Determination of insulin-like growth factor 1 (IGF1) and IGF binding protein levels in swine

A heterologous radioimmunoassay system was validated for the determination of IGF1 concentrations in swine sera. Parallelism, accuracy and response to physiological stimuli were obtained following the incubation of serum samples with 1M glycine-glycine HC1 buffer at a pH of 3.5 +/- 0.2 for 24 hours at 37C. Following acidification and neutralization, circulating IGF1 concentrations were significantly (P less than .05) reduced in hypophysectomized swine and elevated in swine injected with porcine growth hormone (pGH) when compared to IGF1 levels in control hogs. IGF binding protein levels were also increased following GH administration and reduced by hypophysectomy. In addition, circulating IGF1 concentrations were significantly (P less than .05) correlated with body size in three types of swine which differ in growth rate and mature body weight. These data suggest that IGF1 is involved in the regulation of swine growth in vivo and that its physiologic regulation is similar to that in humans.     

Characterization of type II insulin-like growth factor (IGF) receptors in sheep liver plasma membranes

Interactions of insulin-like growth factors (IGFs) from recombinant human and natural ovine sources with sheep liver plasma membranes have been studied. Total specific binding of 125I-hIGF-II (40%) to liver plasma membranes greatly exceeded that of 125I-hIGF-I (1.5%) after incubation at 20 C for 90 min. Binding of 125I-hIGF-II to the plasma membranes was dependent upon time, temperature and membrane concentration of the incubation. Binding of 125I-hIGF-II was only partially reversed by addition of 100 nM IGF-II (18%) or by dilution with excess buffer (36%). Competitive inhibition studies of 125I-hIGF-II binding demonstrated that IGF-II from ovine or recombinant human sources was more effective at inhibiting binding than ovine or human IGF-I. Insulin did not affect binding of 125I-hIGF-II. Plasma membranes were affinity cross-linked to 125I-IGF-II followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Following autoradiography, radioactive bands were localized at 274,000 Mr and 210,000-215,000 Mr in the presence and absence of reducing agent, respectively. This pattern was unaffected by 100 nM human or ovine IGF-I or 1,000 nM insulin, but coincubation with 100 nM human or ovine IGF-II eliminated the radioactive band. These data indicate that an IGF-II specific receptor is present in sheep liver plasma membranes which has characteristics similar to those of nonruminant Type II receptors.     

Porcine insulin-like growth factor (IGF)-binding protein-3 elicits bi-phasic effects on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts

The present study was undertaken to investigate the effects of porcine IGFBP-3 on IGF-I stimulated DNA synthesis in neonatal porcine skin fibroblasts. IGF-1 stimulated DNA synthesis in skin fibroblasts in a concentration dependent manner. DNA synthesis was maximally stimulated by 5 to 20 fold at 5 nM IGF-I; half-maximal stimulation was observed at approximately 1 nM IGF-I. Co-incubation of IGFBP-3 with a maximally effective dose of IGF-I (10 nM) did not inhibit the stimulatory effects of IGF-I on DNA synthesis. In contrast, when IGFBP-3 at concentrations of 0 to 20 nM was co-incubated with 1 nM IGF-I, a bi-phasic dose response was observed with IGFBP-3 being inhibitory only at a 10 to 20 fold molar excess to IGF-I. Based on the approximately equal molar ratio of IGFBP-3:IGF-I present in the circulation of control and pST-treated pigs our results suggest that IGFBP-3 does not inhibit the mitogenic effects of IGF-I. In summary, these results indicate that the combination of IGFBP-3 with IGF-I optimizes mitogenic signalling via the type I IGF receptor and suggest that IGFBP-3 does not inhibit the effects of ST that are mediated by IGF-I.     

Follicular fluid concentrations of free insulin-like growth factor (IGF)-I during follicular development in mares

The objective of the present study was to evaluate changes in concentrations of free insulin-like growth factor (IGF)-I in follicular fluid (FFL) during follicle development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6–15 mm; n = 54), medium (16–25 mm; n = 23) or large (>25 mm; n = 15) and FFL was collected. Free IGF-I levels in FFL in large follicles of follicular phase mares were greater (P < 0.05) than in large follicles of luteal phase mares and small or medium follicles of luteal and follicular phase mares. Free IGF-I concentrations were greater (P < 0.05) in large follicles of luteal phase mares than small but not medium follicles of luteal phase mares. FFL ratio of estradiol:progesterone paralleled changes in free IGF-I. Free IGF-I concentrations were negatively correlated (P < 0.05) with insulin-like growth factor binding protein (IGFBP)-2, -4 and -5 but not IGFBP-3 levels. In addition, free IGF-I concentrations in FFL were positively correlated (P < 0.01) with FFL estradiol, progesterone, androstenedione, estradiol:progesterone ratio, total IGF-I and total IGF-II. We conclude that increases in intrafollicular levels of bioavailable (free) IGF-I are associated with increased steroidogenesis in developing mare follicles.     

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.     

Basic principles of muscle development and growth in meat-producing mammals as affected by the insulin-like growth factor (IGF) system

This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.     

Polymorphism and expression of insulin-like growth factor 1 (IGF1) gene and its association with growth traits in chicken

1. The objectives of the study were to detect polymorphism in the coding region of the IGF1 gene, explore the expression profile and estimate association with growth traits in indigenous and exotic chickens.

2. A total of 12 haplotypes were found in Cornish, control layer and Aseel breeds of chicken in which the h1 haplotype was most frequent.

3. Nucleotide substitutions among haplotypes were found at 21 positions in the IGF1 gene in which 4 substitutions resulted in non-synonymous mutations in the receptor binding domain of the IGF1 protein.

4. The haplogroup showed a significant effect on body weight at 24 and 42 d of age in the control layer line, body weight at 42 d and daily weight gain between 29 and 42 d in the control broiler line, daily weight gain between 29 and 42 d in Cornish, and body weights at 42 d as well as daily weight gain between 29 and 42 d in Aseel birds.

5. IGF1 expression varied among the breeds during embryonic and post-hatch periods. The expression among the haplogroups varied in different chicken tissues. The effect of haplogroup on myofibre number in pectoral muscle was non-significant, although there was significant variation in numbers between d 1 and d 42, and between broiler and layer lines.

6. It was concluded that the coding region of the IGF1 gene was polymorphic, expressed differentially during the pre-hatch and post-hatch periods, and haplogroups showed significant association with growth traits in chicken.     

Expression and localization of IGF family members in bovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and pregnancy

The objectives of the study were to monitor the detailed pattern for mRNA expression (RT-PCR and RPA) of IGFs, IGFR-1, IGFBPs, GHR and localization of protein (immunohistochemistry) for IGF-1 and IGFR-1 in bovine follicle classes during final maturation and different corpus luteum (CL) stages during estrous cycle and during pregnancy. A relative high expression of IGF-1 in theca interna (TI) was observed before selection (E<0.5ng/mL). In GC, mRNA expression increased after selection. In contrast, IGF-2 was mainly expressed in the TI. The IGFR-1 mRNA was present in the TI and GC with increasing levels during final development. The expression results were confirmed by localization of IGF-1 and IGFR-1 proteins in GC and TI. There is clear evidence for the local expression of IGFBPs in TI and GC compartment with clear regulatory differences. In CL, the highest mRNA expression of IGF-1, IGF-2 and IGFR-1 was observed during early luteal phase, followed by a decrease, and then by a tendency of an increase during the mid and late luteal phases of the cyclic CL. This level remained low during pregnancy. Intense immunostaining for IGFR-1 in CL was observed mainly in large luteal cells. Evidence for a mRNA for all six IGFBPs were obtained with distinct differences for BP-3, -4 and -5. In conclusion, this comprehensive study gives clear evidence for an important role of the IGFs and IGFBPs in bovine follicular development and CL function. The relative amounts of IGFBPs may ultimately determine ovarian IGF action.     

Moderate doses of porcine somatotropin do not increase plasma insulin-like growth factor-I (IGF-I) or IGF binding protein-3.

The growth rate of the young pig is generally much less than its potential and may be constrained by endocrine status as well as by nutrient intake. The aim of this study was to determine whether porcine somatotropin (pST) could increase growth in the nursing pig. Fourteen sows nursing litters of 6 (n = 7) or 12 (n = 7) piglets were utilized to establish a high and low plane of nutrition for sucking pigs. On Day 4 of lactation, the median two male pigs from each litter were randomly allocated to one of two doses of pST (0 or 60 micrograms/kg/d) until weaning on Day 31. Pigs were bled on Days 4, 13, 22, and 31 of lactation and the plasma was analyzed for insulin-like growth factor (IGF)-I, IGF-II, and IGF binding protein-3 (IGFBP-3). Pigs were weaned into conventional accommodation and further weighed on Days 63, 91, and 119. Pigs from litters of 6 grew more quickly and weighed 2.2 kg (P = 0.01) and 3.5 kg (P = 0.04) more than pigs from litters of 12 at 31 and 63 d of age, respectively. There was no effect of pST on preweaning growth of sucking pigs (261 vs. 258 g/d, P = 0.68), although growth rate increased in the final 3 d before weaning at 31 d (241 vs. 294 g/d, P = 0.01). IGFBP-3 was greater (1.09 vs. 0.78 micrograms/ml, P < 0.001), whereas IGF-I tended to be greater (206 vs. 176 ng/ml, P = 0.14), in pigs from the small litters. There was no effect of pST on plasma IGF-I (182 vs. 195 ng/ml, P = 0.454) or IGFBP-3 (0.93 vs. 0.94 microgram/ml, P = 0.85) concentrations. Plasma IGF-I and IGFBP-3 were highly correlated with the growth rate of nursing pigs (R = 0.638 and 0.756, respectively). There were no effects of pST (340 vs. 328 ng/ml, P = 0.48) or litter size (336 vs. 333 ng/ml, P = 0.88) on IGF-II. In conclusion, pST had no little or no effect on growth performance or plasma IGF-I, IGF-II, or IGFBP-3 in sucking pigs on either a high or low plane of nutrition.     

Involvement of connective tissue growth factor (CTGF) in insulin-like growth factor-I (IGF1) stimulation of proliferation of a bovine mammary epithelial cell line

The objective of this study was to determine the mechanism by which insulin-like growth factor-I (IGF1) stimulates proliferation of mammary epithelial cells, using the bovine mammary epithelial cell line MAC-T as a model. IGF1 significantly up- or down-regulated the expression of 155 genes in MAC-T cells. Among the most significantly suppressed was the gene for connective tissue growth factor (CTGF), a secretory protein that has both proliferative and apoptotic effects and is also a low-affinity binding protein of IGF1. IGF1 inhibited CTGF expression through the PI3K-Akt signaling pathway. Administration of growth hormone (GH), a strong stimulator of IGF1 production in vivo, decreased mammary CTGF mRNA in cattle; however, GH did not affect CTGF expression in MAC-T cells, suggesting that IGF1 may also inhibit CTGF expression in the mammary gland. Added alone CTGF stimulated proliferation of MAC-T cells, but in combination with IGF1 it attenuated IGF1's stimulation of proliferation of MAC-T cells. Excess IGF1 reversed this attenuating effect of CTGF. Despite being an IGF binding protein, CTGF did not affect IGF1-induced phosphorylation of IGF1 receptor (IGF1R) or IGF1R expression in MAC-T cells, indicating that the attenuating effect of CTGF on IGF1 stimulated proliferation of MAC-T cells was not mediated by decreasing IGF1's ability to bind to IGF1R or by decreasing IGF1R expression. Overall, these results suggest a novel biochemical and functional relationship between CTGF and IGF1 in the bovine mammary gland, where IGF1 may inhibit CTGF expression to reduce the attenuating effect of CTGF on IGF1 stimulated proliferation of epithelial cells.     

Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures.

Insulin-like growth factor-binding proteins (IGFBP) regulate the biological functions of insulin-like growth factors (IGF) and may affect cell growth through IGF-independent actions. Growth factors and hormones have been shown to alter IGFBP production by target cells suggesting that the effects of these factors may be partially mediated by the local production of IGFBP. Growth factors, including IGF-I, transforming growth factor-beta1 (TGF-beta1), and basic fibroblast growth factor (bFGF) have potent effects on satellite cell proliferation and differentiation, and some of these factors have been shown to alter IGFBP production in various cell types. Consequently, some of their actions on muscle satellite cells may be mediated by the local production of IGFBP. In this study, we measured the effects of IGF-I, bFGF, and TGF-beta1 on IGFBP production by primary porcine satellite cell (PSC) cultures after first determining physiologically active concentrations of these growth factors to use according to [3H]thymidine incorporation dose responses. There is little information on the effects of these growth factors on IGFBP production in primary porcine myogenic cells due to the confounding affects of contaminating nonmuscle fibroblasts. Comparative studies show that primary porcine satellite cells produce IGFBP-3 and -5 whereas porcine muscle-derived nonfusing cells (FIB) produce IGFBP-2 and -4 but not IGFBP-3 or -5. Because of this, our investigations have focused on growth factor-induced production of IGFBP-3 and -5 in primary porcine satellite cells cultures. Both IGF-I and bFGF exhibited dose-dependent increases in [3H]thymidine incorporation with increasing concentration from 1 to 50 ng/mL (P < 0.05), whereas TGF-beta1 caused a dose-dependent decrease from 0.01 to 0.5 ng/mL (P < 0.05). When 20 ng/ mL of IGF-I was added to the media, IGFBP-3 was increased approximately 65% (P < 0.05) and IGFBP-5 was increased approximately twofold (P < 0.05). The addition of 0.5 ng/mL TGF-beta1 caused more than a two-fold increase in IGFBP-3 (P < 0.05) and approximately an 80% increase in IGFBP-5 (P < 0.05), whereas 50 ng/ mL of bFGF caused approximately 40% (P < 0.05) and 70% (P < 0.05) increases in IGFBP-3 and -5, respectively. Neither IGFBP-3 nor -5 was detectable in the conditioned media from fibroblasts whether or not IGF-I, TGF- beta1 or bFGF were present. These data suggest that the effects of IGF-I, TGF- beta1 and bFGF on porcine satellite cells may in part be through the autocrine/ paracrine production of IGFBP-3 and -5 by porcine satellite cells.     

突变型猪表皮生长因子在毕赤酵母中的表达

使用α因子做为信号肽的酵母系统分泌表达猪表皮生长因子(pEGF)时,由于对信号肽末端Glu-Ala氨基酸残基切除不完全,表达产物是Glu-Ala-pEGF和pEGF的混合物.本研究为了获得单一表达的pEGF产物,对pEGF基因进行适当的突变修改,构建缺失Glu-Ala重复基因序列的重组载体.把重组载体pPIC9-pEGF1-48电转化毕赤酵母,用同位素标记随机引物斑点杂交法筛选多拷贝整合转化子,并诱导大量表达.用硫酸铵沉淀及超滤的方法浓缩和纯化蛋白,并用Bradford检测方法对蛋白浓度进行了初步测定.结果表明,构建的重组载体经过BglⅡ线性化后稳定转入毕赤酵母中,转化子表型主要为MutS型;选的多拷贝MutS型转化子经诱导后成功表达约6 000的pEGF1-48蛋白,经检测蛋白表达水平约为34 mg/L.     

生命早期铅暴露对小鼠海马内IGF1表达的影响

雌性小鼠自妊娠第1天开始经饮水染铅(0.1%、0.5%、1%浓度溶解在去离子水中,对照组饮蒸馏水)至仔鼠出生后21d断乳为止。在出生后的第21天,分别采用石墨炉原子吸收光谱法测定血铅和海马组织中铅的水平,免疫组织化学和免疫印迹法检测海马组织中胰岛素样生长因子1(insulin-like growth factor 1,IGF1)的表达。结果显示:与对照组相比,铅暴露组血铅水平和海马组织中铅的水平显著增高(P0.05);铅暴露组IGF1的表达比对照组明显下降(P0.05)。结果表明:母体铅暴露使铅在仔鼠体内蓄积,使仔鼠海马组织中IGF1蛋白的表达下调,从而造成海马神经元损伤,引起神经毒性。     

Effect of feed intake on antimicrobially induced increases in porcine serum insulin-like growth factor I

This study was conducted to determine whether an antimicrobially induced (ASP-250) increase in serum IGF-I was the result of differences in feed intake. Serum IGF-I concentrations were measured in crossbred pigs that were fed a control diet or a diet supplemented with ASP-250 either for ad libitum consumption or limited to 85% of the control pigs' consumption. The pigs that consumed either diet ad libitum, control or ASP-250, consumed similar quantities of feed. The ASP-250 ad libitum-intake pigs had serum IGF-I concentrations that were greater (P<.01) than those of their ad libitum-intake control littermates. Similarly, the ASP-250 limit-fed pigs had serum IGF-I concentrations that were greater (P<.01) than those of the controls. Although the serum IGF-I concentrations of pigs fed the ASP-250-supplemented diet for ad libitum intake were greater than the serum IGF-I concentrations of the pigs limit-fed the ASP-250-supplemented diet, the differences were not significant (P<.08). The ASP-250-fed pigs had higher serum IGF binding protein (BP)-3 concentrations than did their control littermates (P<.003). A time course of antimicrobially induced alterations in serum IGF-I concentrations revealed that the effect of increased serum IGF-I levels in ASP-250-supplemented pigs (P<.02) was observed within 4 d and was maintained throughout the 4-wk study. These findings show that feed intake is not responsible for the increase in serum IGF-I observed with ASP-250 supplementation. Additionally, the antimicrobially induced increase in serum IGF-I concentrations occurs within a few days after initiation of the treatment.     

Effect of feed restriction on serum somatotropin, insulin-like growth factor-I-(IGF-I) and IGF binding proteins in cyclic heifers actively immunized against growth hormone releasing factor

Feed restriction often increases serum somatotropin (ST) and decreases insulin-like growth factor-I (IGF-I) in ruminants; however, the mechanisms responsible for this change in ST and IGF-I are not well defined. We investigated the effects of feed restriction on serum ST, IGF-I, IGF binding proteins (IGFBP), insulin and nonesterified fatty acids (NEFA) in cyclic Angus and Charolais heifers (n=15) previously immunized against growth hormone releasing factor (GRFi) or human serum albumin (HSAi). Cows were fed a concentrate diet ad libitum (AL) or were restricted to 2 kg cotton seed hulls (R) for 4 d. Each heifer received each dietary treatment in a single reversal design. As anticipated, GRFi decreased ST, IGF-I and insulin (P<.05). In addition, GRFi decreased serum IGFBP-3 (P<.01), but increased IGFBP-2 (P<.01). Feed restriction resulted in an increase in serum ST in HSAi, but not in GRFi heifers. Regardless of immunization treatment, feed restriction decreased serum IGF-I and insulin, and increased NEFA (P<.01). In conclusion, the increase in serum ST levels observed during feed restriction was blocked by active immunization against GRF. However, feed restriction resulted in decreased serum IGF-I in GRFi heifers in spite of initial low levels of IGF-I (due to GRFi). Although GRFi decreased levels of IGFBP-3 and increased levels of IGFBP-2, feed restriction for 4 d did not alter serum IGFBP.