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DATE DUE DATE DUE DATE DUE 6/07 p:lClRC/DateDue.indd-p.1 INVESTIGATION OF ADIPOGENIC DIFFERENCES BETWEEN BOVINE INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES By Guillermo Ortiz-Colon A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 2006 ABSTRACT INVESTIGATION OF ADIPOGENIC DIFFERENCES BETWEEN BOVINE INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES By Guillermo Ortiz-Colén Our objective was to examine signaling mechanisms most likely to explain the lower adipogenic capacity of bovine intramuscular (IM) preadipocytes when compared to bovine subcutaneous (SC) preadipocytes. We hypothesized that the lower adipogenesis of IM preadipocytes was caused by decreased glucocorticoid receptor expression (GR), sensitivity to glucocorticoids, peroxisome proliferator-activated receptor 72 (PPARyz) expression, and(or) PPARyz ligand synthesis. Stromal-vascular cells, containing preadipocytes, were isolated from IM and SC adipose tissue of 3 Angus-cross steers. Immunoblot analysis detected GR immunoreactive bands of ~97, ~62, and ~48 kDa, which were equally expressed in IM and SC cells (P > 0.50). Intramuscular preadipocytes were less adipogenic than SC preadipocytes as determined by glycerol-3- phosphate dehydrogenase (GPDH) activity and oil red O staining (P < 0.05). Dexamethasone (DEX), a synthetic glucocorticoid, increased GPDH activity similarly in preadipocytes from both depots (P < 0.05). Dexamethasone increased the percentage of SC preadipocytes with lipid droplets Z 10 um (P = 0.002), but had no effect on IM preadipocytes (P > 0.27). Immunoblot analysis revealed a PPARyz immunoreactive band of ~53 kDa, which was expressed equally in IM and SC cells (P = 0.39). Conversely, IM cells secreted more of the presumptive PPAR ligand prostacyclin (PGIz), than SC cells (P = 0.046). Because exposure of SC preadipocytes to an inhibitor of PGIz synthesis had no effect on adipogenesis (P = 0.99), and exogenous cPGIz (PGI; analog) tended to enhance adipogenesis (P = 0.06), the greater secretion of PGI; by IM preadipocytes does not explain their lower adipogenesis. Exposing IM and SC preadipocytes to the cyclooxygenase (COX) inhibitor/PPARyz ligand, ibuprofen (IBU) for 48 h or 12 d resulted in a treatment by depot interaction (P = 0.002). Ibuprofen exposure for 48 h enhanced DEX stimulation of GPDH activity only in IM cells (P = 0.009). Exposure to 100 uM and 500 uM IBU for 12 d enhanced DEX induction of differentiation in IM preadipocytes, whereas only 100 uM IBU enhanced DEX induction of differentiation in SC preadipocytes (P S 0.05). In the absence of DEX, exposure to IBU for 12 d maximally increased GPDH activity in [M preadipocytes by 12-fold, but only increased GPDH activity by 1.5-fold relative to control in SC preadipocytes (P < 0.001). Contrary to IBU, 500 uM aspirin (a COX inhibitor) did not affect GPDH activity either alone (P > .37), or combined with DEX (P > 0.60) in either cell population. Because IBU diminished adipogenic differences between IM and SC preadipocytes, it is suggested that these adipogenic differences may be partially related to differences in the endogenous activation of PPARyz. The use of selective PPARyz agonists or antagonists offers potential to selectively alter adipogenesis in economically-important bovine adipose tissue depots. Copyright by GUILLERMO ORTIZ-COLON 2006 I dedicate this dissertation to my wife, Catherine Mazak-Tumminia, whose support was essential for the completion of this project. I am deeply indebted to you for keeping my hopes up when the end was not even seen as a possibility. Gracias. .. de verdad que ”somos mucho mds que dos”. ACKNOWLEDGEMENTS I would like to recognize the people that gave me the opportunity to pursue this research project, and to everybody that provided me the guidance, encouragement, and assistance that was essential for my completion of the dissertation. Firstly, I would like to express my gratitude to Dr. Dan Buskirk, for giving me the opportunity to broaden my education and research capabilities under his supervision. His encouragement, mentorship, as well as his respectful critiques, were undoubtedly essential for my successful completion of this project. The autonomy that I enjoyed while pursuing my research, accompanied by Dr. Buskirk’s personal involvement in the project was certainly greatly appreciated. I would also like to acknowledge the essential support of Dr. Matthew Doumit, whose immeasurable assistance in all aspects of this project undoubtedly assured its completion. In addition, I would like to express gratitude Dr. Steven Rust for informing me about Michigan State University’s Animal Science program and opportunities, and for inviting me to attend this university. In addition, I would like to thank Dr. Jeanne Burton and Dr. Dale Romsos, also members of my graduate committee and whose expertise, criticisms, and direction were invaluable for the completion of this dissertation, but also for my improvement as a scientist. I would also thank Dr. Rob Tempelman for his help with the statistical analysis. vi I would like to thank Aaron Grant, Jason Scheffler, Chuck Allison and Michelle Martinez, fellow graduate students that helped me considerably throughout this research project. I would also like to thank Emily Helman for her assistance with my experiments, particularly in the latter part of the project. I would like also to recognize the assistance that I received from Drs. Patty Weber and Sally Madsen as they trained me in RNA work, as well as, Sue Sipkovsky for her help with the microarray analysis. I also recognize Tom Forton, Jennifer Dominguez, as well as, the rest of the Meat Lab workers for their assistance with the sample isolations. At last, I thank the Department of Animal Science at Michigan State University for giving me the opportunity to pursue my graduate studies at this institution. I am certainly greatly satisfied with the education and training I have received for the last five years. In addition, I would like to acknowledge the Graduate School at Michigan State University, specifically the Office of ALANA students’ affairs for assisting me with the fellowship that financially supported me for part of my tenure here. vii TABLE OF CONTENTS LIST OF ABBREVIATIONS .......................................................................................... xx INTRODUCTION ............................................................................................................ 1 Chapter I: Review of Literature .................................................................................... 3 Biological Importance of Adipose Tissues ....................................................... 3 Marbling ................................................................................................................ 5 Marbling and Beef Palatability Traits ............................................................... 6 Adipogenic Potential of Intramuscular Adipose Tissue ................................ 8 Fatty acid and triacylglyceride synthesis ............................................. 9 Uptake of dietary fatty acids ................................................................ 11 Hypothetical function of intramuscular adipose tissue ................... 12 Maturity of intramuscular adipocytes ................................................ 13 Preadipocyte recruitment ..................................................................... 14 Adipogenesis: The Mechanism of Preadipocyte Differentiation ................ 15 Commitment of mesodermal stem cells ............................................. 15 Clonal amplification .............................................................................. 17 Growth arrest .......................................................................................... 18 Preadipocyte development and the mature adipocyte phenotype 24 Post-natal adipogenesis ......................................................................... 24 Adipogenic Regulators of Preadipocyte Differentiation .............................. 25 Glucocorticoids ....................................................................................... 25 Ligands of PPARyz ................................................................................. 37 Conclusion .......................................................................................................... 41 Literature Cited .................................................................................................. 44 Chapter H: Bovine intramuscular, subcutaneous, and perirenal preadipocytes express similar glucocorticoid receptor isoforms, but exhibit different adipogenic capacity ............................................................................................................................ 62 Abstract ................................................................................................................ 62 Introduction ........................................................................................................ 63 Materials and Methods ..................................................................................... 65 Results .................................................................................................................. 73 Discussion ........................................................................................................... 76 Implications ........................................................................................................ 81 Literature Cited .................................................................................................. 90 viii Chapter IH: Differences in adipogenesis between bovine intramuscular and subcutaneous preadipocytes are not related to expression of peroxisome proliferator-activated receptor gamma two or secretion of prostacyclin .............. 94 Abstract ................................................................................................................ 94 Introduction ........................................................................................................ 95 Materials and Methods ..................................................................................... 97 Results and Discussion .................................................................................... 106 Implications ...................................................................................................... 113 Literature Cited ................................................................................................ 120 Chapter IV: Ibuprofen preferentially enhances adipogenesis in bovine intramuscular preadipocytes when compared to subcutaneous preadipocytes 124 Abstract .............................................................................................................. 124 Introduction ...................................................................................................... 125 Materials and Methods ................................................................................... 127 Results ................................................................................................................ 132 Discussion ......................................................................................................... 135 Implications ...................................................................................................... 140 Literature Cited ................................................................................................ 146 Chapter V: Interpretive Summary ............................................................................ 150 Appendix A: Clonal efficiency and adipogenic capacity of cells isolated from bovine intramuscular, subcutaneous, and perirenal adipose tissue .................... 160 Abstract .............................................................................................................. 160 Introduction ...................................................................................................... 162 Materials and Methods ................................................................................... 163 Results ................................................................................................................ 167 Discussion ......................................................................................................... 168 Implications ...................................................................................................... 172 Literature Cited ................................................................................................ 176 Appendix B: Influence of dexamethasone on the gene expression of bovine intramuscular preadipocytes ...................................................................................... 179 Abstract .............................................................................................................. 179 Introduction ...................................................................................................... 180 Materials and Methods ................................................................................... 181 Results and Discussion .................................................................................... 186 Summary ........................................................................................................... 191 Literature Cited ................................................................................................ 195 ix Appendix C: Immunocytochemical detection of glucocorticoid receptor in clonal bovine preadipocytes isolated from intramuscular, subcutaneous, and perirenal adipose tissue ................................................................................................................ 198 Objectives .......................................................................................................... 198 Materials and Methods ................................................................................... 198 Results ...................................................................... 200 Discussion ......................................................................................................... 200 Literature Cited ................................................................................................ 206 Appendix D: Effect of nordihydroguaiaretic acid on the activity of glycerol-3- phosphate dehydrogenase in bovine subcutaneous preadipocytes ..................... 207 Appendix E: Effect of ibuprofen on the activity of glycerol-B-phosphate dehydrogenase in bovine heterogeneous intramuscular and subcutaneous preadipocytes ............................................................................................................... 208 Appendix F: Cells utilized within this dissertation ............................................... 210 LIST OF TABLES APPENDIX B Table B-1. Selected genes related to preadipocyte adipogenesis that upon cDNA microarray analysis were determined to be differentially expressed (P < 0.05) in bovine intramuscular preadipocytes upon dexamethasone treatment. .................................................................................................... 194 xi LIST OF FIGURES Images in this dissertation are presented in color CHAPTER I Figure 1-1: Simplified diagram of adipogenesis. Stages of differentiation are shown as black rectangles with white letters. Genes expressed in the different stages of adipogenesis are shown in white rectangles and black letters ..................................................................... 43 CHAPTER II Figure 2-1. Glucocorticoid receptor immunoblot of bovine intramuscular , subcutaneous, and perirenal preadipocytes. Cells were grown to confluence and then exposed to 0 or 250 nM dexamethasone (DEX) for 48 h. Twenty micrograms of protein per sample were separated by gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Membranes were subsequently incubated overnight with a polyclonal antibody raised against the glucocorticoid receptor, which detected major immunoreactive bands of ~97, ~66, and ~48 kDA. Positions of molecular weight standards are indicated to the left ........................... 83 Figure 2—2. Effect of dexamethasone (DEX) on the activity of glycerol-3- phosphate dehydrogenase (GPDH) in bovine preadipocytes isolated from bovine intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of three steers. Bovine preadipocytes were grown to confluence and subsequently exposed to DEX for 48 h, and differentiation media for 10 additional days. Glycerol-B-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Bars represent means i SEM. Means with different superscripts differ (P < 0.05) ............................................................................................ 84 Figure 2-3. Effect of dexamethasone (DEX) concentration on the percentage of differentiated preadipocytes (cells with a lipid droplet 2 10 pm). Bovine preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of three steers were grown to confluence and subsequently exposed to xii the indicated DEX concentrations for 48 h and differentiation media for 10 additional days. The percentage of differentiated preadipocytes was determined by microscopy 12 d after addition of treatments. Bars represent means 1: SEM. There was an interaction (P = 0.03) between DEX concentration and depot. Means with different superscripts differ (P < 0.05) ............................... 85 Figure 24. Effect of dexamethasone (DEX) concentration on the percentage of preadipocytes with a lipid droplet < 10 um. Bovine preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of three steers were grown to confluence and subsequently exposed to the indicated DEX concentrations for 48 h and differentiation media for 10 additional days. Preadipocyte lipid droplets were evaluated by microscopy 12 d after addition of treatments. Bars represent means 1 SEM. Means did not differ (P > 0.20) ....................................... 86 Figure 25. Effect of dexamethasone (DEX) concentration on morphological differentiation. Bovine preadipocytes isolated from intramuscular (1M; a, b, c), subcutaneous (SC; d, e, f ), and perirenal (PR; g, h, i) adipose tissue of three steers were grown to confluence and subsequently exposed to 0 (a, d, g), 25 (b, e, h) or 2500 (c, f, i) nM DEX for 48 h and differentiation media for 10 additional days. Photomicrographs were taken 12 d after addition of treatments. Lipid droplets in cells were stained with oil red O and cell nuclei were counterstained with giemsa. Photomicrographs shown represent average fields of view. Bar = 100 uM (Panel i) ...................................................................................... 88 Figure 2—6. Relationship between the specific glycerol-3-phosphate dehydrogenase (GPDH) activity and percentage of cells with a lipid droplet 2 10 um (r = 0.95, P < 0.001) in preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissues .................................................................. 89 CHAPTER III Figure 3-1. Western immunoblot of PPARyz from cultured bovine preadipocytes. Preadipocytes isolated from intramuscular (IM) and subcutaneous (SC) adipose tissue were grown to xiii confluence and then exposed to 0 or 25 nM dexamethasone (DEX) for 48 h. Twelve days after addition of treatments, total protein was collected and 50 pg of protein per sample were separated by gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Membranes were cut above the 45 kDa band and the appropriate portions were incubated with an antibody raised against peroxisome proliferator-activated receptor yz (PPARyz) or B-actin, which resulted in immunoreactive bands of ~53kDa (PPARyz) and ~451 0.50). Dexamethasone exposure decreased the abundance of the ~97 and ~62 kDa GR immunoreactive bands in preadipocytes from the three depots (P < 0.001), but did not affect the expression of the ~48 kDa band (P = 0.96). Preadipocytes isolated from three steers were grown in culture, and upon confluence, were exposed to 0, 25, or 2500 nM DEX for 48 h. After an additional 10 d in differentiation media, the propensity to differentiate, was determined by glycerol-3-phosphate dehydrogenase (GPDH) specific activity 62 and oil red O staining. The propensity to differentiate was PR > SC > IM (P < 0.05). Compared with control, 2500 nM DEX increased GPDH activity in preadipocytes from all depots (P < 0.05). There was no interaction between adipose tissue depot and DEX concentration for GPDH activity, (P = 0.99). However, the percentage of PR preadipocytes with lipid droplets greater than 10 pm-diameter increased in response to DEX in a linear manner (P < 0.02), but only increased above control in SC cells exposed to 2500 nM DEX (P = 0.002). However, we failed to detect an increase in the percentage of IM preadipocytes with large (2 10 pm-diameter) lipid droplets upon DEX exposure (P > 0.27). These observations reflect an adipose tissue depot by DEX concentration interaction (P = 0.03). Relative differences in adipogenic capacity among preadipocytes isolated from IM, SC, and PR bovine adipose tissue were evident, although they express GR similarly. Dexamethasone enhanced adipogenic enzyme activity in all three depots, but did not significantly enhance morphological differentiation of IM preadipocytes. Introduction Glucocorticoid induction of adipose tissue development has anatomical specificity. In swine, glucocorticoid stimulation of subcutaneous (SC) preadipocyte differentiation was greater in cells isolated from the shoulder than those from the ham (Ramsay et al., 1989). Additionally, preferential accretion of 63 abdominal adipose tissue has been seen in humans treated with glucocorticoids (Rebuffé-Scrive et al., 1988; Fried et al., 1993; Boschmann, 2001). In cattle, glucocorticoid treatment was shown to increase intramuscular (IM) adipose tissue development (Brethour, 1972). In humans and rats, the differing abundance of glucocorticoid receptors (GR) among adipose tissue depots is associated with the differential development of adipose depots following glucocorticoid supplementation (Feldman and Loose, 1977; Brbnnegard et al., 1990; Rebuffé-Scrive et al., 1990). Although preadipocytes are also considered a target for glucocorticoids, and glucocorticoids are commonly added to the differentiation media for cultured preadipocytes (Sato et al., 1996; Ailhaud, 2001; Brandebourg and Hu, 2005), to our knowledge, no previous studies have documented GR expression in bovine preadipocytes. If differences in GR expression exist between bovine preadipocytes from different adipose tissues, it could present a possibility to selectively alter adipose tissue accretion. Because IM adipose tissue accretion is positively associated with beef palatability and is the main determinant of beef quality within a carcass maturity Classification, any treatment that could selectively enhance IM adipose tissue development, or selectively reduce SC fat, would potentially benefit the beef industry. 64 Therefore, the objectives of this study were to compare GR expression among bovine IM, SC, and perirenal (PR) preadipocytes, and to compare the effects of a glucocorticoid on the differentiation of these distinct cell populations. We hypothesized that bovine IM, SC, and PR preadipocytes would present differences in GR expression, and differences in the adipogenic response to glucocorticoids. Materials and Methods Isolation of bovine preadipocytes Preadipocytes from IM, SC, and PR adipose tissues were isolated using a modification of a protocol previously described (Forest et al., 1987). Briefly, adipose tissue samples were collected immediately after exsanguination of one Angus steer and two Angus x Simmental steers (13.5 mo. old, 558 to 563 kg). Procedures were approved by the Michigan State University Committee on Animal Use and Care (AUF No. 10/03-130-00). Portions of the longissimus muscle (LM) between the 12th and 13th ribs and adipose tissue surrounding the kidneys were removed and immediately placed in sterile, ice-cold PBS (pH 7.2) and transported to the laboratory. Under sterile conditions, SC adipose tissue was separated from the LM and visible fibrous connective tissue surrounding SC adipose tissue was removed. Intramuscular adipose tissue was carefully excised from LM. For each adipose tissue depot, samples were minced and 65 approximately 3 g were aliquoted into 50 mL conical tubes and digested in 6 mL Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen Corp, Carlsbad, CA) supplemented with 12 mg of collagenase (from Clostridium histolyticum, Type II, >125 collagen digestion units/mg solid and 0.5 to 5.0 furylacryloyl-Leu-Gly-Pro- Ala hydrolyzing units/mg solid) and 2% BSA. Samples were incubated in a 37°C water bath, and were inverted at 0, 5, 10, and 15 min. After 15 min, samples were transferred to an incubator (Lab-Line Instruments Inc. Melrose Park, IL) and further digested with shaking (230 rpm) for 45 min at 37°C. The digested tissue was then sequentially filtered through 1000, 500, and 53 um nylon mesh and the filtrates were centrifuged for 10 min at 800 x g. Resulting supematants were collected and centrifuged again for 10 min at 800 x g. The pellets were resuspended in growth medium (DMEM [5.5 mM glucose] supplemented with antibiotic-antimycotic (Final concentration: 100 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate and 0.25 ug/mL amphotericin B), 0.05 mg/mL gentamicin, 33 uM biotin, 17 pM pantothenate, 200 pM ascorbate, 1,000 pM octanoate, and 10% fetal bovine serum [FBS]). Unless otherwise stated, all reagents were of tissue culture grade and were purchased from Sigma (St. Louis, MO). The resulting suspension of preadipocytes was seeded at 0.05 g equivalent per cm2 in 35 mm-diameter cell culture wells (Corning lnc., Corning, NY). 66 Alternatively, cells were suspended in freezing medium (base medium supplemented with a final concentration of 20% FBS and 10% dimethyl sulfoxide) and aliquoted into 1.8 mL cryogenic vials. Cells were placed at -20°C for two hours, and overnight at -80°C, before storage in liquid nitrogen for later use. Analysis of glucocorticoid receptor expression Cell culture. Primary preadipocytes from the IM, SC, and PR adipose tissue of two steers were propagated in culture and in their 4th passage, preadipocytes were seeded at a density of 3,600 cells/cm2 in 35 mm-diameter cell culture wells. Cells were allowed to proliferate to confluence (5 d) in growth medium, while incubated in a humidified atmosphere (37°C, 95% air and 5% C02). Growth medium was replaced every 2 d. After reaching confluence, plates were washed twice with PBS and the preadipocytes were exposed to modified growth medium (1% FBS) containing 0 or 250 nM dexamethasone (DEX) for 48 h. Identical procedures were followed in four individual trials. Immunoblot analysis. After 48 h of exposure to 0 or 250 nM DEX, preadipocyte monolayers were washed twice with ice-cold PBS and subsequently solubilized by the addition of hot (95°C) electrophoresis sample buffer (62 mM Tris-HCL (pH 6.8), 2% SDS, and 10% glycerol). Cell lysates from two wells per treatment were pooled and immediately stored at -20°C. Protein 67 concentrations were determined using the bicinchoninic acid assay (BCA, Pierce Biotechnology Inc., Rockford, IL). Prior to electrophoresis, protein samples were diluted to equal concentrations (0.33 ug/uL) by the addition of electrophoresis sample buffer supplemented with B-mercaptoethanol (5%) and bromophenol blue (0.01 %). Samples were then boiled for 3 min and 20 pg of protein per sample were subjected to SDS-PAGE using 7.5% (37.5:1 acrylamide/bis acrylamide) (Bio-Rad Laboratories, Hercules, CA) separating mini-gels, with 4% (37.5:1 acrylamide/bis acrylamide) stacking gels. After PAGE, proteins were transferred to polyvinylidine difluoride membranes at 4°C for 2 h at 100 V in a buffer containing 25 mM Tris, 193 mM glycine, and 15% methanol using a using a Bio Rad Mini-Trans-Blot electrophoretic transfer cell. Lanes with the molecular weight standards were cut from the membrane and stained with amido black. The remaining membranes were then cut just above the 45 kDa band to allow for individual B-actin and GR antibody labeling. Nonspecific antibody binding was prevented by incubating the membranes for 1 h in blocking solution (Tris buffered saline [198.2 mM Tris, 1.3 M NaCl, 26.8 mM KCl; pH 7.2] containing 0.1% Tween-20 [Bio-Rad Laboratories, Hercules, CA] and 5% non-fat dry milk). The membranes were then incubated overnight at 4°C in blocking solution containing 1 ug/mL of a polyclonal (rabbit) anti-GR antibody (PA1-511A, Affinity BioReagents, Inc., Golden, CO). After the primary antibody incubation, 68 membranes were washed three times with blocking solution and incubated for 1 h in blocking solution containing 1:1000 (vol/vol) of an alkaline phosphatase conjugated goat anti-rabbit IgG antibody (A3683, Sigma, Inc.). Membranes were washed three times with blocking solution and three times with TBS (0.1% Tween-20), and immunoreactive bands were detected upon the addition of 5- bromo-4-Chloro-3-indoyl phosphate/nitroblue tetrazolium (Bio-Rad Laboratories, Hercules, CA). Utilizing a similar procedure, the appropriate membrane sections were incubated for 1 h in blocking solution containing 0.16 pg/mL of a monoclonal anti-B-actin antibody (ab6276, Ab Cam, Inc., Cambridge, UK) and subsequently incubated for 1 h in blocking solution containing 1:1000 (vol/vol) of an alkaline phosphatase conjugated goat anti-mouse IgG antibody (A3562, Sigma, Inc.). Images of immunoblots were acquired using a Fluor-S Multilmager (Bio-Rad Laboratories) and analyzed with Discovery Series Quantity One 1-D Analysis Software (Bio-Rad Laboratories). Abundance of GR immunoreactive bands was normalized based on B-actin immunoreactive band intensity to account for loading differences. Analysis of the effects of glucocorticoids on bovine preadipocyte differentiation Cell culture. Primary preadipocytes from the IM, SC, and PR adipose tissue of three steers were propagated in culture and secondary cultures were seeded at a density of 4,600 cells/cm2 in 35 mm-diameter cell culture wells. Cells 69 were allowed to proliferate to confluence (4 d) in growth medium. Growth medium was replaced every 2 d. After reaching confluence, plates were washed twice with PBS and differentiation treatments were applied. Differentiation medium was supplemented with 280 nM bovine insulin and 5 uL/mL bovine serum lipids (Ex-Cyte; Serologicals Corp, Norcross, GA). Preadipocytes were exposed to 0, 25, or 2500 nM DEX for 48 h. Previously, DEX optimized bovine Clonal SC preadipocyte differentiation at a concentration of 2500 nM (not shown). Each treatment was applied to two wells of a 6-well plate, in two replicate plates for each of the three steers. After 48 h, treatment media were replaced with basic differentiation medium supplemented with 280 nM bovine insulin and 5 uL/mL bovine serum lipids and fresh medium was provided every 2 d for 12 d. Glycerol-3-phosphate dehydrogenase activity. Cell differentiation was quantified biochemically by measuring glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity using a modification of a method previously described (Adams et al., 1997). Cells were washed twice with ice-cold PBS. Contents of two wells per treatment were harvested and combined and in a total volume of 200 pL of ice-cold Tris (5 mM, pH 7.4) containing 1 mM EDTA and 50 pM dithiothreitol (extraction buffer). Each sample was transferred into prechilled 1.5 mL microcentrifuge tubes, and then disrupted by sonification three times at 70 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 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 for GPDH activity in duplicate, within 30 min of isolation. The final concentration of the assay mixture was: 100 mM triethanolamine—HCL (pH 7.4), 2.5 mM EDTA, 50 pM 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 (Immulon 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 3 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 200 s and the Vmax used to calculate GPDH activity was obtained from the linear range. Enzyme activity was expressed as nanomoles of NADH oxidized 0 min'1 0 mg protein‘. Protein concentrations of soluble cell lysates were determined by BCA protein assay (Pierce Biotechnology Inc., Rockford, IL). Analysis of morphological difi‘erentiation. Cell differentiation was morphologically assessed by counting the number of cells containing lipid droplets stained with oil red O (ORO). The ORO solution was prepared using a 71 protocol previously described (Ramirez-Zacarias et al., 1992). Cells were fixed by addition of 3.7% formaldehyde (Mallinckrodt Baker Inc., 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 and the cells were washed twice with distilled water (15 min incubation/wash). Cell nuclei were stained by adding 1 mL giemsa solution (1 g Giemsa, 66 mL glycerol, 66 mL methanol) to each well for 1 h, after which the cells were washed twice in distilled water, and stored dry at 4°C. Cells were visualized within 8 h of staining. Digital photographs were taken using a Nikon CoolPix 5000 digital camera (Nikon Inc., Melville, NY) fitted to a Zeiss inverted microscope (Carl Zeiss Inc., Thomwood, NY). Five fields of view, selected a priori, were photographed for each treatment replicate. Total cells were counted, and the percentage of ORO positive cells and differentiated cells were determined. Differentiated cells were defined as having one or more lipid droplet(s) with a diameter of 10 pm or larger, determined using an electronically generated ruler. Statistical analysis Data were analyzed using the Mixed Model procedure of SAS (SAS, Cary, NC). For the GPDH and immunoblot data, pooled cells from two wells of a six- well plate were considered the experimental unit, while for morphological 72 differentiation a single well was the experimental unit. For the immunoblot data, means were calculated using the fixed effects of DEX, depot, and DEX x depot, with steer included as a random variable. To satisfy the conditions of normality and homogeneity of variance, GPDH data were loge transformed. For GPDH and morphological differentiation data, means were calculated using the fixed effects of DEX, depot, and DEX x depot, with steer and steer x replication included as random variables. When the main effects were significant (P < 0.05), mean differences were analyzed utilizing Tukey’s multiple comparisons. Correlation analysis was perform utilizing the CORR procedure of SAS where the variables analyzed were GPDH activity, percentage of differentiated cells, and percentage of ORO positive cells. Results Glucocorticoid receptor (GR) expression The immunoblot analysis of protein isolates from IM, SC, and PR bovine preadipocytes revealed three GR immunoreactive bands of ~97, ~66, and ~48 kDa (Figure 2-1). No differences in the abundance of these immunoreactive bands across the three depots were found (P > 0.50). The relative expression of the ~97 kDa and ~48 kDa bands were approximately 2-fold more abundant than the ~66 kDa band in these cells. Dexamethasone exposure reduced the level of the ~97 kDa isoform by 36.5 .+_ 8.9%, and reduced ~66 kDa isoform expression by 73 72.5 i 23.7% in preadipocytes from all depots (P < 0.001). However, the level of expression of the ~48 kDa immunoreactive band was not affected by DEX treatment (P = 0.96). DEX eflects on adipogenic enzyme activity Supplementation of differentiation media with 25 nM DEX increased GPDH activity in preadipocytes from all depots (P < 0.001), and there was no interaction (P = 0.99) between DEX concentration and depot of origin of the preadipocytes (Figure 2-2). However, 2500 nM DEX did not significantly increase GPDH activity over 25 nM DEX levels (P = 0.45). Intrinsic differences existed in the propensity of the preadipocytes from different depots to undergo adipogenic differentiation. Independent of treatment, PR preadipocytes were the most adipogenic, followed by SC preadipocytes with GPDH activities 37.5% lower, while IM preadipocytes had GPDH activities 74.5% lower than PR preadipocytes (P < 0.001). Morphological assessment of lipid accumulation The percentage of PR preadipocytes with lipid droplets greater than 10 um—diameter increased in response to DEX in a linear manner (P < 0.02), but only increased above control in SC cells exposed to 2500 nM DEX (P = 0.002; Figure 2-3). However, we failed to detect an increase in the percentage of IM preadipocytes with large (2 10 jam-diameter) lipid droplets upon DEX exposure 74 (P > 0.27). These observations reflect an adipose tissue depot by DEX concentration interaction (P = 0.03). The percentage of preadipocytes with lipid droplets smaller than 10 um-diameter averaged 21.3% and was not influenced by depot (P = 0.18) or DEX exposure (P = 0.84; Figure 2-4). Photomicrographs representing average fields of view are shown in Figure 2—5. To evaluate our criterias for adipogenic differentiation, we compared percentage of preadipocytes with lipid droplets 2 10 pM—diameter with biochemical differentiation data (GPDH activity). Correlation analysis revealed linear relationships (r = 0.95, P < 0.001) between the percentage of morphological differentiated cells and GPDH activity in the three depots (Figure 2-6). Independently, the correlation between the percentage of morphological differentiated cells and GPDH activity was: IM preadipocytes (r = 0.97, P < 0.001); SC preadipocytes (r = .90, P < 0.001); and PR preadipocytes (r = 0.98, P < 0.001). The percentage of of cells containing lipid droplets smaller than 10 nM- diameter was not correlated (P = 0.19) with GPDH activity in SC preadipocytes. However, the percentage of cells containing lipid droplets smaller than 10 BM- diameter was negatively correlated with GPDH activity in IM preadipocytes (r = -0.54, P = 0.02), and tended to be negatively correlated with GPDH activity in PR preadipocytes (r = -0.44, P = 0.07). 75 Discussion To our knowledge, this is the first study that documents the expression of GR in cultured bovine preadipocytes. Immunoblot analysis of protein homogenates resulted in distinct GR immunoreactive bands of ~97, ~66, and ~48 kDa, which were similar among preadipocytes isolated from different bovine adipose tissues. Although only one GR gene has been identified, multiple promoters, alternative splicing, alternative translation initiation, and posttranslational modifications result in production of various isoforms that fractionate into several bands upon electrophoresis (Bronnegard et al., 1995; Yudt and Cidlowski, 2002). The ~97 kDa isoform is the best Characterized GRa isoform, and presumably the primary mediator of glucocorticoid action. We found that upon DEX exposure the ~97 kDa GR immunoreactive band was downregulated, which is similar to previous results in human adipocytes (Bronnegard et al., 1995). Upon glucocorticoid binding, GRot itself can inhibit GRa expression by repressing the transcription of the GR gene (Nobukuni et al., 1995) and by reducing GRa mRNA stability or translatability (Bamberger et al., 1996). The ability of DEX to downregulate the ~97 kDa GR immunoreactive band detected in our study suggests that cultured bovine preadipocytes express a functional GRot. 76 The function of the ~66 kDa putative GR isoform is uncertain. Although the abundance of the ~66 kDa immunoreactive band was not different among preadipocytes isolated from different adipose depots, its abundance was also decreased by DEX exposure, suggesting a possible role in glucocorticoid signaling. The specific identity of the ~48 kDa putative isoform is not known. However, this immunoreactive band may be functionally equivalent to a splice variant named GRp which has been identified in myeloma tumor cells (Yudt and Cidlowski, 2002). In some cells, the level of expression of this protein may represent 10 to 50% of the total GR proteins (de Lange et al., 2001). In our preadipocytes, the relative expression of the ~48 kDa immunoreactive band was 40% of the total GR immunoreactive bands. The GRp isoform has been documented to be constituted of only 676 amino acids, compared to 777 amino acids in GRa. Although GRp lacks a ligand binding domain (Yudt and Cidlowski, 2002), it may be involved in the upregulation of GRa mediated gene expression (de Lange et al., 2001). Bovine preadipocyte differentiation, as measured by GPDH activity, was induced by DEX, independent of the adipose tissue depot from which the cells were isolated. The responsiveness to glucocorticoids is closely related to the abundance of GR in many tissues (i.e. hepatic, lymphatic) (Rosseau et al., 1972; 77 Bamberger et al., 1996). The proportionally comparable induction of differentiation among IM, SC, and PR preadipocytes by DEX exposure is consistent with our observation that cultured bovine preadipocytes express similar quantities of GR. We hypothesized that glucocorticoids would preferentially enhance adipogenic differentiation in IM preadipocytes. However, the inferior GPDH activity of IM preadipocytes was not selectively improved by DEX exposure. This implies that differences in adipogenic capacity among these cell populations may be due to signaling pathways unrelated to GR. Although DEX increased GPDH activity in IM preadipocytes, we failed to detect an increase in the percentage of IM preadipocytes with large (2 10 um-diameter) lipid droplets upon DEX exposure. The lower adipogenic capacity of IM preadipocytes may suggest that cell cultures isolated from IM adipose tissue may contain a lower proportion of adipogenic cells. Nevertheless, under similar culture conditions (i.e. supplementation with bovine serum lipids) clonal analysis of cells isolated from IM and SC adipose tissue revealed no differences in the percentages of colonies that were adipogenic, although the number of cells accumulating lipid within a colony was higher in SC cells (Grant, 2005). Almost all mammalian cells accumulate minuscule lipid droplets that mainly serve as Cholesterol ester reservoirs used in the synthesis and 78 maintenance of membranes (Murphy and Vance, 1999; Wolins et al., 2005). Conversely, energy storage is primarily found in adipocytes that package triacylglycerides in large (10 to 100 pm in diameter) lipid droplets (W olins et al., 2005). Other cell types, have a limited capacity to store triacylglycerides and seldom accumulate large lipid droplets (Wolins et al., 2005). We used the presence of lipid droplets _>_ 10 tam-diameter as a morphological indicator of preadipocyte differentiation. The percentage of cells with lipid droplets 2 10 um-diameter was highly correlated with GPDH activity among the preadipocytes of the three depots, validating our criterias for determining adipogenic differentiation. In fact, the percentage of cells with lipid droplets < 10 um-diameter was not correlated with GPDH activity in SC preadipocytes, and was negatively correlated with GPDH activity in PR and IM preadipocytes. Glucocorticoid influence on preadipocyte differentiation has been well documented in various species (Ramsay et al., 1989; Xu and Bjorntorp, 1990) and cell lines (Gaillard et al., 1991; Smas et al., 1999). Interestingly, porcine PR preadipocytes have been reported as unresponsive to glucocorticoid exposure, while SC preadipocytes isolated from the shoulder and ham regions exhibited dose-dependent increases in GPDH activity in response to hydrocortisone (Ramsay et al., 1989). In our study, bovine PR preadipocytes were responsive to DEX and exhibited the highest propensity to differentiate. 79 Our results do not reveal the mechanism of action of DEX stimulation of bovine IM adipose tissue development observed by Brethour (1972). However, it is important to acknowledge that glucocorticoids have dramatic effects on whole body nutrient metabolism and an increase in IM fat accretion may be the indirect result of glucocorticoids creating a state of insulin resistance in SC adipose tissue and(or) other organs (Rebuffé-Scrive et al., 1988; Corah et al., 1995; Cortright et al., 1997), which could increase substrate availability for IM preadipocytes, and as a consequence, augment IM fat development. Dexamethasone equally promoted adipogenic enzyme activity in IM, SC, and PR bovine preadipocytes, and consequently did not abolish their adipogenic differences. It is then suggested that adipogenic differences among these cells are controlled by factors unrelated to GR expression or activation. Peroxisome proliferator-activated receptor yz (PPARyz) is considered the master regulator of adipogenesis (Schoonjans et al., 1996; Knouff and Auwerx, 2004). Therefore, differences in adipogenic capacity between bovine preadipocytes could be related to differences in PPARyz expression. However, the activity of PPAR‘yz is regulated by the availability of lipophilic molecules that could be different between preadipocyte populations. Consequently, adipogenic differences among bovine preadipocytes isolated from distinct adipose depots may result 80 from intrinsic differences in the endogenous activation of PPARyz among distinct bovine preadipocyte populations. We conclude that cultured bovine preadipocytes from IM, SC, and PR adipose tissue express similar quantities of GR immunoreactive bands. Dexamethasone exposure resulted in downregulation of ~97 and ~62 kDa GR immunoreactive bands in preadipocytes from all depots, suggesting similarity in at least one aspect of GR function. Bovine preadipocytes exhibited different propensities to biochemically differentiate (GPDH: PR > SC > IM), but DEX induced proportionally similar increases in GPDH activity in preadipocytes from all depots. In contrast with SC and PR preadipocytes, we failed to detect an increase in the proportion of IM preadipocytes with lipid droplets 2 10 um upon DEX expossure. Because differences among the preadipocyte populations may be related to signaling pathways unrelated to GR, future studies aiming to understand the observed differences among preadipocytes from different depots should focus on studying the expression of other regulators of adipogenesis and(or) differences in secretion of adipogenic molecules. Implications Cultured preadipocytes isolated from bovine intramuscular adipose tissue have a limited ability to accumulate lipid when compared to perirenal and subcutaneous preadipocytes. Because glucocorticoid receptor is equally 81 expressed in intramuscular, subcutaneous, and perirenal preadipocytes, and dexamethasone increased adipogenic enzymatic activity in equal proportions in these cell populations, the observed differences in adipogenic capacity among these cell populations appears to be unrelated to glucocorticoid receptor function. Experiments aiming to compare the expression of other proteins known to be involved in the regulation of preadipocyte differentiation may help decipher what is unique about intramuscular preadipocytes and facilitate the discovery of methods to selectively increase intramuscular lipid accretion in cattle. 82 Intramuscular Subcutaneous Perirenal Negative Control DEX Control DEX Control DEX Control 97 kDa mm: 66 kDa 45 kDa Figure 2-1. Glucocorticoid receptor immunoblot of bovine intramuscular , subcutaneous, and perirenal preadipocytes. Cells were grown to confluence and then exposed to 0 or 250 nM dexamethasone (DEX) for 48 h. Twenty micrograms of protein per sample were separated by gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Membranes were subsequently incubated overnight with a polyclonal antibody raised against the glucocorticoid receptor, which detected major immunoreactive bands of ~97, ~66, and ~48 kDA. Positions of molecular weight standards are indicated to the left. 83 .IM CISC .PR 01 l D. n Log GPDH specific activity, mU/mg protein OJ R\\\\\\\\\\\\\\\\\\\\\\W~% \\\\\\\\\\\\\\ 0 25 2500 Dexamethasone concentration, nM Figure 2-2. Effect of dexamethasone (DEX) on the activity of glycerol-3- phosphate dehydrogenase (GPDH) in bovine preadipocytes isolated from bovine intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of three steers. Bovine preadipocytes were grown to confluence and subsequently exposed to DEX for 48 h, and differentiation media for 10 additional days. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Bars represent means 4.- SEM. Means with different superscripts differ (P < 0.05). 84 ph 01 m 40- .IM 6 E _ EISC _§‘\° ::_ IPR d TEE- 25J % g c: 20-1 / c E X: 15‘ bc % .3 10_ ab% / 3‘ a ab a Q A 0 25 2500 Dexamethasone concentration, nM Figure 2-3. Effect of dexamethasone (DEX) concentration on the percentage of differentiated preadipocytes (cells with a lipid droplet _>_ 10 um). Bovine preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of three steers were grown to confluence and subsequently exposed to the indicated DEX concentrations for 48 h and differentiation media for 10 additional days. The percentage of differentiated preadipocytes was determined by microscopy 12 d after addition of treatments. Bars represent means i SEM. There was an interaction (P = 0.03) between DEX concentration and depot. Means with different superscripts differ (P < 0.05). 85 IIM USC IPR Cells with lipid droplets < 10 um, % 0 25 2500 Dexamethasone concentration, nM Figure 2-4. Effect of dexamethasone (DEX) concentration on the percentage of preadipocytes with a lipid droplet < 10 um. Bovine preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of three steers were grown to confluence and subsequently exposed to the indicated DEX concentrations for 48 h and differentiation media for 10 additional days. Preadipocyte lipid droplets were evaluated by microscopy 12 d after addition of treatments. Bars represent means i SEM. Means did not differ (P > 0.20). 86 Figure 2-5. Effect of dexamethasone (DEX) concentration on morphological differentiation of bovine preadipocytes. Bovine preadipocytes isolated from intramuscular (IM; a, b, c), subcutaneous (SC; (1, e, f), and perirenal (PR; g, h, i) adipose tissue of three steers were grown to confluence and subsequently exposed to 0 (a, d, g), 25 (b, e, h) or 2500 (c, f, i) nM DEX for 48 h and differentiation media for 10 additional days. Photomicrographs were taken 12 d after addition of treatments. Lipid droplets in cells were stained with oil red O and cell nuclei were counterstained with giemsa. Photomicrographs shown represent average fields of view. Bar = 100 uM (Panel i). 87 Figure 2-5. Effects of dexamethasone on morphological differentiation of bovine preadipocytes 60 55 - 50 q A [M 45 - I 40 - 35 - 0 SC 30 - | o 25 - c 20 a I. 15 - 10 '- Cells with lipid droplets 2 10 pm, % 0 I I I I I I I 0 50 100 150 200 250 300 350 400 GPDH specific activity, mU/mg protein Figure 2-6. Relationship between the specific glycerol-3-phosphate dehydrogenase (GPDH) activity and percentage of cells with a lipid droplet 2 10 pm (r = 0.95, P < 0.001) in preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissues. 89 Literature Cited Adams, M., C. T. Montague, J. B. Prins, J. C. Holder, S. A. Smith, L. Sanders, J. E. Digby, C. P. Sewter, M. A. Lazar, V. K. K. Chatterjee, and S. O'Rahilly. 1997. Activators of peroxisome proliferator-activated receptor y have depot-specific effects on human preadipocyte differentiation. J. Clin. Invest. 100:3149-3153. Ailhaud, G. 2001. Development of white adipose tissue and adipocyte differentiation. Page 27-55 in Adipose Tissues. S. Klaus ed. Eurekah Publ., Georgetown,TX. Bamberger, C. M., H. M. Schulte, and G. P. Chrousos. 1996. Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids. Endoc. Rev. 17:245-261. Bishop-Bailey, D., and J. Wray. 2003. 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Endocrin. 16:1719-1726. 93 CHAPTER III: DIFFERENCES IN ADIPOGENESIS BETWEEN BOVINE INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES ARE NOT RELATED TO EXPRESSION OF PEROXISOME PROLIFERATOR- ACTIVATED RECEPTOR GAMMA TWO OR SECRETION OF PROSTACYCLIN Abstract The objectives of these experiments were to determine if intramuscular (IM) and subcutaneous (SC) bovine preadipocytes differ in expression of peroxisome proliferator activated receptor yz (PPARyz) or in secretion of prostacyclin (PG12), a presumptive endogenous PPAR activator. Preadipocytes isolated from IM and SC adipose tissues of three steers were propagated in culture and upon confluence were exposed to 0 or 25 nM dexamethasone (DEX) for 48 h. After exposure to differentiation media for an additional 10 d, cell lysates were subjected to PPARyz immunoblot analysis, which revealed an immunoreactive band of ~53 kDa. There was no interaction between DEX treatment and preadipocyte depot (P = 0.90) in the relative expression of PPARyz. Expression of PPARyz was also equivalent between IM and SC preadipocytes (P = 0.39), and DEX did not affect PPARyz abundance (P = 0.98). Heterogeneous preadipocytes isolated from a steer and clonal preadipocytes derived from a second steer were grown to confluence and exposed to 0 or 25 nM DEX for 48 h. Media were collected every 12 h for 48 h and assayed for the stable PG12 derivatives 6-keto-prostaglandin Fla (Ok'PGFla) and 2,3-dinor-6-keto- 94 prostaglandin Fla (2,3d-6k-PGF1a). After 12 d in differentiation media, glycerol- 3-phosphate dehydrogenase (GPDH) analysis was performed. Intramuscular preadipocytes secreted more PG12 derivatives than SC preadipocytes (P = 0.046) and DEX decreased secretion of the PG12 derivatives equally in cells from both depots (P = 0.001). Although 25 nM DEX increased GPDH activity in both preadipocyte populations (P < 0.001), IM preadipocytes were less adipogenic than SC preadipocytes (P < 0.001). Exposure of clonal SC preadipocytes to 1 pM CPGIz tended (P = 0.06) to enhance differentiation over control conditions, and tended (P = 0.09) to increase the adipogenic enhancement stimulated by DEX. However, supplementation with 0.01 or 0.1 pM CPGIz did not increase differentiation (P > 0.65). Although ibuprofen (IBU), an inhibitor of PG12 synthesis, did not affect adipogenesis in Clonal SC preadipocytes (P = 0.99), 100 pM IBU enhanced (P = 0.01) adipogenesis in the presence of DEX while an intermediate concentration (50 pM) tended (P = 0.08) to enhance DEX effects. We conclude that adipogenic differences between IM and SC bovine preadipocytes are not explained by differences in PPARyz expression or PG12 secretion. Introduction Bovine preadipocytes isolated from intramuscular (IM) and subcutaneous (SC) adipose tissue have different propensities to accumulate lipid (Chapter II). 95 This likely results from differences in the expression or activation of proteins involved in the regulation of adipogenesis. Peroxisome proliferator activated receptor gamma two (PPARyz), a ligand activated transcription factor, is considered the master regulator of adipocyte differentiation and lipid accretion (Schoonjans et al., 1996; Knouff and Auwerx, 2004). In humans, PPARyz mRNA expression is greater in SC preadipocytes than in visceral preadipocytes (Sewter et al., 2002). Although the expression of PPARyz has been documented in bovine perirenal preadipocytes (Ohyama et al., 1998), its expression in bovine preadipocytes from economically important adipose depots has not been reported. Stimulation of adipogenesis by PPARyz depends on its activation by ligands, such as those derived from arachidonic acid (Bishop-Bailey and Wray, 2003; Knouff and Auwerx, 2004). One of these derivatives, prostacyclin (PG12), is considered a PPAR activator (Aubert et al., 1996; Hertz et al., 1996; Wise, 2003) and an inducer of mouse (Ob1771) preadipocyte differentiation (Négrel et al., 1989; Négrel, 1999). We hypothesized that SC bovine preadipocytes are more adipogenic than IM preadipocytes because SC preadipocytes express more PPARyz and secrete more PG12. Therefore, the objectives of this study were to compare PPARyz expression and PG12 secretion between IM and SC bovine preadipocytes. 96 Materials and Methods Isolation of bovine preadipocytes Preadipocytes from IM and SC adipose tissue were isolated using a modification of a protocol previously published (Forest et al., 1987) and described in Chapter II. Preadipocyte cloning Preadipocytes from IM and SC adipose tissue of one steer were seeded at 0.05 g per cm2 in 35 mm-diameter cell culture wells (Corning Inc.) and grown to 70% confluence. Cells were then trypsinized (0.5 g/L trypsin and 0.02 g/L ethylenediaminetetraacetic acid (EDTA) in PBS [pH 7.2]) and seeded at 5 cells/cm2 in 100 mm-diameter culture plates or at one cell/6.2 mm-diameter well in 96-well plates. Cells were then incubated in growth medium for 10 d. Individual colonies were isolated utilizing Cloning-rings in 100 mm-diameter culture plates, or directly from 6.2 mm-diameter wells. Each Clone was individually transferred into 16 mm-diameter culture wells. All surviving Clones were then grown and sequentially transferred into 35 mm-diameter culture plates, then into 100 mm-diameter culture plates (1,800 cells/cmz) after reaching 70% confluence. Cells were cryopreserved after a sub-sample of each Clone (IM n = 48; SC n = 64) was seeded at 10,000 cells/cm2 in 16 mm-diameter cell culture wells for evaluation of their adipogenic capacity. Clones were 97 allowed to proliferate to confluence in growth medium, which was replaced every 2 d. After reaching confluence, cells were washed twice with PBS and exposed to differentiation medium: DMEM (1% FBS) supplemented with 1.74 nM insulin, 20 mM glucose and 10 mM acetate. Differentiation medium was supplemented with 250 nM dexamethasone (DEX) for 48 h. After 48 h, DEX treated medium was replaced with fresh differentiation medium, and subsequently fresh medium was provided every 2 d for 10 d. Adipogenic Clones were identified morphologically as those containing at least two cells stained with oil red O. The oil red O solution was prepared and used following a protocol previously described (Ramirez-Zacarias et al., 1992). An individual adipogenic clone from each adipose depot (IM and SC) was thawed at the time of the experiments. General procedures Cell culture. Preadipocytes from IM and SC adipose tissue were seeded at a density of 4,160 cells/cm2 in 35 mm-diameter cell culture wells. Cells were allowed to proliferate to confluence (4 d) in a humidified atmosphere (37°C, 95% air and 5% C02) while incubated in growth medium (DMEM [5.5 mM glucose] supplemented with antibiotic-antimycotic (Final concentration: 100 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate and 0.25 ug/mL amphotericin B), 0.05 mg/mL gentamicin, 33 pM biotin, 17 PM pantothenate, 200 pM ascorbate, 98 1,000 pM octanoate, and 10% fetal bovine serum [FBS]). Unless otherwise stated, all reagents were of tissue culture grade and were purchased from Sigma (St. Louis, MO). Growth medium was replaced every 2 d. After reaching confluence, plates were washed twice with PBS and differentiation treatments were applied. Differentiation medium was DMEM (5.5 mM glucose), supplemented with antibiotic-antimycotic (Final concentration: 100 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate and 0.25 pg/mL amphotericin B), 0.05 mg/mL gentamicyn, 33 BM biotin, 17 BM pantothenate, 200 pM ascorbate, 280 nM bovine insulin and 5 pL/mL bovine serum lipids (Ex-Cyte; Serologicals Corp., Norcross, GA). Unless otherwise specified, DEX was included in the differentiation media for the initial 48 h, after which, cells were washed twice with PBS and fresh treatment media were provided every 2 d for an additional 10 d. Glycerol-B-phosphate dehydrogenase activity. Cell differentiation was quantified biochemically by measuring glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity using a modification of a method previously published (Adams et al., 1997) and described in Chapter II. Experiment 1: Analysis of PPARyz expression This experiment was conducted to compare the expression of PPARyz between bovine IM and SC preadipocytes. Primary preadipocytes from the IM 99 and SC adipose tissue of three steers were propagated in culture and secondary cultures were grown to confluence and exposed to differentiation medium supplemented with 0 or 25 nM DEX for 48 h. Fresh differentiation medium was provided for an additional 10 d. Each treatment was applied to two 35 mm- diameter wells of a 6-well plate, in two replicate plates for each of the three steers. Immunoblot Analysis. Cell monolayers were washed twice with ice-cold PBS and subsequently solubilized by the addition of hot (95°C) electrophoresis sample buffer (62 mM Tris-HCL (pH 6.8), 2% SDS, and 10% glycerol). Cell lysates from two wells per treatment were pooled and immediately stored at - 20°C. Protein concentrations were determined using the bicinchoninic acid assay (BCA, Pierce Biotechnology Inc., Rockford, IL). Prior to electrophoresis, protein samples were thawed and diluted to equal protein concentrations (0.83 [lg/pl.) by the addition of electrophoresis sample buffer supplemented with B- mercaptoethanol (5%) and bromophenol blue (0.01%). Samples were then boiled for 3 min and 50 ug of protein per sample were subjected to SDS-PAGE using 12.5% (37.521 acrylamide/bis acrylamide) (Bio-Rad Laboratories, Hercules, CA) separating mini-gels (0.75 mm thick), with 4% (37.5:1 acrylamide/bis acrylamide) stacking gels. After SDS-PAGE, proteins were transferred to polyvinylidine difluoride membranes at 4°C for 2 h at 100 V in a buffer 100 containing 25 mM Tris, 193 mM glycine, and 15% methanol, using a Bio Rad Mini-Trans-Blot electrophoretic transfer cell. Pre-stained molecular weight standards were used. The membranes were cut above the 45 kDa band to allow for individual PPARyz and B-actin immunolabeling. Nonspecific antibody binding was prevented by incubating the membranes for 1 h in blocking solution (Tris buffered saline [198.2 mM Tris, 1.3 M NaCl, 26.8 mM KCl; pH 7.2] containing 0.1% Tween-20 [Bio-Rad Laboratories] and 5% non-fat dry milk). The membranes were then incubated for 2 h at room temperature in blocking solution containing 1 ug/mL of a polyclonal (rabbit) anti-PPARyz antibody (PA1-824, Affinity BioReagents, Inc., Golden, CO). After the primary antibody incubation, membranes were washed three times with blocking solution and incubated for 1 h in blocking solution containing 1:1000 (vol/vol) of an alkaline phosphatase conjugated goat anti-rabbit IgG antibody (A3683, Sigma, Inc.). Membranes were then washed three times with blocking solution and three times with TBS (0.1% Tween-20), and immunoreactive bands were detected upon the addition of 5-bromo-4-Chloro-3-indoyl phosphate/nitroblue- tetrazolium (Bio-Rad Laboratories). Utilizing a similar procedure, the appropriate membrane sections were incubated for 1 h in blocking solution containing 0.16 ug/mL of a monoclonal anti-B-actin antibody (ab6276, Ab Cam, Inc., Cambridge, UK) and subsequently incubated for 1 h in blocking solution 101 containing 1:1000 (vol/vol) of an alkaline phosphatase conjugated goat anti- mouse IgG antibody (A3562, Sigma, Inc.). Images of immunoblots were acquired using a Fluor-S Multilmager (Bio-Rad Laboratories) and analyzed with Discovery Series Quantity One l-D Analysis Software (Bio-Rad Laboratories). Abundance of PPARyz immunoreactive bands was normalized based on B-actin immunoreactive band intensity to account for loading differences. Experiment 2. Evaluation of PG12 secretion and GPDH activity. The purpose of this experiment was to compare the secretion of PG12 between bovine IM and SC preadipocytes. Secondary IM and SC preadipocytes from one steer, in addition to clonal preadipocytes (6th passage) derived from IM and SC adipose tissue of a second steer were utilized. Cells were exposed to differentiation medium supplemented with 0 or 25 nM DEX for 48 h. Each treatment was applied to two 35 mm—diameter wells of a 6-well plate, in two replicate plates for each of the two steers. After 12 d in differentiation medium, GPDH analysis was performed. Because equivalent results were obtained from heterogeneous and clonal preadipocytes, and there was no interaction between cell type (heterogeneous or clonal preadipocytes), DEX treatment, depot and(or) time (P > 0.15), the results obtained from heterogeneous and Clonal preadipocytes were pooled. 102 Measurements of PG12. Because PG12 has a half life of only 2 to 3 min in buffer, PG12 secretion was quantified indirectly by the measurement of its non- enzymatic hydration products 6-keto-prostaglandin Fla (6k-PGFia) and 2,3- dinor-6-keto-prostaglandin Fm (2,3d-6k-PGF1a) with an enzyme immunoassay kit (900-025, Assay Designs, Inc., Ann Arbor, MI). Samples of 250 uL of differentiation media supplemented with 0 or 25 nM DEX were collected at 12, 24, 36, and 48 h and immediately frozen at -80°C until assayed. Within one week of sample collection, media samples were thawed and 100 uL per sample were incubated in each of duplicate wells in a 96-well assay plate for 2 h with a polyclonal (sheep) anti-6-keto—PGF1a antibody and alkaline phosphatase- conjugated 6k'PGF1a. The assay plate was placed in a plate shaker (Lab-Line Instruments Inc.) at 500 rpm during incubation. The wells were then emptied and washed 3 times (300 uL wash solution per well). Wells were then incubated for 45 min without shaking, with 200 uL of alkaline phosphatase substrate (p- nitrophenyl phosphate). Finally, 50 pL of stop solution were added to each well and the optical density was immediately read at 405 nm in a spectrophotometer (Versamax Tunable Microplate Reader, Molecular Devices), and the concentration of Ok-PGFla and 2,3d-6k-PGF1a was quantified by using a standard curve developed by the serial dilution of 2,3d-6k-PGFia. The standard curve concentrations utilized were 7.81, 31.25, 125, 500, and 2000 pg/mL. Nonspecific 103 binding and background optical density were calculated and used to correct the spectrophotometer readings. The concentration of PG12 derivatives were determined by interpolation from the standard curve utilizing a 4 parameter logistic curve fitting program (TableCurve 2D, Statistical Solutions, Saugus, MA). Prostacyclin derivatives were not detected in media (0 or 25 nM DEX) before exposure to preadipocytes. Experiment 3: Evaluation of the effects of carbaprostacyclin on preadipocyte differentiation This experiment aimed to evaluate the effects of carbaprostacyclin (CPGIz), at concentrations previously shown to induce adipogenic differentiation in Ob1771 mouse preadipocytes (Catalioto et al., 1991), on the adipogenic differentiation of bovine SC preadipocytes. Clonal SC preadipocytes (6th passage) were grown to confluence and exposed to unsupplemented differentiation media (control), or differentiation media supplemented with 1 uM cPGlz, or 25 nM DEX in combination with 0, 0.01, 0.1, or 1.0 pM cPGIz for 48 h. Carbaprostacyclin was diluted in ethanol (5 mg/mL), and treatments not containing CPGIz were supplemented with equivalent concentrations of ethanol. For this and all subsequent experiments, treatment media were applied for 48 h. Treatment media were then replaced with fresh differentiation medium, and fresh medium was provided every 2 d for 10 d. Each treatment was applied to 104 two wells of a 6-well plate, in two replicate plates. After 12 d in differentiation media GPDH analysis was performed. Experiment 4: Evaluation of the effects of ibuprofen in preadipocyte differentiation This experiment was designed to evaluate the effect of ibuprofen (IBU), a cyclooxygenase (COX) inhibitor, on Clonal SC preadipocyte adipogenesis. Cyclooxygenase is the enzyme that catalyzes the rate-limiting reaction during the biosynthesis of PG12 and other prostanoids (Funk, 2001). Clonal SC preadipocytes (6th passage) were grown to confluence and exposed to unsupplemented differentiation media (control), or differentiation media supplemented with 100 [1M IBU, or 25 nM DEX in combination with 0, 10, 50, or 100 ptM IBU for 48 h. Statistical analysis Data were analyzed using the Mixed Model procedure of SAS (SAS, Cary, NC). In all experiments, pooled samples from two 35 mm-diameter wells of a 6- well-plate were considered the experimental unit. When main effects were significant (P < 0.05), differences between means were evaluated utilizing Tukey’s multiple comparison test. In Exp. 1, means were calculated using the fixed effects of DEX, depot, and DEX >< depot. In Exp. 2, PG12 data were analyzed with repeated measures, where means were calculated using the fixed effects of depot, treatment (0 or 25 nM DEX), time, and their interactions. Means 105 for GPDH data were calculated using the fixed effects of DEX, depot, and DEX x depot. In Exp. 3, means for GPDH data were calculated using the fixed effects of DEX, and CPGIz. In Exp. 4, means for GPDH data were calculated using the fixed effects of DEX and IBU. In Exp. 2, 3, and 4, GPDH data, and in Exp. 2, PG12 data, were loge transformed to satisfy the conditions of normality and homogeneity of variance. In Exp 1. and 2, steer and steer by replication were included as random variables, while in Exp. 3 and 4 replication was included as a random variable. Results and Discussion The objectives of these studies were to characterize differences in PPARyz expression and PG12 secretion between preadipocytes isolated from economically important adipose depots (i.e. IM and SC), and evaluate their relation to the differences in adipogenic capacity exhibited by these cell populations. Experiment 1 We hypothesized that differences in adipogenic capacity between bovine preadipocytes were related to differences in PPARyz expression. Immunoblot analysis revealed a PPARyz immunoreactive band of ~53 kDa in control and DEX treated IM and SC preadipocytes (Figure 3-1). No interaction (P = 0.90) between DEX treatment (0 or 25 nM) and depot (IM or SC) existed for PPARyz 106 protein expression. Expression of PPARyz was not different between IM and SC bovine preadipocytes (P = 0.39) and DEX treatment had no effect on the abundance of this protein (P = 0.98). Human SC preadipocytes express more PPAR-Y2 mRNA than omental (OM) preadipocytes (Sewter et al., 2002). Conversely, no differences in the expression of PPARyz protein have been detected between human SC and OM preadipocytes (Adams et al., 1997). Although they exhibit similar patterns of PPARyz protein expression, human OM preadipocytes are less adipogenic, and have been shown to be less sensitive to a PPARyz agonist (rosiglitazone) than SC preadipocytes (Adams et al., 1997). The expression of PPARyz has been previously reported in bovine perirenal preadipocytes (Ohyama et al., 1998), but our study is the first to establish that PPARyz protein expression is equivalent in bovine IM and SC preadipocytes. Differences in adipogenic capacity between bovine SC and OM preadipocytes were abolished when a PPARyz ligand (indomethacin) was added to differentiation media (Wu et al., 2000). This suggests that adipogenic differences between preadipocytes from different adipose depots may be related to differences in PPARyz ligand synthesis rather than PPARyz expression. The activity of PPARyz may be regulated by the availability of lipophilic molecules 107 like PG12 (Hertz et al., 1996; Bishop-Bailey and Wray, 2003), a potent adipogenic inducer (Négreleta1., 1989; Négrel, 1999). Preadipocyte differentiation has been shown to be dependent on the presence of arachidonic acid (Gaillard et al., 1989). To date, PG12 is the only adipogenic arachidonic acid metabolite demonstrated to be produced by preadipocytes (Aubert et al., 1996; Négrel, 1999; Ailhaud, 2001). Cyclooxygenase catalyzes the rate-limiting reaction in the biosynthesis of PG12 (Figure 3-2). Exposing Ob 1771 preadipocytes to COX inhibitors abrogates adipogenic differentiation. Because the abrogation of Ob 1771 preadipocyte adipogenesis caused by COX inhibitors is prevented by concomitant exposure to CPGlz (Négrel et al., 1989), it has been suggested that Ob 1771 preadipocyte adipogenesis may be regulated through PG12 secretion (Gaillard et al., 1991). Experiment 2 We hypothesized that bovine IM preadipocytes were less adipogenic than SC preadipocytes because they secreted lower levels of PG12. However, it was found that IM preadipocytes secreted greater amounts of PG12 (P = 0.046) (Figure 3-3). There were no 2- or 3-way interactions among DEX, depot, and time for PG12 secretion (P > 0.26). The concentration of PG12 in differentiation media increased with time, up to 36 h, independent of DEX treatment or depot (P < 108 0.001), and exposure to DEX decreased PG12 secretion in IM and SC preadipocytes (P = 0.002). In contrast to our results, DEX was shown to increase PG12 secretion in Ob 1771 preadipocytes (Gaillard et al., 1991). However, consistent with our results, DEX has previously been shown to decrease PG12 secretion in non-adipose cultured cells (Blackwell et al., 1980; Hullin et al., 1989; Rosenstock et al., 1997), and to reduce prostaglandin synthesis through inhibition of phospholipase A2 (Figure 3-2), the enzyme that catalyzes the endogenous release of arachidonic acid, the precursor of prostaglandins (Heiko Miihla et al., 1992). Although DEX decreased PG12 secretion, DEX also stimulated GPDH activity in IM and SC preadipocytes as expected (P < 0.001) (Figure 3-4). There was no interaction (P = 0.64) between DEX concentration and adipose tissue depot. However, IM preadipocytes exhibited less propensity to differentiate than SC (P < 0.001). Experiment 3 Because IM preadipocytes were least adipogenic than SC preadipocytes while secreting higher levels of PG12, we proceeded to investigate if PGI2 may have an inhibitory role in the adipogenesis of bovine preadipocytes. Arachidonic acid, the precursor of PG12 synthesis, has been reported to inhibit lipogenic gene expression (Mater et al., 1998). The arachidonic acid mediated 109 abrogation of adipogenesis is reversed by inhibitors of COX (Casimir et al., 1996; Mater et al., 1998; Petersen et al., 2003). Because COX is the rate limiting enzyme in the synthesis of prostaglandins, these experiments suggest that arachidonic acid anti-adipogenic effects are mediated through prostaglandins (Figure 3-2). Therefore, we evaluated the effects of micromolar concentrations of CPG12 in the adipogenic differentiation of bovine Clonal SC preadipocytes. As expected, DEX enhanced preadipocyte adipogenesis (P = 0.04) (Figure 3-5). Exposure to 1 uM CPG12 tended (P = 0.06) to enhance differentiation over control conditions, and tended (P = 0.09) to increase the adipogenic enhancement stimulated by DEX. However, supplementation with 0.01 or 0.1 pM CPG12 did not increase differentiation (P > 0.65), even though these concentrations are more than 25 times higher than our previous measurements of PG12 secretion (3.52 nM). Prostacyclin (1 nM) may have tended to enhanced differentiation acting as a PPARyz ligand. Contrary to our experiment in which we utilized a lipid supplement (5 pL/mL), previous work performed with Ob 1771 preadipocytes (Gaillard et al., 1989; Négrel et al., 1989; Gaillard et al., 1991) has utilized serum free media supplemented only with arachidonic acid as a source of fatty acid. We speculate that in the absence of a variety of fatty acids, CPG12 may induce adipogenesis more dramatically than when preadipocytes are exposed to a variety of lipids and fatty acids that serve as PPARyz ligands or PPARyz ligand 110 precursors. Although PG12 may play a role in stimulating bovine preadipocyte adipogenesis, lower secretion of PG12 does not explain the greater propensity of SC preadipocytes to differentiate, when compared with IM preadipocytes. Experiment 4 Intramuscular preadipocytes were less adipogenic than SC preadipocytes while secreting higher levels of PG12. Furthermore, micromolar concentrations of CPG12 enhanced Clonal SC preadipocyte adipogenesis. To examine the role of endogenous PG12 in Clonal SC preadipocyte adipogenesis, we exposed the preadipocytes to IBU, a COX inibitor. We found that 10 [1M IBU did not affect DEX enhancement of differentiation (P = 0.99) (Figure 3-6). In contrast, we found that 100 uM IBU enhanced (P = 0.01) DEX induction of preadipocyte differentiation, while an intermediate concentration (50 pM) tended (P = 0.08) to enhance DEX effects. However, exposing the cells to 100 uM IBU without DEX did not have an effect on differentiation when compared to control conditions (P = 0.99). In agreement with our study, Lehmann et a1. (1997) found that, in the absence of DEX, 100 uM IBU failed to promote lipogeneis in C3H10T1/2 murine fibroblasts, even though cells were exposed to IBU for 9 d. High concentrations of IBU (2 100 nM) have been shown to activate PPARyz, suggesting that IBU may stimulate adipogenesis through a COX independent mechanism. 111 Because IBU has been previously shown to inhibit COX activity at a concentration of 10 [1M (Rome and Lands, 1975; Mitchell et al., 1993; Neupert et al., 1997), but this concentration did not affect adipogenesis in our study, the adipogenic role of endogenous PG12 seems unlikely. Previously, the role of endogenous PG12 has been questioned because the concentration of PG12 required to activate PPAR in vitro (Yu et al., 1995; Brun et al., 1996; Hertz et al., 1996; Gupta et al., 2000) and trigger Ob 1771 preadipocyte differentiation is in the micromolar range (Négrel et al., 1989; Catalioto et al., 1991; Gaillard et al., 1991; Aubert et al., 1996; Aubert et al., 2000), which is significantly higher than that measured in Ob 1771 mouse preadipocytes (Catalioto et al., 1991), primary cultures of rat preadipocytes (Shillabeer et al., 1998), and bovine endothelial cells (Rosenstock et al., 1997). Therefore, we hypothesized that other arachidonic acid metabolites could be adipogenic regulators responsible for differences between IM and SC preadipocytes. Indeed, arachidonic acid derivative products of lipoxygenase (LOX) enzymatic action, like 8[s]—HETE (Figure 3-2), are PPAR activators and inducers of 3T3-L1 preadipocyte adipogenesis (Yu et al., 1995). In addition, exposing mouse preadipocytes to nordihydroguaracetic acid (NDGA), a LOX inhibitor, reduces their adipogenesis (Shillabeer et al., 1998; Madsen et al., 2003). However, we found that adding NDGA (10 or 20 pM) to differentiation media had no effect (P > 0.95) on the differentiation of bovine clonal SC 112 SC DEX (nM) 0 PPARyz-t 53kDa ' i ‘g’ I B actinq ; 45 kDa I Negative control Figure 3-1. Western immunoblot of PPARyz from cultured bovine preadipocytes. Preadipocytes isolated from intramuscular (IM) and subcutaneous (SC) adipose tissue were grown to confluence and then exposed to O or 25 nM dexamethasone (DEX) for 48 h. Twelve days after addition of treatments, total protein was collected and 50 pg of protein per sample were separated by gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Membranes were cut above the 45 kDa band and the appropriate portions were incubated with an antibody raised against peroxisome proliferator-activated receptor yz (PPARyz) or B-actin, which resulted in immunoreactive bands of ~53kDa (PPARyz) and ~45kDa (B-actin). Membrane sections used as negative control were not incubated with the antibody raised against PPARyz, but all other procedures were identical among all membrane sections. Positions of molecular weight standars are indicated to the left. 114 preadipocytes (Appendix D). Concentrations of 40 pM or higher were toxic to the preadipocytes. In conclusion, 1M preadipocytes are less adipogenic than SC preadipocytes even though they similarly expressed PPARyz, an important regulator of adipogenesis. Intramuscular preadipocytes secreted higher levels of PG12 than SC preadipocytes, and supplementing SC preadipocytes with CPG12 and(or) IBU, an inhibitor of PG12 synthesis, did not have an effect on SC preadipocyte adipogenesis. Therefore, differences in PG12 secretion do not explain the greater adipogenic capacity of bovine SC preadipocytes. We propose that adipogenic differences between IM and SC preadipocytes are a result of differences in the secretion of a biologically relevant PPARyz ligand that is not PG12. Implications Adipogenic differences between intramuscular and subcutaneous preadipocytes are not explained by differences in the expression of peroxisome proliferator-activated receptor gamma two (PPARyz), an important regulator of adipogenesis, or by differences in the secretion of a presumptive PPAR activator, prostacyclin. Consequently, adipogenic differences between intramuscular and subcutaneous preadipocytes may be a result of differences in the synthesis of a biologically relevant PPARyz ligand that is not prostacyclin. 113 Linoleic acid ' . ‘ ,- Extracellular shirt)” i523 2225281 ‘ 8, AraChldOfllC 301d 1 Glycerophospholipids ’Effifteti itffifixflt’ ,. Intracellular .. Arachidonic acid ’100X1and2 .lllI-IIII- 8[s]-HETE Prostaglandin H2 PGI2 synthase Prostacyclin (PGI2) Figure 3-2. Simplified diagram of the arachidonic acid metabolism cascade. Arachidonic acid is derived from linoleic acid, an essential fatty acid which must be obtained from the diet. Arachidonic acid is normally found esterified to glycerophospholipids found in cellular membranes. Activation of phospholipase A2 (PLA2) results in the release of arachidonic acid and its subsequent metabolism by various enzymes like cyclooxygenase 1 and 2 (COX), lipoxygenase (LOX), and cytochrome P450 (not shown). Arachidonic acid metabolism through COX results in the production of prostaglandin Hz which is the precursor of prostanoids (PGDz, PGE2, PGan, TXA2, and PG12). Arachidonic acid metabolism through LOX results in the production of hydroperoxyeicosatetraenoic (HPETE) acids which are precursors of 8-[s]-HETE and other leukotrienes and lipoxins. Dexamethasone (DEX) inhibits PLA2 activity, ibuprofen (IBU) is an inhibitor of COX activity, and nordihydroguaracetic acid (NDGA) is a LOX inhibitor. 115 S" o + IM Control ' I 'IM DEX + SC Control - A 'SC DEX 1“ 01 l Log concentration of 6k-PGFiot and 2,3d-6k-PGF1a, pg/mL 9: it:- 01 O .- .- ‘- 9’ c -I— 12 24 36 48 Time after DEX addition, h Figure 3-3. Effect of dexamethasone (DEX) on the combined secretion of the prostacyclin (PG12) derivatives 6k-PGFiot and 2,3d-6k-PGF10I from bovine heterogeneous and clonal preadipocytes isolated from intramuscular (IM) and subcutaneous (SC) adipose tissue. Preadipocytes were grown to confluence and exposed to 0 nM (control) or 25 nM DEX. Points represent PG12 derivative concentration as means i SEM (Depot effect, P = 0.046; DEX effect, P = 0.002; Time effect, P < 0.001). 116 . 5- c if Iran __l__ .3 :5 4‘EISC b DO 55 8 I eas- tr ES 2- m8 0 a 22° 1- i—l O... O 25 Dexamethasone concentration, nM Figure 3-4. Effect of dexamethasone (DEX) on the activity of glycerol-3- phosphate dehydrogenase (GPDH) in bovine heterogeneous and Clonal preadipocytes isolated from bovine intramuscular (IM) and subcutaneous (SC) adipose tissue of two steers. Bovine preadipocytes were grown to confluence and exposed to DEX for 48 h. Glycerol-3-phosphate dehydrogenase activity was determined 12 d addition of treatments. Means with different superscripts differ (P 0.54). Conversely, exposure to 1000 or 2000 pM IBU enhanced DEX stimulation of GPDH activity in IM preadipocytes 124 (P = 0.01). In Exp. 3, heterogeneous IM and SC preadipocytes were exposed to 0 or 25 nM DEX and IBU at 0, 10, 100, 500, or 1000 pM for 12 (1. Exposure to 100 pM and 500 pM IBU enhanced DEX stimulation of differentiation in IM preadipocytes, while only 100 pM IBU enhanced DEX induction of differentiation in SC (P S 0.05). In Exp. 4, heterogenous IM and SC preadipocytes were exposed to 0 or 25 nM DEX for 48 h. In addition, cells were exposed to IBU at 0, 10, 100, 500, or 1000 pM, or aspirin (ASP) at 0 or 500 pM for 12 d. Exposure to 10, 100, and 500 pM IBU enhanced GPDH activity in SC preadipocytes (P < 0.001), while 500 pM IBU enhanced GPDH activity in lM preadipocytes (P < 0.004). However, the maximum induction of GPDH activity by 500 pM IBU was much greater in IM than SC preadipocytes (12-fold vs. 1.7- fold over control, respectively). Contrary to the effect of IBU, 500 pM ASP did not affect GPDH activity either alone (P > 0.37), or combined with DEX (P > 0.60) in either cell population. Because IBU diminished adipogenic differences between IM and SC preadipocytes, we conclude that their adipogenic differences are partially related to differences in the endogenous activation of PPARyz. Introduction Bovine intramuscular (IM) and subcutaneous (SC) preadipocytes differ in adipogenic capacity (Chapter II and III) while equally expressing peroxisome 125 proliferator activated receptor yz (PPARyz) (Chapter III), a key regulator of adipogenesis. Because PPARyz function depends on its activation by ligands (Bishop-Bailey and Wray, 2003), adipogenic differences between IM and SC preadipocytes may be related to differences in the synthesis of biologically relevant PPARyz ligands, other than prostacyclin (PG12) (Chapter III). Supplementation with an exogenous PPARyz ligand, troglitazone (TGZ), equally stimulated differentiation of bovine 1M and SC preadipocytes (Grant, 2005). Conversely, supplementation with the PPARyz ligand indomethacin preferentially induced adipogenesis in bovine omental (OM) preadipocytes when compared to SC preadipocytes, which resulted in the elimination of their adipogenic differences (Wu et al., 2000). Furthermore, although human SC and OM preadipocytes similarly express PPARyz, rosiglitazone, another PPARyz ligand, exclusively stimulates adipogenesis in SC preadipocytes (Adams et al., 1997). Consequently, specific PPARyz ligands may selectively enhance adipogenesis of bovine IM preadipocytes, and thereby, selectively stimulate IM fat (marbling) development. Ibuprofen (IBU), a cyclooxygenase (COX) inhibitor that also is a PPARyz ligand (Lehmann et al., 1997), induces adipogenesis in murine preadipocytes (Ye and Serrero, 1998). Ibuprofen has also been shown to marginally increase bovine SC preadipocyte differentiation (Chapter 111). Because bovine IM 126 preadipocytes may have a limited adipogenic capacity as a result of limited synthesis of biologically relevant PPARyz ligands, we hypothesized that IBU, a PPARyz ligand, would preferentially enhance adipogenesis of bovine lM preadipocytes. The objectives of these experiments were to evaluate the effects of IBU on adipogenesis of IM and SC preadipocytes. Materials and Methods Isolation and cloning of bovine preadipocytes Preadipocytes from IM, and SC adipose tissue were isolated using a modification of a protocol previously published (Forest et al., 1987) and described in Chapter II. Preadipocytes from IM and SC adipose tissue of one steer were cloned as described in Chapter 111. General Procedures Cell culture. Preadipocytes from IM and SC adipose tissue were seeded at a density of 4,160 cells/cm2 in 35 mm—diameter cell culture wells. Cells were allowed to proliferate to confluence (4 d) in a humidified atmosphere (37°C, 95% air and 5% C02) while incubated in growth medium (DMEM [5.5 mM glucose] supplemented with antibiotic-antimycotic (Final concentration: 100 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate and 0.25 pg/mL amphotericin B), 0.05 mg/mL gentamicin, 33 pM biotin, 17 pM pantothenate, 200 pM ascorbate, 1,000 pM octanoate, and 10% fetal bovine serum [FBS]). Unless otherwise 127 stated, all reagents were of tissue culture grade and were purchased from Sigma (St. Louis, MO). Growth medium was replaced every 2 (1. After reaching confluence, plates were washed twice with PBS and differentiation treatments were applied. Differentiation medium was DMEM (5.5 mM glucose), supplemented with antibiotic-antimycotic (Final concentration: 100 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate and 0.25 pg/mL amphotericin B), 0.05 mg/mL gentamicyn, 33 pM biotin, 17 pM pantothenate, 200 pM ascorbate, 280 nM bovine insulin and 5 pL/mL bovine serum lipids (EX-Cyte; Serologicals Corp., Norcross, GA). After 48 h of DEX exposure, plates were washed twice with PBS and differentiation medium was replaced with fresh media every 2 d for 10 d. Glycerol-B-phosphate dehydrogenase activity. Cell differentiation was quantified biochemically by measuring glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity using a modification of a method previously published (Adams etal., 1997) and described in Chapter II. Experiment 1: Effects of ibuprofen and troglitazone on bovine preadipocyte adipogenesis The objective of this experiment was to compare the adipogenic effects of IBU with the effects of troglitazone (TGZ), a well studied thiazolidinedione with potent adipogenic properties, partially mediated by PPARyz activation (Takeda et al., 2001 ; Hauner, 2002; Gardner et al., 2005). Clonal bovine SC preadipocytes 128 (6th passage) were grown to confluence and exposed to unsupplemented differentiation medium (control), or differentiation medium supplemented with 25 nM DEX, 100 pM IBU, 40 pM TGZ, or their combinations for 48 h. Troglitazone has been previously shown to optimize bovine preadipocyte differentiation at a concentration of 40 pM (Grant, 2005). Troglitazone was solubilized (2.5 mg/mL) in ethanol, therefore treatments not containing TGZ were supplemented with equivalent concentrations of ethanol. After 48 h, treatment media were replaced with fresh differentiation medium, and fresh medium was subsequently provided every 2 d for 10 d. Each treatment was applied to two 35 mm—diameter wells of a 6-well plate, in two replicate plates. Experiment 2: Efi‘ects of 48 h of ibuprofen exposure on the adipogenesis of bovine IM and SC preadipocytes The objective of this experiment was to compare the effects of IBU exposure for 48 h on adipogenesis in bovine IM and SC preadipocytes. Clonal (6th passage) and heterogenous (2nd passage) IM and SC preadipocytes were grown to confluence and exposed to unsupplemented differentiation medium (control) or differentiation medium supplemented with 25 nM DEX and IBU at 0, 250, 500, 1000, or 2000 pM for 48 h. After 48 h, treatment media were replaced with fresh differentiation medium and fresh medium was subsequently provided, every 2 d for 10 d. In this and subsequent experiments each treatment 129 was applied to two 35 mm-diameter wells of a 6-we11 plate, in two replicate plates for each of two steers. Adipogenesis was quantified by measuring GPDH activity. Because there was no interaction between cell type (heterogeneous or clonal preadipocytes) and treatment (DEX and IBU) (P = 0.62), the results obtained from heterogeneous and clonal preadipocytes were pooled. Experiment 3: Effects of exposure to ibuprofen for 12 d on the adipogenesis of bovine IM and SC preadipocytes The objective of this experiment was to compare the effects of IBU exposure for 12 d on adipogenesis in bovine IM and SC heterogeneous preadipocytes (2“d passage). Intramuscular and SC preadipocytes were grown to confluence and exposed to unsupplemented differentiation medium (control) or differentiation medium supplemented with 25 nM DEX for the initial 48 h and IBU at 0, 10, 100, 500, or 1000 pM for 12 d. Fresh treatment media were provided every 2 d for the 12 d period. Experiment 4: Effects of ibuprofen, aspirin, and indomethacin administration for 12 d in the adipogenesis of bovine IM and SC preadipocytes The objective of this experiment was to compare the effects of IBU, aspirin (ASP), and indomethacin (IND) exposure for 12 d on bovine IM and SC heterogeneous preadipocytes (2nd passage) . Ibuprofen, ASP, and IND are well 130 established COX inhibitors (Neupert et al., 1997; Tegeder et al., 2001). While IBU and IND are PPARyz activators, ASP is not (Lehmann et al., 1997). Alternatively, only IBU and ASP are inhibitors of neural factor kappa beta (NF- icB) (T egeder et al., 2001). Neural factor-KB is involved in the stimulation of the expression of tumor-necrosis factor alpha (TNF-a), a negative regulator of adipogenesis (Ntambi and Kim, 2000). Heterogenous IM and SC preadipocytes were grown to confluence and exposed to unsupplemented differentiation medium (control) or differentiation medium supplemented with 25 nM DEX for the initial 48 h. In addition, preadipocytes were exposed for 12 d to IBU at 0, 10, 100, 500, or 1000 pM, or ASP at 0 or 500 pM, or IND at 0 or 500 pM. Fresh treatment media were provided every 2 d for the 12 (1 period. Statistical analysis Data were analyzed using the Mixed Model procedure of SAS (SAS, Cary, NC). In all experiments, pooled cells from two wells of a 6-well plate were considered the experimental unit. When main effects were significant (P < 0.05), differences between means were evaluated utilizing Tukey’s multiple comparison test. In Exp. 1, data means were calculated using the fixed effects of DEX, IBU and TGZ. In Exp. 2 and 3, data means were calculated using the fixed effects of DEX, IBU, depot, and their interactions, with steer and steer x replication included as random variables. In Exp. 4, data means were calculated 131 using the fixed effects of DEX, IBU, ASP, depot, and their interactions, with steer and steer x replication included as random variables. To satisfy the conditions of normality and homogeneity of variance, GPDH data were loge transformed in Exp. 1, and GPDH data were square root transformed in Exp. 2. Results The primary objective of these experiments was to determine if IBU preferentially enhanced adipogenesis in bovine intramuscular preadipocytes when compared to subcutaneous preadipocytes. Furthermore, we compared the adipogenic effects of IBU to those of ASP, another well established COX inhibitor which, contrary to IBU, does not activate PPARyz. Experiment 1 Addition of 100 pM IBU to differentiation media for 48 h did not enhance Clonal SC preadipocyte GPDH activity over control levels (P = 0.99; Figure 4-1). Conversely, TGZ enhanced GPDH activity over control (P < 0.001). However, concomitant exposure of IBU and TGZ diminished TGZ stimulation of differentiation (P < 0.001). As expected, DEX (25 nM) increased GPDH activity (P < 0.001), and this effect was enhanced by the concomitant exposure to IBU (P = 0.01). Conversely, DEX and TGZ did not have additive effects (P = 0.17). The combination of DEX, IBU and TGZ resulted in higher GPDH activity than 132 obtained with TGZ alone (P = 0.02), but similar to GPDH activity of cells treated with DEX and TGZ. Experiment 2 Subcutaneous preadipocytes were more adipogenic than IM preadipocytes (P < 0.01), and DEX enhanced GPDH activity in both preadipocyte populations (P < 0.001; Figure 4-2). There was an interaction between IBU treatment and depot (P = 0.002). Exposure to IBU for 48 h did not enhance DEX stimulation of GPDH activity in SC preadipocytes (P > 0.54). Conversely, exposure to 1000 or 2000 pM IBU enhanced DEX stimulation of GPDH activity in IM preadipocytes (P = 0.01). Experiment 3 Long term (12 d) exposure of bovine preadipocytes to IBU also resulted in a treatment by depot interaction (P < 0.001) (Figure 4-3). Exposure to 100 pM IBU enhanced (P = 0.03) DEX induction of differentiation in IM preadipocytes, whereas 500 pM IBU tended (P = 0.05) to enhanced DEX induction of differentiation in IM preadipocytes. In SC preadipocytes, 100 pM IBU enhanced DEX induction of differentiation (P < 0.001), but 500 pM IBU did not (P = 0.99). Increasing the concentration of IBU to 1000 pM reduced (P < 0.001) DEX stimulation of GPDH in SC, but not IM preadipocytes (P = 0.99). 133 Experiment 4: Effects of exposure to ibuprofen, aspirin, and indomethacin for 12 d on the adipogenesis of bovine IM and SC preadipocytes In the absence of DEX, exposure of bovine preadipocytes to IBU for 12 d resulted in a treatment by depot interaction (P < 0.001) (Figure 4-4). Exposure to 10, 100, and 500 pM IBU enhanced GPDH activity in SC preadipocytes (P < 0.001), whereas 500 pM IBU enhanced GPDH activity (P < 0.004) in IM preadipocytes. However, at 1000 pM IBU the activity of GPDH was higher than control only in IM cells (P = 0.003). As expected, under control conditions SC preadipocytes had higher GPDH activity than IM preadipocytes (P < 0.001). However, the maximum induction of GPDH activity by IBU (500 pM) was much greater in IM than SC preadipocytes (12-fold vs. 1.7-fold over control, respectively) (Figure 4-4; Appendix E). Based on the observed morphological Changes (vacuoles, protuberances, and frequent stress fibers) caused by 1000 pM IBU in IM and SC preadipocytes, we suggest that 1000 pM IBU was harmful to the cells, resulting in a lower GPDH activity than at 500 pM IBU (not shown). Contrary to the effects of IBU, 500 pM ASP did not enhance GPDH activity either alone (P > 0.37) or in combination with DEX (P > 0.60) in either cell population (Figure 4-5). Indomethacin (500 pM) was toxic to the cells. 134 Discussion We have demonstrated that IBU has the ability to preferentially stimulate adipogenic differentiation in bovine IM preadipocytes when compared with SC preadipocytes. Previously, human (Adams et al., 1997) and bovine (Wu et al., 2000) SC and OM preadipocytes have been shown to exhibit differential responses to supplementation with PPARyz ligands other than IBU. Collectively, these data suggest that adipogenic differences between IM and SC preadipocytes may be, at least in part, related to differences in the ability of cells from these depots to synthesize unique PPARyz ligand(s). Although 1000 pM IBU for 48 h enhanced DEX-induced adipogenesis in IM preadipocytes, IBU failed to enhance SC preadipocyte adipogenesis. Because 1,000 pM IBU is nearly 500 times the IBU concentration required to inhibit COX (Tegeder et al., 2001), we attributed the minor enhancement of adipogenesis to the PPARyz activating capabilities of IBU. Previous studies have found that long term (9 d) exposure to IBU enhanced adipogenic differentiation in 1246 murine preadipocytes (Ye and Serrero, 1998) and C3H10T1/2 clone 8 murine fibroblasts (Lehmann et al., 1997). Therefore, we proceeded to evaluate if longer periods of exposure to IBU could amplify the differential effects of IBU, and further reduce or abolish adipogenic differences between IM and SC preadipocytes. Exposure to IBU for 12 d 135 resulted in subtle stimulation of DEX-induced adipogenesis in both IM and SC preadipocytes, although IM cells were responsive to a broader range of IBU concentrations. In the absence of DEX, IBU at 10 pM enhanced GPDH activity in SC preadipocytes, but not in IM preadipocytes. Furthermore, 100 and 500 pM IBU enhanced GPDH activity by approximately 1.7-fold in SC preadipocytes. Conversely, 500 pM IBU, a concentration at which IBU effectively activates PPARyz (Lehmann et al., 1997), enhanced GPDH activity 12-fold in IM preadipocytes. The minor enhancement of adipogenesis in SC preadipocytes by 10 to 500 pM IBU suggests that IBU action in these cells may be independent of PPARyz activation. In contrast, the dramatic increase in IM adipogenesis elicited by 500 pM IBU suggests that IBU may effectively activate PPARyz, emulating a level of PPARyz activation more near that obtained by natural PPARyz ligand(s) in SC preadipocytes. Ibuprofen is a well established inhibitor of COX activity (Rome and Lands, 1975; Mitchell et al., 1993), and may thereby enhance preadipocyte differentiation. Cyclooxygenase is the enzyme that catalyzes the rate-lirniting reaction during the biosynthesis of prostanoids (Funk, 2001). Prostanoids have been associated positively (Négrel et al., 1989; Négrel, 1999) and negatively (Mater et al., 1998; Petersen et al., 2003; Yan et al., 2003; Xie et al., 2006) with adipogenesis. However, in this and previous studies (Chapter III) we have 136 found that bovine IM preadipocyte differentiation is not affected by IBU exposure at concentrations compatible with COX inhibition (5 10 pM IBU) (Rome and Lands, 1975), but below those required for PPARyz activation (Lehmann et al., 1997). Alternatively, IBU may enhance preadipocyte adipogenesis by indirectly inhibiting the expression of an anti-adipogenic protein, TNF-a. Tumor necrosis factor-a inhibits preadipocyte adipogenesis (Fruhbeck et al., 2001), by decreasing the expression of PPARyz (Gregoire et al., 1998) and lipoprotein lipase (Fried et al., 1998), and by inducing insulin resistance (Fruhbeck et al., 2001). Ibuprofen has been shown to inhibit NF-KB stimulation of gene transcription (Tegeder et al., 2001), and TNF-a is one of the genes stimulated by NF-KB (Clark and Lasa, 2003). Aspirin has also been reported to inhibit NF-KB actions (Tegeder et al., 2001), but does not stimulate preadipocyte differentiation (Gaillard et al., 1989; Catalioto et al., 1991) or activate PPARyz (Lehmann et al., 1997). We found that 500 pM ASP did not affect GPDH activity in either preadipocyte population. These data suggests that IBU may induce bovine IM preadipocyte adipogenesis independently of COX inhibition and NF-KB signaling, acting as a PPAR-Y2 ligand. Previous research in our laboratory demonstrated that the potent PPARyz ligand TGZ, equally increased adipogenesis in bovine IM and SC preadipocytes (Grant, 2005). We initially compared the effects of IBU and TGZ on 137 adipogenesis of Clonal SC preadipocytes. In contrast to TGZ, IBU incubation for 48 h did not enhance the GPDH activity of bovine SC Clonal preadipocytes without concomitant exposure to DEX. Furthermore, IBU suppressed TGZ stimulation of GPDH activity when DEX was not included in the differentiation media. These observations indicate that IBU may stimulate bovine preadipocyte differentiation through a different mechanism than TGZ. Although TGZ is a member of the thiazolidinedione family of antidiabetic drugs Characterized as PPARyz ligands (Kelly et al., 1999; Willi et al., 2002), recent evidence has revealed that TGZ also has non-genomic, PPARyz independent mechanisms of action (Gardner et al., 2005) Specifically, TGZ has been found to activate phosphatidylinositol 3-kinase (PI3K), which subsequently activates various mitogen-activated protein kinases (MAPK) (Gardner et al., 2005). Of particular importance, PI3K activates extracellular signal-regulated kinase (Erk) that in turn can activate MAPK phosphatase-1 (MKP-l) (Takeda et al., 2001). As an inhibitor of various MAPK (i.e. C-Jun N- terminal kinase (JNK), p18) (Clark and Lasa, 2003), MKP-l can trigger cell cycle withdrawal, an essential step for the progression of preadipocyte differentiation (Ailhaud, 2001). Inhibition of PIBK prevents differentiation of 1246 and 3T3-L1 preadipocytes (Xia and Serrero, 1999). The rapid, PPARyz independent activation of P13K by TGZ may be a crucial difference between IBU and TGZ 138 mechanisms of action. Interestingly, glucocorticoids have also been reported to activate MKP-l (Clark and Lasa, 2003), offering a common pathway between DEX and TGZ that may partially explain the absence of an additive adipogenic effect between them under our conditions. However, TGZ also serves as a high affinity PPARyz ligand (Hauner, 2002; Houseknecht et al., 2002), which may explain the greater stimulatory effect of TGZ when compared to DEX. Ibuprofen could function as a conditional antagonist of TGZ. Indomethacin, a PPARyz activator (Lehmann et al., 1997) that stimulates preadipocyte differentiation (Knight et al., 1987), can also act as a PPARyz conditional antagonist in the presence of rosiglitazone, which is a PPARyz ligand of higher activity (Bishop-Bailey and Warner, 2003). Because IBU has been described as a low activity PPARyz ligand (Lehmann et al., 1997), in the absence of a high activity ligand, IBU could act as a PPARyz activator, explaining its enhancement of preadipocyte differentiation observed in our study. However, in the presence of TGZ, a high activity PPARyz ligand (Hauner, 2002; Houseknecht et al., 2002), IBU could act as a conditional antagonist, resulting in the observed suppression of TGZ enhancement of preadipocyte differentiation. Various PPARyz ligands have the ability to induce PPARyz to recruit different coactivators (i.e. PPARy co-activator 1a [PGCla]), and these 139 coactivators determine PPARyz target genes (Knouff and Auwerx, 2004). Thus, IBU may preferentially enhance adipogenesis of bovine IM preadipocytes by recruiting PPARyz coactivators that may be differentially expressed in IM preadipocytes. In conclusion, IBU preferentially increases adipogenesis in bovine IM preadipocytes compared to SC preadipocytes. It is suggested that adipogenic differences between IM and SC preadipocytes may be related to differences in the endogenous activation of PPARyz. Implications Bovine intramuscular and subcutaneous preadipocytes differ in their ability to accumulate lipid. Ibuprofen preferentially increased adipogenesis in intramuscular preadipocytes, under conditions that it likely functions as an activator of peroxisome proliferator-activated receptor yz (PPARyz). Therefore, the observed differences in adipogenic capacity among intramuscular and subcutaneous preadipocytes may be related to differences in the endogenous activation of PPARyz. The preferential enhancement of adipogenesis in intramuscular preadipocytes by ibuprofen may present a mechanism to increase intramuscular lipid accretion in cattle with little or no increase in subcutaneous fat accretion. 140 de C 5 - 333‘ 3“ :3: '5 333. 1:: .l 33:, IControl 3 ,E 4 a :g} IZIIBU OJ 1 i3 *- a 333‘ DTGz -- 8 333: 3 D- 3 _ :1 BIBU+TGz % g3 3: DDEX .1 m 5 :: EIDEX+IBU .4 E a 2 - ”g; BDEX+TGz (.9 .EE} BDEX+IBU+TGZ go 333: n—l 1 - 3331 900‘ .0" OOO‘ 333‘ 0 333. Figure 4-1. Effect of dexamethasone (DEX), ibuprofen (IBU), and troglitazone (TGZ) on the activity of glycerol-3-phosphate dehydrogenase (GPDH) in bovine clonal subcutaneous preadipocytes. Preadipocytes were grown to confluence and exposed to differentiation medium (control) or differentiation medium supplemented with 25 nM DEX, 100 pM IBU, 40 pM TGZ, or their combinations for 48 h. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 141 4- C 3- J— .IM USC 2.. Log GPDH specific activity, mU/mg protein Control DEX DEX+ DEX+ DEX-t» DEX+ ZSOpM 500pM 1000pM 2000pM IBU IBU IBU IBU Figure 4-2. Effect of dexamethasone (DEX) and ibuprofen (IBU) on the activity of glycerol-3-phosphate dehydrogenase (GPDH) in bovine Clonal and heterogeneous preadipocytes isolated from intramuscular (IM) and subcutaneous (SC) adipose tissue. Preadipocytes were grown to confluence and exposed to differentiation medium (control) or differentiation medium supplemented with 25 nM DEX and IBU at 0, 250, 500, 1000, or 2000 pM for 48 h. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 142 Log GPDH specific activity, mU/mg protem 03 LL Control DEX DEX+ DEX+ DEX+ DEX+ 10 pM 100 pM 500 M 1000 pM IBU IBU IBU IBU IIM EISC Figure 4-3. Effect of dexamethasone (DEX) and ibuprofen (IBU) on the activity of glycerol-3-phosphate dehydrogenase (GPDH) in bovine heterogeneous intramuscular (IM) and subcutaneous (SC) preadipocytes. Preadipocytes were grown to confluence and exposed to differentiation medium (control) or differentiation medium supplemented with 25 nM DEX for 48 h, and IBU at 0, 10, 100, 500, or 1000 pM for 12d. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 143 [9. 1‘. 4- 1 IIM a a CISC 21 Log GPDH specific activity, mU/mg proteln W H 1 Q g 0 10 100 500 1000 Ibuprofen concentration, pM Figure 4-4. Effect of ibuprofen (IBU) on the activity of glycerol-3-phosphate dehydrogenase (GPDH) in bovine heterogeneous intramuscular (IM) and subcutaneous (SC) preadipocytes. Preadipocytes were grown to confluence and exposed to differentiation media supplemented with IBU at 0 (control), 10, 100, 500, or 1000 pM for 12d. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 144 6 d d d d 5 - C C be b be C 4 _ IIM DSC Log GPDH specific activity, mU/mg proteln Control 500 pM 500 pM DEX DEX+ DEX+ ASP IBU 500 pM 500 pM ASP IBU Figure 4-5. 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Serrero. 1998. Stimulation of adipose differentiation related protein (ADRP) expression by ibuprofen and indomethacin in adipocyte precursors and in adipocytes. Biochem. J. 330:803-809. 149 CHAPTER V: INTERPRETIVE SUMMARY Because intramuscular (IM) fat (marbling) is the main determinant of USDA Quality Grade within a carcass maturity Classification, beef producers are motivated to feed cattle high energy diets for long periods to attain premiums for highly marbled carcasses. Unfortunately, as cattle are fed for long periods, excessive subcutaneous (SC) fat develops, and results in economic losses for the US. beef industry. Therefore, the beef industry would benefit from knowing how to selectively stimulate marbling development with reliability, or selectively reduce SC adipose tissue accretion. To selectively manipulate beef fat accretion, it is necessary to understand the intrinsic differences between IM and SC preadipocytes, and comprehend how these differences relate to adipogenesis. Accordingly, the objectives of the experiments contained in this dissertation were to determine if the lower adipogenic capacity of bovine IM preadipocytes when compared to bovine SC preadipocytes was related to differences in: 1) glucocorticoid receptor (GR) expression and response to glucocorticoids; 2) peroxisome proliferator-activated receptor (PPARyz) expression and prostacyclin (PG12) secretion, or alternatively; 3) if the adipogenic capacity of IM preadipocytes could be preferentially enhanced by ibuprofen (IBU), a PPARyz activator. 150 I—fl- Glucocorticoid induction of adipose tissue development has been shown to have anatomical specificity in vivo. Therefore, in the first set of experiments we aimed to determine if there were differences in GR expression among bovine IM, SC, and perirenal (PR) preadipocytes. Moreover, we also compared the effects of dexamethasone (DEX), a synthetic glucocorticoid, on adipogenesis among bovine IM, SC, and PR preadipocytes. Preadipocytes from the three depots similarly expressed the ~97 kDa immunoreactive band consistent with the size of GR isoform 0:, presumably the GR isoform responsible for inducing glucocorticoid effects. Accordingly, DEX equally induced adipogenic differentiation in IM, SC, and PR preadipocytes, as determined by the stimulation of glycerol-3-phosphate dehydrogenase (GPDH) activity. The proportionally comparable induction of differentiation among IM, SC, and PR preadipocytes by DEX exposure is consistent with our observation that cultured bovine preadipocytes express similar quantities of GR. Although DEX increased the percentage of SC and PR preadipocytes with large lipid droplets (10 2 pm-diameter), we did not detect an effect of DEX in the percentage of IM preadipocytes with large lipid droplets. Independent of DEX exposure, the propensity for adipogenic differentiation was PR > SC > IM, which suggests that adipogenic differences among these cell populations are due to signaling pathways unrelated to GR. 151 Peroxisome proliferator-activated receptor yz has been Characterized as the master regulator of adipogenesis and accordingly, a pleiotropic regulator of the adipocyte phenotype. Therefore, we determined if there were differences in PPARyz expression between bovine IM and SC preadipocytes. Moreover, we compared the secretion of PG12, a PPAR activator and potent adipogenic molecule, between bovine IM and SC preadipocytes. Preadipocytes from IM and SC adipose tissue expressed PPARyz equally, although they exhibited different adipogenic phenotypes. Because PPARyz is a marker of mid differentiation, this may reflect an uncoupling between mid differentiation and the mature adipocyte phenotype. Perhaps, the mechanisms that initiate preadipocyte differentiation are different from those that regulate the extent of lipid accumulation. For instance, PPARyz may have different roles in distinct phases of adipogenesis, and these different roles may be subject to dissimilar regulation in IM and SC preadipocytes. Alternatively, because PPARyz activity is regulated by the availability of ligands, differences in the synthesis of PPARyz ligands between IM and SC preadipocytes may help explain their adipogenic differences. Fatty acids and metabolites of arachidonic acid (i.e. prostaglandins) have been characterized as PPARyz activators. Of the metabolites of arachidonic acid detected in cultured preadipocytes, PG12 is the only has been Characterized as a 152 potent adipogenic molecule and a PPAR activator. Therefore, we proceeded to test the hypothesis that adipogenic differences between IM and SC preadipocytes could be related to differences in the secretion of PG12. Unexpectedly, IM preadipocytes secreted more PGI2 than SC preadipocytes. Therefore, we speculated that perhaps PG12 and(or) other prostaglandins may actually be a negative regulator of adipogenesis in our conditions. Nevertheless, a PG12 analog, carbaprostacyclin, tended to enhanced adipogenesis of clonal SC preadipocytes, as previously reported for the Ob 1771 mouse preadipocyte cell line. Moreover, IBU (10 pM), a well established inhibitor of prostaglandin synthesis, did not affect adipogenesis of clonal SC preadipocytes. Supplementation with CPG12 at hyperphysiological concentrations may be able to substitute for the endogenous PPARyz ligand, and consequently trigger preadipocyte differentiation. However, in bovine preadipocytes, endogenous PG12 secretion might have a role unrelated to adipogenesis. While using IBU as an inhibitor of prostaglandin synthesis, it was revealed that high concentrations (100 pM) of IBU enhanced the DEX stimulation of adipogenesis in Clonal SC preadipocytes. Review of current literature unveiled that 100 pM IBU can activate PPARyz. Therefore, our objective for the third set of experiments was to compare the adipogenic effects of IBU between bovine IM and SC preadipocytes. 153 Firstly, we compared the adipogenic effects of IBU with those induced by troglitazone (TGZ), a potent PPARyz activator, by incubating bovine Clonal SC preadipocytes with these substances for 48 h. We found that although TGZ enhanced GPDH activity over control, in the absence of DEX, concomitant exposure of IBU and TGZ diminished TGZ stimulation of differentiation. This observation indicated that IBU, being a weak PPARyz activator, may act as a conditional PPARyz antagonist in the presence of T GZ. These further supported the hypothesis that IBU adipogenic induction is mediated through a PPARyz mechanism. However, IBU and TGZ presented different modes of action. For instance, contrary to TGZ, exposure of Clonal SC preadipocytes to IBU for 48 h did not enhance preadipocyte differentiation without concomitant exposure to DEX. Indeed, previous research has revealed that although TGZ has important PPARyz mediated effects, it also has non-genomic actions that could explain the different adipogenic actions between TGZ and IBU. Various PPARyz ligands have the ability to induce PPARyz to recruit different coactivators, and these coactivators determine PPARyz target genes. We speculated that PPARyz coactivators are differently expressed between IM and SC preadipocytes and that IBU could selectively induce IM preadipocyte differentiation. We found that IBU exposure for 48 h selectively enhanced DEX induction of IM preadipocyte differentiation. However, the effect was relatively 154 small, and SC preadipocytes were still considerably more adipogenic than IM cells. Conceivably, activation of PPARyz for only 48 h may not be enough time to greatly enhance adipogenesis in IM preadipocytes. We then hypothesized that continuous exposure (12 d) to IBU could be able to diminish the adipogenic differences between IM and SC preadipocytes by stimulating adipogenesis more effectively in 1M preadipocytes. Exposure to 100 pM IBU (12 d) enhanced DEX induction of differentiation in both IM and SC preadipocytes. Conversely, 500 pM IBU enhanced DEX induction of differentiation in IM, but not in SC preadipocytes. Furthermore, in the absence of DEX, exposure to 10 and 100 pM IBU (12 d) enhanced GPDH activity in SC, but not IM preadipocytes. Conversely, the maximum induction of GPDH activity by IBU (500 pM) was much greater in IM than SC preadipocytes (12-fold vs. 1.7-fold over control, respectively). It is important to note that IBU at 10 pM inhibits cyclooxygenase (COX) activity but does not activate PPARyz. Furthermore, 100 pM IBU has been described as a weak PPARyz ligand. The adipogenic induction by 10 pM IBU and comparable but modest effects of 100 and 500 pM IBU suggest that IBU enhancement of SC preadipocyte adipogenesis might be independent of PPARyz activation. Conversely, in IM preadipocytes, 10 or 100 pM IBU did not enhance adipogenesis, but 500 pM IBU, a concentration at which IBU effectively activates 155 PPARyz, enhanced GPDH activity 12-fold over control. These data suggest that adipogenic differences between IM and SC preadipocytes might be partially explained by differences in the synthesis of ligands for PPARyz. Ibuprofen, acting as a PPARyz ligand, may partially compensate for this difference. Alternatively, both cell populations may secrete PPARyz ligands equivalently, but IM cells may express a different set of coactivators. It is conceivable, that IBU may be able to recruit the particular set of coactivators expressed by IM preadipocytes. The possibility exists that IBU enhancement of preadipocyte differentiation could be partially independent of PPARyz activation. Indeed, IBU is a well established inhibitor of COX activity, and has been reported to inhibit the actions of neural factor kappa beta (NF-KB). However, another well established COX inhibitor, aspirin (ASP; 500 pM), that has also been reported to inhibit the actions of (NF-KB), did not enhance GPDH activity either alone or in combination with DEX in both cell populations. These results further support that IBU adipogenic stimulation is dependent upon PPAR-Y2 activation. To better explain the observed differences in adipogenesis between IM and SC preadipocytes, it would be important to determine if IBU effects are unique. If similar results are obtained with other PPARyz activators like indomethacin or rosiglitazone, the hypothesis that the observed adipogenic 156 differences are related, at least in part, to the synthesis of the physiological PPARyz ligand would be supported. However, if the selective enhancement of IM preadipocyte differentiation is a characteristic unique to IBU, it would suggest that IBU may be able to allow PPARyz to recruit the coactivators available within IM preadipocytes. Alternatively, IBU may downregulate potential PPARyz corepressors expressed by IM preadipocytes, in addition to function as a PPARyz ligand. The function of these corepressors may be to assure that 1M preadipocytes abide by their proposed regulatory role and do not become large energy reservoirs. Therefore, experiments that compare the expression of PPARyz coactivators and(or) corepressors between IM and SC preadipocytes would certainly help clarify the understanding of our observations. Moreover, studies that evaluate how PPARyz interactions with other proteins Change upon IBU exposure would be valuable. While IBU mechanism(s) of selectively inducing IM preadipocyte differentiation still needs to be better delineated, I believe that pilot studies, whose aim is to evaluate IBU effects on marbling development, are worth pursuing. Ibuprofen has been used to treat bovine mastitis and to improve fertilization rate during artificial insemination. The actual dosage that may successfully enhance IM adipose tissue development needs to be determined 157 empirically, but concentrations used in the mentioned studies may offer a reasonable starting point. In addition, because IBU is a widely utilized drug for the treatment of a variety of human ailments, its usage by the beef industry certainly would be more acceptable to consumers than the use of TGZ and other PPAR ligands that have been associated with severe hepatotoxicity in diabetic patients. We have demonstrated that bovine IM and SC preadipocytes have intrinsic adipogenic differences. We propose that these adipogenic differences are partially caused by differences in endogenous PPARyz activation. Even though we cannot rule out an alternative mechanism(s) of action for IBU, we suggest that IBU compensates for a lower secretion of appropriate PPARyz ligands in IM preadipocytes. Future studies should focus in determining if IBU effects are unique, or shared by other PPARyz activators and on better deciphering IBU mechanism(s) of action. Because supplementation of cattle with IBU may offer a strategy to selectively increase marbling with reliability, in vivo studies to determine appropriate IBU dosage to influence marbling development, as well as practical modes of delivery, are also worth pursuing. 158 APPENDICES 159 APPENDIX A: CLON AL EFFICIENCY AND ADIPOGENIC CAPACITY OF CELLS ISOLATED FROM BOVINE INTRAMUSCULAR, SUBCUTANEOUS, AND PERIRENAL ADIPOSE TISSUE Abstract The objectives of this study were to evaluate clonal efficiency, proportion of adipogenic clones, and adipogenic capacity, among cells derived from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue. Adipose tissues were isolated from an Angus steer (556 kg, 13.5 mo. old), and 1 g equivalent was seeded at 0.05 g / cm2 in growth media. Four to seven days after seeding, cells were serially diluted and seeded in six, 96-well plates: 4 plates for control, one plate with 1 ng/mL of fibroblast growth factor (FGF) and one plate with 10 ng/mL FGF. Clonal efficiency was evaluated 8 to 13 d later. There was an interaction between depot and treatment (P < 0.001). Perirenal cells did not respond to FGF treatment (P = 1.00). In control or 1 ng/mL FGF, intramuscular (IM) and subcutaneous cells presented equivalent clonal efficiencies (P = 1.00). Exposure to 1 ng/mL FGF increased Clonal efficiency exclusively in SC cells (P = .003), but 10 ng/mL FGF did not (P > 0.74). In IM cells, 10 ng/mL FGF decreased Clonal efficiency (P < 0.05). Surviving clones from each depot (IM n = 48; SC n = 64; PR n = 11) were seeded at 10,000 cells/cmz. At confluence the cells were exposed to growth media supplemented with 1% FBS, 10 ng/mL insulin, 20 mM glucose, and 10 mM acetate with or without 1 ng/mL 160 It FGF. Cells were also exposed to 250 nM dexamethasone (DEX) for 48 h. Proportion of adipogenic Clones was determined by oil red O staining. There was no FGF treatment effect (P = 0.25) or treatment by depot interaction (P = 0.60). There was no difference in the proportion of adipogenic clones between SC and PR cells (49%) (P = 0.60), but both were higher than the proportion of IM adipogenic clones (12.5%) (P < 0.002). Clonal adipogenic cells isolated from IM, SC, and PR adipose tissue were seeded at 4,600 cells/cm2 and upon confluence, were exposed to 0, 25, or 2500 nM DEX for 48 h. After 12 d, glycerol-3- phosphate dehydrogenase (GPDH) enzymatic analysis was performed. There was an interaction between treatment and depot (P < 0.001). Independent of DEX treatment, IM cells were less adipogenic that SC or PR (P < 0.003). Although SC cells were more adipogenic than PR at 0 (P < 0.001) and 25 nM DEX (P < 0.001), when exposed to 2,500 nM DEX these differences disappeared (P = 0.33). Dexamethasone increased GPDH activity in a dose dependent manner in IM and PR cells (P < 0.004). In contrast, in SC cells, the activity of GPDH was increased by exposure to 25 nM DEX (P = 0.04), but 2,500 nM DEX did not enhance differentiation further (P = 0.37). Differences in Clonal efficiency and adipogenic capacity among cells isolated from distinct bovine adipose tissues are evident in culture. 161 Introduction Adipogenic differences exist between bovine preadipocytes isolated from distinct adipose depots (Chapter II, IH, and IV; Ohyama et al., 1998; Wu et al., 2000). Because adipose tissue is comprised of many different cells (i.e. pericytes, fibroblasts, preadipocytes, etc.) (Ailhaud, 2001), it is not known if adipogenic differences are a result of different proportions of adipogenic cells in the different adipose depots. Knowing the proportion of non-adipogenic to adipogenic cells is needed to determine if there are differences in the adipogenic potential of preadipocytes from different adipose depots. By studying the differentiation of cell colonies derived from a single adipogenic cell (Clones), comparisons of adipogenesis between cells isolated from different adipose depots would not have the confounding factor of different proportions of adipogenic cells being responsible of observed differences. The growth of Clonal cells also presents a Challenge because the ability of cells to survive without the support of the surrounding heterogeneous cell population may be impaired. Consequently, evaluation of the ability of growth factors to improve survival of Clonal cells is important when developing such a model. Fibroblast growth factor (FGF) has been utilized in the cloning of bovine preadipocytes (Aso et al., 1995), but in other studies, FGF has induced differentiation in ovine preadipocytes (Broad and Ham, 1983). Because 162 proliferation and differentiation are mutually exclusive, the usefulness of FGF in bovine preadipocyte cloning success is uncertain. Therefore, the objectives of the study were to evaluate the Clonal efficiency of cells derived from bovine intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue and to study the influence of FGF in clonal efficiency. Furthermore, we aimed to determine if there were differences in the proportion of adipogenic clones, and(or) if there were differences in adipogenic enzyme activity among Clones isolated from IM, SC, and PR adipose tissue, that were previously Characterized as adipogenic. Materials and Methods Isolation and cloning of bovine preadipocytes Preadipocytes from IM, SC, and PR adipose tissue were isolated using a modification of a protocol previously published (Forest et al., 1987) and described in Chapter II. Preadipocytes from IM, SC, and PR adipose tissue of one steer were Cloned as described in Chapter III. Determination of clonal efficiency Four to seven days after the seeding, cells were trypsinized, counted, serially diluted to 5 cells per mL and seeded in 6 mm-diameter wells of 96-well plates at a proportion of 200 pL per well, and allowed to grow while incubated in a humidified atmosphere (37°C, 95% air and 5% C02). Therefore, the seeding 163 was expected to place one cell per well on average. Six, 96-well plates per depot were utilized: 4 plates with growth media (control), one plate with growth media supplemented with 1 ng/mL of FGF and one plate with growth media supplemented with 10 ng/mL FGF. Within each depot and treatment, the proportion of wells that had a single proliferative colony was considered the Clonal efficiency and was evaluated 8 to 13 days after the initial seeding. Wells with more than one colony were not considered in the clonal efficiency analysis. Determination of the proportion of adipogenic clones Surviving Clones from each depot (IM n = 48; SC n = 64; PR n = 11) were isolated and seeded at 10,000 cells/cm2 in 35 mm-diameter cell culture wells. Cells were allowed to proliferate to confluence in growth media. Growth media was replaced every 2 d. After reaching confluence, plates were washed twice with PBS and the preadipocytes were exposed to growth media (1% FBS) supplemented with 1.74 nM insulin, 20 mM glucose and 10 mM acetate with or without 1 ng/mL FGF. Preadipocytes were also exposed to 250 nM dexamethasone (DEX) for 48 h. After 12 d in culture, cell differentiation was morphologically assessed by determining the number of Clones containing at least two cells stained with oil red O (ORO). The ORO solution was prepared using a protocol previously described (Ramirez-Zacarias et al., 1992). Cells were fixed by addition of 3.7% formaldehyde (Mallinckrodt Baker Inc., Phillipsburg, 164 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 and the cells were washed twice with distilled water (15 min incubation/wash). Cell nuclei were stained by adding 1 mL giemsa solution (1g Giemsa, 66 mL glycerol, 66 mL methanol) to each well for 1 h, after which the cells were washed twice in distilled water, and stored dry at 4°C. Cells were visualized within 8 h of staining. Determination of differences in adipogenic capacity Cell culture. Clonal preadipocytes isolated from IM, PR, and SC adipose tissue of a steer were proliferated in culture and in their 6th passage were seeded at a density of 4,600 cells/cm2 in 35 mm-diameter cell culture wells, and allowed to proliferate to confluence (4 d) in growth medium. Growth medium was replaced every 2 d. After reaching confluence, plates were washed twice with PBS and experimental differentiation treatments applied. Differentiation medium was DMEM (5.5 mM glucose), supplemented with 1% antibiotic- antimycotic, 0.1% gentamicyn, 33 pM biotin, 17 pM pantothenate, 200 pM ascorbate, 280 nM bovine insulin and 5 pL/mL bovine serum lipids (Ex-Cyte; Serologicals Corp, Norcross, GA). Preadipocytes were exposed to 0, 25, or 2500 nM DEX for 48 h. Each treatment was applied to two wells of a 6-well plate, in two replicates for each adipose depot. After 48 h, treatment media were 165 replaced with basic differentiation medium supplemented with 280 nM bovine insulin and 5 pL/mL bovine serum lipids and fresh medium was provided every 2 d for 12 d. Glycerol-3-phosphate dehydrogenase activity. Cell differentiation was quantified biochemically by measuring glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity using a modification of a method previously published (Adams et al., 1997) and described in Chapter 11. Statistical analysis Data were analyzed using the Mixed Model procedure of SAS (SAS, Cary, NC). For the evaluation of morphological differentiation and clonal efficiency, a single well was the experimental unit, while for GPDH data, pooled cells from two wells of a six-well plate were considered the experimental unit. For the Clonal efficiency data, means were calculated using the fixed effect of depot. For morphological differentiation data, means were calculated using the fixed effects of depot, FGF and FGF x depot. To satisfy the conditions of normality and homogeneity of variance, GPDH data were log. transformed. For GPDH data, means were calculated using the fixed effects of DEX, depot, and DEX x depot with replication included as a random variable. When the main effects were significant (P < 0.05), mean differences were analyzed utilizing Tukey’s multiple comparison test. 166 Results Clonal efiiciency The obtained Clonal efficiencies showed an interaction between adipose depot (IM, SC, and PR) and treatment (0, 1, or 10 ng/mL FGF (P < 0.001) (Figure A-l). Perirenal cells, showed the lowest Clonal efficiency (13.8%) among the three depots (P < 0.001), and their Clonal efficiency was not affected by FGF treatment (P = 0.99). Under control conditions or when exposed to 1 ng/mL FGF, IM and SC cells presented similar clonal efficiencies (P = 0.99). Compared to control, exposure to 1 ng/mL FGF increased Clonal efficiency from 49.7 to 69.8%, exclusively in SC cells (P = .003), however, when exposed to 10 ng/mL FGF, SC Clonal efficiency (59%) was not different from control or 1 ng/mL FGF (P > 0.74). Although exposure to 1 ng/mL FGF did not enhance IM Clonal efficiency (56.8%) compared to control (P = 0.18), 10 ng/mL decreased Clonal efficiency to 33.7% (P < 0.05). Proportion of adipogenic clones Adipose depot (IM, SC, PR) of origin affected the proportion of adipogenic clones (P < 0.001) (Figure A-2). However, there was no effect of treatment (0 or 1 ng/ml FGF) (P = 0.25) or a treatment by depot interaction (P = 0.60) in the proportion of adipogenic Clones. Although there was no difference in the proportion of adipogenic clones between SC and PR cells (49%) (P = 0.60), 167 both showed a higher proportion of adipogenic clones that IM cells (12.5%) (P < 0.002). Adipogenic capacity There was an interaction between treatment (0, 25, and 2500 nM DEX) and depot (IM, SC, and PR) (P < 0.001) for adipogenic capacity of the Clones (Figure A-3). Although SC Clones were more adipogenic than PR at 0 or 25 nM DEX (P < 0.001), when exposed to 2,500 nM DEX these differences disappeared (P = 0.33) (Figure 3). In IM and PR Clones GPDH activity increased in response to DEX in a dose-dependent manner (P < 0.004). Conversely, in SC Clones GPDH activity was increased at 25 nM DEX (P = 0.04), but 2,500 nM DEX did not further enhance GPDH activity (P = 0.37). Independent of treatment, lM clones were less adipogenic than SC or PR clones (P< 0.003). Discussion Our results support the hypothesis that intrinsic differences exist between cells isolated from IM, SC, and PR adipose tissues. The study reveled that the ability of Clones to survive in culture varies among cells isolated from these adipose depots, and resulted in a lower number of clones obtained from the PR depot. The usefulness of FGF to enhance Clonal efficiency may only be granted in SC cells. Furthermore, it was determined that in cells isolated from the IM adipose tissue, a lower proportion of clones were able to accumulate lipid when 168 compared to clones isolated from SC and PR adipose depots. In addition, an IM adipogenic clone showed lower adipogenic enzyme activity than SC and PR adipogenic clones. Our data shows that adipogenic differences exist between cells isolated from different bovine adipose tissues. It has been determined that hyperplasia in PR adipose tissue is completed earlier than in IM and SC adipose depots (Hood and Allen, 1973; Cianzio et al., 1985). As a result, Clonal efficiency would be expected to be higher in late developing adipose tissues like SC and IM when compared to early developing adipose tissue, like PR, where the population of proliferative/progenitor cells may have been diminished in a nearly mature animal. Indeed, differences in clonal efficiencies have been previously reported. In the rat, clones from PR adipose tissue have higher Clonal efficiencies (34%) than epididimal cells (29.3%), and in both tissues, Clonal efficiencies decreased with age of the donor (Kirkland et al., 1990). Alternatively, our results may reflect the differences in adipose tissue growth in different anatomical locations. In rats, it has been determined that the growth of internal adipose depots depend more on hypertrophy than hyperplasia, while surface adipose depots rely more on hyperplasia for their growth (DiGirolamo et al., 1998). In general terms, the Clonal efficiency results may not only reflect differences in the stage of development of IM, SC, and PR 169 adipose tissue, but also intrinsic differences in the growth patterns of the different adipose depots. Contrary to our observed differences in the proportion of adipogenic Clones among cells isolated from different bovine adipose depots, in humans, independent of adipose depot (subcutaneous, mesenteric, and omental), more than 90% of the isolated clones accumulated lipid when compared after the Clones were maintained in differentiation media for up to 60 days (Tchkonia et al., 2002). However, when clones were compared at 15 days in differentiation media, the proportion of adipogenic Clones were 85% for SC, 75% for mesenteric, and 55% for omental (Tchkonia et al., 2002). Consequently, because we observed the cells for only 12 days, it could be speculated that our results may reflect that bovine preadipocytes from different depots differ in the time required for differentiation to proceed. This assertion is supported by our observations that PPARyz, a transcription factor expressed almost exclusively in adipose cells (Bishop-Bailey and Wray, 2003), is equally expressed among heterogeneous preadipocyte cultures from IM, SC, and PR adipose tissue (Chapter 111). Even though FGF has been previously used in Cloning media for bovine lM preadipocytes (Aso et al., 1995), in retrospect, according to our results this treatment may not have been needed or may even have hampered the Cloning 170 procedure. Although FGF has been described as a mitogen for preadipocytes (Yamasaki et al., 1999), other studies have found that FGF is adipogenic (Broad and Ham, 1983; Gabrielsson et al., 2002). Because different FGF concentrations may have different effects on cells, and 22 different FGF isoforms have been identified (Gabrielsson et al., 2002), the effects of FGF in clonal efficiency and adipogenesis are uncertain. The lesser propensity of bovine IM adipose tissue to develop in vivo may result from a smaller proportion of adipogenic cells and(or) a lower adipogenic enzymatic activity of the cells contained within IM adipose tissue. Various studies have determined that IM adipose tissue has a lower capacity to synthesize lipids (Smith and Crouse, 1984; Smith et al., 1984; Miller et al., 1991). The capacity of SC adipose tissue to synthesize fatty acids from acetate (the main substrate for fatty acid synthesis in ruminants) is greater that in IM adipose tissue (Smith, 1995). In addition, when ruminants are exposed to high energy diets the activities of enzymes involved in adipose tissue metabolism (i.e. ATP Citrate lyase and NADP malate dehydrogenase) are increased in SC adipose tissue but not in IM adipose tissue (Smith and Crouse, 1984). The resistance of IM adipose tissue to accumulate lipid may be a result of a lower proportion of adipogenic cells in this adipose depot, and(or) that these cells have an intrinsic constraint for lipid accumulation. 171 In conclusion, cells isolated from bovine IM, SC, and PR adipose tissue present distinct Characteristics in terms of Clonal efficiency, proportion of adipogenic clones, and adipogenic enzyme activity of adipogenic Clones. The evidence presented suggests that intrinsic differences among cells isolated from different adipose depots, may be responsible for observed developmental differences of distinct adipose depots in the bovine. Implications The utilization of adipogenic cells derived from a single progenitor cell (Clones) isolated form different adipose depots can help in discerning intrinsic differences between preadipocytes from distinct anatomical locations. During the Cloning procedure it should be expected that the number of Clones obtained from adipose depots would be smaller in adipose tissues that develop earlier in the bovine’s life (i.e. perirenal), as the population of proliferative cells may have diminished with time, when compared to intramuscular and subcutaneous adipose tissue. In our culture conditions, the utilization of fibroblast growth factor to enhance Clonal efficiency is not recommended. The study also suggests that the reduce capacity of IM adipose tissue to develop in the bovine compared to other depots may result from a lower proportion of adipogenic cells, combined with the possibility that intramuscular adipogenic cells have a lower adipogenic capacity than subcutaneous and perirenal cells. 172 100 90- IIM so- EISC 7o] IPR 60- 50- 4o- 30- 20- 1o- Clonal efficiency, proliferative colonies/seeded cells, % a 0 1 10 Fibroblast growth factor concentration, ng/mL Figure A-1. Differences in clonal efficiency among cells isolated from different adipose depots. Cells isolated from bovine intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of a steer were Cloned by serial dilution. Cells were incubated with growth media supplemented with 0 (control), 1, or 10 ng/mL fibroblast growth factor (FGF). Clonal efficiency was determined as the proportion of proliferative colonies to the total number of cells seeded. Means with different superscripts differ (P < 0.05). 173 100 90- IIM 30- USC Proportlon of adipogenic clones, adipogenic clones/total clones, % U! c I Control FGF (1 ngmL) Figure A-2. Differences in the proportions of isolated Clones that were adipogenic. Cells isolated from bovine intramuscular (IM; n = 12), subcutaneous (SC; n = 64), and perirenal (PR; n = 11) adipose tissue of a steer were cloned, grown to confluence, and subsequently exposed to differentiation media supplemented with 0 (control) or 1 ng/mL fibroblast growth factor (FGF) for 12 d. The percentage of Clonal colonies containing at least two cells with lipid I I droplets were determined 12 d after addition of treatments. Means with I different superscripts differ (P < 0.05). 174 IIM DSC IPR Log GPDH specific activity, mU/mg protein 0 25 2500 Dexamethasone concentration, nM Figure A-3. Effect of dexamethasone (DEX) on the activity of glycerol—3- phosphate dehydrogenase (GPDH) in Clonal adipogenic cells isolated from bovine intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue of a steer. Cells were grown to confluence and subsequently exposed to DEX for 48 h. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 1 75 Literature Cited Adams, M., C. T. Montague, J. B. Prins, J. C. Holder, S. A. Smith, L. Sanders, J. E. Digby, C. P. Sewter, M. A. Lazar, V. K. K. Chatterjee, and S. O'Rahilly. 1997. Activators of peroxisome proliferator-activated receptor e have depot-specific effects on human preadipocyte differentiation. J. Clin. Invest. 100:3149-3153. Ailhaud, G. 2001. Development of white adipose tissue and adipocyte differentiation. Page 27-55 in Adipose Tissues. S. Klaus ed. Eurekah Publ., Georgetown,TX. ‘ Aso, H., H. Abe, I. Nakajima, K. Ozutsumi, T. 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Commun. 258:109-112. 178 APPENDIX B: INFLUENCE OF DEXAMETHASONE ON THE GENE EXPRESSION OF BOVINE INTRAMUSCULAR PREADIPOCYTES Abstract The objective of this study was to evaluate the Changes in gene expression induced by dexamethasone (DEX) (a synthetic glucocorticoid) in bovine intramuscular (IM) preadipocytes utilizing microarray technology. Intramuscular preadipocytes from a steer were proliferated in culture and at the 6th passage were seeded at a density of 1,300 cells/cm2 in six, 100 mm-diameter cell culture wells per treatment. Preadipocytes were propagated in culture and upon confluence were then exposed to 0 or 250 nM DEX for 48 h. After 48 h, total RNA (10 pg) from control and DEX treated bovine intramuscular preadipocytes were extracted and used as templates in reverse transcription reactions. After first-strand cDNA synthesis, cDNAs from control and DEX treated preadipocytes were differentially labeled using N-hydroxysuccinimide- derivatized Cy3 and Cy5 dyes. The National Bovine Functional Genomics Consortium (NBFGC) microarray library was utilized in this study to interrogate 18,263 unique transcripts. Utilizing the ”dye swap” experimental design, samples were exposed to hybridization for 18 h. After microarray data normalization, it was determined that compared to control values, 48 h DEX treatment of bovine IM preadipocytes caused the differential expression (P < 179 0.05) of 583 genes. Of the differentially expressed genes, 31% (184) were found to have a known function while 68% (399) of the genes’ functions remain undefined. Dexamethasone treatment caused the differential expression of many genes known to be involved in cell cycle regulation, metabolism, transcription, immunity and apoptosis. Genes involved in cell cycle regulation, carbohydrate, and lipid metabolism accounted for 16% of the differentially expressed genes. Among the genes found to be differentially expressed were those coding for enzymes involved in fatty acid metabolism including acetyl- CoA carboxylase and steroyl-CoA desaturase, fatty acid binding protein, and the adipogenic protein angiotensinogen. It can be concluded that DEX had a positive effect on the adipogenic gene expression of bovine IM preadipocytes. Introduction Understanding the biological process underlying intramuscular (IM) adipose tissue accretion in bovines is critical to improve beef production efficiency and meat quality. In particular, the characterization of genes whose expression is altered during IM preadipocyte adipogenesis would help us identify potential targets to selectively increase IM adipose (marbling) development and(or) molecular predictors of marbling development. Microarray technology offers the prospect to obtain a comprehensive appraisal of the transcriptional profile of bovine IM adipogenesis. Although 180 various studies have utilized microarray analysis to Characterize the transcriptional profile of adipogenesis in human (Urs et al., 2004) and mouse (Soukas et al., 2001) preadipocytes, we are unaware of any study that has utilized microarray technology to Characterize the transcriptional profile of differentiating bovine preadipocytes. Even though glucocorticoids are commonly added to the differentiation cocktail for cultured preadipocytes (Sato et al., 1996; Ailhaud, 2001; Brandebourg and Hu, 2005), a comprehensive analysis of glucocorticoid induced Changes in gene expression has not been reported for cultured bovine preadipocytes. Therefore, we hypothesized that glucocorticoids would stimulate the expression of genes involved in the adipogenesis of these IM preadipocytes. The objective of the study was to utilize cDNA microarrays to discern the alterations in the transcriptional profile of bovine IM preadipocytes upon glucocorticoid exposure. Materials and Methods Isolation of bovine preadipocytes Preadipocytes from IM adipose tissue were isolated using a modification of a protocol previously published (Forest et al., 1987) and described in Chapter II. 181 Cell culture Intramuscular preadipocytes were proliferated in culture and at the sixth passage were seeded at a density of 1,300 cells/cm2 in 100 mm-diameter cell culture plates. Six cell cultures plates were exposed to each treatment. Cells were allowed to proliferate to confluence (6 d) in growth media, while incubated in a humidified atmosphere (37°C, 95% air and 5% C02). Growth media was replaced every 2 d. After reaching confluence, plates were washed twice with PBS and the preadipocytes were exposed to modified growth medium (1% FBS) containing 0 or 250 nM dexamethasone (DEX) for 48 h. RNA extraction After 48 h, RNA extraction was performed using a modification of the Tri Reagent-RNA/DNA/protein isolation protocol (Molecular Research Center Inc, Cincinnati, OH). Briefly, confluent preadipocytes were washed 3 times with 37°C PBS, detach from the plate with trypsin (0.5 g/L trypsin and 0.02 g/L ethylenediaminetetraacetic acid (EDTA) in PBS [pH 7.2]), and cells from the six culture plates per treatment were collected and combined in 50 mL centrifuge tubes. The preadipocytes were then pelletized upon centrifugation for 5 min at 3000 x g. The supernatant was discarded and 2 mL Tri Reagent added to the pellets. The preadipocyte homogenate was mixed and stored at room temperature for 5 min after which it was supplemented with 200 pL 182 '.I."_" bromochloropropane and vortexed vigorously for 15 s. Homogenates were centrifuged (11,000 x g) for 15 min at 4°C, after which the supematants were transferred to new vials, mixed with 1.0 mL isopropanol, and centrifuged, (11,000 x g) for 8 min at 4°C. Resulting RNA pellets were washed by addition of 2 mL 75% ethanol in diethyl pyrocarbonate treated distilled water, vortexed briefly, and centrifuged at 8,000 x g for 5 min at 4°C. After the pellet dry, it was dissolved in 50 pL of DEPC treated distilled water. To achieve complete solubilization, the samples were incubated in a water bath for 15 min at ~57°C. The RNA extracts were stored at —80°C until use. Concentration and purity of RNA was determined by analyzing a 1/100 dilution of each sample using a spectrophotometer (Model DU-650, Beckman, Schaumberg, IL). Upon gel electrophoresis the integrity of the RNA samples was evaluated by evaluation of the 188 and 288 rRNA bands after ethidium bromide staining. Quality and integrity of RNA was further evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Preparation of labeled cDNA Complimentary DNA synthesis was performed following a procedure previously published (Coussens et al., 2002). Briefly, total RNA (10 pg) from control and DEX treated bovine intramuscular preadipocytes were used as templates in reverse transcription reactions, utilizing SuperScript III (Invitrogen 183 Corp., Carlsbad, CA) and oligo (dT)15 as a primer. After first-strand synthesis, cDNAs from control and DEX treated preadipocytes were differentially labeled using N-hydroxysuccinimide-derivatized Cy3 and Cy5 dyes (Amersham Pharmacia, Ltd., Piscataway, NJ). The ”dye swap” design was used to compensate for potential dye bias. Labeled cDNAs were purified to remove unincorporated dyes using cDNA labeling purification modules (Invitrogen Corp). Differentially labeled samples were then combined and concentrated to 10 pL by using Microcon 30 spin concentrators (Millipore Corp., Bedford, MA). Microarray hybridization Microarray hybridization was performed for 18 h after addition of 100 pL of SlideHyb-3 (Ambion Inc., Alameda, CA) to the concentrated Cy3—Cy5-labeled probe cDNAs. The procedure was performed in a commercial microarray hybridization station, utilizing a step-down hybridization protocol (GeneTAC; Genomics Solutions Inc., Ann Arbor, MI). The National Bovine Functional Genomics Consortium (NBFGC) microarray library was utilized in this study, and has been previously described in detail (Suchyta et al., 2003). In brief, the NBFGC microarray contains 18,263 unique transcripts, 96 Bos taurus B-actin spots, 96 Bos taurus GAPDH spots, 120 lambda Q spots, 241 negative spots (3x SSC), and 384 blank spots. Accordingly, the number of total spots on the 20 x 20 patch configuration is 19,200 (400 spots in each patch). Information for each 184 NBFGC microarray clone can be found at the NBFGC web site (http://nbfgcmsu.edu). After hybridization, cDNA microarrays were washed, rinsed once in 2x SSC (0.3 M NaCl, 0.03 M sodium citrate) and once in double- distilled water. Lastly, microarrays were dried by centrifugation in a cushioned 50 mL conical centrifuge tube. Microarrays were then scanned using a GeneTAC LS IV microarray scanner. GeneTAC LS analyzer software (Genomic Solutions Inc.) was used to determine total spot intensities for both dyes. Microarray data analysis Two NBFGC microarrays were used to interrogate 18,263 genes against the RNA isolated from control and DEX treated preadipocytes using the ”dye swap” experimental design to account for variation due to dye. Array-specific data normalization was then performed using the LOESS procedure of SAS (SAS, Cary, NC). For each microarray, accomplishment of data normalization was evaluated by plotting log intensity ratio M (log Cy3 - logCy5), versus the mean log intensities A ([logCy3 + log Cy5]/2). The normalized data was back- transformed before statistical analyses. To correct for background dye intensities, the median value of the negative spots was determined and subtracted from sample spot intensity values. The natural log of the resulting values was then calculated and submitted to a Student’s T-test using Excel (Microsoft, Corp., Redmond, WA). Fold Change ratios were calculated using the 185 average intensity value of treated vs. control for each gene. Resulting P-values were used to determine level of significance values for genes of interest. Genes having P-values less than 0.05 were considered significant and were evaluated for known function using the NBFGC gene library search. Results and Discussion When compared to control values, 48 h DEX treatment of bovine IM preadipocytes caused the differential expression (P < 0.05) of 583 genes from a total of 18,263 within the NBFGC library. Of the differentially expressed genes, 31% (184) were found to have a known function while 68% (399) of the genes’ functions remain undefined. Ontological clustering of differentially expressed genes with known functions uncovered several important findings. Although many of the genes with a known function proved difficult to categorize and were thus depicted as ”other”, DEX treatment did cause the differential expression of many genes known to be involved in cell cycle regulation, metabolism, transcription, immunity and apoptosis (Figure B-1). Of most interest within this study, genes involved in cell cycle regulation, carbohydrate, and lipid metabolism accounted for 16% of the differentially expressed genes (Table B-1). Selected genes from this list will be discussed in detail. 186 Within in vitro preadipocyte cell studies, DEX is often used to stimulate adipose differentiation, which is partially controlled through growth arrest and exit from the cell cycle (Gregoire et al., 1998; Soukas et al., 2001). Our study confirms these actions of DEX as genes such as Zinc finger protein ZPRl, Cdt1/DNA replication factor, CDK-activating kinase assembly factor MATI, and MAPK 6, all of which have roles in stimulating or maintaining mitogenesis, were downregulated by DEX (P < 0.03). Because growth arrest is a crucial requirement for adipocyte differentiation (Gregoire et al., 1998), DEX seems to induce differentiation, at least partially, by making preadipocytes withdraw form the cell cycle. During preadipocyte differentiation DEX has been shown to cause an increase in triacylglyceride synthesis in bovine IM preadipocytes (Sato et al., 1996) and in rodent preadipocyte cell lines (Gaillard et al., 1991). Also, glucocorticoid treatment has resulted in an increase in the expression of lipoprotein lipase, an enzyme involved in the uptake of plasma fatty acids, in human adipose tissue (Fried et al., 1993). In our study, genes critical to fatty acid synthesis and accumulation within adipocytes were upregulated by DEX treatment. Acetyl-CoA carboxylase 1, which catalyzes the rate-limiting reaction in the biogenesis of long-Chain fatty acids, was upregulated by DEX (P = 0.01). An up-regulation of Acetyl-CoA carboxylase 1 would help explain the increase 187 in lipid droplet accumulation seen in bovine IM preadipocytes following exposure to DEX (Sato et al., 1996). In addition, acetyl-CoA carboxylase 1 is a key regulator of lipogenesis in the bovine, and interestingly, it has been reported that animals that have a greater tendency to fatten have higher activity of this enzyme in their adipose tissues (Allen et al., 1976). Another enzyme involved in fatty acid metabolism upregulated by DEX within our study was stearoyl-CoA desaturase (P = 0.01). This enzyme converts sterate, palmitate, and myristate into their respective unsaturated fatty acids (Sturdivant et al., 1992). The high concentration of oleate (unsaturated fatty acid of sterate) in bovine adipose tissue reflects the importance of stearoyl-CoA desaturase in ruminant lipid metabolism (Lin et al., 1992). It is interesting to note that cattle breeds with greater propensities to deposit IM fat (i.e. Waygu) exhibit greater stearoyl-CoA desaturase activity in their adipose tissues (Sturdivant et al., 1992; Wang et al., 2005). Additionally, a greater expression of stearoyl-CoA desaturase contributes positively to beef flavor. This is caused by a decrease in the melting point of IM fat as a result of a greater unsaturated/saturated fatty acid ratio (Nishimura et al., 1999). Because unsaturated fatty acids can serve as ligands for peroxisome proliferator activated receptor yz (PPARyz), a crucial regulator of adipogenesis (Bishop-Bailey and Wray, 2003), it can be speculated that DEX may increase IM 188 fat development at least partially, by increasing the availability of PPARyz ligands, as a result of steroyl-CoA desaturase induction. The expression of fatty acid binding protein (FABP) was also increased by DEX (P = 0.04), similar to previous microarray studies in rodent preadipocyte cell lines (Soukas et al., 2001). Fatty acid binding protein constitutes 6% of total cytosolic protein in murine mature adipocytes and is involved in extracellular fatty acid uptake (Amri et al., 1991). Also, FABP is involved in intracellular fatty acid trafficking, targeting of fatty acids towards organelles involved in triacylglyceride synthesis and fatty acid oxidation (Fruhbeck et al., 2001). In murine preadipocyte cell lines, DEX acts directly through the glucocorticoid receptor to induce FABP, causing an increase in FABP mRNA within hours of treatment (Amri et al., 1991). Furthermore, the emergence of FABP is Closely related to induction of fatty acid and triacylglycerol synthesizing enzymes (Fruhbeck et al., 2001). The up-regulation of this gene suggests that DEX is involved in the maturation of preadipocytes by stimulating the biochemical machinery necessary for intracellular lipid accretion. Interestingly, the FABP gene is more highly expressed in cattle breeds with a high propensity to accumulate IM adipose tissue (i.e. Waygu) (Wang et al., 2005). Lipid accumulation within preadipocytes can result from fatty acid uptake or de novo synthesis. Within ruminants, it has been suggested that both 189 glucose and acetate may be utilized as fatty acid precursors (Smith and Crouse., 1984). Our study suggests that as preadipocytes differentiate after DEX induction, their capability for glucose uptake and resulting triacylglyceride synthesis may increase. It was discovered that glucose-transporter 3 (GLUT-3) was upregulated by DEX (P = 0.01), along with allograft inflammatory factor-1 (P = 0.04), a gene known to be associated with both glucose/insulin regulation and inhibition of cell proliferation. It therefore appears that DEX could have the capacity to enhance glucose uptake and use in bovine IM preadipocytes. Angiotensinogen is another gene important to adipose development that was upregulated by DEX in our study (P = 0.007). Glucocorticoids have been previously implicated in an upregulation of angiotensinogen gene expression (Kim and Moustaid-Moussa, 2000). There appears to be a positive relationship between angiotensinogen mRNA level and rate of adipocyte growth (Kim and Moustaid-Moussa, 2000). Angiotensin II, a the physiologically active derivative of angiotensinogen cleavage, is implicated in adipose tissue development by stimulating the production and release of prostacyclin by mature adipocytes (Gregoire et al., 1998), which, in turn, may stimulate preadipocyte recruitment and differentiation (Martin et al., 1998). In addition, angiotensin II may regulate adipose tissue blood supply, and as a consequence substrate availability for 190 lipid accretion. Angiotensin II also promotes triacylglyceride storage and stimulates the secretion of leptin (Kim and Moustaid-Moussa, 2000). It can be concluded that DEX treatment of IM bovine preadipocytes decreased the expression of genes involved in cell proliferation, consequently directing the cells toward terminal differentiation. In addition, important genes involved in lipid synthesis and uptake were up-regulated by DEX, as well as genes involved in carbohydrate metabolism. Dexamethasone also increased the expression of angiotensinogen, a key molecule involved in adipocyte development and function. Thus, DEX treatment resulted in a gene expression pattern consistent with the terminally differentiated phenotype in bovine IM preadipocytes. Future studies comparing DEX effects on gene expression of preadipocytes isolated from different bovine adipose depots would increase our understanding of the biological processes that make bovine adipose depots unique. Summary Dexamethasone exposure resulted. in a comprehensive alteration in the gene expression profile of bovine preadipocytes isolated from intramuscular adipose tissue. Importantly, many genes involved in fatty acid uptake, metabolism, and modification were up-regulated by dexamethasone. Because the expression of an important adipogenic protein, angiotensinogen, was also 191 up-regulated by dexamethasone, an adipogenic role for this glucocorticoid is supported. Future studies comparing the gene expression profile of bovine preadipocytes isolated from distinct adipose depots would aid in elucidation of adipose depot differences in bovine preadipocyte adipogenesis. 192 Protein Apoptosis modification 0 I \\\\\\\\\\\ \\\\\\ \ \ \\\\ \\\\\\\\\\\\ \\\\\ 1 1 % Cell cycle 10% FAICHO metabolism Skeletal Signaling 6% development 1 8% 4% Figure B-1. Ontological clustering of genes with known function that upon microarray analysis were determined to be differentially expressed (P < 0.05) in bovine intramuscular preadipocytes upon dexamethasone exposure (250 nM, 48 h). 193 Table B-1. Selected genes related to preadipocyte adipogenesis that upon cDNA microarray analysis were determined to be differentially expressed (P < 0.05) in bovine intramuscular preadipocytes upon dexamethasone treatment. Gene ID Gene name' Function DEX effect AW656833A Angiotensinogen Cell differentiation Up Reg BE482150 MAP kinase kinase 2 Cell signaling Up Reg BE6653 88 Acetyl-CoA Carboxylase 1 Fatty acid synthesis Up Reg BE665830 Stearoyl-CoA desaturase Fatty acid synthesis Up Reg BF 774305 Rab6 GTPase-activating protein Cell cycle Down Reg BE757609 Arfaptin Cell signaling Up Reg AW657014 Ras-GTPase-activating protein 1 Cell signaling Up Reg BE665566 Triacylglycerol lipase Lipid catabolism Up Reg AW668928 Glut-3 Glucose transport Up Reg AW655845 Retinoic acid receptor alpha Cell differentiation Up Reg AW308502 Zinc finger protein ZPRl Cell proliferation Down Reg AW298838 CDK—activating kinase assembly factor MATl Cell proliferation Down Reg BE684394 P150 (regulatory protein of P13 K) Cell signaling Down Reg BF 706948 MAPK 6 Cell signaling Down Reg BF230835 ARP2/3 complex 16 kDa subunit Cytoskeleton organization Down Reg BE23 7542 Rho-GTPase-activating protein 1 Cytoskeleton organization Down Reg BE483745 Collagen alpha 1(XlV) chain precursor ECM reorganization Down Reg BFO77525 Mouse Ten-m/Odz Inhibitor cell proliferation Up Reg BE665570 Cdtl/DNA replication factor DNA replication Down Reg BFO73071 Tyrosine Phosphatase Cell signaling Down Reg AW659446 Mitotic spindle assem. chkpt protein MAD2B Cell cycle Up Reg AW657666 LDL receptor Lipid metabolism Down Reg AW658776 1P3 receptor type 1 Cell signaling Up Reg BE668078 Aldose reductase Carbohydrate metabolism Up Reg AW652540 Hematopoietic PBX-interacting protein Cell differentiation Up Reg AW652741 Allograft inflammatory factor-l Inhibitor cell proliferation Up Reg BE808535 Phosphatidylserine-specific phospholipase A] Cell signaling Down Reg BE749837 Fatty acid binding protein Lipid metabolism Up Reg aGenes in bold are discussed in the text. 194 Literature Cited Ailhaud, G. 2001. Development of white adipose tissue and adipocyte differentiation. Page 27—55 in Adipose Tissues. S. Klaus ed. Eurekah Publ., Georgetown,TX. Allen, C. E., D. C. Beitz, D. A. Cramer, and R. G. Kauffman. 1976. Biology of fat in meat animals. NCR Res. Pub]. No. 234., Univ. Wisc., Madison. Amri, E., G. Ailhaud, and P. Grimaldi. 1991. Regulation of adipose cell differentiation. 11. Kinetics of induction of the P2 gene by fatty acids and modulation by dexamethasone. J. Lipid. Res. 32:1457—1463. Bishop-Bailey, D., and J. Wray. 2003. Peroxisome proliferator-activated receptors: a critical review on endogenous pathways for ligand generation. Prostaglandins and other Lipid Mediat. 71:1-22. Brandebourg, T. D., and C. Y. Hu. 2005. Regulation of differentiation of pig preadipocytes by retinoic acid. J. Anim. Sci. 83:98-107. Coussens, P. M., C. J. Colvin, K. Wiersma, A. Abouzied, and S. Sipkovsky. 2002. Gene expression profiling of peripheral blood mononuclear cells from cattle infected with mycobacterium paratuberculosis. Infect. Immun. 70:5494-5502. Forest, C. C., A. Doglio, D. Ricquier, and G. Ailhaud. 1987. A preadipocyte Clonal line from mouse brown adipose tissue. Exp. Cell Res. 168:218-232. Fried, S. K., C. D. Russel, N. L. Grauso, and R. E. Brolin. 1993. Lipoprotein lipase regulation by insulin and glucocorticoids in subcutaneous and omental adipose tissue of obese women and men. J. Clin. Invest. 92:2191-2198. Fruhbeck, G., J. Gomez-Ambrosi, F. J. Muruzabal, and M. A. Burrell. 2001. The adipocyte: a model for integration of endocrine and metabolic signaling in energy metabolism regulation. Am. J. Physiol. 280:E827—E847. Gaillard, D., M. Wabistsch, B. Pipy, and R. Négrel. 1991. Control of terminal differentiation of adipocyte precursor cells by glucocorticoids. J. Lipid Res. 32:569-579. Gregoire, F. M., C. M. Smas, and H. S. Sul. 1998. Understanding adipocyte differentiation. Phys. Rev. 78:783-809. 195 Kim, S., and N. Moustaid-Moussa. 2000. Secretory, endocrine and autocrine/paracrine function of the adipocyte. J. Nutr. 130:31105—31158. Lin, K. C., H. R. Cross, and S. B. Smith. 1992. Esterification of fatty acids by bovine intramuscular and subcutaneous adipose tissues. Lipids 27:111- 116. Martin, R. J., G. J. Hausman, and D. B. Hausman. 1998. Regulation of adipose cell development in utero. P.S.E.B.M. 219:200-210. Nishimura, T., A. Hattori, and K. Takahashi. 1999. Structural Changes in intramuscular connective tissue during the fattening of Japanese black cattle: effect of marbling on beef tenderization. J. Anim Sci. 77:93-104. Sato, K., N. Nakanishi, and M. Mitsumoto. 1996. Culture conditions supporting adipose conversion of stromal-vascular cells from bovine intramuscular adipose tissues. J. Vet. Med. Sci. 58:1073-1078. Smith, S. B., and J. D. Crouse. 1984. Relative contributions of acetate, lactate and glucose to lipogenesis in bovine intramuscular and subcutaneous adipose tissue. J. Nutr. 114:792-800. Soukas, A., N. D. Socci, B. D. Saatkamp, S. Novelli, and J. M. Friedman. 2001. Distinct transcriptional profiles of adipogenesis in vivo and in vitro. J. Biol. Chem. 276:34167-34174. Sturdivant, C. A., D. K. Lunt, G. C. Smith, and S. B. Smith. 1992. Fatty acid composition of subcutaneous and intramuscular adipose tissue and M. longissimus dorsi of Wagyu cattle. Meat Sci. 32:449-458. Suchyta, S. P., S. Sipkovsky, R. Kruska, A. Jeffers, A. MCNulty, M. J. Coussens, R. J. Tempelman, R. G. Halgren, P. M. Saama, D. E. Bauman, Y. R. Boisclair, J. L. Burton, R. J. Collier, E. J. DePeters, T. A. Ferris, M. C. Lucy, M. A. McGuire, J. F. Medrano, T. R. Overton, T. P. Smith, G. W. Smith, T. S. Sonstegard, J. N. Spain, D. E. Spiers, J. Yao, and P. M. Coussens. 2003. Development and testing of a high-density cDNA microarray resource for cattle. Physiol. Genomics 15:158-164. Urs, S., C. Smith, B. Campbell, A. M. Saxton, J. Taylor, B. Zhang, J. Snoddy, B. Jones Voy, and N. Moustaid-Moussa. 2004. Gene expression profiling in human preadipocytes and adipocytes by microarray analysis. J. Nutr. 134:762-770. 196 Wang, Y. H., A. Reverter, H. Mannen, M. Taniguchi, G. S. Harper, K. Oyama, K. A. Byme, A. Oka, S. Tsuji, and S. A. Lehnert. 2005. Transcriptional profiling of muscle tissue in growing Japanese Black cattle to identify genes involved with the development of intramuscular fat. Aust. J. Exp. Agric. 45:809-820. 197 .l APPENDIX C: IMMUNOCYTOCHEMICAL DETECTION OF GLUCOCORTICOID RECEPTOR IN CLONAL BOVINE PREADIPOCYTES ISOLATED FROM INTRAMUSCULAR, SUBCUTANEOUS, AND PERIRENAL ADIPOSE TISSUE Objectives To detect the expression of the glucocorticoid receptor on bovine Clonal preadipocytes isolated from intramuscular (IM), subcutaneous (SC), and perirenal (PR) adipose tissue, and to evaluate the subcellular localization of the glucocorticoid receptor in these cells. Materials and Methods Isolation and cloning of bovine preadipocytes Preadipocytes from IM, SC, and PR adipose tissue were isolated using a modification of a protocol previously published (Forest et al., 1987) and described in Chapter II. Preadipocytes from IM, SC, and PR adipose tissue of one steer were Cloned as described in Chapter III. Immunocytochemical detection of glucocorticoid receptor Clonal preadipocyte cultures from IM, SC, and PR adipose tissue in the 6th passage were seeded on glass cover slips at approximately 13,000 cells/cm2 and allowed to attach overnight while exposed to serum free media (SFM). Non- attached cells were then removed by three gentle washes with SFM. Attached cells were then incubated in SFM supplemented with 0 or 250 nM dexamethasone (DEX) for 2 h in a humidified atmosphere (37°C, 95% air and 5% 198 C02). Immunolabeling was performed utilizing a modification of a protocol previously described (Briinnegard et al., 1995). Briefly, after the incubation, cells were fixed with 1% formalin in PBS (30 min). Intrinsic fluorescence was quenched with 0.1 M glycine in PBS (5 min), and cells were permeabilized with 0.05% Triton X-100 in PBS (30 min). To minimize non-specific antibody binding, cells were then incubated with 5% sheep serum in PBS for 30 min. Incubation with 1 pg/mL of a polyclonal (rabbit) anti-glucocorticoid receptor (GR) antibody (PA1-511A, Affinity BioReagents, Inc., Golden, CO) or control rabbit IgG at a concentration of 1 pg/mL, was performed overnight at 4°C. Cells were then washed five times, for 5 min each, with PBS (1% sheep serum). The washes were followed with a 2 h incubation with a secondary antibody, fluorescein isothiocyanate (FITC) conjugated, affinity purified, goat anti-rabbit IgG. Five washes (5 min each) with PBS (1% sheep serum) followed, after which glass cover slips were mounted exposed to 4’, 6-diamidino-2-phenylindole (DAPI), and mounted on glass microscope slides and sealed with nail polish. Fluorescence stained cells were then visualized and photographed with a camera (Leica Micosystems Digital Imaging, Cambridge, United Kingdom) attached to a fluorescence sensitive microscope (Leica Microskopie, Wetzlar, Germany). 199 Results During immunocytochemistry analysis, GR was predominantly and consistently observed in the nuclei of both control preadipocytes, and those exposed to 250 nM DEX (Figure C-1, Panels A and C). Only faint cytoplasmic staining, above background, was visualized and this observation was consistent among cells isolated from the different adipose tissues. Cells incubated with irrelevant rabbit IgG, as a negative control, only presented a very faint, non specific labeling (Figure C-l, Panel E). Discussion It was found that GR immunostaining was predominant in the nuclei of preadipocytes isolated from the three depots independent of ligand. Previous reports have also shown that independent of ligand presence, GR immunolabeling results in intense nuclear staining surrounded by cytoplasmic staining (Brbnnegard et al., 1990; Sackey et al., 1996; Oakley et al., 1997; Yudt and Cidlowski, 2001). Perhaps, removing control cells from exposure to FBS only 24 h before the immunocytochemical staining was not sufficient to eliminate the effects of glucocorticoids on GR localization. However, we have obtained similar nuclear staining of preadipocytes following 12 d exposure to serum-free media devoid of glucocorticoids. Alternatively, because according to imunoblot analysis (Chapter II) the used antibody in this study detected various 200 GR isoforms, GR immunostaining in the nucleus and cytoplasm of bovine preadipocytes may reflect distinct subcellular locations of the different isoforms (Yudt and Cidlowski, 2001, 2002). 201 Figure C-l. Glucocorticoid receptor (GR) immunostaining of bovine preadipocytes, after being exposed to 0 (A) or 250 nM dexamethasone (DEX) for 2 h (C). Incubation with an antibody against GR revealed positive staining relative to incubation with control rabbit IgG (B). Cells with DNA labeled with 4’, 6-diamidino-2-phenylindole (DAPI) are shown in panels B, D, and F. Bar = 100 pM. 202 Figure C-1: A Figure C-1: B 203 Figure C-1: C Figure C-1: D 204 Figure C-1: E Figure C-1: F 205 Literature Cited Bronnegard, M., P. Amer, L. Hellstrdm, G. Akner, and J. A. Gustafsson. 1990. Glucocorticoid receptor messenger ribonucleic acid in different regions of human adipose tissue. Endocrinology 127:1689-1696. Brbnnegard, M., S. Reynisdottir, C. Marcus, P. Stiema, and P. Amer. 1995. Effects of glucocorticoid treatment on glucocorticoid receptor expression in human adipocytes. J. Clin. Endocrinol. Metab. 80:3608-3612. Oakley, R. H., J. C. Webster, M. Sar, C. R. Parker, Jr., and J. A. Cidlowski. 1997. Expression and subcellular distribution of the B-isoform of the human glucocorticoid receptor. Endocrinology 138:5028-5038. Sackey, F. N. A., R. J. G. Hache, T. Reich, J. Kwast-Welfeld, and Y. A. Lefebvre. 1996. Determinants of subcellular distribution of the glucocorticoid receptor. Molec. Endocrin. 10:1191-1205. Yudt, M. R., and J. A. Cidlowski. 2001. Molecular identification and Characterization of A and B forms of the glucocorticoid receptor. Molec. Endo. 15:1093-1103. Yudt, M. R., and J. A. Cidlowski. 2002. The glucocorticoid receptor: Coding a diversity of proteins and responses through a single gene. Molec. Endocrin. 16:1719-1726. 206 APPENDIX D: EFFECT OF NORDIHYDROGUAIARETIC ACID ON THE ACTIVITY OF GLYCEROL-3-PHOSPHATE DEHYDROGENASE IN BOVINE Log GPDH specific activity, SUBCUTANEOUS PREADIPOCYTES 7 I Control CI DEX Z DEX + 10 pM NDGA a DEX + 20 pM NDGA b b b 5 :1 E e 4 9.. 60 E 3 :2 E 2 1 o Figure D-l. Effect of 25 nM dexamethasone (DEX) and nordihydroguaiaretic acid (NDGA) on the activity of glycerol-3-phosphate dehydrogenase (GPDH) in bovine Clonal subcutaneous preadipocytes. Preadipocytes were grown to confluence and exposed to 0 nM (control) or 25 nM DEX and 0, 10 or 20 pM NDGA for 48 h. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 207 APPENDIX E: EFFECT OF IBUPROFEN ON THE ACTIVITY OF GLYCEROL- 3-PHOSPHATE DEHYDROGENASE IN BOVINE HETEROGENEOUS Fold Change in GPDH INTRAMUSCULAR AND SUBCUTANEOUS PREADIPOCYTES 14 12- 3.10- IE 5.: 8‘ IIM i2: 6- EISC 3 3‘ 4i 2- 0 10 100 500 1000 Ibuprofen concentration, pM Figure E-1. Effect of ibuprofen (IBU) on the activity of glycerol-3- phosphate dehydrogenase (GPDH) in bovine heterogeneous intramuscular (IM) and subcutaneous (SC) preadipocytes (fold Change compared to control). Preadipocytes were grown to confluence and exposed to differentiation media supplemented with IBU at 0 (control), 10, 100, 500, or 1000 pM for 12 d. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 208 200 . f 180 f 160 - e _]_ 14o - _[_ 120 - 1n d _‘,‘_ IIM Else b Lit-Lil J._ 0 10 100 500 1000 Ibuprofen concentration, pM Log GPDH specific activity, mU/mg prote H 8 I-' N uh 6‘ on O G D O O l L I l I Figure E-2. Effect of ibuprofen (IBU) on the activity of glycerol-3- phosphate dehydrogenase (GPDH) in bovine heterogeneous intramuscular (IM) and subcutaneous (SC) preadipocytes. Preadipocytes were grown to confluence and exposed to differentiation media supplemented with IBU at 0 (control), 10, 100, 500 or 1000 pM for 12 d. Glycerol-3-phosphate dehydrogenase activity was determined 12 d after addition of treatments. Means with different superscripts differ (P < 0.05). 209 APPENDIX F: CELLS UTILIZED WITHIN THIS DISSERTATION Chapter II: Bovine intramuscular, subcutaneous, and perirenal preadipocytes express similar glucocorticoid receptor isoforms, but exhibit different adipogenic capacity GR Immunoblot analysis: Heterogeneous preadipocytes from steers 3 and 4 (4th passage) GPDH analysis: Heterogeneous preadipocytes from steers 3 and 4, and 5 (2ml passage) ORO analysis: Heterogeneous preadipocytes from steers 3 and 4, and 5 (2Ind passage) Chapter III: Differences in adipogenesis between bovine intramuscular and subcutaneous preadipocytes are not related to expression of PPARyz or secretion of PG12 PPARyz Immunoblot analysis: Heterogeneous preadipocytes from steers 3 and 4, and 5 (2nd passage) 210 PG12 Enzyme immuno assay and GPDH activity: Heterogeneous preadipocytes from steer 5 (2nd passage), and clones IM3-C32-P6 and 8C3- CSI-P6 (6th passage, steer 3) Evaluation of the effects of carbaprostacyclin on preadipocyte differentiation: GPDH analysis: Clone SC3-C31-P6 (6th passage, steer 3) Evaluation of the effects of ibuprofen on preadipocyte difi‘erentiation: GPDH analysis: Clone SC3-C31'P6 (6th passage, steer 3) Chapter IV: Ibuprofen preferentially enhances adipogenesis in bovine intramuscular preadipocytes when compared to subcutaneous preadipocytes. Efi‘ects of ibuprofen and troglitazone on bovine preadipocytes adipogenesis: GPDH analysis: Clone SCB-CBI-Pe (6th passage, steer 3) Effects of 48 h of ibuprofen exposure on the adipogenesis of bovine IM and SC preadipocytes. Heterogeneous preadipocytes from steers 3 and 5 (2nd passage), and Clones IMs-Caz-Pe and SC3-C3'1-P6 (6th passage, steer 3) Effects of exposure to ibuprofen for 12 d on the adipogenesis of bovine IM and SC preadipocytes. Heterogeneous preadipocytes from steers 3 and 5 (2"0' passage) 211 Effects of ibuprofen, aspirin, and indomethacin administration for 12 d in the adipogenesis of bovine 1M and SC preadipocytes. Heterogeneous preadipocytes from steers 3 and 5 (2nd passage) Appendix A: Clonal efficiency and adipogenic capacity of cells isolated from bovine intramuscular, subcutaneous, and perirenal adipose tissue Clonal efficiency: Primary stromal-vascular cells from steer 3 Determination of proportion of adipogenic clones: Surviving clones from steer 3 (4th passage) Determination of differences in adipogenic capacity: Clones lM3-C32-P6, SCa- C31-P6, and PR3-C2-Po (6th passage, steer 3) Appendix B: Influence of dexamethasone on the gene expression of bovine intramuscular preadipocytes Microarray analysis: Heterogeneous IM preadipocytes (6th passage, steer 3) Appendix C: Immunocytochemical detection of glucocorticoid receptor in clonal bovine preadipocytes isolated from intramuscular, subcutaneous, and perirenal adipose tissue 212 Immunocytochemical analysis: Clones lM3-C32-Po, SC3-C31-P6, and PR3-C2-Po (6th passage, steer 3) APPENDIX D: Effect of nordihydroguaiaretic acid on the activity of glycerol-3- phosphate dehydrogenase in bovine subcutaneous preadipocytes GPDH analysis: Clone SC3-C3I-P6 (6th passage, steer 3) 213 AAAAAAAAAAAA Illalllllljill