A an.) in» iiilm“: a ”ulna” fI : why" “.4 KAI.“ If a, L 1' vi. h ”mu .2: $2., at ~25... fiufihwfiwé; . . . , ~33. .. . . . .1 A , (1W1? S; ) ,5. ‘ ‘ 1.. 70 . ..Lwr.wn..._a:. “Mi?” ”ft 3.22 r , . .z _ THESIS- lllllllllllllllllllllllllllllllllllllllmllIllll 293 01050 3302 This is to certify that the dissertation entitled Radioligand Binding of [3H]CGP12177 and Efficacies of ISOproterenol, RactOpamine, and Clenbuterol on the Ligand-RegulatedIQAR-Adenylyl Cyclase Systems in Porcine Satellite and CZClZ Cells. presented by Ernest Benjamin Izevbigie has been accepted towards fulfillment of the requirements for Ph.D. degree in Animal Science 4&W‘6 W Major professor Date 8-30-96 MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State I University PLACE IN RETURN 30X to remove this checkout from your record. To AVOID FINES return on or bdoto date duo. DATE DUE DATE DUE DATE DUE MSU In An Affirmative mum/Equal Opportunity Intuition Mmi RADIOLIGAND BINDING OF [3H]CGP12177 AND EFFICACIES OF ISOPROTERENOL, RACTOPAMINE, AND CLENBUTEROL ON THE LIGAND-REGULATED BAR-ADENYLYL CYCLASE SYSTEMS IN PORCINE SATELLITE AND c2c12 CELLS By Ernest Benjamin Izevbigie A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science 1996 ABSTRACT RADIOLIGAND BINDING OF [3H]CGP12177 AND EFFICACIES OF ISOPROTERENOL, RACTOPAMINE, AND CLENBUTEROL ON THE LIGAND-REGULATED BAR-ADENYLYL CYCLASE SYSTEMS IN PORCINE SATELLITE AND C2C12 CELLS By Ernest Benjamin Izevbigie The mechanism of action of beta-adrenergic agonists (BAA) on adipocytes is believed to be beta-adrenergic receptors (BAR)-mediated. Contrary to adipocytes, the mode of action of BAA on myocytes is unclear. The lack of understanding of BAA actions on myocytes is due, at least in part, to few myocyte models available for biochemical studies. Therefore, the present studies sought to provide more insights to better understand the mode of action of BAA on myocytes. Differentiated C2C12 cell membrane preparations were incubated with increasing concentrations of [3H]CGP12177 (specific activity 42.5 Ci/mmole) in the presence or absence of a 1000-fold concentration of unlabeled CGP12177 to investi- gate if C2C12 cells could be used as a model to study the mode of action of BAA on muscle. The specific binding of [3H]CGP12177 to C2C12 cell membrane prepara- tions was highly specific, saturable, reversible, and of high affinity (K, = 0.2 nM). The binding maximum (BM) value was calculated to be 150 fmole/ mg protein, which is similar to the BM value previously reported (Coutinho et al., 1990; Mersmann & McNeal, 1992) for pig adipose membrane preparations. The 10% non-Specific binding of [’H]CGP12177 to C2C12 cell membrane preparations reported is typical for this ligand (Lacasa et a1, 1986; Mersmann & McNeal, 1992). M2 porcine satellite cells were characterized to investigate the presence of functional BAR in these cells. Non-differentiated porcine satellite cell membrane preparations produced a linear response, but unsaturable [3H]CGP12177 specific binding was observed regardless of the membrane protein concentration used (20-50 pg/ml) in non-differentiated porcine satellite cells. Little or no BAR presence was observed in differentiated cells. The radioligand studies established the presence of BAR in differentiated C2C12 cells and non-differentiated porcine satellite cells. These findings were further strengthened by the BAA-induced cAMP release. Results indieated that all concentrations of BAA (109 to 10’) resulted in extracellular cAMP release higher than the negative control. 105 M ISO, RAC, and CLEN increased extracellular cAMP release (P < .01) on per unit of cellular protein. Forskolin-stimulated extracellular cAMP release was 17-fold that of the negative control. In differentiated and non-differentiated porcine satellite cells, forskolin significantly increased extracellular cAMP release (P < .001) compared to the negative controls in all experiments. 10‘ M ISO significantly increased extracellular cAMP release (P < .001) in differentiated but not in non-differentiated porcine satellite cells. BAR transcripts could not be detected by RT-PCR to explain this difference. This dissertation entitled 'Radioligand binding of [3H]CGP12177 and efficacies of isoproterenol, ractopamine, and clenbuterol on the ligand- regulated BAR-adenylyl cyclase systems in porcine satellite and C2C12 cells" is dedicated to my son, Ernest Osahon Izevbigie, Jr. My son, keep your father’s command, and do not forsake the law of your mother. (Proverbs 6:20) iv ACKNOWLEDGEMENTS First and foremost I thank God for being greater than he who is in the world (I John 4:4). Many people have contributed to get me through this program. Sincere appreciation is extended to Dr. Werner G. Bergen for serving as my mentor. His guidance, encouragement, confidence, and friendship were immeasurable throughout my graduate program. I feel blessed to have had the opportunity to work with such a notable scientist. Appreciation is also extended to the following: To Dr. Richard Balander for serving on my graduate committee and providing invaluable assistance whenever needed. To Dr. Robert Cook for serving on my graduate committee. His guidance, inspiration and support have helped me become a better person, scientist, and edueator. To Dr. Roy Emery for serving on my graduate committee and for his assis- tance whenever requested. To Dr. Dave McConnell for serving on my graduate committee and allowing me to work in his laboratory, and providing untiring instruction on the development of the RT-PCR technique. The RT-PCR experiment reported in this dissertation would have been nearly impossible without the tremendous amount of time and relentless effort contributed by Dr. McConnell. A special thanks goes to Dr. Bill Smith, professor and Head of the Department of Biochemistry, for his unselfish contributions toward the development of the radioligand binding and cAMP experiments; it was really a pleasure, as well as a I privilege, to have had the opportunity to know and work with such a fine person. I am also thankful to Drs. Tom Deits and Jim Ireland for their unlimited assistance. I am indebted to Dr. Dale Romsos for allowing me to work in his laboratory, and for providing assistance whenever needed. Appreciation is extended to Drs. Gretchen Hill and Bill Helferich for allowing me to work in their laboratories. To my loving wife, Karen, I am grateful for your sacrifices, support, patience, and love throughout my graduate program. It is truly a blessing to have and love someone like you. 'Whoso findeth a wife findeth a good thing, and obtained favour of the Lord." (Proverbs 18:22). I thank God for my wonderful parents, Benjamin Ikpomwosa and Esther Izevbigie for their unlimited love, guidance and support. A special thanks goes to Mrs. Margaret McCollum for her support and assistance during this difficult time. I am also thankful to Dr. Matthew Doumit, not only for helping me to develop good cell culture skills, but also for providing the M2 clone of porcine satellite cells used throughout this project. vi My thanks goes to a number of staff and graduate students who helped me in so many ways. My sincere appreciation is extended to Dr. Patty Dickerson-Weber, James Liesman, Bal Krishna Sharma, Sharon DeBar, Jane Link, Dr. Paul Kou, Steve Ekunwe, and Victor Siberio for many and varied contributions. Finally, I would like to thank Dr. James Jay, Assistant Vice Provost, Office of Diversity and Pluralism, and the Department of Animal Science, for giving me the opportunity to study at this fine institution of higher learning. I must say my stay here at Michigan State University has been very rewarding. TABLE OF CONTENTS TITLE LIST OF TABLES ................................... LIST OF FIGURES ................................... CHAPTER I: INTRODUCTION ........................... CHAPTER II: LITERATURE REVIEW ...................... B—Adrenergic Agonists and B—Adrenergic Receptors ............. BAR Covalent Modifications ........................... BAR Sequestration ................................. BAR Isomerization ................................. B—Adrenergic Agonists: Effect on Lean Tissue Deposition ......... B-Adrenergic Agonists: Effect on Adipose Tissue Deposition ....... Exogenous Growth Hormone (GH): Effect on Lean Tissue Deposition . . Exogenous Growth Hormone (GH): Effect on Fat Deposition ....... Additive Effects of BAA and GH ..................... Muscle Biology ................................... Satellite Cell Effects on Skeletal Muscle Growth ............... CHAPTER III: CHARACTERIZATION OF CLONALLY-DERIVED M2 PORCINE SATELLITE CELIS--PROLIFERATION AND DIFFERENTIATION STUDIES ........................... Abstract ....................................... Introduction ..................................... Experimental Procedures ............................. Materials and Methods ............................ Cell Number Determination ......................... DNA Determination ............................. Satellite Cell Differentiation ......................... Results ........................................ Cell Number Determination ......................... DNA Determination ............................. Satellite Cell Differentiation ......................... Discussion ...................................... viii 13 14 15 17 18 20 21 22 23 24 30 30 31 31 31 32 32 33 35 35 40 TABLE OF CONTENTS (cont’d.) TITLE CHAPTER IV: BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF BAR IN PORCINE SATELLITE CELL AND C2C12 CELL MEMBRANE PREPARATIONS ........................... Abstract ....................................... Introduction ..................................... Experimental Procedures ............................. Materials and Methods ............................ Porcine Satellite Culture ........................... C2C12 Cell Culture .............................. Membrane Preparation ............................ Radioligand Binding Assay ......................... Results and Discussion ............................... CHAPTER V: BET A-ADRENERGIC AGONISTS-STIMULATED CAMP ACCUMULATION IN PORCINE SATELLITE AND C2C12 CELLS . . . . Abstract ....................................... Introduction ..................................... Experimental Procedures ............................. Materials and Methods ............................ Porcine Satellite Cell Growth and Differentiation ............ C2C12 Cell Growth and Differentiation .................. BAA-Stimulated cAMP Accumulation ................... Results and Discussion ............................... CHAPTER VI: REVERSE TRANSCRIPTION AND POLYMERIZATION OF MESSENGER RNA IN PORCINE SATELLITE AND C2C12 CELLS Introduction ..................................... Experimental Procedures ............................. Materials and Methods ............................ Porcine Satellite Cell Growth and Differentiation ............ C2C12 Cell Growth and Differentiation .................. Total RNA Isolation and Treatment .................... Homogenization ............................. Extraction ................................. Precipitation ............................... Wash .................................... Oligonuclcotides ................................ Reverse Transcriptase Reaction and cDNA Amplifieation ....... Results and Discussion ............................... Conclusion ...................................... PAGE 42 42 43 45 46 46 47 eaaeessaseassss TABLE OF CONTENTS (cont’d.) TITLE PAGE GLOSSARY ....................................... 84 APPENDIX ........................................ 87 BIBLIOGRAPHY .................................... 91 LIST OF TABLES TABLE PAGE 1 Porcine satellite cell serum-free medium ............... 33 2 Effects of cytosine B-D—Arabinofuranoside on insulin- stimulated M2 porcine satellite cell differentiation ......... 36 3 The oligonucleotide sequences used as primer for the three sub-types of BAR ............................ 68 4 RT-PCR master mix composition ................... 75 LIST OF FIGURES FIGURE PAGE 1 The chemical structure of commonly used BAA in meat animals ................................... 6 2 The mechanism of Beta-Adrenergic Agonist-induced activation of adenylyl cyclase ..................... 8 3 The structure and membrane topology of human B2-AR ..... 12 4 Schematic diagram of BAA action on various tissues ....... l9 5 Cell quantitation studies ......................... 34 6 DNA quantitation studies ........................ 35 7 Early-stage differentiation of M2 porcine satellite cells ...... 37 8 Mid-stage differentiation of M2 porcine satellite cells ....... 38 9 Cells grown in a 10‘ M insulin-only serum-free medium, after 72 h of incubation ......................... 39 10 Cells placed in a 10" M insulin + 10" M ara-c serum-free medium, after 72 h of incubation ................... 40 11 Cells placed in a 105 M Ara-c only serum-free medium, after 72 h of incubation ......................... 41 12 [’H]CGP12177 binding to BAR in non-differentiated porcine satellite membrane preparations of 50, 40, and 20 pg membrane protein/mL, respectively ................. 48 13 [’HJCGP12177 binding to BAR in differentiated porcine satellite cell membrane preparation .................. 51 FIGURE 14 15 16 17 18 19 20 21 22 23 LIST OF FIGURES (cont’d.) [3H]CGP12177 binding to BAR in C2C12 cell membrane preparation ............................... Scatchard Plot of specifically bound [3H]CGP12177 to the differentiated C2C12 cell membrane preparation ......... BAA-Stimulated cAMP accumulation in differentiated porcine satellite cells ......................... BAA-stimulated cAMP accumulation in non-differentiated porcine satellite cells ......................... BAA-stimulated cAMP accumulation in differentiated C2C12 cells ............................... Region of homology between human and bovine BAR ..... The reverse transcription and DNA amplification products . . . . A 1.2% agarose gel showing the 18 and 288 RNA bands Chromatograph of control RNA-derived RT-PCR product . . Chromatograph of differentiated porcine satellite cell- derived RT-PCR product ....................... PAGE 52 53 61 63 69 76 80 82 CHAPTER I: INTRODUCTION In North America, a high percentage of human dietary protein, vitamin, and mineral requirements are met through the consumption of meat and meat products. The United States Department of Agriculture (USDA) prime or high choice quality grade beef (prime refers to most marbled/fat-containing cut) was once preferred and demanded, even at higher price, by consumers. However, during the last 25 years, and due to increased public awareness of the health risks associated with chronic over-consumption of dietary energy, modern consumers prefer leaner meat products. Excess fat deposition by meat-producing animals is an economic waste to both producers and consumers. It has been estimated that it costs the meat animal produc- tion industry about $4 billion annually to deposit fat on animals and remove the fat from meat products (reviewed by Bergen & Merkel, 1991). The meat industry was not able to quickly modify their conventional production practices to meet consumer demand for leaner and less fat products and consequently consumption of red meat products declined for the past 10 years. Despite this decline, the obesity rate in the US. has not declined, which seems to suggest that consumption of red meat may not be the sole cause of human obesity. Unquestionably, excess fat deposition in meat-producing animals is a problem and must be controlled. The industry has responded to this preference for leaner and less fat meat products by developing several strategies toward minimizing fat 1 2 deposition in meat-producing animals; the most effective are the use of large frame, late maturing animals and feed intake control. These practices have resulted in improved leanzfat ratio, although these practices are very cumbersome and may not be cost-effective for US. producers. As it relates to research, there is an increasing quest by animal scientists to develop strategies to mitigate fat while increasing lean tissue deposition in meat- producing animals. There are three ways by which this goal may be achieved: (a) increasing lean tissue deposition, (b) decreasing fat deposition, and (c) a combina- tion of both. Research data generated over the past decade suggests that carcass composition may be manipulated by the use of exogenous growth promotants such as anabolic steroids, beta-adrenergic agonists (BAA), and growth hormone (GH). Anabolic steroids such as estrogen, estrogenic analogs, and trenbolone acetate, approved by the Food and Drug Administration (FDA), are used in ruminants (beef) to improve carcass quality. Furthermore, BAA may enhance lean tissue deposition (Ricks et al. , 1984) while GH, approved for milk production in cows by the FDA, exerts lipolytic effects in steers (Schlegel et al. , 1996). BAA and GH (pending FDA’S approval) are more effective in non-ruminant animals such as pigs. BAA are very effective in promoting lean tissue deposition (Bergen et al. , 1989; Grant et al., 1993), while decreasing fat deposition (Barak et al., 1992) in pigs. Similar results have been reported in other meat-producing animal species such as steers (Ricks et al., 1984) and lambs (Baker et al., 1984). BAA appear to be less efficacious in poultry (Johnson, 1989). Recently, the BAA ractopamine (RAC) was reported to exert a stimulatory effect on satellite cell proliferation (Cook et al. , 1994; 3 Grant et al., 1990). The proliferative activity of satellite cells is important for skeletal muscle growth (Allen et al., 1979). Meat animal-derived satellite cells are of great importance in animal agriculture; therefore it becomes imperative to study and understand the factors that regulate the activation of satellite cells from a dormant stage to proliferation, differentiation and subsequent fusion. Currently, most of what we know about the effects of BAA on satellite cells, particularly porcine-derived satellite cells, has emanated from studies that failed to investigate the presence or absence of BAR; there are no published data, at least to our knowledge, to indicate the presence of a functional BAR in any food animal satellite cells. The present studies seek to establish the involvement of yet another factor, BAA, that enhances the proliferative activity of porcine satellite cells besides basic fibroblast growth factor (bFGF), insulin-like growth factors (IGF-I and II), and platelet-derived growth factor- BB (PDGF-BB) (Doumit et al., 1993). Administration of exogenous GH not only promotes muscle hypertrophy (Grant et al., 1991) but also markedly depresses fat deposition in pigs (Capema et al., 1990; Verstegen et al., 1990) by decreasing lipogenesis (Harris et al., 1993; Kramer et al., 1993; Liu et al., 1994). Taken together, a drastic reduction in fat deposition in meat-producing animals may not be accomplished by the use of any one practice, factor, or exogenous agent, but a combination of practices, factors and exogenous agents may result in the production of meat products in which fat supplies not more than 30% of their total calories. Of course, the overall goal is that these lean meat products may help in decreasing acute clinical eases of high dietary fat-related diseases and lower the health care cost burden on society. CHAPTER II: LITERATURE REVIEW Many traditional concepts of meat quality have assumed that intramuscular adipose tissue enhances meat palatability. The USDA prime or high choice quality grade beef (prime refers to high level of marbling/fat-containing meat cuts), once preferred by consumers even at a higher price, are no longer in demand. During the last 25 years the meat industry has experienced a gradual but major shift in consumer preference for high fat to leaner and less fat meat and meat products. This preference for leaner meat products has been driven by increased awareness by the public that consumption of excessive dietary fat, particularly saturated fat, may contribute to obesity, eardiovascular diseases, and other fat-associated health problems. As a result of this concern, consumption of red meat products has declined during the last decade but the claimed fat-related health problems by health officials have not improved, which tends to suggest that consumption of red meat products may not be the sole contributor to these health problems. No doubt the meat industry must do everything possible to reduce fat deposition in meat-producing animals if it plans to compete in the market for low-fat products. The meat industry has responded by developing and implementing several strategies to depress fat deposition in meat-producing animals; the most successful strategy to date is the use of large, late-maturing animals for meat production. At desired market weights large-frame, late maturing animals are much leaner compared to small frame, early maturing animals. As it relates to research, 4 5 animal scientists have investigated the effects of exogenous growth promotants to enhance carcass lean (high proteinzfat ratio). The use of steroids such as estrogen, estrogen analogs, and trenbolone acetate [TBA] (approved by the FDA) is effective in ruminants (beef) to improve carcass lean (Hayden et al., 1992). BAA such as clenbuterol (CLEN), ractopamine (RAC), and cimaterol (CIM) have been shown to be effective in increasing lean tissue deposition in non-ruminant animals such as pigs. Furthermore, the antilipogenic properties of exogenous GH have been reported (Harris et al., 1993; Kramer et al., 1993; Liu et al., 1994), although exogenous BAA and GH are less effective in poultry (Johnson, 1989). B-Adrenergic Agonists and B-Adrenergic Receptors BAA are structurally similar to the mammalian neurotransmitter epinephrine; they are organic molecules that recognize and bind BAR (see Figure 1 for the chemical structure of commonly used BAA in meat animals). BAR are a member of the seven membrane spanning-receptors; they are glycoprotein receptors that trans- verse the plasma membrane with a stretch of about 20-25 hydrophobic amino acids with extracellular N-termini involved in ligand binding (Suryanarayana & Kobilka, 1993) and cytosolic C-termini involved in receptor desensitization (Hausdorff et al. , 1989) and which interact with G, of the G-protein (Okamoto et al., 1991). Presently, there are 10 sub-classes of known adrenergic receptors: (a) three distinct Beta- adrenergic receptors (Beta 1, 2, and 3), (b) four Alpha l-adrenergic receptors (Alpha l-A, B, C, and D), and (c) three Alpha 2-adrenergic receptors (Alpha 2-A, B, and C), all of which are encoded by different genes. .3255 :3:— E <PKA (inactive) W- WWWW - W -Mmtwm -_W Cellular Response 0 - M UNI! D - "My LID-U The Gag-GTP complex stimulates adenylyl cyclase, resulting in an increase in cAMP, while the Gui-GTP complex inhibits adenylyl cyclase, resulting in a decrease in cAMP. Thus, the interplay between the stimulatory and inhibitory complexes modulates the cellular cAMP level. 51811111 The mechanism of Beta-Adrenergic Agonist-induced activation of adenylyl cyclase. 9 The BAR systems have been studied extensively, and thus have become the paradigm for studying signal transduction mechanisms. Since these receptors are the first molecules in the Signal transduction pathway, they are subject to several levels of regulation. Some of the regulatory points will be discussed later at the appropriate time in this review of literature. Other Signaling pathways involving G-protein are also known. Rhodopsin, for example, is a light-sensitive G-transducin (6,) coupled receptor that undergoes a conformational change upon activation by light to activate cyclic guanosine monophosphate (cGMP) phosphodiesterase to hydrolyze cGMP (Stryer, 1986). Cyclic GMP phosphodiesterase regulates the opening and closing of the Na” Ca2+ channels in the plasma membrane of the retinal cells. The muscarinic receptor is also a member of the seven membrane spanning-receptor coupled to a G- protein, G-potassium (6.), which controls potassium channels (Reuveny et al. , 1994). G-olfactory (Gd!) is involved with the sense of smell. Of particular interest is Fusin, a novel putative G-protein-coupled receptor with the seven transmembrane segment motif, which is important for HIV-1 infection in CD4-expressing, non-human cells (Feng et al., 1996). While these receptors are closely related, they are coupled differently. BAR l, 2, and 3 are coupled to adenylyl cyclase through G-stimulatory (G), hence activation of this subclass of receptors by BAA results in elevation of intracellular cAMP levels (Izebvigie & Bergen, 1996a; Izevbigie & Bergen, 1996b; reviewed by Lefkowitz & Caron, 1988). The effects of a 2-receptors are antagonistic to BAR (Coutinho et al., 1993); they are coupled to G-inhibitory (6-,) protein to attenuate intracellular cAMP levels (Gilman, 1987). NAB-dependent ribosylation of the G, 10 subunit catalyzed by pertussis toxin produces similar effects by inhibiting the alpha- inhibitory (Ola) subunit of the G-protein. Cholera toxin-catalyzed ADP-ribosylation of the alpha-stimulatory ((1,) inhibits the ATPase activity of the or” which sustains 0:, activation. Thus both pertussis and cholera toxins lead to elevation of intracellular cAMP (Schramm & Selinger, 1984). Alpha-l receptors are coupled to phospholipase C (PLC), an enzyme that catalyzes the cleavage of phosphatidylinositol (PI 4,5- bisphosphate) to yield diacylglycerol (DAG) and inositol 1,4,5-triphosphate (1P3) (Kjelsberg et al., 1992). DAG activates phosphokinase C (PKC), an enzyme involved in cell growth and differentiation via the MAP kinase cascade. 1P3 activates the IP, receptor, a Ca2+ release channel on the endoplasmic reticulum (Furuichi et al. , 1989). This activation of the IPa-gated Ca2+ channel leads to increased intracellular Ca2+ concentration. 9 On average, these receptors (BAR) are composed of about 414 amino acids and have a molecular weight ranging fiom 60,000—80,000 (Benovic et al., 1984; Boege et al., 1988; Lomsney, 1986; Regan et al., 1986). These receptors are pharmacologically distinguishable based on their ligand specificities and affinities, although it is possible, for example, for all sub-types of BAR (BAR l, 2, and 3) to be recognized by a single ligand. For example, a hydrophilic, non-selective Beta- adrenergic antagonist CGP12177 (Hosada & Duman, 1993) may recognize all three subtypes of BAR (Bl-AR, B2-AR, and B3-AR) with different affinities. Differences exist among the BAR. Bl-AR has a proline-rich 24-amino acid sequence (PARPPSPSPSPVPAPAPPPGPPRP) present in the third intracellular loop of the receptor, but not present in B2-AR or B3-AR (Green & Liggett, 1994). Proline is 11 known to introduce kinks in proteins, and is thus known as a helix-breaker. Proline is usually found in turns made by polypeptides. The presence of such a proline-rich sequence may confer a rigid conformation in that region of the Bl-AR, a region believed to be important for BAR/G, interaction. Hence Bl-AR has a lower efficiency of agonist-stimulated G, coupling compared to BZ-AR (Green & Liggett, 1994). Deletion of the proline-rich region of the Bl-AR improved coupling efficiency and ability of the receptor to form a high-affinity ternary complex. Insertion of the proline-rich sequence of Bl-AR into B2-AR decreased coupling efficiency of B2-AR (Green & Liggett, 1994). Yet another interesting feature about B l-AR and B2-AR genes is that they are both intronless (Strosberg, 1990), an unusual characteristic of eukaryotic genes. On the other hand, B3-AR is more resistant to desensitization compared to B2-AR beeause B3-AR has fewer serine/threonine phosphorylation residues available for PKA and beta-adrenergic receptor-associated kinase (BARK) phosphorylation, and hence prolonged stimulation of cAMP production (Liggett et al. , 1992) in B3-AR (see Figure 3 for the structure and membrane topology of human B2- AR). This suggests that B3-AR may be important for lipolysis and thermogenesis (Krief et al. , 1993); mice with knockout gene (disruption of the B3-AR gene expres- sion) exhibited reduced BAA-stimulated lipolysis (Susulic et al. , 1995). Therefore, B3-AR specific agonists may be used as anti-obesity and antidiabetic drugs in humans and anti-obesity drugs in animals (Connacher et al., 1994). Another important feature about B3-AR is that multiple cAMP response elements (CRE) have been reported (TGACI‘CCA, TGAGGTCT, and CGAGGTCA located in the 418, 622, and 1125 bases) upstream of the B3-AR coding region. The 12 CHO 00000000000000000000'“: CHO 0 0 0 &u 4.. w. Human Bz-Adrenergic Receptor / 0, owe. 000000000 w... 1 . y/ ,/ 7 1 // EXTRACELLULAR SURFACE .o ooooooooeoooc/ 0.. , .. /../ new .. . 0/. Mam/o. // fl ./ /n 0%. .9 / .e W /A 0 ,Oagq o .. . , ./ . ./ . o o o. .o 6/ ooofl%meWWWo . “Moe.” fio®,, / w t / . 630% _© // /. / ea. I . I 0 9, e 0% mW%W%%WW% / .fl // ., .90 .Jo. ////¢/ 27/ I // /x ; a%%%%fi.o .eoo.b. , o.qeoM 40/.» x, / .. / //. ////.,.,/ . / / 7, n ,/0. /,/ ./ 7/ o... mama ,A.zL%Q/ o. 00/ //////// 00 0 020%6. Be a V . wmgg .. I A 0 / aim ., /2/ 20,05 / fl .. //.// I / I”. . 0 0 .96, 00 000 000 CYIOPLASHIC SURFACE '0000000 0*000'000‘ 00 o oo o. 0 0 V0 0 v0 * v0 0 , o v 0 0 O 0 o . 0 O o o G 0 o 0 0 G o a V o I 0 O 0 0 me o o H 0 mo (Hausdorff et al., 1989). Reproduced with permission. Figure; The structure and membrane topology of human B2-AR. 13 biological implication of these CRE is that B3-AR in humans up-regulates its own expression in the presence of intracellular cAMP (Thomas et al., 1992). Also in humans, Collins et a1. (1990) reported that CRE enhanced transcription of the human B2-AR. There is evidence of B3-AR in rodent adipose tissue. However, evidence for the presence of B3-AR in adult humans is in dispute (Thomas & Liggett, 1993; Walston et al., 1995; Widen et al., 1995). Some naturally occurring mutations in this receptor have been studied. Trp 64 Arg mutation is associated with obesity and may contribute to the onset of non-insulin-dependent diabetes melitus (NIDDM) (Walston et al., 1995; Widen et al., 1995). BAR Covalent Modifications Continuous exposure of BAA to cells containing BAR often results in a rapid desensitization of BAR (Hausdorff et al., 1990, Johnson et al., 1978). Several molecular mechanisms are responsible for the desensitization that is reversible within seconds to minutes (Hausdorff et a1. , 1990). A rapid receptor desensitization results from two distinct mechanisms of phosphorylation (Hausdorff et al. , 1989): PKA, and (BARK). Phosphorylation of the BAR by PKA, BARK, or PKA plus BARK, induces a conformational change that promotes the uncoupling of the BAR-G, system. PKA- mediated phosphorylation occurs at nanomolar isoproterenol (ISO) while BARK- mediated phosphorylation occurs at micromolar ISO (Hausdorff et a1. , 1989). BARK- mediated phosphorylation may be of physiological importance in tissues in which BAR are exposed to high concentrations of catecholarnines such as neural synapses. PKA- mediated phosphorylation does not require receptor occupancy (Clark et a1. , 1988). In contrast, BARK-mediated phosphorylation does (Lohse et al. , 1989). Historically, l4 receptor desensitization has been described in two distinct ways: heterologous and homologous desensitizations. Homologous desensitization occurs due to loss of receptor responsiveness to a desensitizing agonist, whereas heterologous desensitiza- tion is said to occur due to lack of receptor responsiveness to a number of agonists, including the desensitizing agonist. Several lines of evidence have shown the PKA- mediated phosphorylation to be responsible for heterologous desensitization. Liggett et a1. (1989) demonstrated that cells expressing the native B2-AR displayed a rapid decline in agonist-induced cAMP accumulation, while the mutant construct lacking the putative PKA phosphorylation site showed prolonged increase of cAMP accumulation (Liggett et al. , 1989). Other investigators have also reported that deletion of the PKA putative phosphorylation site in B2-AR inhibited heterologous but not homologous receptor desensitization (Clark et al. , 1989). Homologous desensitization is believed to be BARK-mediated based on studies using both wild-type (WT) and Kin' (S49 lymphoma cells lacking PKA activity). For example, Post et al. (1996) reported similar kinetics of cAMP accumulation and agonist-induced cell surface B2-AR loss in WT and Kin' S49 lymphoma cells. These data suggest that homologous desensitiza- tion may be mediated by a B2-AR specific kinase, possibly BARK, which requires a cytosolic cofactor, Beta-arrestin (B-arrestin), to augment its activity. BAR Sequestration Desensitization is believed to precede sequestration (Waldo et a1. , 1993). Chronic exposure of cells to ligands causes a loss in the ability of the receptor to bind hydrophilic, but not hydrophobic, ligands. This phenomenon is referred to as 'sequestration. " Evidence suggests that sequestration occurs due to internalization of 15 the surface receptors (Yu et al., 1993). Hydrophobic ligands can translocate the plasma membrane (hydrophobic environment); in contrast, it is energetically unfavor- able for hydrophilic ligands to do so. Highly conserved tyrosine residues have been implicated as important for BAR sequestration (Barak et al. , 1994). Receptor sequestration or internalization of phosphorylated receptors from cell surface into the cytosol, where phosphatases may be located, promotes dephosphorylation of the phosphorylated receptors so that they may be recycled (Zastrow & Kobilka, 1992). Blocking sequestration by pretreating cells with pharmacological agents such as concanavalin A or phenylarsene oxide had no apparent effect on rapid desensitization (Waldo etal., 1983). This suggests that BAR desensitization does precede sequestra- tion of receptors. BAR Isomerization BAR may assume active and inactive conformations. The binding of agonists may promote the formation of active conformations capable of interacting favorably with G, of the G-protein (Kjelsberg et al. , 1992), whereas the binding of antagonists may promote formation of a partially active or inactive conformation that is incapable of interacting with 6,, hence producing no biological consequences. BAA interact with BAR to form BAA-BAR complexes whose stability depends on the affinities of the agonists for the receptors. Tight binding, referred to as low KD or K, is usually indieative of a stable binding, but not of the ability to induce a biological response (Coutinho et al., 1990; Liu & Mills, 1989). A wild type B2-AR recognizes and binds propranolol and dihydroalprenolol hydrochloride (DHA) as agonists. The amino acid ASN312 is important for ligand binding by participating in hydrogen bond formation 16 with the phenoxy oxygen provided by propranolol and DHA. Substitution of ASN312 with amino acids that cannot form hydrogen bonds (alanine and phenylalanine) prevented binding of the compounds (Suryanarayana & Kobilka, 1993). Furthermore, substitution of ASN312 with glutamine and threonine enabled other compounds to act as agonists (Suryanarayana & Kobilka, 1993). Some natural- ly-occurring polymorphisms have been reported to have occurred in the region of the receptor crucial for the formation of stable agonist-receptor-Ga complexes; serine 164 of the human B2-AR in the fourth transmembrane domain, purported to be the ligand binding pocket, interacts with the B-carbon of adrenergic ligands. Mutations at serine 164 to He decreased binding affinity (1450179 versus 368139 nM), adenylyl cyclase stimulation, and BAR sequestration (Green et al. , 1993). Ligands lacking the hydroxyl groups on their B-carbon were not affected by the serine 164 to He polymor- phism (Green et al. , 1993). Agonist-receptor complexes may interact with specific G- proteins (Jones & Read, 1987). Studies with rat olfactory neuroepithelium indicated the existence of multiple forms of G, (Jones & Read, 1987). The biological impliea- tion of multiple G, species is that there may be no additive response when two agonists coupled to the same G-protein are used simultaneously. For example, in the hepatocytes derived from partially heptectomized male rats, epinephrine and glycogen were found to be coupled to the same G- system (Yagami, 1995). However, the potency of activation for each agonist may be different. Furthermore, when multiple agonists are used concurrently, one agonist may affect the affinity of the other (Yagami, 1995). These data might help to explain, at least in part, why Liu and Mills (1989) observed a concentration-dependent inhibition of epinephrine-stimulated l7 lipolysis by RAC or CLEN in pig adipocytes. On this basis, some investigators suggest that in vivo RAC and CLEN may block the lipolytic responsiveness of the BAR-adenylyl cyclase systems to catecholamines. The preponderance of evidence from in vivo (Bergen et al., 1989; Grant et al., 1993; Helferich et al., 1990) and in vitro studies (Anderson et al. , 1990) showed that BAA increased lean tissue deposition while decreasing adipose tissue deposition (Barak et al., 1992). However, the magnitude of responses vary between laboratories; the reasons for some of the variations may be due to tissue-specific responses to agonists (Spurlock et al. , 1994) species-specific responses to agonists (Mersmann, 1984), and pharmacodynamics of the agonists. Therefore, data obtained using a particular species and agonist must not be extrapolated across species. An intriguing question would be what properties make a given drug an agonist or antagonist. The answer is unclear, but it is known that the ability of some agonists to bind tightly to their receptors is unrelated to their ability to stimulate lipolysis (Liu & Mills, 1989). Rather, the primary distinguishing property between beta-agonist and antagonist resides in their ability to activate adenylyl cyclase to produce cAMP (Jasper et al., 1988). B-Adrenergic Agonists: Effect on Lean Tissue Deposition Skeletal muscle is a major target for BAA actions. BAA-fed animals deposited more lean tissue compared to the control (Bergen et al., 1989; Grant et al., 1993) although the magnitude of the BAA-induced hypertrophy depends on dietary protein intake (Adeola et al., 1990; Adeola et al., 1992; Bergen et al., 1989). The lean tissue-stimulatory effect of BAA was also demonstrated in vitro; this effect was reversed in the presence of propranolol, a beta blocker or antagonist (Anderson et a1. , 18 1990). This tends to suggest that the effects of BAA on skeletal muscle growth may be BAR-mediated (Anderson et al., 1990; Choo et al., 1992); however non-BAR- mediated BAA actions have been proposed (Bergen et al., 1989; Smith, 1989). The mechanism whereby BAA promote lean tissue deposition is a matter of controversy. The mechanism may be via increased protein synthesis (Bergen et al., 1989; Grant et al., 1993; Helferich et al. , 1990) or increased expression of certain protease inhibitors to decrease protein degradation (Pringle et al., 1993). When protein synthesis rate was measured in vivo by continuous infusion with [“C] lysine, Helferich et al. (1990) reported that the fractional synthesis rate (FSR) of skeletal muscle a-actin was enhanced by 55% in RAC-treated pigs compared to the control. Bergen et al. (1989) observed an increase in fractional accretion rate (FAR) due to increased FSR in the semitendinosus muscle of barrows. These data generated from continuous infusion of radioactive amino acids further underscores the lean tissue-enhancement properties of BAA. Liu et al. (1994) reported that RAC increased nitrogen retention in longissimus muscle area, and increased a-actin gene expression. B-Adrenergic Agonists: Effects on Adipose Tissue Deposition Depressed body fat deposition is one of the metabolic consequences of feeding BAA to animals. BAA depress fat deposition by decreasing lipogenesis through the inhibition of lipogenic enzymes (Dickerson, 1990). Furthermore, BAA induced cAMP release which activate cAMP-dependent protein kinase A (PKA) which, in turn, activate or deactivate a series of intracellular enzymes by phosphorylation including hormone—sensitive lipase. Increased lipolysis is due to increased in triacylglycerol lipase activity (Yang & McElligott, 1989). Studies with pigs showed 19 that RAC enhanced lipolysis, while adipose tissue malic enzyme fatty acid synthase activities were decreased (Merkel et al., 1987) (see Figure 4 for the schematic diagram of BAA action on various tissues). Evidence from in vitro studies indicates that BAA stimulated glycerol release and inhibited fatty acid synthase (PAS) activity in a dose-dependent manner in TAl cells (Dickerson-Weber et a1. , 1992). In pig adipose tissue explants, Peterla and Scanes (1990) reported that ISO, CIM, and RAC Extracellular Surface V (Niopiasrnic Surface Gs Hormone Sensilive Upase ATP AC cAMP——> PKA Lipolysis \ / BAA e anacyigiycerol ® ,8 ACC W - Protein synthesis :53 Lipogenesis % Amino Acid 9' Protein degradation OOOOOO Muscle WW 0 o o o o a DNA a o o o a o . Satellite cells Figured, Schematic diagram of BAA action on various tissues. 20 exhibited lipolytic and antilipogenic effects. Pigs fed diets supplemented with 20 ppm RAC showed higher blood non-esterified fatty acids (NEFA) compared to the control (Adeola et al., 1992). These results underscore the contention that BAA depress fat deposition in animals by increasing the lipolytic activity (Adeola et al., 1992; Merkel et al., 1987; Peterla & Scanes), while decreasing lipogenesis (Dickerson, 1990; Merkel et al., 1987; Peterla & Scanes, 1990). Exogenous Growth Hormone (GH): Effect on Lean Tissue Deposition Administration of exogenous OH to animals has resulted in improved pro- teinzfat ratio and feed efficiency (Capema et al., 1995; Grant et al., 1991; Solomon et al., 1988; Verstegen et al., 1990). The effective dose range of GH is 50-100 rig/kg BW (Thiel et al., 1993). Although animals must be fed not only adequate dietary crude protein to show growth hormone-stimulated muscle hypertrophy (Seve et al. , 1993; Solomon et al., 1988), but lysine, a first limiting amino acid in pigs’ diet, must be no less than 1.2% (Hansen et al., 1994). Pigs injected with porcine somatotropin (pST') had improved feed efficiency up to 1.2% lysine (Hansen et al., 1994). Growth hormone-treated pigs tend to have heavier livers, kidneys, and hearts (Fabry et al., 1991). It is not clear whether GH stimulates the growth of these organs directly or indirectly as a result of improved growth rate. In pigs, GH increases protein synthesis by enhancing the rate of amino acid incorporation into proteins (Capema et al., 1995) and fractional synthesis rate (FSR) as measured by flooding dose of L-[1-13C] valine (Seve et al.,1993). The mechanism of GH-mediated muscle hypertrophy is not clearly understood. However, there is a positive correlation between GH administration and liver and adipose tissue IGF-l mRN A (Coleman et 21 al., 1994; Grant et al., 1991). Hence the GH effects on these organs and tissues may be IGF-l-mediated. However, in skeletal muscle, GH and IGF-1 mRNA are not correlated, thus suggesting that the GH effects on IGF-l gene expression may be tissue specific (Coleman et al., 1994). Exogenous Growth Hormone (GH): Effect on Fat Deposition The effects of GH oppose those of insulin on lipid and carbohydrate metabo- lism and may be called counter-regulatory hormone (Davidson, 1989). In barrows, administration of exogenous GH resulted in reduced acetyl CoA carboxylase (ACC) enzyme activity, and protein content and mRNA abundance for ACC in adipose tissue by 40 to 50% (Liu etal., 1994). Grant et al. (1991) and Coleman et a1. (1994) reported an elevated IGF-l mRNA level in the liver, and an increased serum IGF-l concentration following GH treatment. Subcutaneous adipose tissue IGF-l mRNA level was increased in GH-treated pigs (Coleman etal., 1994). Based on these findings, Coleman et al. (1994) concluded that GH actions on adipose tissue may be IGF-l mediated. GH-treated barrows showed a 79% decrease in total activated ACC and a 67% decrease in fatty acid synthase (FAS) activity (Harris et al., 1993). Furthermore, northern blot analysis indicated a 90% decrease in the FAS mRNA (Harris et al., 1993). Mildner and Clark (1991) used a 1.5-Kb cDNA probe, representing the thioesterase domain of the mold-functional porcine FAS to examine the tissue distribution of FAS mRN A within the pig and concluded that porcine GH significantly depressed FAS mRNA in both adipose tissue and liver. It has been suggested that porcine GH may also reduce fat deposition in growing pigs up to 70% by inhibiting lipogenesis and the expression of insulin-responsive gene such as glucose 22 transporter protein 4 (GLUT 4) (Etherton et al., 1993). Taken together, GH depress- es fat deposition by decreasing lipogenesis through pretranslational regulation of lipogenic enzymes: ACC (Harris et al., 1993; Liu etal., 1994), FAS (Harris et al., 1993; Mildner & Clark, 1991), and GLUT 4 (Etherton et al., 1993). ' 'v ff f Research data suggest that BAA are effective in promoting lean tissue deposi- tion (Bergen et al., 1989; Grant et al., 1993; Helferich et al., 1990). The FSR of skeletal muscle a-actin was enhanced by 55 % in RAC-treated pigs (Helferich et al. , 1990) and GH more effective in reducing fat deposition. 1n growing pigs, fat deposition may be decreased up to 70% by OH (Etherton et al., 1993). In 1991, a GH—adipose tissue membrane G, protein complex was reported (Roupas et al. , 1991). Ga, interacts negatively with adenylyl cyclase to attenuate the level of cellular cAMP. Whereas BAA-induced Ga, dissociation from the heterotetrameric G protein stimulates adenylyl cyclase. Thus the interplay between the Ga, and Ga, modulates the level of cellular cAMP. GH hormone may nullify the attenuation function of the G protein to augment cAMP production (Roupas et al. , 1991). Therefore further stimulation of the G protein by BAA may further increase cellular cAMP level. Knowing this, one may envisage that concomitant administration of BAA and GH may result in additive effects. Indeed it does (Hansen et al., 1994). Of interest is the biochemical evidence that muscle and adipose tissues possess BAR which may be activated by BAA. Spurlock et al. (1994) evaluated the effect of feeding RAC, a phenethanolamine, on beta-adrenergic receptor densities and affinities in both adipose and muscle tissues. The investigators reported that RAC did not 23 affect the maximum binding (BM) of [3H]dihydroalprenolol ([3H]DHA) to longissimus muscle membrane preparations. However, in the adipose membrane preparations, RAC reduced B-adrenoceptor density by approximately 50%. Furthermore, RAC feeding did not affect [3H]DHA affinity for BAR in muscle or adipose tissue. In another experiment Spurlock et al. (1993) studied the affinities (K,) with which CLEN, RAC, and L-644,969 bind BAR population in porcine adipose and muscle membranes in the presence of a competitor [3H]DHA. CLEN had the highest affinity, 125 nM (the lowest K,), followed by L-644,969 (350nM), and RAC (856 nM). K, values were similar within a given agonist regardless of tissue type, except that RAC had a higher affinity, 856 nM, for the middle subcutaneous (SQ) adipose tissue. Coutinho et a1. (1992) reported the presence of two subtype BAR (B,-AR and Bz-AR) in porcine adipocyte crude membrane preparations of which 45 % of the receptors had a high affinity for ICI89,406, a B,-AR antagonist, K, = 2.27 :1; 0.68 nM. Muscle Biology Skeletal muscle growth is of great interest to animal agriculture because muscle represents the most economically important tissue in the animal’s body. In the human diet, muscle serves as a source of minerals, vitamins, and high quality protein. For these reasons, animal scientists continue to investigate factors that regulate prenatal and postnatal skeletal muscle growth in their quest to maximize lean tissue deposition while minimizing fat deposition. Embryonic myoblasts arise from mesodermal cells. These stem cells are capable of giving rise to either adipoblasts, chondroblasts, myoblasts or osteoblasts. Various growth factors take part in the determination of the fate of stem cells. The 24 process by which myoblasts arise from mesoderm cells is termed "determination," which results in a population of proliferative cells and their descendants committed to the myogenic lineages (reviewed by Stockdale, 1992). During embryonic develop- ment of muscle tissue, two morphologically distinct myotubes, primary and second- ary, form. Both contain mononucleated cells enclosed by a common lamina. The secondary myotube forms on the surface of the primary myotube and later become free of the primary myotube. Consequently, non-fused mononucleated cells are trapped between the basement membrane and sarcolemma. Myogenic cells prolifer- ate, differentiate, and subsequently fuse with each other or myotubes to become a multi—nucleated myotube. Since fused nuclei in multinucleated myotubes are mitotic- incompetent, how then is the increase in DNA and protein concentrations observed in hypertrophied skeletal muscle accounted for? Satellite Cell Effects on Skeletal Muscle Growth In most species, including meat-producing animals, myotube formation is essentially complete at birth or shortly after. Therefore, beyond birth, muscle fiber number remains virtually constant. During skeletal muscle growth, myofibers increase in size due to either hyperplasia (increase in cell number) or hypertrophy (increase in cell size). The latter is mostly responsible for postnatal muscle growth indicated by increased proteinzDNA and proteianNA ratios (Skjaerlund et al. , 1994). As a common feature among cells, genes need to be transcribed to yield total RNA, and mRNA must be translated to yield proteins required for structural or enzymatic function in the cell. Protein accretion is a dynamic process that is dependent on protein synthesis and degradation. Protein accretion occurs when synthesis is greater 25 than the rate of degradation, as is typical in growing animals, but when protein degradation is greater than synthesis, muscle mass degeneration occurs. Comparing the longissimus dorsi (LD), semitendinosus (ST), and brachialis (BR) muscle fraction- al synthesis rate (FSR) of 45 kg (older) versus 22 kg (younger) pigs, Mulvaney et al., (1985) reported a 20% lower fractional synthesis in the 45 kg pigs compared to the 22 kg pigs. However, the fractional breakdown rate (F BR) derived by difference (FBR = FSR - FAR [fractional accretion rate]) was lower in the older pigs, suggesting muscle growth rate may be modulated by alterations in FBR (Mulvaney et al., 1985). Since myofibers are mitotic-incompetent, this means the number of nuclei present within each myofiber remains constant. An intriguing question then arises: How then are the nuclei, whose numbers remain constant, able to meet the protein synthesis needs of the increasing cytoplasmic volume? CNR, as expressed by Landing et al. (1974), is the cytoplasmic volume to nucleus ratio. Postnatal muscle growth is largely due to increasing CNR (Mozdziak et al. , 1994), thus suggesting little or no change in the number of nuclei present in the myofiber. In contrast, Moss (1968) reported that the DNA unit size (which is the same as CNR) remains constant, indicating that, as fibers undergo hypertrophy, they accumulate more proteins, and DNA from outside source must be added to myofiber in order to maintain a constant CNR. Seven years earlier Mauro (1961) had described satellite cells as mononucleated, mitotic-competent cells trapped between the sarcolemma and base- ment lamina of the muscle fiber, indicating that satellite cells are the source of nuclei or DNA addition to myofibers during postnatal muscle growth (Allen et al., 1979) or regeneration (Mauro, 1961). Satellite cells are myogenic cell precursors; they 26 proliferate, differentiate to become myogenic cells that subsequently fuse either with each other to form new myofibers, or fuse with existing myofibers. It is now fairly well-established that satellite cells contribute nuclei, and consequently more DNA and protein, to myofibers (Appell et al., 1988; Kennedy et al., 1988). Mozdziak et al. (1994) observed an age—related decrease in satellite mitotic activity, and also an age- related increase in CNR in turkey satellite cells. These data suggest that satellite proliferation, differentiation and subsequent fusion are more important for growing animals, while late-phase postnatal growth is largely due to hypertrophy (Mozdziak et al., 1994). Since satellite cells play a key role in postnatal muscle growth and development, it becomes imperative to understand the factors involved in the activa- tion of satellite cells from the quiescence stage to proliferation, differentiation, and fusion, especially meat animal-derived satellite cells. Optimum conditions for isolation, proliferation, and differentiation for porcine primary satellite cells have been described (Doumit & Merkel, 1992). In cell culture studies, Doumit et al. (1993) examined the mitogenic properties of basic fibroblast growth factor (bFGF), insulin-like growth factors (IGF-I and II), platelet-derived growth factors (PDGF-AA and BB), and epidermal growth factor (EGF)-individually and combined-4n basal serum-free medium or minimum essential medium containing 2% fetal bovine serum (MEM-2% FBS). Individually, bFGF, IGFs, and PDGF-BB stimulated the proliferative activity of porcine satellite cells propagated in basal serum free medium or MEM-2% FBS. EGF promoted the proliferative activity of porcine satellite only in MEM-2% FBS (Doumit et al., 1993). Any combination of bFGF, IGF-I, EGF, and PDGF-BB, except EGF and bFGF, 27 produced a synergistic response (Doumit et al., 1993). Transforming growth factor- beta (TGF-B) may promote or inhibit the proliferative activity of porcine satellite cells depending on the presence or absence of other growth factors (Cook et al., 1993). TGF-B inhibited PDGF-BB-stimulated proliferation, enhanced bFGF-stimulated proliferation, but had no effect on [GP-stimulated proliferation of porcine satellite cells grown in serum-free medium (Cook et al., 1993). When two growth factors were used concomitantly, TGF-B depressed PDGF-BB and IGF-1, PDGF-BB and EGF, PDGF-BB and bFGF, and IGF-1 and BOP-proliferative activities of porcine satellite cells (Cook et al., 1993), but TGF-B had no effect on the IGF-I and EGF- stimulated proliferation of porcine satellite cells (Cook et al. , 1993). These investiga- tors concluded that bFGF and TGF-B interact favorably to increase the bFGF- stimulated proliferative activity while TGF-B interacts unfavorably with PDGF-BB to depress the mitogenicity of PDGF-BB. Taken together, bFGF, IGF,, TGF-B, and PDGF-BB are potent regulators of porcine satellite cells. Therefore, alteration in the levels of these growth factors remains a possible mechanism to regulate porcine satellite cell proliferative activities. Similar results have been reported in other species such as chicken and rats. In cultured chicken breast satellite cells, RAC or ISO doubled myotube nuclei number compared to untreated cells; however, the BAA-induced increase in myotube nuclei number was decreased by about 25% in the presence of 10’ M propranolol, a beta-adrenergic antagonist (Grant et al. , 1990). FOP-stimulated the proliferative activity of chicken satellite cells (Grant et al., 1990). 28 In rat studies, Bischoff (1990) reported that mitogens released from injured muscle produced a long-lasting effect that committed dormant satellite cells to proliferate, while serum growth factors were needed to maintain progression through the cell cycle (Bischoff, 1990). Four myogenic basic helix-loop-helix proteins--myogenin, MyoD, Myf-S, and MRF4--are capable individually to stimulate cell differentiation when introduced to non-myogenic cells (reviewed by Rawls et al., 1995). Gene knockout experiments showed that no more than two--MyoD and myogenin, or Myf-S and Myogenin--are required for muscle differentiation, at least in rat studies (Rawls et al. , 1995). Because in normal, uninjured adult muscle, satellite cells are mitotically quiescent, in the recent years researchers have sought to examine the physiological cues that activate satellite cells from dormant stage to proliferation, differentiation, and subsequent fusion with other satellite cells or myofibers. They have also examined the role of each myogenic factor (Myogenin, MyoD, Myf-S , and MRF4) by monitor- ing the time of appearance of each myogenic factor during cell culture. In rat satellite cells derived from injured tissues, the first indication of myogenic cell differentiation, an increase in myogenin mRNA expression, occurred within 4-8 h after injury (Rantanen et al., 1995). Furthermore, the first desmin-, MyoD,-, and myogenin- positive myoblasts were observed after 12 h, but satellite cell proliferation, as measured by bromodeoxyuridine incorporation, was not seen until 24 h (Rantanen et al. , 1995). This schedule of events (differentiation preceding proliferation) which contradicts the general concept that proliferation precedes differentiation, led these investigators to propose that there may be two populations of satellite cells: (a) One 29 population differentiates immediately following injury, and (b) the other population proliferates (Rantanen et al., 1995). These findings may have been corroborated by Yablonka-Reuveni and Rivera (1994). Using immunohistochemical techniques, Yablonka-Reuveni and Rivera (1994) reported that only half of myogenin or alpha- smooth muscle actin (alpha SM actin) positive adult rat satellite cells were positive for developmental sarcomeric myosin, a differentiation index. These results suggest that only a fraction (about 50%) of the satellite cell descendants entered the phase of terminal differentiation (Rantanen et a1. , 1995 ; Yablonka-Reuveni & Rivera, 1994). Taken together, this literature review suggests that the use of exogenous growth promotants such as BAA and GH (pending FDA approval) in combination with current industrial practices, may result in the production meat products in which fat supplies less than 30% of their total calories. However, consumer acceptance of the use of endogenous agents leads to the following issues: (a) safety, and (b) quality and palatability of meat derived from BAA-treated animals. In terms of safety, because of the low concentration of BAA required to manipulate carcass composition coupled with an adequate pre-slaughter withdrawal period, it is prudent to speculate that there is little or no risk associated with consum- ing meat or meat products from BAA-treated animals. Indeed, studies with turkeys indicated that RAC was rapidly eliminated after oral dosing (Smith et al., 1993). CHAPTER III: CHARACTERIZATION OF CLONALLY-DERIVED M2 PORCINE SATELLITE CELLS--PROLIFERA'I'ION AND DIFFERENTIATION STUDIES Abstract Data generated from both growth studies quantifying DNA amount or cell number produced similar results. Insulin- or insulin and ara-c-stimulated differentiation, as measured by creatine kinase, were numerically similar and higher than the lower creatine kinase activity observed in ara-c-stimulated differentiation. Ara-c inhibits myoblast DNA synthesis (Turo & Florini, 1982). Insulin addition to the cultures may have countered these effects. DNA content per M2 satellite cell was estimated to be 8.9 pg using procedures described by West et al., (1985). 30 31 Introduction Optimum conditions for the propagation of clonally-derived (Ml) porcine satellite cells have been described (Doumit & Merkel, 1992; Merkel et al., 1993). When Ml porcine satellite cells were cultured either in chemically-defined or serum- containing media, they proliferated, differentiated and subsequently fused with each other to form multi-nucleated myotubes (Doumit, 1994; Doumit & Merkel, 1992) that expressed some muscle-specific proteins including myosin and creatine kinase protein (Doumit, 1994). The induction of creatine kinase activity may be used as a marker for myogenic cell differentiation (Turo & Florini, 1982). M2 porcine satellite cells used in these studies, not previously characterized, were generously provided by Dr. Matthew Doumit, USMARC, NE. Both differentiated and non-differentiated M2 porcine satellite cells were utilized in the present studies. The objectives of these studies were: (a) to characterize the proliferation rate of these cells by using a hemocytometer, and DNA quantitation as described by West et al. (1985); and (b) to conduct differentiation studies-creatine kinase activity--units/ mg DNA. Experimental Procedures WSW Antibiotic-Antimycotic (ABAM), gentamicin, Minimum Essential Medium (MEM), and Fetal Bovine Serum (PBS) were purchased from GIBCO BRL (Grand Island, NY). Tissue culture (35 mm diameter, 6-well) plates were purchased from Corning Glass Works (Corning, NY). Bovine pancreas insulin (I-l882), bovine transferrin (I'-8027), MCDB-llO medium (M-6520), dexamethasone (D-8893), bovine serum albumin (BSA—RIA grade A-7888), water-soluble linoleic acid (L-5900), 32 porcine skin gelatin (G-1890), bisbenzamide [Hoescht 33258 (B1155)], calf thymus DNA (D-8661), UV-47 kit, cytosine B-D-Arabinofuranoside (C-l768), trypsin-EDTA, and Giemsa stain (6-4507), were obtained from Sigma Chemical Company, St. Louis, MO. Clonally-derived M2 porcine satellite cells previously isolated by Doumit and Merkel (1992) from the semimembranosus muscle of 6- to 8-wk-old pigs were extended to the fifth passage and used in these studies. Cells were suspended in MEM containing 10% FBS, 0.5% ABAM, and 0.1% gentamicin, seeded at a density of 10,000 cells per 35 mm diameter well previously coated with 0.1% gelatin as described by Richler and Yaffe (1970), and propagated in a humidified C02 incubator containing 95% air and 5% CO, at 37°. C ll ll 1 D . . Cells were grown as described in the Materials and Methods section for 8 (I. At 24-h intervals six wells were randomly selected for cell number determination. Medium was aspirated from monolayers, and cell monolayers were then washed with PBS (pH 7.4) and trypsinized. Cells were recovered by centrifugation (1400 x g) for 3 nrin. Pellets were resuspended to 400,000 cells/mL before counting using a hemocytometer. E II! E . . For DNA quantitation, cells were propagated as described in the Materials and Methods section. At 24-h intervals, six wells were randomly selected and assayed for DNA content as described by West et al. (1985) based on the measurement of the 33 relative fluorescence of the DNA-bisbenzamide complex. The amount of DNA per cell was estimated by measuring the relative fluorencence of known cell quantities using calf thymus DNA as a standard. Upon confluence at approximately day 5, cells were switched to a serum-free medium as described by Merkel et a1. (1993) except bFGF and PDGF-BB were not added, and 10'0 M dexamethasone was used, to induce differentiation. This medium was originally formulated to promote growth, but was later modified to promote differentiation (upon verbal discussion with Dr. Doumit). The components of the differentiation-promoting medium are listed in Table 1. Table l. Porcine satellite cell serum-free medium. Component Final Concentration MEM:MCDB-110 medium 4:1 Dexamethasone 10.10 M Bovine Serum Albumin 0.5 mg/mL Insan 10‘ M Transferrin 100 ug/mL Water-soluble linoleic Acid 0.5ug/mL Cells were fed serum-free medium, supplemented with or without 10’ M ara-c for 72 h, and then switched to MEM + 10% FBS (growth medium) for 24 h to support the completion of the myotube formation process. At 24-h intervals, six wells were randomly selected and medium aspirated from monolayers. Cells were washed three Cell # I well 34 times with cold PBS (pH 7.4) and overlaid with 0.5 mL of 0.05 M glycylglycine buffer (pH 6.75). Cells were kept at -20° C for creatine kinase activity analysis (within 5 (1) using 47-UV kit (Sigma) based on the procedure described by Szasz et al. (1976) except a 40 pL sample was used for each assay. Duplicate samples of 100ul were taken from each well for DNA content determination using calf thymus DNA as a standard. DNA content per well was determined as described by West et al. (1985) except a 100 pL sample was used for each assay. Results [2 ll 11 l I! . . Results of the cell quantitations studies are presented in Figure 5. Each data point represents the mean of six incubations. 500.000 ,— 400,000 -» 300,000 ~~ 200.000 4- 100.000 -> 1 2 3 4 5 5 7 8 Days in Culture Figure}, Cell quantitation studies. ng DNA I well 35 Wineries Results of DNA quantitation are shown in Figure 6. Each data point represents the mean 1 SD of six incubations. 6,000 -r 5.000 i 4,000 «~ 3,000 4 2,000 4 1,000 4 1 ' 2 3 4 5 6 7 Days in Culture Eigm DNA quantitation studies. 5 ll' CllD'Efl .. Results of the differentiation studies are shown in Table 2. Data are means j; SD of six incubations. Porcine satellite cells were grown as described in the Materials and Methods section. Cells were differentiated in serum-free medium supplemented with or without 105 cytosine B-D-arabinofuranoside (Ara-c). For the Ara-c-only treatment, serum-free medium was not supplemented with insulin. 36 Table 2. Effects of cytosine B-D-Arabinofuranoside on insulin-stimulated M2 porcine satellite cell differentiation. Creatine kinase activity (unit/mg DNA) Hours since Day in Prediffer— Insulin Insulin + Ara-c differentiation culture entiation Ara-c induction 0 5 3.94 3; 0.77 - -- - 24 6 6.14 i 1.5 6.94 :1; 1.21 4.29 1; 1.18 48 7 6.02 i 0.9 6.24 :t 2.67 4.68 :1: 1.07 72 8 5.15 :1: 2.16 4.54 :1; 1.24 1.85 :l; 0.83 96 9 14.0 :1: 7.39 12.55 :1: 3.12 11.51 :1: 2.35 Figure 7 illustrates the morphology of differentiating satellite cells (early stage) in serum-free medium. Cells were grown in MEM + 10% PBS to confluence at 5 d and cells were placed in serum-free medium (differentiation medium) for 24 b. Figure 8 illustrates the morphological appearance of differentiating satellite cells (mid-stage) in serum-free medium. Cells were grown in MEM + 10% PBS to confluence at 5 d and cells were placed in serum-free medium (differentiation medium) for 48 h. Figures 9 and 10 illustrate insulin-stimulated porcine satellite cell differentia- tion. Figure 9 shows multinucleated myofibers grown in an insulin-only medium. Cells were grown as described in the Materials and Methods section. Upon conflu- ence, cell differentiation was induced by switching cells to a serum-free medium containing 10" M insulin. Cells were incubated in serum-free medium for 72 h. They were then fixed in absolute methanol and stained with 0.03 % Giemsa to visualize the nuclei. 37 Eiguml, Early-stage differentiation of M2 porcine satellite cells. ’fl' 38 Figure 8, Mid-stage differentiation of M2 porcine satellite cells. Figure 2. Cells grown in a 1045 M insulin-only serum-free medium, after 72 h of incubation. Figure 10 illustrates multinucleated myofibers grown in an insulin and ara-c medium. Cells were propagated as described in the Materials and Methods section Upon confluence, cell differentiation was induced by switching cells to a serum-free medium containing 1045 M insulin and supplemented with 105M Mara-c. Cells were incubated In serum- -free medium for 72 h. Cells were fixed In absolute methanol and Stained with 0.03% Giemsa to visualize the nuclei. Figure 11 illustrates the effect of cytosine—B—D-arabinofuranoside on porcine satellite cell differentiation in serum-free medium. Cells were grown as described in the Materials and Methods section. Upon confluence, cell differentiation was induced by switching cells to a serum-free medium with 10'5 M ara-c. Cells were 40 differentiated in serum-free medium without 10" M insulin for 72 h, then fixed in absolute methanol and stained with 0.03% Giemsa for nuclei visualization. Figure 10, Cells placed in a 10" M insulin + 10'5 M ara—c serum-free medium, after 72 h of incubation. Discussion The growth data suggest that M2 porcine satellite cells do proliferate in culture, and the estimated 8.9pg DNA per cell is similar to the value previously reported by Doumit (1994). These cells are also capable of undergoing differentiation, as indicated by creatine kinase induction, upon exposure to serum-free medium. Insulin- or insulin plus ara-c-stimulated creatine kinase activity (units/ mg protein) were similar and numerically higher than the ara—c-stimulated creatine activity which is due to the 41 inhibitory action of ara-c on protein synthesis, which may include creatine kinase protein and DNA synthesis (Turo & Florini, 1982). Figure 11. Cells placed in a 10'5 M Ara-c only serum-free medium, after 72 h of incubation. CHAPTER IV: BIOCHEMICAL EVIDENCE FOR THE PRESENCE OF BAR IN PORCINE SATELLITE CELL AND C2C12 CELL MEMBRANE PREPARATIONS Abstract C2C12 cell membrane preparations were incubated at 37°C for 2 h with increasing concentrations of [3H]CGP12177 (specific activity 42.5 Ci/mmole) in the presence or absence of a 1000-fold concentration of unlabeled CGP12177. The specific binding of [3H]CGP12177 to C2C12 cell membrane preparations was saturable, reversible, and of high affinity (K, = 0.2 nM). The binding maximum (Bm) value was calculated to be 150 fmole/ mg protein, which is similar to the BM values previously reported by other investigators for both myocyte or adipocyte membrane preparations. ' The non-specific binding of [3H]CGP12177 to C2C12 membrane preparations was approximately 10% . In other experiments, incubation of differentiated and non- differentiated porcine satellite cell membrane preparations with increasing concentra- tions of [3H]CGP12177 produced a linear response, but unsaturable [’H]CGP12177 specific binding was observed regardless of the protein concentration used (20-50 pg protein/mL) for non-differentiated porcine satellite cells. Little or no BAR presence was observed in differentiated cells. 42 43 Introduction BAA are organic molecules, structurally Similar to the mammalian neurotrans- mitter epinephrine. They are ligands of the guanine nucleotide-binding protein coupled glycoprotein receptors which belong to the seven membrane Spanning- receptor family. The G protein is, in turn, coupled to either an effector or an enzyme, such as adenylyl cyclase. BAR transverse the plasma membrane with a stretch of about 20 to 25 hydrophobic amino acids with extracellular N-termini involved in ligand binding (Suryanarayana & Kobilka, 1993) and cytoplasmic C— termini involved in receptor desensitization (Hausdorff et a1. , 1989) and G, activation (Okamoto et al., 1991). Several BAA have been shown to promote lean tissue deposition while depress- ing fat deposition in several species (Anderson et al., 1990; Baker et al., 1984; Barak et al., 1992; Bergen et al., 1989; Ricks et al., 1984). The anabolic effects of BAA on skeletal muscle may (Choc, Horan, Little & Rothwell, 1992) or may not (Smith, 1989) be BAR-mediated. Cook et al. (1994) reported that ractopamine enhanced the PDGF-stimulated proliferative activity of porcine satellite cells and thus concluded the presence of BAR in these cells even though BAR presence was not studied directly. Satellite cells play a significant role in muscle fiber growth (Allen et al., 1979) and muscle regeneration during injuries (Mauro, 1961). Several models have been developed to study the mode of action of BAA in adipocytes but only a few for myocytes. Because myogenic C2C12 cells have been used to study other aspects of cell biology, the objectives of these studies were: (a) to 44 determine if C2C12 cells could be used as a model to study the action of BAA, (b) to study the ontogeny of porcine satellite cell BAR, and (c) to utilize a hydrophilic, non-selective beta-antagonist to investigate the presence or absence of BAR in porcine satellite cells (just as researchers have used data derived from radiologand binding studies to establish the presence of BAR [Lacasa et al., 1986; Mersmann & McNeal, 1992]). Experimental Procedures WM Antibiotic-Antimycotic (ABAM), gentamicin, Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), and Fetal Bovine Serum (PBS) were purchased from Gibco BRL (Grand Island, NY). Tissue culture (75 mm2 culture plates) were purchased from Corning Glass Works (Corning, NY). Bovine pancreas insulin (I-1882), bovine transferrin (T-8027), MCDB-llO Medium (M- 6520), dexamethasone (D-8893), bovine serum albumin (BSA, RIA grade, A-7888), water-soluble linoleic acid (L-5900), porcine skin gelatin (G-l890) were obtained from Sigma Chemical Company (St. Louis, MO). [3H]CGP12177, specific activity 42.5 Ci/mmole, was purchased from DuPont (NEN). Scintillation Cocktail (Sentiverse', SXl8-4), Whatrnan GF/F filters and scintillation vials were purchased from Fisher Scientific. E . S 11' C 1 Fifth passage porcine satellite cells were suspended in MEM containing 10% FBS, 0.5% ABAM, and 0.1% gentarnicin. Cells were plated at a density of 300,000 45 cells/75 mm2 flask previously coated with 0.1 % gelatin as described by Richler and Yaffe (1970), and propagated in a humidified CO2 incubator containing 95% air and 5% C02 at 37 °C. Fresh medium was supplied every 24 h. For the non-differentiated porcine satellite cell radioligand binding assay, cells were harvested at the log phase of growth (3 to 5 d) for membrane preparation. For the differentiated porcine satellite cell radioligand binding assay, cells were allowed to become confluent (ap« proximately 6 d). Differentiation was induced by switching cells to a serum-free medium as described by Merkel et al. (1993), except bFGF and PDGF-BB were not added, and 10"0 M dexamethasone was used, for 72 h before returning cells to MEM containing 10% FBS for 24 h to support completion of the fusion process. Cells were then harvested for membrane preparation. W C2C12 cells were suspended in DMEM containing 10% FBS, 0.5% ABAM, and 0.1% gentamicin, and plated at a density of 300,000 cells/75 mm2 flask and grown in a humidified CO, incubator containing 95% air and 5% CO, at 37°C. Fresh medium was supplied every 48 h. Cells attained confluence at about 4 d, and differentiation was induced by switching cells to DMEM containing 2% FBS and 10" M insulin (differentiation medium) for 72 h before returning cells to the growth medium (DMEM + 10% FBS) for 24 h. Cells were then harvested for membrane preparation. 46 Membme Prep_a_ratign Cell membranes were essentially prepared as described by Hausdorff et al. (1989). Medium was aspirated from monolayers at appropriate times (see culture conditions above) except cells were lysed with a polytron homogenizer at maximum setting. Cells were washed three times with ice-cold PBS, flasks were immediately placed on ice, and contents were scraped off into a 30 mL test tube containing 10 mL of a 5 mM Tris (pH 7.4)-2 mM EDTA buffer, and cells were lysed with a polytron homogenizer (four bursts for 5 s at maximum setting) on ice. The lysate was centrifuged at 200 x g for 20 min at 4°C to remove organelles and unbroken cells, and the supernatant was centrifuged at 40,000 x g for 20 min at 4°C. The resulting pellet was washed once with 5 mM Tris (pH 7.4)-2 mM EDTA buffer, and mem- brane pellets were rinsed and resuspended in a buffer containing 10 mM Tris-HCl (pH 7.4), 5 mM MgCl,, 2 mM CaClz, and 10% glycerol. Aliquot samples were taken for protein content determination according to Bradford (1976). The remaining membrane suspension was diluted to 80 ug/mL with 10 mM Tris-HCl (pH 7.4), 5 mM MgC12, and 10% glycerol buffer, and rapidly frozen in liquid nitrogen and maintained at -80°C for use within one week. B l' 1' I 3' 1° ! Radioligand binding assays were conducted according to Boege et a1. (1988) using the hydrophilic non-selective beta-adrenergic antagonist [’H]CGP12177 modified as follows: 40 ug/mL membrane protein, 0.45-15.6 nM [3H]CGP12177, and incuba- tion time of 2 h were used. Briefly, 40ug/mL membrane protein was incubated at 30°C for 2 h in a shaking water bath with increasing concentrations of [3H]CGP12177 47 (specific activity 42.5 Ci/mmole) with or without a 1000-fold concentration of unlabeled CGP12177 in a 200 pL final volume. The incubations were stopped by the addition of 5 mL ice-cold PBS (pH 7.4), followed by rapid vacuum filtration of the suspension through GF/F glass fiber filters. The filters were washed twice with 5 mL ice-cold PBS (pH 7 .4) and placed in scintillation vials containing 10 mL scintillation cocktail. After gentle shaking for 20 min, the vials were counted for 3H activity with a scintillation counter at 40% efficiency. Results and Discussion The ontogeny of the porcine satellite cell BAR was investigated using mem- brane preparations from differentiated and non—differentiated porcine satellite cells. The results obtained from the binding of [’H]CGP12177 to BAR in non-differentiated porcine satellite membrane preparations of 50, 40 and 20 pg membrane protein/mL are presented in Figure 12. Specific binding of [’H]CGP12177 to non-differentiated porcine satellite cells was reversible and linear with the membrane protein concentra- tion of 20 to 50 pg protein/mL. Non-specific binding, defined by the binding observed in the presence of a 1000-fold concentration (15.6 pM) of unlabeled CGP12177, was approximately 10%, which is consistent with the non-specific binding reported for this ligand in previous studies (Lacasa et al., 1986; Mersmann & McNeal, 1992). The specific binding was 90% as determined by delineation of total and non-specific bindings, but was not saturable at the highest ligand concentration (15.6 nM) studied. A 60% reduction in membrane protein concentration still did not result in saturable specific binding. While some investigators have reported a range of 2,000 T 1,500 ~~ 1,000 .. DPM Bound 500 ~- 2,000 -~ 10500 '7 1,000 ~ DPM Bound 500- ‘ L I 1000 ~- DPM Bound 500 . + Total + Specific + Non-specific 48 I I 10 [3H]CGP1 21 77 (nM) + Total +Specific [3H]CGP12177 (nM) +Total +Specll'ic + Non-Specific 10 [aruccprzrrr (nM) 15 ——a 15 20 20 Figural; [’H]CGP12177 binding to BAR in non-differentiated porcine satellite membrane preparations of 50, 40, and 20 pg membrane protein/mL, respectively. 49 W. [3H]CGP12177 binding to BAR in non-differentiated porcine satellite membrane preparations of 50, 40, and 20 pg membrane protein/mL, respectively. Note. Fifty, 40, and 20 pg membrane protein/mL, respectively, har- vested from non-differentiated porcine satellite cells, were incubated with increasing concentrations of [3H]CGP12177 with or without a lOOO-fold concentration of unlabeled CGP12177 at 30°C for 2 h. Each data point represents a mean of duplicate incubations. 50 0.2-5 nM KD values for BAR, others have also suggested higher KD values for BAR. Green et al. (1993) reported a K, value of 368 i 89 nM for human Bz-AR. Thus, a much higher ligand ([3H]CGP12177) concentration may have been required to attain saturation of BAR in non-differentiated cells used here. The binding characteristics of [3H]CGP12177 to differentiated porcine satellite cell membrane at 30°C is depicted in Figure 13. Specific binding was very close to background at a concentration range of 0.49-1.95 nM [3H]CGP12177. At higher [3H]CGP12177 concentrations, non-specific binding, as defined by the binding observed in the presence of 15.6 nM unlabeled CGP12177, equaled total binding, indicating little or no BAR presence. The absence of a noticeable binding observed in differentiated porcine satellite cell membrane preparation may have been due to two reasons. First, the differentiation milieuuserum-free medium- may have been too harsh to sustain viable porcine satellite cells for the 4 d required to complete the differentiation process. The serum-free medium lacks growth factor that may have been required for the expression of the BAR gene. On equal cell number basis, non- differentiated porcine satellite cells produced threefold more total cellular protein than differentiated cells. Of course, it is tempting to speculate that a three-fold less BAR protein will be produced if the individual protein constituting the total cellular proteins are equally sensitive to serum starvation. However, the possibility that some proteins may be more sensitive to serum deprivation than others also exists. Second- ly, an age-associated BAR modification, or differential gene expression, that may impair its affinity for the ligand used in these studies also remains a possible explana- tion for the lack of BAR activity observed in differentiated porcine satellite cell 51 membrane preparation. For example, substitution of a single amino acid in the purported ligand binding pocket of human Bz-AR may either decrease the affinity by approximately four-fold (1450 i 79 versus 368 j; 39 nM) (Green et al., 1993) or allow BAR to recognize an antagonist as agonist (Suryanarayana & Kobilka, 1993). 200 n- 150 ~- 100 .4. DPM Bound ti 5 10 151 20 [3H]CGP12177 (nM) W. [3H]CGP12177 binding to BAR in differentiated porcine satellite cell membrane preparation. Note. 50 pg membrane protein/mL harvested from differentiated porcine satellite cells were incubated, with increasing concentrations of [3H]CGP12177 with or without a 1000-fold concentration of unlabeled CGP12177 at 30°C for 2 h. Cells were differentiated as described under the Materials and Methods section. Each data point represents a mean of three experiments done in duplicate. [’H]CGP12177 binding to BAR in differentiated C2C12 cell membrane preparation was studied and results obtained by incubating 40 pg membrane pro- tein/mL with increasing concentrations of [3H]CGP12177 (0.49-15.6 nM) in the presence or absence of 15.6 pM unlabeled CGP12177 are presented in Figure 14. 52 1 .800 . -o- Total 1 '600 ‘ + Specific + Non-specific DPM Bound 20 [3H]CGP12177 (nM) W- [3H]CGP12177 binding to BAR in C2C12 cell membrane preparation. Note. 40 pg membrane protein/mL harvested from differentiated C2C 12 cell was incubated with increasing concentrations of [’HJCGP12177, with or without a 1000-fold concentration of unlabeled [3H]CGP12177 at 30°C for 2 h. QC” cells were differentiated as described under the Materials and Methods section. Each data point represents a mean of three experiments done in duplicate. The specific binding was saturable, reversible, and of high affinity (K, = 0.2 nM) as determined by the Scatchard Plot. Scatchard plot for specific binding was linear and fit a correlation coefficient of > .96 (see Figure 15). The model showed no evidence for curvilinearity to suggest a two-affinity site receptor. The non-specific binding was approximately 10% which is consistent with the 10-15% previously reported for [3I-I]CGP12177 by Lacasa et a1. (1986) and 10% reported by Mersmann and McNeal (1992). The literature suggests that the KB for BAR is ligand- and cell-type-depen- dent. For example, using a ligand [’H]CGP12177 in human adipocyte membranes, the K0 value was reported to be 0.9 nM (Mauriege ct al., 1988), whereas with the 53 0.006 T 0.005 ~- R2 = 0.9621 0.004 «- 0.003 4 0.002 ~~ Bound [3H]CGP12177 l Free [3H]CGP12177 0.001 ~- 0 i i i a 4. i i i 0 100 200 300 400 500 600 700 800 Bound [3H]CGP12177/8ug protein W- Scatchard Plot of specifically bound [’I-I]CGP12177 to the differentiated C2C12 cell membrane preparation. Nate. C2C12 cells were grown and differentiated as described under the Materials and Methods section. Each data point represents the mean of three experiments done in duplicate. same ligand [’H]CGP12177 in pig adipocyte crude membrane preparation, a KD value of 0.6 nM was reported (Mersmann & McNeal, 1988). Lacasa et al. (1986) suggest- ed a KD value of 1.2 1; .3 using intact human adipocyte. In the present studies, the B.“ was calculated to be 150 fmole/mg protein, which is similar to the B.“ values indicated in previous studies. Mersmann and McNeal (1992) reported a B.“ of >150 fmole/mg protein. Coutinho et al. (1990) reported 170 fmole/mg protein. The Kb (0.2 nM) and B.“ (150 fmole/mg protein) values reported in the present studies are characteristic of typical BAR. Thus, we are reporting here the presence of typical BAR in C2C12 cell membrane preparation. CHAPTER V: BETA-ADRENERGIC AGONISTS-STIMULATED CAMP ACCUMULATION IN PORCINE SATELLITE AND C2C12 CELLS Abstract Two experiments were conducted to measure ISO-, RAC-, and CLEN-stimulated CAMP accumulation in both differentiated and non-differentiated porcine satellite cells, and differentiated C2C12 cells. In Experiment 1, post-differentiated C2C12 cells were washed three times and overlaid with 5 mL PBS (pH 7.4) containing either ISO, RAC, or CLEN (109' '7' ’5 M). Forskolin was added at 10’ M as a positive control; for negative control, no drug was added. Ten pM CAMP phosphodiesterase inhibitor was added to each incubation to prevent the conversion of CAMP to AMP. Results indicate that all concentrations of the BAA ISO, RAC, and CLEN studied resulted in CAMP release higher than the negative control. At 10 '7' '5 M, CLEN, ISO, and 105 RAC equaled or exceeded the Forskolin-induced CAMP release per unit of cellular protein. Release of CAMP was increased (P < .01) by 10 '5 M ISO, RAC, and CLEN; however, the RAC response was somewhat less than ISO or CLEN. In Experiment 2, either differentiated or non-differentiated porcine satellite cells were washed three times and overlaid with 5 mL PBS (pH 7.4) containing either ISO, RAC, or CLEN (109- '7' '5 M). Forskolin was added at 10 '5 M as a positive control; for negative control, no drug was added. Results showed that at 105 M, 180 54 55 significantly increased CAMP release (P < .001) in differentiated but not in non- differentiated porcine satellite cells. The Forskolin—induced CAMP release was significantly higher (P < .001) compared to negative controls in both differentiated and non-differentiated porcine satellite cells. These results suggest the the presence of functional BAR-Adenylyl Cyclase systems in differentiated and non-differentiated porcine satellite cells and differentiated C2C12 cells. 56 Introduction Beta-Adrenergic Receptors (BAR) belong to the Seven Membrane Spanning- receptor family. Sutherland and Rall (1960) reported that BAR are coupled to the enzyme adenylyl cyclase, an enzyme that utilizes ATP and generates CAMP as a product. PKA is activated by CAMP association with its regulatory subunits, thus allowing the catalytic subunits to dissociate from the regulatory subunits to either activate or deactivate a series of intracellular enzymes by phosphorylation. Thus, the binding of a BAA to its receptor, BAR, results in the activation of PKA which evokes a pleiotropic effect on the metabolic processes in animals, including meat-producing animals. The lipolytic and antilipogenic effects of BAA on adipose tissue (Peterla & Scanes, 1990; Weber et al. , 1992) and lean tissue-enhancement effect (Anderson et al., 1990; Bergen et al., 1989; Ricks et al., 1984) have been reported. However, the mechanism of action of BAA is a matter of controversy; BAA actions may (Choo et al., 1992) or may not (Smith, 1989) be mediated by BAR in muscle tissue. At the present time, the preponderance of evidence for functional BAR- adenylyl cyclase systems in meat animal research has emanated from studies that evaluated the effects of BAA on NEFA and glycerol release, and FAS, ACC, and malic enzyme (ME) activities. The present studies seek to investigate the effect of ISO, RAC, and CLEN binding to BAR in porcine satellite cells and C2C12 cells grown in culture, and the subsequent biological consequence-a Change in the CAMP concentration. 57 Experimental Procedures W ABAM, gentamicin, MEM, DMEM, and PBS were purchased from Gibco BRL (Grand Island, NY). Tissue culture plates (100 x 20 mm) were purchased from Coming Glass Works (Corning, NY). Bovine pancreas insulin (1-1882), bovine transferrin (D8027), MCDB—llO Medium (M-6520), dexamethasone (D-8893), BSA (RIA grade A-7888), water-soluble linoleic acid (L-5900), porcine skin gelatin (G- 1890), cyclic AMP phosphodiesterase inhibitor (B-8279), isoproterenol hydrochloride (I-6504), and forskolin (F-6886) were purchased from Sigma Chemical Company (St. Louis, MO). Cyclic AMP kits (KAPHZ) were obtained from Diagnostic Products, Inc. (Los Angeles, CA). E . 5 11' 2115 1 ”fl .. Fifth passage porcine satellite cells were suspended in MEM containing 10% FBS, 0.5% ABAM, and 0.1% gentamicin. Cells were plated at a density of 100,000 cells/100 mm diameter flask previously coated with 0.1 % gelatin as described by Richler and Yaffe (1970), and propagated in a humidified CO, incubator containing 95% air and 5% CO, at 37 °C. Fresh medium was supplied every 24 h. For the non- differentiated porcine satellite cell CAMP assay, cells were utilized at the log phase of growth (3 to 5 d). For the differentiated porcine satellite cell CAMP assay, cells were allowed to become confluent (approximately 6 d). Differentiation was induced by switching cells to a serum-free medium as described by Merkel et al. (1993), except bFGF and PDGF-BB were not added, and 10'0 M dexamethasone was used, for 72 h 58 before returning cells to MEM containing 10% FBS for 24 h to support completion of the fusion process. 2 2 11 r w h Differentia ion C2C12 cells were suspended in DMEM containing 10% FBS, 0.5% ABAM, and 0.1 % gentamicin. Cells were plated at a density of 100,000 cells/ 100 mm diameter flask and grown in a humidified C02 incubator containing 95 % air and 5% C02 at 37°C. Fresh medium was supplied every 48 h. Cells attained confluence at about 4 d, and differentiation was induced by switching cells to DMEM containing 2% FBS and 10‘ M insulin (differentiation medium) for 72 h before returning cells to the growth medium (DMEM + 10% FBS) for 24 h. Cells were then utilized for the CAMP assay. SEE-5° 1 15112! 1' Medium was aspirated from monolayers (porcine satellite cells and C2C 12 cells) and washed three times with PBS (pH 7.4). Monolayers were overlaid with 5 mL PBS (pH 7.4) previously incubated to 37 °C in a water bath; either ISO, RAC, or CLEN was added at a final concentration of 10", 107, and 10’ M. To other monolayers as positive control, forskolin was added at 105 M final concentration, while negative control was achieved by not adding any drugs. Ten pM CAMP phosphodiesterase inhibitor was added to each incubation to prevent enzymatic degradation of CAMP. Cells were incubated in a humidified CO, incubator containing 95% air and 5% CO, at 37°C for 5 min. Following the incubation period, a 100 pL aliquot sample/plate was removed and placed in a 12 x 75 mm polypropylene tube 59 and placed in a 0°C ice bath. The cell monolayers and 100 pL aliquot samples were kept at -20°C for protein determination and CAMP analysis respectively (within 3 d). Total cellular proteins/plate were determined according to Bradford (1976). The extracellular CAMP analysis was performed using the Diagnostic Products Incorporat- ed (DPC) CAMP kits per manufacturer’s instructions. This procedure to measure CAMP is based on the principle of competitive protein binding by keeping the CAMP binding protein and 3H CAMP constant, while adding increasing amounts of unlabeled CAMP, resulting in increasing displacement of 3H CAMP by CAMP. Thus, the CAMP binding protein-3H CAMP counts are obtained as a function of the unlabeled CAMP concentration and are plotted to generate a calibration curve from which unknown samples may be read. Results and Discussion Results from the BAA-induced CAMP studies in differentiated and non- differentiated porcine satellite cell studies are presented in Figures 16 and 17. Differentiated and non-differentiated porcine satellite cells responded to BAA stimula- tion; however, the magnitude of response of differentiated cells was greater. 10’ M 180 significantly increased CAMP release (P < .001) in differentiated but not in non— differentiated porcine satellite cells. 105 M 180 numerically, but not significantly, increased CAMP release in non-differentiated cells. Forskolin-stimulated CAMP accumulation was significant (P < .001) in both differentiated and non-differentiated porcine satellite cells compared to the control. In these studies, 107 and 10" M RAC or CLEN marginally increased extracellular CAMP release in differentiated, but not in non-differentiated, porcine satellite cells, thus suggesting that RAC and CLEN may be Pmole Extracellular CAMPIS min! mg Total Protein 60 El Control & Treatment Forskolin Is010-9 ‘ ISO 10-7 lSO10-5 " Ohm ééé PF!" 22 Timur _I_l_l 000 Control (-) RAC 10.9 _ ., . . 1 .1 3, RAC m ‘ RAC10-5 " " ‘ Concentration (M) W. BAA-stimulated CAMP accumulation in differentiated porcine satellite cells. Nate. Porcine satellite cells were propagated and differentiated as I/z . H/ , ' // / :7, I/y/z / / ’y/ .' J/ I / / Forskolin (+) / ’ ’ / / / r z I 43 15 10 described in the Materials and Methods section. Either ISO, RAC, or CLEN was added at a final concentration of 10", 107, or 10" M as a positive control. Forskolin was added at 10’ M. Monolayers were then incubated at 37°C for 5 min and CAMP quantified as stated under the Materials and Methods section. Data are means :1: SD for three experiments done in duplicate. Pmole Extracellular CAMPIS mini mg Total Protein 61 CLEN 10.0 CLEN 10.7 CLEN 105 3.33 000 222 Cm (.) PM" (+) “‘° B9 Concentration (M) W. BAA-stimulated CAMP accumulation in non-differentiated porcine satellite cells. Nate. Porcine satellite cells were propagated and differentiated as described in the Materials and Methods section. Either ISO, RAC, or CLEN was added at a final concentration of 10’, 10", or 10‘ M as a positive control. Forskolin was added at 10’ M. Monolayers were then incubated at 37°C for 5 min and CAMP quantified as stated under the Materials and Methods section. Data are means 1 SD for three experiments done in duplicate. 62 poorly coupled to the BAR-adenylyl cyclase systems at least in porcine satellite cells. These observations are consistent with previous reports. RAC or CLEN were effective inhibitors (antagonists) of epinephrine-stimulated lipolysis in pig adipocytes (Liu & Mills, 1989). In contrast, RAC or CLEN may also be a weak agonist (Coutinho et al. , 1990) in pig adipocytes. Clenbuterol increased plasma concentration of free fatty acids, suggesting that agonist response occurs as well (Mersmann, 1987). These latter reports are corroborated by in vivo studies (Merkel et al. , 1987). These findings not only indicate the presence of functional BAR-adenylyl cyclase systems in both cell types, but also suggest that either the differentiated cell BAR-adenylyl cyclase system may be more sensitive to agonist (ISO) activation, or that BAR may be coupled to a different species of G protein that is a stronger stimulator of adenylyl cyclase. The existence of multiple forms of Ga-stimulation (6,.) was reported (Jones & Reed, 1987). Results from the C2C 12 cell response to beta-adrenergic Stimulation are presented in Figure 18. The control differentiated C2C12 incubations resulted in negligible CAMP release; forskolin-stimulated CAMP accumulation was 17-fold more than the controls. All concentrations of the BAA tested resulted in CAMP release higher than the controls; at 107' '5 M, CLEN, ISO, and 105 M RAC equaled or exceeded forskolin CAMP release per unit of cellular protein. Release of CAMP was increased (P < .01) by all B-adrenergic agonists at 10’ M; RAC response was somewhat less than ISO or CLEN. 63 84 I I I J ‘ O alaaaa v Pmole Extracellular cAMP/5 min! mg Total Protein at l I Iso 105 l l A I CLEN 10.0 CLEN 10-7 ‘ i A CLEN 105 Career.) PM“) Rama-e 3‘ micro-7 RAC10-5 Concentration (M) W. BAA-stimulated CAMP accumulation in differentiated C2C12 cells. Nate. C2C12 cells were grown and differentiated as described under the Materials and Methods section. Either ISO, RAC, or CLEN was added at a final concentration of 10’, 10", or 10’ M. Monolayers were then incubated at 37°C for 5 min and CAMP was quantified as stated under the Materials and Methods section. Data are means 1: SD for three experiments done in duplicate. CHAPTER VI: REVERSE TRANSCRIPTION AND POLYMERIZATION OF MESSENGER RNA IN PORCINE SATELLITE AND C2C12 CELLS Introduction Our biochemical and physiological studies have suggested the presence of functional BAR-Adenylyl Cyclase Systems in both porcine satellite and C2C12 cells, although the evidence is much stronger for C2C12 cells. However, because of the differences observed in both the binding of [3H]CGP12177 to BAR, and CAMP response studies in differentiated versus non-differentiated cells, it became of interest to evaluate the level of BAR messenger RNA in the cell types of satellite cells by utilizing the reverse transcription-polymerase Chain reaction (RT-PCR) technique. Experimental Procedures W ABAM, gentamicin, MEM, DMEM, and FBS were purchased from Gibco BRL (Grand Island, NY). Tissue culture plates (75 cm2/flask) were purchased from Corning Glass Works (Corning, NY). Bovine pancreas insan (I-1882), bovine transferrin (T-8027), MCDB-l 10 medium (M-6520), dexamethasone (D-8893), BSA (RIA grade A-7888), water-soluble linoleic acid (L-5900), and porcine skin gelatin (G-1890) were purchased from Sigma Chemical Company (St. Louis, MO). RNase inhibitor and DNA molecular weight marker V were obtained from Boehringer Mannheim. RT-PCR Kit (N 808-0179) and PCR reaction micro tubes were purchased 64 65 from Perkin Elmer. Total RNA/mRNA isolation reagent (RNA STAT-60) was obtained from TEL-TEST "B," InC., Friendswood, TX. 1 ' 1 r w i r n ' ti n Fifth passage porcine satellite cells were suspended in MEM containing 10% FBS, 0.5% ABAM, and 0.1% gentamicin, plated at a density of 300,000 cells/75 mm2 flask previously coated with 0.1 % gelatin as described by Richler and Yaffe (1970), and propagated in a humidified CO, incubator containing 95% air and 5% CO, at 37°C. Fresh medium was supplied every 24 h. For the non-differentiated porcine satellite cell RT-PCR assay, cells were harvested at the log phase (3 to 5 d) of growth for total RNA isolation. For the differentiated porcine satellite cell RT- PCR assay, cells were allowed to become confluent (approximately 6 d). Differentia- tion was induced by placing cells in a serum-free medium as described by Merkel et al. (1993), except bFGF and PDGF-BB were not added, and dexamethasone used at 10.10 M, for 72 h before returning cells to MEM containing 10% FBS for 24 h to support completion of the fusion process. Total RNA was isolated from the cells using the RNA STAT-60 Kit, which includes phenol and guarridirrium thiocyanate in a monophase solution, per manufacturer’s protocol (described under the Total RNA Isolation section). CZCIZ CHE l lD’Efl . . C2C12 cells were suspended in DMEM containing 10% FBS, 0.5% ABAM, and 0.1% gentamicin, plated at a density of 300,000 cells/75 mm2 flask and grown in A a humidified C02 incubator containing 95% air and 5% CO, at 37°C. Fresh medium 66 was supplied every 48 h. Cells attained confluence at about 4 d, and differentiation was induced by switching cells to DMEM containing 2% FBS and 104’ M insulin (differentiation medium) for 72 h before returning cells to the growth medium (DMEM + 10% FBS). Total RNA was isolated from the cells at times indicated under the Materials and Methods section. W W Medium was aspirated from cell monolayers, 6 mL RNA STAT reagent/75 cm2 flask was added in a fume hood, and cells were lysed by repetitive pipetting. Following homogenization, cells were stored at room tempera- ture for 5 nrin to permit the complete dissociation of nucleoprotein complexes. The Homogenates were transferred from 75cm2 culture flasks into 30 ml Corex glass tubes. m 1.2 mL chloroform was added to each tube. The tubes were covered tightly and shaken vigorously for 15 s. After 3 nrin at room temperature, the homogenates were centrifuged at 12,000 g for 15 min at 4° C. Following centrifuga- tion, the homogenates separated into two phases: a lower red phenol chloroform phase containing DNA and proteins, and a colorless upper aqueous phase containing RNA. Precipitation, The aqueous phase (approximately 3.5 mL) was removed from each 30 mL Corex tube and transferred to a fresh 30 mL Corex tube, and 3 mL isopropanol was added to each tube. Samples were stored at room temperature for 5- 10 min before centrifugation at 12,000 g for 10 min at 4° C. The supernatant was removed from each tube. 67 Wash, The remaining RNA pellets were washed with 6 mL 75% cold ethanol/tube by vortexing and were subsequently centrifuged at 7,500 g for 5 min at 4° C to recover the RNA pellets. Pellets were resuspended in Diethyl/Pyrocarbonate (DEPL)-treated water. Aliquot RNA samples were taken from each cell type and analyzed by spectrophotometer and electrophoresis gel. The remaining RNA samples were diluted with DEPC-treated water to 0.5 pg/pL and stored at —80° C for use within 1 wk. Prior to use for reverse-transcription-polymerase Chain reaction (RT-PCR), aliquots of RNA samples from all cell types (porcine satellite and C2C12 cells) were DNase l-treated and incubated for 30 nrin at 37° C to degrade all possible contaminating DNA. Following incubation, the residual DNase activity was stopped by the addition of 20 mM EDTA, a Chelating agent, to deactivate DNase I. The RNA samples were further treated with RNase inhibitor (40 unitpr stock) to deactivate all contaminating RNases, if any. 01° 1 . l Porcine satellite and C2C12 cell RNA were probed for all subtypes of BAR. The following 2l-mer primers presented in Table 3 were used in the primer-directed DNA polymerization. These 21-mers were made at the Michigan State University Macromolecule and Structural Facility. 68 Table 3. The oligonucleotide sequences used as primer for the three sub-types of BAR. Primer Receptor Strand Sequence (5’-3’) Predicted Product Size (bp) A B3-AR Sense GAG ACT CCA GAC CAT GAC CAA 325 Anti-sense TCA TGA TGG GGG CAA ACG ACA B B3-AR Sense TGT CGT 'I'I‘G CGC CCA TCA TGA 534 B3-AR Anti-sense TAG AGT ACC GGG 'I'I‘G AAG GCA C B3-AR Sense TGT CGT ’l'l'G CGC CCA TCA TGA 415 B3-AR Anti-sense CAA CCA GCA GAG ACT GAA GGT D Bz-AR Sense GCA GAC GGT CAC CAA CT A C'I'I‘ 464 Anti-sense ACG AAG ACC ATG ATC ACC AGG E Bz-AR Sense CCT GCI' GAT CAT GGT CT'I‘ CGT 372 Anti-sense GGC AAT CCT GAA ATC TGG GCI‘ F B,-AR Sense ACG CT C ACC AAC CT C 'I'I‘C ATC 463 Anti-sense TAC AGG AAG GCC ATG ATG CAC G B.-AR Sense GTG CAT CAT GGC C'I'l‘ CGT GTA 376 B,-AR Anti-sense TGA AGA AGA CGA AGA GCC CCI‘ Figure 19 shows a computer-generated line-up file indicating regions of homology between human and bovine BAR where primer selections were made. The conserved sequences between human and bovine B3-AR are boxed. No conserved regions were found between human and bovine B,-AR and Bz-AR. Therefore, primers for B,-AR and Bz-AR were made against areas of low GC contents in human and bovine BAR l and 2, hoping these regions are conserved in porcine. Each primer pair (sense and antisense) was targeted to amplify specific regions of the cDNA derived from the reverse transcriptase reaction in primer-directed DNA polymerization reaction. 69 Because only the targeted regions of the DNA must be amplified, two primers (sense and antisense) were required. For example, for primer A, B3-AR, the expected RT- PCR product was 325 base pair (bp). The sense primers were designed to anneal at base 271 to 291, and extended from one direction, while its antisense primer annealed at base 596-576, and extended from the opposite direction to meet at a point of termination. Thus base 271 to 596 was amplified, resulting in a RT-PCR product of 325 bp. bovbeta123.msf MSF: 1447 Type: N March 10, 1996 18:05 Check: 599 Name: bovbeta3 Len: 1291 Check: 6510 Weight: 1.00 Name: humbeta3 Len: 1280 Check: 4841 Weight: 1.00 Name: bovbeta2 Len: 447 Check: 2162 Weight: 1.00 Name: humbetaZ Len: 1331 Check: 7721 Weight: 1.00 Name: humbetal Len: 1447 Check: 6466 Weight: 1.00 Name: bovbeta123 Len: 1447 Check: 2899 Weight: 1.00 // 1 50 bovbeta3 .................................................. humbeta3 .................................................. bovbeta2 .................................................. humbetaZ .................................................. humbetal ATGGGCGCGG GGGTGCTCGT CCTGGGCGCC TCCGAGCCCG GTAACCTGTC bovbeta123 ATGGGCGCGG GGGTGCTCGT CCTGGGCGCC TCCGAGCCCG GTAACCTGTC 51 100 bovbeta3 ................... A TGGCTCCGTG GCCTCCTGGG AACAGCTCTC humbeta3 .................. A. TGGCTCCGTG GCCTCACGAG AACAGCTCTC bovbeta2 .................... ..TTTCTCCT CCCCCAGGTG ATATCCACTC humbetaZ .............................. ...ATGGGGC AACCCGGGAA humbetal GTCGGCCGCA CCGCTCCC.. CG ........ ACGGCGCGGC CACCGCGGCG bovbeta123 GTCGGCCGCA CCGCTCCC.. tchtccgtg chtc..Ggg aac.gc.ctc Ejgmlfl, Region of homology between human and bovine BAR. 70 150 TGCCAACGCG CGCCAACACC AGCAGCCATT CGCCGGACCA CTCCCGCCAG chc..c.cg 200 GCGGGGGCGC GCCGGGGCCC TCCTGGAAGG ATGGGCATCG ATGGGTCTGC .cggGg..gc 250 GCTGGTAATC GCTGGTCATC GAAAGCGGAG GCTGGTCATC GCTGGTGATC Gctth.atc 300 GTGTTCGT T GCAG C CTCACCA ACCT TT AT 350 GTCGTGCCCC GTGGTGCCGC GGCAGCCAGC GTGGTGCCGT GTGGTGCCGT Gtggthcgc 400 CGTCACCGGT CGCCACTGGC GGCTTTGTGT CAACTTCTGG CTCCTTCTTC 101 bovbeta3 TGACCCCGTG GCCAGATATC CCCACCCTGG CACCCAATAC humbeta3 TTGCCCCATG GCCGGACCTC CCCACCCTGG CGCCCAATAC bovbeta2 TGTTCCCCTG TGTAGTCAGT CCTGTCATTG CTGTTGCTGG humbetaZ CGGCAGCGCC TTCTTGC..T GGCACCCAAT AGAAGCCATG humbetal CGGCTGCTGG TGCCCGCGTC GCCGCCCGCC TCGTTGCTGC bovbeta123 tggcccCgtg t.cag.catc cccacCctgg cg....ct.c 151 bovbeta3 AGTGGGCTGC CAG....GGG TGCCCTGGGC GGTGGCGCTG humbeta3 AGTGGGCTGC CAG....GGG TTCCGTGGGA GGCGGCCCTA bovbeta2 CCCATAGGCC TTCAATGAAG ACCTGCGCAG GCAGAGAAGC humbetaZ CGACGTCACG CAGCAAAGGG ACGAGGTGTG GGTGGTGGGC humbetal CGAAAGCCCC GAGCCGCTGT CTCAGCAGTG GACAGCGGGC bovbeta123 cg..ggctcc cag....ggg ..c.g.gg.g Gg.ggcg.gc 201 bovbeta3 TGTTGGCGCT AGCGGTGCTG GCCACCGTGG GAGGCAACCT humbeta3 TGCTGGCGCT GGCGGTGCTG GCCACCGTGG GAGGCAACCT bovbeta2 CAATCCTGAA ATCTGGGCTC CGGCAGTAGA TAAGGGGATT humbetaZ TCATGTCTCT CATCGTCCTG GCCATCGTGT TTGGCAATGT humbetal TGATGGCGCT CATCGTGCTG CTCATCGTGG CGGGCAATGT bovbeta123 tgangcgct ..c.GthTg gcca.cthg .achaa..T ByAJIQMBnmhAEanQ (271-291) 251 ‘ 4. bovbetaB GTGGCCATCG CCCGGACGCC'GAGACTCCAG ACCATGACCA A humbeta3 GTGGCCATCG CCTGGACTCC AC CAG ACCATGAC CGTGTTCGT bovbetaz TTGATGTAGC CCAACCAGTT TAGAAGGATG TATATTTCCT TACGGATGAG humbetaZ ACAGCCATTG CCAAGTTCGA GCGTC CAG ACGGTCACC ACTA T humbetal GTGGCCATCG CCAAGACGCC GCGG‘ bavbeta123 gtggccatcg CCaagacgcc ga ctgcaG a .aT.aCCa ac.tgtTcat lie-AR (primer D Sense) B,-AR (primer F Smse) (277-297) (281-301) 301 bovbeta3 GACTTCGCTG GCCACAGCCG ACCTGGTGGT GGGGCTCCTG humbeta3 GACTTCGCTG GCCGCAGCCG ACCTGGTGAT GGGACTCCTG bovbeta2 GTTATCCTTG ATCACGTGCA CAATGTTGAC AATGAAGAAG humbetaZ CACTTCACTG GCCTGTGCTG ATCTGGTCAT GGGCCTGGCA humbeta1::aATGTCCCTG GCCAGCGCCG ACCTGGTCAT GGGGCTGCTG bovbeta123 gactTC.cTG gcCacagccg accTGngat ggggctgctg 351 bovbeta3 CGGCGGCCAC CTTGGCGCTG ACCGGCCACT GGCCCCTGGG humbeta3 CGGCGGCCAC CTTGGCGCTG ACTGGCCACT GGCCGTTGGG bovbeta2 ACAGGGTGAA AGTGCCCATG ATAATGCCTA AAGTCTTGGG humbetaz TTGGGGCCGC CCATATTCTT ATGAAAATGT GGACTTTTGG humbetal TCGGGGCCAC CATCGTGGTG TGGGGCCGCT GGGAGTACGG bavbeta123 ..ggGGccac cttggcgch a.gggccact gg.c.ttgGG M (cont’d). cgccttctg. 71 431 450 bovbeta3 TGCGAG .GT GCACCTCAGT GCACCTGCTG TCTCT CACCC CCASCATCGA humbeta3 TGCGAGC TGT GGACCTCGGT GCACCTGCTC TCTGTGACCC CCASCATCGA bovbeta2 TCCTTC MA AGAACTTGGA GCTCCTCCCT TCTCCTAGAC C'CG CC humbeta2 TCCGAGTTTT GGACTTCCAT TGATGTGCTG TCCGTCACGG CCAGCATTGA humbetal TGCGAGCTGT GGACCTCAGT GGACGTGCTG TCCGTGACGG CCAGCATCGA bovbeta123 TgCgagcth gGAccTc.gt gGachGCtg TGtgtgAc.g CCagCathA 451 500 bovbetaB AACCCTGTGC GCCCTGGCGG TGGACCGCTA CCTGGCCGTG ACCAACCCGC humbeta3 AACCCTGTGC GCCCTGGCCG TGGACCGCTA CCTGGCTGTG ACCAACCCGC humbetaZ GACCCTGTGC GTGATCGCAG TGGATCGCTA CTTTGCCATT ACTTCACCTT humbetal GACCCTGTGT GTCATTGCCC TGGACCGCTA CCTCGCCATC ACCTCGCCCT bovbeta123 .ACCCTGTGC G.c.TgGch TGGACCGCTA CCTgGCc.Tg ACc..cCCg. 501 550 bovbeta3 TGCGCTACGG CGCGCTGGTC ACCAAACGCC GCGCCCTAGC AGCCGTGGTC humbeta3 TGCGTTACGG CGCACTGGTC ACCAAGCGCT GCGCCCGGAC AGCTGTGGTC humbetaZ TCAAGTACCA GAGCCTGCTG ACCAAGAATA AGGCCCGGGT GATCATTCTG humbetal TCCGCTACCA GAGCCTGCTG ACGCGCGCGC GGGCGCGGGG CCTCGTGTGC bavbeta123 T.cgcTAC.. .cCTG.T. ACcaagcgcc g.GCcnggc ag.chggtc fie-AR (primer C Sense) 33'“ (primer A Anti-sense) B3-AR (primer a Sense) (576-596) (596-576) (576-596) 551 - 600 bovbeta3 CTGGTGTGGG TGGTGTCCGC CGCGGTGTCG TTTGCGCCCA TCATGSECAA humbeta3 CTGGTGTGGG TCGTGTCGGC CGCGG TTTGC CCC TCATG CCA humbetaZ ATGGTCTGGA TTGTGTCAGG CCTTACCTCC TTCTTGCCCA TTCAGATGCA humbetal ACCGTGTGGG CCATCTCGGC CCTGGTGTCC TTCCTGCCCA TCCTCATGCA bovbeta123 .tgGTGTGGg tchgTCch C..ggthC. TT.g.GCCCA Tc.tgA..cA 601 650 bovbeta3 ATGGTGGCGC ATCGGGGCCG ATGCCGAGGC GCAGCGTTGC CACTCCAACC humbeta3 GTGGTGCCGC GTAGGGGCCG ACGCCGAGGC GCAGCGCTGC CACTCCAACC humbetaZ CTGGTACCGG GCCACCCACC A...GGAAGC CATCAACTGC TATGCCAATG humbetal CTGGTGGCGG GCGGAGAGCG A...CGAGGC GCGCCGCTGC TACAACGACC bovbeta123 cTGGngCG. g.cggggcCg A...CGAgGC gca.cgcTGC .ActcCaAcc 651 700 bavbeta3 CGCGCTGCTG CACCTTCGCC TCCAACATGC CCTACGCGCT GCTCTCCTCC humbeta3 CGCGCTGCTG TGCCTTCGCC TCCAACATGC CCTACGTGCT GCTCTCCTCC humbetaZ AGACCTGCTG TGACTTCTTC ACGAACCAAG CCTATGCCAT TGCCTCTTCC humbetal CCAAGTGCTG CGACTTCGTC ACCAACCGGG CCTACGCCAT CGCCTCGTCC bovbeta123 cg.gcTGCTG .g.CTTCg.C .CcAAC.tg. CCTAch..T g..cTCcTCC Beer“ . w .4 .4 . 4...... 701 (741-721) 750 bovbeta3 TCGGTCTCGT TCTATCTTfiGCTCCTGGTG ATGCTiTCG TCTACGCACG humbeta3 TCCGTCTCCT TCTACCTTC TCTTCTCGTG ATGCT TCG TCTACGCGCG humbetaZ ATCGTGTCCT TCTACGTTCC|CCTGGTGATC ATGGTCTTCG EETACTCCAG humbetal GTAGTCTCCT TCTACGTGCC CC GTGCATC ATGGCCTTCG TGTACCTGCG bovbeta123 t.cGTcTCcT TCTAC TtC . cCTgct/ BpAR(pdnandknu9 (724-744) £193.12. (cont’d). ATG. tCTNI‘cTACgcch B,-AR (primer F Anti-sense) (744-724) bovbeta3 humbetaB humbetaZ humbetal bovbeta123 bovbeta3 humbeta3 humbetaZ humbetal bovbeta123 bovbeta3 humbeta3 humbetaZ humbetal bovbeta123 bovbeta3 humbeta3 humbetaz humbetal bovbeta123 bovbeta3 humbeta3 humbetaZ humbetal bovbeta123 bovbeta3 humbeta3 humbetaZ humbetal bovbetalZB 751 AGTTTTCGTG GGTTTTCGTG GGTCTTTCAG GGTGTTCCGC gGTtTTc.tg 801 GTCGCTTCCC GCCGCTTTCC GCCGCTTCCA GCCGTTTCCT GcCGcTTcCc 631 CCTGGCCTGG CCGGCCCCGG GGGCATGGAC GTCCCCGCGC .cg.cc.cg. 901 CCGGCGGCCG CCGGCGGCCG CCTCAAGACG CGCCACCGCC ch..ggcC. 951 CCTTGGGGCT CCTTGGGTCT CCTTCTTCAT GCCTCGTGGC cCtT.g.gct 1001 GTGGTCAACG CTGGCCAACG AAGGAAGTTT ATGGGCGTCT atGG.c..c. 72 GTGGCCACGC GTGGCTACGC GAGGCCAAAA GAGGCCCAGA G.GGCca.g. GCCAGAGGAG GCCCGAGGAG TGTCCAGAAC CGGCGGCCCA g.ccgaggag CGGGGCCGTG TGGGGACGTG TCCGCAGATC CCGCGCCGCC ..ggg.cgt. GCGCGCCTTC GCGCGCCTCC TTAGGCATCA CCGCTGGCCA gcgcqcctc. CATCATGGGA CATCATGGGC CGTTAACATT CCTACGCGAG CaTcat.gg. TGGTGCGCGC TGGTGCGCGC ACATCCTCCT TCACGCTCTG t.ath.Cgc GCCAGCTGCG GCCAGCTGCG GGCAGCTCCA AGCAGGTGAA g.CAGchc. TCTCCGCCGG TCTCCGCCGG CTTAGCCAGG GCGCGGCCGC tctc.chGg CGCCTCGCCC CGCTCCGCCC TTCCAAGTTC GCCCGGACCC chc.cgccC TGCCTCTGCG TGCCTCTCCG TCATGGGCAC ACGGGCGTGC t.CC.C.CC. lfluKR(bfinnw(3Anfi4nnmfl (99L9TD CTTGCTGCGC CTTGCTGCGC GAAGATTGAC GAAGATCGAC ...G.Tg..C CTCCTTCTCG CGCCGTCGCG TGGAGCAGGA CCTCGCCCTC cgccg.cgcg GCGGGGGTGC GAAGGGGTGC TGCTTGAAGG CCGCGCCCCG gcgggggtg. GGAACACCGC GGAACACCGG TTTCACCCTC GGGTAAGCGG ggaa.acCg. T 800 CGGGAGCTGG GGGGAGCTGG AAATCTGAGG AGCTGCGAGC agg.ag..Gg 850 CTCCGGATCC CTCTCTGGCC TGGGCGGACG GCCCTCGCCC ctcccgg.Cc 900 CCTCCTACGG CCGCCTGCGG AGCACAAAGC CCGCCGCCGC ccgcCtacG. 950 GCCCTGCGCA GCCCTGTGCA TGCTGGCTGC CGGCCCTCGC g.cctg.g.. 1000 ACCTTCACTC ACCTTCACTC TCTGCTGGTT TCTGCTGGTT CTTTCTTT CCCTTCTTT G GTGCATGTGA CAGAAGGCGC 3C.C.C.C.C CCTCGGGGGC CCTGGGGGGC AAATTGGATA CTGGCTGCCC cctgggGggc (oont’d). TCCAGGATAA TCAAGACGCT TCt..tggtt CCCTCTCTGG CCCTCTCTAG GGCTATGTCA TTCTTCCTGG ccCTctchg CCTCATCCGT GGGCATCATC gccc.TCttt 1050 TGTCCGGCCC TCCCGGGCCC ATTCTGGTTT CCAACGTGGT tctcchcc. bovbeta3 humbetaB humbetaZ humbetal kaovbeta123 bovbeta3 humbeta3 humbetaZ humbetal bovbeta123 bovbeta3 humbeta3 humbeta2 humbetal bovbeta123 bovbeta3 h~umbeta3 humbetaZ humbetal b0vbeta123 laovbetaB humbeta3 ldumbetaz lfiumbetal b O vbeta123 l"lumbetaz Ifiumbetal ’3 OVbeta123 -r111mbetal bovbetaIZB humbetal ‘U'kbeta123 1051 CACTTTCCTC GGCTTTCCTT CAATCCCCTT GAAGGCCTTC .a.tt.CcT. GCCCTTAACT GCCCTGAACT ATCTACTGCC gcCctcaac. CACCGCGAGCgZfiCTGCCCdA 73 63-AR (primer B Anti-sense) (1110-1090) \ TGCCAACleiGCCTTCAACC TGCCAATTCT GCCTTCAACC GGCTGGGCTA GQQTAGGTTA 1100 GdAGCCCAGA Gctg..c.A 132-1111 (pdmér 1a Anti-sense) (1093-1073) 1101 CGCTCATCTQ‘ CTGCCGCAGC CGCTCATCTA AGCTTCTGTG ACTGGCTGGG .gctc.T.t. 1151 CTGTGTCGCT CTGTGCCGCT TCCAGCAACG AGCCCCGACT ct.tgcc.Ct 1201 AGCCCCCTCC CCCGGCCCTC GAAAGAAAAT GGCTGCCCGC ggc.gccc.c 1251 AGCCCCCAGA AGCCCAGGGG GTGGGCCATC GCTGTCTGGC .gc.ccc.gc 1301 GAATTGTAGT GACGACGACG GA ..... A.. 1351 GCCCTGGGCC GCCCTGGGCC 1401 TGGACGAGCC TGGACGAGCC CTGCCGCAGC CCTGCGCAGG CTACGCCAAC CtgcchAgc GCCGGCCGGA GCGGCCGTCG GCAACACAGG TCCGCAAGGC chgc.cggg GGCGCCCCCA TTCCCCTCGG AAACTGCTGT CGGCGCCACG .gcccccc.g GCTCGACGGG CTTCTTGGGG AAGGTACTGT CCGGCCCGGA cc..tachg ACAAATGACT ACGATGTCGT AC.A ..... T GGCTGCAACG GGCTGCAACG GTGCCGCCCC GTGCCGCCCC CCGGACTTTC CCGGACTTTC TCTTCTTTGA TCGGCCTTCA .ng.cTTt. GGAGCACCTC CCTGCCTCCG GGAGCAGAGT CTTCCAGGGA .g.gCagcg. CGGCCCTGAC GCGTTCCTGC GTGAAGACCT CGACCCACGG .ggcccacgc GCTTCCTGCG AGTTTCTTAG GCCTAGCGAT CCCCCGCCAT gc.tc..ga. CACTGCTGTA CGGGGCCACG C...GC.... GCGGGGCGGC GCGGGGCGGC GGCTTCGCCT GGCTTCGCCT TTTCAGGATT GcngCCAGG CCGCCTCTTC GTCTTCTTCX} tgcC3§;1.t GcCTTCaacc BpAprfinwrCIAnfiwnmw) (1100-1080) GGAGCGCCTT GCAGCGCCTT AGGCCTATGG ACCCCATCAT ..a.Cgcctt GCCGCTGCCT GAGCCCTGCG GGATATCACG CTGCTCTGCT g.gcc.th. CAGCCCCGCT GGCCCGGAGC CCCAGGCACG AGACCGGCCG cgcccg.acg GACTTTCTTA AACATTGATT CGCCCGGGGC .aC.ttg.t. A CCGCCCGCGC .CGCCCGCGC GGCGGACAGC GGCGGACAGC CGGAATCCAA CGGAATCCAA mm (cont’d). 1150 CCGCCGCCTG CCGCCGTCTT GAATGGCTAC CTACTGCCGC cc.cchctc 1200 CCCCGCCCCG CCGCCGCCCG TGGAACAGGA GCGCGCGCAG ccgcgccccg 1250 GGCCCCATGC AGCCCAGCGC GAAGACT.TT CGCGCCTCGG ggc.cctcgc 1300 G CACAAGGGAG CGCCTCGGAC C.C...GGA. 1350 GCCTGCTGGA GCCTGCTGGA 1400 GACTCGAGCC GACTCGAGCC 1447 GGTGTAG GGTGTAG 74 Beverg jljmscripgse Reaction and cDNA Amplifigtign RT-PCR assays were performed using the Perkin-Elmer EZthh RNA PCR Kit, N808-0179, according to the manufacturer’s suggested protocols, except as precautionary steps, the experimental RNA samples (porcine satellite and C2C12 RNA) were pretreated with DNase I and RNase inhibitor. The Perkin-Elmer Gene" EZthh RNA PCR Kits were designed to detect gene expression at the mRN A level. The kit provides reagents required to perform the reverse transcription of the mRN A to produce cDNA and the subsequent amplification all in one reaction tube by a single thermostable DNA polymerase r’I‘th DNA polymerase, an enzyme that possesses reverse transcription and DNA polymerization activities under the appropriate buffer conditions. For both reverse transcription and resulting cDN A amplification, master mixes of reagents including buffers, water, dATP, dGTP, dCTP, d'l'I'P, manganese acetate, and l‘Tth DNA polymerase for all samples were prepared and aliquotted (34 11L) to individual 200 ”L micro tubes. All assays were done in duplicate. Using such master mixes minimized reagent pipetting errors and variation among assays. The composition of the master mix is present in Table 4. Twelve ’11.! tube of experimental, pretreated RNA sample from either porcine satellite or C2C12 cells were placed in their respective micro tubes, followed by the addition of 2 11L each sense and antisense primers at a 0.8 uM final concentration (total reaction volume = 50 uL). For the positive control assay, in addition the basal 34 “L master mix, 12 "L H20, 1 14L RNA (provided), and 1.5 [L each sense and antisense primers at 0.45 uM final concentration were added to each control assay tube. Briefly, all tubes were centrifuged to remove all bubbles. All tubes were then placed in the Perkin-Elmer 75 Gene Amp" PCR System 9600 that had been previously checked for functionality. The reverse transcription reactions were conducted at 60° C for 3 min, followed by a two-temperature polymerase chain reaction; cDN A denaturized at 94° C for 15 s and primers annealed and extended at 60° C for 30 s in 40 cycles. The resulting RT-PCR products were delayed at 60° C for 7 min and held at 4° C before removal. Ten uL aliquot samples were taken per cell type per receptor subtype and electrophoresed in a 4% agarose gel in Tris-borate/EDTA running buffer, at 45 amps for 2 h. Table 4. RT-PCR master mix composition. Component uL Final Composition Water 11 - 5X EZ Buffer 10 -- dATP (10 mM) 1.5 300 14M dGTP (10 mM) 1.5 300 uM dCTP (10 mM) 1.5 300 11M d'l'I‘P (10 mM) 1.5 300 M thh DNA polymerase 2.0 5 units/50 uL 25 mM Mn (CAC); soln. 5.0 2.5 mM Results and Discussion The results from the RT-PCR experiments are presented in Figure 20. The positive control RNA and primers (kit provided) produced the expected RT-PCR product of about 300 base pairs (bp) as indicated by its mobility rate compared to the DNA marker V, whose largest band equalled 500 bp. The estimated 300 bp was further confirmed by the DNA sequence chromatograrn starting at base 1 to 296 (arrowed). The experimental RNA (porcine and C2C12 cells) produced smaller than expected fragment size (see Table 4). In contrast, no stretch of DNA sequence could \l O) 61 AR) BrAR) non-diff. PSC (Bl-AR) diff. PSC (fig-AR) vv AAA positive contro non—diff. PSC diff. PSC (IL-AR) diff. psc (B.-AR) DNA marker V DNA marker V b) 8 0’ 'U at 123456789101112 - M. The reverse transcription and DNA amplification products. Note. The reverse transcription and DNA amplification experiments were performed using seven pairs of primers targeted to recognize all the three mRNA transcripts of BAR. The experiment was conducted as described under the Materials and Methods section. Lane 1) DNA molecular weight marker 5, Lane 2) positive control, Lanes 3—5) C2C12 cells (fig-AR, 52-AR and Bl-R), Lanes 6-8) non-differentiated porcine satellite cells (Ba-AR, 6,-AR and Bl-AR), Lanes 9-11) differentiated porcine satellite cells (fig-AR, 8,-AR and Bl-AR), and Lane 12 DNA molecular weight marker V. These products were resolved in a 4% agarose gel in a Tris-borate/EDTA running buffer at 45 amps for 2 h. be seen in the differentiated porcine satellite cell RNA-derived RT-PCR chromato- gram product. C2C12 cells and non-differentiated porcine satellite cells produced signals that were not discemable from background. The reason(s) for not detecting any BAR transcript, despite all precautionary steps taken, is unclear. Prior to use for the RT-PCR, all RNA samples (porcine satellite and C2C12 cells) were DNase 1- treated and incubated at 37° C for 30 min to degrade all possible contaminating DNA, followed by RNase inhibitor treatment (40 units/uL stock) to deactivate possible contaminating RNases, if any. 77 Furthermore, experimental RNA was analyzed qualitatively by spectropho- tometer and quantitatively using spectrophotometer and gel electrophoresis. The 260- 280 absorbance ratio equaled or slightly exceeded 1.8 for all cell types. Ten pg aliquot RNA samples were electrophoresed in a 1.2% agarose gel in l X (3- [1111"; L " L r ”' ' acid) MOPS, as running buffer, at 45 amps for 2 h (see Figure 21). Because the sequences of the porcine BAR are unknown, and to increase the chances of detecting BAR in C2C12 and porcine satellite cells, multiple primer pairs (sense and antisense) were designed to recognize conserved region in human and bovine B3-AR. Sequences of Bz-AR and Bl-AR were found to be less conserved between human and bovine. The detection of mRNA and subsequent amplification of Direction 1 of migration W. A 1.2% agarose gel showing the 18 and 28S RNA bands. Note. The figure illustrates porcine satellite and QC” cells in four lanes: (a) Lane l--differemiated 02C" cells, (b) Lane 2-non-differenti— ated porcine satellite cells, (c) Lane 3--non-differentiated porcine satellite cells (second preparation), and (d) Lane 4— differentiated porcine satellite cells. All cell types were grown or differentiated as described in the Materials and Methods section. 78 the positive control cDNA (see Figure 22) indicated that the thermal cycles and assay conditions worked, which leaves one to speculate that the lack of detection of any BAR transcript in C2C12 and porcine satellite cells (see Figure 23) may be due to the following: (a) Primers may not have been specific enough to detect and discriminate against the BAR subtypes, and (b) BAR were expressed in levels not high enough for detection using this approach. In the future, the sensitivity or efficiency of this assay may be improved by lowering or raising the anneal/extend temperature of 60° used in this study. The selection of 60° for the anneal/extend temperature was based on the manufacturer’s suggested temperature range of 60-72° C. Furthermore, the optimal manganese acetate concentration for RT-PCR using these cells may be determined empirieally by varying the manganese acetate concentration. Of course, most importantly, future investigators should consider designing another set of primers highly specific for the porcine BAR if sequences become known. Conclusion The radioligand experiments were conducted by incubating increasing concen- trations of [’H]CGP12177 with either porcine satellite or C2C12 cell membrane preparations. The specific binding of [’H]CGP12177 to C2C12 membrane prepara- tions was highly specific (90%), saturable, reversible, and of high affinity, as indicated by a low Kb (0.2 nM). Thus the radioligand binding experiment established the presence of BAR in C2C12 cells. For the porcine satellite cell membrane preparation, [3H]CGP12177 binding was linear with increasing protein concentration (20-50 pg protein/mL). This binding, although highly Wific (90%) and TCVCI‘SibIC, 79 was unsaturable. The observed reversibility and specificity of [3H] binding in non- differentiated porcine satellite cells suggested the presence of BAR. These reports were further strengthened by the isoproterenol-induced extracellular cAMP (P < .001). In C2C12 cells, 10" M 180, RAC, and CLEN significantly increased extracel- lular cAMP release (P < .01). Furthermore, these BAA-induced extracellular cAMP results do not only corroborate the findings from the radioligand binding experiment, indicating the presence of BAR in both cell types, but also establish that these BAR are functional. We are presenting both biochemical and physiological evidence to indicate the presence of functional BAR in porcine satellite and C2C12 cells. These findings also suggest that C2C12 cells may be used as a muscle cell model to study the mode of action of BAA. Finally, studies that will elucidate the complete sequences of the porcine BAR and the regulatory mechanisms will be helpful in the future. 80 3885 meet. Béozzx .228 a £83325 3 .Q r , ... . ... v .. .. ....1 . . . ..,. 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Transfer solution to fume hood and slowly add formaldehyde, while mixing, pour gel in hood. Wm 1. 10X MOPS, pH 7.0 2. Dilute 10X MOPS with DEPC-treated milli-Q H20 (1:10 dilution). Make solution in a sterile graduated cylinder and store at room temperature until needed. I! I . M' I I E I 1 1. 10X MOPS, pH 7.0 20.0 uL 2. Deionized formaldehyde 70.0 pl. 3. Deionized formamide 200.0 uL Make solution in sterile microfuge tube and store at room temperature until needed. 87 P.“ HP‘E‘PP’P?‘ 88 DNA l4‘7A roe4 mL Component Volume or Weight Agarose 1.6 g 1X TBE 40.0 mL Ethidium Bromide 1.0 “L 1 l ’ r k 1' 1X TBE as needed. W Samples should contain at least 3 pg total RNA in a volume of 5.5 pL of TE, pH 8.0 or DEPC-treated H20. Sample must be prepared in a sterile microfuge tube. To the RNA samples in 5.5 ”L volume add 14.5 “L denaturing mix and cap microfuge tube. Heat denatured samples at 60° C for 5 min. Add 5.0 “L of 4X-dye and apply 25 uL sample to gel. Electrophorese sample at 45 volts for 2-3 h. “El” 2 . Sl!!°| (Ill [11] Dissolve one pack of MEM mix in 500 mL millipore H20. Add 2.2 g of sodium bicarbonate. Stir solution for about 5 min or until mixture is completely dissolved. Adjust pH to 7.1 with 1 N HCl. Bring volume to 1 L with millipore H20. In a sterile hood, filter medium through 0.2 microns filter paper to sterilize. Keep medium at 4° C until needed. H9999P7‘ 89 r h M di m for P ine S tellit l 500 mL MEM + 10% FBS Component mL MEM 447.0 FBS 50.0 ABAM 2.5 Gentamicin 0.5 li - Component Final Concentration MEMzMCDB-l 10 medium 4:1 Dexamethasone 10” M Bovine Serum Albumin 0.5 mglmL Insulin 10" M Transferrin 100 pg/mL Water-soluble linoleic Acid 0.5ug/mL W Dissolve one pack of DMEM mix in 500 mL millipore H20. Add 3.7 g sodium bicarbonate. Stir medium for about 5 min or until mixture is completely dissolved. Adjust pH to 7.3 with 1 N HCl. Bring volume to 1 L with millipore H20. In a sterile hood, filter medium through 0.2 microns filter paper to sterilize. Keep medium at 4° C until needed. Growth Medium for C2C12 Qelfi 1500 mL DMEM + 10% FBS! 90 Component mL DMEM 447.0 FBS 50.0 ABAM 2.5 Gentamicin 0.5 _!.I ' (n -2 .1! an 1 r 1'" u. __ D U u + . '1' Component Final Concentration or mL DMEM 487.0 FBS 10.0 ABAM 2.5 Gentamicin 0.5 Insan 10° M Component Volume or Weight Millipore H20 500 ml. Gelatin 0.5 g H o Autoclave solution for 45 min. In a sterile hood, apply solution to plates/wells, cover tightly, and allow to cool at 4° C for a minimum of 2 h. Return plates/wells to sterile hood, aspirate solution from plates/ wells, and rinse with MEM (no serum) equal the volumes of gelatin solution used for coating. Allow MEM (no serum) to remain on plates/wells until cells are ready to be plated. BIBLIOGRAPHY BIBLIOGRAPHY Adeola, O., Darko, E. A., He, P., & Young, L. G. (1990). Manipulation of porcine carcass composition by ractopamine. J, Anim, Sci,, 68(11), 3633- 3641. Adeola, O., McBride, B. W., & Young, L. G. (1992). Metabolic responses induced by isoproterenol in ractopamine-fed pigs. Lung“ 122(6), 1280-1286. Allen, R. E., Merkel, R. A., & Young, R. B. (1979). Cellular aspects of muscle growth: Myogenic cell proliferation. W, 42, 115-127. Anderson, P. T., Helferich, W. G., Parkhill, L. C., Merkel, R. A., & Bergen, W. G. (1990). Ractopamine increases total and myofibrillar protein synthesis in cultured myotubes. Lung, 129(12), 1677-1683. Appell, H. 1., Forsberg, S., & Hollmann, W. (1988). Satellite cell activation in human skeletal muscle after training: Evidence for muscle fiber neoformation. W. 2. 297-299. Amer, P. (1995). The beta-3-adrenergic receptor-a cause and cure of obesity? fl, M9 213(6): 382-383° Ashcroft, F. M., Harrison, D. 5., & Ashcroft, s. J. H. (1984). Glucose induces closure of single potassium channels in isolated rat pancratic B-cells. Ha; manndon), 112, 446-448. Baker, P. K. Dalrymple, R. H., Ingle, D. L., &Ricks, C. A. (1984). Use ofaB- Adrenergic Agonist to alter muscle and fat deposition in lambs. LAIQDLSQL. 52(5), 1256-1261. Barak, L. 1., Stahly, T. S., Cromwell, G. L., & Miyat, J. (1992). Influence of genetic capacity for lean tissue growth on rate and efficiency of tissue accre- tion in pigs fed ractopamine. M. 1001), 3391-3400. Barak, L. S., Tiberi, M., Freedman, N. J., Kwatra, M. M., Lefkowitz, R. J., & Caron, M. G. (1994). A highly conserved tyrosine residue in G protein- coupled receptors is required for agonist-mediated Bz-adrenergic receptor sequestration. Wm, 262(4), 2790-2795 . 91 92 Benovic, J. L., Shorr, R. G. L., Caron, M. G., & Lefkowitz, R. J. (1984). The mammalian Bz-adrenergic receptor: Purification and characterization. Biochem. 23. 4510-4518. Bergen, W. G., & Merkel, R. A. (1991). Body composition of animals treated with partitioning agents: Implication for human health. FASEB 1., 5 2951-2957. Bergen, W. G., Johnson, S. E., Skjaerlund, D. M., Babikeri, A. S., Ames, N. K., Merkel, R. A., & Anderson, D. B. (1989). Muscle protein metabolism in finishing pigs fed ractopamine. ,1, Anim, Sci” fl(9), 2255-2262. Bischoff, R. (1990). Cell cycle commitment of rat muscle satellite cells. L_Qe_l_L m, 111(1), 201-207- Blake, W. L., & Clark, S. D. (1990). Suppression of rat hepatic fatty acid synthase and 814 gene transcription by dietary polyunsaturated fat. Lung, 120, 1127. Boege, F., Ward, M., Jurss, R., Hekman, M., & Helmreich, E. J. M. (1988). Role of glycosylation for Bz-adrenorecptor function in A431 cells. Wm, 263(18), 9040-9049. Bradford, M. M. (1976). A rapid sensitive method for the quantification of micro- grarn quantities of protein utilizing the principle of protein-dye binding. Anal, Memo 22. 248-254. Brustis, J. J., Elamrani, N., Balcerzak, D., Safwate, A., Soriano, M., Poussard, S., Cottin, P., & Ducastaing, A. (1994). Rat myoblast fusion requires exterior- ized m-Calpain activity- W. $0). 320-327- Capema, T. J., Campbell, R. G., Ballard, M. R., & Steele, N. C. (1995). Somato- tropin enhances the rate of amino acid deposition but has minimal impact on amino acid balance in growing pigs. 1M, 125(8), 2104-2113. Capema, T. J ., Steele, N. C., Komarek, D. R., McMurtry, J. P., Rosebrought, R. W., Solomon, M. B., & Mitchell, A. D. (1990). Influence of dietary protein and recombinant porcine somatotropin administration in young pigs: Growth, body composition and hormone status. W, 63(12), 4243-4252. Choo, J. J., Horan, M. A., Little, R. A., & Rothwell, N. J. (1992). Anabolic effects of clenbuterol on skeletal muscle are mediated by BZ-adrenoceptor activation. W. 263 W. 26). BSD-56. 93 Clark, R. B., Friedman, J., Dixon, R. A. F., & Struder, C. D. (1989). Identifica- tion of a specific site required for rapid heterologous desensitization of the B- adrenergic receptor by cAMP-dependent protein kinase. Mgl, Ehm” 16, 343-348. Clark, R. B., Kunkel, M. W., Friedman, J., Goka, T. J., & Johnson, J. A. (1988). Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase 1n S49 wild- -type lymphoma cells. Em W 85. 1442- 1446. Clark, S. D., Armstrong, M. K., & Jump, D. B. (1990). Nutritional control of rat fatty acid synthase and 814 mRNA abundance. Lm, 129, 218. Coleman, M. 13., Russell, L., & Etherton, T. D. (1994). Porcine somatotropin (PST) increases IGF-l mRNA abundance in liver and subcutaneous adipose tissue but not in skeletal muscle of growing pigs. M, 12(4), 918- 924. Collins, 8., Altschmied, J ., Herbsman, O., Caron, M. G., Mellon, P., & Lefkowitz, R. J. (1990). A cAMP response element in the Bz-adrenergic receptor gene confers transcriptional autoregulation by cAMP. 1m, 265(31), 19330-19335. Collins, 8., Bouvier, M., Bolanowski, M. A., Caron, M. G., & Lefkowitz, R. J. (1989). cAMP stimulates transcription of the Bz-adrenergic receptor gene in response to short-term agonist exposure. W, 8.6, 4853-4857. Connacher, A. A., Bennet, W. M., & Jung, R. T. (1994). Clinical studies with the B-adrenoceptor agonist BRL 26830A. MW, 55 (Suppl.), 2588- 2618. Cook, D. R., Doumit, M. 13., & Merkel, R. A. (1993). Transforming growth factor-beta-l , fibroblast growth factor and platelet-derived growth factor-BB interact to affect proliferation of clonally-derived porcine satellite cells. L W. .151, 307-312. Cook, D. R., Doumit, M. 15., & Merkel, R. A. (1994). Ractopamine, cAMP and forskolin enhance the mitogenic action of platelet-derived growth factor-BB in cultured porcine satellite cells. mm, 22 (Suppl. 1), 267. Costelli, P., Garcia-Martinez, C., Llovera, M., Carbo, N., Lopez-Soriano, F. J ., Agell, N., Tessitore, L., Baccino, F. M., & Argiles, J. M. (1995). Muscle protein waste in tumor-bearing rats is effectively antagonized by a beta 2- adrenergic agonist (clenbuterol). Role of the ATP-ubiquitin-dependent proteo- lyfic Pathway- 1.3mm. 25(5). 2367-2372. 94 Cottin, P, Brustis, J. J, Poussard, S., Elamrani, N. ,,Bronchard S., & Ducastaing, A. (1994). Ca2*-dependent proteinases (calpains) and muscle cell differentia- tion W l_322 (2). 170178 Coupe, C. D., Perdereau, P., Ferre, Y., Hitier, M., Narkewicz, & Girard, J. (1990). Lipogenic enzymes, activities and mRNA in rat adipose tissue at weaning. Am, 1, Bhysigl,, 258(1), 5126. Coutinho, L. L., Bergen, W. G., Merkel, R. A., & Smith, C. K. II. (1992). Quantitative characterization of Beta-Adrenergic Receptor Subtypes in porcine adipocthS- W. 1.012(3), 481-485- Coutinho, L. L., Bergen, W. G., Merkel, R. A., & Smith, C. K. II. (1990). Use of radioligand binding assay to determine the affinity of isoproterenol (ISO), ractopamine (RAC), and clenbuterol (CLE) for the B-adrenergic receptor m (BAR) subtypes in porcine adipocytes. mm, 68(Suppl. 1), 234 . (Abstr.). Davidson, T. D. (1989). Effect of growth hormone on carbohydrate and lipid ‘J metabolism. m, 8, 115-131. 2 De Lean, A., Stadel, J. M., & Lefkowitz, R. J. (1980). A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase- coupled B-adrenergic receptor. Mm, 255, 7108-7117. Dickerson, P. S. (1990). WEE—ML Doctoral dissertation I Michigan State University, E. Lansing. Dickerson-Weber, P. S., Helferich, W. G., Merkel, R. A., & Bergen, W. G. (1991). Pretranslational control of adipogenic enzyme activity in TAl cells by ractopamine- EASEBJ... 52, A1307. tion, MiChigan State University, E1. Lansing. Doumit, M. 13., Cook, D. R., & Merkel, R. A. (1993). Fibroblast growth factor, epidermal growth factor, insulin-like growth factors, and platelet-derived growth factor-BB stimulate proliferation of clonally derived porcine myogenic satellite cells. W, 151, 326-332. Doumit, M. E. , & Merkel, R. A. (1992). Isolation and culture of porcine myogenic satellite cells. 11M“, 24, 253-262. 95 Etherton, T. D. (1989). The mechanisms by which porcine growth hormone im- proves pig growth performance. In R. G. Heap, C. G. Prosser, & G. E. Lamming (Eds), i hn l r w ti n (pp. 97-105). London: Butterwords. Etherton, T. D., Louveau, 1., Sorensen, M. T., & Chandhuri, S. (1993). Mecha- nism by which somatotropin decreases adipose tissue growth. Am, 1, Clin, ME"... 58 (Suppl. 2), 2878-2958. Fabry, J ., Demeyer, D., Theilemans, M. F., Deroanne, C., VandeVoorde, G., Deroover, E., & Dalrymple, R. H. (1991). Evaluation of recombinant porcine somatotropin on growth performance, carcass characteristics, meat quality, and muscle biochemical properties of Belgian landrace pigs. Lm 5521,, 52(10), 4007-4018. Feng, Y. U., Broder, C. C., Kennedy, P. E., & Berger, E. A. (1996). HIV-1 Entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled recptor. Science, 212, 872-876. Furuichi T., Yoshikawa, S., Miyawaki, A., Wada, K., Macda, N., & Midoshiba, K. (1989). Primary structure and functional expression of the inositol 1,4,5- diphosphate-binding protein Pm. Nature, £2, 32-38. Gilman, A. G. (1987). G proteins: Transducers of receptor-generated signals. mm, W. 56. 615-649. Grant, A. L., Helferich, W. G., Kramer, S. A., Merkel, R. A., & Bergen, W. G. (1991). Administration of growth hormone to pigs alters the relative amount of insulin-like growth factor-1 mRNA in liver and skeletal muscle. L W, 139(3), 331-338- Grant, A. L., Helferich, W. G., Merkel, R. A., & Bergen, W. G. (1990). Effects of phenethanolamines and propranolol on the proliferation of cultured chick breast muscle satellite cells. W, 58, 652-658. Grant, A. L., Skjaerlund, D. M., Helferich, W. G., Bergen, W. G., & Merkel, R. A. (1993). Skeletal muscle growth and expression of skeletal muscle alpha- actin mRNA and insulin-like growth factor-1 mRNA in pigs during feeding and withdrawal of ractopamine. mm, 11(12), 3319-3326. Green, E. A., & Allen, R. E. (1991). Growth factor regulation of bovine satellite cell growth in vitro. mm, 62(1), 146-152. 96 Green, S. A., Cole, G., Jacinto, M., Innis, M., & Liggett, S. B. (1993). A poly- morphism of the Bz-adrenergic receptor within the fourth transmembrane domain alters ligand binding and functional properties of the receptor. L_Bi_m_. Chem“ 2_6_8(31), 23116-23121. Green, S. A., & Liggett, S. B. (1994). A proline-rich region of the third intracellu- lar loop imparts phenotypic 13, versus Bz-adrenergic receptor coupling and sequestration. L_B_i_QL__C_l_1_e_m_,, 262(42), 26215-26219. Green, S. A., Turki, J., Innis, M., & Liggett, S. B. (1994). Amino-terminal polymorphisms of the human flz-adrenergic receptor impart distinct agonist- promoted regulatory properties. 21m, 53(32), 9414-9419. Hansen, J. A., Nelssen, J. L., Goodband, R. D., & Laurin, J. L. (1994). Interac- tive effects among porcine somatotropin, the beta-adrenergic agonist salbutamol, and dietary lysine on growth performance and nitrogen balance of finishing swine. LAnijSgL, 12(6), 1540-1547. Harris, D. M., Dunshea, F. R., Bauman, D. E., Boyd, R. D., Wang, S. Y., John- son, P. A., & Clark, S. D. (1993). Effect of in vivo somatotropin treatment of growing pigs on adipose tissue lipogenesis. W, 2102), 3293- 3300. Hausdorff, W. P., Bouvier, M., O’Dowd, B. F., Irons, G. P., Caron, M. G., & Lefkowitz, R. J. (1989). Phosphorylation sites on two domains of the B,- adrenergic receptors are involved in distinct pathways of desensitization. L W, 261(24), 12657-12665. Hausdorff, W. P., Caron, M. G., & Lefkowitz, R. J. (1990). Turning off the signal: Desensitization of the B-adrenergic receptor function. W, 4, 2881-2889. » Hausman, D. B., Martin, R. J., Veenhuizen, E. L., & Anderson, D. B. (1989). Effect of ractopamine on insulin sensitivity and response of isolated rat adipocytes. LAM. 62(6). 1455-1464- Hayden, J. M., Bergen, W. G., & Merkel, R. A. (1992). Skeletal muscle protein metabolism and serum growth hormone, insulin, and cortisol concentrations in growing steers implanted with estradiol-17B, Trenbolone Acetate, or Estradiol- 175 plus Trenbolone Acetate. W, 10, 2109-2119. Hayes, V. Y., Isackson, P. J., Fabrazzo, M., Follesa, P., & Mocchetti, I. (1995). Induction of nerve growth factor and basic fibroblast growth factor mRN A following clenbuterol: Contrasting anatomical and cellular localization. Em, Neurol..122. 33-41. 97 Hedeskov, C. J ., Capito, K., & Thomas, P. (1987). Cytosolic ratios of free [NADPH]/[NADP*] and [NADH]/[NAD+] in mouse pancreatic islets, and nutrient-induced insulin secretion. Bighem, 1,, 2_4_1, 161-167. Helferich, W. G., Jump, D. B., Anderson, D. B., Skaerlund, D. M., Merkel, R. A., & Bergen, W. G. (1990). Skeletal muscle alpha-actin synthesis is increased pretranslationally in pigs fed phenethanolamine ractOpamine. Wu, 126(6), 3096-3100. Hoffstedt, J ., Shimizu, M., Sjostedt, S., & Lonnquist, F. (1995). Determination of B3-adrenoreceptor mediated lipolysis in human fat cells. ML, 1(5), 447-457. Hosada, K., & Duman, R. S. (1993). Regulation of B,-adrenergic receptor mRNA and ligand binding by antidepressant treatments and norepinephrine depletion in rat frontal cortex. 1m, EM). 1335-1343. Hu, C. Y., Novakofski, J., & Mersmann, H. J. (1987). Hormonal control of porcine adipose tissue fatty acid release and cyclic AMP production. Lm 55211. 54(4), 1031-1037. Izevbigie, E. B., & Bergen, W. G. (1996a). Properties of B-receptors and response to B-agonists in C2C12 cells. W, 14(Suppl. 1), 139 (Abstr.). Izevbigie, E. B., & Bergen, W. G. (1996b). Ontogeney of beta-adrenergic receptors in cultured porcine satellite cells. mm, 14(Suppl. 1), 139 (Abstr.). Jasper, J. R., Michel, M. C., & Insel, P. A. (1988). Molecular mechanism of B- adrenergic receptor blockers with intrinsic sympathomimetic activity. Bed, 12m. 2. 2891. Jezek, P., & Garlid, K. D. (1990). New substrates and competitive inhibitors of the Cl' translocating pathway of the uncoupling protein of brown adipose tissue mitochondria. LEW, 265(31), 19303-19311. Johnson, G. L., Wolfe, B. B., Harden, T. K., Molinoff, P. B., & Perkin, J. P. (1978). Role of B-adrenergic receptors in eatecholamine-induced desensitiza- tion of adenylate cyclase in human astrocytoma cells. mm, 253, 1472-1480. Johnson, R. J. (1989). Growth physiology and biotechnology: Potential to improve broiler Production. W. 46. 228-240. Jones, D. T., & Reed, R. R. (1987). Molecular cloning of five GTP-binding protein cDN A species from rat olfactory neuroepithelium. M, 262, 14241-14249. 98 Kang, C. W., Sunde, M. L., & Swick, R. W. (1985). Characteristics of growth and protein tum-over in skeletal muscle of turkey poults. ngltg 891., 64, 380- 387. Kennedy, J. M., Eisenberg, B. R., Reid, S. K., Sweeney, L. J., & Zak, R. (1988). Nascent muscle fiber appearance in overloaded chicken slow-tonic muscle. M, m: 203-215 . Kjelsberg, M. A., Cotecchia, S., Ostrowski, J., Caron, M. G., & Lefkowitz, R. J. (1992). Constitutive activation of the arm-adrenergic receptor by all amino acid substitutions at a single site. m, 2&0), 1430-1433. Kobilka, B. K., Frielle, T., Dohlman, H. G., Bolanowski, M. A., Dixon, R. A. F., Keller, P. Caron, M. G, & Lefkowitz, R. J. (1987). Delineation of the intronless nature of the genes for the human and hamster Bz-adrenergic receptor and their putative promoter regions. LBW, 262, 7321. Kramer, S. A., Bergen, W. G., Grant, A. L., & Merkel, R. A. (1993). Fatty acid profiles, lipogenesis, and lipolysis in lipid depots in finishing pigs treated with recombinant porcine somatotropin. 1M, 11(8), 2066-2072. Krick, E. J., Boyd, R. D., Roneker, K. R., Beermann, D. H., Bauman, D. E., Ross, D. A., & Meisinger, D. J. (1993). Porcine somatotropin affects the dietary lysine requirement and net lysine utilization for growing pigs. Lung” 12301), 1913-1922. Krief, S. Lonnqvist, F., & Raimbault, S. (1993). Tissue distribution of B3— -adrener- gic receptor mRNA in man. mm, 23, 344-349. Lacasa, D., Mauriege, P., Lafontan, M., Berlan, M., & Guidicelli, Y. (1986). A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand [3H]CGP-12177. Mid—395s. 21, 368-376. Landing, B. H., Dixon, L. G., & Wells, T. R. (1974). Studies on isolated human skeletal muscle fibers including a proposed pattern of nuclear distribution and a concept of nuclear territories. 11mm, 4, 441-461. Lefkowitz, R. J ., & Caron, M. G. (1988). Adrenergic receptors: Models for the study of receptors coupled to guanine nucleotide regulatory proteins. L_Big1, Chm. 263(11). 4993-4996. Liggett, S. B., Bouvier, M., Hausdorff, W. P., O’Dowd, B., Caron, M. G., & Lefkowitz, R. J. (1989). Altered patterns of agonist-stimulated cAMP accumulation in cells expressing mutant BZ-adrenergic receptors lacking phosphorylation sites. mm. 16, 641-646. 99 Liggett, S. B., Freedman, N. J., Schwinn, D. A., & Lefkowitz, R. J. (1992). Structural basis for receptor subtype specific regulation revealed by a chimeric B.,/BZ-adrenergic receptor. Pree. Nag]. Aead. 8ci,, 20, 3665-3669. Liggett, S. B., Schwinn, D. A., & Lefkowitz, R. J. (1992). Structural basis for receptor subtype specific desensitization revealed by a chimeric film-adrener- gic receptor. Clin, Res, 40, 252A. Liu, C. Y., Boyer, J. L., & Mills, 8. E. (1989). Acute effects of beta-adrenergic agonists on porcine adipocyte metabolism in vitro. L__A_njm_,_SeL, 61(6), 1453-1464. Liu, C. Y., Grant, A. L., Kim, K. H., Ji, S. Q., Hancock, D. B., Anderson, D. B., & Mills, S. E. (1994). Limitations of ractopamine to affect adipose tissue metabolism in swine. L_Am'_m_._Sei_,, 12(1), 62-67. Liu, C. Y, Grant, A. L., Kim, K. H. &Mills, S. E. (1994). Porcine somatotropin decreases acetyl-CoA carboxylase gene expression in porcine adipose tissue. W. 1.10). 125- 132 Liu, C. Y, & Mills, S. E. (1989). Determination of the affinity of ractopamine and clenbuterol for the beta-adrenoceptor of the porcine adipocytes. W, 6101), 2937-2942. Liu, C. Y., & Mills, S. E. (1990). Decreased insulin binding to porcine adipocytes in vitro by beta-adrenergic agonists. mm, 68(6), 1603-1608. Lohse, M. J. Lefkowitz, R. J, Caron, M. G, & Benovic, J. L. (1989). Inhibition of B-adrenergic receptor kinase prevents rapid homologous desensitization of ,B,-adrenergic receptors MW. 8.6. 3011-3015 Lomasney, J. W., Leeb-Lundberg, L. M., Cotecchia, S., Regan, J. W., DeBernardis, J. F., Caron, M. G., & Lefkowitz, R. J. (1986). Mammalian a,-Adrenergic Receptor. Mm. 261. 7710-7716. Lonnqvist, F., Krief, S., Strosberg, A. D., Nyeberg, B., Emorine, L. J., & Agner, P. (1993). Evidence for a functional Ba-adrenergic receptor in man. ELL W9 11.0. 929'936- Matthews, E. K. , & Sakamoto, Y. (1975). Eletrieal characteristics of pancreatic islet cells. LMQL. 246, 421-437. Mauriege, P., Pergola, G. D., Berlan, M., & Lafontan, M. (1988). Human fat cell B-adrenergic receptors: B-agonist-dependent lipolytic responses and character- ization of B-adrenergic binding sites on human fat cell membranes with highly selective Bl-antagonists. M1396... 22, 587. 100 Mauro, A. (1961). Satellite cells of skeletal muscle fibers. 1. Biephys. Bighem. Cytel,, 2, 493-495. Merkel, R. A., Dickerson, P. S., Johnson, S. E., Burkett, R. J., Burnett, R. J., Schroeder, A. L., Bergen, W. G., & Anderson, D. B. (1987). The effect of ractopamine on lipid metabolism in pigs. Fee, Prm” 46 (Abstr.), 1177A. Merkel, R. A., Doumit, M. E., & Cook, D. R. (1993). Proliferation of porcine satellite cells in serum-free medium. FASEB1,, 1, A584. Mersmann, H. J. (1984). Adrenergic control of lipolysis in swine adipose tissue. Wile.” Mersmann, H. J. (1987). Acute metabolic effects of adrenergic agent in swine. Am..1..£hx661..252. E85- Mersmann, H. J., Akanbi, K., Shparber, A., & McNeel, R. L. ’(1993). Binding of agonists and antagonists to the porcine adipose tissue beta-adrenergic recep- tOT(S)- WM. 9M0). 725-732. Mersmann, H. J ., & McNeal, R. L. (1992). Ligand binding to the procine adipose tissue B-adrenergic receptors. L_An1m,_§e'1,, 10, 787-797. Mildner, A. M., & Clark, S. D. (1991). Porcine fatty acid synthase: Cloning of a complementary DNA, tissue distribution of its mRN A and suppression of expression by somatotropin and dietary protein. L_Nu_tr_,, 121(6), 900-907. Mills, S. E., & Liu, C. Y. (1990). Sensitivity of lipolysis and lipogenesis to dibutyryl-cAMP and beta-adrenergic agonists in swine adipocytes in vitro. L Arum. 68(4). 1017-1023. Mills, S. 13., Liu, C. Y., Gu, Y, & Schinckel, A. P. (1990). Effects of ractopamine on adipose tissue metabolism and insulin binding in finishing hogs. Interaction with genotype and slaughter weight. W W. 2(2). 251-263 Moss, F. P. (1968). The relationship between the dimensions of fibres and the number of nuclei during normal growth of skeletal muscle in the domestic fowl. 13mm, 122, 555-565. Moustaid, N. , & Heisook, S. (1991). Regulation of expression of the fatty acid synthase gene in 3'1‘3-Ll cells by differentiation and triiodothyronine. L_BigL 62116111.. 266(28). 18550- Mozdziak, P. B., Schutz, E., & Cassens, R. G. (1994). Satellite cell mitotic activity in posthatch turkey skeletal muscle growth. W, 18, 547-555. 101 Mulvaney, D. R., Merkel, R. A., & Bergen, W. G. (1985). Skeletal muscle protein turnover in young pigs. 1, N11111, 115(8), 1057-1064. Murachi, T., Murakami, T., Ueda, M., Fukui, 1., Hamakubo, T., Adachi, Y., & Hatanaka, M. (1989). The calpain-calpastatin system in hemotopoietic cells. Agv, Exp, Med, 8161,, 255, 445-454. Okamoto, T., Murayama, Y., Hayasha, Y., Inagaki, M., Ogata, E., & Nishimoto, I. (1991). Identification of a G, activator region of the Bz-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation. Cell, 61, 723-730. Perdereau, D., Foufelle, F., Gouhat, B., Ferre, P., & Girard, J. (1992). Influence of diet on the development and regulation of lipogenic enzymes in adipose tissue. m, 51, 387. Peterla, T. A, & Scanes, C. G. (1990). Effect of B-adrenergic agonists on lypolysis and lipogenesis by porcine adipose tissue in vitro. 1m, 68(4), 1024- 1029. Post, S. R., Aguila-Buhain, O., & Insel, P. A. (1996). A key role for protein kinase A in homologous desensitization of the Bz-adrenergic receptor pathway in S49 lymphoma cells. L_Bje],_C_hem1, 211(2), 895-900. Pringle, T. D., Calkins, C. R., Koohmarie, M., & Jones, 8. J. (1993) Effects over time of feeding a beta-adrenergic agonist to wether lambs on animal perfor- mance, muscle growth, endogenous muscle proteinase activities and meat tenderness. 1._Anim._861.. 11(3). 636-644. Rantanen, J ., Harme, T., Lukka, R., Heino, J., & Kalimo, H. (1995). Satellite cell proliferation and the expression of myogenin and desmin in regenerating skeletal muscle: Evidence for two different populations of satellite cells. 1.611. 111166... 22(3). 341-347. Rawls, A., Morris, J. H., Rudnicki, M., Braum, T., Arnold, H. H., Klein, W. H., & Olson, E. N. (1995). Myogenin’s functions do not overlap with those of MyoD or Myf-5 during mouse embryogenesis. M1161” 112(1), 37-50. Regan, J. W., Nakata, H., DeMarinis, R. M., Caron, M. G., & Lefkowitz, R. J. (1986). Purification and characterization of the human platelet az-adrenergic receptor. Mm. 261. 3894-3900. Reuveny, E., Slesinger, P. A., Inglese, J., Morales, J. M., Iniguez-Lluhi, J. A., Lefkowitz, R. J., Bourne, H. R., Jan, N., & Jan, L. Y. (1994). Activation of the cloned muscarinic potassium channel by G protein B subunits. Name, 310, 143-146. 102 Richler, C., & Yaffe, D. (1970). The in vitro cultivation and differentiation capaci- ties of myogenic cell lines. Dev, Biel., 28, 1-22. Ricks, C. A., Dalrymple, R. H., Baker, P. K., & Ingle, D. L. (1984). Use of 6- agonist to alter fat muscle deposition in steers. 1, Agim. 8ei,, 52(5), 1247- 1255. Roupas, P., Chow, S. Y., Town, R. J., & Kostyo, J. L. (1991). Growth hormone inhibits activation of phosphatidylinositol phospholipase C in adipose mem- branes: Evidence for a growth hormone-induced change in G protein function. 233;, N611, Aead, SQi. 118A, 88, 1691-1695. SAS. (1985). SAS user’s guide. Cary, NC: Statistical Analysis System Institute, Inc. Schelegel, M. L., Bergen, W. G., Schroeder, A. L., & Rust, S. R. (1996). Bovine somatotropin increases in vivo lipolytic potential in finishing holstein steers. L_Anim._S.ci..Z4(SuPP1- 1). 143 (Abstr.). Schramm, M., & Selinger, Z. (1984). Message transmission: Receptor-controlled adenylate cyclase system. Sejenee, 225, 1350-1356. Seve, B., Ballevre, O., Ganier, P., Noblet, J., Prugnand, J., & Obled, C. (1993). Recombinant porcine somatotropin and dietary protein enhance protein synthe- sis in growing pigs. Lung” 128(3), 529-540. Skjaerlund, D. M., Mulvaney, D. R., Bergen, W. G., & Merkel, R. A. (1994). Skeletal muscle growth and protein turnover in neonatal boar and barrows. L Anim._S6i.. 22(2). 315-321. Smith, C. K., II. (1989). Affinity of phenethanolamines for skeletal muscle B- adrenoceptors and influence on receptor downregulation. m, 61 (Suppl. 1), 190 (Abstr.). Smith, C. K. 11, Lee, D. E, & Coutinho, L ..L (1990). Quantitative analysis of the selectivity of ractopamine for B-adrenergic receptor subtypes. m, 68(Suppl. 1), 137 (Abstr.). Smith, D. J., Feil, V. J., Huwe, J. K., & Paulson, G. D. (1993). Metabolism and disposition of ractopamine hydrochloride by turkey poults. mm 12151513., 21(4). 624—633- Solomon, M. B., Campbell, R. G., & Steele, N. C. (1990). Effect of sex and exogenous porcine somatotropin on longissimus muscle fiber characteristics of growing pigs. W. 68(4). 1176-1181. 103 Solomon, M. B., Campbell, R. G., Steele, N. C., Caperna, T. J., & McMurtry, J. P. (1988). Effect of feed intake and exogenous porcine somatotropin on longissimus muscle fiber characteristics of pigs weighing 55 kilograms live weight. J, Anim, Sei., @(12), 3279-3284. Spurlock, M. E, Cusumano, J. C., Ji, S. Q., Anderson, D. B., Smith, C. K. 2nd, & Hancock, D. L. (1994). The effect of ractopamine on beta-adrenoceptor density and affinity in porcine adipose and skeletal muscle tissue. L_Anim1 $61.. 12(1). 75- 80. Spurlock, M. E., Cusumano, J. C., & Mills, S. E. The affinities of ractopamine, clenbuterol, and L-644,969 for the beta-adrenergic receptor population in porcine adipose tissue and skeletal muscle membrane. 1, Anim, 8e1',, 11(8), 2061-2065. Stiles, C. R., McKeith, F. K., Singh, S. D., Bechtel, P. J., Mowrey, D. H., & Jones, D. J. (1991). The effects of ractopamine hydrochloride on the carcass cutting yields of finishing swine. L_Anim,_s_ei_,, 62(8), 3094-3101. Stockdale, F. E. (1992). Myogenic cell lineages. W61... 154, 284-298. Strosberg, A. D. (1990). Biotechnology of B-adrenergic receptors. MeLNeumL, 4, 211. Stryer, L. (1986). Cyclic GMP cascade of vision. W, 2, 87-119. Suryanarayana, S., & Kobilka, B. K. (1993). Amino acid substitutions at position 312 in the seventh hydrophobic segment of the Bz-adrenergic receptor modify ligand-binding specificity. mm, 44, 111-114. Susulic, S., Frederich, R. C, Lawitts, J. A., et al. (1995). Knockout of the B3- adrenergic receptor gene In IhLZZthAnMMccfimmeEndmrinc 5582161! (Abstract 36), Bethesda, MD. Sutherland, E. W., & Rall, T. W. (1960). The relation of adenosine-3’,5’-phosphate and phosphorylase to the action of catecholamines and other hormones. WM. 12. 265-299. Szasz, G., Gruber, W., & Bemt, E. (1976). Creatine kinase in serum 1. Deter- mination of optimum reaction conditions. W, 22, 650-656. Tanaka, K, Harioka, T., & Murachi, T. (1985). Changes 1n contents of calpain and mlpastatin m rat liver during growth W 11(4), 357-363. 104 Tatsurou, Y. (1995). Differential coupling of glucagon and B-adrenergic receptors with the small and large forms of the stimulatory G protein. Mel, Phgm“ 48, 849-854. Tessitore, L., Bonelli, G., & Baccino, F. M. (1987). Early development of protein metabolic perturbations in the liver and skeletal muscle of tumor-bearing rats (A model system for cancer cochexia). WELL. 241, 153-159. Thiel, L. F., Beermann, D. H., Krick, B. J., & Boyd, R. D. (1993). Dose-depen- dent effects of exogenous porcine somatotropin on the yield, distribution, and proximate composition of carcass tissues in growing pigs. 1, Anim, 8ei,, 11(4), 827-835. Thomas, R. F., Holt, B. D., Schwinn, D. A., & Liggett, S. B. (1992). Long-term agonist exposure induces upregulation of B3-adrenergic receptor expression via multiple cAMP response elements. MW, 82, 4490- 4494. Thomas, R. F., & Liggett, S. B. (1993). Lack of 63-adrenergic receptor mRNA expression in adipose and other metabolic tissues in the adult human. Mel, 2116611.. 43(3). 343-348. Toussant, M. J ., Wilson, M. D., and Clark, S. D. (1981). Coordinate suppression of liver acetyl CoA Carboxylase and Fatty acid synthase by polyunsaturated Fat. LNDIL. 1.1.1. 146- Turo, K. A. , & Florini, J. R. (1982). Hormonal stimulation of myoblast differentia- tion in the absence of DNA synthesis. W, 248(Cell Physiol. 12), C278-284. Uttaro, S. B., Ball, R. 0., Dick, P., Rae, W., Vessie, G., & Jeremiah, L. E. (1993). Effects of ractopamine and sex on growth, carcass characteristics, processing yield, and meat quality characteristics of crossbred swine. L My fl(9)r 2439-2449- Verstegen, M. W., Vanderhel, W., Henken, A. M., Huisman, J., Kanis, E., Vanderwal, P. , & VanWeerden, E. J. (1990). Effect of exogenous porcine somatotropin administration on nitrogen and energy metabolism in three genotypes of pigs. Waldo, G. L., Northup, J. K., Perkins, J. P., & Harden, T. K. (1983). Character- ization of an altered membrane form of the B-adrenergic receptor produced during agonist-induced desensitization. mm, 258, 13900-13908. 105 Walston, J., Silver, K., Bogardus, C., Knowler, W. C., Celi, F. S., Austin, 8., Manning, B., Strosberg, A. D., Stern, M. P., Raben, N., Sorkin, J. D., Roth, J ., & Shuldiner, A. R. (1995). Time of onset on non-insulin-dependent diabetes mellitus and genetic variation in the B3-adrenergic receptor gene. 12, My 313(6): 343-347- Weber, P. S., Merkel, R. A., & Bergen, W. G. (1992). Adipogenic cell line TAl: A suitable model to study the effects of beta-adrenergic agonist on lipid metabolism. WM. 291(1). 47-53. West, D. C., Satter, A., & Kumar, S. (1985). A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells. W, 141, 289-295. Widen, E., Markku, L., Kanninen, T., Walston, J ., Shuldiner, A. R., & Groop, L. C. (1995). Association of a polymorphism in the B3-adrenergic-receptor gene with features of the insulin resistance syndrome in Finns. M, 523, 348-351. William, N. H., Cline, T. R., Schinkel, A. P., & Jones, D. J. (1994). The impact of ractopamine, energy intake, and dietary fat on finisher pig growth perfor- mance and carcass merit. mm, 12(12), 3152-3162. Xu, X., De-Pergola, G., Erickson, P. S., Fu, L., Carlsson, B., Yang, 8., Eden, 8., & Bjomtorp, P. (1993). Post receptor events involved in the up-regulation of the beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes. Endocrinology. 112(4). 1651-1657. Yang, Y. T., & McElligott, M. A. (1989). Multiple action of B-adrenergic agonists on skeletal muscle and adipose tissue. Bieehem,, 261, 1-10. Yen, J. T., Mersmann, H. J., Hill, D. A., & Pond, W. G. (1990). Effects of ractopamine on genetically obese and lean pigs. L_Amm,_SeL, 68(11), 3705- 3712. Yen, J. T., Nienaber, J. A., Klindt, J., & Crouse, J. D. (1991). Effects of ractopamine on growth, carcass traits, and fasting heat production of U.S. contemporary crossbred and chinese Meishan pure and crossbred pigs. L Anim._S.61.. 62(12). 4810-4822. Yoblonka-Reuveni, Z. , & Rivera, A. J. (1994). Temporal expression of regulatory and structural muscle proteins during myogenesis of satellite cells on isolated rat fibers. M, 13(2), 588-603. 106 Yu, S. S., Lefkowitz, R. J ., & Hausdorff, W. P. (1993). B-adrenergic receptor sequestration: A potential mechanism of receptor resensitization. 1, Biel, gm.” 2.28. 337-341- Zastrow, M. V., & Kobilka, B. K. (1992). Ligand-regulated internalization and recycling of human Bz-adrenergic receptors between plasma membrane and endosomes containing transferrin receptors. 1, Big] chem” 261(5), 3530- 3538. Zhou, G. H., & Han, 2. K. (1994). Effects of dietary supplementation of beta 2- adrenergic agonist clenbuterol on carcass characteristics and some metabolites in ducks. mm, 85(3), 355-361. HICHIGnN STATE UNIV. LIBRARIES ll!IlllllllMlllll\WlllllllllllIllIllllllllllllllllll 31293010503302