Biomedical subjects

P J Fielder

Publications and source records attributed to P J Fielder.

At least 37 records · Page 2Linked to original sources

PubMed1995

Long-term effects of insulin-like growth factor (IGF)-I treatment on serum IGFs and IGF binding proteins in adolescent patients with growth hormone receptor deficiency.

OBJECTIVE: The aim of this investigation was to study the effect of relatively high dose IGF-I therapy given for several months, on serum levels of IGF-I, IGF-II and IGFBP-3, and on IGF-I pharmacokinetics in patients with growth hormone insensitivity due to GH receptor dysfunction. DESIGN AND PATIENTS: Two adolescent subjects from Ecuador were treated with recombinant IGF-I at a dosage of 120 micrograms/kg s.c. twice daily, in combination with a GnRH analogue for 8 months. MEASUREMENTS: Serum was sampled at baseline and at 3-8 months, for determination of IGF-I, IGF-II and IGFBP-3 by radioimmunoassay, and for evaluation of IGFBPs and IGFBP-3 protease activity by Western ligand blot and protease assay, respectively. RESULTS: Peak serum IGF-I levels ranged from 272 to 492 micrograms/l. Mean serum IGF-II levels were decreased concurrently with the increase in IGF-I. Serum IGFBP-3 levels failed to rise with prolonged IGF-I treatment. There was no apparent change in the half-life of IGF-I during the treatment period. CONCLUSIONS: IGF-I administration does not increase serum levels of IGFBP-3 or significantly alter IGF-I pharmacokinetics.

Adolescent↗

PubMed1995

Adaptation of the growth hormone and insulin-like growth factor-I axis to chronic and severe calorie or protein malnutrition.

The hierarchy of diet components (e.g., protein, carbohydrate, vitamins, and minerals) influencing growth hormone (GH), insulin-like growth factor-I (IGF-I), and their binding proteins (BP) is not well defined. Young adult rats were fed diets for 1 mo that included low protein or 60% and 40% of carbohydrate calories. We hypothesized that levels of both hormones, their dominant BPs and liver IGF-I mRNA would fall, and that part of the mechanism for decreasing serum IGF-I would be enhanced IGFBP-3 protease activity. By day 30, caloric deprivation to 40% lowered serum GH, GHBP, IGF-I and IGFBP-3, and liver IGF-I mRNA. This was the only condition resulting in body weight loss (-15%) vs 39% gain in controls. Restriction to 60% calories had no impact on BP levels, slightly lowered IGF-I (-12%) in the face of a 95% inhibition of GH levels, while allowing a modest 9% body weight gain. Protein deprivation lowered serum GH, IGF-I and IGFBP-3, and liver IGF-I mRNA, while GHBP levels were normal. The reduced total IGF-I under these dietary conditions could not be explained by an increase in IGFBP-3 protease activity, or a decrease in the association of IGF-I with IGFBP-3 and the acid labile subunit.

Amino Acid Isomerases↗

PubMed1995

Interleukin-1 and tumor necrosis factor-alpha increase insulin-like growth factor-binding protein-3 (IGFBP-3) production and IGFBP-3 protease activity in human articular chondrocytes.

IGF-I is the major anabolic factor for cartilage matrix production. Chondrocytes and cartilage treated with interleukin-1 alpha (IL-1 alpha), and chondrocytes from several models of inflammatory joint disease, exhibit reduced responsiveness to IGF-I. Since the IGF-binding proteins (IGFBPs) modulate the effects of IGF-I, we examined the effect of IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) on IGFBP production by normal human articular chondrocytes in primary culture. Western ligand blots and immunoprecipitation of conditioned medium samples showed that articular chondrocytes produced IGFBPs-2, -3 and -4 and glycosylated IGFBP-4. Both IL-1 alpha and TNF-alpha increased chondrocyte production of IGFBP-3, but did not alter IGFBP-4 production. The activity of a neutral metalloprotease with the ability to cleave IGFBP-3 was also increased by IL-1 alpha. These data suggest that the cytokines IL-1 alpha and TNF-alpha may act to reduce IGF-I access to chondrocytes by increasing production of IGFBP-3. This may be a factor in the decreased matrix production in the inflammatory arthritides.

Blotting, Western↗

PubMed1995

Characterization of the insulin-like growth factors (IGF) axis in a cultured mouse Leydig cell line (TM-3).

Characterization of the insulin-like growth factor (IGF) axis in a cultured mouse Leydig cell line (TM-3) was performed. Radioimmunoassayable IGF-I and IGF-II concentrations in TM-3 conditioned media (CM) were below the assay detection levels. Affinity cross-linking of IGF-I and IGF-II to crude membranes prepared from TM-3 cells revealed both type 1 and type 2 receptors and a 31 kDa IGF binding protein (IGFBP). Immunoprecipitation of solubilized crude membranes indicated that the 31 kDa protein was membrane associated IGFBP-4. Western ligand blots of CM from TM-3 cells demonstrated the presence of 24 and 28 kDa bands as major IGFBPs, and 25, 40, and 44 kDa as minor ones. The 28 kDa band could be invisible after Endoglycosidase-F treatment, suggesting that the 28 kDa IGFBP was a glycosylated form of IGFBP. The 24 kDa and 28 kDa bands were immunoprecipitated with an antibody to IGFBP-4. Treatment of TM-3 cells with IGF-I increased the levels of the 24 kDa IGFBP-4, as well as 25, 28, 40, and 44 kDa IGFBPs. IGF-I treatment also resulted in the appearance of a 29 kDa IGFBP. Neither the 25 nor 29 kDa bands were deglycosylated with Endoglycosidase-F nor immunoprecipitated by antibodies to rIGFBP-1 and -2. On the other hand, IGF-II treatment resulted in a significant decrease in the 24 kDa IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Is there heterozygote expression of growth hormone receptor deficiency?

Expression of heterozygosity for the defect in the growth hormone (GH) receptor has been proposed to be reflected in stature, and in GH binding protein (GHBP) and insulin-like growth factor I (IGF-I) levels in parents and other relatives of patients with GH receptor deficiency (GHRD; Laron syndrome). The Ecuadorean population with GHRD, in which heterozygosity can be accurately determined in clinically unaffected relatives of probands, offers a unique opportunity to consider this issue. It has previously been demonstrated that 17 parents heterozygous for the Ecuadorean mutation of the GH receptor differed little in biochemical measures (GHBP, IGF-I, IGF-II, IGFBP-2 and IGFBP-3) from Ecuadorean controls. Mean height SDS of 24 non-carrier siblings (-1.3 +/- 0.95 SD) and 41 heterozygote siblings or offspring of probands (-1.8 +/- 1.15) did not differ significantly (p = 0.08). Thus, although there may be slight heterozygote expression of the defective gene for the GH receptor, there is no rationale for counselling based on such minimal variation.

Carrier Proteins↗

PubMed1994

Effects of insulin-like growth factor I treatment on the molecular distribution of insulin-like growth factors among different binding proteins.

The molecular distribution of insulin-like growth factor I (IGF-I) and IGF-II among the IGF binding proteins (IGFBPs) was studied before and during IGF-I therapy in Ecuadorean adults with growth hormone receptor deficiency (GHRD). Of the total circulating IGF-I and IGF-II, 70% was carried by the 150 kDa complex in normal subjects, while in patients with GHRD, 50% of serum IGF-I, but only 30-35% of serum IGF-II, was measured within the 150 kDa IGFBP-3 region. Administration of IGF-I altered the concentration of IGF-I and IGF-II, although the percentage of total IGF measured within each IGFBP region was not affected, as the increase in IGF-I and the decrease in IGF-II were proportional. Similarly, serum concentrations of IGFBP-3 and the acid-labile subunit, measured by radioimmunoassay, were unaltered. Thus, administration of IGF-I to patients with GHRD was unable to correct the aberrant distribution of IGFs among the IGFBPs.

Adult↗

PubMed1994

Biochemical analysis of prostate specific antigen-proteolyzed insulin-like growth factor binding protein-3.

Prostate specific antigen (PSA) has been shown to proteolyze IGFBP-3. However, the cleavage sites and mechanism of proteolysis are unknown. In this study, we proteolyzed recombinant human IGFBP-3 with PSA bound to a solid phase support. The reaction mixture was separated by centrifugation, with PSA remaining in the solid phase and the proteolyzed IGFBP-3 in the aqueous phase. The IGFBP-3 fragments were functionally analyzed by affinity labeling and Western ligand blotting (WLB). Further biochemical analyses were provided by silver staining of total protein and Western immunoblotting (WIB) of immunoreactive fragments with an IGFBP-3 specific antiserum (alpha-BP-3 g1). N-terminal sequence analysis was performed on filter-immobilized IGFBP-3 fragments, following size separation by SDS-polyacrylamide electrophoresis. PSA proteolyzed IGFBP-3 into at least 7 fragments (M(r) of 26 kDa to 13 kDa) as identified by silver staining and WIB. At least 3 fragments were visible by affinity labeling with radiolabeled IGF-I or IGF-II and 4 were weakly visible by WLB. These data indicate that some IGFBP-3 fragments retain their ability to bind IGF. N-terminal sequence analysis revealed at least 5 different proteolytic recognition sites for PSA in IGFBP-3. Three of the 5 sites were consistent with a 'kallikrein-like' enzymatic activity and 2 sites were consistent with a 'chymotryptic-like' enzymatic activity. The chymotryptic activity of PSA was further confirmed by the ability of alpha-1-antichymotrypsin and chymostatin to block PSA cleavage of radiolabeled IGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Serum profiles of insulin-like growth factors and their binding proteins in adults with growth hormone receptor deficiency treated with insulin-like growth factor I.

Six adult patients with growth hormone receptor deficiency (GHRD) (2 men, 4 women) with an identical defect in the growth hormone receptor (GHR) gene, were treated with recombinant human insulin-like growth factor I (IGF-I), 40 micrograms/kg s.c. twice daily, for 7 days. Serum concentrations of IGF peptide and IGF binding protein-3 (IGFBP-3) were measured by specific radioimmunoassays; serum IGFBPs were also measured by Western ligand blotting. The size distribution of both IGF-I and IGF-II was measured in serum following size-exclusion fast-performance liquid chromatography. IGF-I treatment resulted in a normalization of serum IGF-I levels on days 1-7 of treatment and a decrease in serum IGF-II levels. The fall in IGF-II levels and the simultaneous rise in IGF-I levels, however, resulted in an unchanged total serum IGF level. The low IGFBP-3 values did not significantly change during treatment, whereas there was a slight increase in IGFBP-2 levels. Preliminary analysis of size-fractionated sera suggested an increase in IGF-I levels in the 40 and 150 kDa regions at the expense of IGF-II levels. The results suggest that despite the failure of IGF-I treatment to increase IGFBPs significantly, serum IGFBP concentrations were sufficient to maintain normal levels of IGF-I.

Adult↗

PubMed1993

Characterization of the affinities of insulin-like growth factor (IGF)-binding proteins 1-4 for IGF-I, IGF-II, IGF-I/insulin hybrid, and IGF-I analogs.

Insulin-like growth factor (IGF)-binding proteins (BPs) bind IGF-I and IGF-II with high affinity and modify the activity of IGF peptides in a complex manner. We have characterized the affinities of IGFBP-1-4 for IGF-I and -II by employing 1) purified IGFBP preparations, 2) both [125I]IGF-I and [125I]IGF-II as radioligands, and 3) multiple IGF analogs designed to have altered affinities for IGFBPs. To this end, human (h) IGFBP-1, hIGFBP-2, and rat (r) IGFBP-4 have been purified to homogeneity from human amniotic fluid, human prostate epithelial cell culture, and B104 rat neuroblastoma cells; for human IGFBP-3, the glycosylated recombinant form (rec-hIGFBP-3), produced in Chinese hamster ovary cells, was employed. The IC50 values of IGF-I for hIGFBP-1, hIGFBP-2, rec-hIGFBP-3, rIGFBP-4, and human serum IGFBPs were 0.05 +/- 0.01, 5.0 +/- 0.01, 0.25 +/- 0.20, 0.6 +/- 0.4, and 0.1 +/- 0.01 ng/ml, respectively. While hIGFBP-1 and rIGFBP-4 had virtually equivalent affinities for IGF-I and IGF-II, hIGFBP-2 and rec-hIGFBP-3 demonstrated 2- to 5-fold higher affinities for IGF-II than for IGF-I. Studies with [Gln3,Ala4,Tyr15,Leu16]IGF-I and Des-(1-3)-IGF-I indicate that specific residues in the first 16 amino acids of the B domain of IGF-I appear to be critical for binding to all of the IGFBPs tested, but not to IGF receptors. However, severe modifications in the B domain disrupt binding affinity, not only for IGFBPs, but also for receptors (IGF-I/insulin hybrid and B-chain mutant). Interestingly, modifications in the A domain of IGF-I, which is believed to contain residues critical for binding to IGF-I and insulin receptors, show differential effects on binding affinity to BPs. [Thr49,Ser50,Ile51]IGF-I, which has normal affinity for the type I IGF receptor, shows at least a 500-fold decreased affinity for hIGFBP-1 and recombinant hIGFBP-3, in contrast to 50- to 100-fold reduced affinity for hIGFBP-2 and rIGFBP-4, and 5- to 10-fold reduced affinity for purified human serum IGFBP-3. More significantly, [1-27,Gly4,38-70]IGF-I shows a 30-fold decreased affinity for the type I IGF receptor and 10- and 2.5-fold reduced affinities for hIGFBP-1 and rec-hIGFBP-3, respectively, but no reduction in affinity for hIGFBP-2 or rIGFBP-4.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1993

Insulin-like growth factors (IGFs) block FSH-induced proteolysis of IGF-binding protein-5 (BP-5) in cultured rat granulosa cells.

Rat granulosa cells (GC), in vitro, express IGFBPs under the influence of both FSH and IGF-I. The major IGFBP produced by GC is a 28-29 K IGFBP, presumed to be IGFBP-5. When GC-conditioned medium (GC-CM) was assessed by Western Ligand Blotting (WLB), FSH appeared to decrease IGFBP-5, whereas IGF-I appeared to increase IGFBP-5 and to partially block the effects of FSH treatment. When GC-CM from FSH-treated cells was incubated with pure IGFBP-4 and IGFBP-5, the amount of IGFBP-5 (measurable by WLB) was decreased. Similarly, when GC-CM from FSH-treated cells was incubated with iodinated IGFBP-4 and IGFBP-5, IGFBP-5 (but not IGFBP-4) was proteolyzed into fragments of approximately 18 and 14 K. The ability of FSH-treated GC-CM to proteolyze IGFBP-5 was reduced by the addition of IGF-I to the reaction mixture. When the IGFBPs in GC-CM were evaluated by affinity crosslinking, GC-CM from control cultures contained one band with an apparent M(r) of approximately 34 K, whereas GC-CM from FSH-treated cultures displayed a decrease in the intensity of the 34 K band, as well as a new band of approximately 24 K. These data suggest that rat GC cells produce an FSH-inducible IGFBP-5 protease activity, and reveal that the ability of this protease to cleave IGFBP-5 is blocked by IGFs.

Animals↗

PubMed1993

Growth hormone receptor deficiency in Ecuador: clinical and biochemical phenotype in two populations.

We have identified 56 patients with GH receptor deficiency (Laron syndrome) from two provinces in southern Ecuador, one group of 26 (Loja province) with a 4:1 female predominance and 30 patients from neighboring El Oro province with a normal sex ratio. There were no significant differences between the Loja and El Oro populations in stature (-5.3 to -11.5 standard deviation score), other auxologic measures, or in biochemical measures. GH binding protein, the circulating extracellular domain of the GH receptor, was measured by ligand immunofunction assay and found to be comparably low in children and adults. Levels of insulin-like growth factor (IGF)-I and -II and the GH-dependent IGF binding protein-3 (measured by RIA) were significantly greater, and GH and IGF binding protein-2 levels significantly lower in adults than children. Levels of IGF-I (adults) and IGF binding protein-3 (children and adults) correlated inversely with statural deviation from normal (P < 0.01). School performance was at an exceptionally high level, 41 out of 47 who had attended school being in the top 3 in classes of 15-50 persons.

Adolescent↗

PubMed1993

Hormonal and metabolic effects and pharmacokinetics of recombinant insulin-like growth factor-I in growth hormone receptor deficiency/Laron syndrome.

Profound growth failure despite elevated GH levels in GH receptor deficiency (GHRD) results from reduced insulin-like growth factor-I (IGF-I) synthesis. Recent reports of improved growth velocity in children with GHRD during IGF-I therapy indicate growth-promoting potential in humans. We evaluated the pharmacokinetics and metabolic/hormonal effects of recombinant human IGF-I (40 micrograms/kg every 12 h) given sc for 7 days to six adults with GHRD. Hypoglycemia (< 2.5 mmol/L) did not occur, and mean 2 h postprandial insulin levels were reduced. Urinary calcium increased 2-fold (P < 0.01), and serum calcium was unchanged. The mean integrated 24-h GH level was suppressed (6.5 +/- 2.1 to 1 +/- 0.2 micrograms/L), as were the number of peaks, area under the curve, and clonidine-stimulated GH release (all P < 0.05). The mean pretreatment IGF-I level (36 +/- 2 micrograms/L) was 19% of the Ecuadorian control value (190 +/- 15 micrograms/L), it achieved a peak (253 +/- 11 micrograms/L) between 2-6 h after IGF-I injection, and at 12 h it was 137 +/- 8 micrograms/L. There were no significant changes in the half-life (8.2 +/- 1.5 to 9.7 +/- 1.9 h) or metabolic clearance (0.35 +/- 0.1 to 0.24 +/- 0.05 mL/kg.min) between days 1 and 7; however, distribution volume increased (183 +/- 10 to 266 +/- 36 mL/kg; P < 0.03). Baseline IGF-II levels were 47% of the control value and decreased during IGF-I therapy (273 +/- 10 to 178 +/- 9 micrograms/L; P < 0.01), correlating inversely with IGF-I levels (r = -0.3; P < 0.001). Although IGF-binding protein-3 (IGFBP-3) levels were not significantly influenced, baseline IGFBP-2 levels (153% of the control) increased 45% (P < 0.01). We conclude that IGF-I (40 micrograms/kg every 12 h) given sc to adults with GHRD is safe; achieves normal levels of IGF-I; reduces insulin, IGF-II, and GH levels; and increases IGFBP-2 concentrations and urinary excretion of calcium.

Adult↗

PubMed1993

The composition and distribution of insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) in the serum of growth hormone receptor-deficient patients: effects of IGF-I therapy on IGFBP-3.

We have previously reported that adult GH receptor-deficient (GHRD) patients treated subcutaneously with recombinant human insulin-like growth factor (IGF)-I have increased serum IGF-I levels and decreased IGF-II levels, whereas IGF-binding protein-3 (IGFBP-3) levels were unchanged. To further investigate the effects of IGF-I administration upon the IGF-IGFBP axis in GHRD, we have examined: 1) the molecular distribution of IGF-I and IGF-II among the IGFBPs; 2) the composition and distribution of the IGFBPs, in particular IGFBP-3; and 3) the acid labile subunit (ALS). Serum samples from adult GHRD patients who were treated sc with recombinant human IGF-I (40 micrograms/kg, sc, twice a day) or from normal Ecuadorian adults were incubated with [125I]IGF-II and subjected to neutral size-exclusion chromatography. The fractions were then subjected to Western ligand blot, Western immunoblot, IGFBP-3 RIA, and IGF RIAs. Serum of healthy adults incorporated [125I]IGF-II into the 150- and 44-kilodalton (kDa) IGFBP region. The 150-kDa IGFBP region contained most of the circulating IGFBP-3, whereas the 44-kDa IGFBP region contained mainly IGFBP-1, 2, and 4. The 150-kDa region also contained a unique 28-kDa immunoreactive form of IGFBP-3, which was not detectable by Western ligand blot. Endogenous IGF-I and IGF-II were distributed equally in the 150- and 44-kDa IGFBP regions. Sera from GHRD patients mainly incorporated [125I]IGF-II into the 44-kDa IGFBP region. Similar to control sera, the 150-kDa IGFBP region contained IGFBP-3, albeit at lower concentrations. The 44-kDa IGFBP region contained all IGFBPs including 50% of the total immunoreactive IGFBP-3. The two immunoreactive forms of IGFBP 3 (40- to 45-kDa doublet and 28-kDa band) were present in both IGFBP regions. The IGF size-distribution study revealed that the 150-kDa IGFBP region carried half of the circulating endogenous IGF-I, but only 30% of the IGF-II. Concentrations of the ALS were consistently low. Administration of IGF-I to GHRD patients was unable to increase concentrations of the molecular forms of IGFBP-3, correct the aberrant distribution of IGFs among the IGFBPs, or increase serum concentrations of ALS. In conclusion, we have found two forms of IGFBP-3 associated with IGF and ALS, which are capable of forming the ternary 150-kDa complex in healthy adult serum. The ratio of these two forms of IGFBP-3 and their distribution in serum was different between GHRD and control patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1993

Insulin-like growth factors (IGFs) stimulate and dexamethasone inhibits IGF binding protein (BP)-5 expression in a mouse pituitary cell line.

The mouse pituitary cell line AtT-20 was found to secrete two low MW IGFBPs into conditioned medium (CM). The major IGFBP migrated at approximately 29 kDa and a minor IGFBP of 24 kDa was also present on western ligand blots (WLB). Both IGFBPs were purified from CM by IGF-affinity chromatography followed by reverse phase-FPLC. N-terminal analysis revealed that the first 10 amino acids of the 29 kDa and the 24 kDa IGFBPs were homologous to corresponding sequences of both human and rat IGFBP-5 and IGFBP-4, respectively. The 24 kDa IGFBP also crossreacted with a new antiserum specific for rodent IGFBP-4. The concentrations of both IGFBPs were increased by the addition of IGF-I, IGF-II, or insulin to the cell cultures, with IGFBP-5 demonstrating the greatest hormonal stimulation. The effects of IGF-I on IGFBP-5 expression were both time and dose dependent, with IGF-I being more potent than IGF-II, and IGF-II more potent than insulin. The relative potencies of these hormones in stimulating IGFBP-5 production were consistent with the peptides acting through the type-I IGF receptor. Similarly, the IGF-II analog [Leu 27]-IGF-II, which has very low affinity for the type-I receptor, only slightly stimulated an increase in IGFBP-5. Addition of dexamethasone to the cultures decreased both basal and IGF-stimulated IGFBP-5 production. Northern blotting demonstrated that IGF-I increased the expression of the mRNA for IGFBP-5, whereas dexamethasone decreased it. Together, these data suggest that the IGFs can increase IGFBP-5 production at both the protein and mRNA level.

Amino Acid Sequence↗

PubMed1992

The insulin-like effects of mouse growth hormone on adipose tissue from virgin and pregnant mice.

The abilities of mouse prolactin (mPRL), growth hormone (mGH), placental lactogen (mPL)-I, and mPL-II to stimulate glucose oxidation were investigated, using adipose tissue from both virgin and pregnant mice. In separate experiments, adipose tissue segments from the parametrial fat pads of virgin and pregnant mice were collected on days 7, 13, and 17 of gestation. Tissue segments were first preincubated at 37 degrees C for 3 hours in Krebs-Ringer-HEPES (KRH) buffer without any added hormones. Following the preincubation, mPRL, mGH, recombinant mPL-I, mPL-II (1 microgram/mL), and porcine insulin (plns) (1 ng/L) were added to the incubations, along with 0.5 microCi/mL D-[U-14C]glucose, and incubated for an additional 2 hours. After the second incubation, the 14CO2 produced by the oxidation of the D-[U-14C]glucose was collected and counted. Both mGH and plns significantly stimulated glucose oxidation in adipose tissue from virgin and pregnant mice. However, mPRL, mPL-I, and mPL-II failed to stimulate glucose oxidation in adipose tissue from either virgin or pregnant mice. Preincubating adipose tissue, from day 13 of gestation, with 0.5 microgram/mL epinephrine (Epn) increased basal glucose oxidation and decreased both mGH- and plns-stimulated glucose oxidation. Preincubating adipose tissue, from day 13 of gestation, with 0.11 microgram/mL dexamethasone (Dex) significantly decreased the ability of the tissue to oxidize glucose. However, the Dex-treated tissue retained its ability to increase glucose oxidation in response to mGH and plns. These results indicate that mGH, but not mPRL, mPL-I, or mPL-II, has insulin-like effects on adipose tissue, and that mGH and plns may affect glucose oxidation through similar pathways.

Adipose Tissue↗

PubMed1992

Insulin-like growth factors (IGFs): implications for aging.

The insulin-like growth factors (IGF)-I and IGF-II are peptides with structural homology to insulin and potent mitogenic and anabolic actions in vitro and in vivo. IGF-I levels are growth hormone (GH)-dependent and vary strikingly with age. IGF-I levels are typically low in infancy and childhood, increase dramatically during puberty, and then gradually decline with advancing age. Whether age-associated changes in GH production or sex steroid secretion, or other unknown factors, cause diminished IGF production in the elderly remains to be determined. In the brain, IGF-II appears to be the most prevalent IGF, but a truncated form of IGF-I also has been recognized. IGF actions are mediated by binding to a family of receptors, which includes the insulin receptor, the structurally homologous type I IGF receptor, and the IGF-II/M-6P receptor, all of which are found in the central nervous system. Additionally, the IGFs bind with high affinity to a family of IGF-binding proteins (IGFBPs). Of the six known IGFBPs, IGFBP-2 appears to be the major one in the mammalian brain and is a major component of CSF. Immunoreactive IGFBP-2 has been identified in astrocytes, and its mRNA has been identified in fetal and adult brain and choroid plexus. The IGFBPs transport the IGFs in serum and other body fluids and appear to regulate IGF access to receptors. In vivo regulation of IGFBPs includes tissue-specific proteases, which cleave specific IGFBPs, altering their affinities for IGF peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1992

Regulation of insulin-like growth factor-binding protein expression by thyroid hormone in rat GH3 pituitary tumor cells.

Rat pituitary GH3 tumor cells express GH and insulin-like growth factor-I (IGF-I) mRNAs and possess specific IGF-I and IGF-II receptors which mediate the inhibitory effect of IGF on GH secretion. T3 increases the rate of cell replication and GH gene transcription, and causes an increase in IGF-I binding to cell membranes. Since the IGFs circulate in association with specific binding proteins (IGFBPs) that appear to modulate the access of IGFs to their receptors, we have investigated the effect of T3 treatment on the expression of IGFBPs in GH3 cells. Cells were grown in serum-free defined medium, and IGFBP secretion was determined by Western ligand blotting of conditioned medium after the addition of T3 and/or various peptides for 48-72 h. The conditioned medium of GH3 cells revealed a complex of bands migrating at 40-45 Mr, a pattern typical of rat (r) IGFBP-3. T3 treatment resulted in an increase in rIGFBP-3. IGF-I, IGF-II, and insulin did not alter rIGFBP-3 levels. After concentrating (10-fold) conditioned medium samples, two additional bands at 24K and 28K mol wt were also seen. These bands corresponded in size to rIGFBP-4 (24K) and its glycosylated form (28K). The mRNAs for both rIGFBP-3 and rIGFBP-4 were present in GH3 cells; T3 treatment increased steady state levels of rIGFBP-3 mRNA, but did not affect BP-4 mRNA levels. To learn whether the increased expression of IGFBPs could be responsible for the increased IGF-I binding seen after T3 treatment, [125I]IGF-I was cross-linked to GH3 membranes, and the proteins were separated on a 5-15% gradient sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. T3 treatment induced a large increase in the intensity of bands migrating at 135K and 280K, representing the alpha-subunit of the IGF-I receptor and an incompletely reduced alpha-alpha-dimer, respectively. No membrane-associated IGFBPs were detected. In conclusion, GH3 cells express two IGFBPs, rIGFBP-3 and rIGFBP-4, which are differentially regulated by T3. The increased binding of IGF-I to GH3 cell membranes after T3 treatment indicates that thyroid hormone induces an up-regulation of IGF-I receptors and that the increased IGF-I binding to GH3 membranes is not due to increased expression of membrane-associated IGFBPs.

Animals↗

PubMed1992

Expression of a lactogen-dependent insulin-like growth factor-binding protein in cultured mouse mammary epithelial cells.

The ability of normal mouse mammary epithelial cells (MECs) to express insulin-like growth factor-binding proteins (IGFBPs) was examined. MECs were isolated from day 11 pregnant mice and cultured on floating collagen gels in serum-free basal medium. After 24 h, the medium was replaced with fresh medium with/or without mouse PRL (mPRL), mouse placental lactogen-I (mPL-I), mPL-II, mouse GH (mGH), IGF-I, and IGF-II, either alone or in combinations. The MECs were cultured for an additional 5 days before collection of conditioned medium (CM). The relative amount of IGFBPs present in the CM was determined by Western ligand blotting, and alpha-lactalbumin content was determined with a specific RIA. The CM of the MECs contained two IGFBPs, with approximate mol wt of 29K and 40-45K. The 40-45K IGFBP appears to be the mouse equivalent of IGFBP-3, but the identity of the 29K IGFBP is not presently known. The 29K IGFBP was not N-glycosylated and did not cross-react with antiserum to rodent IGFBP-2 or human IGFBP-1. Basal IGFBP expression was very low, but the addition of mPL-I, or mPL-II stimulated a marked increase in the amount of 29K IGFBP that was released into the CM and a lesser increase in the release of IGFBP-3. This increase in the release of 29K IGFBP was dose dependent, with increases found at concentrations as low as 1 ng/ml lactogen. mGH also stimulated the release of 29K IGFBP, but was less potent than any of the three lactogens. Treatment of MECs with either IGF-I or IGF-II increased the amount of both the 29K IGFBP and IGFBP-3 in the CM, with relative potencies similar to those of the lactogenic hormones. However, when either IGF-I or IGF-II was added together with one of the lactogenic hormones, the release of 29K IGFBP was increased in an additive manner. While the IGFs acted additively with the lactogenic hormones on the expression of 29K IGFBP, they did not stimulate alpha-lactalbumin production by the MECs or act to enhance the effects of the lactogenic hormones in stimulating alpha-lactalbumin production. This study demonstrates that IGFBPs are expressed in normal mouse MECs, and the release of these IGFBPs into the CM is hormonally regulated by both lactogenic hormones and IGFs.

Animals↗