Can the Insulin Sensitivity Index (ISI) in Association with Insulin-like Growth Factor Binding Protein-1 Identify Insulin Resistance Early in Overweight Children?

Association between insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) has been reported. This prompted us to evaluate the power of the insulin sensitivity index (ISI) in association with IGFBP-1 to identify IR early in obese children/adolescents. OGTT was performed in 34 obese/overweight children/adolescents. Glucose, insulin and IGFBP-1 were measured in serum samples and ISI was calculated. Considering the presence of three or more risk factors for IR as a criterion for IR, ISI <4.6 showed 87.5% sensitivity and 94.5% specificity in diagnosing IR. IGFBP-1 was lower in the group with ISI <4.6 (p <0.01). In this group, three patients had higher than expected IGFBP-1, suggesting hepatic IR, while three patients with ISI >4.6 showed very low IGFBP-1 levels. Conclusion: ISI <4.6 is a good indicator of early peripheral IR and, associated with IGFBP-1, can identify increased risk of hepatic IR. Low IGFBP-1 levels among non-IR children may indicate increased portal insulin levels.

Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5%) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells

Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. We find that the Caenorhabditis elegans protein DAF-16 binds the IGFBP-1⋅IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1⋅IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). We show that DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1⋅IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, we conclude that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site.

Avaliação de IGF-1 (Insulin-like growth factor-1), IGFBP-1 e IGFBP-3 (Insulin-like to growth binding protein-1 e 3) no fluído folicular de pacientes infertéis com endometriose

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Assessing the benefits of rosiglitazone in women with polycystic ovary syndrome through its effects on insulin-like growth factor 1, insulin-like growth factor-binding protein-3 and insulin resistance: a pilot study

Federal University of Sao Paulo

Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-kappa B and sterol regulatory element-binding protein-1 in adipocytes

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 mu M arachidonyl-2'-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 mu M AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-kappa B and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (similar to 2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-kappa B at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-kappa B and SREBP-1 transcriptional regulation. Journal of Molecular Endocrinology (2012) 49, 97-106

Domains of transcription factor Sp1 required for synergistic activation with sterol regulatory element binding protein 1 of low density lipoprotein receptor promoter.

Feedback regulation of transcription from the low density lipoprotein (LDL) receptor gene is fundamentally important in the maintenance of intracellular sterol balance. The region of the LDL receptor promoter responsible for normal sterol regulation contains adjacent binding sites for the ubiquitous transcription factor Sp1 and the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs). Interestingly, both are essential for normal sterolmediated regulation of the promoter. The cooperation by Sp1 and SREBP-1 occurs at two steps in the activation process. SREBP-1 stimulates the binding of Sp1 to its adjacent recognition site in the promoter followed by enhanced stimulation of transcription after both proteins are bound to DNA. In the present report, we have defined the protein domains of Sp1 that are required for both synergistic DNA binding and transcriptional activation. The major activation domains of Sp1 that have previously been shown to be essential to activation of promoters containing multiple Sp1 sites are required for activation of the LDL receptor promoter. Additionally, the C domain is also crucial. This slightly acidic approximately 120-amino acid region is not required for efficient synergistic activation by multiple Sp1 sites or in combination with other recently characterized transcriptional regulators. We also show that Sp1 domain C is essential for full, enhanced DNA binding by SREBP-1. Taken together with other recent studies on the role of Sp1 in promoter activation, the current experiments suggest a unique combinatorial mechanism for promoter activation by two distinct transcription factors that are both essential to intracellular cholesterol homeostasis.

The-202 A Allele of Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Promoter Polymorphism Is Associated with Higher IGFBP-3 Serum Levels and Better Growth Response to Growth Hormone Treatment in Patients with Severe Growth Hormone Deficiency

Context: Genetic factors that influence the response to recombinant human GH (rhGH) therapy remain mostly unknown. To date, only the GH receptor gene has been investigated. Objective: The aim of the study was to assess the influence of a polymorphism in the IGF-binding protein-3 (IGFBP-3) promoter region (-202 A/C) on circulating IGFBP-3 levels and growth response to rhGH therapy in children with GH deficiency (GHD). Design and Patients: -202 A/C IGFBP3 genotyping (rs2854744) was correlated with data of 71 children with severe GHD who remained prepubertal during the first year of rhGH treatment. Main Outcome Measures: We measured IGFBP-3 levels and first year growth velocity (GV) during rhGH treatment. Results: Clinical and laboratory data at the start of treatment were indistinguishable among patients with different -202 A/C IGFBP3 genotypes. Despite similar rhGH doses, patients homozygous for the A allele presented higher IGFBP-3 SD score levels and higher mean GV in the first year of rhGH treatment than patients with AC or CC genotypes (first year GV, AA = 13.0 +/- 2.1 cm/yr, AC = 11.4 +/- 2.5 cm/yr, and CC = 10.8 +/- 1.9 cm/yr; P = 0.016). Multiple linear regression analyses demonstrated that the influence of -202 A/C IGFBP3 genotype on IGFBP-3 levels and GV during the first year of rhGH treatment was independent of other variables. Conclusion: The -202 A allele of IGFBP3 promoter region is associated with increased IGFBP-3 levels and GV during rhGH treatment in prepubertal GHD children. (J Clin Endocrinol Metab 94: 588-595, 2009)

Konstruktion einer nicht-IGF-bindenden Insulin-like Growth Factor Binding Protein-2-Variante und Untersuchung ihrer Wirkung auf das Tumorzellwachstum

Magdeburg, Univ., Medizin. Fakultät, Diss., 2008

Amniotic fluid insulin-like growth factor binding protein 3 concentration as early indicator of fetal growth restriction.

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is an important regulator of fetal growth and its bioavailability depends on insulin-like growth factor binding proteins (IGFBPs). Genes coding for IGF-I and IGFBP3 are polymorphic. We hypothesized that either amniotic fluid protein concentration at the beginning of the second trimester or genotype of one of these two genes could be predictive of abnormal fetal growth. STUDY DESIGN: Amniotic fluid samples (14-18 weeks of pregnancy) from 123 patients with appropriate for gestational age (AGA) fetuses, 39 patients with small for gestational age (SGA) fetuses and 34 patients with large for gestational age (LGA) were analyzed. Protein concentrations were evaluated by ELISA and gene polymorphisms by PCR. RESULTS: Amniotic fluid IGFBP3 concentrations were significantly higher in SGA compared to AGA group (P=0.030), and this was even more significant when adjusted to gestational age at the time of amniocentesis and other covariates (ANCOVA analysis: P=0.009). Genotypic distribution of IGF-I variable number of tandem repeats (VNTR) polymorphism was significantly different in SGA compared to AGA group (P=0.029). 19CA/20CA genotype frequency was threefold decreased in SGA compared to AGA group and the risk of SGA occurrence of this genotype was decreased accordingly: OR=0.289, 95%CI=0.1-0.9, P=0.032. Genotype distribution of IGFBP3(A-202C) polymorphism was similar in all three groups. CONCLUSIONS: High IGFBP3 concentrations in amniotic fluid at the beginning of the second trimester are associated with increased risks of SGA while 19CA/20CA genotype at IGF-I VNTR polymorphism is associated with reduced risks of SGA. Neither IGFBP3 concentrations, nor IGF-I/IGFBP3 polymorphisms are associated with modified risks of LGA.

Amniotic fluid insulin-like growth factor binding-protein 3 concentration as early indicator of fetal growth restriction

Rapport de synthèse : Introduction : La croissance foetale infra-utérine dépend d'un grand nombre de facteurs maternels, placentaires et foetaux. Une inadéquation d'un ou plusieurs de ces facteurs peut induire un retard de croissance infra-utérin (RCIU) ou au contraire une macrosomie. Les principales causes de RCIU comprennent les infections maternelles, l'éclampsie, les cardiovasculopathies maternelles, la toxicomanie, les malformations foetales et les insuffisances placentaires. Les facteurs endocriniens constituent un petit pourcentage des causes de RCIU, mais méritent que l'on s'y intéresse de plus près. Les facteurs hormonaux les plus importants pour la croissance fatale sont l'insuline et les insuline-like growth factors (IGFs) et non l'hormone de croissance (GH) qui joue un rôle majeur dans la croissance postnatale. Notre attention s'est portée sur IGF-1 qui joue un rôle important dans la croissance intrautérine. Sa biodisponibilité dépend de plusieurs protéines plasmatiques, les IGF-binding proteins (IGFBP 1 à 9). IGFBP-3 est la principale de ces IGFBPs, autant d'un point de vue quantitatif que fonctionnel. Nous avons cherché à déterminer si les concentrations d'IGF-1 et d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre étaient prédictives de la croissance infra-utérine. Les gènes codant pour IGF-1 et IGFBP-3 contenant certaines séquences polymorphiques, nous avons également étudié leur influence sur la croissance foetale. L'analyse du liquide amniotique présente l'avantage de pouvoir être effectuée dès la 14ème semaine d'aménorrhée alors que la biométrie foetale échographique ne permet pas à ce stade de déceler des anomalies de la croissance infra-utérine. Méthode : Nous avons analysé des échantillons de liquide amniotique prélevés entre la 14ème et la 18ème semaine de grossesse chez 196 patientes. Les concentrations d'IGF-1 et d'IGFBP-3 ont été dosées par ELISA, les polymorphismes analysés par PCR. Ces résultats ont été ensuite analysés en fonction du poids de naissance des nouveaux-nés, répartis en trois groupes normal pour l'âge gestationnel (AGA), petit pour l'âge gestationnel (SGA) et grand pour l'âge gestationnel (LGA). Résultats : Les concentrations d'IGFBP3 dans le liquide amniotique sont significativement plus élevées (p = 0.030) dans le groupe SGA par rapport au groupe AGA, d'autant plus quand les taux sont ajustés en fonction de paramètres tels que l'âge gestationnel lors de l'amniocentèse (ANCOVA analysis : p = 0.009). La distribution du polymorphisme VNTR (variable number of tandem repeat) dans la région promotrice d'IGF-1 au sein du groupe SGA est significativement différente de celle du groupe AGA (p = 0.029). En effet, la fréquence de l'association allélique 19CA/20CA est diminuée dans le groupe SGA. Nous n'avons pas identifié de différence de distribution des séquences polymorphiques d'IGFBP-3 entre les différents groupes. Conclusion : Une concentration élevée d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre est associée à un risque plus élevé de retard de croissance alors que l'association allélique 19CA/20CA dans la région polymorphique IGF-1 VNTR est un facteur protecteur.

Prospective study of insulin-like growth factor-I, insulin-like growth factor-binding protein 3, genetic variants in the IGF1 and IGFBP3 genes and risk of coronary artery disease.

Although experimental studies have suggested that insulin-like growth factor I (IGF-I) and its binding protein IGFBP-3 might have a role in the aetiology of coronary artery disease (CAD), the relevance of circulating IGFs and their binding proteins in the development of CAD in human populations is unclear. We conducted a nested case-control study, with a mean follow-up of six years, within the EPIC-Norfolk cohort to assess the association between circulating levels of IGF-I and IGFBP-3 and risk of CAD in up to 1,013 cases and 2,055 controls matched for age, sex and study enrolment date. After adjustment for cardiovascular risk factors, we found no association between circulating levels of IGF-I or IGFBP-3 and risk of CAD (odds ratio: 0.98 (95% Cl 0.90-1.06) per 1 SD increase in circulating IGF-I; odds ratio: 1.02 (95% Cl 0.94-1.12) for IGFBP-3). We examined associations between tagging single nucleotide polymorphisms (tSNPs) at the IGF1 and IGFBP3 loci and circulating IGF-I and IGFBP-3 levels in up to 1,133 cases and 2,223 controls and identified three tSNPs (rs1520220, rs3730204, rs2132571) that showed independent association with either circulating IGF-I or IGFBP-3 levels. In an assessment of 31 SNPs spanning the IGF1 or IGFBP3 loci, none were associated with risk of CAD in a meta-analysis that included EPIC-Norfolk and eight additional studies comprising up to 9,319 cases and 19,964 controls. Our results indicate that IGF-I and IGFBP-3 are unlikely to be importantly involved in the aetiology of CAD in human populations.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Insulin-like growth factor binding protein 2 (IGFBP2) promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression and poor survival in many types of human cancer (such as prostate, ovarian, adrenocortical, breast, colorectal carcinomas, leukemia, and high-grade gliomas). We therefore hypothesize that IGFBP2 is a key regulator of tumor progression. We tested our hypothesis in gliomas using the somatic gene transfer RCAS-tva mouse model system, which permits the introduction of specific genes into specific, cell lineages, in this case glial cells (RCAS: Replication competent avian sarcomavirus, tv-a: avian RCAS virus receptor). Mice are transgenic and harbor the tv-a receptor under the control of a glial-specific promoter and study genes are cloned into the RCAS vector for post-natal intracranial delivery. For these experiments, the study genes were IGFBP2, platelet-derived growth factor B (PDGFB), K-Ras, Akt, and IIp45 (invasion inhibitory protein 45 kDa; known to bind and block IGFBP2 activity), which were delivered separately and in combination. Our results show that PDGFB signaling leads exclusively to the formation of low-grade (WHO grade II) oligodendrogliomas. PDGFB delivered in combination with IGFBP2 results in the formation of anaplastic oligodendrogliomas (WHO grade III), which are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. IIp45 injected in combination with PDGFB and IGFBP2 ablates IGFBP2-induced tumor progression, which results in formation of low-grade oligodendrogliomas, and an overall reduction in tumor incidence. K-Ras expression was required to form astrocytomas with either IGFBP2 or Akt, indicating the activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells co-expressing IGFBP2 versus PDGFB expression alone. This study provides definitive evidence, for the first time, that: (1) IGFBP2 plays a role in activation of the Akt pathway, (2) IGFBP2 collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma, and (3) IGFBP2-induced tumor progression can be ablated by IIp45 or by specific inhibition of the Akt pathway. ^