Enhancement of the insulin antagonistic effect of porcine somatotropin in swine using a monoclonal antibody.

A monoclonal antibody (mAb), PS-7.6, to porcine somatotropin (pST) significantly enhanced the growth responses to pST injections in hypophysectomized (hypox) rats but could not be tested in pigs because of the large quantity of antibody required for a growth trial. Because pST inhibits the hypoglycemic effects of insulin, an insulin tolerance test procedure was established to measure pST activity in jugular-catheterized pigs. Doses of 0, 30, 100, and 300 micrograms/kg per day of pST were split and administered subcutaneously (sc) in equal portions twice daily for 2 d. After a 17-hr fast, plasma samples were obtained at 10-min intervals for 30 min before an intravenous injection of insulin (0.08 IU/kg) and then for an additional 50 min. Because pST increased fasting plasma glucose concentrations, preinsulin glucose values were used as a covariate to adjust the postinsulin concentrations. pST caused a dose-dependent increase in resistance to the insulin injection in these pigs. The areas under the curves (AUC), for plasma glucose were 22.1, 29.0, 39.0, and 47.2 mg/dl per min for the 0, 30, 100, and 300 micrograms/kg pST doses, respectively. Because different doses of pST could be detected, the PS-7.6 enhancement of pST treatment was evaluated. In the first experiment, five pigs/group each received sc injections of either vehicle, pST (75 micrograms/kg; approximately 3.0 mg/d), pST (75 micrograms/kg) + PS-7.6 at 3.75 mg/kg, or pST (75 micrograms/kg) + PS-7.6 at 15 mg/kg for 2 d before the insulin test. The pST and PS-7.6 were combined and incubated for at least 1 hr at room temperature before being injected. The injection of pST alone did not significantly change insulin tolerance activity (23.1 vs. 21.1, AUC), but insulin resistance was enhanced when this dose of pST also included PS-7.6 (27.4 and 29.5, AUC, respectively; P < 0.05). In a second experiment, the effects of PS-7.6 and PS-4.2, a mAb that did not potentiate the pST-stimulated growth of hypox rats, were compared. The five pigs/treatment received either vehicle, pST (75 micrograms/kg), pST (75 micrograms/kg) + PS-7.6 (3.75 mg/kg), or pST (75 micrograms/kg) + PS-4.2 (3.75 mg/kg) for 2 d. The administration of pST increased the resistance to insulin (26.7 vs. 18.8, AUC; P < 0.01), which was markedly potentiated by PS-7.6 (54.3, AUC, P < 0.001) but not affected by PS-4.2 (27.6 AUC). The injection of PS-7.6 at 7.5 mg/kg without exogenous pST did not alter the sensitivity to insulin. These results indicate that PS-7.6, but not PS-4.2, enhanced the insulin antagonistic activity of pST in swine, suggesting that an enhancement of pST-stimulated growth would also occur in PS-7.6-treated pigs.

Effects of alpha2-adrenoceptor antagonists on metabolic processes of swine: I. Effects on nonesterified fatty acid and plasma urea nitrogen concentrations in jugularly catheterized pigs.

The presence of alpha2-adrenoceptors in membranes from omental and s.c. adipose tissue from gilts and barrows was shown in saturation binding assays with [3H]yohimbine. Four trials tested effects of alpha2-adrenoceptor antagonists (A2AA) on plasma concentrations of NEFA and urea nitrogen (PUN). In Trial 1, barrows were given i.v. injections of saline, 200 microg/kg BW of one of three A2AA (efaroxan, idazoxan, or RX821002), or 25 microg/kg BW of isoproterenol. Concentrations of NEFA were measured in plasma harvested every 15 min from 1 h before to 2 h after treatment. Compared with results for saline-treated pigs, areas under the curve (AUC) for NEFA were increased (P < .05) by efaroxan, RX821002, and isoproterenol. In Trial 2, barrows received i.v. doses of saline, efaroxan (200 or 400 microg/kg BW), or RX821002 (200 or 400 microg/kg BW). Levels of NEFA were quantified in plasma obtained at 15-min intervals through 2 h after treatment. Among pigs treated with RX821002 at 400 microg/kg BW, mean NEFA AUC was more than three times greater (P < .05) than that for saline-treated animals. Trial 3 tested whether NEFA responses to A2AA were due to direct effects on alpha2-receptors or involved beta-adrenoceptor mediation. Pigs were first treated i.v. with saline or propranolol (1 mg/kg BW). One hour later, pigs were treated i.v. with RX821002 (400 microg/kg BW) or the beta-adrenoceptor agonist cimaterol (25 microg/kg BW). Compared to values for pigs treated with saline at both injections, NEFA AUC among pigs treated with saline at the first injection and RX821002 at the second doubled (P > .05). Plasma NEFA AUC among pigs treated with saline then cimaterol rose nearly fourfold (P < .05) compared with saline-treated controls. Mean NEFA AUC among propranolol-treated pigs was similar to values for saline-treated pigs, suggesting beta-adrenoceptor involvement in the effect of A2AA on NEFA. In Trial 4, pigs were treated s.c. 10 times at 8-h intervals with saline, RX821002 (400 microg/[kg BW x injection]), cimaterol (20 microg/[kg BW x injection]) or recombinant porcine somatotropin (rpST; 1 mg/[pig-injection]). After the 10th treatment, only cimaterol increased NEFA AUC compared to saline-treated controls (P < .05). Mean PUN AUC was reduced by RX821002 and rpST compared to controls; PUN among rpST-treated pigs was lower than that among RX821002-treated pigs (P < .05). In summary, A2AA increase lipolysis in swine by potentiating lipolytic effects of endogenous catecholamines on beta-adrenoceptors. Reduced PUN suggests improved nitrogen efficiency may result from treatment with A2AA.

Effects of alpha2-adrenoceptor antagonists on metabolic processes of swine: II. Nitrogen balance responses.

We studied the effects of alpha2-adrenoceptor antagonists (A2AA) on nitrogen (N) partitioning. The diets fed contained 19.8% CP and 1.15% lysine. Pigs were fed the diet as a percentage of BW equaling approximately 90% of voluntary intake. In Trial 1, pigs (n = 11/treatment) were fed a basal diet and injected s.c. at 8-h intervals for 11 d with saline, RX821002 (25 mg/injection), or cimaterol (.6 mg/injection). Compared to saline-treated pigs, urinary N, as a percentage of N eaten, decreased among pigs injected with RX821002 (15%, P < .05) or cimaterol (17%, P < .05). In Trial 2, pigs got saline (n = 6) or 25 mg RX821002 (n = 6) as s.c. injections three times daily, or they were fed a diet containing 150 ppm RX821002 and injected thrice daily with saline (n = 6) for 11 d. The RX821002 lowered apparent DM and N digestibility (P < .05). Compared to controls, RX821002 lowered urinary N, as a percentage of N eaten, 15 and 18% when given by injections or per os, respectively, but effects were not significant. Trial 3 evaluated the effects of RX821002 fed at levels of 0 (n = 6), 37.5 (n = 5), 75 (n = 6), or 150 ppm (n = 6). Contrasts showed linear dose-dependent decreases in gain and apparent N digestibility (P < .05). Compared to untreated controls, urinary N, expressed as a percentage of N consumed, decreased 2, 12, and 10% among pigs fed diets with 37.5, 75, or 150 ppm RX821002, respectively, but effects were not significant. Trial 4 compared N balance in pigs (n = 6/treatment) fed basal diet or diet with 100 ppm RX821002 to that of pigs fed diets with 25 or 100 ppm yohimbine. Treatments reduced apparent N and DM digestibility (P < .05). Urinary N, as a percentage of N consumed, decreased 16 (P > .05), 18 (P < .05), and 24% (P < .05) for 100 ppm RX821002, 25 ppm yohimbine, or 100 ppm yohimbine, respectively. Data from Trials 2, 3, and 4 from control pigs (n = 18) or pigs fed A2AA (all A2AA sources and doses; n = 41) were pooled and analyzed. Feeding A2AA decreased apparent N and DM digestibility (P < .01). The fact that fecal moisture content was higher in pigs fed A2AA suggests rate of digesta passage increased and offers an explanation for reduced N and DM digestibility in treated pigs. Despite adverse effects of A2AA, efficiency of postabsorptive N metabolism increased. As a percentage of N consumed and compared to control pigs, urinary N decreased 15% (P < .01) and retained N increased 12% (P < .05) in animals fed A2AA. Data from these studies show net efficiency of N metabolism is improved in swine given A2AA.

Augmentation of hormonal activities with antibodies from cattle immunized with a combination of synthetic and recombinant growth hormone peptide.

Antibodies generated against a synthetic growth hormone (GH) peptide in a number of animal species were shown to enhance the efficacy of GH. However, the ability to produce the effective antibodies diminished over the time and repeated boosters failed to overcome the hurdle. Therefore, this study was designed to address the issue on the fallen antibody responses by employing different GH peptide antigen preparations in cattle. Holstein steers were repeatedly immunized with a synthetic peptide corresponding to an amino acid sequence 54-95 of porcine GH (pGH). The peptide was conjugated to ovalbumin (OVA) as a carrier. Animals initially responded to the antigen well and elicited antibodies specific to the peptide. However, the 4th challenge with the same OVA-peptide antigen rendered animals unresponsive, resulting in a decline in antibody production. This unresponsiveness was overcome by switching the antigen at the 5th immunization from OVA-peptide to a recombinant peptide preparation which was composed of maltose binding protein (MBP) as a carrier. Antibodies generated in cattle after the 5th immunization recognized not only the pGH(54-95) peptide, but also bovine GH (bGH) and pGH. These antibodies were not immunoreactive with an unrelated control peptide. Hypophysectomized (hypox) rats were used for functional analysis and bGH was active in promoting the growth of these GH-deficient rats. The growth-promoting effect of bGH was significantly enhanced by mixing with bovine anti-peptide antibodies prior to administration. Therefore, the present findings suggest that peptide 54-95 induces cattle to elicit antibodies capable of not only recognizing bGH but also augmenting the somatogenic effectiveness of bGH in hypox rats.

Augmentation of hormonal activities with antibodies from cattle immunized with a combination of synthetic and recombinant growth hormone peptide.

Antibodies generated against a synthetic growth hormone (GH) peptide in a number of animal species were shown to enhance the efficacy of GH. However, the ability to produce the effective antibodies diminished over time and repeated boosters failed to overcome the hurdle. Therefore, this study was designed to address the issue on the failed antibody responses by employing different GH peptide antigen preparations in cattle. Holstein steers were repeatedly immunized with a synthetic peptide corresponding to an amino acid sequence 54-95 of porcine GH (pGH). The peptide was conjugated to ovalbumin (OVA) as a carrier. Animals initially responded to the antigen well and elicited antibodies specific to the peptide. However, the 4th challenge with the same OVA-peptide antigen rendered animals unresponsive, resulting in a decline in antibody production. This unresponsiveness was overcome by switching the antigen at the 5th immunization from OVA-peptide to a recombinant peptide preparation which was composed of maltose binding protein (MBP) as a carrier. Antibodies generated in cattle after the 5th immunization recognized not only the pGH(54-95) peptide, but also bovine GH (bGH) and pGH. These antibodies were not immunoreactive with an unrelated control peptide. Hypophysectomized (hypox) rats were used for functional analysis and bGH was active in promoting the growth of these GH-deficient rats. The growth-promoting effect of bGH was significantly enhanced by mixing it with bovine anti-peptide antibodies prior to administration. Therefore, the present findings suggest that peptide 54-95 induces cattle to elicit antibodies capable of not only recognizing bGH but also augmenting the somatogenic effectiveness of bGH in hypox rats.