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This study focused on the expression of somatotropic axis genes in the skeletal muscle of dairy cattle. A slow-release recombinant bovine growth hormone (GH) (rbGH) formulation was administered to 5 cows, and saline solution (control) was administered to another 5 cows every 2 wk for a total of 10 wk, starting from the peak of lactation. Tissue and blood samples were collected on days 2 and 14 after each rbGH injection. As target genes insulin-like growth factor (IGF)-1, IGF-2, IGFBPs (1, 2, 3, 4, 5, 6), acute labile subunit (ALS), IGF-1 receptor (IGF-1R), GH receptor (GHR), and the known GHR 5'-UTR variants were selected as target genes, and their relative expression was measured using real-time polymerase chain reaction. In GH-treated cows, an increase in expression was observed for GHR 5'-UTR variant 1I on day 14 (P < 0.05), whereas a significant down-regulation of GHR (P < 0.05) was found after comparing values of treated cows between day 2 and day 14. However, only IGF binding proteins (BP)-5 was found to be appreciably up-regulated in GH-treated cows (P < 0.001), which may indicate the importance of this gene in the overall molecular response to GH administration. Our study indicated that GH treatment did not affect the expression of most somatotropic axis genes, despite the marked increase in GH and IGF-1 in blood (P < 0.001). Nor did it have a large impact on the proportion of GHR 5'-UTR variants in the skeletal muscle of lactating cows. Finally, although we observed a significant variation in the expression of some genes, it would appear that the differences between GH-treated cows and controls were not great enough to be considered as reliable indirect indicators of GH treatment in dairy cattle.

The effects of fasting on insulin-like growth factor (IGF)-I, IGF-II, and IGF-binding protein (IGFBPs) mRNA in channel catfish were examined. Fed control fish (Fed) were compared to fish that had been fasted for 30 d followed by 15 d of additional feeding (Restricted). Sequence alignment and similarity to orthologous proteins in other vertebrates provided structural evidence that the 3 catfish sequences identified in the present research were IGFBP-1, -2, and -3. Prolonged fasting (30 d) reduced body weight approximately 60% (P<0.001) and decreased IGF-I mRNA in the liver and muscle (P<0.01). Fifteen days of re-feeding restored concentrations of hepatic and muscle IGF-I mRNA. Liver IGF-II mRNA was not affected by fasting but was increased 2.2-fold after 15 d of re-feeding (P<0.05). Abundance of muscle IGF-II mRNA was similar between the fed control group and the restricted group throughout the experimental period. Fasting also increased liver IGFBP-1 mRNA (P<0.05) and decreased IGFBP-3 mRNA (P<0.01), whereas abundance of IGFBP-2 mRNA was not significantly affected. Interestingly, re-feeding for 15 d did not restore concentrations of IGFBP-1 and IGFBP-3 mRNA relative to fed control concentrations. The IGF results suggest that IGF-I and IGF-II are differently regulated by nutritional status and probably have a differential effect in promoting muscle growth during recovery from fasting. Similar to mammals, IGFBP-1 mRNA in catfish is increased during catabolism, whereas IGFBP-3 mRNA is decreased during inhibited somatic growth. The IGFBP results provide additional evidence of the conserved nature of the IGF-IGFBP-growth axis in catfish.

Although cDNA microarray studies have examined gene expression in human and rodent adipose tissue, only one microarray study of adipose tissue from growing pigs has been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) from gilts at 90, 150, and 210 d (n=5 age(-1)). Dye labeled cDNA probes were hybridized to custom porcine microarrays (70-mer oligonucleotides). Gene expression of insulin-like growth factor binding proteins (IGFBPs), hormones, growth factors, neuropeptide Y (NPY) receptors (NPYRs) and other receptors in OSQ and MSQ changed little with age in growing pigs. Distinct patterns of relative gene expression were evident within NPYR and IGFBP family members in adipose tissue from growing pigs. Relative gene expression levels of NPY2R, NPY4R and angiopoietin 2 (ANG-2) distinguished OSQ and MSQ depots in growing pigs. We demonstrated, for the first time, the expression of IGFBP-7, IGFBP-5, NPY1R, NPY2R, NPY, connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes in pig adipose tissue with microarray and RT-PCR assays. Furthermore, adipose tissue CTGF gene expression was upregulated while NPY and NPY2R gene expression were significantly down regulated by age. These studies demonstrate that expression of neuropeptides and neurotrophic factors in pig adipose tissue may be involved in regulation of leptin secretion. Many other regulatory factors were not influenced by age in growing pigs but may be influenced by location or depot.

Rapid uterine involution in the postpartum period of dairy cows is important to achieve a short interval to conception. Expression patterns for members of the insulin-like growth factor (IGF) family were determined by in situ hybridisation at day 14+/-0.4 postpartum (n=12 cows) to investigate a potential role for IGFs in modulating uterine involution. Expression in each uterine tissue region was measured as optical density units and data were analysed according to region and horn. IGF-I mRNA was localized to the sub-epithelial stroma (SES) of inter-caruncular and caruncular endometrium. Both IGF-II and IGF-1R expression was detected in the deep endometrial stroma (DES), the caruncular stroma and myometrium. IGFBP-2, IGFBP-4 and IGFBP-6 mRNAs were all localised to the SES of inter-caruncular and caruncular uterine tissue, and in the DES and caruncular stroma, with IGFBP-4 mRNA additionally expressed in myometrium. IGFBP-3 mRNA was only detectable in luminal epithelium. IGFBP-5 mRNA was found in myometrium, inter-caruncular and caruncular SES and caruncular stroma. These data support a role for IGF-I and IGF-II in the extensive tissue remodelling and repair which the postpartum uterus undergoes to return to its non-pregnant state. The differential expression of binding proteins between tissues (IGFBP-3 in epithelium, IGFBP-2, -4, -5 and -6 in stroma and IGFBP-4 and -5 in myometrium) suggest tight control of IGF activity within each compartment. Differential expression of many members of the IGF family between the significantly larger previously gravid horn and the previously non-gravid horn may relate to differences in their rate of tissue remodelling.

In dairy cows, negative energy balance (NEB) during the early post-partum period is associated with major alterations in the growth hormone-insulin-like growth factor (GH-IGF) axis. Since the liver mediates nutrient partitioning during lactation, we aimed to determine how NEB alters the endocrine regulation of the insulin-like growth factor (IGF) system by investigating the expression of IGF family members and related steroid receptors. On the second day of lactation, cows were allocated to one of two treatments designed to produce mild (MNEB) or severe NEB (SNEB). MNEB cows (n=5) were fed ad lib grass silage supplemented with concentrate and milked x1 daily and SNEB cows (n=6) were restricted in dietary intake and milked x3 daily. Energy balance (EB) status was monitored until the second week of lactation when plasma and liver samples revealed a markedly divergent metabolic profile. At this time, plasma protein and hepatic mRNA for IGF-I was reduced in SNEB cows compared with MNEB cows. Both levels of expression correlated highly when data from all animals was pooled (r=0.963; P<0.01). SNEB cows also exhibited reduced hepatic expression for transcripts encoding IGF-1R, IGF-2R, IGF binding proteins (IGFBPs) -3, -4, -5, -6, acid labile subunit, and receptors for oestrogen (ERalpha) and growth hormone (total GHR and 1A variant), while the expression of IGFBP-2 was elevated. Expression of mRNA for IGF-II, IGFBP-1 and receptors for insulin (A/B) and glucocorticoid (alpha) was unaffected by EB. Results demonstrate that SNEB affects hepatic synthesis of IGF-I, and other components known to modulate the bioavailability and stability of circulating IGF-I.

The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-alpha. Madin-Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF-alpha for 24h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF-alpha increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF-alpha effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1mg/ml), the effect of TNF-alpha was to decrease (P<0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF-alpha consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF-alpha on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 microg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF-alpha affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.

Perturbations in endocrine functions can impact normal growth. Endocrine traits were studied in three dwarf calves exhibiting retarded but proportionate growth and four phenotypically normal half-siblings, sired by the same bull, and four unrelated control calves. Plasma 3,5,3'-triiodothyronine and thyroxine concentrations in dwarfs and half-siblings were in the physiological range and responded normally to injected thyroid-releasing hormone. Plasma glucagon concentrations were different (dwarfs, controls>half-siblings; P<0.05). Plasma growth hormone (GH), insulin-like growth factor-1 (IGF-1) and insulin concentrations in the three groups during an 8-h period were similar, but integrated GH concentrations (areas under concentration curves) were different (dwarfs>controls, P<0.02; half-siblings>controls, P=0.08). Responses of GH to xylazine and to a GH-releasing-factor analogue were similar in dwarfs and half-siblings. Relative gene expression of IGF-1, IGF-2, GH receptor (GHR), insulin receptor, IGF-1 type-1 and -2 receptors (IGF-1R, IGF-2R), and IGF binding proteins were measured in liver and anconeus muscle. GHR mRNA levels were different in liver (dwarfs

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.

We have used a porcine model of spontaneous differential fetal growth to investigate the effects of fetal size on muscle development. We hypothesized that altered muscle development may occur in small fetuses as a consequence of modified expression of selected genes of the insulin-like growth factor system. We examined the development of the Longissimus muscle (m. Longissimus) in small fetuses and their average sized littermates. We collected small for gestational age fetuses and their average sized sibling on days 45, 65 and 100 of gestation (term is 113-116 days). Small fetuses had significantly lower body weight at all three stages of gestation (p<0.05) and significantly reduced secondary to primary muscle fibre ratio in m. Longissimus on day 100 (p<0.05) compared to their littermates. On day 65, the expression of insulin-like growth factor receptor 1 and insulin-like growth factor binding protein 3 were significantly higher (p<0.05) in m. Longissimus of the small fetuses compared with their average sized littermates. On day 100, the expression of insulin-like growth factor receptor 1 remained significantly higher (p=0.001), in addition to significantly higher levels of insulin-like growth factor receptor 2 and insulin-like growth factor binding protein 5 in the small fetuses (p<0.05). No difference in levels of myogenin was observed between the small and average sized littermates. In conclusion, we demonstrate that reduced fetal muscle development is associated with an increased expression of several genes of the insulin-like growth factor system in small fetuses in mid to late gestation.

The aim of this study was to examine whether exogenous somatotropin (ST) can alter the insulin-like growth factor (IGF) axis in the porcine epitheliochorial placenta. Crossbred gilts were injected either 6 mg of recombinant porcine ST or vehicle from days 10 to 27 after artificial insemination (term day 116). Control and ST-treated gilts were euthanized on day 28 (8 control/5 treated), day 37 (4 control/6 treated), and day 62 (4 control/6 treated) of gestation. Endometrium and placental tissue samples were collected and subjected to mRNA analyses. In control gilts, somatotropin receptor (STR) and IGF-I mRNA abundance in the endometrium decreased with gestation. Conversely, the amounts of IGF-II mRNA and of IGF binding protein (BP)-2 and -3 mRNA, which were analyzed in endometrium and placental chorion, increased with gestation. The endometrium contained less IGF-II mRNA but more IGFBP-2 and-3 mRNA than the placental chorion. In response to pST treatment, the amounts of endometrial STR and IGF-I mRNA were lower at days 28 and 37, but higher at day 62 of gestation. The content of IGF-II mRNA was higher in the endometrium of pST-treated than control gilts on day 37. The amount of IGFBP-2 mRNA was increased on day 37 in endometrium and placenta of pST-treated gilts, whereas no changes in IGFBP-3 mRNA were observed. The IGF-II/IGFBP-2 ratio was higher in the placenta in response to pST on day 28 of gestation. Results show that pST treatment of pregnant gilts during early gestation alters IGF axis in maternal and fetal placental tissues and suggest pST may exert an effect on fetal growth by altering the relative amount of IGFBPs and IGFs at the fetal-maternal interface.

To determine if (1) levels of pregnancy-associated plasma protein-A (PAPP-A) mRNA and insulin-like growth factor binding protein (IGFBP) (-2, -3, -4 and -5) mRNAs differ between the dominant and subordinate follicles during the follicular phase of an estrous cycle, and (2) these differences are associated with differences in follicular fluid (FFL) concentrations of steroids (estradiol, androstenedione, and progesterone), total and free IGF-I, or IGFBPs, estrous cycles of non-lactating Holstein dairy cows (n = 16) were synchronized with two injections of prostaglandin (PGF2 alpha) 11 days apart. Granulosa cells and FFL were collected either 24 h or 48 h after the second injection of PGF2 alpha. FFL from dominant follicles had lower concentrations of progesterone (P < 0.08) and higher concentrations of estradiol (P < 0.05), androstenedione (P < 0.0001), estradiol:progesterone ratio (P < 0.0001), free IGF-I (P < 0.0001), and calculated percentage free IGF-I (P < 0.01) than large subordinate follicles. Levels of IGFBP-2, -4, and -5 in FFL were 3.0- (P < 0.05), 2.4- (P < 0.06), and 3.4-fold (P < 0.05) greater, respectively, in subordinate than in dominant follicles. IGFBP-3, IGFBP-4 and PAPP-A mRNA expression and IGF-II concentration did not differ (P > 0.10) between dominant or subordinate follicles. Levels of IGFBP-2 and -5 mRNA were severalfold greater (P < 0.05) in subordinate than dominant follicles. IGFBP-5 mRNA in granulosa cells decreased (P < 0.05) 62% to 92%, between 24h and 48 h post-PGF2 alpha. We conclude that decreased levels of IGFBP-2 and -5 mRNA in granulosa cells may contribute to the decrease in FFL IGFBP-2 and -5 protein levels of preovulatory dominant follicles, and that changes in granulosa cell IGFBP-3 and -4 mRNA and PAPP-A mRNA levels do not occur during final preovulatory follicular development in cattle.

The effects of estradiol, insulin, and gonadotropins on levels of insulin-like growth factor binding protein (IGFBP)-2, -3, -4, and -5 mRNA levels in bovine granulosa and theca cells were evaluated in vitro using serum-free medium containing various hormone treatments arranged in four different experiments. Amounts of IGFBP-2, -3, -4 and -5 mRNA were quantitated using fluorescent quantitative real-time RT-PCR. In small-follicle (1-5 mm) granulosa cells, follicle-stimulating hormone (FSH) in the presence or absence of insulin increased (P<0.05) IGFBP-3 mRNA but did not change IGFBP-2, -4, or -5 mRNA levels; estradiol was without effect on IGFBP-2, -3, -4, or -5 mRNA levels in the absence of insulin but increased (P<0.05) IGFBP-2 mRNA levels in the presence of insulin. Luteinizing hormone (LH) in the absence (but not presence) of insulin increased (P<0.05) small-follicle granulosa cell IGFBP-3 mRNA levels. In large-follicle (>7.9 mm) granulosa cells, insulin alone increased (P<0.05) IGFBP-2 gene expression while LH, FSH, and estradiol were without effect (P>0.10). Estradiol (3 and 300 ng/ml) decreased (P<0.05) IGFBP-5 mRNA levels in large-follicle granulosa cells. In theca cells, insulin decreased (P<0.05) IGFBP-4 expression, but had no effect (P>0.10) on IGFBP-2, -3, or -5 mRNA levels. Estradiol decreased (P<0.05) IGFBP-2, -3, and -4 mRNA levels but had no effect on IGFBP-5 mRNA levels in theca cells. LH had no effect on levels of IGFBP-2, -3, -4, or -5 mRNA in theca cells. These results indicate that expression of IGFBP-2, -3, -4, and -5 mRNA by granulosa and theca cells are differentially regulated by estradiol, insulin and gonadotropins, therefore discretely modulating the amount of bioavailable IGFs to these cells depending upon the specific hormonal stimuli. In particular, these studies are the first in cattle to show that estradiol selectively inhibits IGFBP-2, -3, and -4 gene expression in theca cells, inhibits IGFBP-5 gene expression in large-follicle granulosa cells, and stimulates IGFBP-2 gene expression in small-follicle granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) have been shown to affect proliferation of several cell types via insulin-like growth factor (IGF)-dependent and IGF-independent mechanisms. The goal of this study was to determine if levels of IGFBP-2, -3, -4 and -5 mRNA changed during differentiation of cultured porcine embryonic myogenic cells. Total RNA was isolated from muscle cultures at various stages of differentiation and Northern blots of this RNA were probed with 32P-labeled cDNA probes specific for individual IGFBPs. Fusion, myogenin mRNA, and creatine phosphokinase activity were used as markers of differentiation. The level of IGFBP-3 mRNA in differentiating cultures (120 h in culture) was only one-third of the level in myogenin negative, nonfused cultures (72 h in culture) (P < 0.05, n = 4). In contrast, the level of IGFBP-3 mRNA in extensively fused cultures (144 h in culture) was increased by three-fold as compared to the level in myogenin negative, nonfused cultures (P < 0.05, n = 4) and approximately seven-fold as compared to the 120-h cultures (P < 0.05, n = 4). No significant change in the level of IGFBP-5 mRNA was observed during differentiation of myogenic cultures. IGFBP-2 mRNA levels were not significantly different at 72, 96 and 120 h, but at 144 h IGFBP-2 mRNA level was increased three-fold as compared to nonfused cultures (72 h) (P < 0.05, n = 4). IGFBP-4 mRNA was not detectable on Northern blots of total RNA from porcine myogenic cultures at any stage of differentiation. Changes in IGFBP-3 and IGFBP-2 mRNA levels are associated with differentiation of embryonic porcine myogenic cells in culture and this may indicate that these IGFBPs play a role in differentiation of these cells., (Copyright 2002 Elsevier Science Inc.)

The metabolism in mammalian is regulated by multiple levels of hormone action, with complex feedback and control mechanisms. The somatotropic axis, essentially consisting of growth hormone (GH), insulin-like growth factors (IGF-I and -II), their associated carrier proteins, and receptors, plays a key role in the control of the regulation of metabolism and physiological process. Among this axis, other hormones like insulin, leptine, glucocorticoids or thyroid hormones are involved in this mechanism by modulating GH and/or IGF-I synthesis and availability. This review summarizes the complexity of the regulation of the metabolism by the somatotropic axis using different examples such as special nutritional situations or growth promoters administration.

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.

Plasma insulin-like growth factor-I (IGF-I) concentrations were related to hepatic levels of IGF-I mRNA measured by competitive reverse transcription polymerase chain reaction (PCR) (RT-PCR) in neonatal (8 d old) calves, veal calves, fattened castrated bulls and mature intact bulls. Furthermore, the presence of mRNAs of IGF-II and of receptors for IGF-I (IGF-IR), growth hormone (GHR) and insulin (IR), as well as mRNAs of IGF binding proteins (IGFBP-1, -2 and -3) were assessed by RT-PCR. Hepatic IGF-I mRNA levels and plasma IGF-I concentrations in veal calves, fattened castrated bulls and in intact bulls were 4 to 8 times higher than in 8-d old calves and were 2 to 3 times higher in calves fed colostrum than in calves fed only milk replacer. Hepatic IGF-I mRNA concentrations were closely correlated (r = 0.92) with plasma IGF-I concentrations, suggesting that hepatic IGF-I production largely determines plasma IGF-I levels. The presence of IGF II, IGF-IR, GHR, IR and IGFBP-1, -2 and -3 mRNA was confirmed in the liver of 8-d old calves and older cattle as well, and among newborn calves their presence was independent of differences in nutrition. In conclusion, the major hepatic components of the GH-IGF axis were present in neonatal calves, but the IGF-I expression and therefore also plasma IGF-I levels were relatively low.

A large amount of research, primarily in mammals, has defined to a great extent the pleiotropic effects of the IGF system on growth, development, and intermediary metabolism. Similar elucidations in poultry were hindered to some extent by the absence of native peptides (IGF-I and IGF-II) until their purification, followed by the production of recombinant chicken IGFs. In many ways IGF physiology in birds is similar to that in other species, including but not limited to the fact that IGF-I synthesis is both GH- and GH-independent, and that autocrine-paracrine IGF action is evident. However, it is clear that several unique differences in IGF physiology exist between birds and mammals. For example, more IGF is present in the free form in chickens, and the biological responses to the IGFs is different in several metabolic pathways in birds compared to mammals. To date, no unique IGF-II receptor has been identified in birds. Despite an increasing understanding of the IGFs in aves, several important questions remain to be answered. What is the role of IGF-II in embryo development and posthatch growth? Does an IGF-II receptor entity exist in nonmammalian species? How does nutrition affect IGF-I and IGF-II gene expression, and can this information be used to enhance poultry production? What is the biochemical composition of the IGFBPs, and what are their roles in birds? Can the genetic variation present in poultry be used to positively modify IGF gene expression and physiology? How do the IGFs regulate intermediary metabolism? What is the role of the IGFs in the etiology of several disease states associated with rapid growth in poultry, including tibial dyschondroplasia, obesity, ascites, and spiking mortality syndrome? Answers to these questions are relevant to our understanding of the basic mechanisms of IGF physiology as well as possibly assisting in the amelioration of problems found in modern poultry production.