Disparity in fetal growth between twin and singleton gestation: the role of adipokines.

OBJECTIVE: Twin pregnancies are characterized by unique pattern of attenuated fetal weight gain during late gestation compared with singleton gestation. The mechanism(s) responsible for regulating twin growth has not yet elucidated. Leptin and adiponectin are two adipocytokines implicated in metabolism and energy balance of fetuses, newborns and adults. Moreover, these hormones have been suggested to play a role in fetal growth. The objective of the study was to determine cord blood adiponectin and leptin in twins and singletons, with and without growth impairment. STUDY DESIGN: This was a case-control study. It included two groups of newborns, matched for gestational age and birth weight percentile: singleton (n=60 newborns) and twins (n=44 newborns). Adiponectin and leptin were determined in cord blood, and compared between the groups according to clinical and demographic characteristics. Non-parametric and parametric statistical methods were employed. RESULTS: Median adiponectin and leptin concentrations were lower in twins vs singletons (P<0.001 for both comparisons). Among small for gestational age newborns (SGA), median concentration of adiponectin (P=0.04), but not leptin (P=0.1), was lower in twins compared to singletons. In pooled analysis (singleton plus twins), cord blood adiponectin and leptin were strongly correlated with gestational age (P<0.001 and P=0.005, respectively) and birth weight (P<0.001 and P<0.001, respectively). Regression analysis revealed that plurality (P=0.02) was significantly and independently associated with cord blood adiponectin concentrations, after adjustment for confounding variables. Similar regression in which leptin was the independent variable revealed that only birth weight (P=0.01) was significantly and independently associated with cord blood leptin concentrations. CONCLUSIONS: Twin pregnancies are associated with lower cord blood concentrations of adiponectin and leptin compared with singleton gestations. However, only cord blood adiponectin, but not leptin, was lower in SGA neonates. Collectively, these data suggest that adiponectin may be implicated in the mechanism accounting for the growth disparity between twins and singletons.

Postoperative adiponectin levels in pediatric patients undergoing open heart surgery.

BACKGROUND: Adipose tissue is an important endocrine organ that secretes cytokines, including adiponectin, levels of which are negatively correlated with the severity of the inflammatory process. Aim. To assess the time course of adiponectin levels following open heart surgery with cardiopulmonary bypass and its correlation with early postoperative outcomes. MATERIALS AND METHODS: Blood samples were obtained from 24 children undergoing cardiac surgery and analyzed for adiponectin, C-reactive protein, and other inflammatory markers. RESULTS: Baseline adiponectin levels were negatively correlated with patients' preoperative weight and age. Postoperative adiponectin levels decreased compared to baseline (P = 0.01) and correlated negatively with duration of cardiopulmonary bypass (r = -0.438, P = 0.037), length of stay in the pediatric intensive care unit (r = -0.457, P = 0.025), and the inotropic score (r = -0.471, P = 0.02). Adiponectin levels were positively correlated with sVCAM 1 levels; however, there was no correlation between adiponectin levels and sP selectin, tPA, MCP1, and sCD40. CONCLUSIONS: The inflammatory response after open heart surgery with cardiopulmonary bypass is associated with a reduction in adiponectin levels. Prolonged or more complicated surgery induced a more substantial inflammatory process characterized by a significant reduction in adiponectin levels over time and a delayed return to baseline levels.

Leptin and the post-operative inflammatory response. More insights into the correlation with the clinical course and glucocorticoid administration.

BACKGROUND: Cardiac surgery involving cardiopulmonary bypass (CPB) causes a systemic inflammatory process which can lead to multiple organ failure and postoperative morbidity. Recent animal and human studies suggested a possible involvement of leptin in the systemic inflammatory response. AIM: To characterize the response of leptin to open heart surgery (OHS) and the relationship between the time course of leptin levels and the post-operative clinical course, and to examine the effect of exogenous glucocorticoids. PATIENTS AND METHODS: Forty-seven pediatric patients, undergoing OHS for congenital heart disease were studied. Thirty-four patients (Group 1) received methylprednisolone during CPB while 13 (group 2) did not. Serial blood samples were collected perioperatively and up to 24 h after surgery, and assayed for leptin and cortisol. RESULTS: All patients' leptin levels decreased significantly during CPB (to 44-48% of baseline, p<0.001); they then increased, peaking at 12 h post-operatively. The levels of groups 1 and 2 were similar up to 8 h post-operatively; thereafter, those of group 1 were significantly higher. Recovery of leptin levels in patients with a more complicated post-operative course was comparatively slower. Cortisol levels of all patients increased significantly during CPB (p<0.001), gradually decreasing afterwards. Cortisol and leptin levels were inversely correlated in both patients' groups. CONCLUSIONS: CPB is associated with acute changes in circulating leptin levels. A complicated postoperative course is associated with lower leptin levels which are inversely correlated with cortisol levels. Leptin may participate in post-CPB inflammatory and hemodynamic responses.

Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer.

Klotho is an anti-aging gene, which has been shown to inhibit the insulin and insulin-like growth factor 1 (IGF-1) pathways in mice hepatocytes and myocytes. As IGF-1 and insulin regulate proliferation, survival and metastasis of breast cancer, we studied klotho expression and activities in human breast cancer. Immunohistochemistry analysis of klotho expression in breast tissue arrays revealed high klotho expression in normal breast samples, but very low expression in breast cancer. In cancer samples, high klotho expression was associated with smaller tumor size and reduced KI67 staining. Forced expression of klotho reduced proliferation of MCF-7 and MDA-MB-231 breast cancer cells, whereas klotho silencing in MCF-7 cells, which normally express klotho, enhanced proliferation. Moreover, forced expression of klotho in these cells, or treatment with soluble klotho, inhibited the activation of IGF-1 and insulin pathways, and induced upregulation of the transcription factor CCAAT/enhancer-binding protein beta, a breast cancer growth inhibitor that is negatively regulated by the IGF-1-AKT axis. Co-immunoprecipitation revealed an interaction between klotho and the IGF-1 receptor. Klotho is also a known modulator of the fibroblast growth factor (FGF) pathway, a pathway that inhibits proliferation of breast cancer cells. Studies in breast cancer cells revealed increased activation of the FGF pathway by basic FGF following klotho overexpression. Klotho did not affect activation of the epidermal growth factor pathway in breast cancer cells. These data suggest klotho as a potential tumor suppressor and identify it as an inhibitor of the IGF-1 pathway and activator of the FGF pathway in human breast cancer.

Acute psychotic stress is associated with decreased adiponectin serum levels.

OBJECTIVE: To determine the effect of acute psychotic stress on adipokine secretion in non-diabetic subjects. RESEARCH DESIGN AND METHODS: Adiponectin, leptin, and cortisol serum levels were determined in 39 non-diabetic patients with acute psychotic stress reaction admitted to a psychiatric ward. The clinical global impression (CGI) score was used to evaluate the level of psychotic stress. Insulin sensitivity (IS) was determined by the homeostasis model assessment (HOMA). Patients were re-assessed 2 weeks after admission. During hospitalization patients were treated for variable times with either phenothiazines or thioxanthenes. RESULTS: The mean CGI score decreased significantly with time: 5.3+/-0.8 and 2.6+/-0.8 on admission and after 2 weeks respectively (p<0.001). On admission, the mean adiponectin level was significantly lower in patients compared to normal controls: 15.3+/-8.2 mug/ml and 26+/-12.8 mug/ml, respectively (p=0.02). It increased significantly after 2 weeks to 18.2+/-10 mug/ml (p=0.003). By contrast, the leptin and cortisol levels did not change significantly. No correlation was found between the changes in individual CGI scores and adiponectin levels. However, female patients with the highest stress on admission demonstrated the lowest adiponectin levels and insulin sensitivity: p=0.002 and 0.03 respectively. CONCLUSIONS: These data suggest a link between acute psychotic stress reaction and decreased serum adiponectin levels. Further studies are recommended to determine the strength of this association.

Circadian rhythm sleep disorders following mild traumatic brain injury.

OBJECTIVE: To describe the physiologic and behavioral characteristics of circadian rhythm sleep disorders (CRSDs) following minor traumatic brain injury (mTBI) in patients complaining of insomnia. METHODS: Forty two patients with insomnia complaints following mTBI were screened. Those suspected of having CRSD underwent actigraphy, saliva melatonin and oral temperature measurement, and polysomnography. All patients also filled out a self-reported questionnaire to determine their circadian preference. RESULTS: Fifteen of the 42 patients (36%) with complaints of insomnia following mTBI were diagnosed with CRSD. Eight patients displayed a delayed sleep phase syndrome (DSPS), whereas seven displayed an irregular sleep-wake pattern (ISWP). Whereas all patients with DSPS exhibited a 24-hour periodicity of oral temperature rhythm, three of seven patients with ISWP lacked such a daily rhythm. In addition, ISWP patients exhibited smaller amplitude of oral temperature rhythm vs the DSPS group. Subjective Morningness-Eveningness Questionnaire scores were in accordance with the clinical diagnosis of DSPS or ISWP based on actigraphy. CONCLUSIONS: Minor traumatic brain injury might contribute to the emergence of circadian rhythm sleep disorders. Two types of these disorders were observed: delayed sleep phase syndrome and irregular sleep-wake pattern. The types differed in the subjective questionnaire scores and had distinct profiles of melatonin and temperature circadian rhythms.

Maternal serum adiponectin levels during human pregnancy.

OBJECTIVE: Pregnancy is a unique situation characterized by insulin resistance. The role of adiponectin, an insulin-sensitizing hormone, has not been completely clarified during pregnancy. The aim of this cross-sectional study was to evaluate adiponectin levels during pregnancy and postpartum. STUDY DESIGN: Adiponectin and leptin levels were tested in 80 pregnant women, 20 in each trimester (mean gestational age 10.5+/-1.9; 19.3+/-4.9; 39.3+/-0.8 weeks,) as well as 4 days postpartum. RESULTS: Adiponectin levels during first (13.3+/-3.6 micro g/ml), second (12.6+/-4.4 micro g/ml) and third trimester (11.2+/-3.7 micro g/ml) did not differ and were significantly higher than postpartum levels (8.8+/-2.1 micro g/ml; P<0.0001, P<0.004 and P<0.02, respectively). CONCLUSION: Despite increased insulin resistance during pregnancy, no significant alterations in adiponectin levels were observed. This may imply that the regulation of adiponectin during gestation is altered. The elevated gestational adiponectin levels are consistent with increased 'adiponectin resistance' during pregnancy.

Circulating leptin levels after cardiopulmonary bypass in children.

OBJECTIVE: To characterize the dynamics of circulating leptin in children after cardiac surgery with cardiopulmonary bypass (CPB), which is known to induce a systemic inflammatory response. DESIGN: Investigative study. SETTING: University-affiliated tertiary care hospital. PARTICIPANTS: Eight children (age range, 3 months to 13 years) undergoing CPB to correct congenital heart disease. INTERVENTIONS: The time courses of leptin and cortisol levels were determined. Serial blood samples were collected from the arterial catheter or from the CPB circuit preoperatively; on termination of CPB; and at 2, 4, 8, 12, 18, and 24 hours postoperatively. Plasma was recovered immediately, divided into aliquots, and frozen at -70 degrees C until use. Leptin was measured by a human leptin radioimmunoassay kit. MEASUREMENTS AND MAIN RESULTS: Leptin levels during CPB decreased to 50% of pre-CPB levels (p < 0.01). After termination of CPB, levels increased gradually and peaked at 12 hours postoperatively (10 P.M. to 1 A.M.). Cortisol levels were inversely correlated to leptin levels (p = 0.016). CONCLUSION: CPB is associated with acute changes in circulating leptin levels. These changes parallel those in cortisol, showing an inverse relationship between leptin and cortisol, suggesting a relationship between the neurobiology of these systems that could be important for the neuroendocrine response to CPB. A prognostic role of leptin and its relationship to cortisol after CPB warrant further study.

Activation of the insulin-like growth factor 1 signaling pathway by the antiapoptotic agents aurintricarboxylic acid and evans blue.

Aurintricarboxylic acid (ATA), an endonuclease inhibitor, prevents the death of a variety of cell types in culture. Previously we have shown that ATA, similar to insulin-like growth factor I (IGF-I), protected MCF-7 cells against apoptotic death induced by the protein synthesis inhibitor cycloheximide. Here we show that ATA and a polysulfonated aromatic compound, Evans blue (EB), similar to IGF-I, promote survival and increase proliferation of MCF-7 cells in serum-free culture medium. This may suggest a common signaling pathway shared by the aromatic polyanions and IGF-I. Therefore, the ability of these aromatic compounds to activate the signal transduction pathway of IGF-I was examined. We found that ATA and EB mimicked the IGF-I effect on tyrosine phosphorylation of the IGF-I receptor (IGF-IR) and its major substrates, insulin receptor substrate-1 (IRS-1) and IRS-2; induced the association of these substrates with phosphatidylinositol 3-kinase and Grb2; and activated Akt kinase and p42/p44 mitogen-activated protein kinases. ATA and EB competed for IGF-I binding to the IGF-IR. ATA was found to be selective for the IGF-IR, whereas EB also activated the insulin receptor. Upon fractionation of commercial ATA by size exclusion chromatography, we found that fractions that enhanced the intensity of tyrosyl-phosphorylated IRS-1/IRS-2 also increased the survival of MCF-7 cells in the presence of cycloheximide, whereas fractions devoid of IRS phosphorylation activity had no survival ability. Taken together, these results suggest that the survival/proliferation-promoting effects of ATA and EB in MCF-7 cells are transduced via the IGF-IR signaling pathway.

Circulating leptin and the perioperative neuroendocrinological stress response after pediatric cardiac surgery.

OBJECTIVE: Leptin may be involved in the acute stress response, regulating inflammatory parameters of major importance after cardiopulmonary bypass (CPB) surgery. Critically ill patients demonstrated significant increases in leptin levels in response to stress-related cytokines (tumor necrosis factor, interleukin [IL]-1) and abolishment of the circadian rhythm of leptin secretion. We characterized the pattern of leptin secretion in the acute postoperative period in children undergoing cardiac surgery and compared the changes in leptin levels with concomitantly occurring changes in cortisol levels, IL-8, and clinical parameters. DESIGN: Investigative study. SETTING: University-affiliated tertiary care hospital. PARTICIPANTS AND INTERVENTIONS: Twenty-nine consecutive patients, aged 6 days to 15 yrs, operated upon for the correction of congenital heart defects were studied. Surgery in 20 patients (group 1) involved conventional CPB techniques, and 9 (group 2) underwent closed-heart surgery. The time courses of leptin, cortisol, and IL-8 levels were determined. Serial blood samples were collected preoperatively, on termination of CPB, and at six time points postoperatively. Plasma was recovered immediately, aliquoted, and frozen at -70 degrees C until use. MEASUREMENTS AND MAIN RESULTS: The leptin levels in group 1 decreased during CPB to 51% of baseline (p <.001), then gradually increased, reaching 120% of baseline levels at 12-18 hrs postoperatively (p <.001), returning to baseline levels at 24 hrs (p <.01). In patients undergoing closed-heart surgery (group 2), leptin levels displayed a pattern resembling the first group: they decreased during surgery to 71% of baseline levels (p =.002) and showed a tendency to return to baseline thereafter. All group 1 patients' cortisol levels increased significantly during the first hour of surgery, then decreased, returning to baseline levels at 18-24 hrs postoperatively. There was a significant negative correlation between leptin and cortisol levels (r = -2.8, p <.01). In group 2, cortisol levels increased during and after surgery, peaking 4 hrs postoperatively and decreasing thereafter. IL-8 levels determined in 15 group 1 patients increased significantly during CPB, peaked at the end of surgery, and then decreased but remained slightly elevated even at 48 hrs postoperatively. There was a significant correlation between cortisol and IL-8 levels (r = 2.55, p <.05). Children with leukocytosis, tachycardia, and hypotension had lower leptin levels and less variation over time as opposed to those with an uncomplicated course. CONCLUSIONS: CPB is associated with acute changes in circulating leptin levels. These changes parallel those in cortisol, demonstrating an inverse relationship between leptin and cortisol. Further studies of the prognostic and therapeutic roles of leptin after CPB should be investigated.

Phosphorylation of insulin receptor substrate-1 (IRS-1) by protein kinase B positively regulates IRS-1 function.

Incubation of cells with insulin leads to a transient rise in Tyr phosphorylation of insulin receptor substrate (IRS) proteins, accompanied by elevation in their Ser(P)/Thr(P) content and their dissociation from the insulin receptor (IR). Wortmannin, a phosphatidylinositol 3-kinase inhibitor, selectively prevented the increase in Ser(P)/Thr(P) content of IRS-1, its dissociation from IR, and the decrease in its Tyr(P) content following 60 min of insulin treatment. Four conserved phosphorylation sites within the phosphotyrosine binding/SAIN domains of IRS-1 and IRS-2 served as in vitro substrates for protein kinase B (PKB), a Ser/Thr kinase downstream of phosphatidylinositol 3-kinase. Furthermore, PKB and IRS-1 formed stable complexes in vivo, and overexpression of PKB enhanced Ser phosphorylation of IRS-1. Overexpression of PKB did not affect the acute Tyr phosphorylation of IRS-1; however, it significantly attenuated its rate of Tyr dephosphorylation following 60 min of treatment with insulin. Accordingly, overexpression of IRS-1(4A), lacking the four potential PKB phosphorylation sites, markedly enhanced the rate of Tyr dephosphorylation of IRS-1, while inclusion of vanadate reversed this effect. These results implicate a wortmannin-sensitive Ser/Thr kinase, different from PKB, as the kinase that phosphorylates IRS-1 and acts as the feedback control regulator that turns off insulin signals by inducting the dissociation of IRS proteins from IR. In contrast, insulin-stimulated PKB-mediated phosphorylation of Ser residues within the phosphotyrosine binding/SAIN domain of IRS-1 protects IRS-1 from the rapid action of protein-tyrosine phosphatases and enables it to maintain its Tyr-phosphorylated active conformation. These findings implicate PKB as a positive regulator of IRS-1 functions.

Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.

Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.

A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation.

Tumor necrosis factor alpha (TNFalpha) or chronic hyperinsulinemia that induce insulin resistance trigger increased Ser/Thr phosphorylation of the insulin receptor (IR) and of its major insulin receptor substrates, IRS-1 and IRS-2. To unravel the molecular basis for this uncoupling in insulin signaling, we undertook to study the interaction of Ser/Thr-phosphorylated IRS-1 and IRS-2 with the insulin receptor. We could demonstrate that, similar to IRS-1, IRS-2 also interacts with the juxtamembrane (JM) domain (amino acids 943-984) but not with the carboxyl-terminal region (amino acids 1245-1331) of IR expressed in bacteria as His6 fusion peptides. Moreover, incubation of rat hepatoma Fao cells with TNFalpha, bacterial sphingomyelinase, or other Ser(P)/Thr(P)-elevating agents reduced insulin-induced Tyr phosphorylation of IRS-1 and IRS-2, markedly elevated their Ser(P)/Thr(P) levels, and significantly reduced their ability to interact with the JM region of IR. Withdrawal of TNFalpha for periods as short as 30 min reversed its inhibitory effects on IR-IRS interactions. Similar inhibitory effects were obtained when Fao cells were subjected to prolonged (20-60 min) pretreatment with insulin. Incubation of the cell extracts with alkaline phosphatase reversed the inhibitory effects of insulin. These findings suggest that insulin resistance is associated with enhanced Ser/Thr phosphorylation of IRS-1 and IRS-2, which impairs their interaction with the JM region of IR. Such impaired interactions abolish the ability of IRS-1 and IRS-2 to undergo insulin-induced Tyr phosphorylation and further propagate the insulin receptor signal. Moreover, the reversibility of the TNFalpha effects and the ability to mimic its action by exogenously added sphingomyelinase argue against the involvement of a proteolytic cascade in mediating the acute inhibitory effects of TNFalpha on insulin action.

Intronic mutation in the growth hormone (GH) receptor gene from a girl with Laron syndrome and extremely high serum GH binding protein: extended phenotypic study in a very large pedigree.

Laron syndrome (LS) is a hereditary form of GH resistance due to molecular defects in the GH receptor (GHR). Most of the identified mutations are located in the extracellular domain of the receptor, resulting in a lack of serum GHBP in the majority of LS patients. We present an LS patient with supranormal levels of serum GHBP, in addition to 35 of her relatives. The proband is a 3.5 year-old Druse girl with severe short stature (height SDS -5.1), high GH (250 micrograms/l), low IGF-I (2.7 nmol/l) and IGFBP-3 (410 micrograms/l), both unresponsive to exogenous GH. The binding capacity of the serum GHBP was 22 nM (adult reference serum, 0.7 nM), with an affinity constant Ka = 1.9 x 10(9) M-1 comparable to that of normal sera (Ka = 1.7-2.1 x 10(9) M-1). The apparent MW of the GHBP was approximately 60-80 kDa, similar to that of control sera. In the proband's sister, parents, grandparents and uncles, extremely high GHBP values were observed (43.0 +/- 4.8 RSB, n = 10) compared with normal adults (0.81 +/- 0.06 RSB) (p << 0.001). The remaining subjects had normal or moderately elevated GHBP levels. Serum GH in adults with high GHBP was significantly elevated above control values (6.0 +/- 0.9 micrograms/l vs 0.76 +/- 0.13 microgram/l, p < 0.001). Serum IGF-I and IGFBP-3 levels were normal in all the subjects, with the exception of an aunt (IGF-I 3.9 nmol/l) and the proband's sister (IGFBP-3 460 micrograms/l). All the subjects' heights were within the normal range. Analysis of the GHR gene performed in the proband revealed an as yet undescribed homozygous intronic point mutation. It consists of a G-->T substitution at nucleotide 785-1 preceding exon 8, a sequence that encodes the transmembrane domain. This mutation, which destroys the invariant dinucleotide of the splice acceptor site, is expected to alter GHR mRNA splicing and to be responsible for skipping exon 8. The resulting truncated protein that retains GH binding activity is probably no longer anchored in the cell membrane, affecting signal transmission in the homozygous patient and causing high GHBP levels in the heterozygous relatives.

Long-term effects of insulin-like growth factor (IGF)-I on serum IGF-I, IGF-binding protein-3 and acid labile subunit in Laron syndrome patients with normal growth hormone binding protein.

A minority of patients with Laron syndrome have normal serum GH binding protein (GHBP), indicating that the defect is elsewhere than in the extracellular domain of the GH receptor. We have evaluated the effect of long-term IGF-I treatment on serum IGF-binding protein (IGFBP)-3 and the acid-labile subunit (ALS) in three sibling with Laron syndrome caused by a GH post-receptor defect and with normal GHBP. The children (a boy aged 3 years, a girl aged 4 years and a boy aged 10 years) were treated by daily s.c. injection of IGF-I in a dose of 150 micrograms/kg. IGFBP-3 was measured by RIA and Western ligand blotting, ALS by RIA. Based values of IGFBP-3 and ALS were low. During IGF-I treatment, the IGFBP-3 concentrations in the girl gradually increased, whereas in the boys there was a 60% decrease during the first week, followed by gradual increase towards baseline. The ALS concentrations followed a similar pattern. We conclude that IGF-I treatment induces and initial suppression and then an increase in the IGFBP-3 and ALS concentrations, confirming data from animal experiments that IGFBP-3 synthesis is not solely under GH control. The differences in responsiveness between the female and male siblings may reflect genetic differences, or lower circulating concentrations of IGF-I in the boys compared with the girl.

Insulin-like growth factor-I and IGF binding protein-3 remain high after GnRH analogue therapy in girls with central precocious puberty.

OBJECTIVE: IGF-I rises in normal adolescence and in central precocious puberty (CPP), secondary to a rise in sex steroids and GH. The aim of this study was to examine changes in serum IGF-I and its major binding protein IGFBP-3 after pharmacological arrest of puberty. PATIENTS AND MEASUREMENTS: Ten girls diagnosed for CPP were evaluated before and during the first 3 months of GnRH analogue (GnRHa) therapy aimed at suppression of the gonadal axis. Serum IGF-I was measured by immunoradiometric assay (IRMA) and IGFBP-3 by both IRMA and Western ligand blotting (WLB). RESULTS: Serum IGF-I was markedly higher in patients with CPP as compared with age matched controls (48.8 +/- 6.5 vs 23.1 +/- 4.9 nmol/l, P < 0.01). While GnRHa therapy caused serum oestradiol levels to return to prepubertal levels in all 10 patients, serum IGF-I levels decreased only minimally after 1, 2 or 3 months of therapy (43.2 +/- 5.6, 42.3 +/- 6.4 and 44.1 +/- 7.2 nmol/l respectively). Serum IGFBP-3 levels as determined using IRMA were also higher in CPP compared with age matched controls (4.70 +/- 0.37 vs 3.71 +/- 0.42 mg/l, P < 0.01). These differences were also evident when measured by WLB. GnRHa therapy caused a small and insignificant decrease in serum IGFBP-3 levels after 1, 2 or 3 months of therapy (4.57 +/- 0.33, 4.48 +/- 0.4 and 4.42 +/- 0.3 mg/l respectively). CONCLUSIONS: The lack of suppression of both IGF-I and IGFBP-3, despite therapy which halts puberty and slows growth velocity, suggests that steroids may be involved in the induction of changes in the GH/IGF-I axis but not in their subsequent maintenance.

Increased insulin-like growth factor binding protein-2 (IGFBP-2) gene expression and protein production lead to high IGFBP-2 content in malignant ovarian cyst fluid.

Expression of insulin-like growth factor-I (IGF-I), its receptor and IGF-binding proteins (IGFBPs) by ovarian cancer cells and its mitogenic effect on these cells in vitro, suggest that IGF-I may have a role in regulation of human ovarian cancer. We have recently shown IGFBP-2 to be markedly elevated in malignant ovarian cyst fluid in vivo. To identify the origin of increased IGFBP-2 in these cyst fluids, the gene expression and protein content of IGFBP-2 were investigated in 14 malignant and four benign epithelial ovarian neoplasms. IGFBP-2 mRNA was detected in all ovarian specimens and was 2- to 30-fold higher in malignant than in benign neoplasms. Within the malignant tissues IGFBP-2 mRNA levels correlated with the aggressiveness of the tumour and were higher in invasive tumours than in those with borderline pathology. Southern blot analysis revealed no amplification of IGFBP-2 gene in the DNA samples from ovarian tumours regardless of their nature. IGFBP-2 was the major binding protein in tissue extracts, as measured by both Western ligand blotting and immunoblotting, and was significantly higher in malignant than in benign neoplasms. These findings were further supported by immunohistochemical detection of IGFBP-2 in tumour sections. Our data suggest that increased local production by the tumour in vivo is responsible for the increased IGFBP-2 levels in the cyst fluid bathing the ovarian malignancy. This may represent an autocrine regulatory mechanism for IGF-I proliferative effect of ovarian cancer.

Insulin-like growth factor I and its binding proteins 3 and 4 are increased in Human inflammatory synovial fluid.

OBJECTIVE: To measure insulin-like growth factor I (IGF-I) and its binding proteins (IGFBP) in human synovial fluid (SF), as IGF-I resistance in the chondrocyte was implicated in the pathogenesis of inflammatory arthritis. METHODS: Ten patients with inflammatory arthritis and 7 controls were evaluated. IGF-I and IGFBP-3 were measured by radioimmunoassay while all IGFBP were evaluated by Western ligand blotting. RESULTS: Serum IGF-I did not differ between patients and controls (22.3 +/- 2.9 vs 22.0 +/- 3.0 nmol/l), while significantly higher values were found in inflammatory SF compared with noninflammatory SF (14.7 +/- 1.5 vs 10.2 +/- 1.2 nmol/l; p = 0.05). IGFBP-3 and IGFBP-4 were the most prominent bands found in inflammatory SF by Western ligand blotting. The 24 kDa IGFBP-4 band appeared faintly in 2 noninflammatory effusions and was nearly absent in others. IGFBP-3 levels in sera from the 2 groups did not differ (2.9 +/- 0.3 vs 2.5 +/- 0.3 mg/l), but were significantly higher in inflammatory compared with noninflammatory effusions (1.5 +/- 0.2 vs 0.9 +/- 0.2 mg/l; p = 0.05). CONCLUSION: The pattern of IGFBP in SF differs in inflammatory versus noninflammatory joints.

Sphingomyelinase and ceramide suppress insulin-induced tyrosine phosphorylation of the insulin receptor substrate-1.

The sphingomyelin pathway is a newly described signal transduction pathway mediating the action of several cytokines including tumor necrosis factor-alpha (TNF). TNF was recently shown to interfere with insulin-induced tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1). In this work we examined the possible effect of direct activation of the sphingomyelin pathway on insulin-induced tyrosine phosphorylation of IRS-1. Incubation of the insulin-sensitive rat hepatoma Fao cells with bacterial sphingomyelinase (SMase) that causes membrane hydrolysis of sphingomyelin led to a time- and dose-dependent decrease in insulin-induced tyrosine phosphorylation of IRS-1. The effect was apparent after 10 min of incubation and with a dose of 10 milliunits/ml SMase. It was not associated with a decrease in insulin receptor autophosphorylation. In addition, SMase treatment interrupted the association of the 85-kDa catalytic subunit of phosphatidylinositol 3-kinase with IRS-1. A similar impact on IRS-1 tyrosine phosphorylation was observed after addition of cell-permeable ceramide analogs (C2 and C6). Comparable changes in IRS-1 tyrosine phosphorylation and electrophoretic mobility were found after exposure of cells to either TNF, SMase, or ceramide. Our findings suggest that TNF may utilize the sphingomyelin pathway in its effect on the insulin-stimulated tyrosine phosphorylation of IRS-1.

Cytokines and growth factors modulate cell growth and insulin-like growth factor binding protein secretion by the human salivary cell line (HSG).

The human salivary cell line (HSG) was investigated for the effect of various growth factors and cytokines on cellular proliferation and on the production of insulin-like growth factor binding proteins (IGFBPs). IGF-I increased cell growth by approximately 25%, and induced the appearances of three distinct protein bands on ligand blot of the cell culture. Two bands with molecular weights of 43 and 45 Kda, respectively, proved to be IGFBP-3 using a specific antibody, and the third was a 24 Kda species (probably, IGFBP-4). Similar IGFBPs were released by the cells following stimulation by EGF and insulin as well as following incubation with the IGF-I receptor antibody alpha IR3. Retinoic acid had an inhibitory effect (50%) on IGF-I-induced cellular proliferation and an attenuative effect on the 24 Kda band when it was combined with IGF-I, and to a lesser effect EGF; however, it enhanced IGFBP-3 production when incubated with IGF-I. The IGF-I receptor antibody had an agonistic effect on IGFBPs production when applied alone or together with IGF-I. TNF-alpha and INF-gamma had minimal effects on cell growth when added alone but when applied in combination, a marked inhibition of cellular proliferation was noted. These cytokines caused increased accumulation of IGFBP-3, -4, and -5. Addition of IGF-I to these cytokines enhanced the expression of these bands. These data demonstrate that growth factors and cytokines which modulate HSG cell growth, induce specific IGFBPs which may play a role in their effects on cell growth.