ELSEVIER

DOMESTIC ANIMAL ENDOCRINOLOGY Vol. 14(4):251-262, 1997

PARADOXICAL INCREASES OF CIRCULATING NONESTERIFIED FATTY ACIDS IN SOMATOTROPIN TREATED

CATTLE UNDERGOING MILD DISTURBANCES 1'2

Y.R. Boisclair, K.B. Johnston, 3 D.E. Bauman, 4 B.A. Crooker, 5 F.R. Dunshea, 6 and A.W. Bell

Department of Animal Science, Cornell University, Ithaca, NY 14853

Received December 31, 1996 Accepted February 21, 1997

Effects of various doses of bovine somatotropin (bST) on plasma concentrations of nonesterified fatty acids (NEFA) were studied in a 14-d Latin square with six Holstein heifers. Animals were given daily injections of excipient or bST at 12:00 p.m. and fed twice daily at 7:00 a.m. and 7:00 p.m. On Day 14, plasma NEFA remained low through the day except around the 7:00 p.m. feeding when they were substantially elevated. The elevation was significantly greater in bST-treated animals and corresponded to the excitement of the animals in anticipation of the evening feeding. To further investigate this phenomenon, a second experiment was conducted in which nine growing Holstein steers were fed hourly and received either daily intramuscular (i.m.) injection of excipient or bST (120 mg/kg BW) for 15 d in a crossover design. Daily profiles of NEFA were obtained under undisturbed conditions or concurrently with intensive handling. Although no elevations could be detected in any case in control animals, bST caused a substantial rise in NEFA concentration only when animals were subjected to intensive handling. This suggested that NEFA peaks noted in bST-treated heifers in the first experiment resulted from increased ability of adipose tissue to respond to adrenergic stimulation associated with the anticipation of feeding. Consistent with this hypothesis, plasma NEFA concentrations in bST-treated steers were increased to a greater extent during a challenge involving i.v. injection of epinephrine. This amplification of adipose tissue response by bST must be considered when conducting intensive studies. Even the minimal excit- ment associated with blood sampling can confound the results regarding lipid mobilization, and this may have contributed to the notion that ST is a lipolytic hormone. © Elsevier Science Inc. 1997

I N T R O D U C T I O N

Chronic administration of somatotropin (ST) to growing animals leads to significant gains in productivity (1,2). The most dramatic results have been described in the pig in which reciprocal effects occur for accretion rates of protein in skeletal muscle (increase) and lipid accumulation in adipose tissue (decrease; 1,3). The biochemical basis for these actions have been studied most intensely in adipose tissue. A series of in vivo and in vitro studies have demonstrated that ST decreases l ipid accumulation by reducing the rate of l ipogenesis (3-5). ST induces these changes in adipose tissue metabol ism by altering the ability of the tissue to respond to homeostatic signals such as insulin and catecholamines (1,3).

Changes in the circulating levels of nutrients are often used to obtain clues t o the metabolic changes caused by treatments. However, for a metabolic regulator such as ST, which operates by changing the responses of tissues to homeostatic Signals, the informa- tion obtained from circulating levels of metabolites could be confounded by the experi- mental procedures used. For instance, adipose tissue of ST-treated animals displays an

© Elsevier Science Inc. 1997 655 Avenue of the Americas, New York, NY 10010

0739-7240/97/$17.00 PII S0739-7240(97)00021- 0

252 BOISCLAIR ET AL.

increased ability to respond to lipolytic signals such as catecholamines (see reviews, 1,3,6). Therefore, unless extra precautions are taken, circulating levels of nonesterified fatty acids (NEFA) may not reflect the metabolic status of adipose tissue of resting animals, but rather the increased propensity of adipose tissue to mobilize lipids when the animal is confronted with an unaccustomed procedure (e.g., blood sampling; 7,8).

To address this issue, we examined the effect of chronic ST treatment on the profiles of plasma NEFA in growing cattle experiencing short bouts of excitement. In addition, the effects of ST on the daily pattern of metabolites reflecting primarily carbohydrate (glu- cose) and N (urea) metabolism were also characterized.

MATERIALS AND METHODS

Experiment 1: Effect of Dose of bST on Plasma NEFA Levels. This experiment was designed to study the effect of bST dose on nitrogen retention, and these experimental details and results were described previously (9). Briefly, Holstein heifers [initial body weight (BW) = 105 kg] were acclimated to experimental procedures for 5 wk. Treatments consisted of administration of varying doses of recombinant bovine somatotropin (bST; lot #40,479, The Upjohn Co., Kalamazoo, MI USA) in a 6 X 6 Latin square with 14-d periods. Treatment doses were 0, 6.7, 33.3, 66.7, 100, and 200 mg/kg BW administered via daily intramuscular (i.m.) injection at 12:00 p.m. Experimental periods were separated by a 7-d interval to allow animals to return to basal conditions and to adapt to changes in feed allowances.

Animals were fed a complete mixed ration in equal amounts at 7:00 a.m. and 7:00 p.m. Allowances were calculated to sustain a gain of 0.75 kg/d (9). On Day 14 of each period, blood samples were collected at frequent intervals over the 24-hr period. An aliquot of each sample was processed to plasma by adding heparin (30 U/ml blood). A second blood aliquot was processed to serum after overnight refrigeration. Plasma and serum were stored at - 2 0 ° C until analyzed for ST and NEFA.

Experiment 2: Effect of Excitement on Circulating Metabolites in bST-Treated Steers. Nine Holstein steers (240-250 kg BW, 8-9 months old) were used. Seven of these had chronic catheters in both jugular veins, both external iliac arteries, and one external iliac vein. They also were used to study the metabolic actions of bST on the hindlimb (10). Two additional steers were added for the epinephrine challenge, and these animals had catheters in their jugular veins only. Steers were housed in a controlled environment at 20 ° C with light between 5:00 a.m. and 9:00 p.m. and were familiar with experimental conditions before samples were collected.

Animals were used in a single reversal design with 15-d periods separated by a 6-d interval. Treatments were daily i.m. injection of excipient (control, 75 mM sodium bicarbonate) or 120 mg/kg BW of recombinant bST (lot #40,479; The Upjohn Company) given at 8:00 a.m. Steers received a complete mixed ration consisting of 60% concentrate and 40% hay at levels needed to sustain a gain of 1.2 kg/d (see 10). The daily feed allowance was distributed in 24 hourly meals.

On Day 8 of each period, blood was obtained from four animals at 2:15, 6:15, and 7:15 a.m., and thereafter every 15 min from 7:45 a.m. to 8:15 a.m., every 30 min from 8:15 a.m. to 10:45 a.m., every hour from 10:45 a.m. to 3:45 p.m., and every 2 hr from 3:45 p.m. to 11:45 p.m. Serum and plasma were prepared and stored until analyzed for serum insulin and plasma urea, glucose, and NEFA. Because animals did not experience excitement and were not subjected to any unfamiliar procedures during sampling, Day 8 will be referred to as the "undisturbed condition."

Blood sampling was repeated on Day 13 on seven animals, during the measurements of nutrient utilization by the hindlimb (10). Consequently, animals were subjected to inter-

SOMATOTROPIN MODULATES ADIPOSE TISSUE RESPONSE 253

mittent handling (e.g., verification of catheter patency, installation of infusion lines) from 10:00 a.m. to 12:15 p.m. in preparation for the hindlimb experiment. They were then required to remain standing during blood sampling by a team of four individuals (from 12:15 p.m. to 4:15 p.m.). Therefore, this profile will be referred to as the "disturbed condition." The sampling schedule was identical to Day 8 except that samples were collected every 30 min between 12:15 p.m. and 4:15 p.m. Blood samples were processed and stored as described previously (10).

On Day 16, eight animals received an i.v. injection of epinephrine (1.2 ~zg/kg BW) at 10:15 a.m., 26.25 hr after the last treatment injection. The stock solution of epinephrine- HC1 (1 mg/ml, The Butler Company, Columbus, OH USA) was diluted with 5 ml sterile saline 20-30 min before injection and kept in the dark at 4 ° C until used. The epinephrine challenge was delivered within 3-5 s through a jugular catheter, followed by 5 ml of sterile saline. Blood samples were obtained at - 30 , - 15, - 10, - 5 , - 1, 2.5, 5, 10, 15, 20, 30, 45, 60, 120, 130, and 140 min relative to injection. Plasma was harvested and stored at - 2 0 ° C until analyzed for NEFA. Serum levels of insulin were also determined at - 30 , - 5 , 120, and 140 min relative to challenge.

Chemical Analyses. Commercial kits were used to determine plasma NEFA (WAKO Chemicals, Dallas, TX USA), glucose (glucose oxidase peroxidase method; Sigma Chem- ical Co., St. Louis, MO USA), and urea (hypochlorite-phenol nitroprusside method; Sigma) as described (10).

Plasma ST and serum insulin were analyzed by specific double antibody radioimmu- noassays (RIAs) as described previously (10). The RIA for plasma somatotropin was performed using antibody supplied by NIADDK [anti-oGH-2 (AFP-C0123080)]. Stan- dards and iodination grade hormone were prepared from bovine somatotropin (lot 12, 1.3 IU/mg protein; Miles Laboratories, Naperville, IL USA). Range of the assay was 0.2-8 ng/tube, and intra- and interassay coefficients of variation (CV) were 4.1% and 7.1%, respectively. The serum insulin RIA was based on a guinea pig anti-porcine antibody (catalog #299-230P, Cambridge Medical Technology Corporation, Billerica, MA USA). Porcine insulin was used for iodination (Cambridge Medical Technology) and standards (Micromedic System, Inc., Horsham, PA USA). The assay was linear from 0.02 to 0.12 ng/tube. Intra- and interassay CV were 6.4% and 6.0%, respectively.

Calculations and Statistics. Average daily concentrations (ADC) of hormones and metabolites were calculated by weighing the different time points of the profile. They were analyzed by a model accounting for animal, period, and treatment. Effects of treatment on the various profiles (treatment X time interaction) were analyzed by split plot analysis with animal as the block, treatment as the whole plot, time as the subplot, and animal x treatment X time as the error term. This analysis was adopted after a preliminary ANOVA indicated the absence of period effects for any variables.

Temporal response to epinephrine was identical across treatments and animals, with NEFA peaking at 5-10 min and returning to basal levels at 30 -40 min after challenge. Thus, the response area was integrated from 0 to + 15 min to minimize the effects of clearance and counterregulatory mechanisms. Response areas were subtracted from the basal area defined by the mean concentration of NEFA at -30 , - 1 5 , - 10 , - 5 , 0, 120, 130, and 140 rain from time of challenge. Corrected response areas were analyzed by a model accounting for animal, period, and treatment.

RESULTS

Experiment 1. Nitrogen retention increased, and plasma urea N and urinary N excre- tion decreased in a dose-dependent manner with bST treatment (9). Plasma ST peaked within 2 hr of injection and gradually returned to basal concentrations after 10-14 h

2 5 4 B O I S C L A I R ET AL .

300 -

200

E

~L ~ 100

0 -4

Dose (lag/kg • d)

uL= O s ~ ~i~ ' - 6 . 7 ; ; " ' q ' " 33.3

i ~" . . . . "A'-.. 66.7 . . . . "e . . . 100

i ,

0 ; ; l i 1; 2'0 21 T i m e P o s t I n j e c t i o n (h)

Figure I. Plasma concentration of somatotropin (ST) in heifers on Day 14 of treatment during a 24-hr period after injection of various doses of bovine ST. Variation was similar across time and treatments, with the SE averaging 16%.

(Figure 1). There was a strong linear relation between the ADC of circulating ST (y) and exogenous dose (x) (y = 0.29, x + 4.10; P < 0.001) (9).

The profiles of plasma NEFA exhibited a marked postinjection pattern (Figure 2). Levels were low between 12 and 24 hr after bST injection, but dramatic elevations occurred around the 7:00 p.m. feeding time in all groups, including the control animals. Circulating concentrations of NEFA peaked at the 7:00 p.m. feeding, 7 hr after the daily bST injection and approximately 5 hr after the highest plasma levels of ST were achieved (compare Figures 1 and 2). Although these elevations in plasma NEFA were accentuated by bST, they were not related to the dose of exogenous bST (P > 0.5 for linear and quadratic contrasts; 0 a, 6.7 a, 33.3 ab, 66.7 ab, 100 ab, 200 b for P < 0.05).

As we reported previously, exogenous bST did not alter the ADC of insulin but

PM F ding Dose (l~g/kg • d)

- - 0 400 ~. .. . . . ~ - - - 6.7

.;;~. . . . . 33.3 . . . . . k- - - 66.7 /"

3001 ~ i . . . . . . 100 . . . . . . "it"-" 2 0 0

Z AM d ing 2OO-

, o o , . . . . . . . . . . . . . . . . . . ~ ~ , ~ : - ~ . : - . . . . . . . . . . . . . . ~-.

0 -4 0 ; 8 ,2 ;6 2o 24

T i m e P o s t I n j e c t i o n (h)

Figure 2. Plasma concentration of nonesterified fatty acids (NEFA) in heifers on Day 14 of treatment during a 24-hr period after injection of various doses of bovine somatotropin. Animals were given two equal meals 7 hr (7:00 p.m. feeding) and 19 hr after injection (7:00 a.m. feeding). The pooled SE was 4.6 I-~M.

SOMATOTROPIN MODULATES ADIPOSE TISSUE RESPONSE 255

200'

A

150"

u.I 100' Z

E _~ 50' Q.

5 -4

Control • b S T

o E

E

Q.

8"

6"

4-

2 -

0 -4

t~ E

Q.

4-

3 -

2- o---o

8 1-

0 -4 5 ', ~ 1'2 15 20 2',

T ime Post Injection (h)

Figure 3. Daily plasma profiles of nonesterified fatty acids (NEFA), glucose, and urea in steers in the absence of excitement (undisturbed conditions, Day 8). Treatments were daily injection of excipient (control) or of bovine somatotropin (bST, 120 txg/kg BW). The pooled SE for NEFA, glucose, and urea averaged 4.5 ~zmol/liter, 0.10 mmol/liter, and 0.05 mmol/liter, respectively•

increased the ADC of plasma glucose and serum insulin-like growth factor I (9). Their profiles, however, were not affected by bST, time of injection, or time of feeding.

Experiment 2. This study was designed to identify the basis for the increased circu- lating NEFA concentrations observed at the 7:00 p.m. feeding time in the first experiment. The effect of feeding was eliminated by providing 24 meals evenly distributed throughout the day. As before, bST substantially improved nitrogen retention [30.6 v s . 42.9 g/day for control v s . bST; P < 0.005 (10)]. Also, daily injection of bST produced a temporal pattern of circulating ST, which essentially mimicked that of Experiment 1, with ST levels peaking at 2 hr and returning to baseline by approximately 12 hr after injection (data not presented). Overall, the ADC of plasma ST was increased 10-fold (3.6 vs. 34.8 ng/ml for control vs. bST treatment; P < 0.001) in agreement with the elevation predicted by the dose-response relationship established in Experiment 1.

During the undisturbed condition (Day 8), plasma concentrations of glucose and NEFA were constant throughout the day in the untreated animals (Figure 3). With bST treatment,

256 BOISCLAIR ET AL.

TABLE 1. EFFECTS OF SOMATOTROPIN TREATMENT ON THE CONCENTRATION OF METABOL1TES AND HORMONES

DURING THE DAILY PROFILE

Treatment ~' Significance Level b

Variable Control bST SD" Treatment Time Treatment x time

Undisturbed conditions d Plasma NEFA (~xmol/liter) 75 95 9 0.06 0.005 0.005 Plasma glucose (mmol/liter) 5.48 5.93 0.19 0.05 0.005 0.001 Plasma urea (mmol/liter) 1.69 1.21 0.09 0.005 NS NS

Disturbed conditions ~ Plasma NEFA (p~mol/liter) 102 156 29 0.03 0.0001 0.0001 Plasma glucose (mmol/liter) 5.06 5.58 0.13 0.001 0.0001 0.0001 Plasma urea (mmol/liter) 1.92 1.45 0.23 0.01 NS NS Serum insulin (ng/ml) 0.8 1.7 0.3 0.005 0.0003 0.0001

a Treatments were daily intramuscular injection of excipient (control) or recombinant bovine somatotropin (bST, 120 txg/kg BW) for 15 d. b Significance level for treatment (control or bST) used average daily concentration of the variable in a model accounting for period, animal, and treatment. Significance level for time and treatment × time using the residual error mean square from the split plot analysis of the data with treatment as main plot and time as subplot. NS, nonsignificant (P > 0.1). c SD obtained by submitting the average daily concentration of the variable to a model accounting for period, animal, and treatment. d Data obtained (n = 4) in the absence of any disturbances on Day 8. NEFA, nonesterified fatty acids. e Data obtained (n = 7) in conjunction with the study of hindlimb metabolism on Day 13.

NEFA levels were elevated slightly within 4 hr of injection (treatment x time, P < 0.005) and remained above control values for the next 12 hr, resulting in a significant elevation in the ADC of NEFA (P < 0.06; Table 1). Nevertheless, continuous feeding, coupled with minimal excitement, eliminated the dramatic elevation of NEFA observed in Experiment 1 (compare Figures 2 and 3). ADC of plasma glucose were elevated with bST (P < 0.05) mostly because of a modest elevation occurring 6 - 8 hr after bST injection (treatment x time; P < 0.001; Figure 3). Consistent with the increase in nitrogen retention, ADC of plasma urea was lowered by bST treatment and the temporal pattern was relatively constant throughout the day (P < 0.005; Table 1 and Figure 3).

In contrast, bST treatment caused major pertubations of the NEFA profiles during the disturbed condition (Day 13; Table 1, treatment X time, P < 0.0001; Figure 4). NEFA levels gradually increased during the intermittent handling period (2-4.25 hr after injec- tion) and plateaued at levels 2.5-fold greater than basal during intensive blood sampling (4.25-8.5 hr after injection). Once the handling associated with blood sampling was over (8.5 hr after injection), plasma concentrations of NEFA declined to baseline levels. Although procedures and animal handling were exactly the same, this pattern did not develop in these animals during the control period when NEFA levels remained low and constant throughout the entire day (Figure 4). Therefore, the period of intensive handling was responsible for the 1.5-fold increase in ADC of NEFA observed with bST treatment (P < 0.03, Table 1).

ADC of plasma glucose and serum insulin during the disturbed conditions were also elevated with bST (P < 0.005, Table 1). These effects were chronic, but again the profiles of bST and excipient treated animals were not parallel (treatment x time, P < 0.0001; Figure 5). During the control period, circulating levels of glucose and insulin remained constant throughout the day. In contrast, during the bST treatment period, glucose and insulin levels started to rise 4 - 6 hr after injection, reached a definite peak 1.5 hr after completion of the hindlimb study (5.75 hr after injection) and gradually returned to baseline thereafter (Figure 5). Plasma concentrations of urea were constantly lowered by bST treatment throughout the day (P < 0.01; Table 1; Figure 5).

SOMATOTROPIN MODULATES ADIPOSE TISSUE RESPONSE 257

350 ¸

300'

~ 250' E

200 LL U.I Z m 150' E

Q- 100'

50'

,~ ~ Control • bST

0 • , . , . , , , ,

-4 0 4 8 12 16 20 24

Time Post Injection (h)

Figure 4. Daily plasma profiles of nonesterified fatty acids (NEFA) in steers obtained in conjunction with the study of hindlimb metabolism (disturbed conditions, Day 13). Treatments were daily injection of excipient (control) or bovine somatotropin (bST, 120 p~g/kg BW). The period of study of hindlimb metabolism is shown by a horizontal arrow and includes a period of preparation (2-4.25 hr after injection) and a period of intensive sampling (4.25-8.5 hr after injection). The pooled SE averaged 11 ~mol/liter.

Treatment with bST increased the NEFA response to an epinephrine challenge. This is illustrated by considering the full profile (Figure 6) as well as the area under the 0-15 min response curve (1423 vs. 1942 mmol/liter • min for control and bST; P < 0.02). Higher concentrations of plasma insulin prevailed in the bST-treated animals at the time of the challenge (1.0 and 1.6 ng/ml for control and bST; P < 0.007), indicating that the chronic adaptations to bST persisted 26.25 hr after administration of the last dose.

DISCUSSION

Lipid Metabolism. In well fed growing ruminants, bST treatment did not alter circu- lating levels or daily profiles of NEFA (7,11-13). With feed restriction, NEFA levels were increased but were devoid of a post-injection pattern (7,8). Elevations of NEFA levels were observed in bST-treated dairy cows only when increased milk production resulted in negative energy balance (14). Therefore, the marked increase in NEFA we observed between 3 and 10 hr after injection in the dose-response study was unexpected (Exper- iment 1, Figure 2). As in other bST-treated animals, these changes probably reflect increased lipolysis rather than decreased reesterification of NEFA (14-16). However, because peaks in circulating NEFA and ST were separated by approximately 5 hr (Figures 1 and 2), a direct stimulation of lipolysis by ST seems unlikely. This interpretation is supported by recent in vitro studies in which no lipolytic action of recombinant bST was detected (for review, see 1,3,17). In addition, smaller but detectable elevations of NEFA also occurred during the control treatment period, suggesting an interaction between bST treatment and some other factor (Figure 2).

In the dose-response study, circulating NEFA levels peaked at the 7:00 p.m. feeding time (Figure 2). We observed that animals became noticably excited in anticipation of the evening feeding, whereas animals were typically resting before the 7:00 a.m. feeding. In the second experiment, animals were fed hourly to avoid these influences of behavior. Hourly feeding and the absence of excitement eliminated the postinjection increase in circulating NEFA (Figure 3). Repeating the blood sampling during the hindlimb study provided the opportunity to establish whether feeding pattern alone or excitement asso-

258 BOISCLAIR ET AL.

c

o E E

8 =

E

0 -

5 o

4 "

3 -

2 -

Contro l • b S T

0

2"

0 -4

i , , i ,

4 ~ ,2 16 20 24

=o

13.

3"

0 -4 0 ~ ~ 1'2 1'~ 2'0 2',

Time Post Iniection (h~

Figure 5. Daily profiles of circulating insulin, glucose, and urea in steers obtained in conjunction with the study of hindlimb metabolism (disturbed conditions, Day 13). Treatments were daily injection of excipient (control) or bovine somatotropin (bST, 120 Ixg/kg BW). The period of study of hindlimb metabolism is shown by the horizontal arrow (2-8.5 hr after injection). The pooled SE for insulin, glucose, and urea averaged 0.11 ng/ml, 0.05 mmol/liter, and 0.09 mmol/liter, respectively.

ciated with feeding or other activity caused the postinjection elevation of circulating NEFA in the dose- response study. During the 2 hr preceding the hindlimb study (2-4 .25 hr after injection), animals were exposed to handling and various preparatory procedures (e.g., verification of catheter patency and installation of extension lines and infusion pumps)• During the study itself, an intense blood sampling regime involving multicath- eters was followed, and steers remained standing at all times (4.25-8.50 hr after injec- tion). Animals were familiar with these procedures and cooperated readily with the obligation to remain standing. On no occasion did the animals manifest physical (e.g., refusal to eat, cessation of rumination, behavior traits) or physiological (e.g., t ime-related increase in hindlimb blood flow or oxygen consumption) signs of stress, and this is reflected by the absence of NEFA elevation during the control period (Figure 4). How- ever, during bST treatment, NEFA levels progressively increased during the preparatory period and plateaued during the intensive sampling period. After this interval, animals

SOMATOTROPIN MODULATES ADIPOSE TISSUE RESPONSE 259

o

¥ IJ .

E

350"

399"

250"

200"

150"

100-

59"

9 -69

/ ~ ~ Control

-~0 0 3o 6'0 9'0 "1'2o 1 ~ 0 Time Post Challenge (rain)

Figure 6. Plasma concentration of nonesterified fatty acids (NEFA) during an epinephrine challenge in steers. Treatments were daily injection of excipient (control) or bovine somatotropin (bST, 120 Ixg/kg BW). The epinephrine challenge (1.2 ixg/kg BW) was administered i.v. on Day 16 of treatment, 26.25 hr after the last injection. The pooled SE averaged 12 ixmol/liter.

were left alone, and NEFA levels gradually returned to basal levels. Therefore, in bST-treated steers there was a temporal correlation between the intensity and duration of the disturbance on one hand and the elevation in circulating NEFA on the other. This observation suggests that excitement caused the elevation of NEFA around the 7:00 p.m. feeding time in the dose-response study (Experiment 1).

This phenomenon likely reflects the enhancement by ST of the response of adipose tissue to adrenergic signals (1,3,6). The mild disturbance associated with the hindlimb study probably increased the stimulatory influence of the sympathetic nervous system (18). Because of the highly labile nature of NEFA and sensitivity to adrenergic stimula- tion, it is imperative that in studies of NEFA metabolism animals become accustomed to handling and blood sampling before studies commence (19). This was certainly the case for the present studies because there were no outward behavioral signs of excitement or stress and the stimulation was modest as indicated by the unchanged NEFA levels during the control period (Figure 4). Nevertheless, it was sufficient to cause a 2.5-fold increase in circulating NEFA in bST-treated steers because of the increased ability of their adipose tissue to respond to adrenergic signals. A similar mechanism probably explains the increase in NEFA observed at the 7:00 p.m. feeding time in Experiment 1 (Figure 2). The increased ability of ST-treated steers to respond to adrenergic stimulation was directly demonstrated by the greater release of NEFA on epinephrine challenge (Figure 6). Chronic ST treatment has been shown previously to cause similar amplification of NEFA responses to epinephrine challenges in growing animals (7,20) and lactating cows irre- spective of the prevailing energy balance (14,21,22). Cellular mechanisms responsible for these effects of ST have been investigated and include attenuation of the G-inhibitory system (23,24).

C a r b o h y d r a t e and Prote in Metabolism. ST caused chronic elevations of circulating glucose and insulin under all conditions in Experiment 2 (Table 1). These observations agree with previous results in growing ruminants (7-9,11-13,25,26) and pigs (15,27). This probably reflects a decreased response of organs and tissues to insulin in ST-treated animals. Direct evidence for this view has been obtained for key tissues involved in

260 BOISCLAIR ET AL.

glucose metabolism. On the supply side, indices of glucose production by the liver were either increased in ST-treated animals when insulin levels were unchanged (28,29) or remained constant when insulin levels were increased (30,31). On the disposal side, ST treatment reduced the uptake of glucose by hindlimb tissues irrespective of prevailing insulin levels (10,28,32,33).

Chronic ST treatment also caused modest increases in circulating levels of glucose and insulin in the hindlimb study (Table 1, Figures 3 and 5). These daily postinjection oscillations in levels of glucose and insulin were most obvious under disturbed conditions where they could be correlated with the activity of the steers. They evolved simulta- neously in bST-treated steers with peak levels for both variables observed 90 min after completion of the 4-hr blood sampling period when all animals were lying, after the standing period required during blood sampling. Presumably, tissues such as skeletal muscles moderated their utilization of glucose during the resting period. These results suggest that the homeostatic system in bST-treated animals is more sluggish in matching glucose production to demand when challenged with dynamic changes. These postinjec- tion oscillations in bST-treated steers are minor, however, when compared with the elevations of NEFA occurring over the same interval or compared with the elevations in glucose and insulin in pST-treated pigs (16).

Treatment with ST caused a reduction in circulating urea. However, in contrast with glucose and insulin, plasma urea nitrogen levels were constant throughout the day and unaffected by the various conditions (feeding pattern or disturbances). This is consistent with the hypothesis that the stimulation of protein accretion by ST occurs via a signal providing constant stimulation throughout the day such as insulin-like growth factor I (9,10).

In summary, these studies provide additional evidence for the view that ST does not exert an acute lipolytic action in growing ruminants. Rather, increases in plasma NEFA in bST-treated steers were temporally related to specific disturbances, implying that ST amplified the response of adipose tissue to adrenergic agents released by the sympathetic nervous system. Increased glucose and insulin concentrations accompanied by minor transitory postinjection elevations of glucose and insulin were also observed in ST-treated steers. These data confirm that ST exerts chronic control over lipid and carbohydrate metabolism. They also raise the possibil i ty that as in the pig, these metabolic processes in growing ruminants are subject to short term regulation by ST, perhaps via an attenuation of insulin action (15). Finally, these studies illustrate the need to design studies carefully when probing mechanistic aspects of ST action. Chronic ST treatment can generate paradoxical results under certain conditions because it alters the response of tissues to homeostatic signals. In the case of lipid metabolism, a determination of NEFA kinetics during the hindlimb study would have led to the conclusion that ST chronically increases lipid mobilization in well fed growing steers. Indeed, excitement may have confounded the results of some previous investigations and contributed to the notion that ST is a l ipolytic hormone.

A C K N O W L E D G M E N T S / F O O T N O T E S

1 This work was supported in part by Cornell University Agricultural Experiment Station, USDA/National Research Initative Competitive Grant 93-37206-9025, and The Upjohn Company.

2 Authors appreciate the technical assistance of Marie Harkins, Bill English, Mark McGuire, Ramona Slepetis, Dottie Ceurter, and Debbie Dwyer.

3 Present address: 49 Commonwealth Drive, Basking Ridge, NJ 07920. 4 Reprint requests. 5 Present address: Depamnent of Animal Science, 130 Haecker Hall, University of Minnesota, 1364 Eckles

Avenue, St. Paul, MN 55108. 6 Present address: Animal Research Institute, Werribee, Victoria 3030, Australia.

SOMATOTROPIN MODULATES ADIPOSE TISSUE RESPONSE 261

REFERENCES

1. Boyd RD, Bauman DE. Mechanism of action for somatotropin in growth. In: Animal Growth Regulation, Campion DR, Hausman GJ, Martin RJ (eds). Plenum Press, New York, pp. 257-293, 1989.

2. National Research Council. Metabolic Modifiers: Effects on the Nutrient Requirements of Food-Producing Animals. National Academy Press, Washington DC. 1994.

3. Etherton TD, Donkin SS, Bauman DE. Mechanisms by which porcine somatotropin (pST) decreases adipose tissue growth in growing pigs. In: The Biology of Fat in Meat Animals: Current Advances, Smith SB, Smith DR (eds.). American Society of Animal Science, Champaign, IL pp. 53-69, 1995.

4. Vernon RG. Influence of somatotropin on metabolism. In: Use of Somatotropin in Livestock Production, Serjsen K, Vestergaard M, Neimann-Sorensen A, (eds.). Elsevier Applied Science, New York, pp. 31-50, 1989.

5. Dunshea FR, Harris DM, Bauman DE, Boyd RD, Bell AW. Effect of porcine somatotropin on in vivo glucose kinetics and lipogenesis in the growing pig. J Anim Sci 70:141-151, 1992.

6. Bauman DE, Vernon RG. Effects of exogenous somatotropin on lactation. Annu Rev Nutr 13:437-461, 1993.

7. Peters JP. Consequences of accelerated gain and growth hormone administration for lipid metabolism in growing beef steers. J Nutr 116:2490-2503, 1986.

8. Eisemann JH, Hammond AC, Bauman DE, Reynolds PJ, McCutcheon SN, Tyrrell HF, Haaland GL. Effect of bovine growth hormone administration on metabolism of growing hereford heifers: Protein and lipid metabolism and plasma concentrations of metabolites and hormones. J Nutr 116:2504-2515, 1986.

9. Crooker BA, McGuire MA, Cohick WS, Harkins M, Bauman DE, Serjsen K. Effect of dose of bovine somatotropin on nutrient utilization in growing dairy heifers. J Nutr 120:1256-1263, 1990.

10. Boisclair YR, Bauman DE, Bell AW, Dunshea FR, Harkins M. Nutrient utilization and protein turnover in the hindlimb of cattle treated with bovine somatotropin. J Nutr 124:664-673, 1994.

11. Zainur AS, Tassell R, Kellaway RC, Dodemaide WR. Recombinant growth hormone in growing lambs: Effects on growth, feed efficiency, body and carcase characteristics and on wool growth. Aust J Agric Res 40:195-206, 1989.

12. Zainur AS, Tassell R, Kellaway RC, McDowell GH, Dodemaide WR, Kirby RC. Recombinant growth hormone in growing lambs: Effects on growth, body and carcase characteristics, hormones and metabolites. Aust J Agric Res 40:663-674, 1989.

13. Pell JM, Elcock C, Harding RL, Morrell DJ, Simmonds AD, Wallis M. Growth, body composition, hormonal and metabolic status in lambs treated long-term with growth hormone. Br J Nutr 63:431-445, 1990.

14. Sechen S J, Dunshea FR, Bauman DE. Somatotropin in lactating cows: Effects on response to epinephrine and insulin. Am J Physiol 258:E582-E588, 1990.

15. Dunshea FR, Bauman DE, Boyd RD, Bell AW. Temporal response of circulating metabolites and hormones during somatotropin treatment of growing pigs. J Anim Sci 70:123-131, 1992.

16. Dunshea FR, Harris DM, Bauman DE, Boyd RD, Bell AW. Effect of somatotropin on nonesterified fatty acid and glycerol metabolism in growing pigs. J Anim Sci 70:132-140, 1992.

17. Houseknecht KL, Bauman DE, Vernon RG, Byatt JC, Collier RJ. Insulin-like growth factors I and II, somatotropin, prolactin and placental lactogen are not acute effectors of lipolysis in ruminants. Domest Anim Endocrinol 13:239-249, 1996.

18. Fain JN, Garcia-Sainz JA. Adrenergic regulation of adipocyte metabolism. J Lipid Res 24:945-966, 1983. 19. Pethick DW, Dunshea FR. Fat metabolism and turnover. In: Quantitative Aspects of Ruminant Digestion

and Metabolism, Forbes JM, J. France (eds.). CAB International, United Kingdom, pp. 291-311, 1993. 20. Wray-Cahen D, Boyd RD, Bauman DE, Ross DA, Fagin K. Metabolic effects of porcine somatotropin (pST)

in growing swine. J Anita Sci 65 (Suppl 1):261, 1987. 21. McCutcheon SN, Bauman DE. Effect of chronic growth hormone treatment on responses to epinephrine and

thyrotropin-releasing hormone in lactating cows. J Dairy Sci 69:44-5 l, 1986. 22. Sechen S J, McCutcheon SN, Bauman DE. Response to metabolic challenges in early lactation dairy cows

during treatment with bovine somatotropin. Domest Anim Endocrinol 6:141-154, 1989. 23. Lanna DPD, Houseknecht KL, Harris DM, Bauman DE. Effect of somatotropin treatment on lipogenesis,

lipolysis and related cellular mechanisms in adipose tissue of lactating cows. J Dairy Sci 78:1703-1712, 1995.

24. Houseknecht KL, Bauman DE. Regulation of lipolysis by somatotropin: Functional alteration of adrenergic and adenosine signalling in bovine adipose tissue. J Endocrinol 1996, 152:465-475, 1996.

25. Eisemann JH, Hammond AC, Rumsey TS, Bauman DE. Nitrogen and protein metabolism and metabolites in plasma and urine of beef steers treated with somatotropin. J Anim Sci 67:105-115, 1989.

26. Johnsson ID, Hart IC, Butler-Hogg BW. The effects of exogenous bovine growth hormone and bromocrip-

262 BOISCLAIR ET AL.

tine on growth, body development, fleece weight and plasma concentrations of growth hormone, insulin and prolactin in female lambs. Anim Prod 41:207-217, 1985.

27. Etherton TD, Wiggins JP, Evock CM, Chung CS, Rebhun JF, Walton PE, Steele NC. Stimulation of pig growth performance by porcine growth hormone: Determination of the dose-response relationship. J Anim Sci 64:433-443, 1987.

28. Eisemann JH. Bovine somatotropin alters glucose production and uptake by tissues of beef steers. FASEB J 3:A1258, 1989.

29. Cohick WS, Slepetis R, Harkins M, Bauman DE. Effects of bovine somatotropin (bST) on net flux rates of glucose and insulin across splanchnic tissues of lactating cows. FASEB J 3:A938, 1989.

30. Reynolds CK, Lapierre H, Tyrrell HF, Elsasser TH, Staples RC, Gaudreau P, Brazeau P. Effects of growth hormone-releasing factor and feed intake on energy metabolism in growing beef steers: Net nutrient metabolism by portal-drained viscera and liver. J Anim Sci 70:752-763, 1992.

31. Dunshea FR, Boisclair YR, Bauman DE, Bell AW. Effects of bovine somatotropin and insulin on whole-body and hindlimb glucose metabolism in growing steers. J Anim Sci 73:2263-2271, 1995.

32. Lakehal F, Crompton LA, Lomax LA. The effect of growth hormone on the hindlimb metabolism in growing lambs. In: Biotecbnology in Growth Regulation, Heap RB, Prosser CG, Lamming GE (eds.). Butterworths, London, Great Britain, p. 231 (abstract), 1989.

33. Wray-Cahen D, Bell AW, Boyd RD, Ross DA, Bauman DE, Krick B J, Harrell RJ. Nutrient uptake by the hindlimb of growing pigs treated with porcine somatotropin and insulin. J Nutr 125:125-135, 1995.