Programming of adipose tissue miR-483-3p and GDF-3 expression by maternal diet in type 2 diabetes.

Nutrition during early mammalian development permanently influences health of the adult, including increasing the risk of type 2 diabetes and coronary heart disease. However, the molecular mechanisms underlying such programming are poorly defined. Here we demonstrate that programmed changes in miRNA expression link early-life nutrition to long-term health. Specifically, we show that miR-483-3p is upregulated in adipose tissue from low-birth-weight adult humans and prediabetic adult rats exposed to suboptimal nutrition in early life. We demonstrate that manipulation of miR-483-3p levels in vitro substantially modulates the capacity of adipocytes to differentiate and store lipids. We show that some of these effects are mediated by translational repression of growth/differentiation factor-3, a target of miR-483-3p. We propose that increased miR-483-3p expression in vivo, programmed by early-life nutrition, limits storage of lipids in adipose tissue, causing lipotoxicity and insulin resistance and thus increasing susceptibility to metabolic disease.

Bases adjacent to mononucleotide repeats show an increased single nucleotide polymorphism frequency in the human genome.

Mononucleotide repeats (MNRs) are abundant in eukaryotic genomes and exhibit a high degree of length variability due to insertion and deletion events. However, the relationship between these repeats and mutation rates in surrounding sequences has not been systematically investigated. We have analyzed the frequency of single nucleotide polymorphisms (SNPs) at positions close to and within MNRs in the human genome. Overall, we find a 2- to 4-fold increase in the SNP frequency at positions immediately adjacent to the boundaries of MNRs, relative to that at more distant bases. This relationship exhibits a strong asymmetry between 3' and 5' ends of repeat tracts and is dependent upon the repeat motif, length and orientation of surrounding repeats. Our analysis suggests that the incorporation or exclusion of bases adjacent to the boundary of the repeat through substitutions, in which these nucleotides mutate towards or away from the base present within the repeat, respectively, may be another mechanism by which MNRs expand and contract in the human genome.

The transcription factors Egr1 and Egr2 have opposing influences on adipocyte differentiation.

The zinc finger-containing transcription factors Egr1 (Krox24) and Egr2 (Krox20) have been implicated in the proliferation and differentiation of many cell types. Egr2 has earlier been shown to play a positive role in adipocyte differentiation, but the function of Egr1 in this context is unknown. We compared the roles of Egr1 and Egr2 in the differentiation of murine 3T3-L1 adipocytes. Egr1 protein was rapidly induced after addition of differentiation cocktail, whereas Egr2 protein initially remained low before increasing on days 1 and 2, concomitant with the disappearance of Egr1. In marked contrast to the effects of Egr2, differentiation was inhibited by ectopic expression of Egr1 and potentiated by knockdown of Egr1. The pro-adipogenic effects of Egr1 knockdown were particularly notable when isobutylmethylxanthine (IBMX) was omitted from the differentiation medium. However, knockdown of Egr1 did not affect CCAAT/enhancer binding protein (C/EBP)beta protein expression or phosphorylation of CREB Ser133. Further, Egr1 did not directly affect the activity of promoters for the master adipogenic transcription factors, C/EBPalpha or peroxisome proliferator-activated receptor-gamma2, as assessed in luciferase reporter assays. These data indicate that Egr1 and Egr2 exert opposing influences on adipocyte differentiation and that the careful regulation of both is required for maintaining appropriate levels of adipogenesis. Further, the pro-differentiation effects of IBMX involve suppression of the inhibitory influence of Egr1.

Contextual Assessment of the Maternity Experience: development of an instrument for cross-cultural research.

BACKGROUND: There is evidence that stressors may trigger the onset of a depressive episode in vulnerable women. A new UK interview measure, the Contextual Assessment of the Maternity Experience (CAME), was designed to assess major risk factors for emotional disturbances, especially depression, during pregnancy and post-partum. AIMS: With in the context of a cross-cultural study, to establish the usefulness of the CAME, and to test expected associations of the measure with characteristics of the social context and with major or minor depression. METHOD: The CAME was administered antenatally and postnatally in ten study sites, respectively to 296 and 249 women. Affective disorder throughout pregnancy and up to 6 months postnatally was assessed by means of the Structured Clinical Interview for DSM-IVAxis I Disorders. RESULTS: Adversity, poor relationship with either a partner or a confidant, and negative feelings about the pregnancy all predicted onset of depression during the perinatal period. CONCLUSIONS: The CAME was able to assess major domains relevant to the psychosocial context of the maternity experience in different cultures. Overall, the instrument showed acceptable psychometric properties in its first use in different cultural settings.

Can we prevent postnatal depression? A randomized controlled trial to assess the effect of continuity of midwifery care on rates of postnatal depression in high-risk women.

OBJECTIVE: To evaluate the effectiveness of continuous midwifery care in reducing rates of postnatal depression in women with histories of depression. METHODS: Fifty-one women from a sample of 98 pregnant women with histories of major depressive disorder were randomly allocated, at antenatal booking, to continuous midwifery care. The remaining 47 women received standard maternity care. A total of 87 women (44 treatment, 43 control) completed baseline assessments (after randomization) and outcome assessments (at 3 months postpartum). RESULTS: Of the women allocated to continuous midwifery care, 88% complied in full with their allocated treatment protocol. Forty-nine percent of women had an episode of illness in pregnancy (DSM-III-R case of major or minor depression), 26% developed a new episode of illness after antenatal booking, and 23% were depressed in the first 3 months postpartum. There were no differences between treatment conditions in total rates, or rates within the context of social adversity, of antenatal depression, depressions which occurred post-booking, postnatal depression, and the duration of depressive episodes. CONCLUSIONS: While continuous midwifery care had no impact on psychiatric outcome, it was highly successful at engaging women in treatment and therefore has an important contribution to make in the care of child-bearing women with mental health problems.

Deletion of V335 from the L2 domain of the insulin receptor results in a conformationally abnormal receptor that is unable to bind insulin and causes Donohue's syndrome in a human subject.

An infant with Donohue's syndrome (leprechaunism) was found to be homozygous for an in-frame trinucleotide deletion within the insulin receptor gene resulting in the deletion of valine 335. When transiently transfected into Chinese hamster ovary cells, mutant receptor was produced in a mature form, but at significantly lower levels compared with wild-type receptor. Cell surface biotinylation experiments revealed that significant amounts of the DeltaV335 receptor were expressed on the cell surface. Despite this, cells expressing this receptor showed no significant insulin binding or ligand-induced receptor autophosphorylation. Although the DeltaV335 receptor was capable of being immunoprecipitated with antibodies directed against the beta-subunit of the receptor, the mutant receptor could not be recognized by a panel of antibodies directed against different epitopes of the alpha-subunit, suggesting that the loss of V335 results in a major conformational alteration in the receptor alpha-subunit. This would be predicted by the positioning of V335 at a critical location within a strand that provides the main rigid scaffold for the two beta-sheet faces of the L2 domain of the receptor. The severe biochemical and clinical consequences of this novel mutation, which occur despite substantial expression on the cell surface, emphasize the crucial role of the L2 domain in ligand binding by the insulin receptor.

Specificity in ligand binding and intracellular signalling by insulin and insulin-like growth factor receptors.

The physiological roles of insulin and insulin-like growth factors (IGFs) are distinct, with insulin acting to regulate cellular uptake and metabolism of fuels, whereas IGFs promote cell growth, survival and differentiation. The only components of signalling pathways known to be unique to insulin and IGFs are their respective receptors, and even these display substantial structural and functional similarity. Specificity of action in vivo must in part reflect relative levels of receptor expression in different tissues. The extent to which the receptors differ in intrinsic signalling capacity remains unclear, but specificity might in principle arise from differences in ligand-binding mechanism or properties of intracellular domains. To identify ligand binding determinants we expressed receptor fragments as soluble proteins. Both N-terminal domains and a C-terminal peptide sequence from the alpha-subunit are essential for ligand binding with moderate affinity. However, binding of ligand with high affinity and specificity requires higher-order structure. To compare signalling capacities, we constructed chimaeras containing intracellular domains of insulin or IGF receptors fused to the extracellular portion of TrkC. Expression and activation of these chimaeras in cell lines reveals subtle differences in signalling and end-point responses, which may depend on cell background.

Adipose depot-specific expression of cIAP2 in human preadipocytes and modulation of expression by serum factors and TNFalpha.

BACKGROUND: Visceral obesity is more closely associated with many deleterious metabolic sequelae than obesity per se. The identification of properties that distinguish fat cells of the omentum from adipocytes situated elsewhere in the body may lead to the development of therapeutic strategies targeting visceral obesity. We have previously demonstrated that cIAP2 mRNA is significantly overexpressed in omental (Om) compared with subcutaneous (Sc) adipocytes. This molecule is involved in the TNFalpha signalling pathway and may inhibit apoptosis. OBJECTIVE: To examine the effect of serum agents and TNFalpha upon cIAP2 mRNA expression in human primary culture preadipocytes. DESIGN: Paired omental and subcutaneous adipose tissue biopsies were obtained from 11 patients, nine female and two male, with ages ranging from 29 to 82. These were cultured in either serum containing medium or serum-free medium with or without the addition of TNFalpha for 4 h and mRNA levels analysed by quantitative reverse-transcription polymerase chain reaction. RESULTS: When human preadipocytes were cultured in a defined medium containing foetal calf serum the Om cells had a greater level of expression of cIAP2 mRNA than Sc cells from the same individual (mean 3.5-fold higher Sc>Om; P<0.01). However, when serum was removed from this media for a transitory period the level of cIAP2 mRNA decreased in the omental depot such that Sc preadipocytes had greater cIAP2 expression than their Om counterparts. Addition of TNFalpha induced a large increase in mRNA levels of cIAP2 (mean 20-fold). CONCLUSIONS: These results demonstrate that cIAP2 is expressed in a depot-specific manner in human preadipocytes and that levels of expression are regulated by serum factors and TNFalpha. Thus there may be intrinsic differences between preadipocyte cells from different adipose depots and this may play a role in the regulation of body fat distribution via the modulation of fat cell apoptosis.

Defect in insulin-like growth factor-1 survival mechanism in atherosclerotic plaque-derived vascular smooth muscle cells is mediated by reduced surface binding and signaling.

Apoptosis of vascular smooth muscle cells (VSMCs) is increased in atherosclerosis compared with normal vessels, where it may contribute to plaque rupture. We have previously found that human plaque-derived VSMCs (pVSMCs) are intrinsically sensitive to apoptosis and not responsive to the protective effects of insulin-like growth factor-1 (IGF-1). We therefore examined the mechanism underlying this defect. Human pVSMCs showed <25% (125)I-IGF-1 surface binding, <20% IGF-1 receptor (IGF-1R) expression than that of normal medial VSMCs, and <40% Akt kinase activity in response to IGF-1. pVSMCs expressed and secreted high levels of IGF-1 binding proteins (IGFBPs), and the IGF-1 analogues, long R3 and Des 1,3 IGF-1, which do not bind to IGFBPs, were able to increase pVSMC survival to normal medial VSMC levels. The long R3 survival effect was phosphatidylinositol 3-kinase-mediated, but it was not dependent on Akt activity alone. Intimal pVSMCs in vivo showed reduced IGF-1R expression compared with medial VSMCs, in particular at the shoulder regions of plaques. We conclude that human pVSMCs show an intrinsic sensitivity to apoptosis caused in part by defective expression of IGF-1R, impaired IGF-1-mediated survival signaling and increased IGFBP secretion. This impaired IGF-1 protection against apoptosis may promote VSMC loss and plaque instability in atherosclerosis.

Comparison of anti-apoptotic signalling by the insulin receptor and IGF-I receptor in preadipocytes and adipocytes.

We compared the effectiveness of insulin receptor (IR) and type I insulin-like growth factor (IGF) receptor (IGFR) cytoplasmic domains in mediating anti-apoptotic effects in 3T3-L1 preadipocytes and adipocytes. We used TrkC/IR and TrkC/IGFR chimeras, stably expressed in these cells and stimulated with neurotrophin-3 (NT-3), so as to avoid interference from endogenous receptors. After 24-h serum deprivation, 10% of preadipocytes and 2% of adipocytes appeared apoptotic as determined by fluorescence-activated cell sorter (FACS) analysis of cells stained with propidium iodide (PI) and Annexin V. When NT-3 was added, the two chimeras inhibited apoptosis to the same extent by 80% in preadipocytes and 50% in adipocytes. Mutation of juxtamembrane tyrosines (IR Y960F, IGFR Y950F) abrogated these anti-apoptotic effects. Qualitatively similar results were obtained by determination of viable rather than apoptotic cells. We conclude that IR and IGFR have equal potential to inhibit apoptosis in cell backgrounds, which are normally responsive to either IGF-I or insulin.

The role of insulin dissociation from its endosomal receptor in insulin degradation.

Mechanisms that terminate signals from activated receptors have potential to influence the magnitude and nature of cellular responses to insulin. The aims of this study were to determine in rat liver endosomes (the subcellular site of insulin signal termination) whether dissociation of insulin from its receptor was a pre-requisite for ligand degradation and whether the state of receptor phosphorylation influenced the dissociation and hence endosomal degradation of insulin and/or receptor recycling. Following in vivo administration of 125I-[A14]-insulin or analogues (native, X10 or H2, relative binding affinities 1:7:67) livers were removed and endosomes prepared. In the endosomal preparations a significantly greater percentage of both analogues were receptor-bound than native insulin with concomitantly less ligand degradation. When rats were injected with protein-tyrosine phosphatase inhibitors (peroxovanadium compounds bpV(phen) or bpV(pic)) before insulin, endosomal insulin receptor phosphotyrosine content, assessed by Western blotting, was increased as was receptor-bound 125I-[A14]-insulin, whilst insulin degradation was decreased. Peroxovanadiums also completely inhibited recycling of insulin receptors from endosomes. However, treatment of freshly isolated endosomes with acid phosphatase which completely dephosphorylated the insulin receptor, did not return the rate of insulin dissociation and degradation to control values, suggesting that peroxovanadium compounds elicit their effect on binding and degradation via a mechanism other than as protein-tyrosine phosphatase inhibitors. We conclude that promotion of sustained receptor binding decreases endosomal insulin degradation and extends the half-life of the activated endosomal receptor, which in turn would be expected to potentiate insulin signalling from this intracellular compartment.

IGF-I inhibits apoptosis induced by serum withdrawal, but potentiates TNF-alpha-induced apoptosis, in 3T3-L1 preadipocytes.

We have previously shown that human preadipocytes in primary culture undergo apoptosis in response to serum deprivation and addition of tumour necrosis factor alpha (TNF-alpha), and have proposed that regulation of preadipocyte apoptosis in vivo may contribute to the overall control of adipose mass. In the present study we have investigated both pro- and anti-apoptotic factors, and the signalling pathways by which they act, in murine 3T3-L1 preadipocytes. Apoptotic indices (fraction of cells undergoing apoptosis) were determined by microscopic examination of acridine orange-stained cells, fluorescence-activated cell sorting of propidium iodide-stained cells, or phase-contrast video microscopy. Murine 3T3-L1 cells were more susceptible to apoptosis than human preadipocytes. In medium containing 10% newborn calf serum, the basal apoptotic index was very low (<2%), but the number of apoptotic cells increased significantly following serum withdrawal (10% after 24 h). Addition of TNF-alpha (6 nM) stimulated apoptosis in both serum-containing and serum-free media (apoptotic indices of 12% and 20% respectively after 24 h). IGF-I inhibited by approximately 50% the apoptosis induced by serum withdrawal, but increased by 25% the apoptosis induced by TNF-alpha in serum-free medium. It was shown by using specific inhibitors of lipid and protein kinases (LY294002, rapamycin, PD98059, SB203580) that both phosphoinositide 3-kinase and MAP kinase pathways contribute to the anti-apoptotic action of IGF-I on serum-starved cells, while phosphoinositide 3-kinase but not MAP kinase activity is required for the paradoxical pro-apoptotic action of IGF-I in the presence of TNF-alpha. We conclude that, in addition to its previously described anti-apoptotic action, IGF-I can also be pro-apoptotic in 3T3-L1 cells in the presence of TNF-alpha, and that both the anti- and pro-apoptotic effects of IGF-I require the activation of phosphoinositide 3-kinase.

Structural domains of the insulin receptor and IGF receptor required for dimerisation and ligand binding.

We investigated structural requirements for dimerisation and ligand binding of insulin/IGF receptors. Soluble receptor fragments consisting of N-terminal domains (L1/CYS/L2, L1/CYS/L2/F0) or fibronectin domains (F0/F1/F2, F1/F2) were expressed in CHO cells. Fragments containing F0 or F1 domains were secreted as disulphide-linked dimers, and those consisting of L1/CYS/L2 domains as monomers. None of these proteins bound ligand. However, when a peptide of 16 amino acids from the alpha-subunit C-terminus was fused to the C-terminus of L1/CYS/L2, the monomeric insulin and IGF receptor constructs bound their respective ligands with affinity only 10-fold lower than native receptors.

The alpha-isoform of class II phosphoinositide 3-kinase is more effectively activated by insulin receptors than IGF receptors, and activation requires receptor NPEY motifs.

Little is known about the physiological role and mechanism of activation of class II phosphoinositide 3-kinases (PI3Ks), although it has been shown that the PI3K-C2alpha isoform is activated by insulin. Using chimaeric receptor constructs which can be activated independently of endogenous receptors in transfected cells, we found that PI3K-C2alpha activity was stimulated to a greater extent by insulin receptors than IGF receptors in 3T3-L1 adipocytes. Activation of PI3K-C2alpha required an intact NPEY motif in the receptor juxtamembrane domain. We conclude that PI3K-C2alpha is a candidate for participation in insulin-specific intracellular signalling.

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.

Insulin and insulin-like growth factor-I (IGF-I) receptor overexpression in breast cancers leads to insulin/IGF-I hybrid receptor overexpression: evidence for a second mechanism of IGF-I signaling.

The insulin receptor (IR) form hybrids with the closely related insulin-like growth factor-I (IGF-I) receptor (IGF-I-R). Because most human breast carcinomas overexpress both the IR and the IGF-I-R, we evaluated whether the insulin/IGF-I hybrid receptor (Hybrid-R) is also overexpressed in these tumors and what role it plays in breast cancer biology. Using specific ELISAs and Western blots, we measured Hybrid-R content and function in 8 human cultured breast cancer cell lines and 39 human breast cancer specimens. Hybrid-R content and function were also compared to the content and function of the IR and the IGF-I-R. Hybrid-R content exceeded the IGF-I-R content in >75% of breast cancer specimens and was directly related to the molar ratio of both the IR and IGF-I-R content, suggesting that Hybrid-R formation occurred by random assembly of IR and IGF-I-R half-receptors. Hybrid-Rs became tyrosine autophosphorylated when breast cancer cells were exposed to IGF-I but not when they were exposed to insulin. In cells with an elevated Hybrid-R content, Hybrid-R autophosphorylation in response to IGF-I exceeded IGF-I-R autophosphorylation, suggesting that most of the IGF-I effect occurred via the Hybrid-R. Furthermore, Hybrid-Rs mediated growth in response to IGF-I, as indicated by experiments with blocking antibodies to the IGF-I-R. These data indicated therefore that: (a) Hybrid-Rs are present and play a major role in mediating the IGF-I signal in breast cancer; (b) their expression is directly related to IR overexpression; and (c) potential therapies designed to block IGF-I actions in breast cancer must take into account the role of these Hybrid-Rs.

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

The insulin receptor: from protein sequence to structure.

Sequences of the insulin receptor (IR), the type-I insulin-like growth-factor receptor (IGFR) and the insulin-receptor-related receptor show that they belong to a homologous family but, until recently, have given few clues about their structures. Three repeats of fibronectin type III have been identified close to the membrane. Although the N-terminal L1, Cys-rich and L2 domains of the IGFR have been identified from their sequences and their structures determined by X-ray crystallography, little is known of their relative positions in the complete receptor in vivo. Here, we ask what can be learnt further from the analysis of sequences, about the structure, organization and function of the extracellular regions of the IR family.

Corrosion of lead shielding in a radiology department.

Corrosion of lead sheet used for structural radiation protection in the Princess Alexandra Hospital radiology department has been identified. The corrosion is thought to have been caused by organic acid vapours released from oregon timber wall panelling. Non-destructive testing (NDT) and X-ray transmission measurements were used to define the extent and severity of damage, and subsequently to provide the data necessary for estimation of staff and public radiation doses. Although radiation dose limits have not been exceeded, corrective actions including structural modifications and staff information sessions have been undertaken.

Human preadipocytes display a depot-specific susceptibility to apoptosis.

Adipose tissue mass is determined by both the number and volume of adipose cells. Adipose cell number reflects the balance of cell acquisition and cell loss, whereas adipose cell volume represents the balance of lipolysis and lipogenesis. It is well recognized that insulin resistance, NIDDM, and other metabolic disorders are associated more strongly with increased omental adiposity than with subcutaneous adiposity. Depot-related differences exist in adipocyte responses to lipolytic and lipogenic stimuli, in adipocyte apoptosis, and in the potential for preadipocyte replication and differentiation. In the present study, we address the question of whether there might also be a site-specific difference in the susceptibility of human preadipocytes to apoptosis. Paired samples of human omental and subcutaneous preadipocytes from 12 individuals were cultured, and apoptosis was induced by serum deprivation or treatment with tumor necrosis factor (TNF)-alpha for 4 h. Cells were then stained with acridine orange, and apoptotic indices were calculated as the fraction of cells showing nuclear condensation. Under both conditions, in 9 of 11 subjects, apoptotic indices were substantially greater in preadipocytes from the omental depot than in those from the subcutaneous depot, and mean apoptotic indices were more than twofold higher in omental cells (serum-free medium: P < 0.05; TNF-alpha: P < 0.02; paired t test). Omental preadipocytes are therefore more susceptible to two different apoptotic stimuli than subcutaneous preadipocytes, demonstrating another intrinsic site-specific difference between human adipose cells of the two depots. These results suggest that the regulation of adipose tissue distribution in humans could involve depot-specific differences in rates of preadipocyte apoptosis.