Insulin-like growth factor-II is produced by, signals to and is an important survival factor for the mature podocyte in man and mouse.

Podocytes are crucial for preventing the passage of albumin into the urine and, when lost, are associated with the development of albuminuria, renal failure and cardiovascular disease. Podocytes have limited capacity to regenerate, therefore pro-survival mechanisms are critically important. Insulin-like growth factor-II (IGF-II) is a potent survival and growth factor; however, its major function is thought to be in prenatal development, when circulating levels are high. IGF-II has only previously been reported to continue to be expressed in discrete regions of the brain into adulthood in rodents, with systemic levels being undetectable. Using conditionally immortalized human and ex vivo adult mouse cells of the glomerulus, we demonstrated the podocyte to be the major glomerular source and target of IGF-II; it signals to this cell via the IGF-I receptor via the PI3 kinase and MAPK pathways. Functionally, a reduction in IGF signalling causes podocyte cell death in vitro and glomerular disease in vivo in an aged IGF-II transgenic mouse that produces approximately 60% of IGF-II due to a lack of the P2 promoter of this gene. Collectively, this work reveals the fundamental importance of IGF-II in the mature podocyte for glomerular health across mammalian species.

Role of the IGF axis in prostate cancer.

The major issue currently being faced in the management of prostate cancer is the inability to distinguish between indolent prostate tumors that will not present clinically from more aggressive and metastatic prostate cancers that will impact on men's lives. Only a small proportion of prostate cancers can be accounted for by unmistakable hereditary cancer syndromes and the predominant contribution to the progression of most sporadic cancers is thought to be environmental, with nutrition having the greatest influence. Population studies have clearly implicated metabolic factors as contributors to disease progression and poor response to therapy. It is well established that the IGF system is key in regulating growth and metabolism and mediates the effects of nutrition on these processes. It consists of two ligands (IGF-I and IGF-II), two receptors [type 1 IGF-IR and IGF-II/mannose 6-phosphate receptor], and six high affinity IGF-binding proteins (IGFBP-1 to -6). This review provides evidence from in vitro, in vivo, clinical and epidemiology studies that indicates an important role for the IGF axis in the development of prostate cancer and the likely role that it plays in mediating the effects of nutrition on disease progression. We suggest that the IGF axis is central to understanding how lifestyle impacts on prostate cancer and we highlight this by describing numerous strategies being developed to target this axis.

Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel.

BACKGROUND: The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments. AIM: The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel. METHODS: DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT-PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting. RESULTS: The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with ß-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel. CONCLUSION: The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel.

IGF-II and IGFBP-2 differentially regulate PTEN in human breast cancer cells.

The dual-function phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is the second most frequently mutated gene in human cancers. PTEN counteracts the functions of many growth factors, the most prevalent of which is insulin-like growth factor II (IGF-II). PTEN expression is stimulated by IGF-II forming a feedback loop. Investigating IGF-binding protein (IGFBP) modulation of IGF-II actions on MCF-7 breast cancer cells, we found that IGFBP-2 also regulates PTEN. The MCF-7 cells were not responsive to high doses of IGF-II due to induction of PTEN, which was not observed with an IGF-II-analog that does not bind to IGFBPs or in the presence of an inhibitor that prevents IGFs associating with IGFBPs. These cells predominantly produce IGFBP-2: blocking IGFBP-2 with a specific antibody, or preventing IGFBP-2 binding to integrins, restored the induction of PTEN and the cells were non-responsive to high doses of the IGF-II-analog. Our findings indicate that breast cancer cells do not respond to high doses of IGF-II due to induction of PTEN, but IGFBP-2, when free from IGF-II can suppress PTEN. Levels of IGFBP-2 are elevated frequently in human tumors: its ability to regulate PTEN could have important implications in relation to therapeutic strategies targeting growth factor pathways.

Inhibition of FASN and ERα signalling during hyperglycaemia-induced matrix-specific EMT promotes breast cancer cell invasion via a caveolin-1-dependent mechanism.

Since disturbed metabolic conditions such as obesity and diabetes can be critical determinants of breast cancer progression and therapeutic failure, we aimed to determine the mechanism responsible for their pro-oncogenic effects. Using non-invasive, epithelial-like ERα-positive MCF-7 and T47D human breast cancer cells we found that hyperglycaemia induced epithelial to mesenchymal transition (EMT), a key programme responsible for the development of metastatic disease. This was demonstrated by loss of the epithelial marker E-cadherin together with increases in mesenchymal markers such as vimentin, fibronectin and the transcription factor SLUG, together with an enhancement of cell growth and invasion. These phenotypic changes were only observed with cells grown on fibronectin and not with those plated on collagen. Analyzing metabolic parameters, we found that hyperglycaemia-induced, matrix-specific EMT promoted the Warburg effect by upregulating glucose uptake, lactate release and specific glycolytic enzymes and transporters. We showed that silencing of fatty acid synthase (FASN) and the downstream ERα, which we showed previously to mediate hyperglycaemia-induced chemoresistance in these cells, resulted in suppression of cell growth: however, this also resulted in a dramatic enhancement of cell invasion and SLUG mRNA levels via a novel caveolin-1-dependent mechanism.

Childhood diet and insulin-like growth factors in adulthood: 65-year follow-up of the Boyd Orr Cohort.

OBJECTIVE: High levels of insulin-like growth factor-I (IGF-I) are associated with an increased cancer risk and reduce risk of diabetes and coronary heart disease. We investigated associations of diet in childhood, in particular energy intake, with the IGF system in adulthood to determine if IGF-I - disease associations could be linked to early nutrition. DESIGN: Retrospective cohort study. SETTING: Sixteen survey centres in England and Scotland that originally participated in the Carnegie (Boyd Orr) Survey of Diet and Health in Pre-War Britain, 1937-1939. SUBJECTS: Seven hundred and twenty-eight participants (679 with complete data) in the Boyd Orr cohort. METHODS: Participants were originally surveyed between 1937 and 1939 (at median age 5.8 years; inter-quartile range: 2.9-9.6) and were followed up for 65 years. Dietary exposure in childhood was assessed from 7-day household food inventories. Outcomes are expressed as regression coefficients for the change in IGF per standard deviation increased childhood nutrient or food intake, as derived from levels of household consumption. RESULTS: In fully-adjusted models, energy-rich family diets in childhood were not associated with IGF-I (regression coefficient: 0.9 ng/ml; 95% confidence interval (CI): -1.8, 3.7), IGF-II, IGF binding proteins (IGFBP)-2 or IGFBP-3 in adulthood. IGF-I was associated inversely with childhood family-diets high in milk (-2.5 ng/ml; -5.1, 0.1; P=0.05) and positively with vegetable-rich diets (3.5 ng/ml; 0.9, 6.1; P=0.009). IGF-I was not associated with family diets rich in protein, carbohydrates, fats, calcium, meat or fruit. IGF-II, IGFBP-2 and IGFBP-3 were not related to childhood family diet. CONCLUSIONS: This study suggests that energy-rich family diets in childhood do not program the IGF system in adulthood. As childhood diet was based on household consumption, however, measurement error may obscure individual-level diet-IGF associations. The associations of milk- and vegetable-rich family diets in childhood with IGF-I could be chance findings, but nevertheless are consistent with recent publications and warrant further investigation.

Hyperglycaemia-induced resistance to Docetaxel is negated by metformin: a role for IGFBP-2.

The incidence of many common cancers varies between different populations and appears to be affected by a Western lifestyle. Highly proliferative malignant cells require sufficient levels of nutrients for their anabolic activity. Therefore, targeting genes and pathways involved in metabolic pathways could yield future therapeutics. A common pathway implicated in energetic and nutritional requirements of a cell is the LKB1/AMPK pathway. Metformin is a widely studied anti-diabetic drug, which improves glycaemia in patients with type 2 diabetes by targeting this pathway. We investigated the effect of metformin on prostate cancer cell lines and evaluated its mechanism of action using DU145, LNCaP, PC3 and VCaP prostate cancer cell lines. Trypan blue dye-exclusion assay was used to assess levels of cell death. Western immunoblotting was used to determine the abundance of proteins. Insulin-like growth factor-binding protein-2 (IGFBP-2) and AMPK genes were silenced using siRNA. Effects on cell morphology were visualised using microscopy. IGFBP-2 gene expression was assessed using real-time RT-PCR. With DU145 and LNCaP cells metformin alone induced cell death, but this was reduced in hyperglycaemic conditions. Hyperglycaemia also reduced the sensitivity to Docetaxel, but this was countered by co-treatment with metformin. LKB1 was required for the activation of AMPK but was not essential to mediate the induction of cell death. An alternative pathway by which metformin exerted its action was through downregulation of IGFBP-2 in DU145 and LNCaP cells, independently of AMPK. This finding could have important implications in relation to therapeutic strategies in prostate cancer patients presenting with diabetes.

Insulin-like growth factor binding protein 3 has opposing actions on malignant and nonmalignant breast epithelial cells that are each reversible and dependent upon cholesterol-stabilized integrin receptor complexes.

IGF-binding protein (IGFBP)-3 is generally considered to have actions that counterbalance those of IGFs and is therefore being developed as a cancer treatment. In breast tumors, however, high levels are associated with aggressive tumors and poor prognosis. Consistent with this we have demonstrated that although IGFBP-3 and a non-IGF-binding fragment (serine phosphorylation domain peptide) reduced attachment and enhanced apoptosis of Hs578T breast cancer cells cultured on collagen or laminin, it promoted their attachment and survival on fibronectin, which is abundant in the matrix of aggressive tumors. We have now examined the factors that determine whether IGFBP-3 has positive or negative actions on breast epithelial cells. IGFBP-3 also promoted survival of Hs578T cells in the presence of an antibody to the beta1-integrin subunit or when cholesterol-stabilized complexes were disrupted. These actions were blocked by IGF-I or a MAPK inhibitor. Serine phosphorylation domain peptide had similar actions on MCF-7 cells that were again reversed on fibronectin or with disruption of cholesterol-stabilized complexes and blocked by the beta1-integrin antibody. In contrast, IGFBP-3 promoted growth and survival for nonmalignant MCF-10A cells, but these effects were again reversed on fibronectin and blocked by the beta1 antibody or a MAPK inhibitor or by disruption of cholesterol-stabilized complexes. On Hs578T cells, IGFBP-3 bound to caveolin-1 and beta1-integrins, enhancing their aggregation, the recruitment of focal adhesion kinase, and the activation of MAPK. In summary, with three breast epithelial cell lines, IGFBP-3 had positive or negative effects on growth and survival dependent upon the status of cholesterol-stabilized integrin receptor complexes.

Increased p53-dependent apoptosis by the insulin-like growth factor binding protein IGFBP-3 in human colonic adenoma-derived cells.

We investigated the expression of insulin-like growth factor binding protein 3 (IGFBP-3) in normal human colonic epithelium and whether IGFBP-3 is involved in the induction of apoptosis in colonic epithelial cells. A gradient of IGFBP-3 protein expression was observed within the normal colonic crypt, and increased IGFBP-3 expression was coincident with the region of increased differentiation and apoptosis. Treatment of human colonic tumor cell lines with IGFBP-3 alone was shown to have no effect on growth. However, an increase in p53-dependent apoptosis was observed in the presence of 100 ng/ml IGFBP-3 24 h after the induction of DNA damage by gamma-irradiation. These results suggest that IGFBP-3 enhances the p53-dependent apoptotic response of colorectal cells to DNA damage.

Hyperglycaemia-induced chemoresistance in breast cancer cells: role of the estrogen receptor.

Breast cancer patients with diabetes respond less well to chemotherapy; in keeping with this we determined previously that hyperglycaemia-induced chemoresistance in estrogen receptor (ERα) positive breast cancer cells and showed that this was mediated by fatty acid synthase (FASN). More recent evidence suggests that the effect of metabolic syndrome and diabetes is not the same for all subtypes of breast cancer with inferior disease-free survival and worse overall survival only found in women with ERα positive breast cancer and not for other subtypes. Here we examined the impact of hyperglycaemia on ERα negative breast cancer cells and further investigated the mechanism underlying chemoresistance in ERα with a view to identifying strategies to alleviate hyperglycaemia-induced chemoresistance. We found that hyperglycaemia-induced chemoresistance was only observed in ERα breast cancer cells and was dependent upon the expression of ERα as chemoresistance was negated when the ERα was silenced. Hyperglycaemia-induced an increase in activation and nuclear localisation of the ERα that was downstream of FASN and dependent on the activation of MAPK. We found that fulvestrant successfully negated the hyperglycaemia-induced chemoresistance, whereas tamoxifen had no effect. In summary our data suggests that the ERα may be a predictive marker of poor response to chemotherapy in breast cancer patients with diabetes. It further indicates that anti-estrogens could be an effective adjuvant to chemotherapy in such patients and indicates the importance for the personalised management of breast cancer patients with diabetes highlighting the need for clinical trials of tailored chemotherapy for diabetic patients diagnosed with ERα positive breast cancers.

Still more questions than answers: report on the 5th International Symposium on Insulin-like Growth Factors, Brighton, UK, 31 October-4 November 1999.

The diverse backgrounds of the 550 scientists attending the 5th International Symposium on IGFs reflected the increasingly multidisciplinary approach to the field of insulin-like growth factor research. The presentations covered every aspect of the regulation, structure, physiology and pathology of not only IGF-I and IGF-II, but also their cell receptors and intracellular-signalling components, their specific-binding proteins (IGFBPs), as well as factors that bind to or modify these IGFBPs.

Altered insulin-like growth factor-II (IGF-II) level and IGF-binding protein-3 (IGFBP-3) protease activity in interstitial fluid taken from the skin lesion of psoriasis.

In the present study, we have investigated insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in serum and artificially raised blister fluid from uninvolved and involved areas of nine patients with psoriasis. Both levels of IGFs and IGFBP-3, and profiles of IGFBP in serum and fluid from the uninvolved areas of these patients were comparable to those seen in normal subjects. In fluid from the involved areas, the IGF-II but not IGF-I level was significantly elevated. Among five molecular forms of IGFBP, the density of 41.5- and 38.5-kDa forms of IGFBP-3 were apparently increased in fluid from the involved areas, shown by Western ligand blotting. Radioimmunoassay further showed that the IGFBP-3 concentration in the involved areas was significantly raised. Immunoblotting revealed that the predominant form of IGFBP-3 in fluid from the uninvolved areas was a 29-kDa proteolytically modified product. In contrast, intact doublet IGFBP-3 was the main form of IGFBP-3 in fluid from the involved areas. Fluid from the involved areas but not the matched serum concentration-dependently inhibited the degradation of 125I-labeled nonglycosylated IGFBP-3 (ngIGFBP-3) caused by fluid from the uninvolved areas, suggesting the presence of an IGFBP-3 protease inhibitor(s) in psoriatic skin lesion. These findings suggest that the alterations in IGF/IGFBP system may contribute to the pathogenesis of psoriasis.

Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity.

Although expression of the IGF-II has been demonstrated within the central nervous system (CNS), past studies have failed to reveal its precise roles or responses subsequent to a traumatic injury. To demonstrate that IGF-II, IGFBP, and IGF receptor (-R) expression alters in response to a penetrating CNS injury, we used the techniques of ribonuclease protection assay, in situ hybridization, immunohistochemistry, Western blotting, and RIA. Under normal physiology, IGF-II expression is restricted to the mesenchymal support structures of the brain, including the choroid plexus, where its expression is coincident with that of IGFBP-2. Between 1-7 days post lesion (dpl), in the acute phase following a penetrant wound to the CNS, IGF-II and IGF-IIR protein, but not messenger RNA, were colocalized, with IGF-I, IGF-IR, and IGFBP-1, -2, -3, and -6, to neurons, macrophages, astrocytes, and microglia within the damaged tissue. Within the cerebrospinal fluid (CSF), levels of IGF-II peptide increased to peak at 7 dpl. IGFBP-2, -3, and -6 were also observed within the CSF, with IGFBP-2 predominating and exhibiting an increase in binding efficiency from 7-10 dpl. In the chronic phase of injury (7-14 dpl), an increase in both IGF-II, IGF-IIR and IGFBP-5 messenger RNA and protein was observed specifically and focally in the marginal astrocytes forming the limiting glial membrane of the wound. Thus, our evidence suggests that there are two mechanisms of action for IGF-II within the injured rat brain. During the acute phase, the secretion of IGF-II from the choroid plexus into the CSF is up-regulated, resulting in increased transport of the peptide to the wound. In the CSF, transported IGF-II is complexed to IGFBP-2 and essentially demonstrates an endocrine mode of action with a balance of locally produced IGFBPs modulating its bioactivity in the wound. Later in the wounding response, levels of IGF-II decline in the CSF and the wound neuropil, possibly with the aid of increased IGFBP-5 levels that may help to locally sequester and down-regulate IGF-II activity. Hence, in the chronic phase of the injury response, IGF-II reasserts itself to a predominantly autocrine/paracrine role restricted to the mesenchymal support structures, including the glia limitans, which may help reestablish and maintain tissue homeostasis.

Acetylcholine and norepinephrine stimulate the release of corticotropin-releasing factor-41 from the rat hypothalamus in vitro.

The effects of the two putative neurotransmitters acetylcholine and norepinephrine on immunoreactive CRF-41 release from incubated rat hypothalami were studied. Acetylcholine at concentrations of 10(-11) to 10(-7) M stimulated CRF-41 release. This effect was blocked in a dose-dependent manner by the muscarinic antagonist atropine (10(-9) to (-7) M). The nicotinic antagonist hexamethonium was ineffective at a dose of 10(-7) M, but produced slight inhibition of this response at 10(-5) M. Norepinephrine at concentrations of 10(-10) to 10(-6) M also produced a dose-dependent stimulation of CRF-41 release. The beta-adrenoceptor antagonists propranolol (10(-5) M) and timolol (10(-6) M) blocked norepinephrine-induced CRF-41 release. The alpha 1-adrenoceptor antagonists thymoxamine (10(-5) M), prazosin (10(-5) M), and corynanthine (10(-4) M), and the alpha 2-antagonist idazoxan (10(-5) M), were ineffective. Potassium depolarization (56 mM) caused stimulation of CRF-41 release which was dependent on the presence of calcium in the incubation medium. Authenticity of immunoreactive CRF-41 released was demonstrated by chromatographic criteria using gel filtration and reversed phase HPLC. These results provide evidence for a stimulatory role of acetylcholine and norepinephrine on CRF-41 release, and consequently on hypothalamo-pituitary-adrenal axis in the rat, through actions at a hypothalamic level. The stimulatory effect of acetylcholine is mediated principally through muscarinic receptors and that of norepinephrine through beta-adrenoceptors.

The role of cell surface attachment and proteolysis in the insulin-like growth factor (IGF)-independent effects of IGF-binding protein-3 on apoptosis in breast epithelial cells.

We have recently reported that insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can significantly increase ceramide-induced apoptosis in an Hs578T breast carcinoma cell line in an IGF-independent manner. It was observed in that study that IGFBP-3 added to the cultures was proteolytically modified, generating a specific pattern of fragmentation. We have also previously reported that almost all of the IGFBP-3 outside the circulation in extravascular fluids is in a fragmented form, apparently due to the activity of a cation-dependent serine protease. The aim of this study was to investigate the role of proteolysis in the IGFBP-3 enhancement of C2-induced apoptosis. In this study we confirmed that preincubation of Hs578T cells with IGFBP-3 enhances the apoptotic effect of the ceramide analog C2. The presence of IGF-I completely inhibited the enhancement effect, apparently by inhibiting cell surface association and proteolytic modification. The presence of a serine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely inhibited the enhancement effect of IGFBP-3, and Western immunoblotting of conditioned medium and cell surface-associated IGFBP-3 revealed that proteolytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibility of Hs578T cells to C2. The effect of these fragments could, however, also be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact that IGFBP-3 was already fragmented. This suggests additional roles for serine proteases in the IGFBP-3 effect on C2-induced apoptosis in addition to the cleavage of the binding protein.

Active and inhibitory components of the insulin-like growth factor binding protein-3 protease system in adult serum, interstitial, and synovial fluid.

Circulating insulin-like growth factor binding protein-3 (IGFBP-3) proteolytic activity is normally low but increases in serum from pregnant women and from patients with various pathologies. In contrast, we have recently reported that outside the circulation, such activity is normally high but decreases in various pathologies. We have now compared components of the IGFBP-3 proteolytic system revealed after size fractionation of serum and extravascular fluids with different intrinsic levels of such activity. Normal serum, serum from pregnant women, and synovial fluid from patients with rheumatoid arthritis revealed high and low molecular weight (MW) areas of activity. However, only the low MW activity was apparent in interstitial fluid from normal skin (N Inst F) or psoriatic lesions (P Inst F) and in synovial fluid from normal volunteers (N Syn F) or patients with osteoarthritis (OA Syn F). Addition of inhibitors revealed both areas to comprise more than one enzyme, including serine proteases and metalloproteinases; both could also be inhibited by P Inst F, NS, RA Syn F, and inhibitory fractions from the separation of the latter two. These findings demonstrate low and high MW regions of proteolytic activity, which may contribute to the IGFBP-3 protease system, the former always present, whereas the latter seems to be retained within the circulation apart from inflammatory conditions. The variations apparent in IGFBP-3 protease activity in the intact samples related to the presence of an inhibitor, which may protect IGFBP-3 from proteolysis, rather than to changes in the component proteases.

Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells.

Although abundant mRNA for IGF-II has been detected in the human ovary, a role for IGF-II in steroidogenesis has not yet been established. In rat adipocytes, incubation with insulin greatly increases cell-surface IGF-II receptor (5-fold) and the receptor is rapidly internalised in the absence of insulin. We have therefore investigated the effects of insulin preincubation on the response of granulosa cells from unstimulated ovaries to a range of doses of IGF-II. In the absence of insulin, IGF-II stimulated steroidogenesis in only one of three experiments. After incubation with 10 ng/ml insulin, there was a dose-dependent response to IGF-II in all experiments. Cells incubated with insulin produced 5-10 fold more estradiol in response to IGF-II than those incubated without. In contrast, insulin produced only a small increase of estradiol in response to IGF-I. These results demonstrate a synergistic interaction of insulin with IGF-II in human granulosa cells and suggest that there is an important role for IGF-II in human ovarian steroidogenesis.

Potent inhibition of human ovarian steroidogenesis by insulin-like growth factor binding protein-4 (IGFBP-4).

Several studies have now documented the existence of IGFBPs in follicular fluid and their correlation with the health of the follicle. In particular, increased levels of IGFBP-4 have been reported in androgen-dominant atretic follicles and those from polycystic ovaries. The aim of this study was to elucidate the role of IGFBP-4 in ovarian steroidogenesis. Granulosa cells and theca tissue were incubated with or without LH or FSH in the presence or absence of IGFBP-4 (0.5-50 ng/ml). Inhibition by IGFBP-4 of estradiol production in the presence of testosterone alone was seen in three of four experiments. IGFBP-4 completely inhibited FSH-stimulated estradiol production in three experiments and caused 67% inhibition in a fourth. Similar results were obtained for theca, in which concurrent incubation with IGFBP-4 completely negated the stimulatory effects of LH on androstenedione production. The mechanism by which IGFBP-4 exerts these potent effects and the possibility that this may by IGF-independent are currently being investigated.

Changes in circulating insulin-like growth factor-binding protein-1 (IGFBP-1) during prolonged exercise: effect of carbohydrate feeding.

Plasma levels of glucose, insulin, the insulin-like growth factor (IGF-I and -II) and IGFBP-1 were determined in four young healthy males performing cycle exercise to fatigue while being fed either placebo (trial C) or glucose polymer solution (trial G). There was a significant decline in glucose and insulin from rest to fatigue in C (P < 0.01 and P < 0.05, respectively), but not in G. IGF-I or IGF-II levels did not change significantly in either of the trials. IGFBP-1 levels increased 12-fold in C (11.4 +/- 1.6 ng/ml at rest to 136.5 +/- 19.7 ng/ml at fatigue P < 0.01), and 5.6-fold in G (11.0 +/- 2.3 ng/ml to 62.2 +/- 15 ng/ml, P < 0.05). In C a significant negative correlation was found between IGFBP-1 and glucose (r = 0.69, P < 0.01) and IGFBP-1 and insulin (r = -0.612, P < 0.05) in C, but not in G. These results suggest that during prolonged exercise factors other than insulin or glucose may regulate IGFBP-1 and that IGFBP-1 may serve a role other than to prevent the hypoglycaemic action of the IGFs.

Insulin-like growth factor (IGF) I and II, IGF-binding proteins, and IGF-binding protein proteases are produced by theca and stroma of normal and polycystic human ovaries.

There is increasing evidence for an important regulatory role for the insulin-like growth factor (IGF) system in the human ovary. IGF-I and -II and IGF-binding proteins (IGFBPs)-1 to -4 have been identified by analysis of follicular fluid and granulosa cell-conditioned medium and by in situ hybridization and Northern and dot blot analyses of ovarian tissues. It has been suggested that abnormalities of intraovarian IGF-I or IGFBPs may play a part in the pathogenesis of polycystic ovary syndrome. The aim of this study was to identify production of IGF-I and -II and IGFBP-1 to -4 by unstimulated normal and polycystic ovaries. IGF-I and -II were measured by RIA after acid-gel exclusion chromatography in medium conditioned by incubation for 48 h with granulosa cells or explants of theca or stroma. Both IGF-I and -II were present in the low nanograms per mL range in theca- and stroma-conditioned medium (T+SCM). IGFBPs in T+SCM were initially analyzed by Western ligand blotting, which revealed that low mol wt IGFBPs were predominant, especially IGFBP-2 (35 kDa). There was a band corresponding to 26 kDa with smaller amounts of a 31-kDa band, but only a trace of IGFBP-3 (44 and 40 kDa, confirmed by immunoblot). We found no consistent differences between normal and polycystic ovary syndrome ovaries, and although there was a trend toward increased IGFBP accumulation in response to LH, this was not consistent. We were unable to detect IGFs or IGFBPs by Western ligand blotting in granulosa cell-conditioned medium. In further studies we attempted to measure IGFBP-3 by RIA using two different antisera (alpha-BP-3gl and 1287-2-14) that detect different epitopes of IGFBP-3 and allow the presence of proteolytic activity to be demonstrated. Results obtained using alpha-BP-3gl were lower than those using 1287-2-14, suggesting proteolysis of IGFBP-3 in the medium. There was no evidence of proteolysis of serum IGFBP-3 after incubation with conditioned medium, but in contrast, radiolabeled [125I]IGFBP-3 was cleaved after incubation with T+SCM. Immunoblotting revealed intact IGFBP-2 (35 kDa) and bands of various sizes between 16-33 kDa. Immunoreactive fragments of IGFBP-3 between 13-40 kDa were seen. In conclusion, T+SCM contained IGF-I and -II. IGFBP-2 and -4 were the predominant species of IGFBP in T+SCM. T+SCM also contained significant protease activity directed toward IGFBP-2 and -3. Proteolytic activity may be an important mechanism by which bioactive IGFs are made available to these tissues.