J. Anim. Sci. 2003. 81:1456-1463
© 2003 American Society of Animal Science
Effects of antimicrobials and weaning on porcine serum insulin-like growth factor binding protein levels1
M. R. Hathaway2,
W. R. Dayton,
M. E. White and
M. S. Pampusch
University of Minnesota, Department of Animal Science, St. Paul 55108
2 Correspondence:
350 ABLMS, 1354 Eckles Ave. (phone: 612-624-1771; fax: 612-624-3677; E-mail:
hathaway{at}umn.edu).
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Abstract
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The effects of subtherapeutic antimicrobial supplementation and weaning on serum levels of IGF-I and insulin-like growth factor binding proteins (IGFBP)-2, -3 and -4 were determined in crossbred weanling pigs. At weaning, pigs were allotted to a diet containing 21.8% crude protein and 1.15% lysine with or without Aureozol (110 mg/kg of Aureomycin chlortetracycline, 110 mg/kg of sulfathiazole, and 55 mg/kg of penicillin) for 4 wk. Insulin-like growth factor-binding proteins and IGF-I analyses were performed on blood samples that were drawn weekly. Weaning decreased serum IGFBP-3 levels in both control and Aureozol-treated groups on d 6 and d 14 (P < 0.05) relative to preweaning levels. The IGFBP-3 values returned to preweaning levels by d 21. Although the circulating levels of both the 43-kDa and the 39-kDa glycosylation variants of IGFBP-3 were affected by weaning, the level of the 39-kDa IGFBP-3 was affected relatively more than that of the 43-kDa IGFBP-3 (P < 0.05). Compared with circulating IGFBP-3 levels in control pigs, Aureozol-treated pigs had higher circulating IGFBP-3 levels on d 21 (43%, P < 0.05) and d 27 (46%, P < 0.05). In direct contrast to the effect of weaning on serum IGFBP-3 level, serum IGFBP-2 levels increased on d 6 and d 14 after weaning (P < 0.05) and decreased to preweaning levels by d 21. The IGFBP-2 levels continued to decline and were less than preweaning levels by d 27 (P < 0.05). Aureozol treatment had no effect on serum IGFBP-2 levels at any time. Serum levels of nonglycosylated IGFBP-4 were not affected by either weaning or Aureozol supplementation. Weaning decreased circulating IGF-I concentration on d 6 in both control and Aureozol-treated pigs (76 and 73%, respectively, P < 0.05) and on d 14 (62%, P < 0.05) and d 21 (32%, P < 0.05) in control pigs. Aureozol-supplemented pigs had higher serum IGF-I concentrations than control pigs on d 14 (82%, P < 0.05), d 21 (55%, P < 0.05), and d 27 (36%, P < 0.05). The Aureozol-fed pigs had a 14.2% increase in BW gain (P < 0.05) and a 59.6% increase in ADG (P < 0.05) compared with pigs fed the control diet. Both Aureozol-supplementation and weaning cause changes in serum IGFBP levels and IGF-I concentrations that might be involved in regulating rate and efficiency of growth.
Key Words: Binding Proteins • Chlortetracycline • Insulin-Like Growth Factor • Pigs • Weaning
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Introduction
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Previous studies have shown that weanling pigs fed subtherapeutic levels of an antimicrobial grow faster, more efficiently, and have higher circulating concentrations of insulin-like growth (IGF)-I than pigs fed the same diet without the antimicrobial (Hathaway et al., 1996; Hathaway et al., 1999). Additionally, our studies and those of others have shown that circulating levels of IGF-I drop dramatically after weaning and then gradually return to preweaning levels over three to four weeks (Hathaway et al., 1996; Hathaway et al., 1999). Thus, both weaning and antimicrobial supplementation have been shown to affect circulating IGF-I concentrations.
Insulin-like growth factor-I is a very potent mitogenic growth factor that has been shown to affect proliferation and differentiation of a wide variety of cell types (D’Ercole, 1996). In vivo, IGF-I is bound to one of six different IGF binding proteins (IGFBP) (Clemmons, 1997). Binding of IGFs to IGFBP extends the half-life of IGFs and regulates their biological activity (Clemmons, 1997). In addition to binding IGF-I and affecting its biological activity, some IGFBP affect cells via IGF-independent mechanisms presumably involving binding to specific cell surface receptors (Oh, 1998; Wu et al., 2000; Ricort and Binoux, 2001). Taken together these data suggest that the IGFBP may make a significant contribution to the growth process, both via IGF-dependent and IGF-independent mechanisms, and that changes in circulating levels of IGFBP may impact rate and efficiency of growth. Changes in circulating IGFBP levels often accompany changes in circulating IGF-I concentrations such as those observed at weaning or upon antimicrobial supplementation (Underwood, 1996). Consequently, the goals of the experiments reported here were to examine the effects of weaning and of feeding subtherapeutic levels of the antimicrobial Aureozol on circulating levels of IGFBP-2, -3, and -4.
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Materials and Methods
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Materials
Globulin-free bovine serum albumin (BSA; cat. no. A-7638) was obtained from Sigma Chemical (St. Louis, MO). Rabbit anti-human IGF-I polyclonal antibody, AFP4892898, was provided by the National Hormone and Pituitary Program (Baltimore, MD). Insulin-like growth factor-I was purchased from Bachem (Torrance, CA). 125I-Insulin-like growth factor-I was prepared in our laboratory using previously reported procedures (Frey et al., 1994). Aureozol was kindly provided by Roche Vitamins, Inc. (Parsippany, NJ).
General Methods
An experiment involving 14 crossbred pigs (Hampshire x Cambrough) was conducted to evaluate the effect of Aureozol (110 mg/kg of Aureomycin chlortetracycline, 110 mg/kg of sulfathiazole, and 55 mg/kg of penicillin) on serum IGFBP levels. The University of Minnesota Animal Care Committee approved all experimental procedures performed in this study. The baby pigs were not allowed access to creep feed prior to weaning. One week before weaning, pigs were weighed and a single blood sample was collected from the jugular vein of each pig by venipuncture. Serum was harvested 24 h after blood collection and then assayed for IGF-I. At weaning, pigs were bled, weighed again and allotted to either the control or Aureozol diet. Littermate pigs of the same gender and of similar body weight, preweaning serum IGF-I concentrations and average daily gain for the preceding week were paired. Subsequently, one pig from each pair was randomly allotted to a treatment. The pigs were weaned at 25 ± 1 d of age with an average initial weight of 7.83 kg (SEM = 0.22, n = 14). The pigs were housed in a temperature-controlled nursery building in 1.25 x 1.25 m, elevated flat-deck pens with expanded metal floors. Each pen was equipped with a stainless steel self-feeder and a nipple waterer. Pigs were given ad libitum access to a corn-soybean meal-based diet based on NRC (1988) recommendations with or without the addition of Aureozol (basal diet, Table 1
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The study used a randomized complete block design with two treatments. The treatment groups consisted of 1) a control group fed the basal diet for 4 wk and 2) an antimicrobial group fed the basal diet supplemented with Aureozol for four weeks. Pigs were not given any other antimicrobial agents, before or during the trial. Pigs were weighed at weekly intervals. Pigs were bled between 1100 and 1200 h on d 0, 6, 14, 21, and 27. Based on previous studies (Hathaway et al., 1999) one blood sample pig-1•wk-1 was considered adequate to reflect treatment effects. Blood samples were allowed to clot at 4°C for 24 h. Serum was harvested and stored at -80°C.
[125I] - Insulin-Like Growth Factor-I Ligand Blot.
[125I]-Insulin-like growth factor-I ligand blotting (Hossenlopp et al., 1986) was used to detect any functional IGFBP in serum. Serum samples were diluted 2:1 with 3x nonreducing tracking dye and heated in a boiling water bath for 5 min before being loaded onto discontinuous polyacrylamide gels consisting of a 3.5% stacking gel and a 10% separating gel. Samples were subjected to nonreducing (without mercaptoethanol) SDS-PAGE and subsequently transferred to a nitrocellulose membrane by electroblotting. After blocking, membranes were stored at -20°C until all samples were electrophoresed and transferred. Each membrane was then incubated with 5 mL of blocking buffer containing 200,000 cpm of 125I-labelled IGF-I/mL and then washed. All membranes were incubated simultaneously and the incubation solution was prepared utilizing the same 125I-IGF-I preparation to ensure uniformity. The membranes were allowed to dry and then placed in contact with Kodak X-Omat AR film for 116 h at -80°C. Even though it was necessary to use multiple gels to accommodate the number of samples in this study, the fact that nonglycosylated IGFBP-4 levels did not change with time or treatment establishes that observed differences in the quantities of IGFBP were not the result of variations in loading or transfer efficiency between gels or individual samples. Using this methodology, there is a linear dose-response relationship between increasing loads of serum and optical density obtained by scanning laser densitometry (Hembree et al., 1996).
Insulin-Like Growth Factor-I RIA.
Porcine serum samples were diluted 1:1 (vol/vol) with 0.1 M glycyl-glycine, pH 2, so that the pH of the final solution was 3.6 to 3.8. The samples were incubated at 37°C for 48 h (Frey et al., 1994). Subsequently, IGF-I levels were measured using a heterologous RIA as previously described (Frey et al., 1994). Serum samples were diluted in RIA buffer, a rabbit anti-human IGF-I polyclonal antibody (1:18,000 dilution) (AFP4892898 provided by the National Hormone and Pituitary Program) and approximately 12,000 cpm 125I-labeled IGF-I were added and the samples were incubated for 24 h at 4°C. Bound and free IGF-I were separated by the addition of a preprecipitated second antibody, goat anti-rabbit gamma globulin (1:5 dilution) and normal rabbit serum (1:50 dilution), in RIA buffer. Based upon the ability of the assay to detect the IGF-I standard, the effective detection range of the RIA was 10-640 pg IGF-I. The IGF-1 RIA had an intra-assay coefficient of variation of less than 3.0% and all serum samples were analyzed within the same assay.
Statistical Analysis.
A randomized complete block design with two treatments was used. Data were analyzed with analyses of variance using the GLM procedures of PC SAS. Serum IGFBP-2, -3, and -4 levels and serum IGF-I concentrations were analyzed using a repeated measures model. Pair, day (day of bleeding) and treatment were included as main effects. The treatment x pair interaction served as the error term for testing the effect of treatment and pair. For ADG the model included treatment as a main effect. When significant, treatment means were separated using the LSD test. Comparisons between treatments were considered significantly different if P < 0.05.
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Results
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Effect of Aureozol Treatment and Weaning on Growth Rate of Weanling Pigs.
Growth rates of control and antimicrobial-treated groups were evaluated to ensure that the pigs were responding as expected to subtherapeutic antimicrobial supplementation. The ADG for the 4-wk study are presented in Table 2. Predictably, growth rate in both the control and treated groups dropped to near zero during the first week after weaning (d 6) and then increased during the remainder of the study. Overall, the pigs fed the Aureozol-supplemented diet gained more weight per day than the pigs fed the control diet (P < 0.05).
Effect of Aureozol Treatment and Weaning on Circulating IGF-I Levels.
Previous studies have shown that feeding young pigs subtherapeutic levels of an antimicrobial resulted in an increase in serum IGF-I concentrations. Consequently, to corroborate earlier reports and verify the antimicrobial effect, the serum samples obtained in this study were assayed for IGF-I using a RIA. Weaning resulted in a significant reduction in circulating IGF-I concentration in both control and Aureozol-treated pigs by d 6 (Figure 1). Insulin-like growth factor-I levels began to rebound by d 14 and continued to increase throughout the duration of the study. Aureozol-treated pigs had elevated levels of serum IGF-I on d 14 (P < 0.05), d 21 (P < 0.05) and d 27 (P < 0.05) compared to control pigs (Figure 1). Overall, the pigs fed Aureozol had higher serum concentrations of IGF-I (P < 0.05) than did the pigs fed the control diet.
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Figure 1: Serum IGF-I level in control and Aureozol treated pigs before weaning (d 0) and on d 6, 14, 21 and 27 after weaning. Within a treatment (Control or Aureozol) time points with different letter designations differ (P < 0.05, n = 7). * indicates that the control and Aureozol values differ (P < 0.05, n = 7) on the indicated day. SEM = 4.18.
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Effect of Aureozol Treatment and Weaning on Circulating IGFBP Levels
In light of the weaning- and Aureozol-induced changes in circulating IGF-I concentrations, it was of interest to determine whether circulating IGFBP levels were altered by these treatments. 125I-Insulin-like growth factor-I Western ligand blots were done on serum samples from each pig at each time point and relative densities of bands corresponding to IGFBP-2, IGFBP-3 (both the 43- and 39-kDa bands), and nonglycosylated IGFBP-4 were determined. Figure 2 is a representative 125I-IGF-I Western ligand blot showing the IGFBP banding pattern obtained from paired control and Aureozol-treated pigs on d 27. The IGFBP labeled in this figure have been identified by immunoprecipitation utilizing antibodies specific for each IGFBP. Insulin-like growth factor binding protein-3 consists of two glycosylation variants that resolve at apparent molecular weights of 43 and 39 kDa on a nonreducing, SDS gel (Hembree et al., 1996). Insulin-like growth factor binding protein-2 migrates as a single band at 32 kDa and nonglycosylated IGFBP-4 migrates at 24 kDa (Hembree et al., 1996). It is not possible to resolve IGFBP-5 and glycosylated IGFBP-4, both of which band at approximately 28 kDa.
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Figure 2: 125I-Insulin-like growth factor-I Western ligand blot showing IGFBP in 1 µL of serum obtained from paired control (C) and Aureozol-treated (T) pigs 27 d after weaning. Pigs were paired before weaning as described in the Materials and Methods. In each pair, one pig was randomly assigned to the control diet (C), and one pig was assigned to the Aureozol-containing (T) diet at weaning.
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Effect of Aureozol Treatment and Weaning on Circulating IGFBP-3 Levels.
Ligand blots were done to measure the relative quantity of IGFBP-3 present in 1 µL of serum from each pig and time point in the study and the results of densitometric scans of these ligand blots are shown in Figure 3. The 43-kDa and 39-kDa bands were scanned and evaluated separately. Additionally, the total IGFBP-3 level (sum of the 43-kDa and 39-kDa band densities) was evaluated. On d 6 and d 14 the densities of both the 43- and 39-kDa bands were dramatically decreased in both control and Aureozol-treated pigs, indicating that circulating levels of IGFBP-3 were significantly (P < 0.05) reduced by weaning in both groups (Figure 3A and 3B). Weaning, but not Aureozol treatment, affected the ratio of the 43-kDa band to the 39 kDa band (Figure 3C). On d 6 and d 14 the 43 kDa/39 kDa ratio was nearly two times greater than on d 0 (P < 0.05); however, by d 21 and 28 the ratio had returned to the level observed on d 0 (the day of weaning and the day Aureozol treatment began).
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Figure 3: Effect of Aureozol treatment and weaning on the levels of total IGFBP-3, 43-kDa IGFBP-3, 39-kDa IGFBP-3 and on the ratio of the 43-kDa to 39-KDa IGFBP-3 variants in serum. Values were obtained from densitometric scans 125I-IGF-I Western ligand blots done on serum obtained from seven control and seven treated pigs at the indicated times. A) Relative 43-kDa IGFBP-3 level in serum from control and from Aureozol-treated pigs at various times post-weaning. Within a treatment (Control or Aureozol), time points with different letter designations differ (P < 0.05). * indicates that the control and Aureozol values are different (P < 0.05) on the indicated day. SEM = 0.539. B) Relative 39-kDa IGFBP-3 level in serum from control and from Aureozol-treated pigs at various times after weaning. Within a treatment (Control or Aureozol), time points with different letter designations differ (P < 0.05). * indicates that the control and Aureozol values differ (P < 0.05) on the indicated day. SEM = 0.356. C) Effect of weaning on the ratio of the 43-kDa IGFBP-3 to the 39-kDa IGFBP-3 in serum from control pigs. Values are the ratio of the quantity of 43-kDa IGFBP-3 to the quantity of the 39-kDa IGFBP-3 on the indicated days of the study. Values with different letter designations differ (P < 0.05; SEM = 0.149). Aureozol treatment did not significantly affect the 43-kDa to 39-kDa ratio (data not shown). D) Total relative IGFBP-3 level in serum from control and from Aureozol-treated pigs at various times after weaning. Values were obtained by summing the densities of the 43-kDa and the 39-kDa IGFBP-3 bands on the 125 I-IGF-I Western ligand blots. Within a treatment (Control or Aureozol), time points with different letter designations differ (P < 0.05). * indicates that the control and Aureozol values differ (P < 0.05) on the indicated day. SEM = 0.826.
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By d 21, the total level of IGFBP-3 (43-kDa plus 39-kDa IGFBP-3) in the serum obtained from pigs fed the Aureozol diet was significantly greater than the level of IGFBP-3 in the serum obtained from pigs fed the control diet and this difference was maintained throughout the remainder of the study (Figure 3D
). Individual examination of the levels of the 43-kDa and 39-kDa IGFBP-3 revealed that by d 21 both the 43- and 39-kDa variants of IGFBP-3 were significantly (P < 0.05) higher in the serum of Aureozol treated pigs than in the serum of control pigs (Figure 3A and 3B).
Effect of Aureozol Treatment and Weaning on Circulating IGFBP-2 Levels.
Ligand blots were done to measure the relative quantity of IGFBP-2 present in 1 µL of serum from each pig and time point in the study and the average densities of the IGFBP-2 bands for each group at each time point are shown in Figure 4. Six days post-weaning (d 6), serum IGFBP-2 levels were increased (P < 0.05) in both control and Aureozol-treated pigs. Thereafter, IGFBP-2 levels gradually decreased to near preweaning levels. There was no effect of Aureozol treatment on the level of circulating IGFBP-2.
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Figure 4: Relative IGFBP-2 level in serum from control and from Aureozol-treated pigs at various times post-weaning. Values were obtained from densitometric scans 125I-IGF-I Western ligand blots done on serum obtained from seven control and seven treated pigs at the indicated times. Within a treatment (Control or Aureozol), time points with different letter designations differ (P < 0.05). * indicates that the control and Aureozol values differ (P < 0.05) on the indicated day. SEM = 0.196.
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Effect of Aureozol Treatment and Weaning on Circulating Nonglycosylated IGFBP-4 Levels.
Serum levels of unglycosylated IGFBP-4 remained unchanged in both control and Aureozol-treated pigs throughout the duration of the study (Figure 5). The fact that nonglycosylated IGFBP-4 levels did not change establishes that observed differences in the quantities of IGFBP-3 and -2 are not the result of variations in loading or transfer efficiency between gels or individual samples.
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Figure 5: Relative nonglycosylated IGFBP-4 level in serum from control (Control) and from Aureozol-treated (Aureozol) pigs at various times post-weaning. Values were obtained from densitometric scans 125I-IGF-I Western ligand blots done on serum obtained from seven control and seven treated pigs at the indicated times. Neither weaning nor Aureozol treatment had a significant on serum IGFBP-4 level. SEM = 0.165.
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Discussion
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Our results show that immediately after weaning circulating levels of IGF-I and IGFBP-3 are decreased, IGFBP-2 levels are increased and IGFBP-4 levels remain constant. Additionally, the temporal effects of weaning on quantities of 43-kDa and 39-kDa IGFBP-3 are different. Results of the current study also support our previous finding that supplementation with subtherapeutic levels of antimicrobials significantly increases circulating levels of IGF-I. In addition, antimicrobial supplementation results in increased circulating levels of the 43-kDa IGFBP-3 and the 39-kDa IGFBP-3 by d 21 following weaning. In contrast, antimicrobial supplementation has no effect on circulating IGFBP-2 or nonglycosylated IGFBP-4 levels.
The significant reduction in circulating IGF-I concentration observed immediately after weaning in this study confirms data reported previously for pigs supplemented with ASP-250 (100 ppm chorotetracycline, 100 ppm sulfamethazine, 50 ppm penicillin), and is not surprising in view of the well-documented reduction in feed intake during the first few days of the weaning period. Fasting has been shown to decrease circulating IGF-I levels in several species including pigs (Smith et al., 1995; Estivariz and Ziegler, 1997; Hathaway et al., 1999). The Aureozol-induced increase in circulating IGF on d 14, 21, and 27 confirms our previous observations and establishes that the antimicrobial used in this study is affecting the IGF system. Our previous studies have also shown that increased feed intake resulting from subtherapeutic antimicrobial treatment is not solely responsible for the increase in circulating IGF-I level observed in weanling pigs receiving subtherapeutic levels of antimicrobial (Hathaway et al., 1999).
Insulin-like growth factor-I is a potent, anabolic growth factor that is involved in the regulation of multiple cellular functions including proliferation and differentiation. However, the role of circulating IGF-I in growth has recently been placed in question by studies showing that conditional knockout of the IGF-I gene in the liver of mice resulted in a 70 to 75% reduction in serum IGF-I concentration but did not result in reduced growth rate or altered carcass composition (Sjogren et al., 1999). Although the total IGF-I concentration is significantly reduced in these knockout mice, the concentration of free IGF-I in the serum is unchanged. Thus, it is possible that circulating free IGF-I plays a role in stimulating growth. Additionally, recent studies with rats in which production of the 50-kDa acid labile subunit (ALS) and hepatic IGF-I have been knocked out have shown that a threshold level of circulating IGF-I is necessary for normal bone growth (Yakar et al., 2002). Tissue production and utilization of IGF-I in an autocrine and/or paracrine manner also appear to be critical elements in determining normal growth processes. However it has been shown that growth-promoting treatments such as growth hormone and anabolic steroids cause increases in serum IGF-I levels that parallel increases in muscle IGF-I mRNA levels (Grant et al., 1991; Johnson et al., 1998). Consequently, at the very least, circulating IGF-I may provide a marker for identifying treatments that cause increased or decreased tissue production of IGF-I. Thus, although the specific role that serum IGF-I plays in the growth process is unclear, it may still be a useful indicator of growth promotion.
When added to cultured cells, IGFBP-3 has been shown to affect proliferation, differentiation and apoptosis (Baxter, 2000; Butt and Williams, 2001; Baxter, 2001). It is hypothesized to mediate the actions of growth inhibitors such as TGF ß, TNF 
and retinoic acid (Baxter, 2001). Additionally, levels of IGFBP-3 mRNA and secreted protein are dramatically reduced during differentiation of porcine embryonic myogenic cells in culture, suggesting that IGFBP-3 levels may need to be reduced in order for differentiation to occur (Johnson et al., 1999). In the circulation, IGFBP-3 binds to IGF-I and the ALS to form a complex that carries the majority of the IGF-I present in the blood (Baxter, 2000). Binding of IGF-I to IGFBP-3 and ALS increases its half-life in the circulation from minutes to hours (Baxter, 2000). Additionally, infusion of IGF-I/IGFBP-3 complexes has been shown to cause anabolic responses in humans and animals (Debroy et al., 1999; Svanberg et al., 2000; Boonen et al., 2002). Consequently, IGFBP-3 is thought to play a significant role in regulating both the quantity and biological activity of IGF-I. The reduction in IGFBP-3 levels observed immediately after weaning are not surprising in view of the reduction in feed intake typically observed immediately after weaning (Hathaway et al., 1999) and the reduction in IGFBP-3 levels observed in fasted animals (Goya et al., 1996; Underwood, 1996; Simmen et al., 1998). Consequently, it is likely that the reduction in IGFBP-3 on d 6 and d 14 results from decreased feed intake. It is of interest to note that temporal effect of weaning on the levels of the 43-kDa and 39-kDa IGFBP-3 glycosylation variants is different. Although, it is not clear whether these glycosylation variants serve different functions, our data suggest that their synthesis and (or) degradation are (is) affected differently by weaning. This finding suggests that the body is able to discriminate between these two forms of IGFBP-3. By d 21 the levels of IGF-I, 43-kDa IGFBP-3, and IGFBP-2 have returned to preweaning levels. Additionally, the ratio of the 43-kDa IGFBP-3 to the 39-kDa IGFBP-3 has returned to values observed prior to weaning. Consequently, the effects of weaning on the IGF-IGFBP system appear to be largely over by d 21.
Data obtained in the current study are consistent with preliminary data in an earlier report suggesting that IGFBP-3 levels were higher in ASP-250-supplemented pigs than in nonsupplemented control pigs after 4 weeks of supplementation (Hathaway et al., 1999). Significant effects of Aureozol treatment on circulating IGFBP-3 levels are not observed until d 21 when the weaning-related effects on the IGF-IGFBP system appear to be over. The mechanism by which Aureozol treatment increases IGFBP-3 levels is not clear. Since IGFBP-3 levels are sensitive to feed intake and subtherapeutic antimicrobial supplementation has been shown to increase feed consumption, it is possible that increases in IGFBP-3 result from increased feed intake. However, we have shown that antimicrobial induced increases in circulating IGF-I levels are not solely the result of increased feed intake (Hathaway et al., 1999). Additionally, preliminary studies have shown that ASP-250-induced increases in circulating IGFBP-3 levels are not the result of increased feed intake (Hathaway et al., 1999). Thus, it is probable that mechanisms other than or in addition to increased feed intake are responsible for increased IGFBP-3 levels observed in Aureozol-treated, weanling pigs in the current study.
The Aureozol-induced elevation in serum IGFBP-3 levels in this study appeared to lag slightly behind the Aureozol-induced increase in serum IGF-I levels that occurred by d 14. Since other studies have shown that IGFBP-3 levels are positively correlated with circulating IGF-I levels, it is possible that the increased level of circulating IGF-I in Aureozol-treated pigs as compared to controls on d 14 is responsible for the increase in circulating IGFBP-3 in these pigs on d 21. It is also possible that changes in IGF-I levels are detected earlier than changes in IGFBP-3 levels because the IGF-I RIA is more sensitive than the 125I-IGF-I ligand blots used to detect IGFBP-3. The inability of Aureozol supplementation to affect the levels of IGFBP-2 or nonglycosylated IGFBP-4 suggests that the effects of the antimicrobial on IGFBP-3 are somewhat specific and not simply general effects on the IGFBP system.
In contrast to circulating IGFBP-3 levels, circulating levels of IGFBP-2 are increased immediately after weaning and then gradually decrease to near or below preweaning levels. In view of reports that fasting increases circulating IGFBP-2 levels in several species, it is likely that the increased circulating IGFBP-2 level observed after weaning is the result of decreased feed intake (Clemmons and Underwood, 1991; McCusker et al., 1991; Estivariz and Ziegler, 1997). Since IGFBP-2 generally inhibits the actions of IGF-I, increased levels of this binding protein during fasting may help to reduce the anabolic effects of IGF-I when nutrient levels are low.
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Implications
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Both weaning and supplementation with subtherapeutic levels of the antimicrobial Aureozol affected growth rate of weanling pigs. Additionally, these treatments caused alterations in circulating levels of insulin-like growth factor-I and insulin-like growth factor binding proteins. Because both insulin-like growth factor-I and the insulin-like growth factor binding proteins have significant effects on proliferation and differentiation of many cell types, alterations in their levels may affect growth rate and efficiency changes observed as a result of weaning or antimicrobial supplementation.
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Footnotes
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1 This research has been supported in part by the Minnesota Agricultural Experiment Station, by a USDA National Research Initiative Competitive Grant # 00-03271 and by a gift from Roche Vitamins, Inc. 
Received for publication December 10, 2002.
Accepted for publication February 26, 2003.
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Abstract
Introduction
