Insulin-like Growth Factor Bioavailability Research Articles

Serum proteolysis of IGFBP-3

Insulin-like growth factor (IGF) bioavailability is modulated by a family of six IGF binding proteins (IGFBPs) that binds IGF with affinities similar to that of the type 1 IGF receptor. Proteolytic degradation of IGFBP-3, the major serum IGFBP, was first reported in pregnancy serum and suggested to be an additional mechanism involved in the regulation of IGF bioavailability. In this paper, the presence of serum IGFBP-3 proteolysis and its potential role in the regulation of IGF bioavailability is discussed partly in view of our recent finding of IGFBP-3 proteolysis by the tissue plasminogen activator (tPA)-plasminogen-plasmin system in human serum.

Proteolytic activity of IGFBP-3 in various clinical conditions during childhood studied by means of western immunoblotting.

Insulin-like growth factors (IGFs) have 6 types of binding proteins (IGFBPs), and IGFBP-3 is the major IGFBP in human sera. A proteolytic enzyme for IGFBP-3 has recently been reported to be present in human and animal pregnant sera. Although the physiological significance of a pregnancy-associated IGFBP-3 protease remains to be established, the proteolysis could result in lowering the affinity for IGFs, thereby enhancing their delivery to target tissues by increasing free IGFs in the circulation. The methods for detection of IGFBP-3 protease which have been widely used so far are a method reported by Lamson et al. which used affinity crosslinking or western ligand blotting. These methods need radioactive materials (iodinated IGFs and IGFBP-3) and it takes at least a few days to get the results. We have now developed a simple assay for the proteolysis of IGFBP-3. The method is western immunoblotting without radioactive materials. The results can be obtained in a day. With this method, we proved the absence of significant proteolytic activity in sera from rapidly growing children (early stage of puberty or precocious puberty), and sera from a severe type of growth hormone deficiency. Significant proteolytic activity, as in pregnant women, was detected in 6 out of 11 patients with acute disorders such as measles, Kawasaki disease, bacterial meningitis and mycoplasma pneumonia, some of whom were probably in a catabolic condition. These data suggests that the proteolysis of IGFBP-3 might also be important in modulating IGF action in some acute diseases during childhood. The increased bioavailability of IGFs by IGFBP-3 proteolysis may play a role in overcoming catabolic conditions.

A proteolytic fragment of insulin-like growth factor (IGF) binding protein-3 that fails to bind IGF is a cell growth inhibitor

Limited proteolysis of insulin-like growth factor binding protein-3 (IGFBP-3) is now recognized as a normal process in the regulation of insulin-like growth factor (IGF) activity, its major effect being to increase IGF bioavailability. In order to characterize the proteolytic fragments of IGFBP-3, we reproduced this proteolysis in vitro using plasmin which provokes cleavages that are similar to those induced in vivo by (unidentified) specific IGFBP-3 proteases. Two major peaks were purified by RP-HPLC. One contained a 16 kDa fragment and the other comprised two fragments of 22 and 25 kDa. Competitive binding experiments showed that the 16 kDa material had no affinity for IGFs. The 22–25 kDa fragments had considerably reduced affinity, particularly for IGF-I. In a chick embryo fibroblast assay where DNA synthesis was stimulated by IGF-I or insulin, the 22–25 kDa fragments weakly inhibited IGF-I-induced cell proliferation and had no effect on stimulation by insulin. The 16 kDa fragment unexpectedly proved to be a potent inhibitor of both IGF- and insulin-induced cell growth. This proteolytic fragment of IGFBP-3 therefore exhibits intrinsic inhibitory activity.

Reversible effects of cessation and recommencement of thyroxine treatment on insulin-like growth factors (IGFs) and IGF-binding proteins in patients with total thyroidectomy.

There is a complex relationship between the thyroid and pituitary GH/insulin-like growth factor (IGF) axes. IGFs circulate in association with six specific high affinity binding proteins (IGFBPs) that modulate their bioactivity and bioavailability. Recent evidence suggests that gene expression and circulating levels of IGFBPs are related to prevailing thyroid hormone status. We have investigated the effects of both withdrawal and reinstitution of thyroid hormone replacement on circulating IGF and IGFBP levels in athyreotic patients (n = 10). The mean IGF-I concentration fell from a basal level of 191.8 +/- 12 micrograms/L to a nadir of 136.4 +/- 17.8 micrograms/L (P = 0.026) 5 weeks after stopping T4 treatment and returned to normal values 3 weeks after recommencement of replacement treatment. The fall in IGF-II levels followed a similar pattern from a basal mean level of 649 +/- 33.7 to 547 +/- 42.7 micrograms/L (P = 0.026) at 5 weeks. These changes paralleled the fall in free T3 and free T4. Similarly, IGFBP-1 levels fell after stopping T4 treatment from a basal level of 54.8 +/- 4.0 to 24.6 +/- 7.0 micrograms/L (P = 0.001) 5 weeks later. After T4 treatment was restarted, IGFBP-1 levels rose and were not significantly different from basal values by week 8. There were strong positive correlations between paired data sets for IGFBP-1 and free T3 (r = 0.488; P = 0/0037) and free T4 (r = 0.56; P = 0.0006), and a strong negative correlation with TSH (r = -0.515; P = 0.0001). Insulin is known to be important in the regulation of IGFBP-1, but no changes in fasting insulin levels during T4 withdrawal were noted, and levels of IGFBP-1 did not exhibit the normal inverse relationship with circulating insulin levels. Levels of IGFBP-2, assessed by Western ligand blotting, increased during the development of hypothyroidism, peaked 5 weeks after stopping T4 replacement, and declined on reinstitution of replacement treatment. A further level of regulation of the IGF-IGFBP axis is afforded by the presence of specific circulating IGFBP proteases. Proteases directed against IGFBP-3 proteolytically cleave the major carrier BP in the circulation and reduce its binding affinity, possibly resulting in increased tissue IGF bioavailability. Despite the marked reduction in circulating IGF levels and the generation of significant biochemical hypothyroidism, IGFBP-3 protease activity was not apparent during the 10-week period of the study.(ABSTRACT TRUNCATED AT 400 WORDS)

Insulin-like growth factor-binding protein-2 concentrations in cerebrospinal fluid and serum of children with malignant solid tumors or acute leukemia.

Many tumor cell lines express insulin-like growth factors (IGFs) as autocrine growth factors and IGF-binding protein-2 (IGFBP-2) as a major IGFBP, which, in turn, regulates the bioavailability and bioactivity of IGFs. The aim of our study was to investigate 1) whether children with malignancies have elevated IGFBP-2 levels in cerebrospinal fluid (CSF) and serum, and 2) whether IGFBP-2 levels in these biological fluids could be useful markers for the diagnosis and follow-up of certain tumor types. We, therefore, measured IGFBP-2 levels in the CSF and serum of children with malignancies by Western ligand blot analysis; RIA with alpha IGFBP-2, a polyclonal antibody for human IGFBP-2; and immunoprecipitation with alpha IGFBP-2 and alpha Hec-1a, a polyclonal antibody that recognizes IGFBP-2 and -3. Furthermore, the expression of IGFBP-2 messenger ribonucleic acid in tumor tissue from three central nervous system (CNS) tumor patients was analyzed by Northern blot analysis. We examined CSF from 21 children with malignant CNS tumors, 25 patients with acute leukemia, and 4 patients with peripheral solid tumors and compared the IGFBP-2 levels with those in CSF from 21 patients who received a lumbar puncture to exclude meningitis. Serum was obtained from 7 patients with solid tumor, 12 patients with malignant CNS tumor, and 16 patients with acute leukemia. The serum IGFBP-2 levels were compared to serum levels in 5 patients with sarcoma who had reached complete remission and 13 normal control children. CSF and serum were collected at the same time, before initiation of therapy. Patients with malignant CNS tumors showed elevated IGFBP-2 levels in CSF (P < 0.001), whereas patients with solid peripheral tumor or acute leukemia had normal IGFBP-2 levels in CSF. CNS tumor patients with microscopically detectable malignant cells in the CSF had the highest CSF IGFBP-2 levels. Serum IGFBP-2 levels were increased in patients with solid peripheral tumors (P < 0.05), whereas patients in complete remission had normal serum IGFBP-2 levels. In summary, IGFBP-2 was elevated specifically in CSF from patients with CNS tumor, whereas IGFBP-2 serum levels were elevated in children with various peripheral tumors. We conclude that IGFBP-2 in CSF could be a specific marker for malignant CNS tumors. We detected high IGFBP-2 messenger ribonucleic acid expression in 1 of 3 CNS tumor tissues analyzed.

Human osteoarthritic chondrocytes possess an increased number of insulin-like growth factor 1 binding sites but are unresponsive to its stimulation. Possible role of IGF-1-binding proteins.

To characterize the insulin-like growth factor 1 (IGF-1) receptor in human osteoarthritic (OA) and normal adult chondrocytes. The biologic response of chondrocytes to IGF-1 stimulation was examined, as was the presence and synthesis of IGF binding proteins (IGFBP) in these cells. Binding studies, Northern blot, immunohistochemical analysis, and affinity cross-linking experiments were performed for characterization of the IGF receptor, and the latter method was also used for IGFBP determination. The biologic response was estimated via the incorporation of radiolabeled proline into a newly synthesized protein. Binding experiments revealed a single class of binding sites. The mean +/- SEM affinity (Kd) of normal chondrocytes was 1.4 +/- 0.4 nM, with 26.8 +/- 5.5 x 10(3) binding sites/cell. OA chondrocytes had a lower affinity (Kd 15.4 +/- 4.7 nM) and a higher density (1,178.3 +/- 299.5 x 10(3) binding sites/cell) compared with normal cells (P < 0.004 and P < 0.001, respectively). Immunohistochemical studies with a monoclonal antibody (MAb) against the type 1 IGF receptor (alpha IR3) showed increased staining in OA cartilage compared with normal tissue. Biologic responses of chondrocytes after IGF-1 stimulation revealed that OA chondrocytes were unresponsive, whereas a 2.5-fold increase in new protein synthesis was observed in normal cells. Competition studies in normal chondrocytes revealed that both IGF-1 and IGF-2 displaced radiolabeled IGF-1 in a comparable manner; however, insulin at high concentration weakly competes. Moreover, MAb alpha IR3 effectively blocked specific binding in normal chondrocytes (77%), but not in OA chondrocytes (26%). Northern blot and covalent cross-linking analyses revealed the specific band characteristic of type 1 receptor. With the latter technique, other bands corresponding to the IGFBPs were also detected. Comparison between normal and OA chondrocytes showed increased intensity of the IGFBP bands, particularly those corresponding to the IGFBP-3 doublet. It is shown that type 1 IGF receptor is expressed in human articular cartilage and that the level of binding sites is significantly increased in OA chondrocytes. Interestingly, despite the higher level of binding sites in OA cells, no response to IGF-1 stimulation was found in these cells. Our data suggest that this increase in specific binding may involve not only the type 1 IGF receptor but also IGFBP on the cell surface. The latter, by binding the IGF-1, will diminish the bioavailability of IGF-1 and thus prevent its anabolic action.

Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3.

Insulin-like growth factor-binding protein-3 (IGFBP-3) forms a 140-kilodalton (kDa) complex with an acid-labile glycoprotein after complexing with IGF-I or IGF-II. To investigate the bioavailability of circulating IGFs in the rat, we determined the molecular distribution and disappearance of an iv bolus of human IGFBP-3 (130 micrograms/kg). Serum fractions were assayed after size-separation chromatography for human (h) and rat (r) IGFBP-3 with specific RIAs. Within 2 min of injection, 52.9 +/- 1.6% of the hIGFBP-3 appeared in a 140-kDa complex. This form remained in the circulation for hours, whereas hIGFBP-3 in 50- and 30-kDa forms disappeared within 30-60 min. The rapid complexing of hIGFBP-3 to the 140-kDa form did not occur in vitro. Measurement of endogenous rIGFBP-2 indicated that there was minimal exchange of hIGFBP-3 with rIGFBP-3 in the 140-kDa complex within the 60 min of the in vivo study. To determine the importance of IGF-I in regulating the molecular distribution of hIGFBP-3 in serum, the experiment was repeated in streptozotocin-diabetic rats, with total IGF-I levels 50% lower than control values. In these animals, 40.1 +/- 5.2% of the hIGFBP-3 appeared in the 140-kDa complex 2 min after injection, significantly less than that in controls (P < 0.05), and hIGFBP-3 disappeared more quickly from the circulation than in controls (P < 0.05). After coinjection of recombinant human IGF-I, the retention of hIGFBP-3 in the circulation was prolonged in both control and diabetic animals. Because IGFBP-3 must bind IGF before it can associate with the acid-labile subunit, these findings indicate a much greater availability of IGFs to the circulation than previously estimated.

Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3

Bioavailability of insulin-like growth factors (IGFs) in rats determined by the molecular distribution of human IGF-binding protein-3

Pregnancy in growth hormone-deficient rats: assessment of insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs) and IGFBP protease activity

Pregnancy in rodents is associated with important maternal metabolic changes. Early pregnancy is considered to be an anabolic phase of nutrient storing, while in late pregnancy, a catabolic phase develops to help meet the metabolic demands of the rapidly growing conceptus. Similarly, major changes also occur in the IGFs, IGF-binding proteins (IGFBPs) and GH axis. In the rat, maternal serum IGF-I levels increase from early to mid-pregnancy, after which IGF-I levels decline. Conversely, as IGF-I levels decline, pituitary-derived rat GH increases twofold. This coincides with a decrease in IGFBP-3 and the appearance of an IGFBP protease. However, the physiological role of these changes is unclear. The aim of our study was to examine the roles of GH, IGFs and IGFBPs during pregnancy in a unique isolated GH-deficient rat model, spontaneous dwarf rats. Pregnancy in GH-deficient dams resulted in a significantly reduced litter number, and maternal weight gain (day 21-day 1) was reduced by 28% when compared with dams with normal GH levels (GH-normal dams). In the sera of GH-normal dams, IGF-I levels increased 2.6-fold by day 4 of pregnancy and then progressively declined to below non-pregnant levels. Serum IGF-II levels were low and remained unchanged. Western-ligand blot analysis showed that IGFBP-3 was present in non-pregnancy and early pregnancy sera, but declined dramatically after day 12. This decline in IGFBP-3 coincided with the detection of an IGFBP protease. In contrast, in non-pregnancy sera from GH-deficient rats, serum IGF-I concentrations were 10% of the levels seen in GH-normal females, and neither IGF-I nor IGF-II concentrations changed with pregnancy. Furthermore, in GH-deficient dams, serum IGFBP-3 (as assessed by Western-ligand blot analysis) was low in non-pregnancy and early pregnancy sera, and became undetectable by day 12 of pregnancy. The low concentrations of IGFBP-3 in early pregnancy serum from GH-deficient rats coincided with IGFBP-proteolytic activity, and the decline in serum IGFBP-3 after day 16 was the result of increased protease activity. In conclusion, isolated GH deficiency results in: (1) reduced maternal weight gain and correspondingly smaller litter sizes; (2) low and unchanged maternal serum IGF-I levels; (3) low, but declining, serum IGFBP-3 levels; and (4) activity of IGFBP protease(s) detectable in early and late pregnancy, which may modulate the bioavailability and bioactivity of IGFs by regulating IGFBP-3.

Immunohistochemical localization of insulin-like growth factors (IGFs) and IGF binding proteins-1, -2 and -3 in human placenta and fetal membranes

Insulin-like growth factor (IGF) I and II are synthesized within the placenta and are believed to play an important role in the regulation of placental growth and endocrine function. IGF bioavailability is determined at a cellular level by several specific binding proteins (IGF BPs), which are widely but selectively distributed in all developing tissues. We have used immunohistochemistry to localize IGF I and II peptides, and IGF BP-1, -2, and -3 in human placentae, fetal membranes and umbilical cord at 6-8 weeks after therapeutic termination and at term after spontaneous delivery. Primary antisera were directed against human IGF I, human IGF BP-1, bovine IGF BP-2, and human IGF BP-3 respectively. Immunoreactive IGF BP-2 was found in association with the syncytiotrophoblast, intermediate trophoblasts of the fetal villi and chorion, amnion and decidua; while weaker staining was seen in some but not all cytotrophoblasts. A similar but less intense staining pattern was observed for IGF BP-1 and IGF peptides in placenta and amnio-chorion. Strong immuno-staining for IGF BP-1 was seen in decidual cells. No immunoreactive IGF BP-3 was found in placenta or membranes. A co-distribution of IGF BP-2, BP-1 and IGF peptides in placenta suggests a role for these IGF BPs in determining the localization of the IGFs for actions on target tissues.

Can "big" insulin-like growth factor II in serum of tumor patients account for the development of extrapancreatic tumor hypoglycemia?

The pathogenesis of extrapancreatic tumor hypoglycemia has been related to the secretion of big insulin-like growth factor (IGF) II by the tumor. In 25 of 28 patients with this type of hypoglycemia we found 1.5-8-fold elevated serum levels of immunoreactive big (15-25 kD), but decreased levels of normal IGF II. After removal of the tumor, big IGF II disappeared and normal IGF II increased. Tumors contained elevated levels of IGF II, 65-80% in the big form. The insulin-like bioactivity of big IGF II and its affinity towards IGF-binding proteins (IGFBP)-2 and -3 are similar to those of normal IGF II, but two- to threefold higher on a molar basis. Big IGF II is mainly bound to the 50-kD IGFBP complex. The latter contains approximately 10 times more of this peptide than in normal serum and displays three- to fourfold increased insulin-like bioactivity. The formation of the 150-kD IGFBP complex with 125I-recombinant human IGFBP-3 is impaired in tumor serum. This results in sequestration of IGFBP-3 and predominant association of big IGF II with IGFBP-2 and -3 in the 50-kD complex. Increased bioavailability of big IGF II in this complex due to unrestricted capillary passage and enhanced insulin bioactivity of this big IGF II pool provide a continuous increased insulin-like potential available to insulin and type 1 IGF receptors of insulin-sensitive tissues and thus may lead to sustained hypoglycemia.

Growth hormone in renal transplantation--the mode of action, animal studies, and clinical use.

During circulation, growth hormone (GH) is bound to about 50% by the high-affinity, low-capacity GH-binding protein (BP). GHBP represents the extracellular binding domain of the GH receptor and modulates the action of GH. After binding to its receptor, GH induces the local production of insulin-like growth factor 1 (IGF-1) by autocrine and paracrine mechanisms. In uremia, the plasma GH-binding activity is low and does not get up-regulated by recombinant human GH treatment, which is in contrast to the experience in short, normal children. There is evidence that hepatic IGF-1 production is low, whereas the serum concentration of IGF-binding protein 3 (IGF-BP3) and other IGFBPs is increased because of the reduced renal clearance of the low-molecular-weight fragment of IGF-BP. This result in the reduced bioavailability of free IGF-1 and reduced IGF bioactivity. There is a strong interaction between GH and corticosteroids. Corticosteroids suppress growth by reducing the food efficiency ratio (weight gain per food intake), reduce pituitary GH secretion, and decrease the local production of and cell responsiveness for IGF-1. The growth-depressing and catabolic effects of corticosteroids can be counterbalanced dose dependently by recombinant human GH in animal experiments, and growth can be improved in corticosteroid-treated renal allograft recipients with and without normal renal function. It is not clear at this time to what extent GH may induce acute or chronic rejection crises.

Similar distribution of insulin-like growth factor binding proteins-1, -2, -3 in human fetal tissues.

The actions of insulin-like growth factors (IGFs) are modulated by several specific binding proteins (IGF BPs). Since the anatomical distribution of IGF BPs is likely to dictate IGF bioavailability we used immunocytochemistry to localize IGF BP-1, -2, and -3 in early second trimester human fetal tissues. Primary antisera were directed against hIGF BP-1, bIGF BP-2 and hIGF BP-3 respectively, and showed less than 5% cross-reaction with heterologous IGF BP species. The distribution of immunopositive staining was similar for each IGF BP in many tissues being prominent in the epithelial lining of the gut, kidney and lung; in epidermis, adrenal cortex and pancreatic endocrine tissue; and in association with membranes of skeletal muscle fibres and cardiocytes. Unlike IGF BP-1 -2, IGF BP-3 was barely detectable in liver and absent from epiphyseal chondrocytes. Conversely, IGF BP-3 alone was visualized within neurons of the cerebral cortex. The co-distribution of IGF BPs in many human fetal tissues suggests that they may co-ordinately regulate IGF bioavailability in target tissues and modify IGF: receptor interactions.