARy protein has been reported in undifferentiated bovine and porcine S-V cells. As well, the expression of PPARy protein was not increased by DEX during porcine S-V cell differentiation. These findings, along with preliminary results from our lab, lead to speculation that DEX may not be involved in the upregulation of PPARy expression in the s.c. clone (unpublished observations). Prostacyclin is an arachidonic acid metabolite thought to be Involved in activating PPARy. Since prostacyclin production may be stimulated by glucocorticoids through increased arachidonic acid metabolism, this may be another way in which glucocorticoids enhance differentiation. Thus, future studies may attempt to measure the effects of glucocorticoids on prostacyclin production. To further determine if increased prostacyclin enhances differentiation of bovine cells, studies using agents that block its production may also be employed. Alternatively, glucocorticoids have been shown to increase the abundance of insulin receptor mRNA in cell types such as lymphocytes. If glucocorticoids increase insulin receptor abundance in preadipocytes, differentiation may be enhanced via this pathway. Therefore, future studies that attempt to define the adipogenic effects of glucocorticoids on the differentiation of bovine preadipocytes are of interest. Troglitazone appeared to play a role in the differentiation of the s.c. clone, as its removal from media (Trt 2 vs. Trt 3) reduced differentiation. Therefore, the reduced activity observed upon removal of TRO further indicates that PPARy ligands are important in the differentiation of the s.c. clone. 115 The reduced GPDH activity observed upon insulin removal from media (Trt 5 vs. Trt 3) demonstrate a role for insulin and(or) insulin-like growth factor (IGF)-1 in preadipocyte differentiation. Although not fully understood, insulin has been found to enhance differentiation by various means. For instance, insulin is known to stimulate the translocation of GLUT4 and fatty acid transport proteins from intracellular pools to the cell membrane, thus enhancing cellular glucose and FA uptake. Insulin has also been found to stimulate the cellular expression of adipogenic genes such as GPDH and(or) lipoprotein lipase, in addition to increasing glucocorticoid receptor number in S-V cells. In our study, the removal of insulin appeared to reduce lipid accumulation within differentiated cells. The observed increase in lipid accumulation observed in cells treated with insulin suggests that cellular uptake of FA and(or) glucose may have been improved by insulin addition. As it is not known if the SL supplement used in our experiments contains insulin and(or) IGF 1, we cannot state that the s.c. clone was capable of differentiation in the absence of insulin. Future experiments testing various concentrations of insulin would aid in determining the effect of insulin and(or) IGF-1 on the differentiation of our cells. The removal of SL from Trt 3 caused substantial cell detachment from the plates 2 d after exposure to differentiation media. Although not defined, SL appear to contain components that aid in maintaining cell viability and(or) attachment during differentiation in the presence of DEX and(or) TRO. Preliminary studies found differentiation medium without SL maintianed cell 116 viability in the presence of DEX. However, TRO addition to differentiation media in the abscence of SL has not been tested. Since thiazolidinediones have been found to have depot specific effects on human and ovine S-V cell differentiation, we tested the effects of TRO on the differentiation of i.m. and s.c. preadipocytes. Although TRO addition increased differentiation in all clones, variation in adipogenic capacity within and between depots existed, however, no depot differences between i.m. and s.c. clones were recorded. Previous research using human and rat preadipocytes also found variation in differentiation among clonally-derived colonies originating from the same depot. As we were only able to successfully propagate and differentiate two viable i.m. clones, future attempts should be made to identify more i.m. clones using optimized conditions, or clone more i.m. preadipocytes to increase sample size. This may allow for possible differences between depots to be tested with greater accuracy. Differences in the extent of differentiation between individual clones may be regulated via variations in PPARy protein expression and(or) the ability to produce endogenous PPARy ligands. Therefore, future experiments may be conducted to measure the expression of PPARy of each clone, which may aid in determining the differences in adipogenic capacity among clones. Overall, these studies aid in defining appropriate procedures for the isolation and cloning of i.m. and s.c. bovine preadipocytes, in addition to determining factors that enhance their differentiation. Although no differences between depots were observed, the results of our experiments point to possible 117 factors that could be used for the manipulation of cellular adipose tissue development in beef cattle. Based on results determined from the previous experiments, a second set of studies was conducted to compare the response of s.c. and i.m. S-V cells isolated from three Angus steers to differentiation media containing TRO and(or) DEX. Initially, the effects of TRO were tested on the differentiation of i.m. and s.c. S-V cells in media containing SL and insulin. Although no significant differences between depots were found, the capacity for enhanced differentiation of both i.m. and s.c. S-V cells in response to a PPARy agonist were apparent in this experiment. Due to the variability found in response to treatment in this study, future trials using a greater number of animals may be of benefit in further characterizing treatment and(or) depot differences. Since both DEX and TRO have been found to have differential depot effects on differentiation in S-V cells of other species, we tested their effects both individually, and in combination on the differentiation of i.m. and s.c. S-V cells. Both DEX and TRO enhanced differentiation when compared to non-treated cells while no significant depot effects or interactions were found. Thus, their effects were assumed to be additive. This suggests that these compounds may exert their effects via separate pathways. If DEX did influence differentiation through increasing PPARy in our studies, monitoring PPARy mRNA or protein expression throughout the course of differentiation in DEX treated and non-treated cells may aid in clarifying this. 118 Individual colonies derived from S-V cells were next analyzed to determine treatment effects on the percentage of adipogenic colonies, and the proportion of differentiated cells within adipogenic colonies. When added individually, DEX and TRO were both found to increase the percentage of differentiated colonies relative to those not treated with DEX or TRO, respectively. Although the percentage of adipogenic colonies tended to be greater in s.c. than i.m. isolates, they did not significantly differ. This may indicate that the isolation procedures used were capable of isolating a similar percentage of adipogenic cells from both depots. To further study the inherent characteristics of adipogenic cells between i.m. and s.c. depots, we next determined the percentage of differentiated cells present within adipogenic colonies using a sub-sample of the adipogenic colonies. Regardless of depot, TRO addition increased the proportion of differentiated cells 10-fold over those not treated with TRO, while the percentage of differentiated cells was not significantly increased by DEX. The increase in the percentage of differentiated cells in response to TRO suggests that cells from both i.m. and s.c. depots were at a stage of development where PPARy was already expressed. Alternatively, it is possible that DEX recruited cells to differentiate that were not previously expressing transcription factors such as CIEBP. The percentage of differentiated cells within adipogenic colonies was found to be 6.4-fold greater in s.c. isolates compared with i.m. isolates, regardless of treatment. This suggests that inherent depot differences in the 119 ability to differentiate exist. As TRO appeared to enhance the relative increase in i.m. and s.c. S-V cell differentiation similarly, these depot differences may not be readily explained by variations in the expression of PPARy, or response to PPARy ligands. If these differences were simply a result of an increased inherent ability of s.c. cells to produce endogenous PPARy ligands, we should have observed a decreased response to TRO when compared to i.m. cells. Alternatively, if i.m. cells inherently expressed lower amounts of PPARy compared to s.c., a reduced response to TRO may have resulted in these cells. Therefore, it is possible that differences between s.c. and i.m. S-V cells are caused by factors involved in differentiation and(or) lipid accumulation which are not directly associated with PPARy expression and(or) activation. This would enable i.m. cells to respond to PPARy ligands, while still showing a reduced ability to differentiate than s.c. Components of SL or insulin used in these studies may have been causative to the depot differences observed. For instance, s.c. cells may be more sensitive to the effects of insulin compared to i.m. S-V cells. Insulin was shown to enhance differentiation of a s.c. clone in our previous studies, suggesting that bovine s.c. cells are responsive to the hormone. However, the effect of insulin on i.m. S-V cell differentiation has not been tested. Thus, it would be of interest to examine the response of S-V cells from both depots to varying insulin concentrations. Additionally, s.c. cells may differentiate more readily in the presence of SL than i.m. Therefore, further analysis of the effects of individual SL components on the differentiation of both cell types may be of importance. Although addition of individual FA to serum-free 120 media may not stimulate GPDH activity, it may be possible to test the effects of FA through clonal analysis experiments. A reason as to why depot differences were found using clonal analysis techniques and not in mass culture experiments are not readily apparent. However, the relatively small sample size, combined with the variability in GPDH activity between clones and steers may have affected enzyme experiments more so than the clonal analysis studies. In addition, enzyme activity in some of the control treatments proved to be below the sensitivity of the GPDH assay. Therefore, clonal analysis studies may have been more sensitive in detecting differences between depots than GPDH. The results of these experiments indicate that preadipocytes and S-V cells isolated from bovine i.m. and s.c. adipose tissues are capable of enhanced differentiation in response to adipogenic stimuli. A selective PPARy agonist appeared to have the greatest effect on the differentiation of both cell types while having no apparent depot specific effects. However, differences between adipogenic i.m. and s.c. cells appear to exist, as s.c. cells were shown to have an increased ability to differentiate under our culture conditions. As TRO did not appear to have depot specific effects in our experiments, other media additives such as SL and insulin may have induced differential responses between i.m. and s.c. adipogenic cells. Therefore, continued studies investigating the effects of specific compounds involved in bovine preadipocyte differentiation may lead to the discovery of the mechanisms responsible for the differences between depots. Knowledge of specific differences that exist between i.m. and s.c. preadipocytes 121 may lead to strategies that influence individual adipose tissue development, which may ultimately result in the production of more highly marbled beef with less subcutaneous fat trim. 122 APPENDICES 123 APPENDIX A Adipogenic cloning efficiency and clone inventory All clones isolated from steer #3 (Harvest date 6-30-03) Subcutaneous: % Adipogenic clones: 47%; n = 30 of 64 96-well plate procedure: 22%; n = 8 of 36 Cloning-ring procedure: 79%; n = 22 of 38 Adipogenic clones (CR = Isolated using cloning ring procedure; 96 = Isolated using 96-well plate procedure): C3(CR), C4(CR), C5(CR), C7(CR), C9(CR), C10(CR), C11(CR), C12(CR), C13(CR), C14(CR), C15(CR), C16(CR), C17(CR), C18(CR), C19(CR), C20(CR), C21(CR), C22(CR), C31(CR), C32(96), C34(96), C35(96), 036(96), C38(CR), C39(CR), C40(CR), C41(96), 042(96), 047(96), 048(96). Intramuscular: % Adipogenic clones: 12.5%; n = 6 of 48 96-well plate procedure: 7.7%; n = 2 of 26 Cloning-ring procedure: 18%; n = 4 of 22 Adipogenic clones (CR = Isolated using cloning ring procedure; 96 = Isolated using 96-well plate procedure): C7(CR), 09(CR), CI 1(CR), C14(CR), C32(96), C38(96). Clones used in this dissertation: lM3-C32, lM3-C38, SC3-020, 803-021, SC3-C31 124 APPENDIX B Effects of adipogenic stimuli on the differentiation of preadipocyte clones Figure B-1. Photomicrographs of a bovine subcutaneous preadipocyte clone (SC3-C31) exposed for 12-d to differentiation media containing: 1 mM octanoate, 10 mM acetic acid, and 0.25 uM dexamethasone (DEX) (A); 20 uUmL serum lipids (SL) and 0.25 (M DEX (B); 20 uUmL SL and 40 pM troglitazone (T RO) (C); 20 uUmL SL, 0.25 pM DEX, and 40 uM TRO (D). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. 125 _._ IM3-C32 + lM3-C38 _ . -sc3-c2o _ . -scs-c21 _ .- -sca-ca1 Log. GPDH nmoI-min"-mg protein" Troglitazone, pM Figure B-2. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH), activity. Values are least squares means and SEM for two independent experiments of three subcutaneous (s.c.), and two intramuscular (i.m.), bovine preadipocyte clonal cell lines. 126 I—I—SC Loge GPDH nmol-min’I-mg protein" 0 10 20 30 40 50 Troglitazone, P M Figure B-3. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH), activity. Values are pooled least squares means and SEM for two independent experiments of three subcutaneous (s.c.), and two intramuscular (i.m.), bovine preadipocyte clonal cell lines. 127 APPENDIX C Effects of adipogenic stimuli on the differentiation of stromal-vascular cells and clonally-derived adipogenic colonies - — .- _SC -. 4- _._IM C E . 2 _. Q. 3_ ———— U! .5 Ts ' .E 2- 'o E - C E 1 a . c a? n O —| o. 0 1o 20 30 4o 50 so Troglitazone, uM Figure C-1. Effect of troglitazone supplementation on glycerol-3-phosphate dehydrogenase (GPDH), activity. Values are pooled least squares means and SEM of subcutaneous (s.c.), and intramuscular (i.m.), stromal-vascular cells from three steers. 128 WDIM .SC No DEX Loge GPDH nmol-min'I-mg protein" N 7: 6 - -- IM 8 E] 0 .SC L- 5 - O. D) g 4. 'c E 3 - '5 E 2. C E 1. (‘9 on; 0 3 No TRO TRO Figure C-2. Effect of 0.25 uM dexamethasone (DEX) (upper panel), and 40 uM troglitazone (TRO) (lower panel), on glycerol-3-phosphate dehydrogenase (GPDH) activity. Values are pooled least squares means and SEM of subcutaneous (s.c.), and intramuscular (i.m.), stromal-vascular cells from three steers. 129 Figure C-3. Photomicrographs of subcutaneous clonally-derived adipogenic colonies exposed for 10-d to differentiation media containing: 20 uL/mL serum lipids (SL) (A); 20 uL/mL SL and 0.25 uM DEX (B); 20 uL/mL SL and 40 uM troglitazone (T R0) (C); 20 uleL SL, 0.25 [M DEX, and 40 pM TRO (D). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. 130 Figure C-4. Photomicrographs of intramuscular clonally-derived adipogenic colonies exposed for 10-d to differentiation media containing: 20 pUmL serum lipids (SL) (A); 20 pUmL SL and 0.25 uM DEX (B); 20 uUmL SL and 40 uM troglitazone (T R0) (C); 20 uL/mL SL, 0.25 pM DEX, and 40 pM TRO (D). Cells are stained with oil red O and lightly counterstained with giemsa. Bar = 100 pm. 131 APPENDIX D Characteristics of steers used for stromal-vascular cell isolations Table D-1. Identification, live, and carcass characteristics of steers used for stromal-vascular cell isolations Steer code Steer #3 Steer #4 Steer #5 ID M408 N140 N144 Breed Angus Ang x Simm Ang x Simm . Sire MSU Easy Bando, or SRS Fortune Seldom Rest Network SRS Fortune 500 500 Reg # AAA# 12836291, or 13637205 ASA# 1901357 ASA# 1901357 Dam identification 1304 J715 G520 Age at harvest, d 411 409 408 Days on feed 209 219 249 , Implanta Synovex-S Synovex-S Synovex-S ' Implant admin., days on feed 18 and 87 35 and 105 35 and 105 Harvest date 6-30-03 6-21-04 7-20-04 Live wt., kg 558 560 563 Carcass wt., kg 345 346 346 Dressing, % 62.0 61.8 61.4 Ribeye area, cm2 82.6 74.8 74.8 U.S.D.A.Yield Grade 3.69 3.54 4.40 Fat thickness, mm 20.3 12.7 21.6 Kidney, pelvic, heart fat, % 2.0 3.0 3.0 Marbling score Moderate80 Small80 Moderate90 U.S.D.A. Quality Grade Choice+ Choice‘ Choice+ Time of: Exsanguination 0600 0600 0555 1"t collagenase digestion 0700 0730 0730 2"d collagenase digestion 0830 0900 0900 Initial freezing] seeding 1030 1022 0922 Final freezing/ seeding 1210 1127 1110 aImplant composition: 200 mg progesterone, and 20 mg estradiol benzoate. APPENDIX E StromaI-vascular cell extraction protocol Adapted from Forest et al. (1987) Fat cell extraction preparation (1-2 days prior to extraction) These materials should be appropriate for the isolation of 15 g equivalents each from intramuscular (i.m.), subcutaneous (s.c.), and perirenal (p.r.) fat depots from one steer. a—S . Make up 8 to 10 L PBS (pH 7.4) and refrigerate. Make 150 mL freezing media. Make 1 L low glucose DMEM (LGDMEM) with no additives. . Make 1 L of growth media (for plating freshly isolated cells). . Autoclave approx. 80, 50 mL tubes. . Autoclave approx. four of each: #3 & #4 scalpels, hemostats, alligator and regular tweezers, and tissue shears. Have #11 and #20 scalpel blades readily available. Have a digital scale in close proximity to work space (To weigh tissue samples into 50 mL vials). Autoclave at least 4 glass pyrex dishes (To use as tissue dissection plates). Make 6, 1000 um nylon screens (2 I depot) (Lab-Pak 06-1000/44 Nitex; SEFAR America Inc. 111 Calumet, Depew, NY, Fax 716-706-0154, Phone 716-601-3165). Make filter by tracing out circumference using a 133 large funnel and cut. Tape screens inside funnels, wrap in aluminum foil, and autoclave (Figure D-1). 10. Make 20, 500 um (03-500/47 Nitex; SEFAR America Inc.) and 20, 53 pm (3-53/30 Nitex; SEFAR America Inc.) screens. Remove inside of 50 mL tubes lids using a hammer and flat head screwdriver. Cut 50 mL tubes in half with a hacksaw. Cut out appropriate sized screen and screw lid back on tube with screen fastened in between the tube and the lid. Should use 5 of each screen size per depot (Figure D-1). 11.Autoclave at least 2, 2L beakers (for tissue transport) and about 8, 1L beakers (for tissue washes and storage). 12.Autoclave approx. 15 small funnels (to use with 500 and 53 pm filter units). 13.Clean 1 to 2 cutting boards using bleach and ethanol (for i.m. tissue extraction). Place boards in hood and use UV light to further sterilize. 14. Label at least 15, 1.8 mL cryogenic vials / depot. Label vials with: depot, animal identification, 1 9 equivalent I vial, and date. Refer to APPENDIX F for cell inventory identification system. 15. Have 1 to 2 sharp knives available to cutaway s.c. from i.m. tissue Sterilize with ethanol prior to use. 16. Have approx. 2 g of bovine serum albumin (BSA; Sigma A-7888), 5 mL dimethyl sulfoxide (DMSO), and 2, 100 mg bottles Type II collagenase stock (Sigma C6885) on hand. 134 17.Clip hair on animal using sheep shears and surgical shears to expose hide. Fat cell extraction checklist (morning of extraction) 1. Set incubator and water baths to 37°C. 2. Turn on RC3 centrifuge and make sure it is set to 4°C. 3. Make Type II collagenase stock (C6885-Sigma): 2 mglmL collagenase and 2% (weightzvolume) BSA in LGDMEM. a. Add 1 9 BSA to 50 mL LGDMEM in a 50 mL tube. Gently invert. Mug BSA takes time to go into solution. Do not vortex. Media will acidify somewhat and turn pale yellow in color. b. Add 5 mL DMEM/BSA solution directly to a 100 mg collagenase bottle. Solubilize. c. Add resulting collagenase solution back to the initial 50 mL tube containing 45 mL DMEM/BSA solution. d. Store tube at 4°C until you begin tissue isolation. 50 mL solution is enough for 25 g of tissue as it is used at 2 mL per g of isolated fissue. 4. Place all tools, trays and plates in sterilized hoods. 5. Place 2 L of PBS on ice for isolation and transport sample to laboratory. Fat cell extraction protocol 1. Record time of exsanguination, gender and weight of animal. Record times of collagenase digestions and freezing of cells. 135 .'- 'I-- .1. 2. Immediately after exsanguination, wash and sterilize incision area on carcass with alcohol. 3. Make an incision through the hide and excise a sample of muscle tissue containing s.c. adipose tissue from between the 12th and 13th rib (Figure D-2). Place sample in 1L cold PBS and transport to lab. 4. As soon as possible in the harvest process, collect a sample of pr. adipose tissue and place in 1L PBS for transport to the lab (Figure D-2). 5. Warm collagenase solution in water bath to 37°C. 6. Wash samples twice in cold PBS. 7. Using sterile technique and knife, separate s.c. adipose tissue from muscle tissue and place in a fresh beaker of cold PBS. 8. For s.c. and pr. samples, remove visible connective tissue from samples using scissors and scalpels. Cut clean pieces into more manageable sizes and place in fresh beaker filled with cold PBS. 9. For i.m. tissue, cut muscle into manageable pieces and place in beaker filled with cold PBS. Take muscle pieces and dissect fat away from muscle (Figure D—3). Place fat samples in 10-cm culture plates on ice. Add a small volume of cold PBS to keep samples from drying out. 10. For s.c. and pr. tissues, take clean pieces out of PBS, place in a sterile glass plate, and mince into 2 mm sections (Figure D-3). Place minced pieces into 10 cm-plates on ice. Add a small volume of cold PBS to keep samples from drying out. 136 11.Aliquot samples into 50 mL tubes and weigh out approx. 3 9 into each tube. Once weighed, place sample tubes on ice while weighing out others. 12.Add 1 mL collagenase stock I 0.5 g wet weight to each tube. 13. Incubate samples at 37°C in water bath for 15 min with gentle inversion of tubes at t = 0, 5, 10, and 15 min. 14. Place tubes in Lab Line incubator at 37°C for 45 min at 230 rpm (Figure D- 3). 15.Sequentially filter samples through 1000 pm, 500 um and 53 um screens, into new 50 mL conical vials (Figure D-1). Rinse tubes with LGDMEM. Place non-filtered tubes in water bath at 37°C while filtering others. 16. Balance tubes to an equal volume using LGDMEM (usually 15 mUtube). 17. Centrifuge tubes at 800 x g (approx. 1750 rpm, in RC3 centrifuge with HL- 8 swing bracket rotor) for 10 min. 18. Carefully pour out supernatant into another tube, retaining pellet, and centrifuge supernatant a second time. Resuspend pellets in 1 mL freezing media immediately after centrifugation. 19.Combine cell suspensions into one tube. Retain both pellets. 20.Add appropriate volume of freezing media to tube (Figure D-3) (Freeze at 1 gram equivalent of tissue I vial): Total freezing media DMSO 1 vial 1.62 mL 0.18 mL 2 vials 3.24 mL 0.36 mL 3 vials 4.86 mL 0.54 mL 4 vials 6.48 mL 0.72 mL 137 21.Slowly add 10% DMSO dropwise to freezing media cell suspension while swirling. Add 1.8 mL of the final cell suspension / 2.0 mL cryogenic vial. Incubate at room temperature for 10 to 12 min with vial caps loosened. Tighten caps and place vials in styrofoam container. Incubate overnight at -80°C, then place in liquid nitrogen for long-term storage. ALTERNATIVELY: Pellets may be resuspended in growth media and immediately seeded at a concentration of 1 9 equivalent / 2 wells of a 6-well tissue culture plate (35 mm diameter; Corning Inc., Corning, NY) and propagated. Reference: Forest, 0., A. Doglio, D. Ricquier, and G. Ailhaud. 1987. A preadipocyte clonal line from mouse brown adipose tissue. Exp. Cell. Res. 168:218-232. 138 Figure E-1. Photos of 1000 pm screen taped in funnel (A); 500 and 53 pm screens secured in modified conical tube tops (B); filtration technique using 1000 pm screen (C); and filtration technique using 500 and 53 um screens (D). 139 Figure E-2. Extraction process of longissimus muscle, subcutaneous (A), and perirenal adipose tissue samples (B). Carcass following extraction (C). 140 Figure E-3. Excision of i.m. adipose tissue from muscle (A); mincing of s.c. adipose tissue (B); incubation of 50 mL tubes in Lab Line incubator (C); and addition of final cell suspension to cryogenic vials (D). APPENDIX F GcheroI-3-phosphate dehydrogenase cell solubilization and assay protocol Adapted from Adams et al., 1997 DHAP fli—p Glycerol-3-Phosphate NADH + H NAD“ Important: Assays must always be conducted on freshly extracted samples. 1. Prepare TRIS-EDTA extraction buffer. a. 5 mM Tris (lnvitrogen, Carlsbad, CA, Cat# 15504-020; FW 121.1) (0.061 g TRIS / 100 mL H20). b. 1 mM EDTA (Sigma, St. Louis, MO, E-5134; FW 372.2) (0.03722 9 EDTA/ 100 mL H2O). c. Reduce temperature of buffer to 4°C (TRIS is temperature sensitive). d. Adjust pH of buffer to 7.4. Make sure buffer temperature is 4°C and that pH meter temperature setting is at 3 to 4°C. e. Store buffer at 4°C. 2. Prepare TEA-EDTA assay buffer a. 133.4 mM Triethanolamine-HCL (Sigma T-1502; FW 185.7) (2.47 g TEA-HCL / 100 mL H2O). b. 3.33 mM EDTA (Sigma E-5134; FW 372.2) (0.124 9 EDTA/ 100 mL H20). c. Adjust pH of buffer to 7.4 and store at 4°C. These buffers are stable when stored at 4°C. 142 Buffer preparation on day of assay (Normally make 20 mL of each buffer). Extraction Buffer 1. Prepare 1 M stock solution of dithiothreitol (DLDTT) (Sigma D-9163, FW 154.2) (0.015 g l 100 uL extraction buffer). 2. Add 1 pL DLDTT I 20 mL extraction buffer. Assay Buffer 1. Prepare 58.8 mM DHAP stock (Sigma D-7137; FW 170.1) (If the formula weight of DHAP is 170.1, this stock can be made by addition of 500 uL assay buffer to 5 mg bottle of DHAP). This has to be made fresh daily. 2. Add 15 mL of assay buffer to 50 mL tube. Add: 1.3 pL of 1 M DTT stock. Conc. in this buffer = 66.67 pM Add: 361 pL DHAP stock. Conc. in this buffer = 1.06 mM Add: 0.006 g NADH (Sigma N8129; FW 709.4) Conc. in this buffer = 0.423 mM 3. Bring to volume (20 mL) using graduated cylinder. When 150 pL of assay buffer/substrate mixture is combined with 50 uL of sample/extraction buffer, the final concentrations of each component in the 200 [AL will be: TEA-HCL 100 mM EDTA 2.5 mM DLDTT 50 uM DHAP 0.8 mM NADH 0.317 mM 143 4. 5. Prepare glycerol-3-phosphate dehydrogenase (GPDH; Sigma G6751) standards. Serially dilute from 1:500 to 1:32000 concentration. Add 4 uL GPDH to 1996 uL extraction buffer (containing DLDTT) (1:500). Add 500 pL of that solution to a new vial containing 500 pL extraction buffer (1 :1000). Continue diluting. Assay standards immediately to get an initial reading as enzyme activity of standards begin to degrade quickly (within 1 h). Extraction protocol 2 wells of a 6 well plate are combined to assay GPDH activity. 1. 2. Aspirate media from cells. Wash cells twice with ice-cold PBS (pH 7.4). Aspirate PBS from top well sequentially using 1000 and 200 uL pipetman. Leave last PBS wash in bottom well to keep cells hydrated while scraping the top well. Add 100 uL ice-cold extraction buffer to top well. Scrape well using a plastic scraper, rinse once, and scrape solution to bottom of well. Aspirate PBS from bottom well using 1000 and 200 uL pipet. Aspirate solution from top well and add to bottom well using 100 uL pipetman. Scrape well. Add 100 pL fresh extraction buffer to well and scrape well again. Using 200 uL pipetmen, aspirate solution (200 uL) and add to a pre-chilled microfuge vial. Place on ice as you scrape other wells. 144 9. Disrupt cells by sonicating 3 times (3 second bursts each time) with 1 min cooling time on ice between bursts at 40 watts using homogenizer. After the first sonification, spin for 5 sec. in microcentrifuge. 10.Centrifuge samples at 16,000 x g for 15 min at 4°C. During centrifugation process, turn on Versamax Tunable Microplate Reader. Computer settings: Set temperature to 30°C. Select Experiment then I select Setup. Set L1 to 340 nm. Select Kinetic, select Run time 6 min. Change intervals to 15 seconds. Select Show Detail, select Automix (before and between reads). Go back to template and enter samples J and standards as they will appear on the 96-well plate. Select Reduction and make sure absolute values icon is checked. 11.After centrifugation, transfer sample supernatants into pre-chilled micro centrifuge vial being careful to avoid the lipid layer at the top of the vial and the pellet at the bottom. 12.Transfer 50 pL of each sample supernatant, GPDH standards, and reagent blanks (extraction buffer containing DLDTT) in duplicate into wells of a 96 well plate (lmmulon 18; Fisher Scientific, Hampton, NH). 13. Using multipipetmen, transfer 150 pL of assay/substrate mixture into wells immediately prior to analysis. As soon as possible, transfer the plate into the plate reader and select read. 14. Follow M340 in a spectrophotometer at 30°C over a 1 cm light path for 6 min to obtain an initial reaction. 145 Raw Vmax values for each sample must first be subtracted from the Vmax values for the reagent blanks. To obtain the AA340 value for each sample, divide the Vmax value by 1000. GPDH, uM I min I mL = M2440 x Total volume LP x SV x DC LP = Light Path = 0.622 (Total volume in mL (0.2) I area of well (0.3217cm2)). TV = Total volume = 200 uL SV = Sample volume = 50 uL DC = Dissociation constant of NADH = 6.22 ._ . ":51. OR: For the above conditions: GPDH, [M l min I mL = AAm X1034 (correction factor). GPDH, nMI min/ mL = ((GPDH, pMImin/mL)/1000) I (Protein, mg/ mL) Reference: Adams, M., C. T. Montague, J. B. Prins, J. C. Holder, S. A. Smith, L. Sanders, J. E. Iigby, C. P. Sewter, M. A. Lazar, V. K. K. ChatterjeeJand S. O'Rahilly. 1997. Activators of peroxisome proliferator-activated receptor 9 have depot-specific effects on human preadipocyte differentiation. J. Clin. lnvest. 100:3149-3153. 146 Protein assay (Using BCA Reagent) BCA Protein Assay Reagent Kit Pierce, Rockford, Illinois Immediately after analyzing samples for GPDH activity, analyze samples for protein content. 1. Make up BCA reagent. Add 1 mL Assay Reagent B to 49 mL Assay Reagent A. Make up only as much as you need to add 200 pL of BCAI well of plate. 2. Thaw protein standards. Computer settings: Select experiment, select setup, select endpoint. Change L1 setting to 562 nm. Select show detail, select automix before first read. Select template and enter samples and standards as they will appear on the 96-well plate. Change fit from no fit to linear. 1. Load all standards and samples in duplicate on 96 well plate. Add 20 uL of sample to each well, then using repeater digital pipetman. add 200 uL Iwell of BCA reagent to all samples/standards. Lightly vortex standards prior to adding to plate. 2. Place plate in plate reader and read. Once reading is complete, place plate in incubator at 37°C for 30 min. After 30 min, place plate back in Microplate reader and read the protein samples. Check standard curve R2 value. To read computer results select unknown and use the average column to obtain results. 147 If making up a new BSA Stock solution it needs to be run through the spec to get an accurate protein concentration Protein Standard Preparation 1. Make stock BSA solution at approx. 10 mglmL concentration in dd H2O. Assay an aliquot of this solution using a spectrophotometer to determine the exact protein concentration. 2. Make up standards of 0, 125, 250, 500, 750, 1000, 1500, and 2000 pg/mL. Prepare the protein standards using extraction buffer containing DLDTT to ensure the standards are in exactly the same buffer as the samples. 148 APPENDIX G Cell inventory identification system Cryogenic vials were labeled according to a three part naming system. 1. SC, IM, or PR indicates subcutaneous, intramuscular or perirenal adipose tissues the cells were isolated from. The number following indicates which steer in harvest order the cells were derived from. 2. C Indicates “clone”, while the number following indicates the individual clone number. “Het” indicates heterogeneous cell population comprised of stromal-vascular cells. 3. “P” indicates the passage number the cells were frozen at. E.g. SC3-CS1-P9 = Subcutaneous cells from steer the third steer harvested, 31st clone isolated, at passage 9. lM4-Het-P5 = Intramuscular cells from the fourth steer harvested, heterogeneous cells, passage 5. IMPORTANT: All cells isolated and propagated between 6-2001 and 6-2005 are labeled according to the following naming convention. Initial Namingfionvention +O+O +0 Primary Passage 1 Passage 2 Passage All experiments conducted in this dissertation used clonal cell lines at passage 8 or 9, and heterogeneous cells at passage 2 according to this naming convention. 149 HOWEVER: In retrospect, the correct naming convention for these cells should have been: Correct Naming Convention Isolation -> O —> + 0 Primary Passage 2 Passage 3 Passage 1&8; All experiments conducted in this dissertation used clonal cell lines at passage 9 or 10, and heterogeneous cells at passage 3 according to the correct naming convention. ALL FUTURE CELL ISOLATIONS AND PROPAGATIONS SHOULD BE LABELLED USING THE CORRECT NAMING CONVENTION . 150 APPENDIX H Media components Low glucose DMEM Dulbecco’s modified Eagle’s medium (DMEM; 5.5 mM glucose) (lnvitrogen 31600-034) Base medium DMEM; 5.5 mM glucose Gentamicin, 0.1% (Sigma G1397) Antibiotic-antimycotic, 1% (Sigma 5955) Biotin, 33 pM (Sigma B-4639) Pantothenate, 17 uM (Sigma P-5155) Ascorbic acid, 200 uM (Sigma A4034) Growth medium DMEM; 5.5 mM glucose Gentamicin, 0.1% (Sigma G1397) Antibiotic-antimycotic, 1% (Sigma 5955) Biotin, 33 (M (Sigma B-4639) Pantothenate, 17 uM (Sigma P-5155) Ascorbic acid, 200 pM (Sigma A4034) FBS, 10% (Sigma F2442) Freezing medium DMEM; 5.5 mM glucose Gentamicin, 0.1% (Sigma G1397) Antibiotic-antimycotic, 1% (Sigma 5955) Biotin, 33 pM (Sigma B-4639) Pantothenate, 17 uM (Sigma P-5155) Ascorbic acid, 200 pM (Sigma A4034) FBS, 20% (Sigma F2442) Dimethylsulfoxide, 10% (Sigma D5879) 151 Differentiation medium (280 nM Insulin) DMEM; 5.5 mM glucose Gentamicin, 0.1% (Sigma G1397) Antibiotic-antimycotic, 1% (Sigma 5955) Biotin, 33 [M (Sigma B-4639) Pantothenate, 17 uM (Sigma P-5155) Ascorbic acid, 200 pM (Sigma A4034) Glucose, 20 mM (Sigma 68769) Insulin, 280 nM (Sigma I-1882) Differentiation medium (1 mM Octanoate, 10 mM Acetate, 8.7 nM Insulin) DMEM; 5.5 mM glucose Gentamicin, 0.1% (Sigma G1397) Antibiotic-antimycotic, 1% (Sigma 5955) Biotin, 33 uM (Sigma B-4639) Pantothenate, 17 uM (Sigma P-5155) Ascorbic acid, 200 uM (Sigma A4034) Glucose, 20 mM (Sigma 68769) Acetic acid, 10 mM (EM Science AX0073-75) Capryllic acid/octanoate, 1 mM (Sigma C2875) Insulin, 8.7 nM (Sigma l-1882) Differentiation media additives: Dexamethasone, 0.25 [M (Initial 48 h) (Range 0.0025 to 2.5 uM) (Sigma D2915) Ex-Cyte, 20 uUmL (Range 0 to 20 uLImL) (Serologicals Corp. 81-129) Troglitazone, 40 uM (Range 0 to 60 pM) (Calbiochem 648469) 152 APPENDIX I Ex-Cyte media supplement components Table I-1. Fatty acid composition of Ex-Cyte lipid supplement‘ Concentration, mglmL Fatty acid supplement Myristic (14:0) 0.067 Palmitic (16:0) 1.13 Palmitoleic (16:1) 0.028 Stearic (18:0) 1.968 Oleic (18:1) 0.797 Linoleic (18:2) 3.01 Linolenic (18:3) 0.086 Homo-2-Iinolenic (18:3) 0.46 Arachidonic (20:4) 0.47 Eicosapentaenoic (20:5) 0.096 Docosenoic (22:1) or Docosenoic (22:0) 0.048 Docosatetaenoic or Docosatetraenoic (22:4) 0.105 Docosapentaenoic (22:5) 0.096 Docosahexanoic (22:6) 0.029 Others 0.912 Total Fatty acid 9.33 Unsaturated fatty acids 57.5% Polyunsaturated fatty acids 14.7% Concentration, glL, Other Components supplement Cholesterol 9.0 - 11.0 Protein 13.0 -18.0 “Data obtained from: Serologicals Corp., Norcross, GA, 1-800-227-9412. Ex-Cyte is an aqueous lipoprotein concentrate containing a mixture of fatty acids, cholesterol, and phospholipids derived from bovine serum. 153 VITA Aaron Christopher Grant was born on September 4, 1975, in Calgary, Alberta, Canada, son of Don and Lori Grant. Aaron was raised on a grain and cattle farm and was active in sports, 4-H, and the purebred cattle industry throughout his youth. In 1993, Aaron graduated from Olds Junior-Senior High School and began his secondary education at Northeastern Oklahoma A&M College in Miami Oklahoma. After receiving his Associates Degree in Animal Science, Aaron transferred to Texas A&M University in 1995. Aaron received a BS. in Animal Science in 1997, and a MS. in 2000. While at Texas A&M, Aaron was an active participant, and coach of the livestock judging team. In August of 2000, Aaron began work on his Ph.D. at Michigan State University. That fall, Aaron assisted Jason Rowntree in coaching the 2000 National Champion Livestock Judging Team. Aaron is a member of the American Society of Animal Science. After completion of his Ph.D., Aaron will return to Alberta to become a Consulting Nutritionist in the fed beef industry. 154