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R C Baxter

Publications and source records attributed to R C Baxter.

At least 73 records · Page 4Linked to original sources
PubMed1998

The regulation of acid-labile subunit gene expression and secretion by cyclic adenosine 3',5'-monophosphate.

Circulating acid-labile subunit (ALS) is mainly hepatocyte derived and is GH dependent. ALS buffers the metabolic effects of the insulin-like growth factors by sequestering them in a ternary complex with insulin-like growth factor-binding protein-3. Nutritional regulation of ALS may be mediated by cAMP and changes in circulating GH levels or tissue GH sensitivity. Therefore, we examined the regulation by cAMP of ALS steady state messenger RNA (mRNA) levels and secretion in isolated hepatocytes under basal and GH-induced conditions. Increasing intracellular cAMP in primary hepatocytes produced a dose-dependent suppression of ALS mRNA levels and secretion. This effect was not related to a reduction in mRNA stability. In the presence of GH there was a parallel suppression of mRNA levels and secretion. However, under basal conditions cAMP had less effect on ALS mRNA levels than on secretion. Thus, in the absence of GH, expression of ALS may be predominantly posttranscriptionally regulated by cAMP. Our study suggests that cAMP affects ALS gene transcription, perhaps by interrupting the GH signaling pathway, and also inhibits posttranscriptional events in ALS expression.

Animals↗
PubMed1998

Developmental changes in serum levels of free and total insulin-like growth factor I (IGF-I), IGF-binding protein-1 and -3, and the acid-labile subunit in rats.

We have recently described a competitive binding assay for rat insulin-like growth factor-binding protein-3 (IGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid-labile subunit (ALS) in the presence of IGF-I. Using this assay we studied groups of male (n = 6) and female rats (n = 6) at 20, 30, 40, 50, 60, 80, and 130 days of age. Nonfasting serum levels of IGFBP-3 were compared with those of total (extractable) IGF-I (tIGF-I) and ALS as well as IGFBP-3 determined by ligand blotting. Additionally, we studied the relationship between ultrafiltered free IGF-I (fIGF-I) and immunoassayable IGFBP-1. IGFBP-3 was dependent on age only (P < 0.0001), but tended to be higher in males than in females (P = 0.06); between 20-130 days levels increased from 6.5 +/- 1.7 to 73.6 +/- 7.2 nmol/liter in males and from 5.4 +/- 1.6 to 51.3 +/- 8.0 nmol/liter in females. IGFBP-3 correlated positively with tIGF-I (r = 0.90; P < 0.0001), ALS (r = 0.92; P < 0.0001), and IGFBP-3, as determined by ligand blotting (r = 0.88; P < 0.0001). The molar ratio of IGFBP-3 to tIGF-I increased from 0.23 +/- 0.04 to 0.76 +/- 0.04 (P < 0.0001) without any sex dependence. An age- and sex-dependent decrease in IGFBP-1 was observed (P < 0.0001), from 10.9 +/- 2.5 to 1.2 +/- 0.2 nmol/liter in females and from 8.9 +/- 0.7 to 0.2 +/- 0.04 nmol/liter in males. Free IGF-I (fIGF-I) increased with age (from 0.7 +/- 0.2 to 7.1 +/- 0.5 nmol/liter; P < 0.0001), and levels were inversely correlated with IGFBP-1 (r = -0.80; P < 0.0001). In young rats, IGFBP-1 circulated in a 10-fold molar excess over the level of fIGF-I, whereas in older rats, fIGF-I exceeded IGFBP-1 by an average of 9-fold in females and by up to almost 60-fold in males. We conclude that in rats 1) IGFBP-3 and fIGF-I are strongly age dependent; 2) IGFBP-3 correlates positively with ALS and tIGF-I; and 3) fIGF-I and IGFBP-1 are inversely correlated. This is in accordance with clinical findings. However, in humans the adult level of fIGF-I rarely exceeds 0.3 nmol/liter, and IGFBP-1 usually circulates in excess of fIGF-I. Thus, our results also imply species differences in the IGF systems of humans and rats.

Age Factors↗
PubMed1998

Competitive binding assay for determination of rat insulin-like growth factor binding protein-3.

We describe a novel competitive assay for rat insulin-like growth factor (IGF)-binding protein-3 (rIGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid labile subunit (ALS) in the presence of IGF. Human (h)ALS was bound to test tubes pre-coated with anti-human ALS antibody. The assay depends on competition between a covalent complex of 125I-hIGF-I and hIGFBP-3, added as tracer, and hIGFBP-3 or rIGFBP-3 in standards and test samples, for binding to the immobilized hALS. Purified natural hIGFBP-3 served as standard. Human IGFBP-3 and rIGFBP-3 were able to compete for tracer binding in the presence, but not in the absence, of IGF-I. Before assay, rat serum samples were acidified to denature endogenous ALS. Standards ranged from 0.10 (lower detection limit) to 20 ng/tube. Rat serum, semipurified rIGFBP-3, human serum and purified hIGFBP-3 diluted in parallel. The level of rIGFBP-3 was 1.63+/-0.28 mg/l (mean+/-SEM) in young rats and increased to 3.41+/-0.26 mg/l (p < 0.05) in old rats (n = 5-6). Fasting for 3 days reduced rIGFBP-3 from 2.41+/-0.27 to 1.33+/-0.14 mg/l (p < 0.05). Levels of rIGFBP-3 were reduced in hypophysectomized (0.16+/-0.04 mg/l; p < 0.05) and diabetic rats (1.04+/-0.30 mg/l; p < 0.05), and normal in insulin-treated diabetic rats (2.49+/-0.04 mg/l; ns), when compared to controls (2.79+/-0.22 mg/l). Changes in levels of IGFBP-3 parallelled those of immunoreactive rALS. We conclude that this assay provides a novel method of quantitating functional IGFBP-3 in rat serum.

Animals↗
PubMed1998

Neuroendocrinology of prolonged critical illness: effects of exogenous thyrotropin-releasing hormone and its combination with growth hormone secretagogues.

The catabolic state of prolonged critical illness is associated with a low activity of the thyrotropic and the somatotropic axes. The neuroendocrine component in the pathogenesis of these low activity states was assessed by investigating the effects of continuous intravenous infusions of TRH, GH-releasing peptide-2 (GHRP-2), and GHRH. Twenty adult patients, critically ill for several weeks, were studied during two consecutive nights. They had been randomly allocated to one of three combinations of peptide infusions, each administered in random order: TRH (one night) and placebo (other night), TRH + GHRP-2 (one night) and GHRP-2 (other night), or TRH + GHRH + GHRP-2 (one night) and GHRH + GHRP-2 (other night). The peptide infusions were started after a 1-microgram/kg bolus and infused (1 microgram/kg per h) until 0600 h. Blood sampling was performed every 20 min, and pituitary hormone secretion was quantified by deconvolution analysis. Reduced pulsatile fraction of TSH, GH, and PRL secretion and low serum concentrations of T4, T3, insulin growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) were documented in the untreated state. Infusion of TRH alone or in combination with GH secretagogues augmented nonpulsatile TSH release 2- to 5-fold; only TRH + GHRP-2 increased pulsatile TSH secretion (4-fold). Average rises in T4 (40-54%) and in T3 (52-116%) were obtained with all three combinations, whereas reverse T3 levels did not increase, except when TRH was infused alone. Pulsatile GH secretion was amplified > 6- and > 10-fold, respectively, by GHRP-2 and GHRH + GHRP-2 infusions, generating mean increases of serum IGF-I (66% and 106%), IGFBP-3 (50% and 56%), and ALS (65% and 97%) within 45 h. The addition of TRH did not alter the GH secretory patterns. TRH infusion increased PRL release only when combined with GH secretagogues. No effects on serum cortisol were detected. In conclusion, the pathogenesis of the low activity state of the thyrotropic and somatotropic axes in prolonged critical illness appears to have a neuroendocrine component, because these axes are both readily activated by coinfusion of TRH and GH secretagogues.

Adult↗
PubMed1998

Leptin levels in protracted critical illness: effects of growth hormone-secretagogues and thyrotropin-releasing hormone.

Prolonged critical illness is characterized by feeding-resistant wasting of protein, whereas reesterification, instead of oxidation of fatty acids, allows fat stores to accrue and associate with a low-activity status of the somatotropic and thyrotropic axis, which seems to be partly of hypothalamic origin. To further unravel this paradoxical metabolic condition, and in search of potential therapeutic strategies, we measured serum concentrations of leptin; studied the relationship with body mass index, insulin, cortisol, thyroid hormones, and somatomedins; and documented the effects of hypothalamic releasing factors, in particular, GH-secretagogues and TRH. Twenty adults, critically ill for several weeks and supported with normocaloric, continuously administered parenteral and/or enteral feeding, were studied for 45 h. They had been randomized to receive one of three combinations of peptide infusions, in random order: TRH (one day) and placebo (other day); TRH + GH-releasing peptide (GHRP)-2 and GHRP-2; TRH + GHRH + GHRP-2 and GHRH + GHRP-2. Peptide infusions were started after a 1-microgram/kg bolus at 0900 h and infused (1 microgram/kg.h) until 0600 h the next morning. Serum concentrations of leptin, insulin, cortisol, T4, T3, insulin-like growth factor (IGF)-I, IGF-binding protein-3 and the acid-labile subunit (ALS) were measured at 0900 h, 2100 h, and 0600 h on each of the 2 study days. Baseline leptin levels (mean +/- SEM: 12.4 +/- 2.1 micrograms/L) were independent of body mass index (25 +/- 1 kg/m2), insulin (18.6 +/- 2.9 microIU/mL), cortisol (504 +/- 43 mmol/L), and thyroid hormones (T4: 63 +/- 5 nmol/L, T3: 0.72 +/- 0.08 nmol/L) but correlated positively with circulating levels of IGF-I [86 +/- 6 micrograms/L, determination coefficient (R2) = 0.25] and ALS (7.2 +/- 0.6 mg/L, R2 = 0.32). Infusion of placebo or TRH had no effect on leptin. In contrast, GH-secretagogues elevated leptin levels within 12 h. Infusion of GHRP-2 alone induced a maximal leptin increase of +87% after 24 h, whereas GHRH + GHRP-2 elevated leptin by up to +157% after 24 h. The increase in leptin within 12 h was related (R2 = 0.58) to the substantial rise in insulin. After 45 h, and having reached a plateau, leptin was related to the increased IGF-I (R2 = 0.37). In conclusion, circulating leptin levels during protracted critical illness were linked to the activity state of the GH/IGF-I axis. Stimulating the GH/IGF-I axis with GH-secretagogues increased leptin levels within 12 h. Because leptin may stimulate oxidation of fatty acids, and because GH, IGF-I, and insulin have a protein-sparing effect, GH-secretagogue administration may be expected to result in increased utilization of fat as preferential substrate and to restore protein content in vital tissues and, consequently, has potential as a strategy to reverse the paradoxical metabolic condition of protracted critical illness.

Adult↗
PubMed1998

The effects of recombinant human IGF-I administration on concentrations of acid labile subunit, IGF binding protein-3, IGF-I, IGF-II and proteolysis of IGF binding protein-3 in adolescents with insulin-dependent diabetes mellitus.

The long term therapeutic potential of recombinant human (rh) IGF-I administration in insulin-dependent diabetes mellitus (IDDM) may be determined by changes in the IGF binding proteins (IGFBPs) and thus the bioavailability of IGF-I. We have therefore studied the effects of a single subcutaneous dose of rhIGF-I (40 micrograms/kg at 1800 h), when compared with an untreated control night, in 17 subjects with IDDM, on serum concentrations of IGF-I, IGF-II, IGFBP-3, acid labile subunit (ALS), and IGFBP-3 proteolysis. Mean (+/- S.E.M.) IGF-I levels increased from 242 +/- 30 ng/ml to 399 +/- 26 ng/ml (P = 0.01) after rhIGF-I whereas IGF-II levels declined from 600 +/- 45 ng/ml to 533 +/- 30 ng/ml. There was a small overnight reduction in baseline ALS levels from 48 +/- 2.8 to 44.5 +/- 3.2 micrograms/ml (P = 0.04) after rhIGF-I administration. An early fall in IGFBP-3 concentrations on the control night was not seen after rhIGF-I and overall mean levels were increased (5.2 +/- 0.2 micrograms/ml vs 4.9 +/- 0.2 micrograms/ml, P = 0.04, on the control night). On the baseline night, IGFBP-3 levels correlated with the sum of IGF-I and IGF-II (r = 0.73, P = 0.02) and with levels of the ALS (r = 0.7, P = 0.002). However after rhIGF-I, the sum of IGF-I and IGF-II no longer correlated with IGFBP-3, whereas the relationship with ALS was maintained. Immunoblot studies in six subjects indicated that 60%-70% of the IGFBP-3 was detected as a low molecular weight fragment at 1900 h on both study nights, but the amount of fragment declined to approximately 50% at 0100 h and 45% at 0700 h. In conclusion, despite a slight but significant fall in ALS, IGFBP-3 levels rise after rhIGF-I administration in IDDM. This cannot be explained by alterations in IGFBP-3 proteolysis, and may relate to the relative stability of ALS/IGFBP-3 when complexed principally with IGF-I rather than IGF-II.

Adolescent↗
PubMed1998

Regulation of insulin-like growth factor-binding protein-3 ternary complex formation in pregnancy.

The IGFs are believed to be important in pregnancy and are implicated in the pathophysiology of pre-eclampsia. In adults the IGFs circulate primarily with IGF-binding protein-3 (IGFBP-3) and an acid-labile glycoprotein (ALS) in a 140 kDa complex which limits IGF bioavailability. Less than 10% of IGFBP-3 is in lower molecular weight forms. We have investigated the developmental regulation of the IGF/IGFBP system in normal and pre-eclamptic pregnancies with particular emphasis on the IGFBP-3 ternary complex. Circulating levels of IGF-I, IGFBP-3 and ALS, and their degree of association in the ternary complex in the fetus increased with gestational age. In neonatal serum from deliveries <35 weeks' gestation IGFBP-3 was predominantly in 30-50 kDa form(s) and ALS was a limiting factor for ternary complex formation. In serum from deliveries >35 weeks both ALS and IGFs were limiting but approximately 25% of IGFBP-3 was unable to form the ternary complex even in the presence of excess ALS and IGF-I. Serum IGFBP-1, -2 and -6 concentrations tended to decrease with increasing gestational age. In pre-eclamptic pregnancies, amniotic fluid IGFBP-2, -3 and -6 levels decreased with gestational age while IGFBP-1 levels did not show the normal decline. We speculate that the endocrine IGF system develops in the fetus during the third trimester of pregnancy when ALS levels increase.

Adult↗
PubMed1997

Phosphorylation of insulin-like growth factor binding proteins.

Insulin-like growth factor (IGF) binding proteins (IGFBPs) play a key role in regulating the availability of IGFs in the circulation and the extracellular environment. Three of these proteins-IGFBP-1, IGFBP-3 and IGFBP-5-are known to be serine-phosphorylated in their central domains, and the others have possible target sites for serine/threonine kinases. Whereas nonphosphorylated IGFBP-1 may potentiate IGF action in certain cells, phosphorylation increases its affinity for IGFs, and converts the protein to an inhibitory form. The highly phosphorylated protein predominates in the circulation, where it may acutely regulate IGF bioavailability. IGFBP-3 is also secreted as a phosphoprotein, and can be phosphorylated in vitro by protein kinases A and C, and casein kinase II. De-phosphorylation has no effect on IGF-binding, but may increase its ability to bind to the acid-labile subunit and to associate with cell surfaces. Although no specific functions have yet been ascribed to phosphorylated forms of the other IGFBPs, current evidence supports the proposal that IGFBP phosphorylation plays an important role in the regulation of IGFBP function.

Animals↗
PubMed1997

Insulin-like growth factors (IGFs) and IGF binding proteins, serum acid-labile subunit and growth hormone binding protein in nephrotic children.

We hypothesized that the increased glomerular permeability to serum proteins in the nephrotic syndrome might lead to alterations of the somatotropic hormone axis, thereby contributing to growth failure and catabolism in the nephrotic state. The insulin-like growth factors (IGF)-I and -II and the IGF binding proteins (IGFBP)-1, -2 and -3 were analyzed in serum and urine of 21 children with the nephrotic syndrome and normal glomerular filtration rate. Mean age-related serum IGF-I levels by RIA (-0.53 +/- 0.34 SD) were slightly, but significantly (P < 0.05) decreased compared with the reference population, whereas mean age-related serum IGF-II levels (0.68 +/- 0.21 SD) were slightly, but significantly (P < 0.005) increased. The urinary excretion rate of both peptides was enhanced fivefold. By RIA, mean age-related serum IGFBP-1 (2.05 +/- 0.19 SD) and, even more pronounced, IGFBP-2 (5.97 +/- 0.65 SD) were clearly elevated despite a 12-fold and 2-fold increase of the respective urinary excretion rate. There was a tight and specific correlation between age-related serum IGFBP-2 levels and the degree of the nephrotic syndrome, as estimated by serum albumin levels (r = -0.78, P < 0.0001). Serum immunoreactive IGFBP-3 levels were also elevated (1.79 +/- 0.33 SD) in nephrotic serum, due to an increase of low-molecular weight IGFBP-3 fragments. By FPLC analysis, there was a decrease of the 150 kDa IGFBP ternary complex in nephrotic serum, which in the presence of normal concentrations of the acid-labile subunit by RIA appears to be due to a reduction of intact IGFBP-3. Serum levels of the high-affinity GH binding protein that presumably reflects GH receptor status in tissues were normal. In summary, total serum IGFs in children with the nephrotic syndrome are normal, but the binding of IGFs to IGFBPs in the circulation is altered with a shift from the 150 kDa IGFBP complex to an excess of low molecular weight IGFBPs. Because increased unsaturated high-affinity IGFBPs in nephrotic serum have the ability to inhibit IGF action on target tissues by competing with the type 1 IGF receptor for IGF binding, this alteration is likely to contribute to growth failure and tissue catabolism in the nephrotic state.

Carrier Proteins↗
PubMed1997

Human renal fibroblasts modulate proximal tubule cell growth and transport via the IGF-I axis.

To determine the paracrine interactions involved in the tubulointerstitial response to progressive renal disease, the role of insulin-like growth factor-I (IGF-I) and its binding proteins (IGFBPs) in in vitro interactions between human proximal tubule cells (PTC) and renal cortical fibroblasts (CF) were studied in primary cell culture. PTC growth and transport were increased in the presence of CF-conditioned media (CF-CM), as shown by increased thymidine incorporation, cellular protein content and sodium-hydrogen exchange (NHE) activity, to 185 +/- 31% (P < 0.01), 150 +/- 18% (P < 0.05) and 195 +/- 27% (P < 0.01) of the control values, respectively. IGF-I was produced by cultured CF at a rate of 64.6 +/- 7.5 ng/mg protein/day. Exogenous IGF-I applied to PTC provoked similar enhancement of growth and NHE activity as CF-CM and the stimulatory effect of CF-CM was blocked by specific immunoneutralization of IGF-I receptors. These receptors were threefold more abundant on PTC basolateral versus apical membranes. IGF binding proteins (IGFBP)-2 and IGFBP-3 were secreted by CF at rates of 694 +/- 88 and 1769 +/- 45 ng/mg/day, with the release of IGFBP-3 being enhanced in the presence of PTC-CM (120.0 +/- 9.7% of control, P < 0.01). Moreover, the addition of CF-CM to PTC increased cell-associated IGFBP-3 on PTC surfaces, without changes in IGF-I receptor numbers or affinity and without changes in PTC mRNA for IGFBP-3. Des(1-3)IGF-I, an analog that binds to the IGF-I receptor but not to IGFBPs, provided a less potent stimulus for PTC growth compared with IGF-I, indicating that cell-associated IGFBP-3 facilitates the action of IGF-I on PTC. The results support important paracrine roles for both IGF-I and IGFBPs in the interstitial regulation of proximal tubule growth and transport.

Antibodies↗
PubMed1997

The effect of an intravenous infusion of IGF-I and insulin on IGFBP-1, IGFBP-3, acid labile subunit, free and bound IGF-I, catecholamines and potassium in normal volunteers during an amino acid and glucose clamp.

OBJECTIVES: To investigate the effects of IGF-I and insulin at doses equipotent with respect to hypoglycaemic effect on IGF-I concentrations (free and bound), IGF binding proteins, catecholamines and potassium levels. DESIGN: A glucose and amino acid clamp technique was used to investigate the effects of a 3 h intravenous infusion of either IGF-I 43.7 pmol/kg/min (20 micrograms/kg/h) or insulin 3.4 pmol/kg/min (0.5 mU/kg/min). MEASUREMENTS: Circulating levels of total IGF-I, free and bound IGF-I, Insulin, IGFBP-1, IGFBP-3, ALS, catecholamines and potassium were measured. PATIENTS: 6 normal human volunteers aged 21-49. RESULTS: During the IGF-I infusion, IGF-I levels increased (P < 0.01) (26.6 +/- 2.8-88.9 +/- 14.2 nmol/l) and insulin levels fell (P < 0.05) (16.7 +/- 2.9-7.2 +/- 1.6 mu/l). During the insulin infusion, insulin levels increased (P < 0.01) (11.7 +/- 1.6-56.7 +/- 16 mu/l) and there was no change in IGF-I. There was no significant change in IGFBP-3 or ALS during the IGF-I or insulin infusions. There was a significant (P < 0.05) fall in IGFBP-1 levels from 27.1 +/- 4.1-8.06 +/- 1.6 micrograms/l during the insulin infusion and a significant (P < 0.05) rise during the first 120 min of IGF-I infusion from 28.6 +/- 6.3-67.9 +/- 10.6 micrograms/l. There was a significant (P < 0.05) increase in free IGF-I and IGF-I bound to the 150 and 50 kD plasma fractions during the IGF-I infusion. At basal and the end of the IGF-I infusion adrenaline levels (pmol/l) were: 173 +/- 22 and 174 +/- 28; dopamine levels (pmol/l) were: 415 +/- 87 and 470 +/- 87; plasma potassium (mmol/l) was 4.2 +/- 0.06 and 3.56 +/- 0.13. At basal and the end of the insulin infusion adrenaline levels (pmol/l) were: 160 +/- 38 and 163 +/- 21; dopamine levels (pmol/l) were: 238 +/- 26 and 316 +/- 31; plasma potassium (mmol/l) was 4.2 +/- 0.07 and 3.92 +/- 0.17. There were no significant changes in plasma catecholamine concentrations with either infusion but potassium concentrations were significantly (P < 0.05) reduced during the IGF-I infusion. CONCLUSIONS: We conclude that an infusion of IGF-I resulted in increased levels of both free and bound IGF-I. IGF-I and insulin under conditions of adequate substrate supply have acute effects on IGFBP-1 and potassium physiology, but have little effect on IGFBP-3, ALS or catecholamines.

Adult↗
PubMed1997

Decreased hepatic insulin-like growth factor (IGF)-I and increased IGF binding protein-1 and -2 gene expression in experimental uremia.

The imbalance between normal insulin-like growth factor-I (IGF-I) and markedly increased IGF binding protein (IGFBP) plasma levels plays a pathogenic role for growth retardation and catabolism in children with chronic renal failure. To investigate the mechanism of these alterations, experiments were performed in an experimental model of uremia in rats (5/6 nephrectomy) and in pair-fed and ad libitum-fed sham-operated controls Using a specific solution hybridization/RNase protection assay, we observed a marked reduction of hepatic IGF-I messenger RNA (mRNA) abundance at steady state in uremic animals (37 +/- 5% of control) compared both with pair-fed (65 +/- 10%) and ad libitum-fed controls (100 +/- 11%) (P < 0.001). Reduced IGF-I gene expression was clearly organ-specific; it was most pronounced in liver (significant vs., pair-fed controls) and lung and muscle tissue (significant vs., ad libitum-fed controls); no change was observed in kidney and heart tissue. To determine a potential mechanism of reduced hepatic IGF-I gene expression in uremia, the hepatic GH receptor gene expression in the same experimental animals was analyzed by specific solution hybridization/RNase protection assay. Uremic animals had a 20-30% reduction of hepatic GH receptor mRNA abundance compared with controls. Hepatic GHBP expression in uremia was decreased in parallel. Despite the reduction of hepatic IGF-I mRNA abundance, plasma IGF-I levels in uremia were not different from ad libitum-fed controls. This discrepancy is explained by an increased concentration of IGFBPs in uremic plasma. By RIA, plasma IGFBP-1 levels in uremia were increased 4-fold; by Western immunoblot, plasma IGFBP-2 levels were increased 7-fold and plasma IGFBP-4 levels were increased 2-fold compared with both control groups. Intact IGFBP-3 (M(r), approximately 48 kDa) and low molecular IGFBP-3 fragments were not significantly different among the three groups. By Northern blot analysis, hepatic IGFBP-1 mRNA levels in uremia were 2-fold higher than in controls. IGFBP-2 mRNA abundance in liver tissue was increased 4-fold, whereas in kidney there was a significant reduction of IGFBP-2 mRNA (30% of control). IGFBP-4 mRNA was increased by 50% in kidney but not in liver. Plasma insulin and corticosterone levels were not different among the groups. Our study shows that hepatic IGF-I gene expression was specifically reduced in uremia, partially as the consequence of a reduced hepatic GH receptor gene expression. One of the mechanisms contributing to increased IGFBP levels in uremia is increased hepatic gene expression of IGFBP-1 and IGFBP-2. The imbalance between reduced hepatic IGF-I production and increased hepatic IGFBP-1 and 2 production is likely to play a pathogenic role for catabolism and growth failure in CRF.

Animal Nutritional Physiological Phenomena↗
PubMed1997

Insulin-like growth factor binding protein (IGFBP)-3 protease activity secreted by MCF-7 breast cancer cells: inhibition by IGFs does not require IGF-IGFBP interaction.

The proliferative action of insulin-like growth factors (IGFs) on breast cancer cells is regulated by IGF binding proteins (IGFBPs). This study characterizes the proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 human breast cancer cells. Proteolysis of IGFBP-3 by incubation at 37 C with serum-free medium from MCF-7 cells was maximal at pH 5.0-5.5, with no activity detected below pH 4.5. This enzyme activity resulted in the disappearance of the 40- to 45- and 30-kDa bands of pure plasma-derived IGFBP-3, detectable by immunoblotting after SDS-PAGE, and the appearance of a single 21-kDa immunoreactive species. The 21-kDa protein did not bind IGF-I or IGF-II by ligand blotting. The enzyme activity appeared at 25- to 30-kDa by gel chromatography at pH 6.5 and was inhibited by EDTA and leupeptin, an inhibitor of cysteine and serine proteases, but not by the serine protease inhibitors aprotinin and benzamidine. IGFBP-3 protease activity was inhibited in medium conditioned by cells incubated with 50 ng/ml IGF-I. A similar inhibitory effect was seen under cell-free conditions by adding IGF-I to medium harvested from cells incubated without IGFs. The cell-free inhibition of IGFBP-3 proteolysis by IGFs did not require IGF interaction with the binding protein, because [long Arg3]IGF-I, which binds to IGFBP-3 with less than 0.2% of the potency of IGF-I, inhibited IGFBP-3 proteolysis with 20% of the potency of IGF-I. These results suggest that IGFs may regulate their own activity in breast cancer cells, preventing IGFBP-3 proteolysis by a mechanism that is not receptor mediated and does not require IGF-IGFBP interaction.

Breast Neoplasms↗
PubMed1997

Characterization of truncated insulin-like growth factor-binding protein-2 in human milk.

Truncated forms of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) have been purified from human milk and shown to retain partial IGF-binding activity. By affinity chromatography on agarose-IGF-I and HPLC, truncated IGFBP-2 of apparent Mr 14,000-16,000 resolved into two peaks. Both peaks bound radioiodinated IGF-II on ligand blotting. Within both peaks, two sequences were identified, starting at Gly169 and Lys181 of hIGFBP-2 (predicted Mr, 13,786 and 12,502, respectively, if both extend to Gln289). Mass spectrometry of a fraction predominantly containing Gly169 peptides yielded two major species, 13,840 and 13,425 Mr. Prolonged incubation of radioiodinated recombinant human (rh) IGFBP-2 with human milk failed to reveal any degradation, suggesting the formation of the fragments within the mammary gland. By solution binding assay, truncated IGFBP-2 showed less than 10% binding of [125I]IGF-I and 25% binding of [125I]IGF-II at pH 7.0 compared with rhIGFBP-2. No binding activity was seen at pH 4.0, in contrast to intact IGFBP-2, which showed peak binding from pH 4.0 to at least pH 9.0. The IGF-II association constant for truncated IGFBP-2 (6.5 nM(-1)) was 10-fold lower than that for intact IGFBP-2 (58 nM(-1)). Des(1-6)-IGF-II was totally inactive in displacing IGF-II tracer from the IGFBP-2 fragment, but displaced tracer from rhIGFBP-2 with 10% the activity of IGF-II. Thus, the amino-terminal hexapeptide of IGF-II is required for interaction with the carboxy-terminal domain of IGFBP-2. The presence of active IGFBP-2 fragments in milk suggests a role for milk IGFBP-2 in modifying IGF activity in the neonatal gut.

Amino Acid Sequence↗
PubMed1997

Serum insulin-like growth factor I (IGF-I), IGF-binding protein-1 and -3, and the acid-labile subunit as serum markers of body composition during growth hormone (GH) therapy in adults with GH deficiency.

Serum levels of insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), the acid-labile subunit (ALS), insulin, and IGFBP-1 were evaluated as indicators of body composition during GH replacement therapy in 20 GH-deficient patients (9 women and 11 men), aged 22-57 yr, with IGF-I levels below -2 SD. The mean GH dose was 0.128 +/- 0.003 IU/kg.week during the first month and thereafter 0.23 +/- 0.01 IU/kg.week, divided into daily doses (0.7-4.3 IU/day). Serum levels of IGF-I, ALS, and IGFBP-3 above the normal range were reached in seven, five, and three subjects, respectively, after 12 months of GH therapy. IGF-I and ALS levels, but not IGFBP-3 levels, correlated with the total daily GH dose (r = 0.676; P = 0.001 and r = 0.631; P = 0.003). The mean increase in lean body mass (LBM) measured by dual energy x-ray absorptiometry was 3.0 +/- 0.5 kg (P < 0.001). At 12 months, the LBM values were significantly correlated to the IGF-I levels (r = 0.718; P < 0.001), but not to ALS or IGFBP-3 levels. No correlation was found before therapy, and the increase in LBM at 12 months correlated with the IGF-I increase (r = 0.514; P = 0.029) only after exclusion of two nonresponders. Both before and during therapy, LBM was inversely related to IGFBP-1 (r = -0.715; P < 0.004 at 12 months). None of the GH-induced proteins could be used as indicators of body fat changes. In conclusion, both IGF-I and ALS can be used as indicators to avoid GH excess during replacement therapy, but only IGF-I relates to changes in LBM.

Adipose Tissue↗