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R C Baxter

Publications and source records attributed to R C Baxter.

At least 109 records · Page 6Linked to original sources
PubMed1995

Modulation of human IGF binding protein-3 activity by structural modification.

To delinate regions of IGFBP-3 involved in ligand and cell-surface binding. DNAs encoding human IGFBP-3 [1-264] and several variants were transfected into CHO cells. Of three deletion (delta) mutants. IGFBP-3 [1-88], [1-184], and [delta 89-184], none bound IGF-I tracer by ligand blotting, although all were detectable by immunoblotting. No ALS binding was detectable, as predicted by the lack of IGF binding. Normal-sequence IGFBP-3 associated with the CHO cells and was partly displaceable by IGF-I. Whereas IGFBP-3 [1-88] and [1-184] failed to cell-associate, the non-IGF-binding central deletion variant [delta 89-184] did associate with CHO cells but was not displaced by IGF-I. To further examine the role of the carboxy-terminal domain in cell-association, the basic sequence IGFBP-3 [228-232] (KGRKR) was altered to the corresponding IGFBP-1 residues MDGEA, a major charge reversal. This variant showed reduced IGF-I binding, and bound ALS with decreased affinity as determined by Scatchard analysis. It showed no cell binding, implicating the basic domain in cell-association. We conclude that, whereas the central and carboxy-terminal domain deletions fail to bind IGF-I, the ability to cell associate requires the carboxy-terminal but not the central domain. Specifically, the basic region [228-232] is essential for cell binding, and also affects IGF-I binding, and independently, ALS affinity.

Amino Acid Sequence↗
PubMed1995

The role of glycosylation in the action of IGFBP-3.

There are three potential N-glycosylation sites (Asn-X-Ser/Thr) located in the nonconserved central region of the human IGFBP-3 sequence (Asn89, Asn109, Asn172-sites 1, 2 and 3, respectively). Upon ligand blotting with IGFs, IGFBP-3 appears as two bands (40-45 kDa) representing different glycosylated forms. We have mutated the N-glycosylation sites in permutations of three single, three double and one triple mutations and expressed these variant cDNAs. Each mutant protein was detected by radioimmunoassay, indicating that glycosylation is not required for the secretion of the protein from CHO cells. Ligand blotting using [125I]IGF-I indicated that all seven mutants retained IGF-I binding. Based on the molecular weights of the variant proteins, there are approximately 4, 5 and 6 kDa of carbohydrate on sites 1, 2 and 3, respectively. Furthermore, the two forms of IGFBP-3 represent the protein glycosylated either at all three sites or at Asn89 and Asn109 only. There appears to be no difference between the mutants and the fully-glycosylated rhIGFBP-3 in their acid-labile subunit (ALS) binding. Analysis of variance confirmed that the association constant for ALS was not significantly changed by any mutation [Ka (fully-glycosylated) = 12.5 +/- 4.1 nM-1; mean Ka (all mutants) = 22.1 +/- 3.0 nM-1]. While glycosylation does not appear to play a role in IGFBP-3 ligand binding, it may affect the turnover rate of the protein or be involved in rendering the protein resistant to proteolysis.

Animals↗
PubMed1995

Characterisation of the rat acid-labile subunit gene.

The acid-labile subunit (ALS) of the ternary insulin-like growth factor binding protein (IGFBP) complex has a central role in regulating the bioavailability of circulating IGF. We have shown that gene expression of ALS in vivo and in vitro is regulated by a variety of factors, including growth hormone (GH). Our aim was to isolate and characterise the ALS gene as a step in defining the mechanism of its regulation. Southern analysis of rat genomic DNA suggests that the ALS gene exists as a single copy in the rat genome. In order to isolate this gene we screened 5 x 10(5) clones and selected fragments of two genomic clones were sequenced. Comparison of this sequence with the cDNA identified two exons and a single approximately 1.1 kb intron. Primer extension experiments suggest two major transcription initiation sites at -539 and -396 nts relative to the translational initiation codon, although there are no consensus TATA-boxes in this region. Analysis of 2.3 kb of 5' flanking sequence identified two LF-A1 sites which may confer the liver-specific expression of the ALS gene. In addition, there are several elements that may be involved in regulation by growth hormone and cytokines.

Adaptor Protein Complex alpha Subunits↗
PubMed1995

Developmental regulation of circulating insulin-like growth factor-binding proteins in normal pregnancies and in pre-eclampsia.

The insulin-like growth factors (IGFs) and their binding properties (IGFBPs) are believed to play important roles in the growth and development of the human fetus. They have been implicated in the pathophysiology of pre-eclampsia. In this study we have characterized the developmental regulation, in normal and pre-eclamptic pregnancies, of IGFs and IGFBPs in maternal serum, neonatal serum and amniotic fluid. In neonatal cord serum IGFBP-1, -2 and -6 decreased with increasing gestational age. In contrast, the ternary complex and its components, IGF-I, IGFBP-3 and ALS increased with gestation. We show that while ALS is an important limiting factor for ternary complex formation in the fetal circulation, there is a fraction of IGFBP-3 which is unable to form this complex. IGFs and IGFBPs in the maternal and fetal circulation were similar in normal and pre-eclamptic pregnancies.

Amniotic Fluid↗
PubMed1995

Regulation of insulin-like growth factor (IGF) binding protein-3 phosphorylation by IGF-I.

Insulin-like growth factor (IGF) action is modulated by six IGF-binding proteins (IGFBP-1 to -6). IGFBP-3 is the main IGFBP in serum and is produced by many cell types, which can modify it post-translationally to yield glycosylation, proteolysis and phosphorylation products. This study investigates the regulation of IGFBP-3 phosphorylation by IGF-I in human neonatal skin fibroblasts. Fibroblasts were incubated with IGF peptides and 32P-orthophosphate for 4 h, and phosphorylated IGFBP-3 (P-IGFBP-3) was immunoprecipitated from the medium and analysed by SDS-PAGE. Media collected from parallel experiments without radioactivity were assayed for immunoreactive IGFBP-3 (I-IGFBP-3). IGF-I (50 ng/ml) increased levels of P-IGFBP-3 and I-IGFBP-3 in conditioned medium to 205 +/- 9% and 198 +/- 10% of control, respectively (n = 5). Stimulation of I-IGFBP-3 was consistent with IGF-mediated release of cell-associated IGFBP-3, since treatment with an IGF-I analogue with reduced affinity for IGFBPs did not increase I-IGFBP-3 levels, whereas treatment with an analogue with reduced affinity for receptor but normal affinity for binding proteins did. In contrast, stimulation of P-IGFBP-3 occurred independently of IGF binding to IGFBP, instead requiring interaction of IGF-I with its receptor. While the functional significance of IGFBP-3 phosphorylation is unclear, we propose that it plays a regulatory role in IGFBP-3 action.

Cells, Cultured↗
PubMed1995

Insulin-like growth factor-binding protein-3 production by MCF-7 breast cancer cells: stimulation by retinoic acid and cyclic adenosine monophosphate and differential effects of estradiol.

The insulin-like growth factors (IGFs) stimulate the proliferation of human breast cancer cells, including the estrogen-dependent cell line MCF-7. These cells secrete regulatory IGF-binding proteins (IGFBPs) which may enhance or attenuate IGF-stimulated cell proliferation. In this study, we have used RIA to quantify the production and regulation of IGFBP-3 and IGFBP-6 by MCF-7 cells in vitro. Under basal (serum- and phenol red-free) conditions, IGFBP-3 and IGFBP-6 accumulated in 72 h-conditioned MCF-7 medium to concentrations of approximately 0.18 nM and 0.02 nM, respectively. Treatment with retinoic acid (RA, 100 nM) increased medium concentrations of IGFBP-3 to 175 +/- 8% (mean +/- SE, n = 4), and IGFBP-6 to 217 +/- 20% of control values. Forskolin (0.5 microM) or dibutyryl cAMP (db-cAMP, 1 mM) increased both proteins 2- to 3-fold. In the presence of 100 nM RA, the stimulation elicited by these agents was enhanced, with IGFBP-3 levels increasing to 6-fold above that seen with RA alone. IGFBP-6 increased 12-fold with RA + forskolin and 20-fold with RA + dbcAMP. Estrogen (10 nM estradiol) reduced basal IGFBP-3 levels by 25% but increased IGFBP-6 1.5- to 2-fold. The stimulatory effect of RA + forskolin on IGFBP-3 was partially reversed by estrogen, whereas RA + forskolin-stimulated IGFBP-6 levels were further increased by estrogen. Increased IGFBP-3 and -6 production in response to RA + forskolin was accompanied by a decrease in IGF-stimulated thymidine incorporation into DNA; by contrast, the bioactivity of an IGF analog that does not bind with IGFBPs, [Gln3, Ala4, Tyr15, Leu16]IGF-I, was unchanged under these conditions. These data demonstrate that modulating the production of IGFBPs can lead to changes in the sensitivity of breast cancer cells to IGFs, and as a result change the cell proliferative effects of these growth factors. Further, IGFBP-3 and IGFBP-6 are differentially regulated by estrogen. Dissecting the roles of the individual IGFBPs is essential to understanding how such differential regulation will ultimately affect IGF-stimulated cell proliferation in breast cancer.

Breast Neoplasms↗
PubMed1995

Energy metabolism and substrate oxidation in acromegaly.

Short term GH administration increases lipid breakdown and oxidation (lipidox) and reduces glucose uptake and carbohydrate oxidation (CHOox). It is not clear whether similar shifts in substrate oxidation occur in acromegaly, and our aim was to investigate this. Using indirect calorimetry, we compared energy expenditure, CHOox, and lipidox in 20 acromegalic patients and 20 normal subjects pair-matched for sex, age, height, and weight. Investigations were performed in the basal state (12-h fast) and during a 75-g oral glucose tolerance test (OGTT). Acromegalic patients had significantly higher fasting glucose levels and greater glucose and insulin responses during an OGTT than normal subjects. Fasting nonesterified free fatty acid and insulin-like growth factor (IGF)-binding protein-1 levels were similar in the two groups, and both were acutely suppressed by oral glucose to the same degree. Basal energy expenditure was significantly greater in the acromegalic patients (1682 +/- 49 vs. 1540 +/- 45 Cal/24 h; P < 0.05), who showed a trend toward higher basal CHOox. Oral glucose resulted in a significantly higher rise in energy expenditure in the normal compared to the acromegalic subjects. During the OGTT, CHOox significantly increased in both groups, but rose to a higher level in the acromegalic patients (177 +/- 10 vs. 138 +/- 9 mg/min; P = 0.004). Oral glucose significantly reduced lipidox in both groups, but lipidox was reduced to a significantly lower level in the acromegalic patients (32 +/- 4 vs. 46 +/- 3 mg/min; P = 0.004). In acromegaly, basal CHOox (r = 0.56; P = 0.01) and postglucose CHOox (r = 0.79; P = 0.0001) were both positively correlated to IGF-I, but not to insulin and/or glucose. In normal subjects, postglucose CHOox was positively correlated to IGF-I. In summary, hyperinsulinemia in acromegaly was associated with higher glucose levels and a blunted thermogenic response to glucose, and displayed no relationship to the pattern of substrate oxidation. CHOox was increased, and lipidox was reduced in acromegaly, and the extent of IGF-I elevation was related to CHOox in the basal and postglucose states. We conclude that 1) the chronic effects of GH excess on substrate oxidation differ from the short term effects of GH administration; 2) impaired insulin action in acromegaly extends to effects on energy expenditure; and 3) IGF-I may be an important regulator of substrate oxidation in acromegaly.

Acromegaly↗
PubMed1995

Regulation of the insulin-like growth factors and their binding proteins by glucocorticoid and growth hormone in nonislet cell tumor hypoglycemia.

Hypoglycemia in patients with nonislet cell tumors is often secondary to overexpression of tumor insulin-like growth factor (IGF) II. In these patients the formation of serum complexes between IGFs, IGF binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) is impaired. An 87-yr-old woman with nonislet cell tumor hypoglycemia resulting from a localized fibrous tumor of the pleura was treated for 97 days with graded doses of prednisolone (30, 10, and 5 mg/day) followed by GH (1, 4, 8, 4, and 2 U/day). Both prednisolone and GH alleviated the hypoglycemia, concomitantly with increases in IGF-I, IGFBP-3, and ALS levels. Pretreatment serum IGFBP-2 and IGFBP-6 levels were greatly elevated, but as glucose normalized with treatment, only IGFBP-2 decreased, showing an inverse correlation with glucose (r = 0.716). IGFBP-1 gave a variable pattern not clearly related to blood glucose. Both treatments caused a redistribution of serum IGFBP-3 from binary- to ternary-complexed forms. However, only prednisolone improved the ability of IGFBP-3 to bind ALS in vitro. Prednisolone also suppressed IGF-II, the effect being confined to pro-IGF-II forms. Compared with normal IGF-II, pro-IGF-II inhibited ALS binding to IGFBP-3 in vitro. Although prednisolone and GH reverse hypoglycemia by different mechanisms, with only prednisolone suppressing tumor IGF-II secretion, both increase the formation of ternary IGF-IGFBP-3 complexes. We conclude that the failure of serum IGFBP-3 and tumor IGF-II to complex with ALS is a primary cause of hypoglycemia in nonislet cell tumor hypoglycemia.

Aged↗
PubMed1994

Diagnosis of growth-hormone deficiency in adults.

There is no consensus as to the most appropriate method of diagnosing growth-hormone (GH) deficiency in adults. We have evaluated the relative diagnostic merits of measuring peak GH response to insulin-induced hypoglycaemia (insulin tolerance test), mean 24 h GH concentration derived from 20 min sampling, serum insulin-like growth factor I (IGF-I) concentrations, and serum IGF binding protein 3 (IGFBP-3) concentrations. These tests were undertaken in 23 patients considered GH deficient from extensive organic pituitary disease, and in 35 sex-matched normal subjects of similar age and body-mass index. Hypopituitary subjects had significantly lower stimulated peak GH, mean 24 h GH, IGF-I, and IGFBP-3 concentrations than normal subjects. The ranges of stimulated peak GH responses were clearly separated between the hypopituitary (< 0.2-3.1 ng/mL) and normal (5.3-42.5 ng/mL) groups, but mean 24 h GH, IGF-I, and IGFBP-3 concentrations overlapped. Mean 24 h GH concentrations were below assay sensitivity in 80% of hypopituitary subjects and 16% of normal subjects. 70% and 72%, respectively, of the IGF-I and IGFBP-3 values in hypopituitary subjects were within the range for normal subjects. We conclude that GH deficiency in adults is most reliably identified by stimulatory testing, and that IGF-I and IGFBP-3 are poor diagnostic tests of adult GH deficiency.

Adolescent↗
PubMed1994

Regulation of immunoreactive insulin-like growth factor binding protein-6 in normal and transformed human fibroblasts.

Insulin-like growth factor-binding proteins (IGFBPs) have been shown to both potentiate and inhibit IGF bioactivity in vitro; thus, changes to the type or amount of IGFBPs present in the cellular environment will ultimately affect insulin-like growth factor action. In this study, we have investigated the production of immunoreactive IGFBP-6 by normal human fibroblasts (NHF) and an SV-40-transformed human fibroblast line (AG2804). When analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting, IGFBP-6 appeared as a doublet of 32-34-kDa in conditioned medium of both cell lines, with the lower molecular mass band predominating in the NHF cell line. Measured by a specific radioimmunoassay, serum-free NHF, and AG2804 cultures secreted IGFBP-6 at 1.44 +/- 0.09 and 1.23 +/- 0.08 ng/10(4) cells (mean +/- S.E.), respectively. Despite a relatively weak IGFBP-6 signal by ligand blot compared with IGFBP-3, the two proteins were secreted in similar molar concentrations by NHF. Retinoic acid increased IGFBP-6 by 3-fold in NHF and AG2804-conditioned media, maximal at approximately 100 nM retinoic acid. In contrast, IGFBP-6 production was inhibited by transforming growth factor-beta 1 and agents that increase intracellular cAMP concentrations, including dibutyryl cAMP, forskolin, isobutylmethylxanthine, and cholera toxin. This study indicates that IGFBP-6 has a pattern of regulation unique among the IGFBPs, supporting the concept of specific roles for each binding protein in regulating cell growth and metabolism.

1-Methyl-3-isobutylxanthine↗
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

Insulin-like growth factor binding proteins in the human circulation: a review.

The actions and bioavailability of the insulin-like growth factors (IGFs) are regulated by a family of six IGF binding proteins. IGFBP-3, the major circulating IGFBP, is unique in combining with a glycoprotein, the acid-labile subunit (ALS), to form a ternary complex with IGF-I or IGF-II. Each component of this trimer is growth hormone-dependent, and the hypoglycemic potential of circulating IGFs appears neutralized in this form. IGFs in the complex have a greatly extended circulating half-life, their stability being conferred by the presence of ALS, which is itself very stable in the circulation. IGFBP-1 does not appear to be a carrier of IGFs, but to act as an acute modulator of their metabolic activities. In this role it can be viewed as an insulin counter-regulator, blocking 'free' insulin-like activity during fasting or hypoglycemia. IGF-I administration causes complex changes in circulating IGFBPs, so that a detailed knowledge of their regulation is essential if the therapeutic potential of IGF-I is to be optimised.

Adult↗
PubMed1994

Purification and characterization of the acid-labile subunit of rat serum insulin-like growth factor binding protein complex.

Circulating insulin-like growth factor (IGF) binding protein-3 (IGFBP-3), when occupied by IGF-I or IGF-II, combines with an acid-labile glycoprotein subunit (ALS) to form a high molecular weight complex. In this study, ALS from rat serum has been purified and its properties investigated. Purification involved ion-exchange chromatography, and affinity chromatography on an IGF-I-IGFBP-3 column, yielding almost 1 mg ALS from 100 ml serum. Amino-terminal sequencing confirmed the prediction from previous complementary DNA analysis but indicated that the protein may circulate in a truncated form. Rat ALS was almost as potent as human ALS in binding to human IGF-I-IGFBP-3 complex (association constant, 2.3 nM-1). A sensitive RIA was developed, with high specificity for rodent ALS. Serum ALS rose from 3 micrograms/ml in 2-day-old rats to more than 40 micrograms/ml at 10 weeks, with no sex difference. GH-deficient rats showed 60-75% lower values than controls. This study shows rat ALS to have similar binding properties, and age and GH dependence, to human ALS. The new RIA will facilitate studies of IGF and IGFBP regulation in the rat.

Aging↗
PubMed1994

Complex formation by human insulin-like growth factor-binding protein-3 and human acid-labile subunit in growth hormone-deficient rats.

Insulin-like growth factor-binding protein-3 (IGFBP-3), after first associating with IGF-I or IGF-II, is able to associate with the acid-labile subunit (ALS) and form a 140-kDa complex. To investigate the factors regulating ternary complex formation in vivo, human (h) IGFBP-3, hIGF-I, and hALS were administered in various combinations to GH-deficient (dw/dw) rats. hIGFBP-3 had a complex pattern of disappearance from the rat circulation, with an initial phase lasting minutes and a prolonged phase(s) lasting hours. If coinjected with hIGF-I, significantly more hIGFBP-3 was retained over 2 h. The molecular distribution of hIGFBP-3 was determined after size-separation chromatography. After an iv bolus of hIGFBP-3, 36.1 +/- 5.0% was in the 140-kilodalton complex at 5 min; this increased to 55.1 +/- 7.1% if hIGF-I was coinjected (P < 0.05). The 140-kDa complex disappeared slowly over hours, whereas 50- and 30-kDa forms of hIGFBP-3 cleared rapidly, with half-lives of minutes. To determine the importance of ALS in regulating the molecular distribution of hIGFBP-3, hALS was coinjected. Immunoreactive hALS disappeared slowly from the circulation and was shown to retain functional activity after 2 h in vivo. Coadministration of hALS did not influence the pattern of ternary complex formation, consistent with the presence of excess endogenous rat ALS. We conclude that ALS circulates in excess even in GH deficiency, is retained in the circulation for hours, and determines the stability of the 140-kDa complex, whereas IGF-I is a limiting factor in ternary complex formation by hIGFBP-3.

Animals↗
PubMed1994

Cellular localization and regulation of gene expression for components of the insulin-like growth factor ternary binding protein complex.

Insulin-like growth factors (IGFs) are present in the circulation, largely as part of a high mol wt complex including IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit (ALS). This study used in situ hybridization to investigate the cellular sites of synthesis of these factors in the rat and to evaluate changes in transcript levels during development and after hypophysectomy and GH treatment. IGFBP-3 transcripts are considerably more abundant and widely expressed than ALS at birth, but both are present in liver and kidney. Hepatic IGFBP-3 gene expression increases slightly, whereas ALS increases dramatically in the first few weeks after birth. IGFBP-3 mRNA is concentrated in portal venous and sinusoidal endothelium, but is not detected in hepatocytes, whereas ALS mRNA is diffusely expressed by hepatocytes, but is not detected in nonparenchymal cells. Both transcripts are localized in the renal cortex; however, IGFBP-3 mRNA is concentrated in interstitial cells, whereas ALS is expressed in proximal tubule epithelium. Hypophysectomy results in a 90% reduction in hepatic ALS and an approximately 50% decrease in IGFBP-3 mRNA level. ALS, but not IGFBP-3, transcripts were also reduced in the kidney. GH receptor mRNA is coexpressed with ALS in liver and kidney, suggesting that the effects of GH on ALS gene expression may be direct. In summary, the fact that IGFBP-3 gene expression is far more widespread than that of ALS in both spatial and temporal parameters suggests that IGFBP-3 has a role apart from contribution to the ternary complex. We have also shown that IGFBP-3 and ALS are synthesized by distinct hepatic cell types in an anatomical organization that may serve to ensure efficient formation of the ternary complex in the blood passing through the sinusoids. Finally, the present data suggest that regulation of ALS synthesis may be the primary site of GH regulation of ternary complex formation.

Animals↗
PubMed1994

Regulation of the acid-labile subunit of the insulin-like growth factor complex in cultured rat hepatocytes.

The acid-labile subunit (ALS) is a glycoprotein that forms a ternary complex in serum with insulin-like growth factors and insulin-like growth factor-binding protein-3. This study investigates the regulation of ALS production, measured by RIA, and messenger RNA (mRNA) content, measured by Northern analysis, in primary rat hepatocyte monolayer cultures. Hepatocytes produced ALS at a linear rate over 48 h. Exposure to human GH (30 ng/ml) caused a maximum 2.2-fold stimulation of ALS production compared to that in control cultures, giving a rate of 200 ng/10(6) cells.48 h. ALS mRNA appeared as a predominant 2-kilobase band and increased 4-fold by administration of 30 ng/ml GH. Both dexamethasone and epidermal growth factor (EGF) inhibited ALS production, with maximal effects at 100 nM dexamethasone and 50 ng/ml EGF (both approximately 50% inhibition). ALS mRNA levels measured 24 or 48 h after dexamethasone addition were decreased 75-80% compared to the control value. A similar decrease in ALS mRNA was observed 24 h after EGF addition, but a second addition of EGF at 24 h was required to maintain this decrease for 48 h. This study demonstrates that rat hepatocytes secrete immunoreactive ALS under GH regulation, and that EGF and corticosteroid inhibit ALS production and mRNA levels. The quantitative discrepancies between ALS production rates and mRNA levels suggest that posttranscriptional events may have a role in ALS regulation.

Acids↗