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L - IL L.- :III .L I I‘m-LL “.III'I _I..L.I 4AM}, I I IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII IIIIIIIIIIIII IIIIIIIIIIIIIII IIIIIIIIIIIIIIIIIII -— 3 1293 00551 4504 r LIBRARV Michigan State University This is to certify that the dissertation entitled EFFECTS OF CIMATEROL, A BETA ADRENERGIC AGONIST, 0N LIPID AND PROTEIN METABOLISM IN RATS presented by Jyothi Kanakamedala Eadara has been accepted towards fulfillment of the requirements for Ph.D degmm Nutrition Jig/W Major professor Date Feb 21, 1988 M301: mm man'chc‘n lion/£4 urtalOppou awn-mm Mon 042771 IV1E3I_J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from ”— your record. FINES will be charged if book is returned after the date stamped below. EFFECTS OF CIMRIERDI»IA.BETA.ADRENERGIC AGONIST, ON LIPIDMANDIERDTEINIMETABOIISMIIN'RAIS Jyothi Kanakamedela Eadara .A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of IIIHUR.OF'PHIIOSOPHY Department of Food Science and Human Nutrition 1988 M MD? CIMATERDL, ABETAWIC MIST, ON IIPIDANDPEUI‘EINWIISIINRATS By JyothiKanakanedalaEadara Female rats (133 g) fed ad libitmn for 4 wks high-carbohydrate purified diets containing 10 or 100 ppn cimaterol gained «15-75% more skeletalmscleandZS—Bl’t lessabdaninalwhiteadiposetissuethan controls, with a significant acceleration in skeletal nuscle gain evident within the first wk. 'Ib elucidate mechanisMs) of action of cimatenol in these rats, effects of cineterol on energy balance, lipid arxi protein metabolisn were examined. Energy balance was unaffected by cimaterol, indicating that cinatereldoesmtsinplyincreaseenergyexpeniimretoreducebcdy fat. Cineterol failed to influence rates of fatty acid synthesis in liver and white adipose tissue in vivo or in vitro. Activities of fattyacidsyrrthetasearflnelicenzymeintlmetisamwerealso unaffected by cimaterol . Cimaterol stinulated lipolysis in vivo and in vitro, but failed to influence lipoprotein lipase activity in white adipose tissue. Lipoprotein lipase activity was elevated 65-75% in extensor digitorum longus nuscle after cimaterol administration in vivo, and also after addition of cimaterol in vitro indicating its direct effects on nuscle. Total body 3-methy1histidine content and urinary excretion were Jyothi Kanakamedala Eadara measured in these rats in order to calculate fractional rates of accretion, degradation and synthesis of total body 3-methylhistidine containing proteins (actin and myosin). Corsunption of a diet containing 100 mu cimaterol for 1 wk elevated fractional accretion rates of 3-methylhistidine containing proteirs by 120%, this resulted frana25%decreaseinfractional degradationrates, anda32% increaseinfractional synthesisratesoftheseprcteins. In agreaterrtwiththe increasedfractional synthesis rates, therewasa narked increase in RNA gain (165%) and concentration (20%) in hindlimb nuscles of these rats. Plasna insulin, corticosterone and triiodcthyrmine concentrations were unaffected by cimatercl . In emery, cimaterol stinnlated lipolysis in white adipose tissue to reduce the deposition of fat, wittnit affecting either de novo rates of fatty acid synthesis or lipoprotein lipase activity. Cimaterol also stinulated lipoprotein lipase activity in muscle to directenergyawayfrmadiposetissuedepositiontowardskeletal nuscle accretion. The marked increase in rate of accretion of 3-nethylhistidine containing proteins was caused by decreased degradation and increased synthesis. AW I wish to thank Dr. Dale R. Ronsos for his valuable advice and guidence during the course of these studies. Ialsowishtotharflcthemembersofmyconmittee: Dr. Maurice Bemmflc, Dr. Werner Bergen, Dr. Diane Ullrey, and Dr. Maija Zile. 1'1' TABLE OF W5 Page LIST OF TABLES ......................................... iv LIST OF FIGURES ........................................ v REVIB‘? OF LITERATURE.‘ .................................. 1 Introduction ......................................... 1 Body caupcsition ..................................... 3 Energy balance ....................................... 5 Lipid metabolism ..................................... 6 Fatty acid synthesis ............................. 6 lipoprotein lipase .............................. 9 Lipolysis ........................................ 12 Protein metabolisn ................................... 13 Protein degradation .............. . ........ . . ..... 13 Protein synthesis ................................ 14 Dissertation Objective ............ . .................. 16 EFFECIS OF CIMATEROL, A 83178 AIRENERGIC MONIST, ON. LIPID WHSM IN RATS ............................... 17 Introduction ......................................... 17 Materials and methods ................................ 19 Resilts .............................................. 29 Discussion ........................................... 49 EFFECIS OF CMTEROL, A BETA MIC AGONIST, ON HUI'EIN meousu IN RATS ............................. 56 Introduction..... ............................ 56 Materials and methods ................................ 58 ME ......... O O O O O OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 62 Discussion ........................................... 72 (INCLUSIONS ............................................ 79 LIST OF m ..................................... 83 iii LISI‘OFTABLES Table Page 1 Effect of consuming cimaterol on lipogenic enzymes.... 35 2 Rates of fatty acid synthesis in meal-fed rats ........ 38 3 In vitro rates of fatty acid synthesis in the presenceof insulin.. ........ ....... . .......... 40 4 Acute effects of cimaterol on lipoprotein lipase aMViW ....... ......OOOOOOOO. ........... ...... ....... 44 5 Effects of cimaterol on plasma hormones ............... 74 iv LIST OF FIGURES Figure 1 Effects of cimaterol on body composition ............... 2 Energybalanceinrats fed cimaterol............ ....... 3 Effects of cimaterol on rates' of fatty acid synthesis. . 4 Acute effects of cimaterol on fatty acid synthesis ..... 5 Rates of fatty acid synthesis in vitro ................. 6 Body composition in rats fed a high-fat diet ........... 7 Effects of cimaterol on lipoprotein lipase activity. . . . 8 Lipoprotein lipase activity in vitro in muscle ......... 9 Effects of cimaterol on plasma metabolites ............. 10 Acute effects of cimaterol on plasma metabolites ....... 11 Rates of lipolysis in vitro in white adipose tissue. . . . 12 mergy intake and body weight in rats fed cimaterol. . . . 13 Effects of cimaterol on tissue weights. . ............. . . 14 Gain in protein in rats fed cimaterol .................. 15 Effects ofcimaterol onRNAarxim........... ......... 16 3-Methylhistidine content in the body of rats fed’ cmaterol ............ 17 Fractional accretion, degradation and synthesis rates of total body 3-methylhistidine containing proteins. . . . 18 Effects of cimaterol on 3-methylhistidine excretion. . . . 19 Effects of cimaterol on plasma amino acids ............. Page 30 32 33 36 39 41 43 46 47 48 50 63 65 66 68 69 7O 71 73 WWW W mposition of excess fat is a problem not only for humans, but also for meat producing animals. Many people are trying to avoid ca'nsmtption of products with excess fat for reasons related to consumerhealtharrifitness. Thus, thereisincreasedenfliasison productim of leaner meat. Oasiderable research progress has been madeinthisareainthepastseveraldecades. Pecentresearrh approaches imltxiethetseofgrwthhorm, beta-agonists, and imxmlcgical tedmiques to improve the lean to fat ratio in livestock. Administratim of enragenms growth hormone to increase msclemass, arridecreaseadiposetissaeress, of livestockisunder active investigation (37). Several beta adrenergic agonists that alter body cmpcsitim, either by reducing only body fat deposition with little apparent effect an lean body mass or by sinultaneouly decraasirgbodyfatandincreasing leanbodymassaccmzlation, are under extensive investigation for their possible use for runners as antiobesitydru; (5,112) andintheneatiniustryasameansto produceleanerneat (30). Mutharrecentapproaditoreducedbcdy fat deposition has been ins of passive immdzatim with antibodies to fat cells. misapproadinayalsohavepotentialbenefit forincreased protein deposition (40). My research has focused on the effects of a beta-mist, cimaterol m body carpositicn and on possible mechanisus) of action of this W. I will, therefore, focus the literature review on beta-agonists. Beta adrenergic agonists are synthetic carpamis which act specifically at beta receptors and exert their action via increases in intracellular levels of cyclic AMP (38,68). These compounds are unlike the naturally ocwrring catecholamines, epinephrine and mrepinqahrine, whichcanactonbothalphaandbeta receptors. 'Ihe presence of beta receptors is thus a prerequisite for beta-agonist action. Speciesandageoftheaninalnayinfluencethemmberof beta-adrenergic receptors present in a particular organ. For example, thenmberofbetareoeptorsinadultratliverisapproxinatelylo fold lower than in the liver of 7 day old rats (77), whereas the nmberofbetareceptors inrabbit liverdoesnotchangewithage (62). Betareceptorsareftmctionallysubdividedintobetalorbeta 2. 'meproportimoftheseuVOreoeptors fourdwithinanorganinthe bodyisacharacteristicofthatorgan. Forexanple, lunganduterine tissueshaveprimrilybetaz receptors. Stinulatimofthese receptors leads to tissue relaxation (67). ms, beta 2 agonists, of midiclenbrterol isanexanple, areusefulfortreatmentofbrordiial asthma (32). Incontrast, thehearthaspredcninantlybetal receptors, arriactivationofthesereceptorsleadstoarapidincrease in heart rate (67). Various types of beta-agonists, which are know to function specifically at beta 1 or 2 receptor type, are now available. Recently, severalbetaagcnistshavebeenidentifiedthatalterbody cmposition in animals. Among these carpounds, agom'sts with qaecificity for beta 2 receptors are lmown to reduce deposition of body fat: some also pramote the deposition of body protein. Beta-agcnists currently under investigation include clenbuterol (9,30,31,34,36,80,95,97,110), cimaterol (10,11,24,60,85), ractcpamine (3,12,79), Lr-640,033 (34,87,98), 13 104119 (112), BRL 26830 (4,5,6), BRL 35135 (34). Effects of catecholamines, and of these conpouros, on body cmpositim are reviewed below. W Initial studiesweredcnebyadministeringepinephrireand norepinerhrine, the naturally occurring cateoholamines. In pigs, daily injections of epinephrine (0.15 mtg/kg body weight) increased nitrogen retention (29) with no appreciable change in fat deposition. I. “areas in rats, administratim of epinephrine and mrepinerhrine : reduced fat depositicn; effects of the cated'nolamines on muscle aocreticmwere not studied (97). Basedonthese limited data it appearsthattheeffects of catedaolamimsaremtmiformbetween species. the difficulty faced in these studies is that epinephrine arrimrepinefiirinehavevery shorthalf lifes inthebody (25).. My, a single daily injecticn may not be effective. Another difficulty is that these catecholamines interact with both beta and alpha receptors. This interaction results in ccnplex responses, rather than specific effects on a single system. To circumvent these problu, specific agonists have been synthesized that have longer half lifes and act rather specifically with ame receptor type. Anuigthevarimsbeta-agmiststhathavebeensynthesizedto alter body carpositim, same of these carpourds (m. 26830A and LY 104119, for example) reduce body fat deposition with little apparent influenceonleanbodymassaccmlation. mm26830was fedto genetically (ob/ob) mice for 28 days, it prevented weight gain due to reduced body lipid content, with minimal effects on lean body mass (4) . Similar effects with 321. 26830 are also observed in genetically these (db/db) mice ard in obese (fa/fa) rats (5). Another beta-agonist, LY 104119, has been shown to decrease body fat in obese (AW/a) mice (112). Although fat gain is also slightly reduced in normal rats and mice given BRL 26830 and LY 104119, there are no charges in weight gain or carcass protein gain in response to these cmpounds (5, 112). Thus, these campounds are useful in reducing body fatinobeseanimals, buttheydonotimprcve leanbodymassgain. Other beta-agonists , such as clenbuterol , cimaterol , ractopamine f and L-640033 , are under active investigation because they increase lean body mass in association with reduced adipose tissue mass. Clenbuterol is the most extensively investigated beta-agonist to date in terms of altering body composition in meat animals. Clenbuterol, I when fed to finishing lambs at 2 ppm level for 8 wks, decreased fat depthatthe12thribby37%andincreasedlongismusmmscleareaand semitendinosis muscle weights by 42% and 23%, respectively. Body weight gain and feed efficiency were enhanced with no changes in quality grades (9) . Similar reductions in fat deposition and improved muscle accretion have also been achieved by feeding diets containing clenbuterol to finishing steers (97), swine (30), broilers (31) and rats (36,95) . In agreement with the studies on Clenbuterol, other beta-agonists like ractopamine, L 640033 and cimaterol have been shown to also decrease adipose tissue deposition and increase muscle accretion (3,10,11,24,60,85,87,97). The reasons that one group of beta-agonists reduces only body fat, whereas another group of beta-agonists reduces not only fat, but alsoincreasesleanbodymassarenotclear. Itmaybethatthe agonists have more or less affinity for a specific receptor type, or that they interact with different receptor types. The mechanism(s) of action by which beta-agonists alter body omposition has not been extemively evaluated. In the following sections I will review several possible mechanisms of action. W Decreased fat deposition in animals fed diets containing beta-agmistsoalldoocurasadirectresultofincreasedenergy expenditure, for example by stimulating brown adipose tissue metabolism, witrmtaoonoanitantixcreaseinenregyintake. BRL 26830Aislmowntodirectlystimulatebrownadiposetisaie thermogensis(5). Brwnadiposetissue,duetoitshighthermogenic ! capacity (99) , plays an important role in regulation of energy balance. When BRL 26830A is given as a single dose (mung/mouse) to anob/ob mouse, the rate of energyexpenditure remained elevated for 21 h.Afterfeedingthednigdaily (9.5m/kgp.o) for 28 days, it pmeventedfatgainbyircreasedenergyexperdiuire,withmeffecton foodintake(4). Similareffectshavealsobeenobservedin geneticallyobese (fa/fa) Zuckerratsardincafeteria fedobesemice (6). 321.26830Aandclenbuterol alsoincreasesenergyexperfiiturein leananimals,hitthisincreaseinenergyexpendinlreisompensated byincreasesinenergyintake,resultirginminimldangesinbody lipidoontentor weightgain (6,36). 'misixmeaseinenergy expenditure observedinanimalstreatedwithERL26830Aisinlarge partdausedbystimlation of brown adiposetissuemetabolism (5). Similareffectsonenergybalanoearealsoobservedinanimalstreated with the beta-agonist, LY 104119 (112). Effects of cimaterol on energybalanoeareunlmown. Reduced fat deposition associated with increased protein aocretioninrespa'lsetobeta-agaiistsisamedlanismtoirdirectly influenoeenergybalanoe. 'meenergyoostoffatardprotein depositial is the inc-renent of food energy (usually expressed as metabolizableenergy) requiredtopranoteadefinedincranentinbody proteinorfat. Basedmanumberofstudies, itisestimatedthatan intake of 2.25 keel metabolizable energy is required for formation of - ‘1‘ m- I! 1 lean of protein, while ally 1.36 kcal isneeded forthe formation of 1 keel of fat. these values correspond to efficiencies for protein formation of 48%, arri for fat formation of 77% (94). films, decreased body fat deposition, and increased protein accretion, in response to " beta-mists my lead to overall decreases in energy efficiency of the animal. Althoughanincreaseinenergyexpadiuiremayexplainthe reduceddepositionofadiposetissueinanimlstreatedwithm. 26830A, it gamut explain the associated increase in skeletal muscle depositionobservedafteradministratimofsateoftheother beta-magic mists (3,9,10,11,24,30,31,36,60,85,87,95,97,98) . ItisalsoliJcelythebeta-agaiistshavedirecteffectsmwhite adipose tissue metabolism, for example on fatty acid synthesis, lipoprotein lipase activity or lipolysis. 'Ihese possibilitis will be reviewed in the following section. I' . l ! l J . am am mg. Beta-agonists' might decrease the conversion of dietarycarbdiydratestofattyacidsasoremedlanisn' todecreasebody fat. Fatty acid synttmis fran acetyl 00A in mammalian tissues recpires the sequential action of two enzyme systems: acetyl CoA carboxylase arr! fatty acid synthetase (101,107) . Liver and adipose tissue contribute significantly to whole body fatty acid synthesis in rats and mice (54) . Available data on effects of catediolamines and beta-agmistsmtheretflmzttmxghthispathway, andonthe activities of these enzymes, in liver and adipose tissue are discussed below. The actions of catedlolamines on liver fatty acid synthesis is not well defined. It has been reported that epirephrine markedly decreases fatty acid synthesis in rat liver slices, and it was suggested that this inhibition was dependent on cAMP (78). later, it was shown that norepinephrire inhibited fatty acid synthesis in rat liver through an alpha-adrenergic mediated phosphorylation and inactivation of acetyl OoA carboxylase (72) . 'Ihe beta-agonist isoproternol had no effect on carboxylase activity in this study. The cartrastingobservationsintheabovetwostudiesmaybeduetothe differences in body weights, 160-180 g (78) compared to 300-350 g (72) . Maturation of the rat liver appears to be accompanied by a loss of functioral beta-receptors (77) leading to catedmolamine action in adult rat liver primarily through the alpha-receptor system (16,72,88) . 'Ihese effects were also confirmed by the changes observed inthepresenceofthebeta-agonistisoproten'lolardthe beta-antagonist propronalol. In contrast to liver, epinephrine action in white adipose tissue is mainly mediated through beta-adrenergic receptors; epinephrine stimulates the phosphorylation of acetyl CoA carboxylase; this effect can be blocked by propanolol, a beta-antagonist (65,66). In vivo treatment of rats with epinephrine caused phosphorylation of adipose tissue acetyl Ooh carboxylase leading to inhibition of fatty acid synthesis (66). Beta-agonists like Clenbuterol, isoproterenol, 14-640033 “M35135 alsohavebeenshcwntodecreasethe invitro incorporation of CIA-acetate into total lipids in white adipose tissue (34) : ireorporation of CIA-acetate specifically into fatty acids was (not stalled. In sheep adipose tissue, cla'buterol did not ... influence the incorporation of CM-acetate, CIA-lactate or (tn-glucose intofatty acids in vitro (27) . Cautim must be misedininterpretixetheseresults. Incorporationofthe excgereusCM-labeledsubstratesusedintheabovesmdiesdoesnot provide a quantitative measure of lipogensis or fatty acid synthesis because of thepresenoe of erriogernisprecursors (26). Use of. tritiated water to measure rates of fatty acid synthesis would provide aneasureoffattyacidsynthesistratisiniependaatofprecursor source. Aprelimiraryreportsaggeststtattlebeta—agmist ractopaminemay inhibit fattyacidsyntlesisinmiteadiposetissue as measured by tritiated water method (79). Interscapflarbrcxvnadiposetisaieiscapable ofhigherrates of fatty acid synthesis (3-4 times higher than liver or white adipose tissue) (106) . Rates of fatty acid synthesis and the activity of acetleoAcarboxylase ininterscapularbrownadiposetisaieof cold-adapted rats were decreased in vivo by about 70% after injection of norepinephrire (43) . Fatty acid synthesis was also imiibited in vitro in the presence of cAMP, indicating beta adrenergic mediated effects (86) . Data are unavailable on effects of beta-agonists on fatty acid synthesis in brown adipose tissue. In most of the reported studies with beta-agonists that alter body camposition, animals have been fed high-carbohydrate diets (3,6,9,10,11,24,30,31,36,60,85,95,97). Therefore, an important contributor to fattening in these animals would be conversion of dietary carbohydrate to fatty acids. Measurements of rates of fatty acidsynthesisinliverarriadiposetisaleunderinvitroardinvivo NF conditions, and measurement of activities of lipogenic enzymes should 1 provide dataonoremedlanismwhereby fat deposition maybedecreased : in response to cimaterol. 5 w. lipoprotein lipase (in) catalyses the hydrolysis I; of plasma triacylglycerol and helps to transfer plasma triacylglycerol fatty acids from the circulation into adipose tissue for storage, and into muscle for metabolic fuel (28,103). This enzyme is synthesised intheparendmymalcellsofamnmberoftissues, andisthensecreted andtransportedtoits functional siteontheluminal surfaceofthe capillary endothelial cells (27) . Adaptive changes in lipoprotein lipase activity occur in different tissues in response to variations in the physiological state. ‘Ihese charges correlate with the altered rates of triacylglycerol fatty acid uptake into the respective tissue (103). Time, another mechanism whereby beta-agonist might affect body camposition is by altering activity of lipoprotein lipase (LPL) . Insulin has long been recognized as a major regulator of white adipose tissue lipoprotein lipase both in vivo and in vitro. Enzyme activity is increased several-fold after injection of insulin into fasted rats (17) and is strongly correlated with plasma insulin concentrations in a variety of nutritional states (28) . 'Ihis ireulin-induced increase in lipoprotein lipase activity is potentiated 10 by glucocorticoids in vitro (8) . Actions of catedlolamines on irsulin-induoed IPL activity in white adipose tissue are not, however, well established. Irmlin—induced increases in adipose tissue LPL activity in vitro are abolished by addition of Qirghrire. It was suggested that epirephrire inactivated IPL before itwas released frcn the adipocytes (7,8). 'Ihese experiments demonstrate that epirephrine inhibits insulin-induced LPL activity, butitisnotclearifthebasalIPLactivitywaildalsobedecreased ! byepirephrire. Recently ithasalsobeenstmnthatthedegradation of lipoprotein lipase is increased in vitro by epinephrine (93) . If catecholamines play an important role in regulation of white adipose tissue IPL activity, denervation of the tiswe should affect L‘PL activity. hit after microsurgical derervation of rat adipose tissue, IP12. activity is similar to activity in non-operated tissue, evenflngh ttecaeentratimofmrepinfilriiewasatleasttentimeslwerafter denervation (51). Althoughthereissamedisagreementamengthe studies about the role of catecholaminee in regulation of white adiposetissueIPLactivity, underscmecorditionsenzymeactivitywas reducedinthepresenoeof epinephrine. Effects ofbeta- on IPLactivityinwhiteadiposetisaiehavenotbeenreported. films, the measurement of IPL activity in white adipose tissue shmld provide datama'emedlanisnwhereby fatdepcsitionmaybereduced in response to cimaterol. Unlike the tendency of catecholamines to depress white adipose tissue LPL activity, a large elevation in LPL activity in rat brown adipose tissue is observed 3-4 hours after injection of norepirephrine, and also after cold exposure (4°C) for 28 days (21) , 11 which mild ause release of norepinephrine frtml nerve ending in brown adipose tissue. Neither insulin nor glucose could mimic the cold-induced irerease in IPL activity, suggesting that in contrast to whiteadipoeetissue, brwnadipose tissueIPLactivity isnot stimulated by insulin, but rather by mrepinephrine (21) . 'Ihis stimulation of IPL activity by norepinefllrine is mimicked by the beta-agonist iscprenaline, and abolished by the beta-antagonist propramlol, indicating beta-adrenergic hedmdsn (21) . 'lhese data on effects of catecholamines on IPL activity in white and brown adipose tissuesuggesttissuespecificdlangesintleregulationofIPL activity. Relatively little is known about caltrol of skeletal muscle IPL activity. Skeletal muscle represent about 45% of body weight (22) , indicating its quantitative significance in utilizing considerable amounts of lipids as fuel through LPL activity. Epinephrine increasedtheactivityoftheenzymeonly invasois deepest, Mmeasnorepinephrineirereasedtreactivityintlevastus deepest arr! soleus muscles after sirgle injectiae. In vastus aperficial muscle catecholamires did not influence the enzyme activity (49) . Reascms for these differences among various muscles in responsetocated'lolaminesaremtclear. Dataarenotavailableon the effect of beta-agonists, ireludin; cimaterol, on LPL activity in rat tissues. It is possible that cimaterol may have differential effects on LPI. by decreasing activity in white adipose and increasing activity in brown adipose tissue and muscle. Induced enzyme activity in white adipose tissue would help to reduce the storage of lipids and stimulation of IPL activity in skeletal muscle mild facilitate 12 availability of energy for increased muscle metabolism. W. 'Ihe lipolytic system involves triglyceride lipase which catalyses breakdown of triglycerides to free fatty acids and glycerol (58) . lipolytic activity is commonly measured by release of glycerol or free fatty acids into the medium or plasma under in vitro and in vivo conditions, respectively. Beta-agonists might stimulate :- lipolysis and thereby increase the mobilization of stored lipids as one mechanism to reduce body fat. Effects of catecholamines on lipolysis are well documented. When fatpadsframywngmaleratswereincubatedinvitrointhepresence I -. 4 of epinephrine (1.0 uM) , lipolytic activity increased 3-4 fold, as measured by release of free fatty acids into the medium. This increase in release of free fatty acids is correlated with stimulation of intracellular cAMP concentration. Furthermore, beta-adrenergic blocking agents antagonise the effects of epinephrire on both cAMP levels and lipolysis (19) . A number of other studies confirmed these effects using isolated fat cells or fat pads (15,63,74,102) . Dereasesincdeeperdentproteinkiraseisalsoobservedinthe presence of epinephrire in these studies. Infusion of catecholaminee has also been shown to increase plasra levels of free fatty acids and of glycerol (41) . Consistent with the data on the effects of catecholamines on lipolysis, isoproternol at 1 UN concentration also increased the release of glycerol, cAMP and protein kinase suggesting beta-adrenergic stimulation of lipolysis (2) . Similar increases in release of glycerol, in response to Clenbuterol, LY 79771, I;-640,033 am EL 35135 have been reported (34,113). Available data on the 13 effects of mtecholamines and beta-adrenergic agonists on lipolysis, strongly suggest that the beta-agonist cimaterol should also stimulate lipolysis in white adipose tissue. mt, such studies have not been conducted. Results from the experiments I propose will provide evidence for increased mobilization of lipids as one of the mechanism to reduce deposition of fat in response to cimaterol. MEWS.“ Overall rates of protein synthesis and degradation in muscle determine the state of protein balance in the tissue. Generally, during periods of rapid muscle growth rates of protein synthesis and degradation are both accelerated, with the increase in protein synthesis being greater than the increase in protein degradation (83) . Under some conditions, however, such as during recovery phase frum atrophy of immobilized muscle (47) and during work induced-hypertrophy (46) , gain in muscle protein is caused by simultaneous inhibition of protein degradation and stimulation of protein synthesis. These changes represent the most efficient way for muscle accretion to be accelerated. The review of literature on the effects of catecholamines and beta-agonists on protein degradation and synthesis will provide insight into possible mechanisms of action of cimaterol to increase skeletal muscle protein deposition. W. 'Ihereissaleevidencetosuggestthat catedlolamines slow turnover of skeletal muscle proteins. Epinephrine at physiological concentratiore (1 nM) , lowered the release of alanine and glutamine from isolated rat epitrochlaris muscle (42) . Dibutyryl cyclic AMP reproduced the effect of epinephrine and proponolol (a beta-antagonist) , but not phentolamine (an alpha-antagonist) , blocked 14 the effect of catedlolamines, suggesting a beta-adrerergic process. It was concluded that epinephrine inhibited alanine and glutamire release from skeletal muscle by decreasing degradation of muscle. proteire (42). an: no direct evidence for a role for epinephrine in irhibiting degradation of muscle protein was provided in this study. Based on resilts frum infusion of U-MC leucine in pigs (50) , and [15N11e1ciie and [2H3jleucine in humans (81) it was also calcluiedthatqaiiephrireormrqhireghrimedecreasesprotein degradation. Oa'lsistent with the effects of catecholamines, isoporternol also laaeredthereleaseofaminoacidssn'lasalanire, threonine, phenylalanine, tyrosire, lysine, arginire, leucine and valine fran perfused rat hanicorpls muscle preparations (71) . Protein degradation, asmeasuredbythereleaseofphenylalanire inthese preparations, shmed a 20% reductial with addition of 1 um isoprcternol (71) . It was also shown in mice, that daily injections of isoproternol increasedthehalf lifeofparctidglandproteins (55) . Clenbuterol administratial to calves also decreased protein degradatial, as measured by urinary thim of 3-methylhistidine 110). It has been also suggested that clenbuterol may have rapid effectsalreducingtheproteinmrrever, basedontheresults calculated from measured rates of protein accretion and synthesis (95) . All the data suggest that cimaterol may decrease protein degradation, but direct experimental evidence is lacking. mm Data on effects of catecholamines and beta-agonists on protein synthesis are even less available than are data on protein degradation. Epinephrine has been reported to increase protein 15 synthesis in rat diaphragm muscle by increasing incorporation of BH-leucire and 3m-tyrosine into protein (91) . But stimulation of protein synthesis by epirephrine was only observed in diaphragms from hypophysectimized rats; protein synthesis in diaphragms fram intact rats was not affected by epirephrine (91) . Incorporation of C14-labeled amino acids into total rat tibialis muscle protein is stimulated in vivo by chronic administration of isoproternol (0.3 mg/kg body weight) (33) . Maximum stimulation occurs 2-3 hours after the fifth daily injection of isoproternol. This stimulation is greater during the first few days of treatment, and decreases gradually thereafter (33) . Clenbuterol also increased fractional synthesis rates by 34% l h after the 7th daily injection, as measured by phenylalanine incorporaticm into mixed muscle proteins (36) . Similarly, ractopamire, another beta-agonist, has been sham to increase fractional rates of protein synthesis in pigs (12) . But, Reeds et al (95) reported that clenbuterol failed to stimulate the protein synthesis in rats. Others have reported contrasting results; both epinephrire and norepinephrine at a concentration of 0.1 ug/ml decreased incorporation of 14C-amino acids into muscle protein ( 111) . The inhibition occurred whether epinephrine was added in vitro or administered in vivo (111) . Reasonsforthediscrepanciesamungthesesuxiiesarestill unclear. These studies indicate that catedlolamines or beta-agonists may stimulate, inhibit, or result in no change in protein synthesis. The type of carpalnd studied, duration of administration, method of study (in vitro vs in vivo), the type of measursrent (single injection vsconstantinfusion), time ofthemeasurementafterthedose, and 16 type of mesurament (whole body vs specific muscle) probably all mm to the variable reallts. From the above discussion it is possible that catecholamines and beta-agonists may exert significant regulatory actions a1 protein balanceinskeletalmuscle. Althaighthereare discrepanciesamong the studies for the effects of mtecholamines or beta-agonists a1 proteinsynthesis, allthestlxlies indicatethatthesecalpamds decrease protein'degradatia'l. Therefore, an euamdratia'l of the effects of cimaterol on protein degradation and synthesis associated withproteinaccretia'lwill provide evidence forthemechanismof actimtoincreaseproteindepositial, andalsoprovidedataabaltthe extentofcontributionofeachtotheirereasedaccreticn. 11° ! ! . i! i ! . 'meobjectiveofmydissertatialreseardlwastoeaamilethe mechanisms of action of cimaterol in reducing adipose tissue deposition and increasing skeletal muscle accretion in rats. Therefore, the follaving studies on effects of cimaterol on lipid metabolism, and on protein metabolism, were ca'lducted. m OF CIMATEROL, A MWIC MIST, WIIPIDWIISIINRATS M193 Several beta adrenergic agonists have recently been identified that alter body calposition in animals. Sane of these carpalnds (BRL 26830A and LY 104119, for example) reduce body fat deposition with little apparent influence on lean body mass acamulation (5,112), wtereasotherssichasclenbuterolandcimaterolnotmlydecrease fat deposition but also increase lean body mass accretion. Clenbuterol, whenfedtolambs, reducedfatthicknesscverthethhribby37%and increased longissimus muscle cross sectional surface area by 41% (9) . Similar shifts in body carposition in response to clenbuterol or cimaterol have been reported in other species including cattle (97) , chickens (31), pigs (60) and rats (36). Themechanisnofactionofthebeta-agcnistBRL26830Ahasbeen extensively studied. BRL 2683011 increases eter'gy expenditure by stimulating brown adipose tissue metabolism withart a concamitant increaseinenergyintake: thus, animalstreatedwithBRL26830A retainlesserergythancontrolanimals (5). Althalghanincreasein energy expenditure explaire the reduced deposition of adipose tissue in animals treated with Ed. 26830A, it carmot explain the associated increase in skeletal muscle deposition observed after administration of same of the other beta adrenergic agonists (9,10,31,36,60,95,97) . (he hypothesis for the increased skeletal muscle deposition is that beta adrenergic agonists such as clenbuterol and cimaterol shunt dietary energy away from adipose tissue to skeletal muscle (97). C‘atedlolamines and beta agonists influence a number of metabolic pathways in lipid metabolism, however, the specific effects of 17 18 clenbuterol ard cimaterol on lipid metabolism have not been widely explored. In most of the reported studies with beta-agonists animals have been fed high-carbdiydrate diets (S,9,10,3l,36,60,95,97,112) . Therefore, an important contributor to fattening in these animals would be conversion of dietary carbohydrate to fatty acids. Under some ca'ditias catedlolamines inhibit acetyl coenzyme A carboxylase activity (66) , a key regulatory enzyme in fatty acid synthesis. Thus, tieredlceddepositionofadiposetissleinanimalsgiven beta-aga'lists might be explained by irhibition of dietary carbohydrate conversion to fatty acids. Beta-agonists might also influence lipoprotein lipase activity (7,8) . This enzyme facilitates transfer of fatty acids from cirallating triglycerides into adipose tissue for storageandintomuscle formetabolic fuel. Undercertain physiological corditia'ls, lipoprotein lipase activity in adipose tissue is reciprocally related to lipoprotein lipase activity in skeletal muscle (28). Thus, beta agonists may inhibit lipcprotein lipase activity in white adipose tissue to reduce storage of lipids and stimulate lipoprotein lipase activity in muscle to facilitate availability of energy for increased muscle metabolism. The stimulatory effect of catecholamines on white adipose tissue lipolysis is well established (38) and provides another potential mechanism whereby beta agonists may utilize body lipids and thereby reduce adipose tissue deposition. I examined the effects of cimaterol, a beta-agonist that decreases fat deposition and increases skeletal muscle accumulation (10,11,60,85) on the above mentioned measures of lipid metabolism in rats. Body carposition, energy balance, rates of 19 fatty acid synthesis, activities of fatty acid synthetase, malic enzyme and lipoprotein lipase, and rates of lipolysis were measured in rats administered cimaterol aaltely or chronically. W W Female Sprague-Dawley rats (130-180 g) obtained from Harlan Irrlustries, Indianapolis, IN, were based individually at 23°C in metal cages with wire—mesh floors. Roan lights were on from 0700 to 1900 h. All animals were provided a norprrified diet (Wayne RodentBlooc, OmtirentalGrainOalpany, Chicago, IL) andwaterad libitlmm for the first 2 days after arrival in the laboratory. Rats were then fed either a high-carbohydrate or a high-fat purified diet. The high-carbohydrate diet contained (in g/100 g): 66.0 g glucose,5.0 9 corn oil, 20.0 g casein, 0.3 g methionine, 1.0 g vitamin mix (14), 0.2 g choline chloride, 3.5 g mineral mix (14) and 4.0 g cellulose. This diet provided 3.57 kcal metabolizable energy/g with 67% of metabolizable energy as carbohyrate, 13% as fat ard 20% as protein. The high-fat diet contained (in 9/1009): 18.2 9 glucose, 19.6 9 corn oil, 19.6 g tallow, 29.4 g casein, 0.44 g methionine, 1.47 g vitamin mix, 0.29 9 define dlloride, 5.15 g mineral mix and 5.88 g cellulose. This diet provided 5.25 kcal/g with 13% of metabolizable erergy as carbohydrate, 67% as fat and 20% as protein. The amounts of cimaterol (CL 263,780; anthranilonitrile, S-[l-hydroxy-Z-(isopropylamim)ethyl]-) added to these diets is indicated in the experimental design. W. Ebcperiment 1. Thisexperimentwasdesignedto examine effects of cimaterol a1 body carposition and energy balance of rats fed the high-carbohydrate diet ad libitum. Rats were divided 20 into4gralps. Graiplwaskilledatthebeginningoftheexperiment toobtaininitialbodycalpositionvalues, groups 2, 3, and4were fed the high-carbohydrate diet containing 0, 10 or 100 ppm cimaterol, respectively. Food intake and weight gain were recorded twice weekly. Halftheratsframeadlgralpwerekilledafterlwk, andthe renainingratswerekilledafter4wks. carcasseswerefrozen for subsequent analysis. Ecperiment 2. Effects of cimaterol on in vivo and in vitro rates of fatty acid synthesis and on activities of fatty acid synthetase and malic enzyme were examined in liver, parametrial white adipose tissue and interscapular bravn adipose tissue. Chronic effects of cimaterol on fatty acid synthesis were examinedinrats fedthehigh-carbohydratedietcaltainirgo, 10or looppncimateroladlibiummforlor4wks. Animals fromexperiment 1wereusedforthesemeasursnents. Attheendofthelor4wk feeding period, rats were injected with 1 mi of 31420 (51) at approximately 0900 h and killed 15 minutes later. Blood, liver, total dissectableabdaniralwhiteadiposetisseardinterscapllarbram adipose tissue were rapidly collected. Tissues were frozen at -70°C for subsequent determiraticn of 3H incorporation into fatty acids . Analiquotofplasma separatedframbloodwasusedtodeterminethe qecific radioactivity of water. Results were expressed as lmmoles 3I-lincorporated into fattyacidsperminutepertotal tissue. Tissues ralaining after the measurement of rates of fatty acid synthesiswerereturredtothecarcassformeasurslentofbody composition are energy balance. 21 1b verify the effects of cimaterol on fatty acid synthesis, activities of the lipogenic enzymes fatty acid synthetase and malic enzyme were measured in liver, white adipose tissue and brown adipose tissue of additional rats fed 0, 10 or 100 mu cimaterol for 1 or 4 wks. Rats were killed between 0900 and 1100 h. Tissues were rapidly renoved, weighed and aliquots were hanogenized in 8 volumes of cold phosphate—bicarbonate buffer (70 mM 101003, 85 11M 32111304, 9 mm 31121304, and 1 an dithiothreitol) , pH 8.0 (90) and centrifuged at 100,000g at 4° for 45 minutes. The resulting supernatant solution was stored at -70°C for no longer than two days for subsequent measuranent of the enzyme activites. Acute effects of cimaterol on in vivo rates of fatty acid synthesis were examined after administration of cimaterol to rats that had been fed the high-carbohydrate diet ad libitum for 1 wk. Rats were injected intraperitoneally with 0.0 mg, 0.15 mg or 1.5 mg cimaterol in 0.2 ml saline at approximately 0900 h (time 0) . These doses oorrespord to the daily amount of cimaterol consumed by rats fed the high-carbohydrate diet containing 0, 10, or 100 ppn cimaterol. After 15 minutes (time 15), each rat reseived 1 mi 31120 intraperitoneally, after another 15 minutes (time 30) all animals were killed. Blood, liver, white adipose tissue and brown adipose tissue were processed as described for the We in vivo trial. Meal fed rats show an increased lipogenic capacity (69). To increase the possibility of detecting an effect of cimaterol on rates of fatty acid synthesis, rats were trained to eat meals of the high-carbohydrate diet containing 0, 10 or 100 pm cimaterol twice daily (a 1 h meal at 22 0900 h and a second at 1600 h) for 1 wk. Rats were injected with 31120 (1 uni/rat) 15 mimrtes after carpletion of the 0900 h meal, 15 minutes later rats were killed, blood and tissues were collected an'l processed for the determination of 3!! incorporation into fatty acids. To determine effects of cimaterol on in vitro rates of fatty acid synthesis, rats were killed (0900 h) after being fed the high-carbohydrate diet ad libitum for 1 wk. Liver, parametrial white andinterscepdlarbrmnadiposetismeswererapidlyramvedard placed in nor-Ital saline solution (0.9% NaCl) . Liver slices (150-200 mg) werepreparedusingaStadie—Riggshandmicrctane. 'Ihindistal portiom of parametrial white adipose tissue (100-150 mg) and pieces of interscapilarbrwnadiposetiswe (GO-1001113) werecrt, rinsedin saline, gently blotted and weighed. Liver slices and adipose tissue wereimnediatelytransferredtoa flaskcontainingBmlchreb's Ringer biarbcnate buffer, 40 uCi/ml, 31-120, 10 11M glucose. Tissueswereinwbated for2hat37ocinthepresenceorabsenceof cimaterol (1 nM, luMorlnH) inashakingwaterbathwith 95% 02: 5%ngasmixture. mtesof fattyacidsynthesiswere also measured with the addition of 100 uU/‘ml and 100 w/mlinsulin in the presence or absence of 11114 cinaterol. An aliquot of the final incubation media was used to determine specific radioactivity of media water. At the end of incubatiors, tissues were rammed, rinsed with saline sapcnified and fatty acids were extracted. 31-1 incorporation into fatty acids was detemixed. Data were expressed as umoles of 3H incorporated into fatty acids per 2 h per 100 mg tissue. 23 Experiment 3. If cimaterol exerts its primary effect on body cmposition by inhibiting de novo fatty acid synthesis, then cxrunmgmion.of a high-fat diet which has been shown to inhibit de novo fatty acid synthesis (69) should circumvent effects of cimaterol on bodycanposition. ThishypothesiswastestedbyaddimOor 147ppn cimaterol to the high-fat diet. This dose of cimaterol is equal to the amount of cimaterol (per kcal energy) in the high-carbohydrate diet that contained 100 ppn cimaterol. Food intake and body composition measurements were perfOrmed as in experiment 1. Experiment 4. Since uptake of fatty acids by adipose tissue and muscle from circulating triglycerides is facilitated.by the enzyme lipoprotein lipase (28), the possible effects of cimaterol on modulation of lipoprotein lipase activity were examined. Chronic effects of cimaterol on lipoprotein lipase activity were determined in rats fed the high carbohydrate diet containing 0, 10 or 100 ppm: cimaterol ad libitum for 4 wks. White and brown adipose tissue were removed and frozen immediately on dry ice. Both hindlimbs were also removed and frozen quickly in acetonerdry ice. (All tissues were stored at -70°C for subsequent measurement of lipoprotein lipase activity. Upon thawing, extensor digitorum longus (EDL), soleus muscles and remaining hindlimb muscles (removed from the leg bones and separated from the adhering adipose tissue) were selected as representative of white, red and mixed fiber types, respectively. Acute effects of cimaterol on lipoprotein lipase activity in white and brown adipose tissue and muscle were examined in rats that had recieved the highrcarbchydrate control diet ad libitum for 1 week. Rats were injected intraperitoneally with cimaterol (0.0, 0.15 or 1.5 24 mg in 0.2 ml saline) at 0700 n and killed 4 h later. White and brown adipose tissue and both hindliubs (EDL, soleus and total rareininghirriliubnlscle) wereranoved forsubsequentneasuranentof lipoprotein lipase activity. To determine effects of cimaterol m skeletal nuscle lipoprotein lipase activity in vitro, EDL and soleus nuscles frun rats fed the high-carbohydrate diet ad libitum for 1 wk were used. Rats were deapitated at approximtely 0900 h. EDL (70-90 mg) and soles (30-40 mg) mlewereclanpedatbotherflsinsimtomaintaintheirlength an: then removed carefully arri placed in Kreb's Ringer bicarbonate buffer saturated with 95% 02: 5% (:32, pi 7.4. After gentle blotting, nuscles were transferred to vials containing 2.0 ml of Kreb's Ringer bicarbonate buffer, 10 n“ glucose, 0.1 U porcine insulin/ml, 3.0%bovineser1malblminandamimacidsatfivetines the concentrations of rat plasna (73). insoles were incubated for 2 h at 37°Cinthepresenceorabsenceof1nflcimaterol inashaking waterbathwith95%02: 5%ngasmixmre. Attheerdofthe incubatias, nuscles were rawved, separated fran the clanps, rinsed, blotted, weighed and prepared for lipoprotein lipase determination. Analiquotofthemediawesalsoobtaimdforneasmarentof lipwrotein lipase activity. To determine effects of the incubation per se on lipoprotein lipase activity, enzyme activity was measured in representative nuscles at the start of the imubation. Dcperiment 5. Effects of cimaterol on lipolysis in vivo and in vitro and on plasma concentrations of glucose and lactate were studied. Chronic effects of cimaterol were examined by measuring concentrations of plasma glycerol, free fatty acids, glucose and 25 lactate after feeding the high-carbohyderate diets containing 0, 10 or 100ppncimaterolfor4wks. Animlsfrunexperinent4 ' (i.e., those used to examine chronic effects of cimaterol on lipoprotein lipase activity) were used for these metabolite measurements. Rats were deapitated at 1100 h and blood was collected. Acute effects of cimaterol on in vivo lipolysis were examined in rats that had been fed the high-carbohydrate diet ad libitum for 1 wk. Rats were injected inn-aperitoneally with 0, 0.15 or 1.5 ng cimaterol in 0.2 ml saline at approximately 0900 h. Half the rats receivirgeadldoseofcimaterolwerekilledlSminltesafter injectimandtheranainingratswerekilled30mirutesafter injection of cimterol . Blood was collected for measurement of plasma glycerol , free fatty acids, glucose and lactate. To determine effects of cimaterol on in vitro rates of lipolysis in whiteadiposetissme, ratswerekilled (0900b) afterbeingfedthe high-carbohydrate diet ad libitum for 1 wk. Thin distal porticms of parametrial white adipose tissue (100—150 mg) were rapidly removed. Tissue was rinsed in saline (0.9% NaCl), gently blotted, weighed and transferredintoa flaskcontainithmlofKreb'sRingerbicarbonate buffer, #1 7.4, with 3% fatty acid free bovine serum alhnnin (fraction V) inthepresenceorabsenceofcimaterol (1m, luM, 1n“). Tissueswereinolbatedat 37°C forlhinashakingwaterbathunder anatmosphereof95%02 : 5%CD2. Inapreliminarystudy, rates oflipolysiswereshowntobelirear forat leastoneh. Attheend of incubation, glycerol and free fatty acid concentrations in the media were measured. 26 W- Weights of selected tissues (abdaninal white adipose tissue, right hirdlinb nuscle, EDL and soleus nuscle) were determined 'afterthawirqthecarcassee. Himlinbmlscleweightsweredetermined afterbeingstrippedfrunthebamesarfiseparatedfmnadipcse tissue. Lergthsoftibiaardfmweremeasuredusingcalipers. All tissueswererehmedtothecarcassesexceptforthealiqaotsof Wizedtissueusedfor measuring rates of fatty acid synthesis. Afterrenovaloffoodresiduesfrunthestanadycarcasseswere hamgenizedinwater. Alimotsofthehanogenateweremedto determine body fat by dlloroformmethanol extraction. Protein was determinedbythenethodofuarlmelletal(75)afterthehcnogenate hadbeendissclvedinleoditmhydroxide. Anotheraliquotofthe lmgenatewasdriedatSOOCanlusedtodeterminebodyenergyby bmbcalorineteryusingaparrhdiabaticcalorimemr (ParrInstrument co.,1bline, III). Tissueweights,bonelengthanicarcassenergyofratsatthe begilmihgoftheexperinentalperiodwerepredictedfrmlirear regressim equationsbasedonbodyweights, tissueweights, bone lalgthsardcarcassenergyoftheinitialgrmp.cairsintiss.le weig1t,bonelergthardcarmssenergywerecalwlatedfranobserved valuesattheenioftieexperinentalperiodminlspredictedvaluesat the beginning of the experiment. carcass energy density, an indicator of relative prcportions of fat and protein in the body, was calculated bydividingbodyenergyccntentbybodyweight. Energy efficiencywas calculated as body energy gain divided by metabolizable energy ccnsumedduringthe4wkpericd. Energyexpenditurewasoalcnllatedas the difference between metabolizable energy intake and body energy gain. [in i-—'W_-_-1_- .A . l 27 To estinate rates of fatty acid synthesis tram 31120, tissues (loo-200111;) were saponified. Fatty acids were subsequently extracted and counted for radioactivity (70). Fatty acid synthetase and Italic enzyme activities were determined inliver, whiteadiposetissuearrlbrownadiposetiswebyestablished methods (90,92) . Enzyme activities were expressed as rates of utilization of NADPH (fatty acid synthetase) or NADP (malic enzyme) permof cytosolicproteinrandpertisalepermiruteat 30°C. lipoproteinlipaseactivitywasneaslredbyamodifiednethodof Schotz (53). Aweighedamlnt (100 my) ofmincedwhite adiposetissue washanogenizedforlmiruteatasettihgofs (Potter-Elvehjem hamgenizer) using glass and glass in 0.25 M sucrose-1 n“ m (pl-I 7.4) buffer with 20 U/ml heparin (lg tissue/8 ml blffer) . After centrifugihgthehtmogenateat 120009for15minrtesat4oc, the fat-free, post-mitochondrial supernatant was recovered and stored at -70°C for sabseqent lipoprotein lipase assay. A second hmogenizatim -centrifu;ation step increased recovery of lipoprotein lipase activity fran white adipose tissue by only 8%. ‘merefore, I routinely aployed only a single tangenizatim-centrifugatim step forwhiteadiposetissue. nucleardbrownadiposetissuewere rmgalizedindetergart (59) toinproveextractionof lipoprotein lipase. Tissues were weighed, minced and hanogenized in 1:7 (w/v) 0.25 M sucrose-1 nu EDl‘A (pH 7.4) buffer containing 20 U heparin/ml, 1% bovine serum albumin and 0.2% deoxycholic acid. After calm-ifugatim and aspiration of supernatent, the pellet was again stored at -70°C for subsequent lipoprotein lipase assay. 28 Significant amounts of lipoprotein lipase activity ( 15-40%) were recoveredduringthesecordextractimofthesetissues. Recoveries for nuscle and brown adipose tissue lipoprotein lipase activities were 86% and 95%, respectively, for the caubined two extractions carpared to three extractions. Therefore, I routinely used two extractions for these tissues. Lipoprotein lipase activity was determined using 1“'c triolein prepared with human serum (53) as substrate. To control for nmspecific lipolysis the 14c-triolein substrate was also prepared in the absence of serum. Only fatty acid release resulting from serum activation was specified as lipoprotein lipase activity. Using this assay system, at pH 8.0, enzyme activity was linear for 45 minutes of incubation and substrate (3.4 nM triolein) was not rate limiting. The amamtoftissueusedintheassaywasinthelinearrange for lipoprotein lipase activity (amount varied with tissue depending on the ammt of lipoprotein lipase activity). Since lipoprotein lipase activity was characterized by serum activation and NaCl inhibition, effects of 0.66 M NaCl on lipoprotein lipase activity was evaluated. Inhibition of lipoprotein lipase activity in the preserve of 0.66 M NaCl was 92%, 90% and 82% in white adipose tissue, brown adipose tissue and muscle, respectively. Incubations were conducted for 30 mimtes at 37°C, and free fatty acids were extracted as described by Hietanen and Greenwood (53) and counted for radioactivity. Results were expressed as umoles of free fatty acids released per hour per tissue and per g tissue. Glycerol was measured by enzymatic analysis using glycerokinase, pyruvate kinase and lactate dehydrogenase (Boehringer Manneheim, 29 Indianapolis, IN. cat.no. 148270). I-Yee fatty acids were determined by the titrametric method according to Ko and Royer (64) with a standard curve develqaed using palmitic acid. Plasma glucose was measured, which involved glucose oxidase and peroxidase to produce a colored catpcund (Boehringer Manneleim, Indianapolis, IN. cat.no.189197) . lactate was determined enzymatically (56) . a- Data were analysed statistically using analysis of variance and the Bonferonni t test for post hoc treatment carparisons (44) . m1}: Dcperiment 1. Effects of cimaterol on body composition and energy balance were examined in rats fed the high-carbohydrate diet for 4 wks. All rats remained healthy during the study with no visible adverse effects of cimaterol. An increase in rate of body weight gain was observed in rats fed cimaterol (Figure 1). The major weight gain responseoccurredduringthefirstZwkswhenrats fed 100r100ppm cimaterol gained 27 and 42 % more than control rats. By 4 wks rats fed cinaterol were no longer gaining body weight at an accelerated rate. Consumption of cimaterol stimulated weight gain of hindlinb nuscle at an even faster rate than total body weight gain (Figure 1). In4wksrats fed 10and100ppncimaterolgained569cani759snore hindlimb muscle than control rats with a significant acceleration in gain evident within the first wk (80% and 124%). These increases in nuscle gain represented increased protein deposition because protein concentration in hindlimb mscle was uncharged by cimaterol (Figure 1). Stimulation of muscle growth was generalized rather than selective for a specific fiber type because muscles containing predcminantely red (soleus) or white (EDL) fibers responded similarly Figure 1. 30 aoov ‘ amount J 0 rwenom b a "MUSCLE b o I 95» 5 wk“ 0 E )- aas z s 3 i __W* - o "r . = .‘ a '1': 43’ g E "r 0- I- = I I 2 - 7 5 1- / 3 u / /wh / - . / . / 24 / / u g I- / 5 / " ¢ 3 o] ’1 /. /4 l o o ? 112 f 5 E s < o a‘ o n- o- : 5‘ z 0 2 E 28 u 3 3 0 0 1o , o I 5 E z o ‘ E 1- D- < 2 O I- c I. 0 10 100 0 10100 CIMATEROL-PPM Effects of cineterol (:1 body ccuposition. Gain in body ,tissue ,totalbodyfatandptroteininrats fed ad libitum for 4 wks a high-carbohydrate diet containingo,100r100ppncimaterol. Thefoursegments ofthebarsforbodyweightgainrepresentgainperwkfor eachofthe4wks. Gaininhindlimbmuscleforthefirst wkisrepresentedbythelowersegnentofthebar,the uppersegnentrepresentsgainforthelast3wfi- 440 0 a: m 0 2 0 10 100 0 10 100 m ClMATEROL-PPM Energybalanceinrats fedcimaterol. Ratswerefeda high-carbohydrate diet containing 0, 10, or 100 ppm cimaterol ad libitum for 4 wks. The four segnents of the bars for energy intake represent intake per wk for each of the4wks. Fachbaristhemean: SEMof 10 rats. Different letters above the bars indicates significant differences anong the groups (P<0.05) . Initial body energy was 23817 kcal. Bergy gain was calculated as firalbodyenergyminusthepredicted initialbcdy energy. Bergyeucperxiiturewascalculatedasthe difference between metabolizable energy intake and gain. Energy efficiency equals energy gain divided by exergy intake times 100. Figure 3 . 33 0N! will VOUS WEEKS I lIVEII . LIVER I 3000 2400 a 1 O 1 l I000 1000 . AWAT IBAT s III? a nMOL 3l'l T0 FATTY ACIDS/TlSSUE/MIN O 8 nMOL 311 TO FATTY ACIDS/TlSSUE/MIN 400 200 0 10 \OO O 10 ‘00 CIMATEROL - PPM Effects of cimaterol on rats of fatty acid synthsis. Rats were fed a high-carbohydrate diet containing 0, 10 or 100 ppm cimterol ad libitum for 1 or 4 wks . AWA’I‘ inflates abdcminal white adipose tissue and IBAT irriicates interscapilar brown adipose tissue. Values are nears 1 SDI for 10 animals. Different letters above the bars indicate significant differenca between groups (p.uuosmot .a Nu.s « mm.s use a ~s.c « ~n.u uuoetu>o mucus.tssxs x: o.usu a as» to» nuss.ox mama—u unassuu szuss supsssumtous. us_u>.uuusmst .m ~.s a s.s use a ~.= « m.u uomuto>u unsuspeusxo x: s use a use use assess: tu>__ so»: .ssmscamtu as uouumeeoss use: unease: mama.» amuspuu szssa tu—ssuumsous. use cases .m a H mm“ asses: muss .u—u.s_ .Amo.ovs. assessesu use mtouuop aspeumewssm usutueu_u sue: a x: s_ss_3 to a x: cases: assess sm>ps o to» «sum: .mpsspse s— as msoos use mus_e> .Uosm so some?» use mosses cos ass: to zessz es o_ses use es_eu.us~_p.us no unspeuu m. sua>psuo oss~so so o_ss mess -.s ems.e aae.e .hs.e ss.s ass.~ ea-.~ aem.~ assess o_.as as.s aoe.s are." amm.s ~e.s ase.s a~m.s arm.s oaaoosoesa u.ou sauce mama.» umuspuo szstm so.s ass.s a~s.u aus.a n~.s ass.. aae~.h aes.s assess o__as ec.s ass.s aea.e ees.s ~o.s ae_.s as~.s eh~.s ohaooeoesa upon saute sashes . omss_uu uu_s: ss.s ahe.ss ess.s~ asm.~s sn.s can.e ems.o ash.s accuses os_az s..s os~.u aes.m ae~.s ee.s can." aaa.~ ass." sohaooeoesm usuu saute cusps an ass as s on use as s assess osaaae .usouuspu sasuuopc .osuues_u sadness: emu: c goo; a .moEANsu usswassp— so _utmuue.o assasmsuu es suuecm .s m_so» Figure 4. 36 LIVER 6000 4000 2000 AWAT a a 1920 4 1440 960 nMOL 3H TO FATTY ACIDS/TlSSUE/MIN 0 0.15 1.5 CIMATEROL lNJECTED-MG Acute effects of cimaterol on fatty acid synthesis. Effects of a single injection of cimaterol on in vivc rates of fatty acid synthesis in rats fed a high te diet. Rates of fatty acid synthesis were measured 15 to 30 minutes after intraperitoneal injection of cinaterol at 0900 h. AWAT‘ indicates abdcminal white adipose tissue and IBAT indicates interscapular brown adipose tissue. Values are means 1 sea of 10 animals. Different letters above the bars indicates signifiant differences among groups (P<0.05) . Mean body weight, 15311 9. Tissue weights; liver weight, 6.7910.2 9; white adipose tissue weight, 3.1810.24 g; and brown adipose tissue weight, 0.3210.0Zg. 37 meal-fed rats (Table 2) than in ad libitum-fed rats (Figure 3). Even under these conditions of elevated rates of fatty acid synthesis; chronic administration of cimaterol failed to affect fatty acid synthesis in liver, white adipose tissue and brown adipose tissue (Table 2) . Rates of fatty acid synthesis expressed per gram white adipose tissue were elevated in rats fed cimaterol because of the lower tissue weights (data not presented). In agreement with the results of in vivo experinents, cimaterol failed to depress in vitro rates of fatty acid synthesis in liver or .white adipose tissue (Figure 5) . Rates of fatty acid synthesis were depressedbycimaterol inwhite adiposetissue incubated innedia containing a high concentration (100 nU/ml) of insulin (Table 3). Cimaterol inhibited the in vitro rates of fatty acid synthesis in brownadiposetissue (FigureSandTable 3) aswasalsoobservedafter aorta injection of cimaterol (Figure 4). Experiment 3 . Corsmption of high-fat diets depress de novo rates of fatty acid synthesis (69) . To provide further evidence that effects of cimaterol 0n adipose tissue and nuscle accretion do not require modulation of de novo fatty acid synthesis, rats were fed a high fat-diet containing cimaterol . Consmption of cimaterol increased body weight gain (20%) and hindlimb nnscle gain (66%) , and reduced gain in white adipose tissue (66%) (Figure 6). Energy density of the weight gain, an indicator of relative proportions of fat and protein gain, was approximately 32% lower in rats fed 100 ppn cinaterol. These results paralleled those obtained when the high-carbohydrate diet was fed (Figure 1) and support the suggestion 38 Table 2. Rates of fatty acid synthesis in meal-fed rats E. ! . ! J 0 10 100 SE Liver 8387 7680 6745 406 . i Abdominal white adipose tissue 4825 4223 3374 357 Interscapular brown adipose. tissue 636 522 397 53 W Nananoles tritium incorporated into fatty acids per tissueperminute. Valuesaremearsof 10 animals. Noneof the treatment effects were significant (p<0.05) . Initial body weight averaged 141 1 Zg. Food was available only between 0900-1000 11 8.111 between 1600-1700 h daily. After 1 wk rats were injected intraperitoneally with 1 mi of 31120 at 1015 h. AfteramtherlSmimtesratswerekilledtoneasurerates of fatty acid synthesis. Liver and brown adipose tissue weights were unaffected by treatment and averaged 5.39 1 0.13 g and 0.30 1 0.02 g respectively. Mean weights for white adipose tissle: control, 1.17 1 0.11 g; 10 ppn, 0.94 1 0.14 g: and 100 ppm, 0.58 1 0.06 g. Figure 5. 39 4000‘ 3000 2000 1000 0 1nM 1uM 1mM CIMATEROL nMOL 3H To FA/100MG/2H Rates of fatty acid synthesis in vitro. Each point is the nean1smof120bservatims; tissuesfraneadlof 12 rats were divided into 4 pieces and ireubated at each of the four concentrations of cimaterol. Mean body weight of rats was 211129. letter a indicates that the value is significantly lower than the control value (P<0.05) . 40 Table 3. In vitro rates of fatty acid synthesis in the presence of l0;!!' J'ZJ l:;!!' I.” mum of cinatero1 0 10'314 0 10'311 SE Liver 2249 2319 2355 2611 0.10 Abdominal white adipose tissue 2698 2801 4796b 33973 0.17 Interscapular brown adipose tissue 4465 1729a 3551b 3700allD 0.24 Nananoles tritium incorporated into fatty acids per 100 mg per 2 h. Fachvalueistheneanoflz observations; tissues franeachof 12 rats were incubated at each of the four concentrations of insulin and cimaterol. Mean body weights of rats was 21112 g. Superscript a indicates significant effect of drug within one concentration of insulin (P<0.05). superscript b indicates significant effect of concentration of insulin within control or cimaterol treated tissue (P<0.05) . Figure 6. 41 BODY WEIGHT HINDLIMB (5 ’ b 1 ’MUSCLE b ('9 e ' s " < 3 o 1... '5 :1: 0 2 a mu 3 3 <5 ’ ENERGY . a DENSITY) (5 z 3 1 13.0 \ 2 < b 1 -z- 0 G < l- 12.0 (5 IE I: -' 0 9 1.0 5 Eu- “ 3 3 X (0 0 100 0 100 ClMATEROL-PPM Body cmposition in rats fed a high-fat diet. Gain in bodyweight, tissueweightandenergydensity inrats fed a high-fat diet containing, 0 or 100 ppn cinaterol ad libitmn for 4 wks. MT indicates abdaninal white adipose tissue. Values are neans 1 SEM for 10 rats. Different letters above the bars irrlicate significant differences between the groups (P<0.05). Initial mean body weight averaged 16914 g; nuscle weight, 7.9810.13 g; AWAT weight, 2.210.17 g; and energy density, 1.7810.27 kcal/g body weight. 42 that cineterol does not influence body cmpcsition via alterations in fatty acid synthesis. Ebtperinent4. Sinceuptakeof fattyacidsbyadiposetissueand skeletal nuscle frcn circulating triglycerides is facilitated by lipcprotein lipase (28) . mdulatim of lipoprotein lipase activity by cimterolasapotentialnedanismto influencebodycmpositimwas mined. Chra'dc consmptim of cimaterol (4 wks) failed to influa'ice lipoprotein lipase activity inwhiteadiposetissuewhenthe resultswereexpressedpertotal tissueweight (Figure 7): Lipoproteinlipaseactivitypergwhiteadipcsetissuewaselevatedin rats fed cimterol became tissue weights were dqaressed. Chronic cmsunption of cimaterol failed to influence lipoprotein lipase activitiesinbrwnadiposetissleregardlessofnethodof expressim. As expected, lipoprotein lipase activity per gram miscle waslowerinmLthaninsoleuswithaninternediateactivityintotal hirfllinb Insole. These activities are casistent with their white, red ard mixed fiber types, respectively. Lipoprotein lipase activities, expressedpertissue, inbothEDLandintctalhindlinb mascleswereelevatedby67%wtenthedietcmtainirg100mn cimaterol was casmned. This elevation in activity resulted frun increased weights of these mlscles after consuming cimaterol for 4 wks coupled with a trend for stinulation of activity per gram nuscle. Lipoprotein lipase activity in soleus nuscle was not significantly affected by cimaterol (Figure 7). Effects of a single intraperitoneal injection of cimaterol on activities of lipcprotein lipase are shown in Table 4. Lipoprotein lipase activity in white adipose tissue was unaffected by cimaterol Figure 7. 25[ Q . :1: ........ i +55 I 4 so 22 _ u 4 ‘5 18: a.b ‘ 40 ’2‘ A H; Aw"; 35 l HEAT . . 4 E v 130 ~ ' « 70 :r . I:——-¥"‘I , g \ 120 ~ 4 co m g b I + 0) a ‘ 1° : E- ....... ! ...... . : 50 F I“ f ,. . 1 A \ ‘2 a In 3 s EDL . g _' ' b '4 .44 < m 3.0 b ...,J: 1‘ 38 Ill E 2.5 " - ' .l m 2.0 1 ~32 g D ‘ .26 6 1.5 '7 i f 1' m < 9 0 t 0.0 i .56 < '— o.2 « .52 >. E 5.8 1 .48 t -l 5" sorsus 1 °“ :5 O 9 ‘ ‘ s, ‘ fl 5 b 6' am 4 a 3-5; u, ......»I 3 19.5 a 2.5 _ {I :17.2 .o’ « 14.0 ' 9F! + "5517’? 11’1 2.4 o tofaoo ClMATEROL-PPM Effects of cimaterol on lipoprotein lipase activity. Rats werefedahigh tedietoontaining 0, 10 or 100 ppn cimaterol ad libitum for 4 wks. Values are expressed pergtissueandpertissue. mindicatesabdaninal white adipose tissue; IBAT, irrterscapular brown adipose tissue; EDL, extensor digitonm longus. Values are means of i SEM of 10 rats. Different letters by points on the lines indicate significant differences among the groups (P<0.05) . Initial mean body weight 131: g. Rats were killed at 1100 h to measure the lipoprotein lipase activity. 44 Table 4. Acute effects of cimaterol on lipoprctein lipase activity. :. ! J . . ! 1 0 0.15 1.5 SE Abdaninal white adipose tissue 31.23 32.95“ 33.93 2.9 Interscapilar brown adipose tissue 17.2a 25.2b 27.213 0.7 mtensor Digitorum longus muscle 0.09a 0.13ah 0.16b 0.01 80le muscle 0.363 0.343 0.333 0.02 Totalhindlinbmmcle 14.57a 14.32a 17.783 0.88 Micranoles of free fatty acids released per tissue per h. Means with different superscript letters are significantly different (P<0.05) . Values are means of 8-10 rats per group. Rats, fed the high-carbohydrate control diet for 1 wk, were injected intraperitoneally with cimaterol at 0700 h and killed after 4 h. Mean body weight, 175 i 1 9; white adipose tissue weight, 1.9 1- 0.1 g; brown adipose tissue weight, 265 1- 31 mg; extemor digitorum longus weight, 152 1- 4 ng; soleus weight, 69 1 1 mg; and total hindlinb nuscle weight, 7.8 1- 0.1 g. 45 whereas brcwn adipose tissue lipcprctein lipase activity was elevated by 60% in rats injected with 1.5 ng cimatercl. A marked increase (75%) in lipoprotein lipase activity was also observed in EDL unscle, but not in soleus, in rewonse to injection of 1.5 m; of cimaterol. There was a trend for an increase (22%) in lipoprotein lipase activity in total hindlinb nuscle of rats injected with cineterol, although the ixrzrease was not statistically significant. To determine if the elevation in lipoprotein lipase activity in skeletal nuscle observed after administration of cimaterol to rats was acorsewenoeofdirectactimofcimaterclcnskeletalniscle, enzyme activity was measured in EDI. (white) arri soleus (red) unscles incabated in the absence or presence of cimtercl. Lipqarctein lipase activityinuiecartrclmiscleswasessentiallymdiangedduringfliez h imitation (Figure 8). Release of lipoprotein lipase activity into the immationmediawasstimilatedbycimaterol. InEDL, 1m cimaterol elevated enzyme activity by 66% (tissue ard media carbined) (Figure 8). Althaigh there was a trend for stimulation of lipoprotein lipase activity in soleus unscle in response to cimaterol , activity was not significantly elevated. Dcperimerrt 5. Lipolysis, estimted in vivc by the concentration of free fatty acids in plasma, was stinnlated after assuming cimaterol for 4 wks ixdicating a persistent lipolytic activity of cimaterol (Figure 9) . Both plasna free fatty acids and glycerol cacentrations were elevated following a single intraperitoneal injection of cimtercl (Figure 10). Increases in plasma free fatty acid and glycerol concentrations were noted within 15 minutes of injectim, and tended to be even higher after 30 minutes. Lipolysis Figure 8. 46 .EDL 1 5 ,DMEDIA ' “TISSUE ) i b P > JIMOL FATTY ACIDS RELEASED/G/H CIMATEROL Lipoprotein lipase acivity in vitro in nuscle. Each bar isthemeanismolerats. muscles fruncnehindlinb were incubated for 2 hrs at 37°C in absence of cimaterol andnuscles frmtheotherhinlinb immatedwithlufl cimaterol. Body weight of the rats that had been fed a high-carbohydrate diet for 1 wk ranged fran 80 to 100 g. EDLancleweight ranged franBOto loongand soleus unscle weight ranged frun 30 to 40 my. Lipoprotein lipase activity of repraentative nuscles prior to incubation 0.79:0.09 for EDL nuscle and 8.0 10.3 uM free fatty acids released per gram per hour for soleus nnscle. Different letters above the bars indicates significant differences between gruips (P<0.05) . PLASMA GLYCEROL-uM PLASMA GLUCOSE- mM Figure 9. 47 E 144 500 f‘ < 108 ‘°° & 72 300 g 200 U) 36 < 100 _| O O. E a 2.4 IE a a E 1.6 '— U 8.0 S < E 0 10 100 o 10 100 ‘2 ..l ClMATEROL-PPM 11. Effects of cimaterol on plasma metabolites. Plasma metabolites in rats fed ad libitum for 4 wks a high-arbdiydrate diet containing 0, 10 or 100 mu ciuatercl. Values are meats 1- SEM of 10 rats. Different letters above the bars indicates significant differences among the groups. Initial body weight 13111.0 9. 48 800 c 1.5 c E I .7 MG ‘ ’ b 7:53 ‘ 90° (1600’ b.-°'.b15‘* IE ...... ”15‘ 2 S I I; ...... 1 inc. 4 T---"'7'MG < 2 ( m ‘00[ (2 k "I U ' a ‘ fl. 1 a O .1 H. >' I L’JMG ‘300 a LI. 6' 200 r a no 1 ’ ‘V’MG I i o r r A a E " L ‘ ’ "Gerry“: ‘ E .L I (I1: ,0, , I b 6 ('1' 5,0) ' T 1% 1- 9 4 < 58 a ' ‘ 5 r- 0'3 8’ * ’ L‘JMOG ‘ “'2 o I 1 2 J 7 > . 6‘k - - J" 1 - ‘ 0 15 30 15 30 MINUTES AFTER CIMATEROL INJECTION Figure 10. Acme effects of cimaterol on plasna metabolites. Effects of a single injection of cimaterol (O, 0.15 or 1.5 mg) on plasma metabolites in rats fed a high-carbohydrate diet. Values are meals 1 SEN of 10 animals. Different letters above the points irdicates the significant diffrences among the groups either at 15 or 30 urinates after injections (P<0.05). Mean body weight 187112 9. 49 inwhiteadiposetissue, asmeasured invitrobyreleaseof free fatty acid and glycerol, was stimlated by cimaterol in a dose dependent manner (Figure 11). Plasna lactate was increased following the injection of cimaterol (Figure 10) but was unaffected after caasuming cimaterol for 4 wks (Figure 9). Glucose cawoentratims in plasma were maffected by cimaterol (Figure 9 and 10). mm resultsofthepresentstxflydmxstratethatthebeta adrenergic agonist cimterol increase skeletal muscle accretion and conoanitantly dqaresses adipose tissue deposition in rats. These data parallelresporsesofseveralotherspeciestoselelectedbeta adrenergic agonists (9,10,31,36,60,95, 97). Effects of cimaterol on body cmposition were clearly evident within the first wk of achinistratim (Figure 1, Table 1), ashasalsobeenobservedinrats that recieved clenbuterol (96). Cinaterol exerted a less dramatic influernembodycmpositimbeycndthe firstwkcftreatment. Cimateroldepressedfatgainardacoeleratedproteingaininthese rats with no appreciable change in overall energy balance (Figure 2). Althcxghconstmptionofcimatercl increasedenergyintakeoftherats (FigureZ), thisincreaseinintakeappearedtobeanirdirect remorsetotheheavierbodyweightsbecauseenergyintakesofthe treament groups were not increased when expressed per gram body weight (datamtshown). Othershavereported increases, nochangeor decreasesinenergyintakedependingonthespecificagonistused (5,9,10,31,36,60,95,97,112) . Oorsiderim that beta-agonists activate brownadipose tissue thermogenesis in rats (5), andthat the energetic 50 14.0 ----- GLYCEROL a —— FFA 11.2 8.4 5.6 2.8 FFA-uMOL/G/H O 1nM 1uM 1111M CIMATEROL GLYCEROL-UMOL/G/H Figure 11. lates cf lipolysis in vitro in white adipose tissue. EadipointisthemeanisFMOflz observations. Adipose tissnfraneaduoleratswasdevidedintOIIpieoesand incubated at one of four concentrations. The letter a by point indicates that the value is significantly higher than the respective cmtrcl value (P<0.05). Mean body weight of rats 16532.0 9. 51 efficiency for deposition of protein (48%) is normally considered to benxzhlowerthanfordepositicnofthesameamomtofenergyas fat (77%) (94) , I have predicted that the efficiency of energy retention would be lower in rats fed cimtercl than in control rats. Higher dosesofcimaterolthanusedinthepresentstudywill activatebrown adiposetissiethermogensisandlowertheefficiencyofenergy retention in mice (unpublished). The pathways responsible for the observeddangesinbodyoarpositionappearedtobemoresensitiveto cimaterol than are the thermgenic patinays. my working hypothesis was that cinaterol would inhibit conversion ofdietarycarbdmydratestofattyacidsasonemechanismtodirect energyawayfrunadiposetiswetowardskeletalmscle. Resultsof thepresentsuriydonotsirportthishypothesis. Invivc ratesof fattyacid synthesis inliverandwhiteadiposetissue, assessedby using 31120, were unaffected by cimaterol, even in meal-fed rats wherehighratesofdemvofattyacidsynthesiswereobserved (Table 2) . Furthermore, activities of two adaptive lipogenic enzymes, fatty acid synthetase andmalic enzyme (Table 1), and invitro rates of fatty acid synthesis (Figure 5) were also unaffected by cimaterol, exoeptfordepressedfattyacidsynthesisinwhiteadiposetissuein the presence of high concentrations of irsulin (Table 3). I conclude thatthereductionsinfatoontentandtheincreases inprctein content in rats fed cimaterol are not caused by suppressed conversion of dietary carbohydrates to fatty acids. This conclusion is supported by the observation that cimaterol also decreased white adipose tissue gain and increased skeletal muscle gain in rats fed a high—fat diet where de novc fatty acid synthesis would be suppressed (69). 52 The failure of cimaterol to affect the rates of fatty acid synthesis in liver is probably related to the lack of mum beta-receptors in liver of adult rats (77). This is supported by the firdin; that cinaterol also failed to stimulate hepatic glycogenolysis, as indicated by the W plasma glucose concentrations in rats injected with cimaterol (Figure 10). Although catednlamirnshavebeenslmnmrieroertainoaditiastoinhibitthe actvitiy of acetyl coenzyme A car-boucylase arr! glucose conversion to fatty acids in the presence of high concentrations of insulin in white adipose tissue (20,66) , I found no evidence that cineterol inhibited fattyacidsynthesisinvivoinwhiteadiposetissnmderthe corditions where fat deposition was depressed. cimaterol increased concentrations of plasna lactate, a najor lipogenic substrate (26); this substrate may have permitted rats fed diets containing cimaterol tonaintainrates of fattyacid synthesis inwhite adiposetissue. mtes of fatty acid synthesis and lipolysis in adipose tissue are inverselyrelatedinrats fedahighcarbohydratemealorfasted (100). There are several possibilites to explain why fatty acid syntl'xesiswasnotdepressedaslipolysiswasstimlatedinrats fed cimaterol . Beta-receptor activation has recently been shown to stimlate by 5-10 fold the transport of fatty acids fran adipocytes (1) . Stimulated efflux of fatty acicb generated during lipolysis would limit aocmnlaticn and subsequent feedback inhibition of de novc fatty acid synthesis. Another possibility is that cimaterol selectively activated lipolysis without affecting enzymes involved in fatty acid synthesis. Data are available to show that W 53 activation of glycogen rhosphorylase and of hormone sensitive lipase can occur independently (57). ' De novo fatty acid synthesis in browm adipose tissue is thought to contribute minimally to body fattening, but rather to supply a readily utilizable fuel for brown adipose tissue thermogensis (106) . Rates of fatty acid synthesis in brown adipose tissue were inhibited by cimaterol in vitro and also after acute in vivo administration (Figure 4, 5) . This contrasts with findings in white adipose tissue arr! may be related to high concentrations of beta-receptors in brown adipose tissue (104). Fatty acid synthesis in brown adipose tissue became resistant to action of cimaterol within 1 wk, perhaps due to desensitization of the beta-receptors after chronic exposure to the agonist (45) . lipolysis was stimulated in vivo by cimaterol as evidenced by an increase in plasna free fatty acid concentration (Figure 9, 10). Plasma free fatty acid concentrations correlate well with their tumover rates and with body fat content (13) . Therefore, the elevated concentrations of free fatty acids observed in rats fed cimaterol, even after 4 wks when body fat gain was significantly reduced, is indicative of persistent lipolytic activity of cimaterol . The in vitro results indicate that cimaterol has direct lipolytic effects on adipose tissue in rats (Figure 11) . Stimulation of lipolysis in vitro by beta agonists such as cimaterol and clenbuterol nay, hmever, be species-dependent because Mersmann (80) has shown that Clenbuterol fails to stinulate lipolysis in vitro in pigs although it increases plasma glycerol and free fatty acid concentrations. 54 Lipoprotein lipase activity in white adipose tissue was unaffected by either acute or chrmic exposure to cimaterol (Table 4, Figure 7). This observation agrees with the conclusions of Hansson et al (51) and Chernick et a1 (23) that lipoprotein lipase activity in ratadiposetissueismilikelytoberegulatedbyadrenergic mechanisms although Ashby et al (7,8) reported inhibition of insulin stinnlated lipoprotein lipase activity in adipose tissue in W toepinerhrine. Wecmcimethatredmedadiposetissueg’ainobserved in cimaterol fed rats did not result fran ixhibition of lipoprotein lipase activity. Inoontrasttowhiteadiposetisaiewherelipoprotein lipase activity was unaffected, enzyme activity in skeletal mscle was stimlated after administration of cimaterol to rats (Table 4 , Figure 7). This increase inenzyneactivitywasalsodenmstrable invitro (Figure 8), indicating that cimaterol has direct actions on skeletal mscle. 'mencstprotnmcedeffectsofcimaterolwereobsemedinEDL where white fibers predaninate. Stimlation of lipoprotein lipase activityinmeycineterolmaybeanadaptiveresponsetoashift fran carbohydrate utilization to fat as a metabolic fuel for the tissue. Scleisnuscle, whichoontairsahighproportimcfred fibers, and utilizes mainly fat as an energy source (105), has higher lipoprotein lipaseactivitypergramtissuethanmL. ‘Ihismayhave oontributedtothelesserpercentageircreaseinsoleusmscle lipoprotein lipase activity than in EDL. Since EDL nuscle has a lower concentration of beta airenergic receptors, than does soleus (109) , it would appear that postreoeptor events mediated the selective 55 stimulation of lipoprotein lipase in EDL and the equal percentage increases in weight of the two muscles. A large elevation in activity of lipoprotein lipase was observed inbrownadiposetissuewithin4hafterinvivo injections of cineterol (Table 4). Similar responses have been shown in brown adipose tissue after injection of norepinephrine (21). But after chrmic exposure to cimaterol, lipoprotein lipase activity in brcwm adiposetissuewasnolongerelevated (Figure 7). This issimilarto therespaiseoffattyacidsynthesisinbrownadiposetissuewhereit wasinhibitedaflyafteracutebrtnotafterdmricadministratimof cineterol , again suggesting desensitization of the beta receptors afterdironicexpoairetotheagonist (45). In sunmary, cimaterol altered body ccrpositim by reducing fat gainaniinzreasingproteingainwithminineldangesinoverall energybalanoe. Reducedadiposetissuegaininrats fedcimaterol resulted frun increased mobilization of stored triglycerides by stimulation of lipolysis with no najor influence on either de novo fatty acid synthesis or the activity of lipoprotein lipase in white adiposetissue. mtheotherhand, ircreasedskeletalmusclegainwas associated with elevated lipoprotein lipase activity, indicating a preferential utilization of fatty acids as fuel in nuscle of rats treated with cimaterol. WOFCIMATEWL, ammcmrsr, mmmaomsqmms. mm Clenbuterol and cimaterol, beta adrenergic agonists, increase skeletal miscle accretion in rats (36,76,95) and other species (9,l0,ll,3l,60,97) . The mechanisms) responsible for these beta agonist-irrlucedincreasesinskeletalmusclemasshavenotyetbeen clarified. cimaterol stimulates lipoprotein lipase activity in rat skeletal muscle (Figures 7,8 arri Table 4) . This would facilitate availability of energy for increased miscle metabolism, but would not directly stimilate muscle accreticn. There is sane evidence that catecholamines slow skeletal muscle protein turnover. Epinephrine or the beta-adrenergic agonist isoproterenol decreases release of alanine and glutamine fran isolated rat epitroohlaris muscle preparations dringthircubationperiod, andreleaseofphenylalaninefrmrat henioorpis preparations during a 3 h perfusion (42,71). These effects in the epiu'odalaris preparation were blocked by the beta-antagonist propranolol (42) . m et a1 (95) concluded that the beta-agonist clenhrterolprobablyalsodecreasesskeletalmiscleprcteinumiover in rats: a occlusion based on the daservatim that addition of clerhxteroltothedietforlldaysstimilated fractional accretion rates of skeletal miscle without detectable increases in fractional synthesis ratescfmiscleprcteins. 'artothershavereportedthat clerhrterol increases by 34% fractional rates of phenylalanine moorporatimintomiuaedskeletalmiscleproteirslhafterthethe seventh daily injection (36) , ard that isoproterenol stixmlates incorporation of CIA-labeled amino acids into tibialis miscle protein 2-3 h after the fifth daily injection (33) . Thus, effects of beta-agonists on protein metabolisn renain unclear. The timing of the 56 57 measurenent relative to administration of the beta adrenergic agonist appears inportant. Stimulation of skeletal muscle accretion by beta-agonists in rats ismostpranmoedduringthefirstwkofadministratim (Figure land ref 95) . Therefore, protein metabolisn likely charges with time after administration of beta-agonists. The present study was, therefore, designed to evaluate the tarporal relationships between ministration of a beta-agonist, cinaterol, protein accretion, and protein ulrnover in rats. Urinary accretion of 3-methylhistidine was selected as the indicator of protein turnover, or more specifically as an irflex of bn'nover of 3-methylhistidine containing proteirs (114) . 3-Methylhistidine, found exclusively in actin arr! myosin, is guantitativelyexcretedinurineofrats, ardassuchhasbeentsedas anindicatorof skeletalmlscleproteinuirnover (becausemorethan 90% of total body 3-methylhistidine is in skeletal miscle) (114). Although the non-skeletal miscle pool of 3-nethylhistidine containing proteirsissmall, thispoolturnsoverfasterthantheskeletal muscle pool, and thus provide a disprrportionately large fraction of urinary excretim of 3-methylhistidine (82). Corsequently, urinary excretion of 3-methylhistidine best provides an index of turnover of total body 3-methylhistidine containing proteins, not exclusively thoseinskeletalmuscle. I, therefore, measuredthetotalbodypool size of 3-uethylhistidine containing proteirs and urinary excretzcn of 3—methylhistidine at selected times to calculate effects of cimaterol on turnover of total body 3-methylhistidine containing proteins. Totalbodypr'otein, hindlinbnuscleprotein, Mammaoontent, and 58 concentrations of plasma amino acids, insulin, triiodothyrcnine and corticosterone were also measured. W W. Female wrague-Dawley rats (100-110 g) , obtained fron Harlan Industries, Indianapolis, IN, were housed individually at 23°C in metal cages with wire-mesh floors. Roan lights were on fran 0700 to 1900 h. All animals were provided a nonpurified diet (Wayne RodentBlox, ContinentalGrainCmpany, Chicago, IL) andwaterad libitum for the first 2 days after arrival in the laboratory. Rats were then fed a purified high-carbohydrate diet for 6 days and then " divided into experimental groups. The high-carbohydrate diet cartained (in g/100 g): 66.0 g glucose, 5.0 9 corn oil, 20.0 g casein, 0.3 g nethimine, 1.0 g vitamin mix (14), 0.2 g dialine chloride, 3.5 g mineral mix (14) and 4.0 g cellulose. This diet provided 3.57 kcal metabolizable energy/g with 67% of metabolizable energy as carbohyrate, 13% as fat and 20% as protein. The amount of cineterol (CL 263,780; anthranilonitrile, 5-[1-hydro0cy-2-(isopropylamim)ethyl]-) added to these diets is irdicated in the experimental design. W. Ratsweredividedinto4gruups. Groupl (10 rats) was killed at the beginning of the experiment to obtain initial bodycatpositicnvalues; groupsz, 3, and4 (30 ratspergrcup) were fed for 7, 14, or 29 days the high-carbohydrate diet containing 0, 10 or 100 ppm cimaterol, respectively, and then killed. Blood was collected and plasma was stored at -70°C for subsequent measuranent of amino acids and hormones. Hindlinbs were separated and quickly frozen in dry ice and acetone. Carcasses were frozen for subsequent 59 analysis. Food intake and weight gains were recorded daily during the first wk, and twice weekly during the following 3 wks. Urine was collected daily with a funnel below each cage into the bottles containing6NHCLaspreservativeduringthe firstwk, andfortwo days each wk during the following 3 wks. At the end of each 24 h collection, urine was filtered and stored at -20°C for subsequent 3-methylhistidine analyses. To determine acute effects of cimaterol on plasma amino acids and hormones, rats fed the high-carbohydrate control diet for 1 wk were injected intraperitoneally with saline, 0.15 or 1.5 mg cimaterol in 0.2 ml saline at 0700 h and killed at 1100 h. These doses correspond to the daily amount of cimaterol consumed by rats fed diets containing 0, 10, or 100 ppm cimaterol. Plasma, separated fran the blood, was stored at -700C for subsequent amino acid and hormone analyses. M. Weights of the carcass (skeletal muscle and associated bone afterrencvirgthedissectableadiposetissue) andcftherestofthe body (head, skin, tail, removable adipose tissue and viscera) were recorded. Abdaninal white adipose tissue (total dissectable adipose tissue from the abdcnen) and right hindliunb muscle weights were also recorded separately. Hindlimb muscle weights were determined after beingstripped fronthebonesandseparated fromadiposetissue. 'nuesetissueswereretumedtothecarcassesortherostofthebody, except for aliquots of hcrnogenized hindlinb muscle used for measuring protein, RNA and 111A. Carcassesandtherestofthebody (afterremovalofresidues frcm intestines) were harcgenized in eight volumes of cold distilled 60 water. Aliquots of these hamgenates were used to determine protein and B-methylhistidine content. Protein cmtentwasdeterminedbytheprocedure of Marlcoell (75), after the hancgenates had been dissolved in 1 N NacH at 100°C. RNA was determined by a modified Schmidt-Ehannhauser method as described byhmro&?1eck(1969),arrilNAbythenethcdofnurtonua)as I“ modified by Richards (95) . To analyse urinary 3-nethylhistidine, urine was hydrolyzed in 6 N HCL for 2 h at 110°C. HCL was removed by rotary evaporation, and the hydrolysate was dissolved in 0.2 M pyridine. Initial separation u of 3-nethy1histidine was by the method of Haverberg et al (52) using pyridine elution chrunatography with Dowex 50-X8 mesh columns. The fraction containing 3-methylhistidine was collected in 1 M pyridine, dried by rotary evaporation, redissolved in distilled water and anlyzed by the nethod of Ward (108) using EPIC and UV detection. The eluent was water-methanol (60:40) at a flow rate 1.5 m1.min"1. Chrcmatographic separations were performed using a EPIC column (100 x 4.6 m I.D) packed with uBondapack C18, particle size 5 um. Retention time for the 3-nethylhistidine peak averaged 7 min. Peak heights of the samples were recorded, and the amount of 3-nethylhistidine in samples was calculated based on the peak height of standard 3-methylhistidine. Triplicate stardard 3-methylhistidine solutions were also subjected to all the steps (hydrolysis, ion-eucdange columns and HPLC) and used to calculate recovery of 3-methy1histidine, which under these conditions averaged 75% . For the measurement of body pools of 3-nethylhistidine, aliquots ofhamgenatesfruncarcassandtherestofthebodyweretreatedwith 61 trichloroacetic acid to achieve a final concentration of 10%. The mixture was held at 4°C for 10 minutes to allow precipitation of proteins. This preparation was then centrifuged (1100 g) , and the precipitated proteins were washed twice with ice-cold 10% trichloroaceticacid. Tb extract lipids, the protein precipitate was washed twice with ethanol:ether (1:1) followed by ether (84). The H“ residue was then dried to constant weight. Hydrolysis of the residue I was carried out in 6 N HCL (10 ng/ml) for 20 h at 110°C in sealed angules. Samples were then processed as described for urine for measurement of 3—methy1histidine. Plasma amino acid concentrations were determined after derivatization with phenylisothiocyanate to produce menylthiocerbamyl amino acids. These amino acid derivatives were then analyzed by HPIC with a C18 reverse phase column (Waters PIGDJI'PG system, Waters division of Millipore, Milford, MA 01757). Plasma insulin and corticosterone were determined by standard radioiunmunoassay procedures (Novo laboratory, W and Endocrine Sciences, Tarzana, CA, respectively) . Plasma triiodothyronine was measured by the method of Nejad et al (89) . glglations. Cumulative gains in tissue weights, protein, RNA, [NA and 3-nethylhistidine content were calculated frun observed values at the end of the experimental periods minus predicted values at the beginning of the experimental periods. Values at the beginning of an experinental period were predicted frcn ratio of tissue weights, protein, RNA, [NA or 3-methylhistidine content to body weights, or hiurilimb weights, in rats killed at the start of each treatment period. Gains during wk 2, and wks 3 and 4 were calculated from 62 cbservedvaluesattheendofeuquerinentalperiodnfimsthepredicted initialvaluesfrunthecorrespordingtreatmentgrcupsatthe begimingofwkZandwk3. Thatis,valuesobtainedfrunratsfed dietsccntaining 0, 10or100ppncinaterol andkilled attheendof wklaniwkaereutilizedtopredicttheinititalvaluesforeadu respectivegmlp. Fractional accretim rates of 3-nethylhistidine containing proteinswerecalcflatedfrunthetotalbodygainina-nethylhistidim perdaydividedbytheaverageB-methyll'iistidinepool sizeduringthat period,andecpressedasapercentage. I-‘ractionalaccretionratesof 3-methylhistidine were assumed to equal fractional accretion rates of 3-methylhistidine containing protein's because the concentration of 3-uethylhistidineinproteinwasunaffectedbythetreatment. Practical degradation rates of total body 3-methylhistidine cautainingproteinswerecalculatedfrcntheurinaryeuacretimof 3-methylhistidine per day divided by the average 3-methylhistidine pool size durirg that period, andexpressedas apercentage. itactimalsynthesisrateswerecbtainedbytheadditimoffracticral accretim and degradation rates (FAR= FSR-FBR) . Data were analysed statistically using analysis of variance, and theBmferclmittestforposthoctreatmentcmparisonsMM. m Rats fed diets containing 100 ppn cimaterol for 1 day consumed lesserergythancmtrolrats,nutthenincreasedtheirerergyintake tolevelsabcvethoseofcontrolratsbyday3(Figure12). This elevated energy intake was closely associated with increased body weight, which was also detectable by day 3. The major weight gain 63 .1 < o suencv mun: HCONTROL X I ----- 010 ppm CIMATEROL | v-«IOO ppm CIMATEROL g 70 P I 4 7o ( + + + + + ‘,J 60 D ,x““'*.‘ + + ".A..‘ T ##1: ..... 0 . .0 II:- ‘fll chm-co... .‘“‘"'" N‘a. iljffltia """" . - so P .- .Q‘w ...... .0. ccccc ‘ so 3 ‘0 " a", 4‘0 l E 30 > 1. . ‘ CONTROL v0 10ppm . 30 z 4‘ + comnor 1!. 41000011111 “" 1 2 a 4 5 0 7 " 10 14 11 21 24 20 0 23° . 0001! 111510111 + . ‘ 23° ’ + . .-.A ' + e ...-A """" ... '— 210r + 3 ....... r“ 1210 I + "._.-—'..-“,,. 00W.” (5 100 » -' 1100 l; 170 ~ 1170 > 150 . ‘150 8 ‘3" ' t common. 1:: 10ppm ‘ ‘30 m 1* cournor vs 100 ppm : 1234501W1014172124 20 DAYS Figure 12. Energy intake and body weights in rats fed cimaterol. Rats were fed ad libitum for 4 wks a high-carbohydrate diet containing 0, 10, or 100 ppm cimaterol. Each point isthemeaniSEMof 10rats. Asterisksand+abovea point indicates that values for rats fed diets containing 10 and 100 ppn cimaterol, respectively, are significantly different frcn the control value (P<0.05) .- 64 reqacnseocczrredduringthefirS‘tZwkswhenratsfeddiets W10 or 100ppncimdterolgained45ard80%mrethanccntrol rats (Figure 13). Afterbeing fed dietscontainingcimaterol for2 wks, rats mlongergainedbodyweight at an accelerated rate. Consmpticn of cimaterol stimulated weight gain of carcass, which wasprinarilyskeletalmuscleandassociatedbone,atanevenfaster ratethantctalbodyweightgain(Figure13). RatsfeleanleOppn cimaterolgained45%and65%mrecaroassweightthancontrol rats duringthe4wkexperinentwiththegreatestacceleratimingain evidentwithinthefirstwk(80%and131%). Gaininhinilimbmuscle followedasimilar pattern (Figure 13). Gaininweight oftherestof thebody(allcmpa'entsofthebcdyexcluiingcarcass)msmaffected bycinaterol,ccnfirmingthatmcstofthebcdyweightgainwas associated with increased skeletal nuscle accretion. A dose-dependent reductimingaininabdanimlwhiteadipcsetissueweightwas observedinrats fed dietscontainingcimaterol (Figure 13). Unlike theincreasedgainincarcassvheremajordaangesmreobservedmly withinthefirstwk,gaininabdaninalvmiteadipcsetissuewas significantly reduced in response to cimaterol beyond the first wk. Inagreauentwiththeobserveddiangesintissuegairs,gainin proteininratsfelearrilOOppncimaterolwasalsomarkedly elevated in total body (HQ-212%), carcass (131-236%) and hindlinb muscles (61-13015) within the firstwk (Figure 14). Effects of cimaterolmproteingaininthecnrcassweremininalbeyonithefirst wk. Proteingain inthe rest of thebody (ROB) was not significantly increased during the 4 wk experimental period (Figure 14). Figure 13 . 65 BODY WLIOHT WEIGHT GAIN - G CIMATEROL - ppm Effects of cimaterol on body weights and tissue weights. Ratswerefedadlibitumforl, 20r4wksa high—carbd’uydrate diet containing 0, 10 or 100 ppm cimaterol. The lower two segments of the bars represent gain'perwkforeach ofthe firsttwowks. Theurper segnentrepresentsgainforthelastZwksoft'he4wk study. Carcass indicates total skeletal muscle and the associated bone: hindlimb, muscle stripped from one hindlinb; ROB, mtofthebodyexclwiu'ngcaroass; and AWAT‘, abdaninalwhiteadipose tissue. Eachpointisthe mean 1- sum of 10 rats. Bars within a panel with different letters are significantly different (P<0.05) . Asterisks within bars indicate that the value is significantly different from the ccrrapcnding control value within that wk (P<0.05). Initial body weight, 13312 g: carcass weight, 6111 g; hindlimb muscle, 5.15:0.1 9; rest of the body, 6111 g and abdominal white adipose tissue, 0.87:0.08 g. Gains in tissue weights were calculated fmn the final valus minus the predicted initial valua based on initial body weight of each rat. Figure 14 . PROTEIN GAIN - G 66 WHOLE BODY ' CARCASS b b 12’ y y 10.0 9 4 / 17.5 p 4 6’ Q g 5.0 3 g E 2.5 HINDLIMB b ROB 1-3’ . z 0.8 a Z 0.6 § .’ / __ 0.41 .1 g 0.2 s g ' 1 100 ' 0 10 100 CIMATEROL - ppm Gain in protein in rats fed cimaterol. Gain in protein contentoftctalbcdy, carcass, hindlimbunuscleandrest of the body (ROB) of rats fed ad libitum for 1, 2 or 4 wks a high-carbohydrate diet containing 0, 10, or 100 ppm cimaterol. The lower two segments of eadu bar represents gainperwk, ardtheuppersegmentrepresentsgainfor thelast2wksofthe4wkstudy. Eachbaristhemeani- SEN of 10 rats. Bars within a panel with different letters are significantly different (P<0. 05). Asterisks within a bar indicate that the value is significantly different fran the corresponding control value within that period (P<0. 05). Initial total body protein, 15.96-50.7 g; carcass protein, 8.02:0.5 g: hindlimb muscle protein, 0.79:0.09 g; and rest of the body protein, 7.9-_+0.0.4 g. Gain in protein was calculated as final bodyortissueproteinminusthepredicted initialbody or tissue protein. 67 There was a dramatic increase in RNA gain (84-200%) in hindlimb muscle evident within first wk in response to 10 and 100 ppm cimaterol (Figure 15) . Effects of cimaterol on RNA gain were less prcnounced after2and4wks. Maccmulationduringthefirstwkdidnot respondtocineterol, hrtdidincreaseafter2t04wks. Asaresult oftheduangesinRNA, Matrimscleweightmuringthefirstwk, the cam'ttrationofRNAperghinilinbmusclewaszoithigher, andthatof M111: lower, inrats fedthedietcontaining100ppncimaterolthan in control rats. Effects were minimized after 2 or 4 wks (Figure 15). 'nueamuntof3-uethylhistidineinthewholebodyofrats fed cineterolincreasedinproportimtotteircreaseinprotein accumulation (Figure 16) . More than 90% of the 3-methy1histidine presentinthebcdywasinthecarmss, withnosignificantchangein proportion with consmption of cimaterol (Figure 16) . Concentrations of3-nethylhistidineincarcassandrestofthe bodyaveraged, 3.251- 0.11 and 0.540 1 0.03 umoles per g protein, respectively, and were unaffectedbycimaterol. Fractional accretion rates of 3-methylhistidine containing proteirsincreased40-120tinrespmsetocineterolcuringwk1 (Figure17), inagreeuentwiththeincreaseinproteingainduring this period (Figure 14). No major treatment-induced changes in fractional accreticnrateswereevidentbeyordwdrl. Ebtcreticnof 3-uethy1histidineinurirewasselectedasan iuriicetor of the rates of degradation of 3-uethy1histidine containing proteins (82,114). Urinary excretion of 3-methylhistidine, per 100 g body weight, was lowered 33% the first day rats consumed diets containing cimaterol (Figure 18) . This effect of cimaterol persisted Figure 15 . CUMULATIVE GAIN - mg CONCENTRATION 1- mg/g HINDLIMB WEEK 1 WEEK 2 WEEK 4 RNA c RNA RNA 10 ' 1 I 0 ' b 7 1 b 1 b . b 6’ 4 p b ‘ > . 4 ‘ 4 r a ..n «n .n 1 DNA DNA DNA b b b 200 ' 1 r .b J ‘.5 p . 1 . b . . C 100 b ‘ P 0.5 * fl 1 1 1 NA RNA ‘ ‘ 2.5111111 a r: a . 4 I 2.0 ~ ' - g a a 1 r 1 1.5 r 1 1 r . . . 1.0 t 1 I n 0.5 P > 4 b 4 DNA thA . DNA OoGOI g b b .- fl . 1 p 4 Oo‘BI 1 4 * ‘ b .b I 0.361 1 1 r 1 0.24 ' 1 . , 0.12 ’ . 0 10100 0 10 100 70110100 ”baud CIMATEROL - ppm Effects of cimaterol mm RNA and [NA content, andm andum misconcentrations, in hindliuxb muscle of rats fed ad libitum for 1, 2 or 4 wks a high-carbohydrate diet containing 0,10 or 100 ppm cinetero1.Fad1baristhenean¢SEMcf10rats.Bars Marti 2.0 11.5 11.0 10.5 1 2.5 2.0 ‘ 1.5 1 1.0 0.5 0.60 0.48 0.35 1 0.24 1 0.12 Cumulative gain in within a panel with differrent letters are significantly different (p<0.05) . Initial hindlinb INA content, 10.21-0.5 mg: INA content, 2.8¢O.l my; RNA concentration, 2.01-0.1 lug/g: and mm ccrcentration, 0.56 30.02 ng/g hirriliutbmuscle. GaininRNAandmlAcontentswere calculated fran the final values minus the predicted initial values based m initial body weight and hindlinb muscle weight of each rat. Figure 16. 69 wquCARCAss 8 50) .noeb ‘ '5 401 a ab F2 1 E 30. . a 201 * . I- 10» 1 Z t‘.’ WKZ Z . b O 50 ab FE 1 U 40' a i w 30.. * 4 _2_ 20» 1 Q 10- 1 '0'. — b I .WK4 _' 60 ab Q 1 >- 48- a 1 E 36» * I.” 24L 1'? 121 1 (‘9 ,, 0 10100 CIMATEROL 1- PPM 3-Methy1histidine content in the body of rats fed cimaterol. 3-mthylhistidine content of total body, carcassardrestofthebodyofrats fedadlibituumfor 1, 20r4wksahigh-carbchydratedietccntainingo, 10, or 100 ppn cineterol. Total heigut of each bar indicates the amount of 3-1methylhistidu'ne present in the whole body. Two segments of eadu bar represent 3-methylhistidinecontentofcarcassandrestofthebody (Roe). EadivaluueistlueneanismOf 10 rats. Bars within a panel with different letters are significantly different (P<0.05) . Asterisks within bars irdicate that the value is significantly different frun the ° control valuue (P<0.05) . Initial 3-1nethylhistidine cmtent for total body, 21.0-52.0 uncles; carcass, 19.7113 uncles: and rest of the body, 131-0.1 umoles. Figure 17 . FRACTIONAL RATES - %/DAY 70 DAY 1-7 on 0-14 DAY 15-20 G g 7.5» b ‘1 1 » » i: 5.01 1 1 14 1 4 g 4.5» . 1 » a . . 13» c 13 Mn “finnm “ ‘ 1.5 1 » 11 “NH 11 30.0»! »aa:11§1311100 E 48 '7 b b 1 »1‘1 7 1 48 3 3.0» 1 » 1 13.0 52.4» 1 » 1 12.4 m 1.2» » 11.2 O b 212.0» . '1 » » 112.0 $0.0»' 1»o.'1».,s19.0 55 7.2» 1 » 1 » 17.2 E, 4.0» 1 » 1 » 14.0 "’ 2.4; 1 » 1 H” 12.4 0 10 100 010 100 010 100 CIMATEROL - ppm Fractional accretion, degradation and synthesis rates of total body 3-uethy1histidine containing proteins. Rats were fed ad libitum for 1, 2 or 4 wks a high-carbohydrate diet containing 0, 10, or 100 ppm cimaterol. Each bar is treneanismof 10 rats. Barswithinapanelwith different letters are significantly different (P<0.05) . Gain in 3-methylhistidine content was calculated as final 3-methylhistidire. Fractional accretion rates were calculatedfrcntheaveragebodygainin 3-methylhistidine per day divided by the average 3-methylhistidine pool size during that period, an! expressed as a percentage. Fractional accretion rates of 3-methylhistidine were assured to equal fractional accretim rates of 3-methylhistidine containing proteiue, because the concentration of 3-methylhistidine present in protein was unchanged with treatment. Fractional degradation rates were calculated frcn the average urinary excretion of 3-methylhistidine per day divided by the average 3-methy1histidine pool size during that period, and expressed as a percentage. Fractional synthesis rates were obtained by the addition of fractional accretion and degradation rates (FAR = FSR 4'31). umolas 3-MH/1OO 9 BODY WEIGHT Figure 18 . 71 1 1.0 . a-METHYLHISTIDINE excnrnou .3 1.4 » " 1.2 » ‘00 . °-' ’ HCONTROL 0.. , of}: 2 + ......10 ppm cmarenor :1 1g ...-.0100 ppm CIMATEROL 0.4 I i — cannon. vs 10 ”u 0.2 1» CONTROL vs 100 PPM 4A; R J A l I 1 1.0 1 0.0 1 0.0 1 0.4 ‘ 002 12:4507"1014172124 20 DAYS Effects of cimaterol on 3-methylhistidine accretion. Urinary3 lhistidine accretion of rats fed ad libitum for 4 wks a high-carbd'uydrate diet containing 0, 10, orlOOgncineterol. Eachpoint isthenean1sm of 10 rats. Asteriskarri+aboveapoint indicatuesthe values forrats feddietscartainiuglOarleOppn respectively, respectively, are significantly different frcn the cartrol valuue (P<0.05) . 13 u...— 72 for the first 4 days; thereafter, 3-methylhistidine excretion in cimaterol-treated rats was equual to or higher than values in control rats. Fractional degradation rates of 3-methylhistidine containing proteins, expressed per total body pool of 3-nethylhistidine ccntainirgproteins,were25% lowerinrats fed cimaterol during first F1 wk than in control rats (Figuure 17) , with no effects of cimaterol beyond 1 wk. Fractional synthesis rates of 3-methylhistidine "_'.MA.A.... "... containing proteins, calculated frcn fractional accretion and degradatim rates, were elevated by 32 t in rats fed cimaterol for 1 wk, with no effects of cimaterol thereafter (Figure 17) . . Plasna amino acid concentrations 4 h after an injection of cimaterol were markedly reduced (Figuure 19). Similar reductions in totalaminoacidlevelsinplasuewerereportethafterinjecticnof isqaroterenol (33) and also during a 3 h perfusion of the isolated rat henicorpus with isoproterenol (71). This reduction in plasma amino acidswasnestprcnn'eedforthebrandueddlainamimacidsvalire, isoleulcine and lcucine in agreelent with resuults obtained with isoproternol (71). But, by the end ofwk 1 (Figure 19), 2 and 4 (data not presented) plasue amino acid concentrations were unaffected by cimaterol. Consumption of diets containing cimaterol did not affect plasma insulin or triiodothyronine concentrations (Table 5). L" . It is now well documented that several beta-agonists, including cineterol, can increase skeletal muscle mass in a number of species (9,10,31,36,60,76,97) . The present firrlings show that these effects 73 0.15 “G CIIATEROL ,4 HOURS . 30 D 1.50 no cmnnou 30 m 20* , 1 20 5 ¢ _ 10’ / 1 10 g): 0 Z " ” ’ ¢ 0 ..c a ’ 7 z / 7 I 7 7 7 7 ' W-wééélllé 7% /;Ié//1-10 < 3 I 6 g a -20> / “ 1-20 (2)6 1' a / f / 1r - -30» / / / 1-30 0 ¢ /_ /, 2 J L— ' < O C‘O> I | l L 4-40 B u C b - m ..z. 50 1 50 CO 1 “,0 a c 8 s 5 g c e s .‘a z a u a o < 3 '0' a ‘4' 0 o x 1:- ‘c' t g - g f 5 5 O 5 E . 1 WEEK 7’ 10 nu 0111111901. . ; fig 20. D100 nu CIIATEIOL , 2° ._'- 0.: 10 10 > 4 I . ,azfi sea an 8 LJ L '10 M—U'g'g— 0 1-10 0L0 It! A!“ 01.7 (IL! N15 111. ILA 1'. VAL IE? IL! LEI! '11! 7" L70 Figure 19 . Effects of cimaterol cm plasma amino acids. Plasma amino acid concentrations in rats 4 h after a single injection ofcimaterol, arrialsoafterfeeding forlwka high-carbdrydrate diet containin; 0, 10, or 100 gm. Body weights of rats injected with cimaterol averaged 175119. Valuesareneansof1smof10aninels, and are expressed as percentage changes fran the control value foreachaminoacid. Asteriskswithinabar irdicate that the value is significantly different frcn the control valuue (P<0.05). (butrol values (uncles/d1) 4 hafterinjectimofsalireardlwkafterfeedingthe control diet were for GIU, 7.310.6, 7.410.6: SER, 16311.1, 19311.4: ASN, 5.5103, 6310.4; GLY, 10.6103, 10.6-10.6: GIN, 4113, 4113: HIS, 4.410.2, 4.610.43 1m, 18.211, 3715: MA, 26.5113, 3311.7; TYR, 4310.3, 5310.6: VAL, 14.4103, 14.9-11.2: MET, 4310.4, 7.4-0.7; IL, 6.7103, 6.71-0.67 rm, 11310.8, 11.010.73 PHE, 3.6103, 331-03: '13P, 6.6103, 7.7103 ani LYS, 3514, 4113, respectively. 74 Table 5. Effects of cimaterol on plasma W. Diet-ppm cimaterol Insulin-uU/‘ml AcutePA h. 54 63 S6 4 Day 7 81 72 64 6 Day 14 74 70 62 6 Day 29 75 54 54 7 Triiodcthyronine - ng/ml .Acute- 4 h. 1.5 1.5 1.3a 0.04 Day 7 2.7 2.7 2.6 0.08 Day 14 2.0 1.9 1.8 0.10 Day 29 1.7 1.7 1.7 0.08 Corticosteraae - Ira/100ml Day 25 62 40 40 7.1 mts fed a high-carbohydrate control diet for 1 wk, were injected intraperitaaeally with saline, 0.15 or 1.5 ng cimaterol in 0.2 m1 saline at O700handkilledafter4 h. Bloodwascollectedfzunrats killedafter4hof injectiorsandattheaudof 1, 20r4wkof feeding cimaterol in the diet. Plasma, separated fran blood stored at -70°C until analyses. Values are means of 10 rats per group. letter a indicates that the value is significantly different fran control value (P<0.05) . 75 areverypronounceduponinitialeamretothebeta-agonist, but wane with time likely in part at least due to desensitization of beta-recptors (45). Total body protein gain was stimlated by 169-212% the first wk rats were fed diets containing cimaterol, with virtually all the increase associated with skeletal nuscle. Beyord 1 wk, cimaterol failed to have major stimlatory effects on skeletal nusclegain. 'Iheseresiltsagreewitharecentreportdatastrating that clerbuterol fed to rats for 4 days stimlated fractional rates of skeletal nuscle deposition by 40%, with no further significant ' stimlation evident after 11-25 day exposure to the agonist (95) . Effects of cimaterol on rat skeletal nuscle lipoprotein lipase activity, and on brown adipose tissue lipoprotein lipase activity and de-novo rates of fatty acid synthesis, are also most pronounced innediately after initial exposure to cimaterol (Figure 4 and Table 4). I-bwever, lipolysis inwhite adipose tissue of rats appears to ranainelevatedevmafter4 wksofexposuretocimaterol (Figure 9). 'me meohanism(s) reponsible for these apparent time dependent differences in sensitivity of metabolic pathways to cimaterol min to be elucidated. mringthe firstwkofexposuretocimaterol, fractional accretion rates of 3-methy1histidine containing proteim (actin and myosin) increased up to 120% (Figure 17), with virtually all the increaseconfinedtothecarcass. Basedondailymeasurementof 3-methylhistidine excretion in urine and on total body pool size of 3-methylhistidine containing proteins, fractional degradation rates of 3-methymistidine containing proteins averaged 25% lower in rats fed dietscontaininglOOppncimaterol forlwkthanincontrol rats. 76 During the first day of exposure to cimaterol fractional degradation rates of ~3-methylhistidine containing proteins were depressed by 33%, indicating a rapid onset of action. By day 7 fractional degradation rates were no longer depressed in rates fed cimaterol (data not presented). These data are consistent with the finding that cimaterol depressed plasma amino acid concentrations 4 h after a single administration, but not after feeding for 1 wk, and illustrate the inportame of considering the timing of measurement relative to cimaterol administration. The average depression in turnover of 3-methylhistidine containing proteins of 25% in rats fed cimaterol for 1 wk is in reasonable agreement with the calculated value of a 55% reduction in fractional degradation rates of gastrocnanius and soleus muscle proteins in rats fed clenbuterol for 4 days reported by Reeds et al (95) . 'mese data, together with results of earlier in vivo and in vitro studies (33,42,71) , indicate that one mechanisn of action of beta adrenergic agonists to rapidly stimulate skeletal muscle protein accretion is by slowing protein turnover. From the measurements of fractional accretion and degradation rates of 3-methylhistidine containing proteins it was possible to calculate fractional synthesis rates of these proteins. This approach, although indirect, has the advantage of providing an estimate of fractional synthesis rates integrated over time, unlike the use of’r'adiolabeled amino acids which measures rates of protein synthesis over a period of only several hours. Fractional synthesis rates of 3-methylhistidine containing proteins calculated by this approach were elevated by 32% in rats fed diets containing cineterol for 1 wk, with no effect evident beyond 1 wk. In agreement with the 77 increase in fractional synthesis rates, there was a marked increase in RNA gain and concentration in hindlimb muscles of rats that consmed cimaterol for 1 wk (Figure 15) . Others have also noted increases in skeletal muscle RNA in response to clenbuterol and oimaterol (11,95). As noted in the introduction, beta agonist-induced increases in protein synthesis have been observed when measurements were made within several hours after injection of the agonist (33,36). Reeds et 4 a1 (95) failed to observe an increase in fractional synthesis rates of muscle proteins in rats that consumed cimaterol for 4 days, but they measured protein synthesis during the light period when food intake, and consequently clenbuterol intake, would be expected to be low. A stimulation in fractional synthesis rates of muscle protein may have been observed if they had measured protein synthesis after feeding diets containing clenbuterol for only 1 or 2 days and during the dark period when rats normally consume most of their food. Accelerated skeletal muscle growth in young animals is generally caused by W in rates of fractional synthesis rates with lesser increases in rates of fractional degradation rates ( 83) . cimaterol, however, appears to function by simultaneously stimulating fractional synthesis rates and depressing fractional degradation rates of 3-methylhistidine containing proteins. Such action maximizes accretion of protein with minimal changes in rates of synthesis and degradation; accretion increased by 120% in rats fed cimaterol for 1 wk with only a 32% increase in fractional synthee is rates coupled with a 25% decrease in fractional degradation rates. Similar reciprocal changes in protein synthesis and degradation have been observed during 78 work-induced hypertrophy (46) and during the recovery phase fran atrophy of inuobilized Insole (47) . Under these conditions, increased netgaininproteinisalsocausedbystinulationof fractional rates of protein synthesis arr! imibition of protein breakdown. It is unlomn whether beta-agcnists influence protein accretion directly, or indireclty via release of homes (39) . Several lines of evidence point to direct effects of beta-agonists on skeletal nuscle. Isoproterenol imibits protein degradation as measured by release of phenylalanine from rat hemicorpus preparatiore (71) , and release of alanine and glutamine frun isolated rat epitrochloris nuscle preparations (42) . cimaterol stinulates lipoprotein lipase activityinratEDLnnscleinvitro (Figures). 'Ihebeta . agrrfist-irflnedirueaseinproteinaccretimcanoccmteabsa'eeof charges in plasma insulin, triiodothyronine or corticosterone (Table 5 and ref 36,76). cimaterol will alpress hyperinsulinania in lanbs (11) andmice (24), buttrereismevidencethatthisisessential prereqiesite for accelerated nuscle accretion. The specific nechanisne whereby cimaterol depresses protein tun-over and omccnitantly accelerates protein synthesis remain to be elucidated. 79 Cimaterol , when fed to rats, improved skeletal muscle accretion arddecreasedadiposetissuedeposition. ‘Ihepresentfindingsshow that these effects of cimaterol were rapid, and wane with time due to either desensitization of beta receptors (47) or to sane post receptor effects. Agradual irxzreaseovertineindoseofcimateroltothese ratsmightovercaneatleastpartoftheresistamethatdevelops. Alternatively, intermittant rawval of cimaterol frcm the diet might be effective. It would be interesting to see if these manipulations wouldhavelargereffectsonbodyccmpositimthantheobserved changesinthepresentstudy. Cimaterolalteredthebodycarposition without influencing energy balance indicating that it is not nerely redirecting energy frun fat to protein deposition. In an effort to elucidate the mechanism of action of cineterol to reduce deposition of adipose tissue, effects of cineterol on fatty acid synthesis, lipoprotein lipase activity and lipolysis were examinedintheserats. cimaterol failedto influence invivo orin vitro rates of fatty acid synthesis in either white adipose tissue or liver. Consequently, decreasedbodyfatcontentinresponseto cineteroldidmtresiltfrmdecreasedratesofdemvoratesof fatty acid synthesis. This conclusion is supported by the observation thatcimaterolalsodecreasedwhiteadiposetissuegainaniirereased skeletal muscle gain in rats fed a high-fat diet where de novo fatty acid synthesis would be suppressed (69) . Recmced fat deposition observedinratscoramedcimaterolwasalsomtcausedbyreduced transfer of fatty acidsf ran circulating triglycerides, because in white adipose tissue lipoprotein lipase activity was unaffected by cimaterol. 80 cimaterol stimlated lipolysis in vitro in white adipose tissue and in vivo. Cow-sequently, cimaterol decreased fat gain by increasing mobilization of stored lipids. The specific nechanisn involved in stimlating lipolytic activity by cimaterol ranains to be studied. Although it is )mown that beta adrenergic stimlation of lipolysis is nediatedbyincreasesinommardprcteinkinaseactivitywhidi activate hormore sensitive lipase, it has not been demonstrated whether cimaterol also mediates its effects in a similar manner. Fattyacidsynthesisinbrownadiposetissuewasinhibitedby cimaterol , suggesting different control redianisms for fatty acid synthesisinbrownadiposetissueardinwhiteadiposetissue. This mayberelatedtothepresenceofmorebeta—receptorsinbrownadipose tissue than white adipose tissue, but there were sufficient receptors in white adipose tissue to activate lipolysis. Other possibilities sudiasdifferenttypesofreceptorsinbrcwnadiposetissmarriwhite adipose tissue, differences in intracellular pools of cAMP among tissues, or enzymes under different control mechanisms may be involved in these tissue—specific selective responses. Effects of cimaterol on fatty acid oxidation and transport of circulating free fatty acids into nuscle would provide more complete evidenceinternsofhcwcinaterolcanredirecterergyfronfat deposition to protein accretion. But such experiments have yet to be conflicted. In skeletal nuscle, lipoprotein lipase activity was elevated in response to cimaterol, denmstrating a capacity for the increased transfer of fatty acids fruu circulating triglycerides probably for increased utilization of energy fruu fat. cimaterol also stimulated 81 lipoprotein lipase activitiy in brown adipose tissue. These responses inskeletalnuscleardbrownadiposetissueareincontrasttothe lack of effect on white adipose lipoprotein lipase activity and indicate a selective regulation of this enzyme in different tissues. The wecific nechanisns whereby cimaterol stinulates muscle lipoprotein lipase activity retain to be elucidated; change in enzyme activation, turnover and synthesis, specific types of receptor (beta 1, beta2 ormimed) thatnediatetheseeffects, andmeasuretentof cAMPardproteinkinaseactivityareanaigthefactorsthatneedtobe explored. Very little is currently knowm about the beta adrenergic regulation of this enzyme activity in nuscle. The mechanism of action of cimaterol in increasing protein accretion was examined using 3-nethylhistidine as an irdicator of proteinturnover. mtafranthepresentfindings indicatethat cimaterol irducedincreasesinslmletalmscleproteinaccretim by decreasing fractional protein degradation rates, and increasing fractional synthesis rates. This effect of cineterol is rapid, as evidenced by reduced fractional degradation of 3-methy1histidine oontainirgproteinsevenmtlefirstdaythatratsconsmed cimaterol. Consistant with decreased degradation and increased synthesis rates, plasma amino acid concentrations were also depressed 4haftertheadministrationofcimaterol. Supportingtheobserved stimlationinfractional synthesis rates, therewasamarkedincrease inMgainarriconcentratiminhirdlinbmscles of rats that coreunedcimaterol. Additional stlriiesarereededtoexamirethe possibility that cimaterol has direct effects on protein synthesis. Results frun my experinents provide a clear indication that 82 examinationofproteinsynthesisshouldbedonesconafterthe administration of cimaterol. Measurement of protein degradation under in vitro conditions by the release of 3-methylhistidire from nuscle protein inthepresenceof cimaterol mildsrmmthercineterol can directly affect turnover of myofibrillar proteirs. 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