Domestic Animal Endocrinology - Articles in Press

Ceramide accumulation and up-regulation of proinflammatory interleukin-1β exemplify lipotoxicity to mediate declines of reproductive efficacy of broiler hens - Corrected Proof

2012-01-30

Abstract: The study was conducted to delineate fundamental mechanisms that initiate the deleterious effect of fuel overloading on reproductive efficacy of broiler breeder hens. Sixty hens at age 26 wk were fed recommended amounts of feed (160 g/d per hen) or allowed voluntary feeding (approximately 30% more than restriction). At age 35 and 50 wk, hens were sampled for further analyzes. Voluntary feeding resulted in poor egg production, high rate of mortality, and abnormal ovarian structure (mainly overt hierarchical follicle atresia at age 35 wk and ovarian involution at age 50 wk). In contrast to feed-restricted hens, voluntary feeding also induced metabolic dysregulations that comprised enhanced adiposity; hepatic triacylglycerol accumulation; and elevated concentrations of plasma glucose, NEFAs, very low density lipoprotein, triacylglycerol, phospholipids, and sphingomyelin (P < 0.05). Furthermore, hepatic and circulating ceramide and sphingomyelin accumulation, and up-regulation of proinflammatory IL-1β expression in liver and adipose tissues (P < 0.05) systemically manifested the development of lipotoxicity in feed-satiated hens. Lipotoxicity leading to impaired ovarian dysfunctions, including follicle atresia, ovarian regression, and a decline of circulating estradiol levels (P < 0.05) in feed-satiated hens, was further exemplified by ceramide accumulation and up-regulation of IL-1β, serine palmitoyltransferase, and sphingomyelinase transcript abundance, but suppressed protein kinase Akt activation (P < 0.1 to 0.05) within the hierarchical follicles. This study provides the first in vivo evidence of the actions of ceramide and IL-1β in mediating overfeeding-induced follicle atresia and progression of ovarian involution in broiler hens.

New developments on the galactopoietic role of prolactin in dairy ruminants - Corrected Proof

P. Lacasse, V. Lollivier, F. Dessauge, R.M. Bruckmaier, S. Ollier, M. Boutinaud — 2012-01-27

Abstract: In most mammals, prolactin (PRL) is essential for maintaining lactation and its suppression strongly inhibits lactation. However, the involvement of PRL in the control of ruminant lactation is less clear because inconsistent effects on milk yield have been observed with short-term suppression of PRL by bromocriptine. By contrast, in vitro studies have provided evidence that PRL helps to maintain the differentiation state and act as a survival factor for mammary epithelial cells. Therefore, a series of experiments were conducted to assess the galactopoietic role of PRL. In a first experiment, daily injections of the PRL inhibitor quinagolide reduced milking-induced PRL release and induced a faster decline in milk production. Milk production was correlated with PRL released at milking. Quinagolide reduced mammary cell activity, survival, and proliferation. During the last week of treatments, differential milking (1× vs 2×) was applied. The inhibition of milk production by quinagolide was maintained in the udder half that was milked 2× but not in the udder half milked 1×, suggesting that the response to PRL is modulated at the gland level. In a second experiment, cows were injected with quinagolide, quinagolide + injection of bovine PRL at milking time, or water. As in the first experiment, quinagolide reduced milk, protein, and lactose yields. Although PRL injections at milking time were not sufficient to restore milk yield, they tended to increase milk protein and lactose yields and increased the viability of milk-purified mammary epithelial cells. Recently, we investigated the use of quinagolide at drying off. Treating late-lactation cows with quinagolide decreased milk production within the first day of treatment and induced faster increases in somatic cells and bovine serum albumin content in mammary secretions after drying off, which indicates an acceleration of mammary gland involution. In conclusion, these data, combined with data from other studies, provide a good body of evidence indicating that PRL is galactopoietic in dairy cows. However, the response to PRL appears to be modulated at the mammary gland level.

The impact of maternal overnutrition and obesity on hypothalamic-pituitary-adrenal axis response of offspring to stress - Corrected Proof

N.M. Long, P.W. Nathanielsz, S.P. Ford — 2012-01-24

Abstract: We evaluated the effect of maternal obesity before and throughout gestation on offspring hypothalamic-pituitary-adrenal axis function. Multiparous Rambouillet by Columbia crossbred ewes were fed either 100% of National Research Council (NRC) recommendations (control, C) or 150% of NRC recommendations (obese, OB) from 60 d before mating until lambing. Ten lambs born to OB ewes (five males and five females), and eight lambs born to C ewes (three male and five female) were studied. From delivery to weaning lambs were maintained with their mothers, who were all fed 100% NRC recommendations. After weaning, all lambs were group housed and fed the same diet to meet NRC requirements. At 19 mo of age lambs were placed in individual pens and fed a pelletized diet to meet maintenance requirements. Jugular vein catheters were placed and 2 d later lambs received an intravenous (i.v.) adrenocorticotropic hormone (ACTH) challenge followed by an i.v. corticotropin-releasing hormone (CRH)/arginine vasopressin (AVP) challenge 1 d later. Thirty d later offspring were again catheterized and placed into metabolism crates for 2 d before receiving an isolation stress test. ACTH and cortisol responses to the isolation stress test and CRH/AVP challenge and cortisol responses to ACTH challenge were determined. Cortisol was quantified via radioimmunoassay and ACTH was quantified using an Immulite 1000; both were analyzed using repeated measures using the MIXED procedure of SAS. Offspring from OB ewes had elevated basal plasma ACTH and cortisol compared with C offspring before all three challenges (P < 0.05). Offspring from OB mothers tended (P = 0.06) to have a greater ACTH response after an i.v. CRH/AVP injection than offspring from C mothers (12,340 ± 1,430 vs 8,170 ± 1,570 area under the curve, respectively). Cortisol response to the CRH/AVP and ACTH challenges was not influenced by maternal nutrition (P = 0.46) and averaged 4.77 ± 0.2 μg/dL and 1.94 ± 0.01 μg/dL, respectively. The ACTH response following the isolation stress test was also similar (P = 0.82) for OB and C offspring (147 ± 20 pg/mL), and cortisol response during the isolation stress test was similar between C and OB offspring (P = 0.64, 5.25 ± 0.3 μg/dL). These findings suggest that maternal obesity before and during gestation does not affect stress responses by the offspring, but has an impact on hypothalamic-pituitary-adrenal sensitivity. The lack of differences in cortisol release under the influence of difference concentrations of ACTH during the CRH/AVP challenge could indicate adrenal dysfunction in OB offspring.

Yellowtail insulin-like growth factor 1: molecular cloning and response to various nutritional conditions - Corrected Proof

H. Fukada, K. Murashita, T. Furutani, T. Masumoto — 2012-01-24

Abstract: Insulin-like growth factor 1 (IGF1) plays an important role in fish growth. This study investigated the IGF1 response to various nutritional conditions in yellowtail. First, we cloned 1,075 bp of yellowtail IGF1 cDNA, which codes for a protein of 185 amino acids (aa). This is composed of 44 aa for the signal peptide; 68 aa for the mature peptide comprising the B, C, A, and D domains; and 73 aa for the E domain. The mature yellowtail IGF1 showed high identity to IGF1 of other teleosts. Insulin-like growth factor 1 mRNA expression in the liver and white muscle was measured to observe the IGF1 response to various nutritional conditions, because the liver has the highest IGF1 expression and white muscle comprises the largest fraction of the fish body. Only white muscle IGF1 mRNA expression decreased significantly by 3 wk of fasting and recovered by refeeding. In subsequent feeding ratio (1%, 2%, and 3%/BW/d) experiments, significant correlations to growth were observed in white muscle IGF1 mRNA expression at 2- and 6-wk points and in hepatic IGF1 mRNA expression at 4 wk point. These data suggest that IGF1 expression both in hepatic and white muscle is important for somatic growth in yellowtail. Furthermore, white muscle IGF1 mRNA expression showed better responses to somatic growth and nutrition status in our two experiments than hepatic IGF1 mRNA expression.

Disruption of fibroblast growth factor receptor signaling in bovine cumulus-oocyte complexes during in vitro maturation reduces subsequent embryonic development - Corrected Proof

K. Zhang, A.D. Ealy — 2012-01-24

Abstract: Several fibroblast growth factors (FGF) mediate folliculogenesis and oogenesis in cattle but it is unclear whether FGFs are required during the final stages of oocyte maturation. The objectives of this work were to determine whether blocking FGF receptor (FGFR) activity during in vitro maturation (IVM) affects oocyte fertilization and embryo development; examine changes in FGFR transcript profiles in cumulus cells and oocytes during IVM; and evaluate whether gonadotropins modulate FGFR transcript abundance during IVM. In the first set of studies, bovine cumulus-oocyte complexes (COCs) were matured in the presence of one of two FGFR kinase inhibitors (SU5402 or PD173074). After maturation, COCs were washed and cultured without inhibitors. Inhibitors did not affect cleavage rates but the percentage of ≥ 8-cell embryos at d 3 and blastocysts at d 7 and d 8 postfertilization were decreased when COCs were matured with either inhibitor. Profiles of FGFR mRNA variants were examined in cumulus cells and oocytes separated either immediately before (0 h) or at 6 or 21 h after beginning IVM. In cumulus cells, increases in R1b, R2b, and R2c abundance were detected when cultured in the absence of follicle-stimulating hormone (FSH). Supplementing FSH (1 or 25 μM) increased the abundance of R1b, R1c, R2b, and R2c. In oocytes, no time- or FSH-dependent changes in FGFR transcript abundance were detected. These observations implicate FGFs as crucial components of bovine oocyte competency and indicate that FSH augments FGFR mRNA abundance in cumulus cells during the final stages of oocyte maturation.

Cysteamine improves growth performance and gastric ghrelin expression in preweaning piglets - Corrected Proof

G. Du, Z. Shi, D. Xia, X. Wei, L. Zhang, N. Parvizi, R. Zhao — 2012-01-12

Abstract: The aim of the present study was to investigate the effect of cysteamine on growth performance of preweaning piglets and gastric expression of ghrelin mRNA in vivo and in vitro. Twelve litters of newborn piglets were allocated randomly to control and treatment groups. From 15 d of age, piglets in the control group were fed basal creep diet, whereas the treatment group received basal diet supplemented with 120 mg cysteamine per kg of diet until weaning on 35 d of age. Body weight gain, creep feed consumption, and diarrhea rates were recorded, and gastric mucosal tissues were collected for quantifying mRNA expression. To evaluate the direct effect of cysteamine on gastric ghrelin expression, primary cultures of gastric mucosal cells isolated from 35-d-old piglets were exposed to cysteamine for 20 h at 0, 1, 10, and 100 μg/mL, respectively. Dietary cysteamine increased (P < 0.05) average daily creep feed consumption and BW gain in preweaning pigs, which was accompanied by reduction in diarrhea rates. At 35 d of age, piglets treated with cysteamine showed increased (P < 0.05) ghrelin and gastrin and decreased (P < 0.05) somatostatin mRNA expression in gastric mucosa. Moreover, dietary cysteamine treatment increased serum concentration of gastrin (P < 0.05). In vitro, cysteamine significantly increased ghrelin mRNA expression in gastric mucosal cells at the concentration of 10 μg/mL. In conclusion, dietary cysteamine is effective in improving the growth performance and health condition of preweaning piglets, which is associated with its stimulatory effects on gastric ghrelin mRNA expression both in vivo and in vitro.

Stromal-epithelial interactions modulate the effect of ovarian steroids on goat uterine epithelial cell interleukin-18 release - Corrected Proof

X.F. Qi, Z.C. Nan, Y.P. Jin, Y.Y. Qu, X.J. Zhao, A.H. Wang — 2012-01-09

Abstract: A primary role of epithelial-stromal interactions in mediating steroid hormone action in the uterus has been established. The present study was undertaken to determine the mode of ovarian steroid action in regulating IL-18 release by goat endometrial epithelial cells (EECs) in the presence and absence of endometrial stromal cells (ESCs). Primary and telomerase-immortalized goat EECs grown alone or cocultured with ESCs were treated with two ovarian steroids, 17β-estradiol (E2) and progesterone (P4). The IL-18 mRNA and protein expression in EECs were studied by reverse transcript (RT) PCR, ELISA, and Western blot assay. The E2 and/or P4 treatment of EECs led to a significant increase in both IL-18 mRNA and protein expression either in the primary or in the immortalized EECs compared with that in EECs without the steroid treatment. However, in the presence of ESCs, IL-18 expression by EECs treated with steroids was significantly decreased compared with cells untreated with E2 and/or P4. In addition, significantly high abundance of IL-18 mRNA and protein expression by primary and telomerase-immortalized goat EECs was observed in the presence of ESCs compared with those cells without ESCs. These findings suggest that steroids are important for the control of IL-18 expression in goat EECs. Underlying ESCs are needed to mediate the inhibitory effects of steroids on the IL-18 secretory activity of goat EECs in vitro. The IL-18 abundance expressed by goat EECs in vitro are enhanced by underlying ESCs without the treatment of E2 and/or P4.

Effects of weight loss in obese cats on biochemical analytes related to inflammation and glucose homeostasis - Corrected Proof

2011-12-19

Abstract: The aim of the current study was to measure circulating metabolic and inflammation-related biochemical analytes in obese cats before and after weight loss. Thirty-seven overweight neutered cats were studied, median body weight 6.85 kg (range, 4.70 to 10.30 kg), representing a range of ages and both sexes. An individualized weight-loss program was devised for each cat and monitored until completion. Body fat mass was determined by dual-energy x-ray absorptiometry, whereas plasma concentrations of acute-phase proteins (APPs; eg, haptoglobin and serum amyloid A), hormones (eg, insulin, IGF-1, and adiponectin), and enzymes (eg, butyrylcholinesterase and paraoxonase type 1 [PON-1]) associated with inflammation and metabolic compounds (eg, glucose) were also measured. No significant changes were found in APPs after weight loss (P > 0.3), but significant increases in plasma adiponectin (P = 0.021) and IGF-1 (P = 0.036) were seen, whereas insulin (P < 0.001) and homeostasis model assessment (P = 0.005) decreased significantly. Plasma concentrations before weight loss of PON-1 (P = 0.004), adiponectin (P = 0.02), and IGF-1 (P = 0.048) were less in cats that failed to complete weight loss than cats that were successful, whereas glucose concentration was greater. Finally, multivariable linear regression analysis showed that lean tissue loss during weight management was associated with percentage weight loss (greater weight loss, greater lean tissue loss; R = 0.71, P < 0.001) and plasma adiponectin concentration before weight loss (lesser adiponectin, more lean tissue loss; R = −0.52, P = 0.023). In conclusion, various metabolic abnormalities occur in feline obesity, and these can be linked to outcomes of weight-loss programs. The changes that occur with weight loss suggest an improved metabolic status.

Prenatal and neonatal exposure to flutamide affects function of Leydig cells in adult boar - Corrected Proof

M. Kotula-Balak, A. Hejmej, I. Kopera, M. Lydka, B. Bilinska — 2011-12-19

Abstract: In this study, flutamide, an androgen receptor antagonist, was used as a tool to better understand the role of androgen receptor signaling and androgen signaling disruption during fetal and neonatal periods on porcine Leydig cell development and function. Flutamide, 50 mg kg−1 d−1 was administered into pregnant gilts during gestational days 20 to 28 and days 80 to 88 and into male piglets on postnatal days 2 to 10 (PD2). Leydig cells of flutamide-exposed boars, especially those of PD2 males, displayed morphologic alterations, increased size, and occupied increased area (P < 0.001) of the testes when compared with the control. Despite this, testosterone concentrations were reduced significantly in comparison with those of controls (P < 0.05, P < 0.001). Reduced testosterone production in response to flutamide exposure appeared to be related to changes in testosterone metabolism, as shown by increased aromatase mRNA (P < 0.05, P < 0.01), protein expression (P < 0.01, P < 0.001), and elevated estradiol concentrations (P < 0.001). Moreover, impaired Leydig cell responsiveness to LH was indicated by the decreased expression of LH receptor (P < 0.05, P < 0.001). No significant effect of flutamide was found on LH and FSH concentrations. Taken together, our data indicate that flutamide when administered during prenatal or neonatal period have a long-term effect on Leydig cell structure and function, leading to androgen–estrogen imbalance. Leydig cell failure was most evident in adult boars neonatally exposed to flutamide, suggesting that androgen action during neonatal development is of pivotal importance for the differentiation and function of porcine adult Leydig cell population.

The role of kisspeptin and gonadotropin inhibitory hormone in the seasonal regulation of reproduction in sheep - Corrected Proof

J.T. Smith — 2011-12-19

Abstract: Sheep are seasonal breeders, experiencing an annual period of reproductive quiescence in response to increased photoperiod during the late-winter into spring and renaissance during the late summer. The nonbreeding (anestrous) season is characterized by a reduction in the pulsatile secretion of GnRH from the brain, in part because of an increase in negative feedback activity of estrogen. Neuronal populations in the hypothalamus that produce kisspeptin and gonadotropin-inhibitory hormone (GnIH) appear to be important for the seasonal shift in reproductive activity, and the former are also mandatory for puberty onset. Kisspeptin cells in the arcuate nucleus (ARC) and preoptic area appear to regulate GnRH neurons and transmit sex-steroid feedback signals to these neurons. Moreover, kisspeptin expression in the ARC is markedly up-regulated at the onset of the breeding season, as too are the number of kisspeptin fibers in close apposition to GnRH neurons. The lower levels of kisspeptin seen during the nonbreeding season can be “corrected” by infusion of kisspeptin, which causes ovulation in seasonally acyclic females. The role of GnIH is less clear, but mounting evidence supports a role for this neuropeptide in the inhibitory regulation of both GnRH secretion and gonadotropin release from the pituitary gland. Contrary to kisspeptin, GnIH expression is markedly reduced at the onset of the breeding season. In addition, the number of GnIH fibers in close apposition to GnRH neurons also decreases during this time. Importantly, exogenous GnIH treatment can block both the pulsatile release of LH and the preovulatory LH surge during the breeding season. In summary, it is most likely the integrated function of both these neuropeptide systems that modulate the annual shift in photoperiod to a physiological change in fertility.

Apelin is involved in postprandial responses and stimulates secretion of arginine-vasopressin, adrenocorticotropic hormone, and growth hormone in the ruminant - Corrected Proof

K. Sato, T. Takahashi, Y. Kobayashi, A. Hagino, S.G. Roh, K. Katoh — 2011-12-19

Abstract: Apelin and its mRNA are expressed in several tissues, including the supraoptic and paraventricular nuclei in the hypothalamus. Although apelin is reported to be involved in the regulation of fluid homeostasis, little is known about the postprandial dynamics of apelin in plasma and its regulatory effects on the anterior pituitary hormones of ruminants. Therefore, the aims of this study were to investigate the following: (1) changes in plasma apelin concentrations in response to food intake under conditions of hydration (free access to water) or dehydration (water restriction), and (2) the effects of intravenous administration of apelin on plasma concentrations of arginine-vasopressin (AVP), ACTH, GH, and insulin. In Experiment 1 with the use of goats, the postprandial plasma apelin concentration was significantly increased under the dehydration condition compared with the hydration condition, and this increase was accompanied by increased plasma concentrations of AVP and ACTH after 24 h of dehydration. In Experiment 2 with the use of sheep and hydration conditions, the intravenous administration of apelin ([Pyr1]-apelin-13; 0.5 mg/head) caused a tendency to increase or caused a significant increase in plasma concentrations of AVP, ACTH, GH, insulin, and glucose. On the basis of these findings, we concluded that apelin is involved in the feeding process, and it regulates endocrine functions in the anterior pituitary gland via AVP in ruminant animals.

Effects of the phytoestrogen genistein on the porcine anterior pituitary insulin-like growth factor system - Corrected Proof

J. Clapper, A. Tomlin — 2011-12-19

Abstract: Estrogens have profound effects on the serum and anterior pituitary (AP) insulin-like growth factor (IGF) system in pigs. In this study we determined whether administration of the phytoestrogen genistein increased serum and AP concentrations of IGF-I and relative amounts of serum and AP insulin-like growth factor-binding protein (IGFBP). Twenty barrows of similar age (190 d) and weight (110 kg) were stratified by litter into one of four treatments: controls (C), estradiol (E), 200 mg genistein (G200), and 400 mg genistein (G400). Estradiol-treated pigs were injected daily with 2 mg of estradiol-17β intramuscularly (i.m.), whereas the G200 and G400 groups were injected daily with either 200 or 400 mg of genistein i.m., respectively, beginning on d 0 and continuing through d 15. Blood was collected on d 0, 3, 6, 9, and 13. Blood and AP were collected at slaughter on d 16. Serum and AP concentrations of IGF-I and luteinizing hormone (LH) were determined by radioimmunoassay. Relative amounts of serum IGFBP were determined by Western ligand blot analysis. Relative expression of AP IGF-I, IGF-I receptor (IGF-IR), gonadotropin-releasing hormone receptor, and LHβ subunit was determined by real-time reverse transcriptase polymerase chain reaction. Anterior pituitary concentrations of IGF-I were greater (P > 0.05) in E and G400 pigs compared with controls, whereas AP concentrations of LH were greater (P < 0.05) in G400 pigs compared with C and G200 pigs. Relative expression of LHβ was greater in G200 pigs compared with C pigs but did not differ from that in G400 pigs. Relative expression of AP IGF-IR was greater (P < 0.05) in E pigs compared with all other treatments; however, relative expression of AP IGF-IR was greater (P < 0.05) in both G200 and G400 pigs vs C pigs. No differences were detected (P > 0.05) in serum concentrations of IGF-I or relative amounts of serum and AP IGFBP among treatments. These data provide evidence that genistein is capable of modulating components of the AP IGF system that could affect the synthesis and release of LH.

Glucagon-like peptide-1 inhibits insulinotropic effects of oxyntomodulin and glucagon in cattle - Corrected Proof

S. ThanThan, T. Saito, S. Yannaing, H. Zhao, K. Nakashima, H. Kuwayama — 2011-12-12

Abstract: Oxyntomodulin (OXM), glucagon, and glucagon-like peptide-1 (GLP-1), peptide hormones derived from the glucagon gene, play an important role in glucose homeostasis. The insulinotropic action of these three homologous peptides has been well documented in monogastric animals. However, information on the relationships among these peptides in insulin-releasing action, specifically in ruminants, is still insufficient. In this regard, we carried out two experiments in cattle. In experiment 1, effects of glucagon and GLP-1 on plasma insulin and glucose were investigated in 10-mo-old Holstein steers (347 ± 8 kg, n = 8) under normoglycemic conditions. Peptides were administered intravenously at dose rates of 0.12, 0.25, 0.50, and 1.25 nmol/kg body weight (BW). In experiment 2, the relationships among OXM, glucagon, and GLP-1 in the insulinotropic and glucoregulatory actions were elucidated in 3-mo-old Holstein steers (94 ± 2 kg, n = 8) using agonist-antagonist strategy. In agonist strategy, these three peptides were administered alone or coadministered at dose rates of 10 μg of OXM/kg BW, 4 μg of glucagon/kg BW, and 2 μg of GLP-1/kg BW. In antagonist strategy, 2 μg of each peptide was administered alone or in combination with 10 μg of [des His1, des Phe6, Glu9] glucagon amide (a glucagon receptor antagonist) or exendin-4 (5-39) amide (a GLP-1 receptor antagonist). Our results showed that OXM, glucagon, and GLP-1 had insulinotropic actions in ruminants under normoglycemic conditions. Our results also showed that the insulin-releasing effects of OXM and glucagon were mediated through both GLP-1 receptors (GLP-1R) and glucagon receptors. These insulinotropic effects of OXM and glucagon through GLP-1R were inhibited by GLP-1. Our findings expand the relationships among OXM, glucagon, and GLP-1 in the insulinotropic and glucoregulatory actions.

Quantification of the GH/IGF-axis components: Lessons from human studies - Corrected Proof

J. Frystyk — 2011-12-07

Abstract: Originally, the circulating bioactivity of IGF-I was estimated by bioassays measuring the ability of serum to stimulate uptake of labeled sulfate or thymidine in cultures of costal cartilage or by the ability of serum to stimulate the uptake of glucose in fat tissue cultures. However, because of their laborious and unspecific nature, the original bioassays were quickly abandoned with the development of the first RIA for IGF-I in 1977. Consequently, for the past three decades the endogenous IGF-I bioactivity has been almost exclusively estimated by the use of immunoassays. Beyond any doubt, the immunoassays have provided an extensive insight into IGF-I physiology and pathophysiology. However, immunoassays ignore the presence of the IGFBPs, which are important regulators of IGF-I action in vivo. In addition, immunoassays do not consider the presence of IGF-II, which also interacts with the IGF-I receptor (IGF-IR). This aroused our interest to reintroduce the bioassay; therefore, we established a cell-based kinase receptor activation (KIRA) assay based on cells transfected with the human IGF-IR. The output signal of the KIRA assay is IGF-IR phosphorylation, and, as such, it is highly specific. Further, because detection of phosphorylated IGF-IRs is based on modern immunoassay techniques, the overall performance of the assay is close to that of a traditional IGF-I immunoassay. The first part of this review comprises a short description of the bioassay, and a more in-depth presentation of the data that have been obtained so far. It will be demonstrated that the bioassay is indeed able to yield novel information on the IGF system, most likely because it is able to integrate the different components of the IGF system into one signal: IGF-IR activation. As IGF-I, circulating GH is bound to larger proteins, the far most important GH-binding protein (GHBP) is identical to the extracellular domain of the GH receptor (GHR). Because of its origin, GHBP binds GH with the same affinity as GHR and, consequently, GHBP may affect GH bioactivity as well as pharmacokinetics. To improve our knowledge on the complex interaction between GH and GHBP in vivo, we found it of interest to develop a method for determination of free GH. To this end, we developed an ultrafiltration assay that enabled isolation of free GH in undiluted serum during approached in vivo–like conditions. The last part of this review presents our current data on free GH and its interaction with GHBP.