Effects of dietary clenbuterol on net flux across the portal-drained viscera, liver and hindquarters of steers (Bos taurus)

Camp. Biochem.

Physiol.

Vol. 104C.

No. 3, pp. 401-406,

Pergamon Press Ltd

1993

Printed in Great Britain

EFFECTS OF DIETARY CLENBUTEROL ON NET FLUX ACROSS THE PORTAL-DRAINED VISCERA, LIVER AND HINDQUARTERS OF STEERS (BOS TAURUS) JOANH. EISEMANNand GERALDB. HUNTINGTON USDA-AR& Roman L. Hruska U.S. Meat Animal Research Center, Clay Center, NE 68933, U.S.A. and Ruminant Nutrition Laboratory, Livestock and Poultry Science Institute, Beltsville, MD 20905, U.S.A. (Received 23 November 1992; accepted for publication 15 January 1993) Abstract-l.

Addition of the I-adrenergic agonist clenbuterol to the diet of steers increased blood flow in portal-drained viscera, liver and tissues of the hindquarters. 2. Uptake of oxygen increased with clenbuterol feeding in hindquarters but not portal-drained viscera or liver. 3. On day 1 of clenbuterol feeding, the principal source of circulating L-lactate switched from portal-drained viscera to hindquarters. 4. Both net release of a-amino nitrogen by the portal-drained viscera and net uptake by the hindquarters decreased on day 1 of clenbuterol feeding. Over time of clenbuterol feeding, both release of a-amino nitrogen by the portal-drained viscera and uptake by the hindquarters increased to equal or greater than pretreatment values, respectively.

INTRODUCTlON Clenbuterol, a fl-adrenergic agonist, increases lean body accretion and decreases fat accretion in many species, including cattle (Ricks et al., 1984). Previous studies with veal calves (Williams et al., 1987) show that stimulation of nitrogen accretion by clenbuterol is specific for the carcass tissues. Short-term changes in net tIux in the hindquarters (HQ) of beef cattle (Eisemann et al., 1988) are consistent with the observed carcass changes. Metabolism of the productive tissue, muscle, is frequently emphasized in the growing animal, yet we need to understand also the role of supportive tissues due to both their quantitative contributions to whole animal energy expenditures and their potential regulatory role. Clenbuterol increases oxygen use by hindquarters of beef steers (Eisemann et al., 1988). Effects of clenbuterol on oxygen use and metabolite flux of portal-drained viscera (PDV) and liver, metabolically active supportive tissues (Huntington and Reynolds, 1987), are not known. Our first objective with these two experiements was to determine whether oxygen use by PDV and liver was increased following clenbuterol feeding. Second, we hoped to observe chronic changes in net flux of energy and nitrogenous metabolites to describe the Present address: Department of Animal Science, North Carolina State University, Box 7621, Raleigh, NC 27695, U.S.A. Mention of trade name, proprietary products or specific equipment does not constitute a guarantee or warranty of the product by the USDA and does not imply its approval to the exclusion of other products that may also be suitable.

metabolic adaptations among various tissues to support increased nitrogen retention in carcass tissues in response to feeding clenbuterol.

MATERIALSAND

METHODS

Animals and diets In Experiment 1, four Hereford steers (Bos taurus), 11 months of age were gentled and adapted to a diet formulated to meet crude protein (CP) requirements for 1.0 kg daily weight gain at restricted intake (Eisemann et al., 1988) beginning 1 month before surgery. They were housed in individual stalls in an enclosed barn that had skylights to provide a natural daily light pattern. Steers were fasted for 48 hr and water removed for 24 hr before surgery to install chronic indwelling catheters with tips in the abdominal aorta, caudal vena cava, hepatic-portal vein and branches of the cranial mesenteric vein. An ultrasonic flow probe (16mm, Transonics Systems, Inc., Ithaca, NY) was placed around the abdominal aorta for measurement of blood flow to the HQ. Procedures for catheterization of the HQ tissues were as described by Eisemann et al. (1988), except there was a single arterial and venous catheter inserted while the steer was in right lateral recumbency. Hepatic-portal, hepatic and mesenteric catheterizations were as described by Huntington et al. (1989). Beginning 1 week after surgery, which was 1 week prior to treatment initiation, the steers were fed at 6-hr intervals beginning at 0700. Three steers (BW 254 f 4 kg) recovered from surgery and had patent catheters in the appropriate vessels.

402

J. H. EISEMANN and G. B. HUNTINGTON

In Experiment 2, 10 MARC III (Bos taurus; $ each Hereford, Angus, Pinzgauer and Red Poll) steers were gentled, adapted to diet and housed as described for Experiment 1. Chronic indwelling catheters were surgically inserted with tips in the aorta, hepaticportal vein, hepatic vein, and branches of the cranial mesenteric vein as previously described (Huntington et al., 1989). Six steers (BW 266 f 8 kg) recovered from surgeries and had patent catheters in the appropriate vessels. Design

The focus of Experiment 1 was the comparative response of two independent tissue beds, the PDV and HQ, to duration of clenbuterol feeding. In Experiment 1 there were 6 sample days at weekly intervals. The first 2 sample days were control feeding for all steers and designated week 1 and week 2. Clenbuterol(8 mg(hd.d)-‘; 2 mg/feeding) was added to the feed beginning on the third sample day which corresponded to day 1 of clenbuterol feeding. The remaining 3 sample days corresponded to days 8, 15 and 22 of clenbuterol feeding. The focus of Experiment 2 was the comparative response of PDV and liver (tissues in series). In Experiment 2 there were also 6 sample days at weekly intervals. The experimental design was the same as in Experiment 1 except that three steers were fed clenbuterol and three steers served as controls throughout the study. One steer from the clenbuterol treatment group lost its catheter in the cranial mesenteric vein after day 1 of clenbuterol feeding and could not be used for flux measurements on subsequent sampling days. Methods

Indicator dilution (para-aminohippurate, PAH) was used to measure blood flow in PDV and hepatic tissues. On each sample day, a primed (1500 mg PAH) continuous infusion of PAH (sterile solution) was initiated into a branch of the cranial mesenteric vein at least 45 min before the first sample. The infusion rate of PAH was 5400 mg/hr for both experiments. The protocol for sample collection was the same for both experiments. Blood (25 ml from each site) was withdrawn simultaneously from the abdominal aorta, hepatic-portal vein and caudal vena cava (Experiment 1) or hepattc vein (Experiment 2) at 60-min intervals beginning at 0800 for one feeding cycle (6 hr, six sets of samples/steer). Blood samples were collected in heparinized syringes and immediately placed on ice. Aliquots of blood were removed for later analyses and the samples were centrifuged at 15OOg for 20min at 4°C to obtain plasma. Plasma was aliquoted and stored at -20°C until analysis. A second blood sample was taken anaerobically into 3 ml heparinized syringes, capped with a rubber stopper and kept on ice until analyzed for oxygen

saturation and hemoglobin content using a Hemoximeter (Radiometer of America, Dallas, TX). Individual plasma samples were analyzed for glucose, L-lactate cc-amino N, urea N and ammonia N as previously described (Eisemann et al., 1988). Paraaminohippurate was analyzed on individual plasma samples as well as individual samples of whole blood (diluted 1 + 3 with dd H20) by an automated procedure (Technicon Autoanalyzer II Method No. 31672T, Bran + Luebbe, Tarrytown, NY). Equal aliquots of arterial blood from each sample were pooled within steer on each sample day. Arterial plasma concentrations of nonesterified fatty acids (NEFA) were analyzed on the pooled samples by a modification (Ko and Royer, 1967) of a titrimetric method (Dole and Meinertz, 1960). Plasma values for NEFA were corrected for acetate (Eisemann et al., 1988) and L-lactate content following analysis of the respective standards in the NEFA assay. The correction factor for acetate was 2.29% and for L-lactate 0.5%. The ultrasonic flow probe was used for monitoring heart rate and for measurement of blood flow to the HQ during blood sampling (Experiment 1). Blood flow was recorded every 10 set over the blood sampling interval. An average flow was then calculated for each blood sample. Calculations

For PDV and liver: Blood flow, l/hr =

PAH infusion rate, mg/hr

[PAW,- [PAW,

where u is PAH concentration in the hepatic-portal vein (PDV flow) or hepatic vein (hepatic flow) and a is PAH concentation in the artery (background). Oxygen concentration was calculated from oxygen saturation and hemoglobin concentration by the following equation: Oxygen, mM (Hb, g/l000 ml x 1.34 ml oxygen/g Hb x % oxygen saturation/lOO) = 22.4 ml oxygen/mmol oxygen For blood flow and all metabolites measured, where individual samples were assayed, a mean value (from six sample sets) for each steer and sample day was calculated. Net tissue uptake of oxygen and net tissue uptake or release of other metabolites for the HQ, PDV and splanchnic (sum of liver plus PDV) tissues were calculated as the product of arteriovenous concentration difference and whole blood or plasma flow. For liver, net flux, mmol/hr = (PDV flow x (portal-hepatic minus hepatic concentration, mM)) + (HA flow x (arterial minus hepatic concentration, mM)), where HA flow is blood or plasma flow in the hepatic artery. Flow in the hepatic artery was calculated as hepatic flow minus PDV flow.

Clenbuterol and metabolite flux in steers (Bos raurus)

of clenbuterol feeding and gradually resumed to pretreatment intake by day 4. Steers consumed all feed offered for the remainder of the study. Average + SE daily intakes were 4.21 &-0.08 kg DM, 813 + 15 g CP and 12.45 & 0.22 Meal metabolizable energy (ME, calculated, NRC, 1984) for Experment 1 and 4.66 f 0.91 kg DM, 854& 17g CP and 13.81 rf: 0.27 Meal ME for Experiment 2. To our knowledge, this is the first report on the effects of clenbuterol feeding on net flux of metabelites in PDV and liver. Although the number of steers is small, the integrated data on all three tissue beds clearly describe the origin of changes in plasma metabolite concentration in response to clenbuterol feeding, especially on day 1. Heart rate increased and blood flow increased to tissues of the PDV, liver and HQ on day I (Tables 1 and 2) in agreement with previous observations of increased cardiac output following ingestion of clenbuterol by horses (Claussen, 1981) and steers (Huntington et al., 1990). However, blood flow remained elevated in tissues of the HQ but not the PDV in Experiment 1 (Table l), and liver and PDV blood flow increased with a greater relative change in liver than PDV in Experiment 2 (Table 2) suggesting a change in partition of cardiac output with chronic clenbuterol treatment. Heart rate gradually declined from day 1 of clenbuterol feeding to control values. Clearly, in Experiment 1 (Table 3) oxygen uptake by the HQ increased without an increase in oxygen uptake by tissues of the PDV. In Experiment 2 (Table 4) there were no increases (P > 0.10) in oxygen use by the PDV or liver. Whole body energy expenditure in sheep increased following treatment with clenbuterol for up to 3 weeks (MacRae et al., 1988) or cimaterol for 13 weeks (Kim et al., 1989). Although there may be an increase in whole body energy use under chronic treatment with a /I-adrenergic agonist, our data show that there is a change in partition of whole body energy use with clenbuterol as well, such that the PDV, a highly active metabolic tissue, uses a lower percentage of total body oxygen. Thus one can

Statistical analysis A split-plot

ANOVA

403

was used to analyze the data

in Experiment 1. The mode1 used had steer and treatment as main effects, with the effect of treatment tested using the mean square for steer x treatment. The subplot was day within treatment which was tested using day within (treatment x steer) as the error term. The sum of squares for day within treatment was partitioned into four single degree of freedom contrasts-day within treatment 1 and the linear, quadratic and cubic response of day within treatment 2-and tested using day within (treatment x steer) as the error term. Treatments 1 and 2 were control and clenbuterol, respectively. In Experiment 2 the data from the two control periods were combined and the effect of treatment was tested using the mean square for steer within treatment. This was done to determine whether steers assigned to control vs clenbuterol treatments differed before treatment initiation. Where the treatment effect was significant (P < 0.10) the subsequent treatment means were not adjusted because the n was too small to adequately test for a covariate x treatment interaction. The pretreatment means serve as a reference value only. The acute effect of clenbuterol (day 1) was tested using the mean square for steer within treatment for week 3. The chronic effect of clenbuterol was tested by including the remaining 3 weeks (days 8, 15 and 22) in a split-plot ANOVA. The main plot tested treatment against the mean square for steer within treatment. The effect of day and day x treatment was tested against the residual mean square.

RESULTS AND DISCUSSION

Steers in both experiments consumed all feed offered during each 6-hr measurement period. On day 1 of clenbuterol feeding, steers consumed their initial 0700 feed. Starting with the 1300 meal on day 1, dry matter (DM) intake was reduced for the first 3 days

Table 1. Physiological variables and arterial concentrations of metabolites in steers fed control and clenbuterol treatments (Experiment 1) Variable

Control Wkl Wk2

Heart rate, beats/min 78 79 Blood flow, I/hr Hindquarters 260 261 Portal-drained viscera 463 512 Blood, mM Oxygen 6.30 6.20 Plasma, mM Glucose 4.80 4.81 L-lactate 0.56 0.57 NEFA heq/l) 89 83 a-amino N 2.04 1.90 Urea N 8.0 8.4 Ammonia N 0.263 0.262

Dl

D8

Clenbuterol D15 D22

ANOVA SEM

117

89

90

82

2

456 660

421 518

386 509

317 593

23 I5

6.01

5.58

6.20

5.75

6.29 4.14 4.45 4.21 2.74 0.68 0.69 0.72 393 I48 I31 63 1.00 1.60 I .49 1.66 8.4 9.1 8.9 8.4 0.213 0.257 0.261 0.283

T

B’k -

0.10 0.10

0.05

0.15

0.01

-

0.13 0.12 22 0.08 0.6 0.017

0.01 0.01 0.05 _ -

_ -

L

Q

C

0.01

0.01

0.01

0.01 0.05

0.01

0.01 0.01 0.05 0.01 _ 0.05

0.01 0.01 0.05 0.05 _ -

0.05 0.01 0.01 0.05 0.05 _ -

Arterial samples were taken from the abdominal aorta. ANOVA: Analysis of variance. T is a difference between control and clenbuterol treatments; Wk is a difference between weeks on control treatment; L, Q, C are linear, quadratic and cubic responses, respectively, during clenbuterol treatment. Type I error probability, not significant (P > 0.10) unless noted.

J. H. EISEMANNand G. B. HUNTINGTON

404 Table 2. Blood flow in the portal-drained

Variable Blood flow, ljhr PDV Liver

Pretreatment

Treatment Cont Glen

528 f 568 f (630 f 678 f

Cont Glen

Hepatic arterial Blood, mM Oxygen Plasma, mM GllKo% L-lactate m-amino N Urea N Ammonia

viscera (PDV) and liver and arterial concentrations of metabolites in steers fed control (Cont) and clenbuterol (Glen) treatments (Exoeriment 2) Days on treatment D8 DI5

DI

61 61 61) 46

519_+66* 729 + 66’

666 + 46 (693 f 60)

486 638

D22

SEM

416 648

22 21

T P < 0.06

T P < 0.06

608 + 62t

563

581

586

14

871 + 62t

760

748

189

17

I06 108

110 141

20

I22

17

ANOVA

415 640

NS

Cont Glen

15Oi26 91+ 26 (63 + 13)

89 + 48 142+48

Cont Glen

6.80 + 0.40 6.50 f 0.40

6.68 + 0.62 6.48 f 0.62

6.69 5.91

6.66 5.98

6.55 5.72

0.17 0.17

NS NS

Cont Glen Cont Glen Cont Glen Cont Glen Cont Glen

5.17~0.28 4.98 rfr0.28 0.72 f 0.03 0.68 * 0.03 1.88~0.19 1.73 * 0.19 8.5 k I.2 8.5 f 1.2 0.296 i 0.018 0.289 + 0.018

5.26 * 0.51’ 7.11 *0.51* 0.71 + 0.48f 4.35 + 0.48f 1.87 + 0.05$ I.18 +0.05$ 8.8 k 0.8 9.1 f 0.8 0.308 f 0.020t 0.220 * o.ozot

5.12 4.62 0.73 0.70 I .84 1.52 9.1 8.2 0.326 0.305

4.85 4.32 0.75 0.58 I .88 I .67 IO.1 9.5 0.301 0.296

4.02 3.64 0.74 0.66 1.92 1.64 10.5 9.3 0.307 0.313

0.12 0.12 0.04 0.04 0.03 0.03 0.7 0.7 0.020 0.020

D P
24

NS T P = 0.10, D P < 0.05 NS NS

Arterial samples were taken from the abdominal aorta. SEM: standard error of the mean for day x treatment for D8, Dl5 and D22; n = 3 for control steers, for clenbuterol-fed steers n = 2 for blood flow and n = 3 for concentration data; T: treatment; D: day of clenbuterol feeding. Values in parentheses are the pretreatment means for the two steers on clenbuterol feed that were sampled on D8, Dl5 and D22. For DI, the means for control vs clenbuterol-fed steers, within a sampling day, differ: *P < 0.10, tP < 0.05, $P < 0.01; n = 3 for both groups of steers. NS = P > 0. IO.

specifically stimulate oxidative metabolism in carcass tissues without stimulating all body tissues to the same degree. Acute changes in concentration of metabolites (Tables 1 and 2) were consistent with previous observations (Eisemann et al., 1988). The increase in glucose concentration coincided with a 50% increase in glucose production by the liver (Table 4), 50% increase in uptake of glucose by the HQ (Table 3) and across both experiments a relatively small increase in glucose use by PDV (Tables 3 and 4). Plasma L-lactate concentration increased in acute response to clenbuterol (day 1, Tables 1 and 2) and there was a change in site of production of lactate

from the PDV to the HQ (Table 3) demonstrating a shift in peripheral metabolism of glucose. Lactate uptake by the liver contributed a maximum of 63% to glucose output by the liver on day 1 whereas the maximum contribution was 10% for the control group on day 1 (Table 4). The pretreatment value for the clenbuterol group was 20%. Plasma lactate concentration was unchanged after day 1 (Tables 1 and 2) and the PDV again was the primary source of circulating lactate (Tables 3 and 4). In contrast to previous observations (Eisemann et al., 1988) there were no chronic changes in net flux of lactate in the

HQ. Plasma concentration

Table 3. Net flux of oxygen and metabolites (mmol/hr) in hindquarters (HQ) and portal-drained clenbuterol treatments (Experiment I) Variable Blood Oxygen

Tissue bed

HQ PDV

Plasma Glucose L-lactate a-amino N Urea N Ammonia N

HQ PDV HQ PDV HQ PDV PDV PDV

Control Wkl Wk2

Dl

Clenbuterol D8 Dl5

of a-amino

nitrogen

viscera (PDV) of steers fed control and ANOVA

D22

SEM

T

Wk

L

0

c

-

695 731

713 795

983 811

193 656

811 129

154 153

31 38

0.10 -

-

0.01 -

0.05

43

44 10 10 -72 24 -82 48 -131

64 40 -75 -21 I5 -26 41 -119

38 3 6 -69 24 -18 35 -136

45 I2 -8 -58 22 -45 40 - I05

41 I7 20 -62 34 -101 67 -145

6 IO 8 3 5 14 8 8

0.05 -

-

0.01 0.01 0.05 0.05 0.05

0.10 0.10 0.01 0.01 0.05 0.10 -

:, -71 24 -112 53 -Ill

de-

o;o -

-

0.01 0.01 0.05

Net flux = [A - V] x blood or plasma flow where A is concentration in the abdominal aorta, and V is concentration in the caudal vena cava for HQ measurements or hepatic-portal vein for PDV measurements. Positive sign = net uptake, negative sign = net release. ANOVA: Analysis of variance T is a difference between control and clenbuterol treatments; Wk is a difference between wks on control treatment; L, Q, C are linear, quadratic and cubic responses, respectively, during clenbuterol tnatment. Type I error probabilities, not significant (P > 0.10) unless noted.

Clenbuterol and metabolite flux in steers @OS ruvrur)

405

Table 4. Net flux of oxygen and plasma metabolites (mmol/hr) in portal-drained viscera (PDV), liver (L) and splanchnic (SPL) tissues of steers fed control (Cant) and clenbuterol (Glen) treatments (Experiment 2) Variable Oxygen

Tissue bed

Treatment Cant

PDV

Clen

kPL PDV L SPL

Glucose

Cant

Glen

PDV L SPL PDV L SPL

L-lactate

Cant

Glen

PDV L SPL PDV L SPL

Pretreatment 86Ok99 1074 + sot 1934 f 123 948 It 99 (1062 + 80) 898 + 507 (885 + 68) 1846 f 123 (1944 f 145) 4k 18 -183k27 -179+26 6k 18 (-8+21) -148k27 (-164k34) -142f26 (-172k23) -72 + 16 35 + 8t -37* 15 -71 f 16 (-80+20) 60 f 8t w* II) -Ilk 15 (-16+20)

Dl

Days on treatment D8 D15 D22

SEM’

801 +-90 836 ?r. 120 1637 f 158 833 f 90

765 742 1507 873

818 860 1678 972

750 887 1667 938

31 39 57 38

1025 + 120

970

971

1066

48

1858 + 158

1844

1943

2003

70

6k 12 -162k27 -156rt:36 -6k I2

._ 2 -151 -152 11

16 -147 -131 I5

12 -122 -110 27

9 15 IO 11

-230 f 27

-174

-156

-133

19

-236+36

-163

-I41

- 106

13

-68 21 -47 -81

-67 23 -44 -63

-62 16 -45 -66

5 3 8 6

289 f 44$

82

75

70

4

291 f 425

1

12

4

10

-70*9!j 35+44t -35+45 2*9$

ANOVA NS D P < 0.10

D P < 0.10

NS NS

fJ P < 0.05

NS NS NS

Net flux calculations are defined in text. A positive sign = net uptake; a negative sign = net release. Values are x f SEM. The values in parentheses are pretreatment for the two steers on clenbuterol feed sampled on D8, D15 and D22. The means for control vs. clenbuterol-fed steers, within a sampling day, differ: tP < 0.10, SF’ < 0.05, SF’ < 0.01; n = 3 for both groups of steers for pretreatment and Dl. ‘SEM: standard error of the mean for day x treatment for D8, D15, and D22; n = 3 for control steers; n = 2 for clenbuterol-fed steers. T: treatment; D: day of clenbuterol feeding; NS, P > 0.10.

Table 5. Net flux of plasma nitrogenous metabolites (mmol/hr) in portal-drained viscera (PDV), liver (L) and splanchnic (SPL) tissues of steers fed control ICont) and clenbuterol (Clenj treatments (Exwriment 2) Variable a-amino N

Tissue bed

Treatment Cant

Glen

PDV L SPL PDV L SPL

Urea N

Cant

Glen

PDV L SPL PDV L SPL

Ammonia N

Cant

Glen

PDV L SPL PDV L

Pretreatment -106k 1st 42 f 14t -64k 12 -74 + 1st (-86+ 17) 12 * 14t (26 + 15) -62? 12 (-61 + 17) 56+ IO -180+20 -124k 19 41* IO (42 f 14) -191 f20 (-204?25) -150* 19 (-162+24) -129k24 125+24 -4*1 -159k24 (-176+29) 156f24 (173 + 29) -3+1 (-3 f I)

DI

D8

Days on treatment D22 Dl5

SEM*

-71 18 -53 -46

-66 15 -51 -81

-81 23 -58 -77

I4 9 14 17

- 18

-7

-12

11

-16kl2f

-64

-88

-89

17

52 it: 9 -l83?8 -131 k8t 24 + 9

46 -174 -128 49

64 -192 -128 64

51 -207 -156 72

5 20 21 6

179+8

-159

- 194

-188

24

-154+8t

-110

-129

-117

25

-133+20 131 k 24 -2i6 -152k20

-143 141 -2 -165

-145 143 -2 -185

-156 153 -3 -180

II II

148 f 24

155

175

166

-4k6

-9

-9

-75 f lot 13*6 -61 f 12$ -26 f lO$ IO+6

-14

I

ANOVA NS NS NS

D P = 0.05 NS NS

NS NS T P < 0.05

I3 13

I

Net flux calculations are defined in text. A positive sign = net uptake; a negative sign = net release. Values are x + SEM. The values in parentheses are pretreatment for the two steers on clenbuterol feed sampled on D8, Dl5 and D22. The means for control vs. clenbuterol-fed steers, within a sampling day, differ: tP < 0.10, SP < 0.05; n = 3 for both groups of slews for pretreatment and Dl. *SEW standard error of the mean for day x treatment for D8, D15, and D22; n = 3 for control steers; R = 2 for clenbuterol-fed steers. T: treatment; D: day of clenbuterol feeding; NS, P > 0.10.

J. H. EI~EMANN and G. B.HUNTINGTON

406

creased on day 1 (Tables 1 and 2) due to a decrease in net release from the PDV (Tables 3 and 5). Supply of a-amino nitrogen bv splanchnic tissues was lower (Table 5) and thus uptake by the HQ decreased (Table 3). Uptake of a-amino nitrogen by the HQ as a percentage of that supplied by splanchnic tissues increased because the decline in release by the PDV was greater than the decline in upake by the HQ. Thus it is possible that tissues of the HQ could respond with an immediate increase in net uptake of a-amino nitrogen if it were supplied. Although an immediate decrease in urinary nitrogen output was observed in sheep wholly nourished by intragastric infusion in response to clenbuterol (Herbert it al., 1985) the change is not necessarily due to increased use of nitrogen by carcass tissues. Plasma concentration of a-amino nitrogen remained lower than during the control period as PDV release and HQ uptake increased over time of clenbuterol feeding (Tables 1, 2, 3 and 5). Net uptake of a-amino nitrogen by the liver tended toward zero or net release with chronic clenbuterol feeding: thus

there was a tendency for increased splanchnic release of a-amino N. Plasma concentration of ammonia nitrogen

decreased

acutely (Tables 1 and 2); however,

there were no measured changes in flux of ammonia nitrogen to explain the change. An increase in excretion of ammonia nitrogen by the kidney may contribute to the initial decrease in ammonia nitrogen COI'ICentratiOII. OVer thIC,INA splanchnic r&aSC of

ammonia

nitroeen

increased as henatic extraction

declined (P c OylO). Extraction was 52.7, 54.1 and 54.6% (SEM 1.5) for control and 47.8, 49.0 and 46.4% (SEM 1.8) for steers fed clenbuterol on day 8, dav 15 and dav_ 22. respectivelv. A decline in nitrogen _ extraction by the liver is consistent with previous data (Williams et al., 1987) that show stimulation of nitrogen accretion with clenbuterol is specific for

carcass tissues. Fewer amino acids may be involved in transporting nitrogen from peripheral tissues to the liver (Bergman, 1986) due to changes in protein turnover that result in enhanced nitrogen accretion. These data describe acute and chronic metabolic changes in response to clenbuterol feeding in PDV, liver and HQ of growing beef steers. Chronic metabolic response was minimal compared to the acute response. Of key importance is the observation that responses differ in specific body tissues and it is the sum of these responses that gives the whole animal effect. An increase in net uptake of a-amino N by tissues of the HQ, in conjunction with a tendency for

increased splanchnic

supply, describes coordinated

metabolic changes in several body tissues necessary for homeorhetic control. Acknowledgements-The authors thank K. Sorensen for feeding and care of the steers; M. Buschow and R. Jaeger for assistance at surgery; C. Feller and E. Zetina for technical support; and J. Watts and Linda Manson for t yping the manuscript. The authors thank Boehringer Ingelheim Animal Health, Inc., for providing the clenbuterol used in this study. REFERENCES Bergman E. N. (1986) Splanchnic and peripheral uptake of amino acids in relation to the gut. Fed. Proc. 45, 2217-2282. Claussen H. (1981) Bestimmung des Herzminutenvolumens mit der Thermodilutionsmethode hei Pferden vor und nach Varabreichuna von Clenbuterol oder Carazolol. Inaugural Dissertazon, Hanover, Tierarztliche, Hochsch& No. l-7. Dole V. P. and Meinertz H. (1960) Microdetermination of long-chain fatty acids in plasma and tissues. J. Biol.

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