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National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, P. R. China 100094
2 To whom correspondence should be addressed: E-mail: defali{at}public2.bta.net.cn.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • zinc oxide • IGF-I • IGF-I receptor • small intestine • piglets
Dietary supplementation with zinc oxide (ZnO) results in improved growth performance and reduced scours in weanling piglets (1–3). However, the high levels of Zn excreted by supplemented pigs have raised concerns about its potential environmental pollution (4,5). Additionally, the mechanism responsible for the growth-promoting effect of Zn remains unknown. Elucidating such a mechanism is expected to optimize the growth-promotion efficacy of ZnO while minimizing the amount of Zn supplemented to the piglet's diet.
Some studies suggest that ZnO exerts its effect through controlling Escherichia coli–induced scours (6). However, there is evidence showing that dietary ZnO supplementation to pigs had no effect on the killing or number of E. coli in vivo or in vitro (7,8). Thus, some researchers suggested that zinc enhanced the growth performance of piglets through a systemic effect (via the blood), rather than a direct influence on the gastrointestinal (GI)3 tract. Surprisingly, the efficacy of growth promoting is not associated with the bioavailability of Zn (9,10).
Weaning often results in small-intestinal atrophy and dysfunction in piglets (11), which is a major factor responsible for growth retardation and diarrhea in neonates (12). Intestinal atrophy reduces nutrient absorption and the synthesis of semiessential amino acids (e.g., arginine and proline) that are required for supporting the maximal growth of piglets (13). Interestingly, inclusion of high levels of ZnO in the diet is beneficial for maintaining the normal morphology of the GI tract in weaning pigs (14,15), suggesting that ZnO protects the small intestine from weaning-associated damage. However, the underlying mechanism is unknown.
Insulin-like growth factor-I (IGF-I) is an important regulator of intestinal cell growth and differentiation (16). In particular, orally administered IGF-I is beneficial for the integrity and function of the small intestine (17–20). The IGF-I and IGF-I receptor (IGF-IR) genes have a site for SP-1 (21,22), which is a transcription factor and a zinc finger protein (23). Accordingly, ZnO was shown to regulate IGF-I expression directly in granulation tissue from porcine wounds (24). Moreover, adding 2500 mg ZnO/kg to the diet enhances serum IGF-I levels in weanling piglets (25). On the basis of these previous findings, we hypothesized that an increase in the expression of IGF-I and IGF-IR in the small intestine, coupled with elevated serum IGF-I, protects weanling piglets from intestinal atrophy and dysfunction, thereby enhancing growth performance. This hypothesis was tested in the present study by quantifying IGF-I and IGF-IR gene expression and morphology in the small intestine.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiment 1. Thirty-six 28-d-old barrows (8.1 ± 0.6 kg) were housed in 1.25-m x 1.2-m pens with slotted stainless steel floors, and assigned to 1 of 2 dietary groups in a randomized complete-block design based on body weight and litter, with 6 pens/group (3 pigs/pen). Each pen was equipped with a feeder and a nipple waterer to allow the pigs free access to feed and drinking water. Temperatures (25–27°C) and a cycle of 16 h light:8 h dark were maintained in the mechanically ventilated nursery room. One group of piglets was fed the basal diet, which contained 100 mg Zn/kg. The other group was fed the basal diet supplemented with ZnO at the expense of corn to provide 3000 mg Zn/kg. The basal diet met or exceeded NRC nutrient requirements for swine (26) (Table 1).
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Experiment 2. Sixteen 28-d-old barrows (7.6 ± 0.16 kg) were housed individually in metabolism pens (0.8-m x 0.4-m) and assigned randomly to 1 of the 2 dietary groups as in Experiment 1, with 8 pigs/group. The Zn-supplemented piglets were individually pair-fed to the control group of pigs, as described by Swamy et al. (27). In essence, pigs in the control group were had free access to feed, whereas Zn-supplemented pigs were provided with the same amount of feed consumed by the control group of pigs on the previous day. The pigs were fed twice daily and the feed intake was recorded daily. The sampling of blood and the small intestine was performed as described in Experiment 1.
Measurement of IGF-I in the small-intestinal mucosa and serum.
In Experiments 1 and 2, the small intestinal mucosa (
100 mg) was homogenized in phosphate buffer (0.2 mol/L, pH = 7.4, 4°C) containing 50 mg soybean trypsin inhibitor/L and 0.1 mmol phenylmethylsulfonyl fluoride (PMSF)/L. The homogenate was centrifuged at 14,000 x g for 15 min and the supernatant was used for the IGF-I assay. Concentrations of IGF-I in the small-intestinal mucosa and serum were measured using a commercially available human immunoradiometric assay kit (Diagnostic System Laboratories) according to the manufacturer's protocol. An acid/ethanol extraction step was added to separate IGF binding protein (28). The sensitivity of the assay was 0.8 mg/L. The intra-assay and interassay CV were 2.5 and 4.8%, respectively. The mean recovery of IGF-I from porcine serum was 96%, as assessed using known amounts of IGF-I standard.
Total RNA isolation and reverse transcription (RT).
In both Experiments 1 and 2, total RNA was isolated from the small-intestinal mucosa (100 mg) using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The extracted RNA was dissolved in RNA-free water and quantified using UV-clear microplates (TECAN) at OD260. An RNA aliquot was verified for its integrity by electrophoresis in a 1% agarose gel stained with ethidium bromide. Then, 2 µg of total RNA was reverse-transcribed in a 25-µL reaction mixture using random primer Oligo-dT18 (Sangon) and M-MLV reverse transcriptase (Promega) as described by Lai et al. (29). The RT products (cDNA) were stored at –20°C for analysis of IGF-I and IGF-IR mRNA levels by real-time PCR.
Real-time PCR for quantification of IGF-I and IGF-IR. Real-time PCR was performed using DNA Engine Opticon-2 (MJ Research) and DyNAmo SYBR Green qPCR commercial kits (Finnzymes), in which SYBR Green I was a double-stranded DNA-specific fluorescent dye. ß-Actin was used as the reference gene. The primers of IGF-I, IGF-IR, and ß-actin were as follows: forward 5'- GTA ACC ATG AGG CTG AGA AG - 3', reverse 5'-AAC ACA GGT TCC GTC CAT GA-3' for IGF-I (254 bp); forward 5'-ATG ACG AGA GAC ATC TAT GAG AC-3', reverse 5'-CTC GTC GAA GCT GGC TCG CAG CAC-3' for IGF-IR (561 bp); forward 5'-TGC GGG ACA TCA AGG AGA AG-3', reverse 5'-AGT TGA AGG TGG TCT CGT GG-3' for ß-actin (216 bp). The PCR reaction system consisted of 5.0 µL SYBR Green qPCR mix, 1.0 µL of cDNA, 3.6 µL double distilled H2O, and 0.4 µL of primer pairs (25 µmol/L forward and 25 µmol/L reverse) in a total volume of 10 µL. Cycling conditions were 50°C for 2 min, followed by 95°C for 5 min; by 35 cycles (95°C for 30 s, with different annealing temperatures for different target genesfor 30 s, and 72°C for 45 s). The melting curve program was 65–95°C with a heating rate of 0.1°C/s and a continuous fluorescence measurement. The annealing temperatures for IGF-I, IGF-IR, and ß-actin were 63, 62, and 64°C, respectively. All samples were measured in triplicate. The relative mRNA levels of target genes were determined using the relative standard curve methods as described by Lai et al. (29).
Western blotting.
In Experiments 1 and 2, the small-intestinal mucosa (100 mg) was lysed using a protein isolation kit (Kangchen) containing 50 mg soybean trypsin inhibitor/L and 0.1 mmol PMSF/L. The mucosa was homogenized for 30 s at a low speed using a homogenizer (Fluko). The homogenate was centrifuged at 14,000 x g for 15 min and the supernatant was used for Western blot analysis. Protein concentration was determined using a bicinchoninic acid protein assay reagent kit (Kangchen). The sample was boiled for 3 min, and aliquots of 50 µg protein were electrophoresed (Bio-Rad) on SDS-polyacrylamide gels and then electroblotted (Bio-Rad) onto PVDF membranes (Millipore). Membranes were incubated for 1 h in a blocking buffer containing 5% nonfat dry milk in Tris-buffered saline [TBS (20 mmol/L Tris, 500 mmol/L NaCl, pH = 7.6)]. After blocking, the membrane was washed in TBS plus 0.1% Tween-20 (TBST) and incubated for 1.5 h with mouse IGF-IR (1:100) polyclonal antibody (95 kDa) (Neomarkers). The membrane was then rinsed in TBST and incubated for 1 h with horseradish peroxidase-labeled anti-mouse IgG (Kangchen) (1:5000). The membrane was washed 3 times in TBST. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the reference protein. The GAPDH antibody (Kangchen) was diluted 1:10000. Visualization of the reactive bands was accomplished by chemiluminescence and exposure of the blots to the luminescence detection film (ECL Western Blotting detection reagents and Hyperfilm-ECL; Kangchen). The films were analyzed by a gel imaging system (Tanon Science & Technology) with the ImageJ analysis software (NCBI) (Fig. 1 and Fig. 2).
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Statistical analysis. Data on growth performance and IGF-I levels of small-intestinal mucosa and serum were analyzed as randomized complete-block designs, using ANOVA. Data on small-intestinal morphology were analyzed using one-way ANOVA, and data on gene expression were analyzed using unpaired t test. Pen was used as the experimental unit for performance and serum data, and individual pig was used as the experimental unit for small-intestinal morphology and gene expression data analysis in Experiment 1. In Experiment 2, individual pig was the experimental unit for the analysis of all the data. All analyses were performed using the SAS (30). Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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mRNA and protein levels for IGF-I and IGF-IR. In Experiment 1, adding 3000 mg Zn/kg to the diet of weanling pigs enhanced (P < 0.05) mRNA levels for IGF-I and IGF-IR in the small-intestinal mucosa (Table 4). Similar results were obtained in Experiment 2 when feed intake was similar in the control and Zn-supplemented piglets (Table 4). Intestinal mRNA levels for the ß-actin gene did not differ between control and Zn-supplemented pigs in Experiment 1 or 2. Piglets fed the 3000 mg Zn/kg diet had higher (P < 0.05) intestinal IGF-I and IGF-IR protein levels than those fed the control diet in either experiment (Tables 2 and 4).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There is much speculation about the mechanism responsible for the effect of high levels of ZnO in promoting piglet growth. The Zn ion is considered to be a major causative factor on the basis of the observation that a higher bioavailability of Zn was associated with a better growth-promoting effect (1). However, ZnO, whose bioavailability is much lower than that of zinc sulfate (ZnSO4) or zinc methionine (Zn-Met) (31), had a stronger growth-promoting effect than ZnSO4 or Zn-Met (1,32). Further, Marromichalis et al. (10) found that both high (93%) and low (39%) bioavailability of ZnO had the same performance-enhancing effect in piglets. These findings indicate that the bioavailability of a high level of Zn in the diet is not a major factor affecting growth performance in piglets. Interestingly, ZnO was suggested to exert its effect through killing the intestinal E. coli that causes postweaning diarrhea. However, Jensen-Waern et al. (7) found that dietary supplementation with 2500 mg ZnO/kg did not decrease the number of excreted E. coli. in weaned piglets. Additionally, Roselli et al. (8) reported that the addition of ZnO to the medium did not affect either the growth or viability of E. coli. Thus, it is reasonable to surmise that other mechanism(s) are responsible for the beneficial effect of ZnO on the small intestine.
The intestinal tract is the interface between the external and internal environments. It also serves as a crucial physical and immunological barrier against harmful materials. At the time of weaning, piglets undergo a transition from a milk-based to a conventional corn/soybean–based diet, and the small intestine exhibited a marked change in its structure and function (11). In particular, the villous height is reduced and the crypt depth is increased in association with reductions in the specific activity of the brush border enzymes (12). There is evidence that Zn regulates intestinal integrity and function in animals. For example, dietary supplementation with high levels of ZnO increased hydrolase activity in the rat small intestine (33). Also, ZnO promoted wound healing in the porcine gut (14,15). Thus, the beneficial effect of ZnO during weaning may result from the prevention of intestinal damage.
Recently, increasing efforts were made to define the role of growth factors (primarily IGF-I) in the development of the mammalian GI tract. Burrin et al. (17) found that orally administered IGF-I increased intestinal weight, protein, and DNA content, as well as jejunal and ileal villous height in neonatal pigs. Similarly, Alexander and Carey (18) reported that oral IGF-I administration enhanced nutrient and electrolyte absorption in the neonatal piglet intestine. Moreover, IGF-I could protect intestinal cells from apoptosis induced by radiation in rats (34). Thus, IGF-I was implicated as a therapeutic factor for GI tract diseases, such as short bowel syndrome, gastric ulceration, and intestinal mucositis (35). Consistent with this notion, we found that dietary supplementation with 3000 mg ZnO/kg enhanced (P < 0.05) both mRNA and protein levels for IGF-I and IGF-IR (Tables 2, 4) and villous height (Table 3) in the small-intestinal mucosa of weanling pigs, independent of change in feed intake. IGF-I exerts its biological effect through interaction with specific membrane-bound receptors. In the pig small intestine, IGF-I is bound principally to IGF-IR (36). Thus, the increased IGF-IR expression in the small intestinal mucosa of weaned piglets (Table 4) indicates that the IGF-IR has a positive response to IGF-I (37) and supports the view that the IGF system plays a major role in mediating an anabolic effect of Zn in the small intestine of weanling piglets. Further studies are required to elucidate the molecular mechanism whereby Zn regulates gene expression of IGF-I and IGF-IR as well as enterocyte proliferation and apoptosis in the small intestine and to identify the responsible cell types.
Although IGF-I expression increased in the small-intestinal mucosa of Zn-supplemented piglets, circulating IGF-I levels (Table 2) or hepatic IGF-I expression (data not shown) did not change in these piglets. This result suggests that the release of IGF-I from the liver was not altered in response to dietary Zn supplementation and that the response of gene expression to Zn is likely cell specific. In support of this suggestion, Lefebvre et al. (38) reported that increasing extracellular Zn concentrations did not increase IGF-I gene expression in cultured rat hepatocytes. Differential responses of the small intestine and liver to nutritional and hormonal treatments associated with the expression of urea cycle enzymes were also reported for weaned pigs (39). In contrast, Carlson et al. (25) reported that addition of 2500 mg ZnO/kg to the diet enhanced serum IGF-I levels in weanling pigs. The disparity between our findings and those of others may be explained by a difference in the age of weaned pigs or nutritional status. In the study of Carlson et al. (25), blood samples were collected 5–7 d after weaning, whereas in our present work, blood samples were obtained 14 d after weaning. Because circulating IGF-I levels are strongly correlated with the nutritional status (40), malnutrition can dramatically decrease serum IGF-I levels, and these can return to normal concentrations after refeeding (41). At weaning, energy and protein intakes were reduced markedly, leading to decreased plasma IGF-I levels (42,43). Dietary supplementation with a high level of ZnO alleviated the weaning-associated intestinal damage, which may then improve the nutritional status and thus IGF-I production in the whole body. At 2 wk postweaning, the feed intake of piglets usually returns to normal, as does the circulating level of IGF-I (40). At this age, dietary Zn supplementation may not be able to elicit a further increase in IGF-I production in piglets. Nonetheless, an increase in IGF-I and IGF-IR gene expression in the small intestine is expected to result in a direct anabolic effect locally.
In conclusion, dietary supplementation with a high level of ZnO increased expression of the IGF-I and IGF-IR genes at both the mRNA and protein level in the small intestine of weanling pigs, independent of change in feed intake. This novel finding may explain the beneficial effects of ZnO on intestinal integrity, function, and whole-body growth in weanling piglets.
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FOOTNOTES |
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3 Abbreviations used: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GI, gastrointestinal; IGF-I, insulin-like growth factor-I; IGF-IR, insulin-like growth factor-I receptor; PMSF, phenylmethylsulfonyl fluoride.
Manuscript received 24 January 2006. Initial review completed 13 March 2006. Revision accepted 5 April 2006.
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