Interaction Of Insulin-like Growth Factor Research Articles

Liver Expression of IGF2 and Related Proteins in ZBED6 Gene-Edited Pig by RNA-Seq.

Simple SummaryZinc finger BED-type containing 6 (ZBED6), as a regulatory factor, has different regulatory mechanisms in animal development. The intron of insulin-like growth factor 2 (IGF2) regulates the development of animal muscle and adipose by combining with the binding site of ZBED6. As a member of the insulin-like growth factor family, IGF2 plays an important role in embryonic growth and development, cell proliferation, muscle growth and genome imprinting. In order to further study the regulatory mechanism of ZBED6 on IGF2, we detected the expression of IGF2 and related genes in ZBED6 single allele knockout (ZBED6-SKO) pig tissues and analyzed differently expressed genes of the transcriptome of ZBED6-SKO pig liver. The results showed that the partial knockout of ZBED6 could affect the secretion of IGF2 in pig liver but had no significant difference at the protein level. This research provides a new idea for the interaction between IGF2 and ZBED6.Zinc finger BED-type containing 6 (ZBED6), a highly conservative transcription factor of placental mammals, has conservative interaction of insulin-like growth factor 2 (IGF2) based on the 16 bp binding sites of ZBED6 on the IGF2 sequence. IGF2 is related to embryo growth and cell proliferation. At the same time, its functions in muscle and adipose in mammals have been widely mentioned in recent studies. To further investigate the mechanism of ZBED6 on IGF2, we detected the expression of IGF2 and related genes in ZBED6 single allele knockout (ZBED6-SKO) pig tissues and analyzed the transcriptome of ZBED6-SKO pig liver. Through RNA-seq, we captured nine up-regulated genes and eight down-regulated genes which related to lipid metabolism. The results showed that the mRNA of IGF2 had an upward trend after the partial knockout of ZBED6 in liver and had no significant difference in protein expression of IGF2. In summary, ZBED6-SKO could affect the secretion of IGF2 in pig liver and its own lipid metabolism. Our research has provided basic information for revealing the regulatory mechanism of the interaction between ZBED6 and IGF2 in mammals.

Model-Informed Dose Selection for Xentuzumab, a Dual Insulin-Like Growth Factor-I/II-Neutralizing Antibody.

Over the past decade, the insulin-like growth factor (IGF)-signaling pathway has gained substantial interest as potential therapeutic target in oncology. Xentuzumab, a humanized IgG1 monoclonal antibody, binds to IGF-I and IGF-II thereby inhibiting the downstream signaling essential for survival and tumor growth. This pathway is further regulated by circulating IGF binding proteins (IGFBPs). In this work, a mechanistic model characterizing the dynamics and interactions of IGFs, IGFBPs, and Xentuzumab has been developed to guide dose selection. Therefore, in vitro and in vivo literature information was combined with temporal IGF-I, IGF-II, and IGFBP-3 total plasma concentrations from two phase I studies. Based on the established quantitative framework, the time-course of free IGFs as ultimate drug targets not measured in clinics was predicted. Finally, a dose of 1000mg/week-predicted to reduce free IGF-I and free IGF-II at steady-state by at least 90% and 64%, respectively-was suggested for phase II.

The role of insulin growth factors in autoimmune diseases

Insulin-like growth factors (IGFs), their receptors and their binding proteins form a distinct bioregulation system that regulates growth, cell proliferation, apoptosis and tissue remodeling. Recent data highlight a significant interaction of IGFs and the components of the immune system, especially B and T cells. Accumulating data suggest an important role of IGFs in autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), systemic sclerosis (SSc) and Sjogren’s syndrome (SS). Herein, we summarize the current evidence on the role of IGF pathway in immune system regulation as well as its potential involvement in the pathophysiology of systemic autoimmune diseases.

Signalling through the Type 1 Insulin-Like Growth Factor Receptor (IGF1R) Interacts with Canonical Wnt Signalling to Promote Neural Proliferation in Developing Brain

Signalling through the IGF1R [type 1 IGF (insulin-like growth factor) receptor] and canonical Wnt signalling are two signalling pathways that play critical roles in regulating neural cell generation and growth. To determine whether the signalling through the IGF1R can interact with the canonical Wnt signalling pathway in neural cells in vivo, we studied mutant mice with altered IGF signalling. We found that in mice with blunted IGF1R expression specifically in nestin-expressing neural cells (IGF1RNestin−KO mice) the abundance of neural β-catenin was significantly reduced. Blunting IGF1R expression also markedly decreased: (i) the activity of a LacZ (β-galactosidase) reporter transgene that responds to Wnt nuclear signalling (LacZTCF reporter transgene) and (ii) the number of proliferating neural precursors. In contrast, overexpressing IGF-I (insulin-like growth factor I) in brain markedly increased the activity of the LacZTCF reporter transgene. Consistently, IGF-I treatment also markedly increased the activity of the LacZTCF reporter transgene in embryonic neuron cultures that are derived from LacZTCF Tg (transgenic) mice. Importantly, increasing the abundance of β-catenin in IGF1RNestin−KO embryonic brains by suppressing the activity of GSK3β (glycogen synthase kinase-3β) significantly alleviated the phenotypic changes induced by IGF1R deficiency. These phenotypic changes includes: (i) retarded brain growth, (ii) reduced precursor proliferation and (iii) decreased neuronal number. Our current data, consistent with our previous study of cultured oligodendrocytes, strongly support the concept that IGF signalling interacts with canonical Wnt signalling in the developing brain to promote neural proliferation. The interaction of IGF and canonical Wnt signalling plays an important role in normal brain development by promoting neural precursor proliferation.

Blunting type 1 insulin-like growth factor receptor expression exacerbates neuronal apoptosis following hypoxic/ischemic injury

BackgroundAbundant experimental data have implicated an important role for insulin-like growth factor (IGF) in protecting neuronal cells from injury, including hypoxia/ischemia (H/I) injury, a major cause of neuron death. While the specific interaction of IGFs with neuronal or glial type 1 IGF receptors (IGF1R) has been shown to be essential to IGF actions during development, the same has not been directly demonstrated following H/I injury. To directly examine the role of neuronal IGF1R following H/I injury, we utilized conditional mutant nes-igf1r-/Wt mice and determined the impact of IGF1R haplodeficiency specifically in nestin-expressing neuronal precursors and their progeny on H/I-induced neuronal damage and apoptosis in hippocampus.ResultsH/I induced significant damage to the cerebral hemisphere and hippocampus ipsilateral to the ligated right common carotid artery both in control and nes-igf1r-/Wt mice at postnatal day 10. Blunting IGF1R expression, however, markedly exacerbated H/I-induced damage and appeared to increase mortality. In the ipsilateral hemisphere and hippocampus, nes-igf1r-/Wt mice had infarct areas double the size of those in controls. The size of the ipsilateral hemisphere and hippocampus in nes-igf1r-/Wt mice were 15% to 17% larger than those in controls, reflecting more severe edema. Consistent with its effects on infarct area, IGF1R haplodeficiency causes a greater decrease in neurons in the ipsilateral hippocampus of nes-igf1r-/Wt mice. The reduction in neurons was largely due to increases in neuronal apoptosis. Judged by pyknotic nuclei, TUNEL and caspase-3 labeling, nes-igf1r-/Wt mice had significantly more apoptotic cells than that in controls after injury. To determine possible mechanisms of IGF1R actions, the mRNA expression of the pro-survival proteins IAP-1 and XIAP was determined. Compared to controls, the abundance of cIAP-1 and XIAP mRNA was markedly suppressed in mice with blunted IGF1R or IGF-I expression, while was increased in the brain of IGF-I overexpressing transgenic mice.ConclusionIGF1R in neuronal cells is critically important for their survival following H/I injury, and IGF-upregulated expression of neuronal cIAP-1 and XIAP likely in part contributes to IGF-IGF1R protection against neuronal apoptosis following H/I injury.

A Novel Approach to Identify Two Distinct Receptor Binding Surfaces of Insulin-like Growth Factor II

Very little is known about the residues important for the interaction of insulin-like growth factor II (IGF-II) with the type 1 IGF receptor (IGF-1R) and the insulin receptor (IR). Insulin, to which IGF-II is homologous, is proposed to cross-link opposite halves of the IR dimer through two receptor binding surfaces, site 1 and site 2. In the present study we have analyzed the contribution of IGF-II residues equivalent to insulin's two binding surfaces toward the interaction of IGF-II with the IGF-1R and IR. Four "site 1" and six "site 2" analogues were produced and analyzed in terms of IGF-1R and IR binding and activation. The results show that Val(43), Phe(28), and Val(14) (equivalent to site 1) are critical to IGF-1R and IR binding, whereas mutation to alanine of Gln(18) affects only IGF-1R and not IR binding. Alanine substitutions at Glu(12), Asp(15), Phe(19), Leu(53), and Glu(57) analogues resulted in significant (>2-fold) decreases in affinity for both the IGF-1R and IR. Furthermore, taking a novel approach using a monomeric, single-chain minimized IGF-1R we have defined a distinct second binding surface formed by Glu(12), Phe(19), Leu(53), and Glu(57) that potentially engages the IGF-1R at one or more of the FnIII domains.

Structural Insights into the Interaction of Insulin-like Growth Factor 2 with IGF2R Domain 11

The insulin-like growth factor II/mannose-6-phosphate receptor (IGF2R) mediates trafficking of mannose-6-phosphate (M6P)-containing proteins and the mitogenic hormone IGF2. IGF2R also plays an important role as a tumor suppressor, as mutation is frequently associated with human carcinogenesis. IGF2 binds to domain 11, one of 15 extracellular domains on IGF2R. The crystal structure of domain 11 and the solution structure of IGF2 have been reported, but, to date, there has been limited success when using crystallography to study the interaction of IGFs with their binding partners. As an approach to investigate the interaction between IGF2 and IGF2R, we have used heteronuclear NMR in combination with existing mutagenesis data to derive models of the domain 11-IGF2 complex by using the program HADDOCK. The models reveal that the molecular interaction is driven by critical hydrophobic residues on IGF2 and IGF2R, while a ring of flexible, charged residues on IGF2R may modulate binding.

Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx(1-279)IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly 19 and Asp 20 of IGF-I (and the corresponding residues in IGF-II) - which are located in a reverse turn linking N-domain and C-domain interactive surfaces - are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.

Skeletal muscle cytokines: regulation by pathogen-associated molecules and catabolic hormones

This review will update clinicians and basic scientists who study the molecular mechanisms of muscle wasting associated with infection, trauma, cancer cachexia, and AIDS. A special emphasis is placed on recent studies that examine the interaction of insulin-like growth factor 1 and proinflammatory cytokines as positive and negative regulators of muscle mass. Potential mediators of the wasting syndromes include catabolic hormones, such as glucocorticoids, as well as the inflammatory cytokines tumour necrosis factor, IL-1, and IL-6. Cytokines may function either systemically or locally within muscle per se. Lipopolysaccharide and other pathogen-associated molecules stimulate cytokine expression in muscle. The failure to clear pathogen-associated molecules or the introduction of muscle damage may initiate a protracted activation of enzymes and transcription factors that orchestrate a genetic programme that ultimately produces muscle wasting. This review highlights recent advances in our understanding of the expression of the afferent and efferent limbs of the innate immune system in skeletal muscle. A special emphasis is placed on the recognition of pathogen-associated molecules by skeletal muscle cells and how these molecules regulate the expression of inflammatory cytokines and other muscle genes to result in muscle wasting, and when sustained, the erosion of lean body mass.

Bimolecular interaction of insulin-like growth factor (IGF) binding protein-2 with alphavbeta3 negatively modulates IGF-I-mediated migration and tumor growth.

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the alpha v beta 3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and alpha v beta 3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7 beta 3, which overexpressed the alpha v beta 3 integrin. Collectively, these results indicate that alpha v beta 3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

Structural and functional evidence for the interaction of insulin-like growth factors (IGFs) and IGF binding proteins with vitronectin.

Previous studies demonstrated that IGF-II binds directly to vitronectin (VN), whereas IGF-I binds poorly. However, binding of VN to integrins has been demonstrated to be essential for a range of IGF-I-stimulated biological effects, including IGF binding protein (IGFBP)-5 production, IGF type-1 receptor autophosphorylation, and cell migration. Thus, we hypothesized that a link between IGF-I and VN must occur and may be mediated through IGFBPs. This was tested using competitive binding assays with VN and (125)iodine-labeled IGFs in the absence and presence of IGFBPs. IGFBP-4, IGFBP-5, and nonglycosylated IGFBP-3 were shown to significantly enhance binding of IGF-I to VN, whereas IGFBP-2 and glycosylated IGFBP-3 had a smaller effect. Furthermore, binding studies with analogs indicate that glycosylation status and the heparin-binding domain of IGFBP-3 are important in this interaction. To examine the functional significance of IGFs binding to VN, cell migration in MCF7 cells was measured and found to be enhanced when VN was prebound to IGF-I in the presence of IGFBP-5. The effect required IGF:IGFBP:VN complex formation; this was demonstrated by use of a non-IGFBP-binding IGF-I analog. Together, these data indicate the importance of IGFBPs in modulating IGF-I binding to VN and that this binding has functional consequences in cells.

The Insulin-like Growth Factor System in Hematopoietic Cells

The insulin-like growth factor (IGF) system regulates proliferation and differentiation of hematopoietic cells. IGFs exert their effects through specific receptors on growing and differentiating blood cells as they emerge from their small pool of ancestral stem cells. The IGF system is complex as both stimulating and inhibiting effects occur by interaction of IGFs and IGF-binding proteins (IGFBPs). IGFs stimulate erythrocytes and lymphocytes but also promote leukemic hematopoietic cell proliferation. IGF-I appears to be correlated with hemoglobin levels in anemia and could also be of benefit for patients with bone marrow aplasia after transplantation. Hypersensitivity to IGF-I has been implicated as an underlying cause of polycythemia vera. Loss of imprinting of IGF-II is found in acute myeloid leukemia and myelodysplastic syndrome. Apoptosis of hematopoietic cells is significantly reduced by IGF-I involving an intriguing signal transduction pathway. IGFs could therefore, although not classical hematopoietic growth factors, be of benefit for patients with diverse hematopoietic disorders.

The interaction of Insulin-like Growth Factors (IGFs) with Insulin-like Growth Factor Binding Proteins (IGFBPs): a review

The interactions between insulin-like growth factor bindingproteins (IGFBPs) and insulin-like growth factors (IGFs)are important in regulating the availability of IGFs to thetype 1 IGF receptor (IGF1R) but there is still a lack ofinformation regarding the mechanism of IGF binding byIGFBPs. While many studies have defined general regions ofthe IGFBPs that interact with IGFs, only recently havespecific IGF binding residues been described. This reviewwill outline the structural evidence describing residues ofboth the IGFBPs and IGFs involved in the IGFBP-IGFinteraction.

Growth and the initiation of steroidogenesis in porcine follicles are associated with unique patterns of gene expression for individual componentsof the ovarian insulin-like growth factor system.

Ovarian follicular growth and steroidogenesis are controlled by the interaction of insulin-like growth factors (IGFs) and gonadotropins. The objective was to determine the temporal and spatial relationships for gonadotropin receptor, steroidogenic enzyme, and IGF system gene expression during the development of preovulatory porcine follicles. Sows (n = 18) were weaned and follicles were monitored by transrectal ultrasonography. Ovaries were collected from sows when the mean diameter of the preovulatory follicular cohort was approximately 2, 4, 6, or 8 mm. mRNA were measured by in situ hybridization for individual follicles within the preovulatory cohort (3 to 5 follicles per sow). Patterns of gene expression detected by in situ hybridization were confirmed by RNase protection analyses of pooled RNA samples. The amount of LH receptor mRNA and steroidogenic enzyme mRNA (17alpha-hydroxylase and aromatase) increased as the mean diameter of the follicular cohort increased from 2 to 6 mm, but then decreased abruptly for 8-mm follicles. Estradiol concentrations in follicular fluid closely followed the expression patterns of steroidogenic enzymes and LH receptor mRNA. FSH receptor mRNA was present in cohorts of 2-mm follicles but declined in 4-mm follicles and was undetectable in 6- and 8-mm follicles. The expression of IGF-I and type I IGF receptor mRNA were similar for follicles of 2, 4, 6, and 8 mm. In contrast, IGF-II mRNA progressively increased in follicles collected from 2-, 4-, and 6-mm cohorts, and then decreased slightly at 8 mm. Type II IGF receptor mRNA was greatest in 8-mm follicles. IGF binding protein-2 (BP-2) mRNA decreased as follicles achieved progressively larger sizes during the preovulatory period (2 to 8 mm), whereas the IGFBP-4 mRNA remained relatively low for follicles in 2- to 6-mm cohorts but then increased markedly in 8-mm follicles. In summary, temporal and spatial patterns of gene expression for gonadotropin receptor, steroidogenic enzyme, and IGF system genes were characterized in preovulatory porcine follicles by using in situ hybridization and RNase protection analyses. The unique patterns of gene expression suggest interdependence among specific genes that may be essential for preovulatory follicular development.

Insulin-like growth factor I receptors and estrogen receptors colocalize in female rat brain

Several findings indicate that there is a close interaction between estrogen and insulin-like growth factor I in different brain regions. In adult brain, both estrogen and insulin-like growth factor I have co-ordinated effects in the regulation of neuroendocrine events, synaptic plasticity and neural response to injury. In this study we have qualitatively assessed whether estrogen receptors and insulin-like growth factor I receptor are colocalized in the same cells in the preoptic area, hypothalamus, hippocampus, cerebral cortex and cerebellum of female rat brain using confocal microscopy. Immunoreactivity for estrogen receptors α and β was colocalized with immunoreactivity for insulin-like growth factor I receptor in many neurons from the preoptic area, hypothalamus, hippocampus and cerebral cortex. Furthermore, estrogen receptor β and insulin-like growth factor I receptor immunoreactivities were colocalized in the Purkinje cells of the cerebellum. Colocalization of estrogen receptor β and insulin-like growth factor I receptor was also detected in cells with the morphology of astrocytes in all regions assessed. The co-expression of estrogen receptors and insulin-like growth factor I receptor in the same neurons may allow a cross-coupling of their signaling pathways. Furthermore, the colocalization of immunoreactivity for estrogen receptor β and insulin-like growth factor I receptor in glial cells suggests that glia may also play a role in the interactions of insulin-like growth factor I and estrogen in the rat brain. In conclusion, the co-expression of estrogen receptors and insulin-like growth factor I receptors in the same neural cells suggests that the co-ordinated actions of estrogen and insulin-like growth factor I in the brain may be integrated at the cellular level.

Hydrogen Exchange/Electrospray Ionization Mass Spectrometry Studies of Structural Features of Proteins and Protein/Protein Interactions

The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The information obtained is which region within the protein participates in binding. The method includes hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange. After this procedure those backbone amide groups that participate in protein binding are protected from back exchange and therefore still deuterated. These regions were then identified by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and electrospray ionization mass spectrometry. The approach has been applied to the investigation of structural features of insulin-like growth factor I (IGF-I) and the interaction of insulin-like growth factor I with IGF-I binding protein 1. The data show that the approach can provide information on the location of the hydrophobic core of IGF-1 and on two regions that are mainly involved in binding to IGF-I binding protein 1. The data are consistent with results obtained with other approaches. The amount of sample required for one experiment is in the subnanomolar range.

Perinatal hypoxia-ischemia decreased neuronal but increased cerebral vascular endothelial IGFBP3 expression.

In adults, insulin-like growth factor binding protein 3 (IGFBP3) is the main carrier protein for circulating insulin-like growth factors (IGFs) (IGF-I and -II). While most IGFBP3 is synthesized in the liver, it is also expressed locally by many cell types including vascular endothelial cells. The regulation of this endothelial IGFBP3 expression, especially in response to hypoxic-ischemic injury, has not been investigated in vivo. Using in situ hybridization histochemistry, we studied the cellular distribution of IGFBP3 mRNA in rat brains following hypoxic-ischemic injury at 1, 5, 24, and 72 h of recovery. In normal P7 rat brain, IGFBP3 mRNA was found in neurons within the thalamus, hippocampus, and amygdaloid. Low levels of IGFBP3 mRNA were also detected in cerebral vascular endothelial cells. After the hypoxic-ischemic injury, the levels of neuronal IGFBP3 mRNA substantially decreased within 24 h in areas that were normally supplied by the middle cerebral artery. In the meantime, there was an immediate increase in IGFBP3 expression in vascular endothelial cells throughout the affected hemisphere. This vascular IGFBP3 expression was further enhanced with the highest level at 24 h of recovery whereas neuronal IGFBP3 expression was further decreased. By 72 h of recovery, IGFBP3 was no longer expressed in vascular endothelial cells. Taken together, the activation of IGFBP3 is a likely mechanism by which vascular endothelial cells respond to hypoxic-ischemic insult. In addition, increased endothelial IGFBP3 may modulate the interaction of IGFs with IGF-I receptors at the site of injury and/or act independently on endothelial cell growth.

Interactions of insulin-like growth factor (IGF)-II and growth hormone in vivo: circulating levels of IGF-I and IGF-binding proteins in transgenic mice.

To study interactions between insulin-like growth factor-II (IGF-II) and growth hormone (GH) in vivo, we crossed hemizygous transgenic mice carrying phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II fusion genes with hemizygous PEPCK-bovine GH (bGH) transgenic mice. Offspring harbouring both transgenes (IB), the IGF-II transgene (I) or the bGH transgene (B), and non-transgenic littermates (C) were obtained. Blood samples were taken before (end of week 12) and after (end of week 14) the mice had received a diet high in protein and low in carbohydrates to stimulate PEPCK promoter-controlled transgene expression. Mean serum GH concentrations of both B and IB mice corresponded to 900 ng/ml and increased more than twofold (P < 0.001) after 1 week of the high-protein diet. GH concentrations in controls and I mice were less than 20 ng/ml. Serum IGF-II concentrations in I and IB mice were three-to fourfold higher than those in C and B mice. Whereas IGF-II concentrations were not changed by the high-protein diet in the last two groups, serum IGF-II increased significantly in I (P < 0.001) and IB mice (P < 0.05). This increase was significantly (P < 0.05) less pronounced in IB than in C and I mice. Circulating IGF-I concentrations were about twofold (P < 0.001) higher in B and IB than in C and I mice, and showed a tendency to be lower in I than in C and in IB than in B mice when animals were maintained on the standard diet. The high-protein diet did not change circulating IGF-I concentrations in controls and B mice, but resulted in a significant reduction of serum IGF-I concentrations in I (P < 0.05) and IB mice (P < 0.001). Consequently, after PEPCK-IGF-II transgene expression was stimulated, serum IGF-I concentrations were significantly (P < 0.05) lower in I than in C and in IB than in B mice. Serum IGF-binding protein (IGFBP)-2 concentrations were significantly (P < 0.05) higher in I mice than in all other groups when mice were maintained on the standard diet, with a tendency to reduced IGFBP-2 concentrations in B mice. After the high-protein diet, serum IGFBP-2 concentrations did not change in C and I mice, but increased by two- to threefold in B and IB mice (P < 0.001). Serum IGFBP-3 concentrations tended to be greater in B and IB than in C and I mice, but these differences were mostly not significant. IGFBP-4 concentrations were significantly (P < 0.001) increased by GH overproduction in B and IB mice. Our data suggest that the reduction in circulating IGF-I concentrations by increased IGF-II is most probably due to the limited serum IGF binding capacity and the short half-life of free IGFs, rather than to a reduction in GH-dependent IGF-I production. Effects of GH overproduction on serum IGFBP-2 concentrations depend on dietary factors and may be both inhibitory and stimulatory.

Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system.

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.

Messenger ribonucleic acid for insulin-like growth factors-I and -II, insulin-like growth factor-binding protein-2, gonadotropin receptors, and steroidogenic enzymes in porcine follicles.

Several aspects of growth, development, and steroidogenesis in ovarian follicles are controlled by the interaction of insulin-like growth factors (IGF-I and IGF-II) and gonadotropins (LH and FSH). The objective of this study was to determine the developmental stage and location at which mRNA for IGF, IGF-binding protein (IGFBP)-2, gonadotropin receptors, and steroidogenic enzyme genes are expressed within porcine follicles. Ovaries from pigs on Days 10 (n = 3; midluteal phase) and 19 (n = 3; preovulatory phase) of the estrous cycle were collected, frozen, and sectioned. The mRNA for IGF-I, IGF-II, IGFBP-2, LH receptor, FSH receptor, cytochrome P450 side-chain cleavage (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase (P45017 alpha), and aromatase (P450arom) were detected by in situ hybridization, and relative amounts were determined by image analysis. Expression of mRNA for IGF-I and IGF-II was cell specific (granulosa and theca interna cells, respectively) and increased with follicular size. The IGFBP-2 mRNA was expressed in greater amounts for theca interna cells than for granulosa cells. The pattern of expression for LH and FSH receptors diverged as follicles grew. For LH receptor, mRNA was detected in the theca interna cells of small antral follicles and in both the theca interna and granulosa cells of large antral follicles. Amount of LH receptor mRNA increased with follicular diameter. For FSH receptor, mRNA increased as follicles grew to a small antral size. However, FSH receptor mRNA disappeared in large antral follicles on Day 19 of the estrous cycle. Steroidogenic enzyme mRNA for 3 beta-HSD (theca interna cells), P450scc (theca interna cells), P45017 alpha (theca interna cells), and P450arom (granulosa cells) increased with follicular diameter. In summary, coordinated expression of IGF, IGFBP-2, gonadotropin receptors, and steroidogenic enzyme mRNA throughout different stages of follicular development was observed. Changes in mRNA suggested increased IGF-I and -II action and a switch from FSH to LH dependence in porcine preovulatory follicles.