Effects of bovine somatotropin and insulin on whole-body and hindlimb glucose metabolism in growing...

F. R. Dunshea, Y. R. Boisclair, D. E. Bauman and A. W. Bellmetabolism in growing steers

Effects of bovine somatotropin and insulin on whole-body and hindlimb glucose

1995, 73:2263-2271.J ANIM SCI

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Effects of Bovine Somatotropin and Insulin on Whole-Body and Hindlimb Glucose Metabolism in Growing Steers1t2t3

F. R. Dunshea4, Y. R. Boisclaifi, D. E. Baumad, and A. W. Bell

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

ABSTRACT: Six Holstein steers (245 kg initial SW) were surgically prepared with chronic catheters to allow measurement of blood flow and nutrient flux across the hindlimb. Steers were used in a single- reversal design with 16-d treatment periods of daily i.m. injection of either excipient (control) or recom- binantly derived bovine somatotropin (120 pgkg SW). On d 15 of each period, whole-body and hindlimb glucose metabolism were studied during a primed continuous infusion of [6-3Hlglucose, under both basal conditions and during a hyperinsulinemid euglycemic clamp. Somatotropin increased ( P < .O 1) basal blood glucose and serum insulin by 7 and 150% respectively, with no change in glucose irreversible loss rate (ILR). There was no effect of somatotropin on hindlimb blood flow or oxygen consumption. In contrast, hindlimb lactate uptake ( P < .02) and the

ratio of glucose to oxygen uptake ( P < .08) were reduced by somatotropin. Insulin infusion stimulated ( P < .01) glucose ILR and inhibited ( P < .02) endogenous glucose production to a lesser extent during somatotropin treatment. Basal plasma nonesterified fatty acid concentrations were increased ( P < .01) during somatotropin treatment and decreased ( P < .02) during insulin infusion. Insulin infusion increased hindlimb blood flow and glucose uptake to similar extents during both treatment periods. These data demonstrate that somatotropin treatment of growing steers decreases response of tissues to insulin (sensitivity in muscle, sensitivity, and responsiveness in liver and adipose tissue), thereby increasing glucose availability to other tissues.

Key Words: Steers, Hindlimb, Somatotropin, Glucose, Insulin, Gluconeogenesis

Introduction

Treatment of growing cattle with bovine somatotropin (bST) causes increased nitrogen reten- tion (see reviews Boyd and Bauman, 1989; McBride and Moseley, 1991; NRC, 1994). In general, carcasses from cattle and sheep treated with somatotropin

'Work supported in part by Cornell Univ. Agric. Exp. Sta. and USDNNational Research Initiative Competitive Research Grants No. 89-37265-4478 and No. 93-37206-9025.

2We acknowledge The Upjohn Co. (Kalamazoo, MI) for donation of the bovine somatotropin and Eli Lilly (Greenfield, IN) for bovine insulin. Authors wish to thank W. English, R. Dickens, M. Harkins, K. Johnston, and R. Slepetis for technical support.

'Presented in part at the 1989 annual meeting of the Federation of the American Society of Experimental Biology, New Orleans (FASEB J . 3:A938).

'Present address: Victorian Inst. of Anim. Sci., Werribee, Victoria 3030, Australia.

5Recipient of scholarships from the Natural Sciences and Research Council of Canada and Purina Mills Inc.

6To whom reprint requests and correspondence should be addressed.

Received September 30, 1994. Accepted March 29, 1995.

J. h i m . Sci. 1995. 73:2263-2271

contain more lean and less fat. Adaptations in tissue metabolism seem to be mediated, at least in part, by altering response to homeostatic signals, such as insulin. For example, glucose clearance after an intravenous insulin challenge is decreased in ST- treated ruminants (Hart et al., 1984; Sechen et al., 1990). Such responses represent changes in hepatic and(or) peripheral tissue metabolism. We recently developed a surgical preparation for chronic metabolic studies of the steer hindlimb (Boisclair et al., 1993a), a tissue bed that is representative of peripheral carcass tissues. Therefore, the objective of this study was to examine the effects of bST treatment on whole- body and hindlimb glucose metabolism under basal conditions and during a hyperinsulinemic/euglycemic clamp.

Materials and Methods

Animals and Treatment. Chronic catheters were surgically inserted into the external iliac artery and vein of one leg and into the contralateral external iliac artery of six Holstein steers (240 t o 250 kg BW; 7 mo

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2264 DUNSHEA

old) as previously described (Boisclair et al., 1993a). These animals were used in a previous study (Boisclair et al., 1994). Catheters were flushed every 2 d with sterile saline ( 9 g/L NaC1, Travenol Laboratories, Deerfield, IL) and filled with heparinized saline (100 U/mL) fortified with penicil- lin G sodium (50,000 U/mL; E. R. Squibb & Sons, Princeton, NJ). On sampling days, the heparin concentration was reduced to 10 U/mL.

After recovery and familiarization with experimental procedures ( 10 to 12 d) , steers were assigned to a balanced cross-over design with 16-d treatment periods separated by a 6-d interval. Treatments consisted of a daily intramuscular injection of excipient (control ( C ) ; 75 mmol/L of sodium bicarbonate) or 120 pgkg BW of recombinant bovine somatotropin (bST; lot #40.479; The Upjohn Co., Kalamazoo, MI) at 0800. Steers received a complete mixed diet consisting of 63% concentrate and 37% hay (Table 1) at levels sufficient to support a rate of gain of 1.2 kg/d (NRC, 1984). As reported previously, the feeding level was limited t o 90% of ad libitum dry matter intake (Boisclair et al., 1994). As a result, dry matter intake was approximately equal across treatments (6.9 vs 6.8 kg/d for control vs bST treatments). Crude protein allowances were raised to 115% of predicted require- ment throughout this study in an attempt to ensure that response to bST was not limited by supply of protein. The daily allowance was partitioned in 24 hourly meals to ensure steady-state conditions of nutrient absorption and metabolism.

Whole-body and Hindlimb Glucose Metabolism. On d 15 of each period, the effect of treatment on glucose metabolism was investigated under basal conditions and during a hyperinsulinemic/euglycemic clamp (Figure 1). Steers remained standing for the entire period of study. Starting at 1000, steers received a priming dose ( 180 pCi) followed by a continuous infusion ( 1.8 pCi/min) of [6-3Hlglucose (Amersham, Arlington Heights, IL). The infusate (7.2 pCi/mL) was delivered by syringe pump (Model SE 400, Vial

Table 1. Composition of experimental dieta

Ingredient

Cracked corn 483 Chopped hay 372 Fish meal 28 Corn oil 19 Soybean meal 83 Mineral premixb 15

aAll values expressed on a dry matter basis. Diet formulated to contain 153 &g of crude protein and 2.8 Mcal metabolizable energy per kilogram of dry matter as estimated from tabular values for each ingredient (NRC, 1984).

billinera] premix supplement contained 680 g defluorinated phos- phate and 320 g of trace mineralized NaCl per kilogram of dry matter. Trace minerals included iron, manganese, zinc, iodine, co- balt, and selenium.

ET AL.

Medical, Grenoble, France) via a jugular venous catheter previously inserted on d 8. Hindlimb blood flow was estimated by measuring the downstream dilution of para-aminohippuric acid ( PAH; .257 moll L ) administered into the ipsilateral external iliac artery by a syringe pump (Model 915, Harvard Apparatus, South Natick, MA) as a priming dose (6.2 mmol) followed by continuous infusion (.309 mmoll min). Estimates of pool size and turnover rate were used to determine priming doses and infusion rates for glucose (Kronfeld and Ramburg, 1981) and PAH (Katz and Bergman, 1969).

Basal glucose kinetics were studied from 1200 to 1250 because preliminary results indicated that glucose specific radioactivity ( S W ) was at steady state by this time. Six pairs of arterial (8.5 mL) and venous (4.5 mL) samples were drawn simultaneously from the external iliac vessels at 10-min intervals and arterial glucose concentration was determined immediately using an automated analyzer (Model 27, Yellow Springs Instrument, Yellow Springs, OH). Also, a second pair of samples (1.5 mL) was taken at each time point in heparinized 3-mL syringes, sealed, placed on ice, and analyzed immediately for oxygen saturation and hemoglobin concentration in an auto- matic direct-reading photometer (Hemoximeter OSM2, Radiometer Copenhagen, Copenhagen, Den- mark), and packed cell volume ( PCV) by microcapil- lary centrifugation.

Immediately after the sample at 1250, the isotope infusion was interrupted and a single dose of bovine insulin (50 mUkg BW, lot 615-70N-80, 26.6 U/mg, Eli Lilly and Co., Indianapolis, IN) was administered via the jugular vein followed by a continuous infusion of a solution containing insulin (5.0 mU/[min.kg SW]) and [6-3Hlglucose (1.8 pCi/min). Priming dose and infusion rate of insulin were calculated from data of McCann et al. (1985). This solution was prepared by mixing insulin and radioactive glucose in saline supplemented with the animal's own plasma ( 4 mL/ 100 mL infusate). Thereafter, arterial blood (.75 mL) was obtained every 5 min and glucose concentration determined rapidly as before. A glucose solution (dextrose monohydrate, 500 g/L, Baxter, Deerfield, IL) was infused via a contralateral jugular venous catheter at rates needed to maintain glycemia at levels observed during the basal period. When the exogenous glucose infusion rate required to maintain euglycemia had remained constant for at least 15 min, blood sampling was resumed from the external iliac vessels at 10-min intervals over a 50-min period. The average period from initiation of insulin infusion to beginning of the sampling period was 2 h, ensuring that steady-state levels of insulin and glucose SRA were also obtained (Figure 2). When sampling was completed, insulin and [3H]glucose infusions were dis- continued. However, the unlabeled glucose infusion was progressively decreased over the next 45 to 60



SOMATOTROPIN AND GLUCOSE METABOLISM 2265

Priming Dose

[ ~ ~ G I u c o s e infuslon

Continuous infuslon b

Priming DO=

InwWn Infuakon ConUnuous infusion

Glucose Infusion

Varlabk infusion Rat. (to maintain eugtymmla) .

Figure 1. Experimental protocol for the hyperinsulinemic/euglycemic clamp. In addition to prime and infusion of [6-3H]glucose and insulin, exogenous glucose was also infused to maintain euglycemia during the insulin infusion. Metabolic measures were made on blood samples obtained over the last 50 min of each period.

min to prevent the development of hypoglycemia. Analytical Procedures. A portion of arterial blood

was immediately placed in tubes and allowed to clot for 4 h at room temperature. After overnight refrigera- tion (4"C), serum was obtained by centrifugation (3,000 x g ) and frozen at -20°C. The remaining arterial blood and the venous blood were mixed with heparin (60 U/mL) and hrther processed. An aliquot of arterial blood was also centrifuged and plasma stored at -20°C.

Blood ( .5 mL) used for analyses of glucose and [6-3Hlglucose was mixed with water (2.5 mL) t o rupture red blood cells. One milliliter of .3 M Ba(0H)z and 5% ZnS04 were sequentially added with vortexing after each addition and deproteinized filtrates were obtained by centrifugation and stored at -85°C. When analyzed, filtrates were dried under a gentle stream of air at 40°C and reconstituted with distilled water. Glucose concentration was determined by the glucose oxidase method (Kit 510-A, Sigma Chemical, St. Louis, MO) on a small aliquot; the remainder was dissolved in scintillation fluid (Liquis- cent, National Diagnostics, Manville, N J ) and radioactivity was quantified using a liquid scintillation analyzer (model 2200Ca, Tricarb, Packard Instru- ment, Downers Grove, IL). Aliquots of infusate were also dried under a gentle stream of air, reconstituted in water, and mixed with scintillation fluid and radioactivity was quantified.

Perchloric acid extracts were prepared by mixing equal volumes ( 1 mL) of blood and ice-cold perchloric acid (10% wthol). Filtrates were obtained by centrifugation (31,000 x g at 4"C, 30 min) and neutralized with KOH (20% wthol). The potassium perchlorate salts were removed by centrifugation and the superna- tant stored (-85°C) until analyzed for lactate by a spectrophotometric enzymatic method (Boisclair et al., 1993b). Trichloroacetic acid filtrates of blood were also prepared and PAH determined by a modification of the method of Katz and Bergman (1969).

4000

G 1000

0

1000

750 S=! z 2 500 - 250

0

Animal #7231 Control Period

-60 -30 0 30 60 90 120 150

Time, mln

Figure 2. Blood glucose concentrations and specific radioactivity (SW), plasma insulin concentrations, and exogenous glucose infusion rate under basal conditions (-60 to 0 min) and during the hyperinsulinemic/ euglycemic clamp (0 to 180 min) in a control steer.



2266 DUNSHEA

Arterial plasma was analyzed for nonesterified fatty acids (NEFA) by an enzymatic kit (NEFA-C kit, Wako Chemicals USA, Dallas, TX) using the modifications of Sechen et al. (1990). Serum insulin was determined by a specific RIA as previously described (Boisclair et al., 1993b).

Calculations and Sfafisfics. Whole-body glucose irreversible loss rate ( ILR) was calculated by dividing the infusion rate of [6-3H]glucose by arterial blood glucose SRA. Endogenous glucose production (which includes gluconeogenesis and any glucose absorbed from the diet) was calculated by subtracting the exogenous glucose infusion rate from glucose ILR. Net fluxes of nutrients across the hindlimb were calculated as the product of hindlimb blood flow and arteriovenous differences. Because the main interests in this study were the effects of bST on basal metabolism and on metabolism during the hyperinsulinemic/ euglycemic clamp, separate ANOVA were performed on each variable under basal and hyperinsulinemic conditions as well as on the difference between the two states. The model included steer, period, and bST treatment effects. Because there were no period x bST treatment interactions for any variable, the effect of period has not been presented. For basal insulin levels, the variances for the two treatments were found to be heterogenous by Bartlett's test. Therefore, data were transformed on In basis, which yielded homogenous variances; these transformed data were used in the statistical analysis.

Results

Whole Body. Treatment of growing steers with bST resulted in a 7% increase in basal concentration of blood glucose and a 150% increase in serum insulin without altering glucose ILR (Table 2). Basal plasma

ET AL.

NEFA were also elevated, whereas blood lactate was unchanged during bST treatment (Table 2).

Infusion of insulin markedly increased serum insulin concentrations but simultaneous infusion of exogenous glucose ensured that euglycemia was maintained (Table 2, Figure 2). Hyperinsulinemia increased glucose ILR during both treatment periods, although this increase was reduced by bST treatment (Tables 2 and 3) . In part, this was due to that fact that endogenous glucose production was higher during hyperinsulinemia in bST-treated steers (Table 2, Figure 3 ) because insulin tended ( P = .l l) to be less able to inhibit endogenous production in these steers (Table 3). Insulin was antilipolytic, as evidenced by a reduction in plasma NEFA concentrations during hyperinsulinemia (Table 2). However, because basal NEFA concentrations were higher the insulin-mediated decrease was greater during bST treatment (Table 3) . Also, plasma NEFA tended ( P = .12) to remain slightly higher in bST-treated steers during hyperinsulinemia.

Hindlimb. Somatotropin treatment had no effect on hindlimb blood flow (Table 4). Blood flow increased by approximately 16% during hyperinsulinemia, and the magnitude was similar for both treatment periods. Although the basal hindlimb arteriovenous ( A-V) difference for glucose was 37% lower during bST treatment, this was not significant ( P = .16). The net uptake of lactate by the hindlimb in the control period was reversed to a net output during bST treatment. Basal oxygen A-V was similar during the control and bST treatment periods. However, the ratio of A-V difference for glucose (corrected for lactate) to oxygen was reduced during bST treatment. Because hindlimb blood flow was unchanged, hindlimb uptakes of glucose, lactate, and oxygen responded similarly to A-V differences with bST treatment.

Although hyperinsulinemia increased A-V differences for blood glucose by the hindlimb to a similar

Table 2. Effects of bovine somatotropin and hyperinsulinemia on blood glucose concentrations and kinetics and on circulating concentrations of insulin, nonesterified fatty acids, and lactate in growing steersa

Basal Hyper-INSc Effect of bST

Variable Control bST SED^ Control bST SED Basal Hyper-INS

Blood glucose, p m o n 4,280 4,590 65 4,180 4,420 194 .01 .27 Serum insulin, mU/Ld 22 56 13 62 1 652 26 .01 .31 Glucose irreversible loss, pmol/(min.kg) 13.1 13.3 .6 33.8 29.9 .9 .73 .01

Endogenous glucose production, pmol/(min.kg) 13.1 13.3 .6 6.0 8.5 .7 .73 .02

Plasma NEFA, pmol/L 111 190 15 68 87 10 .01 . l2 Blood lactate, pmol/L 550 630 53 594 630 21 .21 .l6

aTreatment involved daily i.m. injections of bovine somatotropin (bST; 120 pgkg of BW) or excipient (control). Studies were conducted from 2 to 5 h (Basal) and 5 to 8 h (hyperinsulinemia) after the 15th daily injection of bST or excipient. Values are based on samples taken over the last 50 min of both the basal and hyperinsulinemic period.

bSED, standard error of the difference. CHyperinsulinemic/euglycemic clamp (Hyper-INS) was achieved by continuously infusing bovine insulin 5.0 mU/(min.kg) while simul-

dFor basal only, variance was heterogenous. Therefore, in this case statistical analyses were conducted on In-transformed values. taneously infusing sufficient glucose t o maintain euglycemia.




Table 3. Effects of bovine somatotropin on the incremental response in blood glucose concentrations and kinetics and circulating insulin, nonesterified fatty acids, and lactate

during a hyperinsulinemic/euglycemic clamp in growing steersab

Delta-INSc

Variable Control bST SED^ Effect of bST

Blood glucose, pmol/L -110 -170 152 6 9 Serum insulin, mU/L 600 596 32 .90 Glucose irreversible loss, ymol/(min.kg) 20.7 16.6 .8 .o 1 Endogenous glucose production, pmol/(min.kg) -7.1 4.8 1.1 . l 1 Plasma NEFA, pmol/L -43 -103 17 .03 Blood lactate, pmoVL 44 0 49 .42

aTreatment involved daily i.m. injections of bovine somatotropin (bST; 120 ygkg of BW) or excipient (Control). Studies were conducted

bHyperinsulinemic/euglycemic clamp (Hyper-INS) was achieved by continuously infusing bovine insulin (5.0 mU/[min.kg]) while

'Samples were taken over the last 50 min of both the basal and hyperinsulinemic period and values represent the difference between the

dSED, standard error of the difference.

from 2 to 5 h (Basal) and 5 to 8 h (hyperinsulinemia) after the 15th daily injection of bST or excipient.

simultaneously infusing sufficient glucose to maintain euglycemia.

two periods (Delta-INS).

extent during both treatment periods (Table 5), the absolute A-V difference during hyperinsulinemia tended to be lower ( P = .11) during the bST treatment period (Table 4). Oxygen A-V decreased in the control period and increased during bST treatment in re-

- C

E S - 0

E,

4 i

8 a is 0 0

30-

m -

10 -

n. v Control bST Control bST

Control Hyperinsullnemla

Figure 3. Effect of bST on endogenous (solid bars) and exogenous (shaded bars) glucose irreversible loss rate (ILR) under basal conditions and during a hyperinsulinemic/euglycemic clamp in steers. Whole-body glucose ILR was determined by isotope kinetic techniques, exogenous glucose infusion rate was measured directly and endogenous glucose production rate was calculated by difference. SE (units equal prnol.min-l.kg BW-') were .6, .7, and .9 for endogenous glucose ILR during the basal period, endogenous glucose ILR during the hyperinsulinemic/euglycemic clamp, and exogenous glucose ILR during the hyperinsulinemic/euglycemic clamp, respectively.

sponse to hyperinsulinemia (Table 5 ) . Consequently, oxygen A-V tended ( P = . l o ) to be lower during hyperinsulinemia in bST-treated steers (Table 4). Hyperinsulinemia increased the ratio of glucose t o oxygen consumed by the hindlimb, although to a lesser extent in the bST-treated steers (Table 5). Consequently, the ratio of glucose to oxygen consumed by the hindlimb was considerably lower in response to hyperinsulinemia in the bST-treated steers. Although hyperinsulinemia increased glucose uptake by the hindlimb to a similar extent in both control and bST- treated steers, glucose uptake by the hindlimb was still lower in the bST-treated steers.

Discussion

Effects of bST on Basal Glucose Metabolism. We and others have observed increased circulating glucose and(or) insulin during bST treatment of growing cattle, consistent with the decreased tissue responses to insulin (Eisemann et al., 1986b, 1989; Crooker et al., 1990). Although basal blood glucose increased 7% and serum insulin more than doubled during bST treatment, glucose ILR was unchanged, suggesting a reduction in the ability of insulin to stimulate glucose uptake by peripheral tissues and(or) inhibit hepatic gluconeogenesis. Possible sites of inhibition of insulin- stimulated peripheral tissue uptake include skeletal muscle and adipose tissue. Because the hindlimb of the steer is composed predominantly of skeletal tissue with very little adipose tissue and catheters can be maintained for prolonged periods, it offers an excellent model of in vivo skeletal muscle metabolism (Boisclair et al., 1993a).

All indices of hindlimb glucose metabolism tended t o decrease during bST treatment. For example, glucose uptake by the hindlimb was 36% lower during bST treatment despite the large increase in basal



2268 DUNSHEA ET AL.

Table 4. Effects of bovine somatotropin and hyperinsulinemia on hindlimb blood flow and glucose, lactate, and oxygen metabolism in growth steersa

Basal Hyper-INSc Effect of bST

Variable Control bST SED^ Control bST SED Basal Hyper-INS

Blood flow, mumin 1,591 1,699 115 1,807 2,003 139 .40 .23 Hindlimb blood arteriovenous

differences Glucose, pmoYL 232 147 49 561 447 55 . l6 .l1 Lactate, pmoVL 20 -9 7 6 -24 26 .02 .32 Glucose + lactate/2, pmol/L 242 142 51 564 435 64 . l2 . l2 Oxygen, pmoVL 2,613 2,533 71 2,375 2,602 107 .33 . l0 G.(Glucose + lactate/2):02 .57 .36 .09 1.43 1.03 . l 1 .08 .02

Glucose, pmoWmin 368 239 78 1,055 889 70 .l7 .08 Lactate, NmoWmin 41 -9 14 15 -49 52 .02 .29 Oxygen, pmoWmin 4,186 4,321 204 4,340 5,200 291 .54 .04

Net hindlimb uptake

aTreatment involved daily i.m. injections of bovine somatotropin (bST; 120 @kg of BW) or excipient (control). Studies were conducted from 2 to 5 h (Basal) and 5 to 8 h (hyperinsulinemia) after the 15th daily injection of bST or excipient. Values are based on samples taken over the last 50 min of both the basal and hyperinsulinemic period.

bSED, standard error of the difference. CHyperinsulinemideuglycemic clamp (Hyper-INS) was achieved by continuously infusing bovine insulin (5.0 mU/[min.kg]) while

simultaneously infusing sufficient glucose to maintain euglycemia.

concentrations of serum insulin. A comparable reduction (22%) was observed in these same steers on d 11 and 13 of bST treatment ( P = .03; Boisclair et al., 1994). Treatment with oST caused a 40% reduction in hindlimb glucose uptake while increasing circulating insulin (Lakehal et al., 1989). Likewise, Eisemann ( 1989) reported a 32% reduction in glucose uptake by the hindquarters of steers treated with bST, although in that study circulating insulin remained unchanged. In one study on growing lambs, ST chronically increased circulating insulin but did not change glucose uptake by the hindquarters (Elcock et al.,

1988). Overall, these data suggest that ST administration reduces skeletal muscle sensitivity and(or) responsiveness to insulin.

Adipose tissue clearly decreases its rate of glucose utilization during ST treatment. Treatment of growing ruminants with ST has almost universally resulted in decreased lipid accretion (Eisemann et al., 198613; Sinnett-Smith and Woolliams, 1989; Zainur et al., 1989). Also, lipogenesis from acetate and fatty acid esterification are dramatically reduced in adipose tissue from ST-treated cattle and sheep (Peters, 1986; Pell, 1989; Sinnett-Smith and Woolliams, 1989).

Table 5. Effects of bovine somatotropin on the incremental response in hindlimb blood flow and glucose, lactate, and oxygen metabolism during a hyperinsulinemic/euglycemic clamp in growth steersab

~ ~

Delta-INSc

Variable Control bST SED^ Effect of bST

Blood flow, mumin 216 304 103 .44 Hindlimb blood arteriovenous differences

Glucose, pmoVL 329 301 44 .55 Lactate, pmoVL -14 -15 26 .96 Glucose + lactate/2, pmol/L 323 293 44 .53 Oxygen, pmol/L -237 68 92 .03 64Glucose + lactate/2):02 .86 .67 .08 .07

Glucose, pmoVmin 688 657 66 .67 Lactate, FmoVmin -26 -40 53 .79 Oxygen, pmoVmin 160 880 300 .07

Net hindlimb uptake

aTreatment involved daily i.m. injections of bovine somatotropin (bST; 120 pgkg of BW) or excipient (Control). Studies were conducted

bHyperinsulinemideuglycemic clamp (Hyper-INS) was achieved by continuously infusing bovine insulin (5.0 mU/min.kg) while simul-

'Samples were taken over the last 50 min of both the basal and hyperinsulinemic period and values represent the difference between the

dSED, standard error of the difference.

from 2 to 5 h (Basal) and 5 to 8 h (hyperinsulinemia) after the 15th daily injection of bST or excipient.

taneously infusing sufficient glucose to maintain euglycemia.

two periods (Delta-INS).




Tissues that may use glucose spared from peripheral tissue include visceral tissues such as heart, kidney, gut, and liver. In general, ST treatment results in increased mass of these tissues in both ruminants and nonruminants. For example, chronic administration of bST t o growing cattle increased liver, gut, heart, lung, and kidney between 15 and 30% (Sandles and Peel, 1987; Early et al., 1990). These tissues all have a high metabolic rate; the liver and portal-drained viscera ( PDV) each account for approximately 25% of whole- body oxygen consumption (Ferrell, 1988). Therefore, even subtle changes in the mass of these tissues can dramatically affect energy expenditure and, possibly, glucose utilization.

The ruminant liver does not generally utilize glucose as an oxidizable substrate (van der Walt et al., 1983), presumably due to the lack of glucokinase (Ballard et al., 1969). Therefore, an increased hepatic demand for oxidizable substrates is unlikely to account for the glucose spared from peripheral use during bST treatment.

Portal-drained viscera, principally gut tissue, can utilize a considerable amount of glucose. Both net utilization and production of glucose by the PDV have been reported for ruminants, with net production generally occurring when concentrate diets were fed. By using isotope dilution across the PDV, van der Walt et al. (1983) and Bergman et al. (1974) showed simultaneous uptake and release of glucose by the PDV and estimated that this accounts for 16 to 22% of whole-body glucose utilization in sheep in a variety of metabolic states. Therefore, it is possible that the PDV may utilize glucose being spared from skeletal muscle during bST treatment. However, ST did not affect net glucose uptake across the PDV of steers (Eisemann, 1989) or lambs (Elcock et al., 1988).

The heart is another glucose-utilizing tissue, but it accounts for only approximately 5% of glucose ILR in the adult sheep (Fisher et al., 1980). Therefore, it is unlikely that the heart will consume much of the spared glucose. It should be borne in mind that glucose uptake by insulin-independent tissues, such as brain and nervous tissue, is concentration-dependent. Also, even insulin-dependent tissues have a component of glucose uptake that is insulin-independent. Thus, Janes et al. (1985) and Petterson et al. (1993) estimated that for the fed adult sheep approximately 80% of basal glucose entry rate can be accounted for by insulin-independent glucose utilization. Therefore, this component of glucose utilization would presumably increase when glycemia is increased during ST treatment (Gottesman et al., 1983).

In well-fed ruminants such as the steers in this study, the principal substrate for hepatic gluconeogenesis is propionate of ruminal origin (Elliot, 1980). Hepatic uptake and glucogenic use of this substrate are determined mainly by portal venous supply, and little by hormonal state (Brockman, 1990). It is

therefore not surprising that glucose ILR was unchanged by bST treatment because feed intake, and presumably rate of ruminal fermentation, were also unaffected. Other potential glucogenic precursors include amino acids, lactate, and glycerol. The lack of change in amino acid flux in bST-treated cattle, including the present animals (Eisemann et al., 1986a, 1989; Boisclair et al., 19941, together with concurrent reduction in amino acid catabolism (Eisemann et al., 1986a, 19891, suggests that gluconeogenesis from amino acids may be reduced by bST treatment. Conversely, small bST-induced increases in net release of lactate by hindlimb tissues (Table 4) and in fat mobilization (Table 2 ) may have modestly increased the contributions of lactate and glycerol to glucose ILR.

Basal concentrations of plasma NEFA were very low and were modestly increased by bST treatment, consistent with increased fat mobilization and use of NEFA as an energy source (Eisemann et al., 1986a,b). The significantly lower glucose to oxygen quotient across the hindlimb in the bST-treated steers together with unchanged quotients for most other metabolites (Boisclair et al., 1994) suggests an increased muscle reliance on NEFA. Due to simultaneous uptake and release of NEFA, net changes of NEFA are generally not observed across the hindlimb of fed, resting ruminants (Pethick and Dunshea, 1993). However, if a value of 20% is assumed for fractional NEFA uptake (Pethick and Dunshea, 19931, then potentially 15 and 27% of oxygen consumption could be accounted for by NEFA in the control of bST periods, respectively.

Effects of bST on Glucose Metabolism During Hyperinsulinemia. The level of insulin infused during the hyperinsulinemic/euglycemic clamp was chosen as that which would maximize whole-body glucose utilization and was based on preliminary studies in our laboratory as well as literature values (Janes et al., 1985; Petterson et al., 1993). Therefore, glucose uptake across the hindlimb under these conditions should represent the maximal capacity of this tissue bed. Because there were no significant differences in the insulin-stimulated increment in glucose uptake across the hindlimb, responsiveness of hindlimb tissues to insulin was not affected by bST. Rather, sensitivity of these tissues to insulin was decreased by bST treatment, thereby making more glucose availa- ble for utilization by other tissues.

Hyperinsulinemia increased glucose ILR to a lesser extent during bST treatment (Table 31, suggesting a decrease in whole-body responsiveness to insulin. However, the incremental response to insulin in glucose uptake by the hindlimb was unchanged by bST, suggesting no change in hindlimb responsiveness (Table 5). Absolute rates of glucose utilization by the whole body and hindlimb were lower during hyperinsulinemia in the bST-treated steers. These results



2270 DUNSHEA ET AL.

suggest that a component of the whole body not abundant in the hindlimb exhibits reduced responsiveness to insulin during bST treatment. One candidate is adipose tissue, which by mass constitutes only 2.5% of the tissue bed being served by our hindlimb preparation (Boisclair et al., 1993a), but 15% of the whole-body mass. In this context we have calculated that the change in whole-body (Dunshea et al., 1992) and hindlimb (Wray-Cahen et al., 1995) glucose utilization during pST treatment of growing pigs, under both basal and hyperinsulinemic conditions, can be accounted for by reduced adipose tissue utilization of glucose.

Somatotropin treatment also seemed to decrease the responsiveness of glucogenic tissues (presumably liver) to insulin because endogenous glucose production was reduced to a lower level during hyperinsulinemia in the control period than during bST administration. Hepatic sensitivity to insulin may also have been decreased by bST because treated steers had unchanged rates of glucose production despite a 2.5-fold increase in plasma insulin during the basal period. Thus, ST-induced alterations in hepatic and peripheral tissue responses to insulin appear to ensure an adequate supply of glucose for tissues not dependent on insulin by maintaining hepatic production and reducing peripheral tissue consumption of glucose.

It is notable that even during the control period, glucose production in these steers was reduced by only 55% in response to very high levels of serum insulin (>600 mU/L; Table 2). This contrasts with our own and others’ observations that similar or lower levels of hyperinsulinemia can virtually abolish endogenous glucogenesis in maintenance-fed sheep (Janes et al., 1985; Petterson et al., 1993). Although one should not totally discount the possible influences of species and stage of maturity, it is likely that most of the discrepancy is due to relative levels of energy intake and associated rates of ruminal propionate production. The importance of propionate as a glucogenic substrate in ruminants increases with energy intake and propionate supply (Elliot, 1980). Hepatic uptake and gluconeogenesis from this substrate are little affected by hyperinsulinemia in sheep (Brockman, 1990), in contrast to significant decreases in utilization of other glucose precursors, including amino acids, lactate, and glycerol (Brockman, 1985). We would therefore expect the endogenous glucose production (gluconeogenesis) of growing steers fed at more than twice maintenance to be less responsive to insulin than that of ewes at maintenance.

Insulin is also acutely anti-lipolytic; infusion of this hormone results in reductions in circulating NEFA in a dose-dependent manner. The circulating insulin concentration at which a 50% reduction in plasma NEFA is produced in maintenance-fed adult sheep in quite low, approximately 21 mU/L (Petterson et al., 1994). In the present study infusion of insulin

decreased plasma NEFA during both treatment periods. Although this reduction was augmented during the bST period, maximally insulin-suppressed plasma NEFA concentrations still tended to be greater during bST treatment (68 vs 87 pmolll, P = .12). Similar responses to insulin challenge occur in the bST- treated lactating dairy cow (Sechen et al. 1990). Maximally inhibited NEFA concentrations are assumed to be the result of insulin-independent, lipoprotein lipase-catalyzed hydrolysis of circulating triglycerides. Slightly higher plasma NEFA concentrations during hyperinsulinemia in steers treated with bST may reflect less NEFA being taken up by adipose tissue after hydrolysis of circulating triglycerides.

Implications

Treatment of growing steers with bST causes major changes in glucose metabolism. Skeletal muscle utilization of glucose was decreased despite maintenance of whole-body glucose production. Because circulating insulin concentrations were more than doubled during bST treatment, these changes in skeletal muscle and liver metabolism represent clear alterations in sensitivity and(or) responsiveness to insulin. By using the hyperinsulinemic/euglycemic clamp technique we have demonstrated that changes in skeletal muscle glucose metabolism are due to decreased sensitivity whereas both sensitivity and responsiveness are decreased in the liver, and possibly adipose tissue. The overall result is an improved glucose economy in the bST- treated steer, although the actual site of utilization of spared glucose is unknown. These findings are consistent with the coordinated effects of bST on tissue metabolism and previously observed effects on tissue growth and carcass composition of cattle. Further- more, results offer fruitful avenues for more fun- damental investigations of mechanisms of action at the cellular and subcellular level.

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