Porcine insulin-like growth factor (IGF) binding protein blocks IGF-I action on porcine adipose tissue

A growth hormone-dependent insulin-like growth factor (IGF) binding protein (IGFBP) purified from porcine serum specifically blocked the acute insulin-like effects of IGF-I on lipogenesis and glucose oxidation in porcine adipose tissue. This inhibition was dose dependent with half-maximal effective concentrations of IGFBP of 530 ng/ml for lipogenesis and 590 ng/ml for glucose oxidation in the presence of 10(-8) M IGF-I. The IGFBP also caused decreased rates of lipogenesis following a 1-hr preincubation of tissue with IGF-I (10(-8) M). The IGFBP had no effect on insulin action on porcine adipose tissue. These findings demonstrate the inhibitory effects of a highly purified porcine serum IGFBP on the biologic effects of IGF-I in vitro, and provide evidence that the growth hormone-dependent IGFBP blocks the acute insulin-like actions of IGF-I in vivo.     

Short chain fatty acid and glucose metabolism in isolated pig colonocytes: modulation by NH4 +

Using monolayer cultures of costal chondrocytes established from four week old Clun Forest lambs, we have demonstrated that, under serum free conditions the cells release three IGFBPs (32, 29 and 21 kDa) into the medium. The most abundant of these—the 32 kDa BP-was shown to be IGFBP-2 by Western blotting. Furthermore we demonstrate that the levels of IGFBP 2 in conditioned medium are acutely increased (6, 12 and 24 h time points) following treatment of cells with bovine GH (1–100 ng/ml).In a parallel set of experiments, using ovine fibroblasts (derived from dermis) we show that IGFBPs of Mr 32, 29 and 21 kDa are also secreted by this cell type. However the relative abundance of these BPs differed from that seen in the chondrocyte cultures, with the 21 kDa species now the most abundant. In addition, prolonged exposure of autoradiographs indicated that fibroblasts secreted a higher Mr IGFBP (most probably ovine BP-3) that was not detected in any of our chondrocyte cultures. Most significant however was the demonstration that bGH did not dramatically affect the levels of IGFBPs in fibroblast cell cultures. We conclude that GH stimulates BP-2 production from chondrocytes and this is a cell-type specific effect in as much as it is not replicated in cultures of dermal fibroblasts.Abbreviations BCIP 5-bromo-4-chloro-3-indolyl phosphate - CM conditioned medium - DMEM Dulbecco's Modified Eagles Medium - FCS foetal calf serum - IGF insulin-like growth factor - IGFBP insulin-like growth factor binding protein - HBSS Hanks' Balanced Salt Solution - GH growth hormone - NBT nitroblue tetrazolium - SFM serum free medium - TBS tris buffered saline     

Growth factor-mediated regulation of aromatase activity in human skin fibroblasts.

We investigated the effects of various hormones and growth factors on aromatase activity in cultured human skin fibroblasts. Several potential trophic factors were tested for their ability to modify basal aromatase activity or the response to dibutyryladenosine 3',5'-cyclic monophosphate and dexamethasone because (i) no endogenous ligand has been identified that is responsible for stimulating aromatase activity in the periphery, and (ii) dexamethasone and cAMP analogs can increase this enzyme's activity in fibroblasts. The effect of insulin and insulin-like growth factors were examined in closer detail because of the clinical association between insulin and hyperandrogenism. Pituitary hormones and hypothalamic releasing factors, such as human ACTH (10 nM), beta-endorphin (10 nM), beta-lipotropin (10 nM), alpha-MSH (10 nM), gamma 3-MSH (10 nM), ovine luteinizing hormone (10 ng/ml), ovine follicle-stimulating hormone (10 ng/ml), ovine thyroid-stimulating hormone (10 ng/ml), rat growth hormone (10 ng/ml), rat prolactin (10 ng/ml), rat corticotropin-releasing factor (10 nM), luteinizing hormone-releasing factor (10 nM), thyrotropin-releasing factor (10 nM), human growth hormone-releasing factor (10 nM), and somatostatin (10 nM), have no significant effects on aromatase activity. Porcine inhibin A (10 ng/ml) and porcine activin AB (10 ng/ml), two ovarian hormones with structural transforming homology to transforming growth factor-beta, also have no effect on aromatase activity. Although basic fibroblast growth factor (1-100 ng/ml), acidic fibroblast growth factor (1 ng/ml), epidermal growth factor (1 ng/ml), platelet-derived growth factor (1 ng/ml), tumor necrosis factor (1 ng/ml), and transforming growth factor-beta 1 (1 ng/ml) have no effect on basal aromatase activity in human skin fibroblasts, all of these growth factors inhibited the ability of dibutyryladenosine 3',5'-cyclic monophosphate to stimulate aromatase activity. In contrast, both insulin (100 pg/ml-10 ng/ml) and insulin-like growth factor-1 (1-100 ng/ml) had no effect on cAMP-stimulated aromatase but potentiated the action of dexamethasone (100 nM). Thus, there is a clear distinction between the effects of dexamethasone and cAMP on peripheral aromatase. On the basis of the results presented here, it is interesting to speculate that the hyperandrogenism that is often associated with insulin resistance may be due to a combination of growth factor-mediated inhibition of aromatase activity and the failure of peripheral tissues to respond to insulin and metabolize androgens to estrogens.     

Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.     

Actions of insulin-like growth factor-I on the B104 neuronal cell line: effects on cell replication, receptor characteristics, and influence of secreted binding protein on ligand binding

Several peptide growth factors influence the growth and differentiation of neural cells. To investigate further the growth-promoting effects of the somatomedins on cells of neural origin, the authors characterized the binding and mitogenic effects of insulin-like growth factor-I (IGF-I) on a functionally differentiated rat neuronal cell line (B104). Specific, high-affinity (Kd approximately equal to 10(-9) M) receptors for IGF-I were abundant (approximately 124,000 binding sites/B104 cell). These IGF-I receptors were similar to those of non-neural tissue in that they contained 135,000 dalton binding subunits (demonstrated by affinity labeling and autoradiography) and recognized insulin at high concentrations. IGF-I was more potent than insulin at stimulating B104 cell replication in serum-free medium and, at an initial concentration of 100 ng/ml, was the only exogenous growth factor needed to maintain growth through several cell divisions. Furthermore, cells of later passage were found to secrete specific IGF binding proteins that produced an unusual, biphasic binding curve in radioligand displacement studies. These binding proteins apparently sequester IGF-I, limiting its access to the cell. Experiments with B104 cells may provide useful information about the role of IGFs and their binding proteins as potential regulators of growth and differentiation of the primitive neuroblast.     

Regulation of renal growth and IGFBP-4 expression by triiodothyronine during rat development.

The insulin-like growth factors (IGFs) and insulin-like growth factor binding proteins (IGFBPs), which regulate IGF activity, play a fundamental role in renal cell proliferation and differentiation. The thyroid hormone is considered to be required for kidney development; excess induces local hypertrophy and hyperplasia. The aim of the present study was to investigate the possible involvement of the IGF/IGFBP system in thyroid hormone-induced renal growth during the development of the rat. Our results show that thyroid hormone withdrawal by 6-propyl-2-thiouracil (PTU)-treatment of rats at all ages had no effect on renal IGFBP-4 mRNA levels, whereas the abundance of the serum protein was decreased compared to controls. Intraperitoneal triiodothyronine (T3) administration to hypothyroid rats resulted in renal hypertrophy associated with a significant upregulation of IGFBP-4 expression with increased levels of renal IGFBP-4 mRNA and serum protein. T3-induced upregulation of IGFBP-4 expression suggests the involvement of the local IGF/IGFBP system in T3-induced renal hypertrophy.     

Insulin-like growth factors.

The insulin-like growth factors I and II are single chain polypeptides homologous to proinsulin. IGF I and IGF II contribute to cell regulation and stimulate protein synthesis via signaling through type I receptors which are homologous to insulin receptors and activate phosphorylation cascades. IGFs enhance the proliferation of chondocytes and the proliferation of their collagen and proteoglycan matrix; IGFs stimulate longitudinal (endochondral) bone growth. Throughout life, IGFs are constitutvely expressed ubiquitous factors which help to maintain the survival of differentiated cells, Increased expression is found during growth and tissue repair, Six specific binding proteins, IGFBP 1-6, allow additional tissue compartment specific control of IGF activity; IGFBP production favours storage and IGFBP cleavage leads to activation.     

Role of insulin-like growth factor binding protein-3 (IGFBP-3) in the differentiation of primary human adult skeletal myoblasts

Although muscle satellite cells were identified almost 40 years ago, little is known about the induction of their proliferation and differentiation in response to physiological/pathological stimuli or to growth factors/cytokines. In order to investigate the role of the insulin-like growth factor (IGF)/IGF binding protein (IGFBP) system in adult human myoblast differentiation we have developed a primary human skeletal muscle cell model. We show that under low serum media (LSM) differentiating conditions, the cells secrete IGF binding proteins-2, -3, -4 and -5. Intact IGFBP-5 was detected at days 1 and 2 but by day 7 in LSM it was removed by proteolysis. IGFBP-4 levels were also decreased at day 7 in the presence of IGF-I, potentially by proteolysis. In contrast, we observed that IGFBP-3 initially decreased on transfer of cells into LSM but then increased with myotube formation. Treatment with 20 ng/ml tumour necrosis factor-alpha (TNFalpha), which inhibits myoblast differentiation, blocked IGFBP-3 production and secretion whereas 30 ng/ml IGF-I, which stimulates myoblast differentiation, increased IGFBP-3 secretion. The TNFalpha-induced decrease in IGFBP-3 production and inhibition of differentiation could not be rescued by addition of IGF-I. LongR(3)IGF-I, which does not bind to the IGFBPs, had a similar effect on differentiation and IGFBP-3 secretion as IGF-I, both with and without TNFalpha, confirming that increased IGFBP-3 is not purely due to increased stability conferred by binding to IGF-I. Furthermore reduction of IGFBP-3 secretion using antisense oligonucleotides led to an inhibition of differentiation. Taken together these data indicate that IGFBP-3 supports myoblast differentiation.     

Involvement of growth hormone (GH) and insulin-like growth factor (IGF) system in ovarian folliculogenesis

During the last decade, involvement of growth hormone (GH), insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in ovarian folliculogenesis has been extensively studied. This review provides an update on the GH, IGF system and their role in ovarian follicular development. In vitro studies and knockout experiments demonstrated an important role of GH in preantral follicle growth and differentiation through their binding with GH receptors, which are located both in the oocyte and follicular somatic tissues. Furthermore, GH stimulates the development of small antral follicles to gonadotrophin-dependent stages, as well as maturation of oocytes. With regard to the IGF system, IGF-I has no effects on primordial follicle development, but both IGF-I and IGF-II stimulate growth of secondary follicles. Depending on the species studies and method used, these proteins have been detected in oocytes and/or somatic cells. In antral follicles, these IGFs stimulate granulosa cell proliferation and steroidogenesis in most mammals. The bioavailability of IGFs is regulated by a family of intrafollicular expressed IGF binding proteins (IGFBPs). Facilitation of IGF can be increased through the activity of specific IGFBP proteases, which degrade the IGF/IGFBP complex, resulting in the production of IGFBP fragments and release of attached IGF.     

Inhibition of growth hormone-stimulated lipolysis by somatostatin, insulin, and insulin-like growth factors (somatomedins) in vitro

The effects of somatostatin, insulin, insulin-like growth factor I (IGF-I), and insulin-like growth factor II (IGF-II)/MSA on growth hormone (GH) (1 microgram/ml)-induced lipolysis were examined employing chicken adipose tissue in vitro. Basal and GH-stimulated glycerol release were inhibited by somatostatin (1 ng/ml) and by IGF-II/MSA (10 and 100 ng/ml). Insulin and IGF-I (10 and 100 ng/ml) completely inhibited the lipolytic response to GH without affecting basal glycerol release. Insulin and IGF-I were equipotent in inhibiting GH-induced lipolysis while IGF-II is only 16% as potent as insulin.     

Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin-C

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8-16 weeks gestation obtained following prostaglandin-induced pregnancy terminations. They were maintained on collagen-coated tissue culture dishes in selective arginine-free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H-thymidine. The labeling index of hepatocytes was consistently enhanced by 25-250 ng/ml human placental lactogen (HPL), 25-250 ng/ml human growth hormone (HGH), 10-50 ng/ml insulin-like growth factor I/somatomedin-C (IGF I/Sm-C), and 10% dialyzed fetal calf serum, reaching a maximum of three- to four-fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF I/Sm-C. Although this proportion did not change following treatment with HGH and HPL, IGF I/Sm-C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF I/Sm-C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF I/Sm-C.     

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes

Thyroid stimulating hormone (TSH) is shown to have definite anabolic effects on skeletal metabolism. Previous studies have demonstrated that Insulin-like growth factors (IGF-I and IGF-II) and their six high affinity binding proteins (IGFBPs 1-6) regulate proliferation and differentiation of bone-forming osteoblasts. The current study was intended to determine whether the anabolic effects of TSH on human osteoblastic (SaOS2) cells are mediated through insulin-like growth factor system components. TSH given at 0.01 ng to 10 ng/ml dose levels for 24 and 48 h significantly increased human osteoblastic (SaOS2) cell proliferation and alkaline phosphatase activity, the differentiation marker. TSH significantly increased IGFs (IGF-I and IGF-II) mRNA expression after 6 and 24 h and their protein levels after 24 and 48 h of treatment, respectively. Unlike the IGFs, the IGFBPs responded differently to TSH treatment. Though there were some inconsistencies in the regulation of stimulatory IGF binding protein-3 and -5 by TSH treatment, there was an overall increase at the mRNA abundance and protein levels. Again, the inconsistency persisted at the regulation of the inhibitory IGFBPs 2, 4, and 6 especially at the level of mRNA expression due to TSH treatment, there is an overall decrease in the levels of IGFBP-2, 4, and 6 in the conditioned media (CM) of SaOS2 cell cultures. The IGFBP proteases which control the availability of IGFs are also regulated by hormones. Pregnancy-Associated Plasma Protein-A (PAPP-A) is responsible for the proteolysis of IGFBP-4. TSH treatment significantly unregulated the expression of PAPP-A both at mRNA and protein levels. In conclusion, TSH promotes human osteoblastic (SaOS2) cell proliferation and differentiation by upregulating IGFs and their stimulatory IGF binding proteins and down regulating the inhibitory IGF binding proteins.     

Insulin-like growth factor (IGF)-binding proteins: interactions with IGFs and intrinsic bioactivities

The insulin-like growth factor (IGF)-binding proteins (IGFBPs) are a family of six homologous proteins with high binding affinity for IGF-I and IGF-II. Information from NMR and mutagenesis studies is advancing knowledge of the key residues involved in these interactions. IGF binding may be modulated by IGFBP modifications, such as phosphorylation and proteolysis, and by cell or matrix association of the IGFBPs. All six IGFBPs have been shown to inhibit IGF action, but stimulatory effects have also been established for IGFBP-1, -3, and -5. These generally involve a decrease in IGFBP affinity and may require cell association of the IGFBP, but precise mechanisms are unknown. The same three IGFBPs have well established effects that are independent of type I IGF receptor signaling. IGFBP-1 exerts these effects by signaling through alpha(5)beta(1)-integrin, whereas IGFBP-3 and -5 may have specific cell-surface receptors with serine kinase activity. The regulation of cell sensitivity to inhibitory IGFBP signaling may play a role in the growth control of malignant cells.     

Free-ligand accelerated dissociation of insulin-like growth factor 1 (IGF-1) from the type I IGF receptor is reduced by insulin-like growth factor binding protein 3

Insulin-like growth factor binding proteins (IGFBP) can inhibit or accentuate the mitogenic activities of insulin-like growth factor 1 (IGF-1) depending upon the experimental model employed. Inhibitory effects may be attributed to sequestration of IGF-1 onto IGFBP rather than the type I IGF receptor. We have demonstrated that the presence of IGFBP in a simple equilibrium binding assay significantly reduces the total amount of IGF-1 bound to the type I IGF receptor and increases the IC₅₀ for IGF-1 binding. On the basis of such an experiment, performed at equilibrium, IGFBP should reduce the mitogenic activity of IGF-1. Recent work has demonstrated an inverse correlation between the dissociation rate of insulin-like molecules from their receptors and their mitogenic activity. It has also been suggested that the increased rate of dissociation of insulin and IGF-1 from their receptors at increased ligand concentrations serves as a ‘dampening’ mechanism to decrease mitogenic signalling. We have demonstrated increased rates of dissociation of IGF-1 from the type I IGF receptor with increasing concentrations of IGF-1. Furthermore, IGFBP-3 inhibits the acceleration of dissociation rates due to increased IGF-1 levels. Thus, under receptor saturating conditions IGFBP-3 may act to increase mitogenesis by increasing the residence time of individual molecules of IGF-1 upon the type I IGF receptor.     

Somatomedin C/insulin-like growth factor 1: an intratesticular differentiative factor of Leydig cells?

By using immature porcine Sertoli cells cultured in serum-free defined medium, we report that medium conditioned by Sertoli cells contained immunoreactive somatomedin C/insulin-like growth factor 1 (SmC/IGF1) measured following acidic gel filtration. The release of this immunoreactive SmC/IGF1 was slightly increased following Sertoli cell treatment with fibroblast growth factor but not with follicle-stimulating hormone or growth hormone. On the other hand, human biosynthetic SmC/IGF1 exerts a potent stimulatory effect on Leydig cell differentiated functions such as LH/hCG-binding (greater than 4-fold) and hCG-stimulated testosterone secretion (greater than 15-fold). This effect was dose and time dependent and the maximal increase of Leydig cell function was observed following 48 h treatment with 50 ng/ml SmC/IGF1. The steroidogenic action of the peptide was not related to Leydig cell growth since both cell number and 3H-thymidine incorporation into DNA were not or slightly (approximately equal to 1.5-fold) increased in the optimal conditions with SmC/IGF1 treatment (100 ng/ml for 48 h). Moreover, the concomitant treatment of Leydig cells by both arabinoside C (10(-5) M), a DNA synthesis inhibitor, and SmC/IGF1 did not modify the stimulating effect of the peptide on LH/hCG-binding and hCG-stimulated testosterone production. Taken together, the present findings support the concept that Sertoli cell derived SmC/IGF1 could be a potent regulator of Leydig cell differentiated functions.     

Growth hormone dependent stimulation of osteoblast-like cells in serum-free cultures via local synthesis of insulin-like growth factor I

Gene-recombinant human growth hormone (rhGH) elicited a dose-dependent stimulation of the proliferation of osteoblast-like cells (OB), when grown in strictly serum-free longterm cultures. A half-maximal effect was observed at concentrations of 15-20 ng/ml and the maximal stimulation was 160% of hormone-free controls. The rhGH-induced effect on proliferation could be inhibited dose-dependently by the addition of an insulin-like growth factor (IGF) I-antiserum to the medium. Moreover, IGF I and rhGH had additive effects only when the exogenous IGF I concentration exceeded that of endogenously produced IGF I by a large margin. Thus, direct stimulation of OB proliferation by rhGH is, at least in part, mediated by IGF I-like immunoreactivity.     

Alteration in pancreatic immunoreactivity of insulin-like growth factor (IGF)-binding protein (IGFBP)-6 and in intracellular degradation of IGFBP-3 in fibroblasts of IGF-II receptor/IGF-II-deficient mice.

The Type-2 insulin-like growth factor receptor (IGF2R) mediates the transport of lysosomal hydrolases to lysosomes and the clearance of insulin-like growth factor II (IGF-II). Mutant mice lacking IGF2R usually die perinatally, but are completely rescued from lethality in the absence of IGF-II. IGF2R/IGF-II-deficient mice have elevated levels of circulating IGF binding protein (IGFBP)-3 and show a strong IGFBP-6 immunoreactivity in all pancreatic islet cells and in secretory granules of different size in acinar cells and interlobular connective tissue of exocrine pancreas. Fibroblasts derived from double mutant mice missort the lysosomal protease cathepsin D, and are able to degrade endocytosed (125I)IGFBP-3 intracellularly, however, with lower efficiency than in control cells. These results show that the deficiency of IGF2R and IGF-II affects the expression and metabolism of IGFBPs in a tissue- and cell type-specific manner.     

Progesterone regulates FGF10, MET, IGFBP1, and IGFBP3 in the endometrium of the ovine uterus

Progesterone (P4) is unequivocally required to maintain a uterine environment conducive to pregnancy. This study investigated the effects of P4 treatment on expression of selected growth factors (fibroblast growth factor 7 [FGF7], FGF10, hepatocyte growth factor [HGF], and insulin-like growth factors [IGF1 and IGF2]), their receptors (MET, FGFR2(IIIB), and IGF1R), and IGF binding proteins (IGFBPs) in the ovine uterus. Ewes received daily injections of corn oil vehicle (CO) or 25 mg of P4 in vehicle from 36 h after mating (Day 0) to hysterectomy on Day 9 or Day 12. Another group received P4 to Day 8 and 75 mg of mifepristone (RU486, a P4 receptor antagonist) from Day 8 through Day 12. Endometrial FGF10 mRNA levels increased between Day 9 and Day 12 and in response to P4 on Day 9 in CO-treated ewes, which had larger blastocysts, and were substantially reduced in P4+RU486-treated ewes, which had no blastocysts on Day 12. Endometrial FGF7 or HGF mRNA levels were not affected by day or reduced by RU486 treatment, but MET mRNA levels were higher in P4-treated ewes on Day 9 and Day 12. Levels of IGF1, IGF2, and IGF1R mRNA in the endometria were not affected by early P4 treatment. Although stromal IGFBPs were unaffected by P4, levels of IGFBP1 and IGFBP3 mRNA in uterine luminal epithelia were increased substantially between Day 9 and Day 12 of pregnancy in CO-treated ewes and on Day 9 in early P4-treated ewes. Therefore, FGF10, MET, IGFBP1, and IGFBP3 are P4-regulated factors within the endometrium of the ovine uterus that have potential effects on endometrial function and peri-implantation blastocyst growth and development.