Insulin-like growth factor-binding protein-1 (IGFBP-1) regulates IGF availability for glucose homeostasis. The IGFBP-1 promoter shares common regulatory response elements with phosphoenol pyruvate carboxykinase (PEPCK), the expression and activity of which is inhibited by lithium chloride, associated with an inhibition of glycogen synthase kinase (GSK)-3 activity, in the rat hepatoma cell line H4-II-E. We therefore determined the effect of lithium chloride on IGFBP-1 expression and secretion in H4-II-E cells. Lithium chloride inhibited IGFBP-1 secretion in a dose response and reversible manner by approx 80% during 5-h and 16-h incubations. An inhibitory effect on IGFBP-1 mRNA expression was observed at 2 h. The inhibitory effect of lithium and insulin were not additive when used alone, but inhibition by lithium occurred when insulin action was blocked by activating AMP-activated protein kinase with 5-aminoimidazole-4-carboxamide-riboside (AICAR). These findings suggest that GSK-3 inhibition, or another pathway activated by lithium, may be involved in a pathway controlling IGFBP-1, inhibiting synthesis when insulin activity is absent or impaired.
MS Lewitt, K Brismar, J Ohlson and J Hartman
MS Lewitt, SJ Hazel, DB Church, AD Watson, SE Powell and K Tan
The 140 kDa ternary complex of insulin-like growth factor-binding protein-3 (IGFBP-3), IGFs and an acid-labile subunit (ALS) has previously been shown to be decreased in diabetes mellitus in humans and rats. We have studied IGF-I levels and ternary complex formation in normal and diabetic cats. Total IGF-I concentrations, measured by RIA using des(1-3)-IGF-I as tracer were (+/-s.e.m.) 54+/-13 nmol/l in eight normal and 227+/-57 nmol/l in eight diabetic cats (P<0.01). The size-distribution of IGFBPs in the cat circulation was determined by incubation with (125)I-IGF-II and Superose 12 chromatography. In normal animals 26+/-2% of the (125)I-IGF-II were in a 140 kDa form compared with 48+/-5% in diabetic cats (P<0.01). When samples from normal and diabetic animals were co-incubated 52+/-3% were at 140 kDa. A similar shift was seen when normal cat and normal human serum were co-incubated. A 2-fold increase in the 140 kDa form in diabetic cats was confirmed first by size-fractionating samples and then performing a ligand-binding assay with (125)I-IGF-I or -II and charcoal separation. SDS-PAGE and Western ligand blotting demonstrated a 45 kDa doublet (presumably IGFBP-3) and 30-35 kDa forms. There were no apparent differences between normal and diabetic profiles on SDS-PAGE, suggesting that a proportion of IGFBP-3 which circulates 'free' in normal cats forms a ternary complex in the diabetic circulation. We conclude that (i) in contrast to humans and rats, ALS is the limiting factor for ternary complex formation in normal cats, (ii) ALS concentrations increase in feline diabetes mellitus and, by promoting ternary complex formation, this leads to an increase in total IGF-I concentrations, and (iii) total IGF-I concentrations may not be reliable in the diagnosis of acromegaly in diabetic cats.
MS Lewitt, FP Scott, NM Clarke, T Wu, MJ Sinosich and RC Baxter
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.
MS Lewitt, D Cameron-Smith, NM Clarke, H Saunders and JL Phuyal
Insulin-like growth factor-binding protein-1 (IGFBP-1) production is increased by somatostatin and its analogues. In order to determine the time course and identify possible mechanisms of this increase in vivo we administered octreotide to rats and determined IGFBP-1 concentrations by RIA. After 60 min of anaesthesia, the mean baseline IGFBP-1 concentrations were 166 (95% confidence interval 123 to 225) ng/ml and increased in saline-infused animals to 729 (488 to 1086) ng/ml after 180 min. IGFBP-1 was stimulated transiently in response to octreotide, with circulating IGFBP-1 concentrations peaking at 1605 (1220 to 2111) ng/ml at 105 min during a continuous infusion of octreotide (100 micrograms/kg per h). In conscious chronically cannulated rats, baseline IGFBP-1 concentrations were 22 (18 to 28) ng/ml, 8-fold less than in the anaesthetised state, and were stimulated in the short term after administration of an octreotide bolus (100 micrograms/kg s.c.) to reach 88 (62 to 126) ng/ml at 60 min. A similar response was seen after i.v. administration to conscious rats. Intravenous bolus of octreotide (100 micrograms/kg) in rats anaesthetised for 3 h resulted in an increase in IGFBP-1 to peak at 1556 (1268 to 1910) ng/ml at 60 min. The IGFBP-1 response to octreotide was diminished in high-fat fed hyperinsulinaemic rats. The pattern of disappearance of iodinated IGFBP-1 from the circulation was not influenced by octreotide. The changes in GH, insulin and glucose concentrations alone did not sufficiently account for the patterns of response observed. We conclude that, in rats, octreotide stimulates IGFBP-1 acutely and this response is potentiated by factors related to anaesthesia.