The Journal of Nutrition Vol. 128 No. 3 March 1998, pp. 505-511

Change in Insulin Sensitivity or Responsiveness Is Not a Major Component of the Mechanism of Action of Ractopamine in Beef Steers^1,2,3

Joan H. Eisemann⁴ and David G. Bristol^*

Departments of Animal Science and ^* Food Animal and Equine Medicine, North Carolina State University, Raleigh, NC 27695

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Our objective was to determine whether the -adrenergic agonist ractopamine altered sensitivity or responsiveness to insulin. We used the hyperinsulinemic, euglycemic clamp approach in five multicatheterized beef steers to evaluate insulin sensitivity (ED₅₀) and responsiveness (R_max or R_min) during control or ractopamine feeding (80 mg/kg feed). Steers had blood vessel catheters and ultrasound flow probes that allowed measurement of net uptake and release of glucose and insulin by portal-drained viscera (PDV), liver and hindlimb. Steers ate meals of equal size every 2 h. Steers were fed at 1.8 times calculated maintenance energy. The design was a single reversal. Two rates of insulin infusion followed a base-line period on each of three sample days. Insulin was infused into a mesenteric vein at 10, 20, 40, 80, 160 and 320 mU/(h·kg body weight). During the base-line period, arterial concentrations of glucose, oxygen, nonesterified fatty acids and insulin were not different between control and ractopamine feeding. Arterial urea was lower during ractopamine than during control feeding (5.02 vs. 6.20 mmol/L, respectively, P < 0.01). Net release of glucose by liver and net uptake of glucose by the hindlimb were not affected by treatment. Similarly, net release of insulin by PDV and net uptake of insulin by liver were not affected by treatment. The R_max and ED₅₀ for steady-state glucose infusion rate, total glucose entry, hepatic glucose production and hindlimb glucose uptake did not differ between treatments. There was a trend for a lower ED₅₀ in hindlimb with ractopamine treatment (P < 0.13). These data do not support a change in sensitivity or responsiveness of tissues to insulin as a major component of the mechanism of action of ractopamine.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The use of pharmacologic agents or growth promotants is widespread in farm animals (Buttery and Dawson 1990). In general, these agents are, or mimic, inherent endogenous regulators. Ractopamine and other -adrenergic agonists partition the nutrient supply to increase muscle growth and decrease fat deposition in animals (Moloney et al. 1991). Use of exogenous agents such as -agonists allows measurement of changes occurring during manipulated growth as a model for physiologic controls that may alter nutrient partitioning during normal growth.

The mechanisms of importance in regulation of nutrient partitioning during normal or manipulated growth may include a change in tissue sensitivity or responsiveness to a hormone or metabolite (Bauman et al. 1982). Insulin is a key metabolic hormone in ruminant and nonruminant animals. Regulation of nutrient partitioning through a change in tissue response to insulin was demonstrated during pregnancy and lactation (Vernon and Sasaki 1991). The effects of insulin are diverse and include aspects of glucose, amino acid and nonesterified fatty acid metabolism (Bell et al. 1987, Brockman and Laarveld 1986a).

Several studies suggest involvement of insulin in the mechanism of action of -adrenergic agonists that stimulate lean tissue growth. In cattle (Vestergaard and Sejrsen 1989) and sheep (Beermann et al. 1987), plasma insulin concentration decreased after 3 and 6 wk, respectively, of cimaterol treatment. A decrease in insulin response to a glucose challenge and a larger decrease in concentration of glucose after an insulin challenge were observed in bulls treated with cimaterol (Vestergaard and Sejrsen 1989). In response to clenbuterol feeding, insulin concentration decreased with no change in uptake of glucose by the hindquarters of cattle (Eisemann and Huntington 1988). The two last-mentioned reports may indicate an increase in sensitivity or responsiveness to insulin after the feeding of -adrenergic agonists.

The euglycemic clamp technique is a useful approach to the study of insulin sensitivity and responsiveness in vivo (Defronzo et al. 1979). The principle of the clamp is to control blood concentration of glucose under conditions of hyperinsulinemia and thus break the insulin-glucose feedback loop. Thus, one is able to study the effect of insulin on metabolic processes without the confounding effects of hypoglycemia. This approach has been used previously to estimate the sensitivity and responsiveness of liver and peripheral tissues to insulin in growing, euglycemic beef steers (Eisemann et al. 1994). In previous studies, the entire hindquarters was used to estimate peripheral response. In this study, the peripheral response is measured in a portion of the hindquarters only and should be more reflective of muscle metabolism (Oddy and Lindsay 1986).

The objective of this study was to use the euglycemic hyperinsulinemic clamp technique in multicatheterized beef steers to determine whether insulin sensitivity or responsiveness of tissues was altered by ractopamine treatment.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Six Hereford × Angus steers [486.7 ± 15.8 kg body weight (BW)⁵] were used in the experiment. They were gentled and adapted to housing in individual stalls for at least 1 mo before the start of the treatment period. They were fed a medium energy diet (Table 1) at 0.979 MJ metabolizable energy/kg BW^0.75. Feed offered and feed refused were recorded on a daily basis. Animals were fed their rations every 2 h in 12 equal portions. Water was freely available at all times. Animals were weighed on a weekly basis and amount of feed offered adjusted as necessary. The experimental protocol was approved by the Institutional Animal Care and Use Committee.

All steers were necropsied at the end of the experiment to verify placement of catheters. The data from one steer was deleted from the study because the iliac catheter was located in the contralateral vein relative to the flow probe on the iliac artery.

Surgery.  Feed was withheld for 48 h and water removed for 24 h before surgery to install chronic indwelling catheters with tips in the abdominal aorta, external iliac vein, hepatic-portal vein, hepatic vein and branches of the cranial mesenteric vein. An ultrasonic flow probe (12 mm, Transonics Systems, Ithaca, NY) was placed around the external iliac artery for measurement of blood flow to the hindlimb. Procedures for catheterization of the hindlimb were as described by Eisemann and Nienaber (1990) except that the venous catheter was threaded caudally in the vena cava until the tip was in the external iliac vein. Hepatic-portal, hepatic and mesenteric catheterizations were as described by Huntington et al. (1989). Steers were allowed a minimum of 2 wk for recovery from surgery before treatment initiation.

Design.  The design of the experiement was a single reversal with two 15-d periods of either control feed or ractopamine-treated feed. The ractopamine dose was 80 mg/kg feed. This dose of ractopamine was chosen to produce marked changes in nutrient partitioning on the basis of dose-response studies in beef cattle (Anderson et al. 1989) and previous metabolic studies (Eisemann et al. 1993). Between each 15-d period there was a 13-d interim period of no treatment to minimize carryover effects. The experiment was conducted in pairs. One steer was assigned to the control treatment for period 1 and the other steer was assigned to ractopamine.

Blood sampling.  Infusions were conducted and blood samples were taken on d 9, 12 and 15 of each period. There were three blood sampling periods on each day (Fig. 1). The first period was a base-line sampling, and the other two were during the two insulin infusions (described below). Four sets of blood samples were taken simultaneously from the abdominal aorta, hepatic-portal vein, hepatic vein and external iliac vein at 20-min intervals during each blood sampling period. Blood was withdrawn anaerobically into 3-mL heparinized syringes, capped with a rubber stopper and kept on ice until analyzed for O₂ saturation and hemoglobin concentration with the use of a Hemoximeter (OSM2; Radiometer, Copenhagen, Denmark). A second blood sample was taken from all four catheters, into syringes containing disodium ethylenediamine tetraacetate as an anticoagulant, for measurement of other variables and immediately placed on ice. The feeding schedule was maintained throughout sampling except when a meal coincided with blood sampling during one of the insulin infusions. On those occasions, the meal was delayed until the completion of sampling at that insulin dose.

View larger version (15K): [in this window] [in a new window]   Fig 1. Diagram of the sampling protocol. Blood samples were taken simultaneously from the aorta, portal vein, hepatic vein and external iliac vein. The first set of blood samples was the base-line blood sampling period. PAH, p-aminohippuric acid.

Glucose concentration was measured in blood from all vessels immediately after each sample time by using a glucose analyzer (Yellow Springs Instruments, Yellow Springs, OH). Blood was centrifuged for 20 min at 2500 × g at 4°C to obtain plasma. Blood and plasma were diluted 1 + 3 with deionized distilled water for analysis of p-aminohippurate (PAH) in blood from all vessels except the external iliac vein on the day of sampling by using automated procedures (Eisemann and Nienaber 1990). Plasma was stored frozen for later analysis of insulin (all vessels except the external iliac vein) with the use of an antibody-coated tube assay (ICN Biomedicals, Costa Mesa, CA); nonesterified fatty acids (NEFA) were analyzed enzymatically (artery only; NEFA-C kit, Wako Chemicals, Dallas, TX).

Indicator dilution (PAH) was used to measure blood flow in the portal-drained viscera (PDV) and hepatic tissues. A primed continuous infusion of PAH was initiated into a branch of the cranial mesenteric vein 60 min before each sampling period; the infusion rate was 8640 mg/h. The infusion was discontinued between sampling periods to conserve PAH. The ultrasonic flow probe was used for measurement of blood flow to the hindlimb. Blood flow was recorded every 10 s over the sampling interval and an average flow calculated.

Hyperinsulinemic, euglycemic clamp.  A primed, continuous infusion of insulin into the mesenteric venous system was begun as soon as the first blood sampling period was completed. The doses of insulin (26.6 IU/mg, Eli Lilly, Greenfield, IN; or 24.4 IU/mg, Sigma Chemical, St. Louis, MO) were 10, 20, 40, 80, 160 and 320 mU/(h·kg BW). The range of insulin doses was chosen in order to construct a complete insulin dose-response curve (Eisemann et al. 1997). The doses were paired: 10, 80; 20, 160 and 40, 320 mU/(h·kg BW) for the three infusion days. The lower dose always preceded the higher dose on a sampling day. The order of receiving the paired doses was randomly assigned for each steer in each treatment period. A stock solution of insulin 0.005 mol/L HCl was diluted as appropriate in a sterile solution of 0.15 mol/L NaCl in 0.01 mol/L phosphate at pH 7.4 with 1 g/L bovine serum albumin (RIA grade, Sigma Chemical). Infusate solutions were prepared the evening before an infusion and sampling day.

On the evening of the day before the first sampling day in a period, a catheter was inserted into the jugular vein for infusion of glucose. Local injection of lidocaine-HCl was used before catheter insertion. A sterile solution of 2.0 mol/L glucose in deionized distilled water was infused from the start of the primer for the lower dose of insulin until ~1 h beyond the end of infusion of the higher insulin dose. The infusion rate of glucose was adjusted (model 22 Pump, Harvard Instruments, Natick, MA) as necessary to maintain euglycemia. Blood samples were taken approximately every 10 min to monitor glucose concentration.

Calculations.  Net flux of variables across the PDV, liver and hindlimb was calculated as described previously (Eisemann and Nienaber 1990). Data on steady-state glucose infusion rate (SSGIR), hindlimb glucose uptake and total glucose entry (TGE; sum of hepatic glucose production, portal glucose absorption and glucose infused) were fitted to a three-parameter rising logistic equation, and estimates of the insulin concentration required for 50% maximal response (ED₅₀) and for maximal response were made for each animal. A similar falling logistic equation was used for hepatic glucose production. An estimate of insulin independent glucose use was made by regressing total glucose entry on arterial insulin concentration over the linear range (<1320 pmol insulin/L).

Statistics.  Statistical analysis of the data was performed using SAS (SAS 1990). Data on variables during the base-line blood sampling period were analyzed as a single reversal with steer, treatment and steer × treatment as the main effects. The effect of treatment was tested using the steer × treatment interaction as the error term. Parameters estimated from the NLIN procedure of SAS for SSGIR, hindlimb glucose uptake, hepatic glucose production and TGE were analyzed by using the same model. Additional variables measured during infusion of insulin were analyzed by using a model containing steer, treatment, steer × treatment, insulin dose, insulin dose × treatment and insulin dose × steer. The insulin dose was converted to a logarithmic value to generate equal spacing between dose levels. The effect of treatment was tested by using steer × treatment as the error term. The effect of insulin and individual contrasts designed to evaluate the surface response to insulin were tested by using insulin dose × steer as the error term. A probability level of P < 0.10 was considered significant.

	RESULTS

Abstract Introduction Methods Results Discussion References

Base-line period.  Arterial concentrations of glucose, insulin, oxygen and NEFA did not differ during the base-line period when steers were fed control compared with ractopamine-treated feed (Table 2). Concentration of urea was lower when steers were fed ractopamine-treated feed than control feed. Blood flow to tissues of the hindlimb increased with ractopamine feeding; however, blood flow to other tissues measured did not differ between treatments.

View larger version (13K): [in this window] [in a new window]   Fig 2. Relationship between steady-state glucose infusion rate (SSGIR) and arterial plasma insulin concentration in steers fed control or ractopamine-treated feed. Values represent the mean of five steers. The pooled SEM was 63.3 µmol/(h·kg body weight).

Uptake of oxygen by liver decreased and by the hindlimb tended to increase during ractopamine feeding (Table 3). Uptake of oxygen by tissues of the PDV was unchanged. Net uptake of glucose by tissues of the PDV decreased during treatment with ractopamine. Neither net release of glucose by the liver nor net uptake by the hindlimb was altered by ractopamine feeding. There was no effect of ractopamine feeding on PDV release or hepatic uptake of insulin. Extraction of both glucose and oxygen by the hindlimb decreased with ractopamine feeding. Extraction of insulin by the liver was ~14% and unaffected by treatment.

Hyperinsulinemic, euglycemic clamp.  Arterial concentration of insulin increased in conjunction with infusion of increasing doses of insulin from base-line values of ~300 pmol/L (Table 2) to >10,600 pmol/L at the highest rate of insulin infusion (Table 4). Similarly, both release of insulin by the PDV (sum of endogenous and exogenous insulin production) and uptake of insulin by the liver increased as the dose of infused insulin increased. Conversely, extraction of insulin by the liver decreased from 14.2 to 4.2% and metabolic clearance rate (MCR) decreased from 499 to 199 mL/(h·kg) as insulin concentration increased.

Arterial concentration of glucose was unchanged during infusion of insulin (Table 4). Extraction of glucose by the hindlimb increased from 7.4 to 19.2%, and MCR increased from 237 to 563 mL/(h·kg). There was no effect of ractopamine feeding on concentration of insulin and glucose and associated kinetic variables listed in Table 4 during infusion of insulin (data not shown). Across both treatments, the estimated insulin-independent glucose use was 77.5% of total glucose entry, i.e., 656 ± 47.6 µmol/(h·kg).

The curvilinear relationship between the steady-state glucose infusion rate required to maintain euglycemia and arterial insulin concentration is shown in Figure 2. The insulin dose-response parameters estimated from the curve for each steer (Table 5) show that ractopamine did not alter the maximum response, R_max, nor the sensitivity, ED₅₀, to insulin.

The curvilinear relationship between the change in total glucose entry and arterial concentration is shown in Figure 3. Treatment with ractopamine did not affect the maximum response nor the insulin concentration required for half-maximal response (Table 5).

View larger version (13K): [in this window] [in a new window]   Fig 3. Relationship between the change in total glucose entry (TGE) and arterial plasma insulin concentration in steers fed control or ractopamine-treated feed. Values represent the mean of five steers. The pooled SEM was 90.7 µmol/(h·kg body weight).

The curvilinear relationships between change in hepatic glucose production (Fig. 4) and change in hindlimb glucose uptake (Fig. 5) relative to arterial insulin concentration show no effect of ractopamine treatment on estimated parameters for sensitivity and responsiveness (Table 5). There was a trend toward increased sensitivity to insulin for hindlimb glucose uptake (P < 0.13) during ractopamine treatment.

View larger version (13K): [in this window] [in a new window]   Fig 4. Relationship between the change in hepatic glucose production (HGP) and arterial plasma insulin concentration in steers fed control or ractopamine-treated feed. Values represent the mean of five steers. The pooled SEM was 45.6 µmol/(h·kg body weight).

View larger version (13K): [in this window] [in a new window]   Fig 5. Relationship between the change in hindlimb (HL) glucose uptake and arterial plasma insulin concentration in steers fed control or ractopamine-treated feed. Values represent the mean of five steers. The pooled SEM was 10.8 µmol/(h·kg body weight).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Our objective was to evaluate the effects of chronic administration of ractopamine on insulin sensitivity in the bovine hindlimb. Previous in vivo studies with clenbuterol (Eisemann et al. 1988) or other -adrenergic agonists (Zimmerli and Blum 1989) show a large difference in metabolic response after initial ingestion of a -adrenergic agonist compared with ingestion after 1-4 wk of adaptation. The change is likely due to desensitization of -adrenergic receptors (Mills and Mersmann 1995). For example, in regard to glucose (Eisemann et al. 1988, Zimmerli and Blum 1989), there was a dramatic increase in concentration acutely; however, there was no effect on glucose concentration after a week or more of treatment in cattle. It is the chronic changes that should be important in enhancing lean tissue growth.

Neither concentration of glucose nor insulin responded to treatment with ractopamine over the 15-d treatment period (Table 2). This observation was surprising in light of previous research with cimaterol (Beermann et al. 1987, Vestergaard and Sejrsen 1989) and clenbuterol (Eisemann and Huntington 1988). Hepatic release of glucose and uptake of glucose by the hindlimb were not altered by ractopamine feeding. Further, release of insulin by PDV, uptake by the liver and MCR were unchanged with ractopamine treatment (Tables 3 and 4). The only change in flux of glucose or insulin under basal conditions was decreased net uptake of glucose by the PDV, which could reflect decreased use of glucose by tissues of the PDV or an increase in the amount of luminal glucose presented for absorption. Decreased use of glucose by adipose tissue in the PDV is one possible explanation consistent with decreased adipose accumulation in animals treated with -adrenergic agonists (Moloney et al. 1991). Plasma insulin concentration decreased with no corresponding change in concentration of glucose in previous studies in which ram (O'Connor et al. 1991) or wether (Beermann et al. 1987) lambs were treated with cimaterol for several weeks, suggesting a possible change in tissue response to insulin. In response to clenbuterol, insulin concentration decreased with no change in glucose uptake by tissues of the hindquarters of cattle (Eisemann and Huntington 1988). However, Chikhou et al. (1991) observed no change in concentration of glucose or insulin after 13 d of cimaterol treatment in steers. Zimmerli and Blum (1989) observed both a similar change in concentration of glucose and insulin after feeding and no difference in glucose or insulin responses to insulin injections between control calves and those receiving -adrenergic agonists for 2-4 wk. It is possible that sheep respond differently than cattle in regard to insulin, as suggested by their differing response to cimaterol. These studies also support the idea that the effect of various -adrenergic agonists on flux of insulin or tissue response to insulin is not the same. Additional factors such as relative dose-response curve for each -adrenergic agonist, time course of response in different species relative to time of sampling, physiologic age of the animals studied and differential metabolism of each compound likely contribute to the lack of a generalized response across both species and -adrenergic agonists.

Studies with rats do not clarify the role of insulin in the response of animals to -adrenergic agonists that also stimulate lean tissue growth. Obese Zucker rats given an oral glucose load showed decreased response in both plasma glucose and insulin after 5 wk of clenbuterol treatment compared with obese controls that did not receive clenbuterol, suggesting enhanced clearance of glucose and insulin sensitivity. However, insulin-stimulated muscle glucose uptake and 3-O-methyl glucose transport were not increased in the perfused hindlimb (Torgan et al. 1993). In normal rats, insulin stimulated uptake of 3-O-methyl glucose and 2-aminoisobutyric acid in muscle from control animals but not rats treated with cimaterol for 11 d. Similarly, protein synthesis increased in response to insulin in muscle from control rats but not rats treated for 11 d with cimaterol (Sainz et al. 1990). These two studies support decreased response of muscle to insulin after cimaterol treatment. The increased whole-body sensitivity observed in the obese animals may not be due to increased sensitivity in skeletal muscle but changes in adipose or liver tissue. Weight of muscle increased in diabetic rats that were treated with clenbuterol for 7 d, suggesting that circulating insulin concentration does not totally mediate the effect of clenbuterol; however, it does not rule out the need for insulin for maximal effect or a role for changes in sensitivity to insulin in the response (McElligott et al. 1987).

There are studies with other adrenergic agonists supporting a role in enhanced insulin sensitivity. Earlier studies showed that epinephrine increased binding of insulin to skeletal muscle in the rat hindlimb (Webster et al. 1986a), which was blocked by the -blocker propanolol in short term in vitro incubations (Webster et al. 1986b). After infusion of norepinephrine for 10 d to rats, sensitivity of peripheral tissues increased without change in the basal concentration of glucose or insulin. Infusion of insulin and propanolol decreased slightly the increase in glucose disposal due to insulin (Lupien et al. 1990). Glucose transport, glycogen synthesis and lactate formation in isolated soleus muscle were increased at a given insulin dose after 120 h of epinephrine implantation. The sensitivity of glucose transport to insulin decreased initially, returned to control levels and then increased (Budohoski et al. 1987).

Observations of interactions between -adrenergic agonists and insulin, which have antagonistic effects in adipose tissue, are variable. Adipocytes isolated from mice receiving clenbuterol or ractopamine in their drinking water for 5 d showed increased insulin binding (Dubrovin et al. 1990). However, Mills et al. (1990) did not find a change in insulin binding to adipocytes isolated from ractopamine-treated pigs compared with control pigs. When exposed to ractopamine in vitro, isolated adipocytes of rats showed decreased sensitivity and responsiveness to insulin (Hausman et al. 1989), and isolated adipocytes from pigs showed decreased insulin binding (Liu and Mills 1990). The contrast between the effect on insulin binding observed after treatment with -adrenergic agonists in vivo or in vitro may be similar to differences cited previously between acute and chronic administration in vivo. In addition, there are many other factors involved in the in vivo system.

Because we did not see a major effect on insulin sensitivity, a logical question is whether ractopamine elicited expected metabolic responses and whether it increased muscle protein accretion. The observed chronic elevation in heart rate has been reported previously in response to other -adrenergic agonists in cattle (Eisemann et al. 1988, Zimmerli and Blum 1990) and ractopamine in particular (Eisemann et al. 1993). Increased blood flow to the hindquarters in cattle (Eisemann et al. 1988 and 1993) or sheep (Beermann 1987) has been reported also in response to -adrenergic agonists. Decreased extraction of glucose and oxygen by the hindlimb is similar to observations in hindquarters of steers receiving clenbuterol (Eisemann et al. 1988) and is probably related to increased blood flow. The converse, increased extraction of oxygen by hindquarters along with decreased blood flow, was observed in food-deprived compared with fed steers (Eisemann and Nienaber 1990). The decline in urea nitrogen during ractopamine treatment indirectly supports increased nitrogen retention and an anabolic response. Long-term treatment with clenbuterol decreased urea nitrogen concentration in steers, coincident with increased protein deposition (Ricks et al. 1984). Although we did not measure changes in amino acid flux, which would relate more directly to protein metabolism, the fact that there were no major changes in glucose metabolism suggests that a major change in tissue response to insulin for amino acid or protein metabolism is unlikely. Estimation of unidirectional amino acid flux in the hindlimb, under hyperinsulinemic conditions while maintaining baseline concentrations of amino acids, would allow a direct test of changes in sensitivity to insulin for amino acid metabolism.

Concentrations of oxygen and several other metabolites and measurement of oxygen flux across tissues were included to better characterize the response to ractopamine. There were no chronic changes in concentrations of oxygen or nonesterified fatty acids. Observations of chronic changes in NEFA in cattle in response to -adrenergic agonists have been variable (Eisemann et al. 1988; Zimmerli and Blum 1989). Oxygen use by the liver decreased, indicating that ractopamine decreased energy-using processes in the liver. The decrease in urea concentration suggests nitrogen catabolism and urea formation as one energy-requiring process that may be decreased.

Insulin-independent glucose utilization accounted for 77% of glucose use during basal conditions. This is similar to previous observations of 72% in both cattle (Eisemann and Huntington 1994) and nonpregnant ewes (Petterson et al. 1993). Uptake of glucose by the hindlimb accounted for ~7% of hepatic glucose release. This value is slightly lower than estimates of ~10% in younger and lighter Holstein steers (Dunshea et al. 1995). Previous estimates for the entire hindquarters of beef steers were ~27% in animals of similar weight (Eisemann et al. 1996).

As observed previously (Dunshea et al. 1995, Eisemann and Huntington 1994, Eisemann et al. 1994 and 1997, Petterson et al. 1993, Weekes et al. 1983), hepatic glucose production decreased in response to infusion of insulin in fed cattle and sheep, but complete suppression of production did not occur. This likely relates to the lack of effect of insulin on propionate (the major carbon source for gluconeogenesis) use by the liver. Neither incorporation of radiolabeled propionate into glucose by liver of sheep (Brockman, 1990) nor net uptake of propionate by liver of steers (Eisemann and Huntington, 1994) was decreased during conditions of hyperinsulinemia and euglycemia. In contrast, uptake of other glucose precursors decreased under similar conditions (Brockman 1985, Brockman and Laarveld 1986b).

These data describe basal metabolic changes in cattle in response to treatment with ractopamine for 15 d as well as tissue sensitivity and response to insulin, indicated by changes in glucose flux, under hyperinsulinemic euglycemic conditions. By using chronic indwelling catheters, we were able to distinguish responses occurring in tissues of the hindlimb, which should contain ~60% muscle (Boisclair et al. 1993), from whole-body responses. Although there was a trend (P < 0.13) toward increased sensitivity of hindlimb tissues to insulin, the data do not support changes in insulin sensitivity or responsiveness as a major component of the mechanism of action of ractopamine in cattle.

	ACKNOWLEDGMENTS

The authors express their appreciation to S. Freeman and G. Ryan for feeding and care of the steers, D. Hardin for assistance at surgery, and G. Huntington, D. Herman, M. Creed, M. Talley and L. Brandon for technical assistance.

	FOOTNOTES

Manuscript received 14 July 1997. Initial reviews completed 4 September 1997. Revision accepted 17 November 1997.

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