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.     

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.     

Insulin-like growth factor binding protein-3: its biological effect on bovine granulosa cells

This study was aimed at testing the hypothesis that insulin-like growth factor binding protein (IGFBP)-3 can modulate hormone-dependent differentiation of granulosa cells in vitro. Granulosa cells from small (1 to 5 mm) follicles were collected from cattle, cultured for 2 d in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 d in serum-free medium with follicle-stimulating hormone (FSH) (50 ng/ml), recombinant human IGF-I (0, 1.3, 4.0, or 13.3 nM), or recombinant human IGFBP-3 (0 to 4.26 nM). In one series of experiments, IGFBP-3 (0.53 and 2.13 nM) inhibited (51% to 92% decreases; P < 0.05) progesterone and estradiol production induced by 1.3 nM of IGF-I, but did not influence (P > 0.10) granulosa cell numbers or steroidogenesis in the absence of IGF-I. Only 4.26 nM of IGFBP-3 inhibited (by 35%) the increase in granulosa cell numbers induced by 1.3 nM of IGF-I. In another series of experiments, 13.3 nM of IGF-I, but not 4.0 nM of IGF-I, was able to completely overcome the inhibitory effect of 4.26 nM of IGFBP-3 on estradiol production. The increase in cell numbers induced by 4.0 and 13.3 nM of IGF-I was attenuated (P < 0.001) by 4.26 nM of IGFBP-3. In a third series of experiments, IGFBP-3 inhibited 125I-IGF-I binding to granulosa cells. These results indicate that IGFBP-3 has a pronounced inhibitory effect on IGF-I action in cultured bovine granulosa cells, and that this inhibitory effect is likely attributable to IGFBP-3 binding/sequestering IGF-I. Thus, IGFBP-3 may play a significant role in regulating granulosa cell proliferation and steroidogenesis during follicular development in cattle.     

Insulin-like growth factor (IGF)-I and IGF binding proteins-2, -3, -4, -5 in porcine corpora lutea during the estrous cycle; evidence for inhibitory actions of IGFBP-3

In this study we measured protein concentrations of insulin-like growth factor (IGF)-I and IGF binding proteins (IGFBPs) 2-5 in porcine corpora lutea (CLs) throughout the estrous cycle (Experiment 1), and examined the effects of IGFBP-3 and IGFBP-3 antibody (AB) on luteal progesterone (P4) secretion in vitro (Experiment 2). For Experiment 1, (CLs) and serum were collected on days (D) 4, 7, 10, 13, 15 and 16 of the estrous cycle (n = 5 animals per day). IGF-I was extracted from CLs and sera, and measured by radioimmunoassay (RIA). IGFBPs were measured in CLs by ligand blots. For Experiment 2, CLs (from Experiment 1) were enzyme dissociated and luteal cells cultured (24 h) in Medium 199 (M199) containing (0-500 ng/ml) IGFBP-3 (+/-IGF-I; 100 ng/ml), or (0-10 microg/ml) IGFBP-3 AB. P4 in media was measured by RIA. In Experiment 1, luteal IGF-I concentrations (ng/g tissue) were maximal on day 4 and gradually decreased thereafter. Serum IGF-I concentrations (ng/ml) were highest on days 4 and 7, compared with days 10-15. Peak levels of luteal IGFBP-3 were also seen on days 4 and 7 of the cycle. Luteal IGFBP-2 concentrations showed a tendency to increase on day 16 (P < 0.05 versus day 10), but no significant changes in IGFBP-4 or -5 were seen. In Experiment 2, IGFBP-3 (w IGF) inhibited the steroidogenic actions of IGF-I, but had no significant actions alone (IGFBP-3 w/o IGF). Finally, IGFBP-3 AB stimulated P4 secretion on days 4 and 7, but not on days 10-16. We conclude that IGFBP-3 inhibits IGF-I actions in the porcine CL.     

Oxytocin mediates some effects of insulin-like growth factor-I on porcine ovarian follicles

The aims of the present study were (1) to investigate the influence of insulin-like growth factor-I (IGF-I) on follicular size, on the secretion of oxytocin (OT), progesterone (P), estradiol (E), IGF binding protein-3 (IGFBP-3), inhibin A, inhibin B and cAMP and on the expression of proliferation-associated peptide PCNA, ERK-related mitogen activated protein kinase (MAPK/ERK1, 2) and protein kinase A (PKA) in cultured porcine ovarian follicles; (2) to examine the effects of OT on IGF-I and on these functions; and (3) to determine whether the effects of IGF-I can be mediated by OT. To define the involvement of OT in mediating IGF-I action, we compared responses of porcine ovarian follicles to IGF-I and OT and examined whether blockade of endogenous OT by specific antiserum can affect IGF-I action. It was observed that IGF-I (1, 10 or 100 ng/ml) was able to prevent a decrease in the size of ovarian follicles during culture and caused an increase in the diameter of some follicles. It also stimulated the secretion of OT, P, IGFBP-3, inhibin A and cAMP, decreased the secretion of E and inhibin B (RIA/EIA/ELISA), and induced the expression of PCNA, PKA, MAPK/ERK1, but not MAPK/ERK2 (Western blotting). Like IGF-1, OT (100 ng/ml) prevented decrease in follicular size and increased the diameter of some follicles. It also stimulated the secretion of P and IGF-I, but not E. Antiserum against OT (1%), when given alone, did not affect the reduction of follicular size but slightly increased the percentage of follicles increasing their diameter during culture. The antiserum also inhibited secretion of OT and cAMP but not the secretion of P, E, IGFBP-3 or the expression of PKA, MAPK/ERK1 or 2. When given together with IGF-I, the antiserum prevented the stimulatory action of IGF-I on the proportion of enlarged follicles and on OT, IGFBP-3 and MAPK/ERK1. It augmented the effect of IGF-I on P, but not the effect on E, cAMP, PKA or MAPK/ERK2. These observations demonstrate the involvement of IGF-I and OT in the control of ovarian follicular size and follicular cell proliferation, progestagen, estrogen, IGFBP-3, inhibin A and B secretion and in cAMP/PKA- and MAPK/ERK1-dependent intracellular mechanisms. Furthermore, the reciprocal stimulation of IGF-I and OT and the similarity of some their effects, together with the prevention or augmentation of some IGF-I effects after OT blockade, suggest that IGF-I action can be mediated by OT.     

In vitro growth-promoting activity of porcine mammary secretions: initial characterization and relationship to known peptide growth factors

We have examined the in vitro growth-promoting properties and growth factor content of porcine mammary secretions. Defatted, porcine colostrum stimulated the proliferation of fibroblast and epithelial-like cell lines of diverse species origins in serum-free medium and cellular DNA synthesis (4- to 119-fold) as monitored by uptake of 3H-thymidine into DNA of quiescent cells in culture. Porcine milk, although mitogenic, had reduced activity when compared with colostrum on an equivalent-volume basis. Furthermore, the relative mitogenic activity of milk, although still detectable at 3 wk, continued to decline with length of the lactation period. Fractionation of pig colostrum on gel-filtration columns revealed multiple peaks of (AKR-2B) fibroblast mitogenic activity (208, 66 and 4.6 kdaltons) and a heterogenous profile of epithelial cell mitogenicity. Polyclonal antibodies (IgG) specific for murine epidermal growth factor (EGF; the major mitogen in human and murine milk) or human platelet-derived growth factor (PDGF) did not inhibit the mitogenic activity of pig colostrum or milk, demonstrating lack of antigenic relatedness between the contributing porcine factors and mEGF or hPDGF. Also, we were unable to demonstrate similarity of the small Mr colostral factor with EGF by use of EGF radioreceptor assay. These results identify porcine colostrum and milk as sources of potentially important in vitro growth-promoting factors. The enhanced expression of these factors in early mammary secretions suggests their possible in vivo involvement in mammary and neonatal tissue growth processes.     

The ontogeny of the somatotropic axis in male and female Hereford calves from birth to one year of age

Insulin-like growth factor-binding proteins-2 and -3 may play a role in age-dependent growth response to bovine ST (bST) treatment in cattle; however, samples have been collected at infrequent intervals and at limited time points. Therefore, the objective of this experiment was to examine the ontogeny of components of the somatotropic axis in Hereford calves from birth to 1 yr of age at weekly intervals to determine whether there is a certain age or time frame when the somatotropic axis may change and/or potentially become more responsive to exogenous bST administration. Blood samples and body weight measurements were collected from eight male and eight female Hereford calves once per week from birth to 1 yr of age. Serum concentrations of ST, IGF-I, IGFBP-2, and IGFBP-3 were determined. Males began to grow faster than females at approximately 16 wk of age (P < 0.05). Average concentrations of ST, IGF-I, and IGFBP-3 were greater in males than females (P < 0.01). Average concentrations of IGFBP-2 were greater in females than in males (P = 0.05). Concentrations of ST decrease with age (P < 0.01); however, the decrease occurred earlier in female calves. Concentrations of IGF-I and IGFBP-3 increased in males and females (P < 0.01), and concentrations of IGF-I began to plateau at approximately the same time as growth rate differences were observed (16 wk of age). Following an initial increase (birth to approximately 16 wk of age), concentrations of IGFBP-3 remained constant until approximately 43 wk of age. Concentrations of IGFBP-2 increased to approximately 10 wk of age (P < 0.05), followed by a decrease, and then, similar to IGFBP-3, remained constant until 43 wk of age. Correlations between average daily gain, ST, IGF-I, IGFBP-2, and IGFBP-3 were determined. Average daily gain was negatively (P < 0.01) correlated with ST and positively (P < 0.1) correlated with IGF-I. In females, ST was negatively (P < 0.01) correlated with IGF-I. Concentrations of ST were positively correlated (P < 0.01) with IGFBP-2 and IGFBP-3. Concentrations of IGFBP-2 were negatively correlated (P < 0.01) with IGF-I and positively correlated (P < 0.01) with IGFBP-3. In conclusion, serum concentrations of ST, IGF-I, IGFBP-2, and IGFBP-3 differed between male and fe-male calves. In addition, changes in components of the somatotropic axis occurred around the same time as males began to grow faster than females.     

Investigation of the insulin-like growth factor system in the avian epiphyseal growth plate

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.     

IGF-I- and EGF-dependent DNA synthesis of porcine myoblasts is influenced by the dietary isoflavones genistein and daidzein

Soy-derived isoflavones have been reported to be specific inhibitors of protein tyrosine kinases like the type 1 insulin-like growth factor receptor (IGF-1R) and the epidermal growth factor receptor (EGFR). This study was conducted to investigate, whether IGF-I and EGF stimulate porcine myoblast growth and whether the responses are influenced by isoflavones. Satellite cell-born myoblasts derived from the semimembranosus muscle of newborn piglets were treated for 26h with IGF-I or EGF alone and in combination with genistein or daidzein. The DNA amount was measured and DNA synthesis was recorded as 6 h-[(3)H]thymidine incorporation during exponential growth in serum-free basal medium. IGF-I and EGF synergistically stimulated DNA synthesis of porcine myoblast with EGF causing a greater response. Genistein (100mumol/l) effectively reduced the growth factor-mediated DNA synthesis, which was associated with an inhibition of growth factor receptor protein expression. In response to daidzein no reduction in growth factor-mediated DNA synthesis was found. Daidzein (1; 10mumol/l) combined with IGF-I caused even a slight increase in DNA amount compared with the untreated control. The expression of the IGF-1R precursor protein was reduced with 10 and 100mumol/l daidzein, whereas the EGFR expression remained unchanged with daidzein. The results suggest that dietary isoflavones may interact with growth factor-induced stimulation of pig skeletal muscle growth.     

Growth factor messenger ribonucleic acid expression during differentiation of porcine embryonic myogenic cells

The growth factors, IGF-I and II, their binding proteins, IGFBP, and members of the transforming growth factor (TGF) superfamily (myostatin and TGFbeta1) are known to regulate proliferation and differentiation of myogenic cells. We hypothesized that changes in the relative expression of members of the IGF and TGFbeta systems play a significant role in regulating myogenesis in porcine embryonic myogenic cell (PEMC) cultures. Therefore, determining the expression patterns of these factors during PEMC myogenesis is important. Consequently, we used real-time PCR to explore the pattern of IGF-I; IGF-II; IGFBP-2, -3, and -5; IGF-type-I receptor; myogenin; myostatin; and TGFbeta1 mRNA expression during PEMC myogenesis. The progression of differentiation was assessed using creatine kinase activity and myogenin mRNA expression. As anticipated, creatine kinase activity was low in PEMC cultures at 48 h and increased 20-fold (P < 0.0001) between 48 h and its peak at 144 h. Similarly, myogenin mRNA was low at 48 h and increased approximately 5-fold (P < 0.0001) as differentiation progressed, peaking at 120 h and decreasing at 144 h. The patterns of IGF-I and IGFBP-2 mRNA expression were similar and were relatively lower in 48-h PEMC cultures, increasing approximately 5-fold (P < 0.0001) to their greatest levels at 120 h. In contrast, IGF-II and IGFBP-5 mRNA levels were relatively high at 48 h, peaking at 72 h, and steadily decreasing by 60 and 80%, respectively (P < 0.001), at 144 h. The level of IGF-type-I receptor mRNA was relatively high until 96 h of culture, after which it decreased 40% (P < 0.01), reaching a low at 144 h. Levels of IGFBP-3 mRNA were relatively high at 48 h, dropped approximately 40% to their lowest level at 72 h (P < 0.001), and then increased approximately 60% (P < 0.001) to their greatest levels at 144 h. Levels of TGFbeta1 mRNA decreased approximately 30% (P < 0.0001) between 48 and 96 h, then quickly rebounded to a peak at 120 h, and by 144 h had dropped to the levels seen at 72 h. Myostatin mRNA was at its greatest level at 48 h and declined rapidly between 72 and 96 h, finally decreasing by approximately 80% at 144 h (P < 0.0001). Our data demonstrate that these factors are differentially regulated during PEMC myogenesis and provide new information about their pattern of mRNA expression in cultured porcine muscle cells.     

Growth hormone suppresses the expression of IGFBP-5, and promotes the IGF-I-induced phosphorylation of Akt in bovine mammary epithelial cells

Growth hormone (GH) plays a specific role to inhibit apoptosis in the bovine mammary gland through the insulin-like growth factor (IGF)-I system, however, the mechanism of GH action is poorly understood. In this study, we show that GH dramatically inhibits the expression of IGFBP-5, and GH along with IGF-I enhanced the phosphorylation of Akt through the reduction of IGF binding protein (IGFBP)-5. To determine how GH affects Akt through IGF-I in bovine mammary epithelial cells (BMECs), we examined the phosphorylation of Akt in GH treated BMECs and found that IGF-I induced phosphorylation of Akt was significantly enhanced by the treatment with GH. We demonstrated that GH reduces mRNA and protein expression of IGFBP-5 in BMECs, but it does not affect the expression of IGFBP-3. To determine that the enhanced effect of the Akt phosphorylation by the treatment of GH is due to the inhibition of the expression of IGFBP-5, we examined the effect of IGFBP-3 and -5 on the phosphorylation of Akt through IGF-I in the GH-treated BMECs. The phosphorylation of Akt was inhibited in a dose-dependent manner when IGFBP-5 was added at varying concentrations and was also inhibited in the presence of IGFBP-3. The results of this study suggest that GH plays an important role on mammary gland involution in bovine mammary epithelial cells.     

Immunohistochemical detection of components of the insulin-like growth factor system during skeletal muscle growth in the pig

The insulin-like growth factor (IGF) system plays an important role in postnatal somatic and skeletal muscle growth in pigs. There is little information on the occurrence and distribution of components of the IGF system in postnatal porcine skeletal muscle. IGF-I, IGF receptor 1 (IGF1R) and the IGF-binding proteins IGFBP-1 and -3 in longissimus dorsi and triceps brachii were localized in muscle biopsies from 12 commercially crossbred pigs aged from 28 to 199 days as well as from the sire generation, by immunohistochemistry. Plasma IGF-I concentrations were also determined using radio-immunoassays. Unlike other species, IGF-I was localized in porcine skeletal muscle fibres. Staining intensity correlated with the highest plasma IGF-I levels and phases of intensive muscle growth from the 11th to 22nd week. The pattern of IGF1R immunostaining, which was strong, correlated with that of IGF-I, IGF1R was also localized in endomysial tissues. IGFBP-1 was not detected within muscle fibres, but was found in the endomysium and vessel walls, while IGFBP-3 was localized with IGF-1 and its receptor. Higher magnification revealed that IGF1R, IGFBP-3 and probably IGF-I appeared in the tubular system. Inhibitory as well as stimulating controls of IGFBP-1 and -3 on IGF functions are discussed, which may maintain a balance between autocrine growth promoting activities of IGF-I and IGF1R.     

Expression of insulin-like growth factor binding protein (IGFBP)-3, and the effects of IGFBP-2 and -3 in the bovine corpus luteum.

The present study was conducted to gain insight into the insulin-like growth factor (IGF) system in the bovine corpus luteum (CL). Specific aims were to measure the levels of IGF binding protein-3 (IGFBP-3) and RNA encoding IGFBP-3 in the CL throughout diestrus, and to investigate the effects of IGFBP-2 and -3 on IGF-I-stimulated progesterone (P4) production and IGF-I-receptor binding. Bovine CL were collected from a local abattoir and classified according to stage of diestrus based on anatomical characteristics. Corpora lutea from early, mid and late diestrus were each analyzed for the presence of IGFBP-3 by ligand blot analysis, and for RNA encoding IGFBP-3 by Northern blot analysis. Dissociated cells from mid-cycle CL were treated with IGF-I, IGFBP-2 or -3, or a combination of IGF-I and IGFBP-2 or -3. The effect of IGFBP-2 and IGFBP-3 on [(125)I] IGF-I binding to its receptor on CL plasma membranes also was investigated. IGFBP-3 protein and RNA expression were higher in early CL, compared to mid or late CL (p < 0.05). IGF-I stimulated P4 production in a dose-dependant manner (p < 0.05). IGFBP-2 and -3 blocked the stimulatory effect of IGF-I on P4 production (p < 0.05). Both IGFBP-2 and -3 inhibited [(125)I]-IGF-I binding to its receptor in a dose-dependant manner. These results demonstrate that IGFBP-3 protein and RNA are expressed predominantly during early diestrus in the bovine CL. Moreover, both IGFBP-2 and -3 can modulate IGF-I actions in the CL by interfering with binding of IGF-I to its receptor.     

Ontogenic maturation of the somatotropin/insulin-like growth factor axis

The ontogeny of the somatotropin/insulin-like growth factor system was examined in well-fed pigs under basal conditions and during a short-term challenge of porcine ST (pST). The study was conducted with two replicates of eight castrate male pigs from 3.8 kg BW (10 d of age) to 92 kg BW (129 d of age). Pigs were reared individually with ad libitum access to milk replacer through 23 d of age. Thereafter, pigs were fed a corn, soybean meal, and dry skim milk diet formulated to exceed nutrient requirements by approximately 30%. Pigs were randomly assigned to receive daily i.m. injections of either 0 (buffer) or 120 microg/kg BW of pST for a duration of 4 d starting at 10, 19, 33, 43, 63, 83, and 125 d of age. Blood was collected via jugular venipuncture on d 0 and 4 of the challenge. Circulating levels of IGF-I were not dramatically affected by age, but levels of IGF-II were low from 10 to 19 d of age and then increased through later stages of growth. Circulating concentrations of IGF binding protein (BP)-3 increased with age (P < .05), but levels of IGFBP-2, a 30-kDa IGFBP, and IGFBP-4 were unchanged (P > .10). The pST challenge reduced plasma urea nitrogen at all ages, but the magnitude of the response was less in younger pigs compared with the maximum response in pigs greater than 30 kg BW (63 d of age). The IGF-I response to the pST challenge also increased from approximately 30% in young pigs to a threefold increase in older pigs. Regardless of age, concentrations of IGF-II were minimally affected by the pST challenge. Circulating levels of IGFBP-3 increased and IGFBP-2 levels decreased in response to the pST challenge, and the magnitude increased with age. The high nutritional status of pigs in the early phases of growth diminished the postnatal changes in IGF-I and IGFBP-2, but not IGF-II or IGFBP-3. Overall, data demonstrate a developmental regulation of the ST/IGF system, with pST challenges altering circulating concentrations of IGF-I, IGFBP-3, and IGFBP-2 coincident with changes in amino acid metabolism.     

Effects of L-carnitine on fetal growth and the IGF system in pigs

The effects of L-carnitine on porcine fetal growth traits and the IGF system were determined. Fourth-parity sows were fed a gestation diet with either a 50-g top dress containing 0 (control, n = 6) or 100 mg of L-carnitine (n = 6). At midgestation, fetuses were removed for growth measurements, and porcine embryonic myoblasts (PEM) were isolated from semitendinosus. Real-time quantitative PCR was used to measure growth factor messenger RNA (mRNA) levels in the uterus, placenta, muscle, hepatic tissue, and cultured PEM. A treatment x day interaction (P = 0.02) was observed for maternal circulating total carnitine. Sows fed L-carnitine had a greater (P = 0.01) concentration of total carnitine at d 57 than control sows. Circulating IGF-I was not affected (P = 0.55) by treatment. Supplementing sows with L-carnitine resulted in larger (P = 0.02) litters (15.5 vs. 10.8 fetuses) without affecting litter weight (P = 0.07; 1,449.6 vs. 989.4 g) or individual fetal weight (P = 0.88) compared with controls. No treatment effect was found for muscle IGF-I (P = 0.36), IGF-II (P = 0.51), IGFBP-3 (P = 0.70), or IGFBP-5 (P = 0.51) mRNA abundance. The abundance of IGF-I (P = 0.72), IGF-II (P = 0.34), and IGFBP-3 (P = 0.99) in hepatic tissue was not influenced by treatment. Uterine IGF-I (P = 0.46), IGF-II (P = 0.40), IGFBP-3 (P = 0.29), and IGFBP-5 (P = 0.35) mRNA abundance did not differ between treatments. Placental IGF-I (P = 0.30), IGF-II (P = 0.18), IGFBP-3 (P = 0.94), and IGFBP-5 (P = 0.42) mRNA abundance did not differ between treatments. There was an effect of side of the uterus for IGF-I (P = 0.04) and IGF-II (P = 0.007) mRNA abundance; IGF-I mRNA abundance was greater in the left uterine horn than in the right uterine horn (0.14 and 0.07 relative units, respectively). Placental IGF-II mRNA abundance was greater (P = 0.007) in the left than in the right uterine horn (483.5 and 219.59, respectively). The abundance of IGFBP-3 was not affected by uterine horns in either uterine (P = 0.66) or placental (P = 0.13) tissue. There was no treatment difference for IGF-I (P = 0.31) or IGFBP-5 (P = 0.13) in PEM. The PEM isolated from sows fed L-carnitine had decreased IGF-II (P = 0.02), IGFBP-3 (P = 0.03), and myogenin (P = 0.04; 61, 59, and 67%, respectively) mRNA abundance compared with controls. These data suggest that L-carnitine supplemented to gestating sows altered the IGF system and may affect fetal growth and development.     

Effects of beta-aminopropionitrile on equine tendon metabolism in vitro and on effects of insulin-like growth factor-I on matrix production by equine tenocytes

OBJECTIVE: To investigate effects of beta-aminopropionitrile and a combination of insulin-like growth factor (IGF)-I and beta-aminopropionitrile on metabolism of equine tendon fibroblasts. SAMPLE POPULATION: Flexor tendon explants from 3 horses. PROCEDURE: Explants received 1 of 4 treatments (control, IGF-I, beta-aminopropionitrile, and IGF-I/beta-aminopropionitrile) for 10 days, and message expression for collagen types I and III was assessed by use of in situ hybridization. Histologic findings, new protein production, and quantitative determinations of glycosaminoglycan, DNA, and de novo collagen synthesis were made. RESULTS: Insulin-like growth factor-I stimulated an anabolic response in tendon. Collagen synthesis and glycosaminoglycan and DNA content of explants were all increased. Beta-aminopropionitrile significantly suppressed collagen synthesis, which was not ameliorated by concurrent IGF-I treatment. Beta-aminopropionitrile caused alterations in cell morphology characterized by large round cells with eccentric nuclei and decreased density of collagen fibers. Protein production and collagen type-III mRNA expression were reduced in these cells. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment with beta-aminopropionitrile resulted in decreased production of protein and collagen synthesis, which could be expected to suppress tendon healing. The negative effects of beta-aminopropionitrile could not be abrogated by addition of IGF-I to the medium. Treatment resulted in alterations in cell morphology and matrix consistency, which could further delay tendon healing. Beta-aminopropionitrile may impair tendon healing at a cellular level by decreasing collagen production or increasing rate of degradation of existing matrix. Because of reduced crosslinking during beta-aminopropionitrile treatment, in combination with transiently decreased tensile strength, alterations in collagen content and structure may weaken the healing tendon.     

Increased degradation of insulin-like growth factor-I in serum from feed-deprived steers

Severe feed restriction decreases serum insulin-like growth factor I (IGF-I) concentration in animals, and this decrease is thought to be due to reduced IGF-I production in the liver. The objective of this study was to determine whether feed deprivation also increases degradation of serum IGF-I and serum levels of IGF binding protein 3 (IGFBP-3) and acid-labile subunit (ALS), which inhibit IGF-I degradation and increase IGF-I retention in the blood by forming a ternary complex with IGF-I, in cattle. Five steers had free access to pasture, and another five were deprived of feed for 60 h. Serum concentration of IGF-I and liver abundance of IGF-I mRNA at the end of the 60-h period were 50% and 80% lower, respectively, in feed-deprived steers than in fed steers. Less ¹²⁵I-labeled IGF-I remained intact after a 45-h incubation in sera of feed-deprived steers than in sera of fed steers, suggesting that serum IGF-I is more quickly degraded in feed-deprived animals. Serum levels of IGFBP-3 and ALS were decreased by 40% and 30%, respectively, in feed-deprived steers compared with fed steers. These decreases were associated with more than 50% reductions in IGFBP-3 and ALS mRNA in the liver, the major source of serum IGFBP-3 and ALS. Taken together, these results suggest that feed deprivation reduces serum concentration of IGF-I in cattle not only by decreasing IGF-I gene expression in the liver, but also by increasing IGF-I degradation and reducing IGF-I retention in the blood through decreasing IGFBP-3 and ALS production in the liver.     

Insulin-like growth factor I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate

Ovine-derived fibroblasts were used to validate an insulin-like growth factor I (IGF-I) membrane-receptor binding assay system. Competitive binding using fibroblasts revealed that half-maximal inhibition of 125I-IGF-I binding by IGF-I was 2.3 nM. SDS-polyacrylamide gel electrophoresis analysis of specific protein-associated 125I-IGF-I was consistent with the migration of 125I-IGF-I-labeled Type I IGF receptor alpha-subunits at Mr 133,000 daltons. Further, the efficiency of two cell solubilization methods was examined and time-dependent binding equilibrium was determined for the membrane assay system. Satellite cell-derived myotubes were subsequently isolated from primary satellite cell cultures established from the semimembranosus muscles of high and low efficiency-of-gain (EOG) Targhee rams, and IGF-I receptor dynamics were measured. A membrane competitive binding study revealed that half-maximal inhibition of 125I-IGF-I binding was achieved by 1-ng IGF-I for low, and 10-ng IGF-I for high, EOG myotube membrane preparations. Kd values were similar between the high EOG (4.78 nM) and low EOG (2.95 nM) groups; however, receptor concentrations (Bmax) appeared to differ between groups. High EOG membrane receptor Bmax was 3.88 pmole/micrograms protein (19.87 pmole/micrograms DNA), whereas low EOG membrane receptor Bmax was 1.22 pmole/micrograms protein (9.28 pmole/micrograms DNA). These preliminary findings support the hypothesis that genetic selection for EOG results in altered satellite cell responsiveness to IGF-I.     

Age-related changes in serum insulin-like growth factor-I, insulin-like growth factor-I binding protein-3 and articular cartilage structure in Thoroughbred horses

REASONS FOR PERFORMING STUDY: Structural changes in articular cartilage associated with the ageing process require definition for investigators performing developmental and age-related studies, for which information is lacking. OBJECTIVES: To 1) determine the onset and end of puberty as defined by serum insulin like growth factor (IGF-I) and IGF-binding protein-3 (IGFBP-3) concentrations and 2) correlate articular-epiphyseal cartilage complex structural changes with the onset and end of puberty. METHODS: IGF-I and IGFBP-3 were measured in serum samples from normal female and male horses age 9-715 days to determine peak and steady-state values for horses transitioning through puberty. Osteochondral tissue sections were obtained from horses age 120-840 days (4-28 months) and examined histologically for cartilage canals and tidemark formation. RESULTS: In male and female horses, serum IGF-I/IGFBP-3 concentrations peaked at approximately 225 days, defining the onset of puberty. Cartilage canals were absent from articular cartilage just prior to this time point. IGF-I/IGFBP-3 concentrations declined to steady-state levels at approximately age 450 days, signalling exit from puberty and therefore the beginning of ageing. This time point correlated to initial formation of a tidemark in the osteochondral tissue sections. CONCLUSIONS: Horses may be considered pubescent at age 225-450 days, and post pubescent and ageing after age 450 days. POTENTIAL RELEVANCE: Defining the normal post natal to post pubescent concentrations for serum IGF-I and serum IGFBP-3 establishes subsets of animals for age-related studies and may be used to monitor horses for abnormally high IGF-I concentrations due to natural disease or subsequent to systemic growth hormone administration.     

Insulin administration suppresses an increase in insulin-like growth factor binding protein-2 gene expression stimulated by fasting in the chicken

1. We examined the changes in plasma IGF-I concentration and tissue IGFBP-2 gene expression of young fasted chickens refed a commercial diet or administered bovine insulin intravenously. 2. Plasma IGF-I concentration was decreased by fasting for 2 d. Although plasma IGF-I concentration was increased by refeeding, it didn't recover to the level of chickens fed a commercial diet ad libitum. 3. Insulin administration lowered plasma IGF-I concentration compared to other groups. 4. Hepatic IGFBP-2 mRNA was increased by fasting for 2 d and decreased by refeeding for 6 h. Insulin administration also decreased hepatic IGFBP-2 gene expression stimulated by fasting to the level of refed chickens. 5. IGFBP-2 mRNA in the gizzard was increased by fasting for 2 d and tended to decrease after refeeding for 6 h. Insulin administration decreased gizzard IGFBP-2 gene expression to less than that in refed chickens. 6. There was no between-treatment difference in IGFBP-2 mRNA in the brain and kidney. 7. These results suggest that the changes in IGFBP-2 gene expression in the liver and gizzard by fasting and refeeding might be partly regulated by the alteration in plasma insulin concentration.