The Insulin-Like Growth Factor Axis in the Lactating Rat and in the Immune System of the Sheep

The insulin-like growth factors (IGFs) are important in several areas of animal production, for example, IGF-1 mediates the growth-promoting effects of growth hormone (GH). GH has effects on a variety of tissues including bone, mammary gland and adipose tissue; GH effects on adipose tissue are direct but its effects on other tissues are thought to be mediated by IGF production. The aim of this study was the investigation of the IGF axis in two areas important to animal performance; lactation and the immune system

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Cumulative mutagenesis of the basic residues in the 201-218 region of insulin-like growth factor (IGF)-binding protein-5 results in progressive loss of both IGF-I binding and inhibition of IGF-I biological action

We have reported previously that mutation of two conserved nonbasic amino acids (G203 and Q209) within the highly basic 201–218 region in the C-terminal domain of IGF-binding protein-5 (IGFBP-5) decreases binding to IGFs. This study reveals that cumulative mutagenesis of the 10 basic residues in this region, to create the C-Term series of mutants, ultimately results in a 15-fold decrease in the affinity for IGF-I and a major loss in heparin binding. We examined the ability of mutants to inhibit IGF-mediated survival of MCF-7 cells and were able to demonstrate that this depended not only upon the affinity for IGF-I, but also the kinetics of this interaction, because IGFBP-5 mutants with similar affinity constants (KD) values, but with different association (Ka) and dissociation (Kd) rate values, had markedly different inhibitory properties. In contrast, the affinity for IGF-I provided no predictive value in terms of the ability of these mutants to enhance IGF action when bound to the substratum. Instead, these C-Term mutants appeared to enhance the actions of IGF-I by a combination of increased dissociation of IGF-IGFBP complexes from the substratum, together with dissociation of IGF-I from IGFBP-5 bound to the substratum. These effects of the IGFBPs were dependent upon binding to IGF-I, because a non-IGF binding mutant (N-Term) was unable to inhibit or enhance the actions of IGF-I. These results emphasize the importance of the kinetics of association/dissociation in determining the enhancing or inhibiting effects of IGFBP-5 and demonstrate the ability to generate an IGFBP-5 mutant with exclusively IGF-enhancing activity

Enhanced bonding of pentagon–heptagon defects in graphene to metal surfaces : insights from the adsorption of azulene and naphthalene to Pt(111)

The performance of graphene-based (opto)electronic devices depends critically on the graphene/metal interface formed at the metal contacts. We show here that the interface properties may be controlled by topological defects, such as the pentagon–heptagon (5–7) pairs, because of their strongly enhanced bonding to the metal. To measure the bond energy and other key properties not accessible for the embedded defects, we use azulene as a molecular model for the 5–7 defect. Comparison to its isomer naphthalene, which represents the regular graphene structure, reveals that azulene interacts more strongly with a Pt(111) surface. Its adsorption energy, as measured by single-crystal adsorption calorimetry (SCAC), exceeds that of naphthalene by up to 116 kJ/mol (or up to 50%). Both isomers undergo hybridization of their frontier orbitals with metal states, as indicated by X-ray and ultraviolet photoelectron spectroscopy (XPS/UPS) and near-edge X-ray absorption fine structure (NEXAFS) spectroscopy combined with molecular orbital (MO) projection analysis through dispersion-corrected, periodic density functional theory (DFT) calculations. Based on the NEXAFS/DFT analysis, the stronger bond of the 5–7 system is attributed to the different energetic response of its unoccupied frontier orbitals to adsorption. Adsorption-induced bond-length changes show substantial topology-related differences between the isomers. Electron transfer occurs in both directions through donation/back-donation, resulting in the partial occupation (deoccupation) of formerly unoccupied (occupied) orbitals, as revealed by periodic energy decomposition analysis (pEDA) for extended systems. Our model study shows that the topology of the π-electron system strongly affects its bonding to a transition metal and thus can be utilized to tailor interface properties

Insulin-like growth factor binding proteins and mammary gland development

Mammary gland development is dependent upon insulin-like growth factors (IGFs) as survival factors. The actions of the IGFs are modulated by a family of IGF-binding proteins (IGFBP1-6). Expression of the IGFBPs is both time-dependent and cell-specific during both the developmental phases and the involution of the mammary gland. Although studied extensively in vitro, understanding the roles of IGFBPs in vivo has been difficult, largely due to the fact that IGFBP knock-out mice have no dramatic phenotypes. This review examines the evidence from in vitro studies and the attempts to examine in vivo actions utilising models with IGFBP deficiency or over-expression. In vitro studies demonstrate that IGFBPs can act by inhibition of the survival effects of IGFs, as well as by enhancing the effects of IGFs. Because the IGFBPs are found associated with the extracellular matrix, a role for IGFBPs as a reservoir of IGFs or, alternatively as a potential barrier to IGFs, thereby restricting their entry into particular tissues or cellular compartments was postulated. We also provide evidence with respect to the IGF-independent actions of the IGFBPs which include receptors, nuclear localization, and interaction with the extracellular matrix and cell surface proteins including integrins. We believe that recent findings place some of the IGFBPs in a larger family of extracellular proteins, the secreted cysteine-rich protein (CCN) family, which have similar structural domains (involved in binding to IGFs, extracellular matrix and integrins) and are heavily implicated in tissue re-modeling and morphogenesis

Insulin-like growth factor binding protein-5 (IGFBP-5) induces premature cell death in the mammary glands of transgenic mice

We have previously demonstrated that IGFBP-5 production by mammary epithelial cells increases dramatically during involution of the mammary gland. To demonstrate a causal relationship between IGFBP-5 and cell death we created transgenic mice expressing IGFBP-5 in the mammary gland using a mammary-specific promoter, ß-lactoglobulin. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Histological analysis indicated reduced numbers of alveolar end buds, with decreased ductal branching. Transgenic dams produced IGFBP-5 in their milk at concentrations similar to those achieved at the end of normal lactation. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. BrdU labelling was decreased, whereas DNA ladders were increased in transgenic animals on day 1 of lactation. On day 2 postpartum, the epithelial invasion of the mammary fat pad was clearly impaired in transgenic animals. The concentrations of the pro-apoptotic molecule caspase-3 and of plasmin were both increased in transgenic animals whilst the concentrations of 2 prosurvival molecules Bcl-2 and Bcl-xLwere both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I we examined IGF receptor phosphorylation and Akt phosphorylation and showed that both were inhibited. We attempted to "rescue" the transgenic phenotype by using growth hormone to increase endogenous IGF-I concentrations or by implanting minipumps delivering an IGF-1 analogue, R3-IGF-1, which binds weakly to IGFBP-5. Growth hormone treatment failed to affect mammary development suggesting that increased concentrations of endogenous IGF-1 are insufficient to overcome the high concentrations of IGFBP-5 produced by these transgenic animals. In contrast mammary development (gland weight and DNA content) was normalised by R3-IGF-I although milk production was only partially restored. This is the first demonstration that over-expression of IGFBP-5 can lead to; impaired mammary development, increased expression of the pro-apoptotic molecule caspase-3, increased plasmin generation and decreased expression of pro-survival molecules of the Bcl-2 family. It clearly demonstrates that IGF-I is an important developmental/survival factor for the mammary gland and, furthermore, this cell death programme may be utilised in a wide variety of tissues

A novel role for IRF-1 as a suppressor of apoptosis

The tumour suppressor IRF-1 is a transcription factor involved in the induction of apoptosis in several in vitro systems. Post-lactational involution of the mammary gland is characterized by extensive apoptosis of the epithelial cells. We have previously shown that signal transducer and activator of transcription (Stat) 3 drives apoptosis and involution in the mouse mammary gland. Since one of the downstream targets of the Stat signalling pathway is IRF-1, we have used IRF-1 knockout mice to address the potential role of this transcription factor in involution. Surprisingly, in the absence of IRF-1 significantly higher numbers of apoptotic cells were found in involuting glands at 48 h compared to control glands. In addition, the alveolar structure in IRF-1 null mammary glands had collapsed whereas in control glands the alveoli remained intact and distended. However, by 72 h control and null glands were morphologically similar suggesting that IRF-1 suppresses apoptosis only during the early, reversible, stage of involution. This suggests a survival role for IRF-1 in mammary epithelia and demonstrates a novel role for IRF-1 in vivo - suppression of premature epithelial apoptosis during mammary gland involution