Meat Biotechnology – Applications in Pork Quality Muscle Targeted Growth Promotants – Mode of Action of Beta Agonists

Deana Hancock, Diane Moody, Dave Anderson Elanco Animal Health

Reciprocal Meat Conference, June 19, 2006 Pharmacological Activity: Beta•Adrenergic Agonist

A beta•adrenergic agonist (b•agonist) binds to and activates beta•adrenergic receptors (b•AR) that are found on the surface of many different types of cells in the body. G A G M L V L V G P A A A L P D G Beta Adrenergic P G L E N S S A S A T A N D P V L R A Y Receptors L L P L C K P A S P S R C P A P R C A A D S E F E S D E Y G V R E P W S S E P L T H L S R F E Q Q N F V W G F A R A P T W C R A K D A E L W Y V R G V V I W A V L (1) M (3) W H (5) I N (7) F G T T S L A L L G A S L M A F V M F V S F F A D I N L P V V P V F V L Shaded AA's are V I L C L F S V L P L W Transmembrane L G L S F V T Y W G conserved across the L I M A V L V P C Y A Domains V L A L V E I S S G A S A D A I S I C L V F T N human b1, b2 and b3 T L M A N A L W V A G M F AR's (31%) V L C V T F I N V M S C V I P I I I V I V Y G A I A L L L I A (2) F L L (4) G R (6) T Y K N D R V K C A L R F A R T T G L L C Y R R K S Q C A R R P L A F A R L E Q P D F R K A L T Q A R A E A T Q R R I K P H T L L S A R R D G A H R R T Q R L V A G L V S C Circled AA's are S K P R A L S P P Q K D conserved across F R Y I R A R G D D D D R P D V V G A D K G G N P P S the b AR of S A P G A S T 1 C A E L P humans, pigs, R L L P R P P E A F G N W R and sheep (80%) L A A A A G C G A G G A A A R P A A T D P G P P P S S S L E S K A P D D E P A S V C R F R P P G A P P P S P S P S V A Mersmann, 1998; J. Anim. P P Moody et al., 2000; Farm Animal Met. & Nutr. Sci. 76:160 J.P.F. D'Mello (Ed.) G A G M L V L V G P A A A L P D G Beta Adrenergic P G L E N S S A S A T A N D P V L R A Y Receptors L L P L C K P A S P S R C P A P R C A A D S E F E S D E Y G V R E P W S S E P L T H L S R F E Q Q N F V W G F A R A P T W C R A K D A E L W Y V R G V V I W A V L (1) M (3) W H (5) I N (7) F G T T S L A L L G A S L M A F V M F V S F F A D I N L P V V P V F V L V I L C L F S V L P L W Transmembrane L G L S F b AR lacks the V T Y W G 3 L I M A V L V P C Y A Domains V L A L phosphorylation site V E I S S G A S A D A I S I C L V F T N T L M A in domain 4 N A L W V A G M F V L C V T F I N V M S C V I P I I I V I V Y G A I A L L L I A (2) F L L (4) G R (6) T Y K N D R V K C A L R F A R T T G L L C Y R R K S Q C A R R P L A F A R L E Q P D F R K A L T Q A R A E A T Q R R I K P H T L L S A R R D G A H R R T Q R L V A G L V S C S K P R A L S P P Q K D F R Y I R A R G D D D D R P D V V G A D K G G N P P S S A P G A S T C A E L P R L L P R P P E A F G N W R L A A A A G C G A G G A A A R P A A T D P G P P P S S S L E S K A P D D E P A S V C R F R P P G A P P P S P S P S V A Mersmann, 1998; J. Anim. P P Moody et al., 2000; Farm Animal Met. & Nutr. Sci. 76:160 J.P.F. D'Mello (Ed.) OH OH H N

Beta Agonist HO

b Adrenergic Receptor b AR Membrane Gs AC

Terminology b AR Beta Adrenergic Receptor

Gs G Stimulatory Protein AC Adenylate Cylase Beta Agonist

OH OH H b Adrenergic Receptor N HO b AR Membrane Gs AC

Terminology b AR Beta Adrenergic Receptor

Gs G Stimulatory Protein AC Adenylate Cylase Beta Agonist

OH OH H b Adrenergic Receptor N HO b AR Membrane Gs AC

Terminology b AR Beta Adrenergic Receptor

Gs G Stimulatory Protein AC Adenylate Cylase Beta Agonist

OH OH H b Adrenergic Receptor N HO b AR Membrane Gs AC

ATP cAMP

Terminology b AR Beta Adrenergic Receptor

Gs G Stimulatory Protein AC Adenylate Cylase ATP Adenosine Triphosphate cAMP Cyclic Adenosine Monophospate Beta Agonist

OH OH H b Adrenergic Receptor N HO b AR Membrane Gs AC

ATP cAMP

PKAinactive PKAactive

Terminology Enzyme Phosphorylation b AR Beta Adrenergic Receptor

Gs G Stimulatory Protein AC Adenylate Cylase ATP Adenosine Triphosphate cAMP Cyclic Adenosine Monophospate PKA Phosphokinase A Beta Agonist

OH OH H b Adrenergic Receptor N HO b AR Membrane Gs AC Metabolic Modification FAT CELL

ATP cAMP Fat synthesis

Fat breakdown

PKAinactive PKAactive MUSCLE CELL

Protein synthesis Terminology Enzyme Phosphorylation b AR Beta Adrenergic Receptor Protein breakdown

Gs G Stimulatory Protein AC Adenylate Cylase ATP Adenosine Triphosphate cAMP Cyclic Adenosine Monophospate PKA Phosphokinase A Beta Agonist Classification Endogenous Beta Agonist

OH HO NH2

· Name: Norepinephrine · Class: Phenethanolamine Catecholamine · Source: Neurotransmitter substance released at central and sypathetic nervous system nerve endings Circulates in the plasma at Moody et al., 2000 Mersmann, 1998 relatively high concentrations Endogenous Beta Agonist

OH H HO N

· Name: Epinephrine · Class: Phenethanolamine Catecholamine · Source: Synthesized in and secreted from the adrenal medulla Circulates at lower concentrations than norepinephrine Moody et al., 2000 Mersmann, 1998 Biosynthesized from norepinephrine Classification of Phenethanolamines

What is a Phenethanolamine?

Phenyl Ethanolamine All phenethanolamines have the Group Group same base structure indicated in green A OH Phenethanolamines differ in their B CH•CH •NH•R substituents at positions A, B, C, 2 and R indicated in orange C Beta Receptor Selectivity of Beta Agonists used as Metabolic Modifiers

OH OH OH H H N N

HO H N Ractopamine (b1) 2 N L•644,969 (b2)

OH OH H H N N

H N 2 HO

Cimaterol (b ) Salbutamol (b )

2 HO 2

N H O

H OH N H

Cl N

N H N

H2N Cl Clenbuterol (b2) O Zilpaterol (b2) Growth and Carcass Summary

Growth Performance Control

68 kg 49 days 109 kg 147.7 kg feed Ractopamine Ractopamine effect 68 kg 45 days 109 kg • 4 days 129.1 kg feed • 18.6 kg feed

Carcass Dressing Yield 82.45 kg 81.27 kg + 1.18 kg

Boneless, Closely Trimmed Cuts 52.34 kg 49.69 kg + 2.65 kg

Total Dissected Carcass Lean 47.42 kg 42.10 kg + 5.32 kg

Note: Calculations based on 20 ppm Ractopamine treatment and equal slaughter weight basis Trials: Twenty trial summary, 6 trial summary, 4 trial summary Factors that influence response to treatment with phenethanolamines

Factor Requirement Compounds Species Studied

Greater response with Clenbuterol Pigs, Broilers, Rats Dietary Protein higher dietary protein BRL47672

Ractopamine

Duration of Greater response Cimaterol Pigs, Cattle, Sheep during final finishing Clenbuterol Treatment phase L•644969

Ractopamine

Dosage Differential effect on Ractopamine Pigs

growth and leanness

Age or Weight Greater response with Cimaterol Pigs, Cattle

older, heavier animals Ractopamine

Genetics Effective in both fat Cimaterol Pigs, Mice and lean genetics Ractopamine Moody et al., 2000 Nutrient Pool With Ractopamine

Adipose Tissue

Lipogenesis Lipolysis Decreased Increased

Fat Deposition rate reduced by 6%

Adapted from Bauman et al., 1982; Fed. Proc. 41:2538 Ricks et al., 1984; Reciprocal Meat Conf. Proc. 37:5 and Dunshea et al., 1993; J. Anim. Sci. 71:2931 Nutrient Pool With Ractopamine

Muscle Tissue

Protein Synthesis Protein Degradation Increased Similar

Protein deposition rate increased 30%

Adapted from Bauman et al., 1982; Fed. Proc. 41:2538 Ricks et al., 1984; Reciprocal Meat Conf. Proc. 37:5 and Dunshea et al., 1993; J. Anim. Sci. 71:2931 Effect of Ractopamine on Empty Body Protein and Fat Deposition of Finishing Swine

RAC Deposition Rate (g/d) ppm Protein Fat

0 144 442 20 188 (+ 30%) 414 (• 6%)

P<.001 NS Starting wt., 60 Kg; Ending wt., 90 Kg Combined data for barrows, boars and gilts

Adapted from Dunshea etal., 1992 Mode•of•Action

Direct · Adipose tissue and/or Increased lipolysis Indirect Decreased lipogenesis · Muscle Increased protein synthesis / no change Decreased protein degradation / no change

Indirect · Endocrine Effects Likely not primary effector · Blood flow Increased Adipose Tissue Fat Metabolism

Serum CAPILLARY Lipoproteins FFA SerumFFA Lipoprotein Glucose Glycerol Lipase

Fatty Acids Glucose Glycerol Fatty Acids Lipolysis Fatty Acids ADIPOSE CELL Triglyceride Lipogenesis Mechanism in Adipose Tissue Inactive HS Lipase B Receptor Beta Agonist Increased Lipolysis

Active HS Lipase Activated Adenyl Cyclase

ATP Active Active TG Synthetase Acetyl CoA cAMP Carboxylase Decreased Triglycerides Decreased Activated Fatty Acids Protein Kinases Inactive Inactive TG Synthetase Acetyl CoA Carboxylase Muscle Protein Metabolism

NUCLEUS

TRANSCRIPTION rRNA mRNA

tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM

PS • PD = PA Muscle Protein Metabolism

Direct vs Indirect?? NUCLEUS

TRANSCRIPTION rRNA mRNA

tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM Direct vs Indirect · Close arterial hindlimb infusion of cimaterol to steers –Increased fractional rate of protein accretion (% per day) • 1.17 vs 1.53 on day 3* • 0.98 vs 1.58 on day 7* • 0.73 vs 1.68 on day 14* • 0.80 vs 1.10 on day 20 Byrem et al., 1998; J. Anim. Sci. 76:988 · Beta agonists are effective in –Animals with inherited muscular dystrophy –Denervation induced muscle wasting –Endotoxemia, food deprivation, diabetic –Hypophysectomized, castrated, adrenalectomized Choo et al., 1992; Am. J. of Physiol. 263:E5 Byrem et al., 1998; JAS 76:988 Muscle Protein Metabolism b1 b2 b NUCLEUS 3

TRANSCRIPTION rRNA mRNA

tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM

Moody et al., 2000 Mersmann, 1998 Muscle Protein Metabolism b1 Species b2 b Pig • b1 NUCLEUS 3 Bovine & TRANSCRIPTION Rat • b2 rRNA mRNA

tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM

Sillence and Nathewam 1994; Br. J. Pharnacol. 111:866 McNeel and Mersmann, 1999; J. Anim. Sci. 77:611 Jensen et al., 1995; Pharmacol. Toxicol. 76:380 Muscle Protein Metabolism b1 b2 b NUCLEUS 3 Muscle TRANSCRIPTION Dark 30% more rRNA mRNA BAR than White in broilers

tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM

Young et al., 2000 Personal Communication Muscle Protein Metabolism b1 b2 b NUCLEUS 3 ATP ? TRANSCRIPTION cAMP rRNA mRNA Protein Kinase A

tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM Initial Dogma

· Early research in the late 1980’s and early 1990’s suggested that the muscle growth effects were independent of the beta adrenergic pathway

–Resistance to blockade by the nonselective beta adrenergic receptor antagonist propranolol

• Maltin et al., 1987; Biosci. Rep. 7:51

• Reeds et al., 1988; Comp. Biochem. Physiol. 89:337 Current Dogma

· Anabolic effects are dependent upon the beta adrenergic pathway –Propranolol blocks muscle protein accretion when administered IP or at higher oral dosages –Selective beta adrenergic antagonists block effects –Muscle cells have beta adrenergic receptors –Beta agonists increase cAMP in muscle cell culture and muscles in vivo • blocked by antagonists cAMP Mediated Mouse C2C12 Cells

Pmol cAMP/mg protein/10 min

50 c Control 40 30 c 20 b a RAC (10uM) 10 b a 0 Myoblast Myotube RAC + PROP

Isoproterenol • General Beta Agonist Ractopamine • Beta1 Selective Agonist Propranolol • Nonselective Beta Antagonist

Izevbigie and Bergen, 2000; P.S.E.B.M. 223:302 Shappell et al., 2000; J. Anim. Sci. 78:699 cAMP Mediated Rat Skeletal Muscle Slices

ISO +PROP (Nonselective) .1 mM (Beta Antagonist) ISO EPI NE ISO

+ICI 118551 (b2 Antagonist) 10 nM 100 nM 1 mM

ISO

+CGP 20712A (b1 Antagonist) 10 nM Forskolin 100 nM

Roberts and Summers, 1998; Eur. J. Pharmacol. 348:53 cAMP Mediated Rat Skeletal Muscle Crude Cell Membranes Biopsy

Growth and Gastrocnemius Weight Increased Blocked by ICI•118551, but not CGP•20712A Sillence et al., 1995; Am. J. Physiol. 268:E159 Effects of Beta Blockers • Route

Rat Skeletal Muscle

Clenbuterol, .125 mg/kg

Saline

Propranolol, 12.5 mg/kg IP 15 minutes prior to Clenbuterol

Time after Injection (h)

MacLennan and Edwards, 1989; Biochem J. 264:573 Effects of Beta Blockers • Dosage Rat Body Weight Gain, g/4 days Control 50 cd bd 40 ab a Clenbuterol, Choo et al., 1992; 30 4 mg/kg Am. J. Physiol. 263:E50 20 Clen + Prop, 10 200 mg/kg 0 Clen + Prop, 1000 mg/kg Gastrocnemius Weight, g Gastrocnemius Protein, mg 1.4 250 b b b 1.3 b 225 a a 1.2 a a 200 1.1 175 1 150 0.9 125 0.8 100 0.7 75 0.6 50 Effects of Beta Blockers • Selectivity Rat Body Weight Gain, g/4days Control 50 b b 40 a a Clenbuterol, Choo et al., 1992; 30 4 mg/kg Am. J. Physiol. 20 Clen + Prop, 263:E50 10 200 mg/kg 0 Clen + ICI • 118,551, 200 mg/kg

Gastrocnemius Weight, g Gastrocnemius Protein, mg b b 1.2 175 a a 1.1 150 b b 1 a a 125 0.9 100 0.8 0.7 75 0.6 50 cAMP Mediated Rat Skeletal Muscle • Blockage of Peripheral NE Release

Remove NE 15 to 25% Increase in Proteolysis

Carlos et al., 1999; Am. J. Physiol. 277:E883 cAMP Mediated Rat Skeletal Muscle • Blockage of Peripheral NE Release

Remove NE 15 to 25% Increase in Proteolysis

Time Muscle Calpain

Carlos et al., 1999; Am. J. Physiol. 277:E883 cAMP Mediated Rat Skeletal Muscle • Blockage of Peripheral NE Release

Remove NE 15 to 25% Increase in Proteolysis

Time Muscle Calpain

Add ISO 13 & 27% Decrease in Proteolysis

Carlos et al., 1999; Am. J. Physiol. 277:E883 Muscle Protein Metabolism

b1 b NUCLEUS 2 "CRE" ATP b3 TRANSCRIPTION ? cAMP rRNA mRNA Protein actin Kinase A myosin ? calpastatin ? tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM Effect of Ractopamine on Muscle Protein Synthesis in Finishing Swine

Percent / Day Control RAC (20 ppm) Fractional Synthesis Rate 4.4 6.7 P<.06

6 hr infusion of 14C•Tyrosine RAC (20ppm) for 21d and 35d n=7 for Control n=8 for RAC

Bergen, et al., 1989; J.Anim. Sci. 67:2255 Effect of Ractopamine on Muscle Protein Synthesis in Finishing Swine

Percent / Day Control RAC (20 ppm) Fractional Synthesis Rate 4.7 6.2 P<.01

6 hr infusion of 14C•Lysine RAC (20ppm) fed 21d, 10 pig / trt

Culham et al., 1990; J. Anim. Sci. 68 (Suppl. 1):318 Muscle Protein Metabolism

· Myosin mRNA abundance was increased in muscle of cattle fed ractopamine and clenbuterol Smith, et al., 1989; J. Anim. Sci. 67:3496 Smith, et al., 1995; Am. J. Physiol. 268:E858

· a•actin mRNA abundance was increased in muscle of pigs fed ractopamine Bergen et al., 1989; J. Anim. Sci. 67:2255 Helferich, et al., 1990; Endo. 126:3096 Grant et al., 1993; J.Anim. Sci. 71:3319 Ji et al., 1991; J. Anim. Sci. 69(Suppl. 1):329

· a•actin mRNA abundance was increased in muscle of sheep treated with L•644,969 Koohmaraie et al., 1991; J. Anim. Sci. 69:4823

· Interpreted as increased rate of transcription or increased stability of the mRNA Muscle Protein Metabolism

b1 b NUCLEUS 2 "CRE" ATP b3 TRANSCRIPTION ? cAMP rRNA mRNA Protein actin Kinase A myosin ? calpastatin ? tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM cAMP Responsive Human Skeletal Muscle a•Actin

Bishopric and Kedes, 1991; Proc. Natl. Acad. Sci. 88:2132 cAMP Responsive

Rat Cardiac a Myosin Heavy Chain

Gupta et al., 1996; Mol. and Cell. Biochem. 163/164:203 cAMP Responsive

Rat Cardiac a Myosin Heavy Chain

Gupta et al., 1996; Mol. and Cell. Biochem. 157:117 cAMP Responsive

Bovine Calpastatin

Cong et al., 1998; Biochimica et Biophysica Acta 1443:186 cAMP Responsive

Bovine Calpastatin

Cong et al., 1998; Biochimica et Biophysica Acta 1443:186 Muscle Protein Metabolism

b1 b NUCLEUS 2 "CRE" ATP b3 TRANSCRIPTION ? cAMP rRNA mRNA Protein actin Kinase A myosin ? calpastatin ? tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM Muscle Protein Metabolism

b1 b NUCLEUS 2 "CRE" ATP b3 TRANSCRIPTION ? cAMP rRNA mRNA Protein actin Kinase A myosin ? calpastatin ? tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM

Calpain / Calpastatin Effects of L•644,969 on Protein Degradation in Steers

· Fractional protein degradation (% per d) of skeletal muscle myofibrillar protein was 27.1% lower (P<.05) in L•644,969 treated steers after 3 weeks on treatment. –Wheeler and Koohmaraie, 1992 Effect of Cimaterol on a-Actin, Calpain/Calpastatin mRNA Expression in Steers with Close Arterial Hindlimb Infusion Arbitrary units on day 20 4 * } 23% 3

1 0 a•Actin mM Calpain Calpastatin uM Calpain SKM Calpain Sun et al., 1994; FASEB J. 8:A178 Byrem et al., 1998; JAS 76:988 Increased Activity • 51% Effects of Cimaterol on Bovine Calpastatin mRNA Expression

Area under peak kb b 1 5.1 0.8 0.6 a 0.4 3.8 0.2 0 Control Cimaterol 2.8

Parr et al., 1992; Eur. J. Biochem. 208:333

Speck et al., 1993; Biochimie 75:917 Effects of L•644,969 on Ovine and Bovine Calpastatin mRNA Expression and Activity

· Increased calpastatin mRNA and / or activity –Kretchmar et al., 1990 –Koohmarie et al., 1991 –Wheeler and Koohmaraie, 1992 –Pringle et al., 1993 –Killefer et al., 1994 Effects of Epinephrine and Clenbuterol on Calpastatin in Pigs

· Epinephrine infusion for 7 days increased calpastatin activity in porcine skeletal muscle – from 7.3 to 12.9 x 107 fluorescence units/kg · Epinephrine increased the 135 kDa calpastatin · Epinephrine infusion for 7 days did not increase calpastatin mRNA in porcine skeletal muscle Parr et al., 2000; Arch. Biochem. and Biophysics 374:299 · Single oral dose of Clenbuterol (.4 mg/kg bwt) increased calpastatin mRNA, protein, and calpastatin activity 16 hours post feeding Parr et al., 1999; JAS 77(Suppl. 1):164 Effect of Dietary Protein and Ractopamine on Calpain/Calpastatin Activity in Pigs uM Calpain, units/5g mM Calpain, units/5g 4 10 (CP P<.05; RAC P>.90; CP*RAC P>.5) (CP P>.10; RAC P<.04; CP*RAC P>.90) 3 8 6 2 4 1 2 0 0 10 10 18 18 CP 10 10 18 18 CP 0 20 0 20 RAC 0 20 0 20 RAC Calpastatin, units/5g * 25 Ji 1993 (CP P>.08; RAC P>.65; CP*RAC P>.90) Ph.D. Thesis, Purdue Univ. 20 15 In addition, no effect on calpains were observed by 10 Bergen et al., 1989; J. Anim. Sci. 67:2255 5 Sainz et al., 1993; Aust. J. 0 of Agric. Res. 44:1441 Effects of Ractopamine on Skeletal Muscle Calpain Expression in Pigs

Ji et al., 1992; JAS 70(Suppl. 1):208 Calpastatin Phosphorylation by PKA

· Phosphorylation sites are located on bovine calpastatin · Phosphorylation occurs via Protein Kinase A

Cong et al., 1998; J. Biol. Chem. 273:660 Parr et al., 2000; Archives of Biochem. And Biophysics 374:299 Muscle Protein Metabolism

b1 b NUCLEUS 2 "CRE" ATP b3 TRANSCRIPTION ? cAMP rRNA mRNA Protein actin Kinase A myosin ? calpastatin ? tRNA + ATP TRANSLATION AA•TRNA AA Protein PROTEOLYSIS AA CYTOPLASM

Calpain / Calpastatin Effect of Dietary Protein and Ractopamine on Growth and Lean Gain in Pigs Nitrogen Retention, g/d Average Daily Gain, kg 40 1 (CP P<.001; RAC P<.001; CP*RAC P<.001) (CP P<.001; RAC P<.009; CP*RAC P<.003) 30 0.8 0.6 20 0.4 10 0.2 0 0 10 10 18 18 CP 10 10 18 18 CP 0 20 0 20 RAC 0 20 0 20 RAC 10th Rib LEA, cm2 Urinary Creatinine N, g/d 50 4 (CP P<.001; RAC P<.05; CP*RAC P>.69) (CP P<.009; RAC P<.084; CP*RAC P>.80) 40 3 30 2 20 Ji et al., 1991 JAS 1 10 69(Suppl.1):329 0 0 Effect of Dietary Protein and Ractopamine on Lean Gain in Pigs 10th Rib LEA, cm2 Urinary Creatinine N, g/d 50 4 (CP P<.001; RAC P<.05; CP*RAC P>.69) (CP P<.009; RAC P<.084; CP*RAC P>.80) 40 3 30 2 20 10 1 0 0 10 10 18 18 CP 10 10 18 18 CP 0 20 0 20 RAC 0 20 0 20 RAC a•Actin mRNA, arbitrary units 50 40

30 Ji et al., 1991 JAS 20 69(Suppl.1):329 10 0 (CP P>.65; RAC P<.02; CP*RAC P>.16) Effect of Ractopamine (b1) on Protein Metabolism Conclusions · Ractopamine increases rate of muscle protein synthesis · Ractopamine increased abundance of mRNA for myofbrillar proteins actin and myosin · Ractopamine has only minimal effect on muscle protein degrading enzyme systems (calpains, calpastatin, cathepsins) · Supplemental substrate (AA) is required to maximize performance and lean growth Effect of Clenbuterol, Cimaterol and L644,969 (b2) on Protein Metabolism Conclusions · Some studies show an increase rate of muscle protein synthesis · Clenbuterol increased abundance of mRNA for myofibrillar proteins • myosin · L•644,969 increased abundance of mRNA for myofibrillar proteins • actin · Clenbuterol, Cimaterol and L•644,969 have a consistent effect of increasing calpastatin, thus decreasing muscle protein degradation Recent Technologies/Tools

· Antisense inhibition of beta1 adrenergic receptor mRNA

· Monoclonal antibody to human beta3 adrenergic receptor · Chinese hamster ovary cells expressing bovine and porcine beta adrenergic receptors · Beta adrenergic receptor knockout and overexpression models and humanized mouse models Conclusions & Research Needs

· Effects on protein accretion in rodent skeletal muscle are direct and mediated by beta adrenergic receptors and activation of cAMP –Needs confirmation in livestock species • beta receptor subtype proportions in various muscles and across species • cAMP effects • beta blockade –Additional pathways, cross talk, or indirect actions may also be involved Conclusions & Research Needs

· Rat skeletal muscle a•actin promoter has cAMP responsive element – Needs confirmation in livestock species • identification of cis and trans activating factors • confirmation that it is PKA mediated · Deletion analysis indicated that the bovine calpastatin gene promoter has at least one cAMP responsive element • identification of trans activating factors • confirmation that it is PKA mediated · Protein Kinase A phosporylation assays indicate that the calpastatin protein is phosphorylated by Protein Kinase A · Need to evaluate effects of beta agonist on protein translation Summary Mode•of•Action

· Adipose tissue –Increased lipolysis –Decreased lipogenesis · Muscle –Increased protein synthesis / no change –Decreased protein degradation / no change · Dependent upon –Species, compound, beta adrenergic receptor selectivity, dosage, duration, sampling relative to dosing (temporal effects), age / weight, nutrition Summary New Technologies

· Exciting new technologies have recently been published which will greatly aide in further evaluations of the mode of action of the beta adrenergic agonists

· Research efforts are needed to further understand the molecular and biochemical processes regulating protein accretion in livestock species administered beta agonists