Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy.

Podocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2 deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2 deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitro and in vivo and exacerbated by tnfaip2 deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2 deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2 overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target.Abbreviations: AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type.

Loss of nephrin expression in glomeruli of kidney-transplanted patients under m-TOR inhibitor therapy.

The development of proteinuria has been observed in kidney-transplanted patients on m-TOR inhibitor (m-TORi) treatment. Recent studies suggest that m-TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney-transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit-diaphragm. In a group of patients on 'de novo' m-TORi-treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m-TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m-TORi-treatment, a protocol biopsy performed before introduction of m-TORi was also available. The expression of nephrin in the pre-m-TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m-TORi(s). Proteinuria increased after the m-TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m-TORi(s) may affect nephrin expression in kidney-transplanted patients, consistently with the observation in vitro on cultured podocytes.

Normal age-dependent values of serum insulin growth factor-I: results from a healthy Italian population.

Serum IGF-I levels were measured in 547 non-hypopituitaric, non-acromegalic healthy subjects of both sexes in Italy to develop reference values in relation to age and sex. Participant subjects were stratified in three age classes (25- 39, 40-59 and >or=60 yr) and IGF-I assay was carried out by double-antibody radio immunoassay. Pearson's correlation coefficient between age and IGF-I values was calculated by sex and predefined age ranges. IGF-I levels significantly decreased with age (p<0.001, Kruskal-Wallis test) while sex was not a significant factor. The median IGF-I levels were 206 ng/ml in the 25-39 yr range, 147 ng/ml in the 40-59 yr range and 103 ng/ml in the >or=60 yr range. Pearson's correlation coefficient confirmed the negative correlation between age and IGF-I levels in the total sample of subjects (r=-0.529). The r coefficient between age and IGF-I levels did not differ between sexes (r=-0.570 in males and r=-0.529 in females), thus reflecting no sex-effect on IGF-I levels decline over years. No correlations were found in the 25-39 yr range (r=-0.036) or in the 40-59 yr range (r=-0.080) either, while in subjects aged >60 yr, IGF-I levels tended to further decrease with increased age (r=0.389). Ranges of normal values set at the 2.5th-97.5th percentile in the three age ranges were 95.6-366.7 ng/ml between 25 and 39 yr, 60.8-297.7 ng/ml between 40 and 59 yr and 34.5-219.8 ng/ml in subjects aged >or=60 yr. This study may contribute to the development of age-specific reference ranges for IGF-I determination in serum of normal subjects of both sexes in Italy.

Effects of recombinant human growth hormone on muscle protein turnover in malnourished hemodialysis patients.

To assess the effect of recombinant human growth hormone (rhGH) on muscle protein metabolism in uremic patients with malnutrition, forearm [3H]phenylalanine kinetics were evaluated in six chronically wasted (body weight 79% of ideal weight) hemodialysis (HD) patients in a self-controlled, crossover study. Forearm protein dynamics were evaluated before, after a 6-wk course of rhGH (5 mg thrice weekly) and after a 6-wk washout period. After rhGH: (a) forearm phenylalanine net balance--the difference between phenylalanine incorporation into and phenylalanine release from muscle proteins--decreased by 46% (-8+/-2 vs. -15+/-2 nmol/min x 100 ml at the baseline and -11+/-2 after washout, P < 0.02); (b) phenylalanine rate of disposal, an index of protein synthesis, increased by 25% (25+/-5 vs. 20+/-5 at the baseline and 20+/-4 after washout, P < 0.03); (c) phenylalanine rate of appearance, an index of protein degradation, was unchanged (33+/-5 vs. 35+/-5 at the baseline and 31+/-4 after washout); (d) forearm potassium release declined (0.24+/-0.13 vs. 0.60+/-0.15 microeq/min at the baseline, and 0.42+/-0.20 microeq/min after washout P < 0.03); (e) changes in the insulin-like growth factor binding protein (IGFBP)-1 levels and insulin-like growth factor-I (IGF-I)/IGFBP-3 ratios accounted for 15.1% and 47.1% of the percent variations in forearm net phenylalanine balance, respectively. Together, these two factors accounted for 62.2% of variations in forearm net phenylalanine balance during and after rhGH administration. These data indicate: (a) that rhGH administration in malnourished hemodialysis patients is followed by an increase in muscle protein synthesis and by a decrease in the negative muscle protein balance observed in the postabsorptive state; and (b) that the reduction in net protein catabolism obtained with rhGH can be accounted for by the associated changes in circulating free, but not total, IGF-I levels.

Biologic activity of tamoxifen at low doses in healthy women.

BACKGROUND: Results of a clinical trial recently completed in the United States indicate that administration of tamoxifen (20 mg/day) to women at risk can reduce breast cancer incidence by approximately 50% but is associated with an increased risk of developing endometrial cancer and venous thromboembolic events. Since these adverse effects may be dose related, we investigated the effect of tamoxifen on several biomarkers when the drug was given at doses lower than those currently in use. METHODS: In two sequential experiments, 127 healthy hysterectomized women aged 35-70 years were randomly assigned to one of the following four treatment arms: placebo (n = 31) or tamoxifen at 20 mg/day (n = 30) (first experiment); or tamoxifen at 10 mg/day (n = 34) or tamoxifen at 10 mg/ alternate days (n = 32) (second experiment). Baseline and 2-month measurements of the following parameters were compared: 1) total cholesterol (primary end point) and other surrogate markers of cardiovascular disease, e.g., low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, and lipoprotein(a); 2) blood cell count; 3) fibrinogen; 4) antithrombin III; 5) osteocalcin; and, 6) in a subgroup of 103 women, insulin-like growth factor-I (IGF-I), a possible surrogate marker for breast cancer. RESULTS: After adjustment for the baseline values, there were reductions in circulating levels of total cholesterol and IGF-I of the same magnitude in all three tamoxifen treatment arms. A similar pattern was observed for most of the other parameters. In the placebo arm, fibrinogen level, which showed a decrease, was the only parameter exhibiting change. CONCLUSIONS: Up to a 75% reduction in the conventional dose of tamoxifen (i.e., 20 mg/day) does not affect the activity of the drug on a large number of biomarkers, most of which are surrogate markers of cardiovascular disease. This study was hypothesis generating, and larger studies are warranted to assess the efficacy of tamoxifen at low doses.

Evidence for autocrine mitogenic stimulation by somatomedin-C/insulin-like growth factor I on an established human lung cancer cell line.

The production of immunoreactive somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) by the established cell line derived from a human lung carcinoma CALU-6 has been evidenced in the serum-free medium in increasing concentrations as a function of the incubation time. Gel filtration in acid conditions of cell-conditioned medium collected after 72 h showed peaks of immunoreactive Sm-C/IGF-I in the elution volume corresponding to the molecular weight of the synthetic Sm-C/IGF-I, and in the high molecular weight region, where specific binding sites for Sm-C/IGF-I could be also demonstrated. These results indicate that this established cell line produces high amounts of immunoreactive Sm-C/IGF-I and of Sm-C/IGF-I carrier protein. The pooled fractions corresponding to the molecular weight of synthetic Sm-C/IGF-I showed a competitive binding curve parallel to the standard in the Sm-C/IGF-I RIA system, and a mitogenic activity on cells from the same line similar to the one observed using two different pure Sm-C/IGF-I preparations, obtained by chemical synthesis or by DNA recombinant technology. When a monoclonal antibody (sm-1.2) raised against Sm-C/IGF-I was added into the medium, the mitogenic effect observed by both synthetic and cell-derived Sm-C/IGF-I peptide was completely abolished; the monoclonal antibody also partially inhibited the effect of 10% fetal calf serum and the thymidine incorporation observed in serum-free medium without growth factors. In serum-free medium the monoclonal antibody produced a 45% reduction of cells in S phase by thymidine labeling index without modification of the growth fraction as determined by primer-dependent alpha-DNA polymerase labeling index. In conclusion it seems that Sm-C/IGF-I has a critical role in the autocrine stimulation of the replication of this cell line.

Evidence for an increased somatomedin-C/insulin-like growth factor I content in primary human lung tumors.

Immunoreactive somatomedin-C/insulin-like growth factor I (SM-C/IGF I) content was measured in human neoplastic lung tissue obtained from surgery on 10 patients (seven epidermoid carcinoma, three adenocarcinoma), and in normal lung tissue obtained from the same excised portion. SM-C/IGF I content in lung tumors was 615 +/- 123 (SE) milliunits/g of tissue (range, 214-1531), significantly higher (P less than 0.01) than normal tissue (234 +/- 51 milliunits/g of tissue; range, 37-537); in particular, every subject showed a clear-cut difference of SM-C/IGF I content between neoplastic and normal tissue (ratio, 3.41 +/- 0.69; range, 1.4-7.2). The results were essentially unchanged when data were expressed relative to hemoglobin or DNA tissue content. By contrast, in peripheral plasma SM-C/IGF I concentration was 0.51 +/- 0.17 units/ml, significantly lower (P less than 0.01) than in 59- to 70-yr-old control subjects (1.10 +/- 0.13 units/ml). In conclusion, the lung tumors studied, irrespective of their histological structure, contain more SM-C/IGF I than does normal tissue. Whether this is due to a primary in situ production of SM-C/IGF I or is secondary to overproduction of other inducers such as platelet derived growth factor-like peptides is yet to be clarified. The reduced circulating SM-C/IGF I concentration seems to be related more to the nutritional status of the patients.

Epidermal growth factor in breast cyst fluid: relationship with intracystic cation and androgen conjugate content.

In recent years, several studies focused on the biochemical analysis of breast cyst fluid composition. It has been shown that breast cysts lined by apocrine epithelium contain higher levels of potassium and dehydroepiandrosterone-sulphate as compared to cysts lined by flattened cells, and that women with apocrine cysts are more likely to develop breast cancer. In the present study, we measured the intracystic levels of sodium (Na+), potassium (K+), dehydroepiandrosterone-sulphate (DHEA-S), and epidermal growth factor (EGF), a factor which could play a role in the autocrine or paracrine control of breast cancer cell growth as recently proposed by some investigators. Breast cyst fluids obtained by fine-needle aspiration from 86 women with gross cystic breast disease were assayed. On the basis of the relative intracystic concentrations of Na+ and K+ two main classes of cysts were defined. An arbitrary cut-off value of 3 for the Na+/K+ ratio seemed adequate to separate these two types of cysts. An inverse relationship was found between the Na+/K+ ratio and DHEA-S concentration, median levels of the androgen conjugate being 3615 micrograms/dl in Na+/K+ less than 3 cysts and 480 micrograms/dl in Na+/K+ greater than 3 cysts (P less than 0.001). EGF levels were found to be significantly higher in Na+/K+ less than 3 cysts as compared to Na+/K+ greater than 3 cysts: 103.26 ng/ml versus 57.22 ng/ml, respectively (P less than 0.001). EGF appeared inversely correlated with total protein concentration in the Na+/K+ greater than 3 cysts, while in the Na+/K+ less than 3 cysts high EGF levels were observed independently of total protein content. In addition, a direct correlation was found between EGF and DHEA-S concentrations. On the basis of these results, the hypothesis can be made that EGF, which is measurable in all breast fluids tested and is nearly undetectable in plasma, is actually produced by the epithelium lining the cyst wall, particularly as far as the Na+/K+ less than 3 cysts are concerned. In view of our results this type of cyst, which has been shown to be lined by apocrine epithelium, appears to be characterized by high DHEA-S and EGF levels. It is suggested that the latter finding could provide a clue for understanding the increased risk of subsequent breast cancer in women bearing apocrine cysts.

Effect of blood tamoxifen concentrations on surrogate biomarkers in a trial of dose reduction in healthy women.

PURPOSE: Tamoxifen administered at 20 mg/d has been shown to decrease breast cancer incidence in at-risk women by 50%, but toxicity may limit its broad use, particularly in postmenopausal women. Because toxicity may be dose-dependent, we studied the biologic activity of low concentrations of tamoxifen to determine the plausibility of a dose reduction. PATIENTS AND METHODS: We measured the blood concentrations of tamoxifen and its main metabolites in a dose titration study in 105 healthy women (placebo, tamoxifen 10 mg on alternate days, tamoxifen 10 mg/d, and tamoxifen 20 mg/d). Drug levels measured after 2 months of treatment were correlated with the changes in surrogate biomarkers of different diseases, including lipid profile, blood cell count, fibrinogen, antithrombin III, osteocalcin, and insulin-like growth factor I, a promising surrogate biomarker of breast cancer. RESULTS: The means (+/- SD) for tamoxifen and N-desmethyltamoxifen (metabolite X) concentrations (ng/mL) were dose-related, being, respectively, 0 and 0 with placebo, 26.8 +/- 15.1 and 43.7 +/- 22.5 with 10 mg every other day, 51.2 +/- 24.1 and 90.7 +/- 48.0 with 10 mg/d, and 136.0 +/- 52.7 and 230.6 +/- 75.0 with 20 mg/d of tamoxifen. At variance, the biomarker changes were of comparable magnitude at any drug concentration except for platelet count and triglycerides levels, the latter showing a trend to an increase with increasing tamoxifen concentrations. CONCLUSION: An 80% reduction in blood concentrations does not seem to affect the activity of tamoxifen on biomarkers of cardiovascular or breast cancer risk and may in fact have a more favorable safety profile. Additional studies are warranted to determine the most appropriate dose of this agent.

Reduction of adiposity with prolonged growth hormone treatment in old obese rats: effects on glucose handling and early insulin signaling.

OBJECTIVES: Growth Hormone (GH) promotes loss of body fat and causes insulin resistance. It is debated whether reduction of body fat mass during long term growth hormone (GH) administration improves carbohydrate metabolism. To answer this question we assessed carbohydrate handling and tissue specific function of the insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) during prolonged GH treatment of obese rats. METHODS: Body fat % estimated by DEXA scanning, plasma IGF-I, glucose and insulin were studied in 17 months old dietary induced obese rats treated for 4, 21 or 41 days (GH: 4 mg/kg/d or saline total n=90). Adipose tissue, muscle and liver samples were obtained after 21 days and expression and tyrosine phosphorylation of IR and IRS-1 proteins and the degree of IRS-1-Janus Kinase-2 (JAK2) interaction were analyzed by immunoprecipitation and immunoblotting. RESULTS: Forty-one days GH treatment caused the body fat to decline significantly to 20+/-3% (Mean+/-SEM), whereas it remained steady on 51+/-4% in the pair fed group. Insulin levels in response to OGTT were significantly elevated throughout the experiment. IR amount was elevated in adipose tissue but decreased in liver after GH treatment while IR phosphorylation was increased in muscle only. IRS-1 amount was elevated in adipose tissue and muscle while IRS-1 phosphorylation was increased only in liver. The association of IRS-1 with JAK-2 was increased in liver and muscle. CONCLUSIONS: An extensive reduction of fat mass did not improved signs of insulin resistance in GH treated old obese rats. The molecular events associated with GH treatment included tissue specific changes in the function of IR and IRS-1 suggesting the liver to be the primary site of insulin resistance. Furthermore, the association of IRS-1with JAK-2 in the course of GH signaling could present a mechanism for GH to directly induce insulin resistance.

A novel patient-derived tumorgraft model with TRAF1-ALK anaplastic large-cell lymphoma translocation.

Although anaplastic large-cell lymphomas (ALCL) carrying anaplastic lymphoma kinase (ALK) have a relatively good prognosis, aggressive forms exist. We have identified a novel translocation, causing the fusion of the TRAF1 and ALK genes, in one patient who presented with a leukemic ALK+ ALCL (ALCL-11). To uncover the mechanisms leading to high-grade ALCL, we developed a human patient-derived tumorgraft (hPDT) line. Molecular characterization of primary and PDT cells demonstrated the activation of ALK and nuclear factor kB (NFkB) pathways. Genomic studies of ALCL-11 showed the TP53 loss and the in vivo subclonal expansion of lymphoma cells, lacking PRDM1/Blimp1 and carrying c-MYC gene amplification. The treatment with proteasome inhibitors of TRAF1-ALK cells led to the downregulation of p50/p52 and lymphoma growth inhibition. Moreover, a NFkB gene set classifier stratified ALCL in distinct subsets with different clinical outcome. Although a selective ALK inhibitor (CEP28122) resulted in a significant clinical response of hPDT mice, nevertheless the disease could not be eradicated. These data indicate that the activation of NFkB signaling contributes to the neoplastic phenotype of TRAF1-ALK ALCL. ALCL hPDTs are invaluable tools to validate the role of druggable molecules, predict therapeutic responses and implement patient specific therapies.

Corticotropin-releasing hormone (CRH) inhibits steroid biosynthesis by cultured human granulosa-lutein cells in a CRH and interleukin-1 receptor-mediated fashion.

The presence of immunoreactive CRH was recently demonstrated in human ovaries. CRH immunoreactivity was localized by immunohistochemistry in the cytoplasm of thecal cells surrounding the ovarian follicles, in luteinized cells of the stroma, and in large granulosa-derived luteinized cells of developing corpora lutea. Also, CRH and its receptors were identified in Leydig cells of the testis where CRH was shown to inhibit testosterone biosynthesis. To examine the role of CRH in the ovary, we studied its effect on estradiol (E2) and progesterone (P4) release by human granulosa cells obtained from women undergoing in vitro fertilization for male factor infertility or uni- or bilateral tubal impatency. In all subjects, superovulation was induced by treatment with gonadotropins. The effects of graded doses of ovine CRH (10[-11]-10[-6] mol/liter) were evaluated in the conditioned medium obtained after 24 h incubation of the cells. All CRH concentrations employed except for the lowest one (10[-11] mol/liter) caused a significant decrease of media E2 and P4 levels. Maximal inhibition for both E2 and P4 production was obtained by 10[-6] mol/liter CRH concentration, which decreased hormone production by 39% and 34%, respectively. The alpha-helical CRH9-41 antagonist at 10(-6) and 10(-7) mol/liter blocked the suppressive effect of 10(-9) mol/liter CRH on both E2 and P4 secretion, while it had no effect when added to the culture media without CRH. Since interleukin (IL-1)-1 mediates certain actions of CRH on leukocytes, we examined whether the CRH effect on ovarian steroidogenesis was IL-1-mediated. Interleukin-1 receptor antagonist at 10(-7) and 10(-6) mol/liter blocked the inhibitory effects of CRH on E2 and P4 secretion, while it had no effect in the absence of CRH. In conclusion, CRH exerts a CRH- and IL-1 receptor-mediated inhibitory effect on ovarian steroidogenesis and might be actively involved in the still enigmatic processes of follicular atresia and luteolysis.

Effect of IL-6 on IGF binding protein-3: a study in IL-6 transgenic mice and in patients with systemic juvenile idiopathic arthritis.

Stunted growth is a common complication of childhood diseases characterized by chronic inflammation or infections. We previously demonstrated that NSE/hIL-6 transgenic mice, overexpressing the inflammatory cytokine IL-6 since early phase of life, showed a marked growth defect associated with decreased IGF-I levels, suggesting that IL-6 is one of the factors involved in stunted growth complicating chronic inflammation in childhood. Here we show that NSE/hIL-6 mice have normal liver IGF-I production, decreased levels of IGF binding protein-3 (IGFBP-3) and increased serum IGFBP-3 proteolysis. Reduced IGFBP-3 levels results in a marked decrease in the circulating 150-kDa ternary complex, even in the presence of normally functional acid labile subunit. Pharmacokinetic studies showed that NSE/hIL-6 mice have accelerated IGF-I clearance. Patients with systemic juvenile idiopathic arthritis (s-JIA), a chronic inflammatory disease characterized by prominent IL-6 production and complicated by stunted growth associated with low IGF-I levels, have markedly decreased IGFBP-3 levels, increased serum IGFBP-3 proteolysis and normal acid labile subunit levels. Our data show that chronic overproduction of IL-6 causes decreased IGFBP-3 levels, resulting in a decreased association of IGF-I in the 150-kDa complex. Decreased levels of IGF-I appear to be secondary to increased clearance.

Effect of the acid-labile subunit on the binding of insulin-like growth factor (IGF)-binding protein-3 to [125I]IGF-I.

In normal subjects, the major form of circulating insulin-like growth factor (IGF) is the GH-dependent 150K complex. This complex is formed by the IGF peptide, the acid-stable binding protein IG-FBP-3, and the acid-labile subunit (ALS), which, although not binding IGF, appears to be necessary to reconstitute the complex in its natural form. The ALS was purified in our laboratory from human serum by ammonium sulfate precipitation, ion exchange chromatography using DEAE-Sephadex A-50, Concanavalin-A-Sepharose-4B chromatography, and two sequential gel filtrations by fast performance liquid chromatography. As demonstrated by gel permeation chromatography on fast performance liquid chromatography, incubation for 2 h at 20 C of this preparation with [125I]IGF-I and recombinant IGFBP-3 (rIGFBP-3) allows the reconstitution of a complex of about 150 kilodaltons. In these experimental conditions, the ALS is not only able to increase the mol wt of the complex, but also to greatly increase the amount of IGF-I bound; in the absence of ALS, radioactivity in the mol wt volume of the complexed forms was lower than that in the mol wt volume of the free form (percentage of total [125I]IGF-I: rIGFBP-3 alone, 15% and 44%; in the presence of ALS, 41% and 24%, respectively). In both charcoal and polyethylene glycol ligand binding assays, competitive binding curves for the displacement of [125I]IGF-I from rIGFBP-3 by increasing concentrations of unlabeled IGF-I showed an increased binding activity of rIGFBP-3 in the presence of ALS. The effect of ALS on rIGFBP-3-binding activity was dose dependent. These data show that the non-IGF-binding ALS subunit of the 150-kilodalton complex can play an important role in the regulation of IGF-I or IGF-II binding to rIGFBP-3 and, therefore, on the levels of free IGF peptide, possibly by inducing conformational changes in rIGFBP-3. In addition, ligand and immunoblot reveal that ALS and rIGFBP-3 are able to form a high mol wt complex in the absence of IGF peptide. On the basis of these data, ALS seems to have a more complex function than that of simply increasing the mol wt of the IGF-IGFBP-3 complex.

Immunoreactive insulin-like growth factor I (IGF-I) and IGF-I-binding protein content in human thyroid tissue.

We measured immunoreactive insulin-like growth factor I (IGF-I) in extracts of normal and nodular thyroid tissue obtained at surgery from patients with nontoxic goiter. The nodular tissues contained a higher concentration [mean, 279.0 +/- 69.7 (+/- SE) mU/g] than paired normal tissues (115.5 +/- 17.9 mU/g; P = 0.024; n = 12); a difference was evident in all but one patient. Sephadex G-50 gel filtration of tissue extracts revealed two immunoreactive peaks, the first in the void volume of the column, and the second in the elution volume of authentic IGF-I. The first peak was identified as IGF-I-binding protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after cross-linking with iodinated IGF-I. Isolated thyroid cell membranes contained high affinity IGF-I-binding sites of similar affinity and numbers in both normal and nodular thyroid tissue. The IGF-I content of six thyroid cancer extracts was higher than that of normal thyroid tissue, but the IGF-I content of thyroid tissue from six patients with Graves' disease and five patients with Hashimoto's thyroiditis was similar to that in normal thyroid tissue. These data suggest that the stimulatory effect of TSH on thyroid cell proliferation could be mediated through IGF-I action and suggest that an increase in IGF-I production could sustain the goitrogenic process.

Diagnostic value of the acid-labile subunit in acromegaly: evaluation in comparison with insulin-like growth factor (IGF) I, and IGF-binding protein-1, -2, and -3.

In normal subjects the main form of circulating insulin-like growth factor (IGF) is the 150-kDa complex. This complex is formed by the IGF peptide, the acid-stable IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Experimental and clinical data have demonstrated that ALS is primarily under the control of GH and plays a critical role in maintaining constant levels of circulating IGF-I. In this study we evaluated ALS, IGF-I, and IGFBP-1, -2, and -3 in 45 acromegalic patients in basal conditions and, in 37 of these, twice after surgical therapy compared with 100 age- and sex-matched control subjects to estimate their value as parameter of GH secretory state. The results demonstrated that in acromegaly before treatment all parameters (ALS, 523 +/- 26; IGF-I, 129 +/- 6; IGFBP-1, 0.7 +/- 0.1; IGFBP-3, 234 +/- 21; nmol/L; mean +/- SEM) but IGFBP-2 were significantly different (P<0.0001) from those in healthy subjects (ALS, 281 +/- 4; IGF-I, 22 +/- 1; IGFBP-1, 1.6 +/- 0.1; IGFBP-3, 91 +/- 3). IGF-I was more sensitive (100%) than ALS (89%), and both were more predictive of disease status than IGFBP-3, in that 27% of the patients had IGFBP-3 levels within the normal range. Considering the ALS/IGFBP-3 molar ratio, almost 55% of ALS circulated in a free form in active acromegaly. Before treatment, the IGF-I/IGFBPs (-1 + -2 + -3) molar ratio, which can be regarded as free, biologically active, IGF-I, was greatly increased (0.77 +/- 0.06; P<0.0001) compared with that in control subjects (0.23 +/- 0.01). After surgery, all 10 patients with controlled disease showed normalization of ALS (100% sensitivity), whereas 9 of them had normal IGFBP-3; reevaluation after varying lengths of time showed all these parameters within the normal range. In the 27 patients with active disease, IGF-I and ALS were more predictive of disease status (91% and 83% negative predictive values, respectively) than IGFBP-3 (53%). The basal ALS concentration correlated only with IGFBP-3 (r = 0.70; P<0.001). In postsurgery samples (first control) a statistically significant (P<0.001) correlation was found between mean GH values as well as minimum GH after oral glucose tolerance test and ALS (r = 0.72 and 0.83, respectively), IGF-I (r = 0.69 and 0.77), IGFBP-3 (r = 0.50 and 0.72), and IGFBP-2 (r = -0.36 and -0.63). Similarly, IGF-I, IGFBP-3, and ALS were positively correlated among themselves and negatively correlated with IGFBP-2 (P<0.001). In conclusion, in the diagnosis of acromegaly, the measurement of total IGF-I appears to be the most sensitive parameter among the subunits of the 150K complex, and IGFBP-3 the least sensitive. For ALS, this subunit is quite sensitive and appears to be a useful parameter in reassessment after surgical treatment.

Increased insulin-like growth factor (IGF)-II and IGF/IGF-binding protein ratio in prepubertal constitutionally tall children.

The height of subjects with constitutionally tall stature (CTS) is at least 2 SD above the mean of subjects of the same age and sex. Apart from a few discordant data on the role of GH and its direct mediator, IGF-I, no studies have been conducted on other components of the IGF system, which also condition the bioavailability and activity of IGF-I. We, therefore, investigated the possibility that other components of the IGF system might play a role in determining the increased growth velocity seen in CTS. To this end, we evaluated the behavior not only of IGF-I but also of IGF-II, IGF-binding protein (IGFBP)-3, and acid-labile subunit, the subunits that constitute the main IGF complex in circulation (150-kDa complex), as well as of IGFBP-1 and IGFBP-2, which are negatively regulated by GH and, like IGFBP-3, able to influence the bioavailability of the IGFs. The study was performed on 22 prepubertal subjects affected by CTS (16 males and 6 females), aged 2.8-13.3 yr (6.8 +/- 0.5 yr, mean +/- SEM). Thirty-seven normal prepubertal subjects (16 males and 21 females) aged between 2.2 and 13.3 yr (6.7 +/- 0.5 yr), who were comparable in socioeconomic and nutritional terms, served as controls. From the auxological point of view, subjects with CTS differed significantly from controls only in terms of growth velocity (HV-SD score; CTS, 1.8 +/- 0.3; controls, 0.4 +/- 0.2; P < 0.0001) and height (H-SD score; CTS, 3.1 +/- 0.1; controls, 0.4 +/- 0.2; P < 0.0001). The results demonstrated that the concentrations of IGF-I (27.3 +/- 2.0 nmol/liter), IGFBP-3 (66.9 +/- 3.8), and acid-labile subunit (216.8 +/- 13.6) in CTS-affected subjects were not significantly different from those determined in controls (25.0 +/- 2.9, 74.4 +/- 4.1, and 241.0 +/- 11.9, respectively). By contrast, IGF-II levels proved significantly higher in CTS subjects (IGF-II: 87.2 +/- 3.4 vs. 52.4 +/- 2.3, P < 0.0001). Chromatographic analysis, performed after acid treatment of pooled sera, showed only the presence of normal 7.5-kDa IGF-II in both CTS subjects and controls. In comparison with controls, CTS children showed a lower concentration of IGFBP-1 (1.6 +/- 0.3 vs. 4.1 +/- 0.7, P = 0.03) and a higher concentration of IGFBP-2 (14.3 +/- 1.8 vs. 9.6 +/- 1.1, P = 0.03). The IGFs (IGF-I and -II)/IGFBPs (-1 + -2 + -3) molar ratio was significantly higher (P < 0.0001) in CTS children than in controls. In particular, the IGF-II/IGFBP ratio (P < 0.0001) was responsible for the excess of the IGF peptide in relation to the concentrations of IGFBPs and, therefore, for the increase in the potentially bioactive free form of the IGFs. Moreover, the IGFBP-3/IGF molar ratio was significantly reduced, being less than 1 in CTS subjects (0.6 +/- 0.1 vs. 1.1 +/- 0.1), so that a quantity of IGF peptides lack sufficient IGFBP-3 to form the 150-kDa complex with which are normally sequestered in the vascular compartment. The data show that in CTS: 1) the most GH-dependent components of the IGF system are normal, consistent with the finding of a normal GH secretory state; 2) the less GH-dependent IGF-II is significantly increased, in agreement with the finding of a relationship between high levels of IGF-II and overgrowth in some syndromes; and 3) the IGF/IGFBP molar ratio is increased, and, therefore, a greater availability of free IGF for target tissues may be responsible for overgrowth in CTS.

In vivo and in vitro effect of growth hormone on estradiol secretion by human granulosa cells.

GH therapy increases the ovarian response to gonadotropin stimulation in women presenting with ovaries that are relatively resistant to conventional gonadotropin therapy. As it is not completely certain whether GH modulates the actions of FSH on granulosa cells directly or via insulin-like growth factor-I (IGF-I) production, we studied its effect on steroid release by human granulosa cells obtained from subjects affected by unexplained or male factor infertility. In all subjects, superovulation for in vitro fertilization/embryo transfer was induced by treatment with gonadotropins or GH plus gonadotropins combined. The effects of the different in vivo treatments were evaluated in the conditioned medium obtained after the first 24 h of incubation; granulosa cells from patients treated with GH released higher amounts of estradiol and progesterone into the medium than did granulosa cells from patients treated with gonadotropins alone. When the release of steroid due to the in vivo treatment was exhausted, cells were subjected to increasing concentrations of GH in the presence or absence of 200 nmol anti-IGF Sm 1.2 monoclonal antibody (MoAb) or the antitype I receptor alpha IR3 MoAb. The results revealed that GH stimulates estradiol production in a dose-dependent fashion, and the presence of the MoAbs drastically reduces the GH effect. These data demonstrate that the established stimulatory effect of GH on ovarian function is dependent not only on the increased levels of circulating IGF-I, but also on a direct effect of GH on granulosa cells, which seems to be mediated at least in part by the autocrine action of IGF, particularly IGF-II. In fact, chromatographic analysis of medium conditioned by human granulosa cells revealed that these cells clearly produce IGF-II and IGF-binding proteins and only small amounts of IGF-I. Since GH appears to be able to increase the in vitro effect of both IGF-I and IGF-II, we can hypothesize a sensitization of the granulosa cells to the IGF-II produced by the cells themselves, which acts through the IGF-I receptor.

Insulin-like growth factors (IGF-I and IGF-II) and IGF-binding protein-3 production by fibroblasts of patients with Turner's syndrome in culture.

Reports indicate that in plasma insulin-like growth factor I (IGF-I) and IGF-binding protein-3 (IGFBP-3) are normal in patients with Turner's syndrome (TS). The aim of our study was to evaluate both the spontaneous and the stimulated synthesis of these peptides by mesenchymal cells obtained from skin biopsies of patients affected with TS. We compared the ability of fibroblasts from six TS patients with that of fibroblasts from six age-matched control (C) subjects to synthesize in vitro IGF-I, IGF-II, and IGFBP-3 under basal and GH-, estradiol (E2)-, or GH- plus E2-stimulated conditions. Furthermore, we evaluated IGF-I, IGF-II, and IGFBP-3 messenger ribonucleic acid (mRNA) expression in fibroblasts from TS and C subjects. Fibroblasts obtained from TS patients release into the medium significantly lower amounts of IGF-I and IGF-II than C fibroblasts (P = 0.0435 and 0.0318, respectively). In TS fibroblasts, GH and E2 are able to induce a similar increase, although not significant, of IGF-I secretion into the medium (163 +/- 75% and 112 +/- 41% of control values). On the contrary, in C fibroblasts, GH is more effective (275 +/- 61%; P = 0.0277) than E2 (75 +/- 46%). In both cell lines, GH and E2 do not significantly modify IGF-II release. Interestingly, the medium conditioned by fibroblasts from TS contains, under basal conditions, significantly higher amounts (273 +/- 79 ng/1 x 10(6) cells) of IGFBP-3 than that from control fibroblasts (67 +/- 19 ng/1 x 10(6) cells; P = 0.0191). GH exerts a stimulatory effect, although it is not statistically significant, on IGFBP-3 secretion, particularly in control fibroblasts. By contrast, the effect of E2 is inhibitory in all TS fibroblast cell lines, although it does not reach statistical significance (P = 0.067). In agreement with these data, a reduced mRNA expression of the genes encoding for IGF peptides was evident in TS fibroblasts, whereas no significant difference could be demonstrated for IGFBP-3 mRNA. The results suggest a reduced autocrine/paracrine action of IGFs in TS and indicate that skin fibroblast cultures can give information on the local responsiveness to the treatment.

Short stature associated with high circulating insulin-like growth factor (IGF)-binding protein-1 and low circulating IGF-II: effect of growth hormone therapy.

We report a case of short stature associated with high circulating levels of insulin-like growth factor (IGF)-binding protein-1 (IGFBP-10 and low levels of IGF-II responsive to pharmacological treatment with GH. Our patient suffered severe growth failure from birth (2.06 SD below the mean for normal full-term boys, and 5.2 and 7.3 SD below the mean at 5 and 10 months). Studies carried out before referral to our pediatric unit included normal 46,XY karyotype and normal encephalic imaging. Other endocrine and metabolic alterations and other systemic diseases were excluded. At 1.7 yr of age (length, 6.1 SD; weight, 4.6 SD; head circumference, 1.4 SD below the mean, respectively) the patient was referred to our pediatric unit. The baseline GH concentration was 31 microg/L, and the peak after an arginine load was 59.6 microg/L. In the same samples GH bioactivity was nearly superimposable (RIA/Nb2 bioactivity ratio = 0.9). Fasting insulin and glucose concentrations were 7.4 microU/mL and 65 mg/dL, respectively, both normally responsive to an oral glucose load. GH insensitivity was excluded by a basal IGF-I concentration (64 ng/mL) in the normal range for 0- to 5-yr-old boys and its increase after 2 IU/day hGH administration for 4 days. IGFBP-3 (0.5 microg/mL) was slightly reduced, whereas IGFBP-1 (2218 and 1515 ng/mL in two different basal samples) was well above the normal values for age and was suppressible by GH (maximum suppression, -77% at 84 h) and glucose load (maximum suppression, -46% at 150 min). The basal IGF-II concentration was below the normal range (86 ng/mL), whereas IGFBP-2 was normal (258 ng/mL). Analysis of the promoter region of IGFBP-1 and IGF-II failed to find major alterations. Neutral gel filtration of serum showed that almost all IGF-I activity was in the 35- to 45-kDa complex, coincident with IGFBP-1 peak, while the 150-kDa complex was absent, although the acid-labile subunit was normally represented. At 2.86 yr (height, 65.8 cm; height SD score, -7.3; height velocity SD score, -5) the patient underwent treatment with 7 IU/week human GH; after 4 months, the patient's height was 68.5 cm (height SD score, -6.9) corresponding to a growth velocity of 8.3 cm/yr (0.3 height velocity SD score). IGFBP-1 was reduced (216 ng/mL), although still in the high range, whereas IGF-I (71 ng/mL), IGFBP-3 (0.62 microg/mL), and IGF-II (111 ng/mL) were only slightly increased. The IGF-I profile showed activity in the 150-kDa region. In conclusion, we speculate that the increased IGFBP-1 values found in this patient produce 1) inhibition of IGF-I biological activity and, therefore, a resistance to IGF-I not due to a receptor defect for this hormone; 2) inhibition of formation of the circulating 150-kDa ternary complex and, therefore, an accelerated clearance rate of IGF peptides; 3) inhibition of the feedback action on GH, leading to increased GH levels, which could suggest the diagnosis of GH insensitivity syndrome; and 4) inhibition of body growth.