Regulation of insulin-like growth factor-I and of insulin-like growth factor binding protein-1, -3 and -4 in cocultures of rat hepatocytes and Kupffer cells by interleukin-6.

BACKGROUND/AIMS: Catabolism is associated with decreased serum concentrations of insulin-like growth factor (IGF)-I and insulin-like growth factor binding protein (IGFBP)-3 associated with elevated IGFBP-3 protease activity and increased concentrations of IGFBP-1 and -4. The effects of the acute phase mediators interleukin (IL)-6, IL-1beta and tumor necrosis factor alpha (TNFalpha) on the biosynthesis of IGF-I and IGFBPs were studied in primary rat liver cells. METHODS: mRNA levels of IGF-I and of IGFBPs were analyzed by Northern blotting, secretion of IGFBPs by [(125)I]IGF-I ligand blotting. Proteolytic activity was measured using iodinated recombinant IGFBP-3 as the substrate. RESULTS: In hepatocytes, Kupffer cells (KC) and cocultures of hepatocytes with KC, IL-6 reduced IGF-I biosynthesis dose-dependently. IL-6 stimulated mRNA expression and protein secretion of IGFBP-1 and -4 in hepatocytes and that of IGFBP-3 in KC, respectively. In cocultures, biosynthesis of IGFBP-1, -3 and -4 was increased dose-dependently by IL-6, while the effects of IL-1beta or TNFalpha were less prominent. At neutral pH, proteolytic activity against IGFBP-3 was not detected in media of cocultures treated with IL-6. CONCLUSIONS: The alterations of IGF-I, IGFBP-1 and -4 observed in catabolism correlate with the effects of IL-6 on the biosynthesis of these components in primary rat liver cells, while a neutral IGFBP-3 protease was not detectable.     

The acid-labile subunit of the ternary insulin-like growth factor complex in cirrhosis: relation to liver dysfunction

BACKGROUND/AIMS: In the circulation, insulin-like growth factor-I (IGF-I) is bound in a trimeric complex of 150 kDa with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). Whereas circulating IGF-I and IGFBP-3 are reported to be low in patients with chronic liver failure, the level of ALS has not been described in relation to hepatic dysfunction. The aim of the present study was therefore to measure circulating and hepatic venous concentrations of ALS in relation to hepatic function and the IGF axis. METHODS: Twenty-five patients with cirrhosis (Child class A/B/C:5/10/10) and 30 controls with normal liver function were studied. During a haemodynamic investigation, blood samples were collected from the hepatic vein and femoral artery, and the plasma concentrations of ALS, IGF-I and IGFBP-3 were determined. RESULTS: Hepatic venous and arterial concentrations of ALS were significantly decreased in the cirrhotic patients compared with the controls (-69% and -68%, respectively, both p<0.001). IGF-I and IGFBP-3 were similarly decreased in the cirrhotic patients (-51%,p<0.001). A significant hepatic extraction of ALS was found in the controls (6%, p<0.01) and in the cirrhotic patients (8%, p=0.08). ALS correlated significantly with indicators of liver dysfunction, including the Child-Turcotte score (r=-0.69, p<0.0001), IGF-I (r=0.82, p<0.0001) and IGFBP-3 (r=0.74, p<0.0001). CONCLUSIONS: Circulating and hepatic venous ALS are decreased in patients with cirrhosis with significant relations to liver dysfunction and other components of the IGF complex. A small hepatic extraction was found in controls, which suggests extrahepatic production of ALS. Future studies should focus on organ-specific removal of ALS.     

The insulin-like growth factor binding protein 3 ternary complex is reduced in cirrhosis

BACKGROUND/AIMS: In healthy adults, serum insulin-like growth factor I (IGF-I), IGF binding protein 3 (IGFBP-3) and acid labile subunit (ALS) form a 150-kDa ternary complex under the control of growth hormone (GH). Approximately 80-90% of circulating IGF-I is bound to the ternary complex. In cirrhosis the GH/IGF axis is severely disturbed and the individual components of the ternary complex are reduced. However, the degree of ternary complex formation in cirrhosis has not previously been described. METHODS: Serum IGF-I, IGFBP-3, ALS, the 150-kDa ternary complex and IGFBP-3 proteolysis were all measured in six compensated and six decompensated cirrhotic patients and compared to six healthy controls. RESULTS: Patients with compensated cirrhosis had decreased levels of IGF-I (55%), IGFBP-3 (64%) and ALS (53%), and in the decompensated patients these levels were decreased even further: IGF-I (32%), IGFBP-3 (37%) and ALS (27%) compared to healthy controls. The levels of the ternary complex followed this pattern, with low levels seen in the compensated patients (66%) and a further reduction in the decompensated patients (27%). Ternary complex levels correlated negatively with the Child-Pugh score. No increase in IGFBP-3 proteolysis was found in cirrhotic patients compared to healthy controls. CONCLUSION: Cirrhosis is associated with reduced levels of the 150-kDa ternary IGFBP-3 complex correlating with the degree of liver disease.     

Short-term infusion of LongR(3) insulin-like growth factor (IGF)-I decreases hepatic IGF-I mRNA but not IGF binding protein-3 mRNA expression in pigs

Infusion of pigs with an insulin-like growth factor-I (IGF-I) analogue (LongR(3)IGF-I) that does not bind to IGF-binding proteins decreases growth rate and the plasma concentration of growth hormone (GH), IGF-I, IGFBP-3, and insulin. This study was designed to determine whether the decrease is due to changes in IGF-I and IGFBP-3 gene expression. IGF-I or LongR(3)IGF-I (180 microg/kg/day) was infused into 55-kg finisher pigs for 4 days using Travenol infuser pumps. Plasma IGF-I concentration was measured by radioimmunoassay and plasma IGFBP-3 and IGFBP-2 were estimated by Western ligand blotting. Steady-state levels of IGF-I and IGFBP-3 mRNA were measured by RNase protection assay. Neither IGF-I nor LongR(3)IGF-I had a significant effect on hepatic IGF-I class 1 mRNA expression, whereas hepatic IGF-I class 2 mRNA expression was significantly reduced by both peptides. Plasma IGFBP-3 levels were unaffected by IGF-I treatment but were reduced by LongR(3)IGF-I treatment. The decrease in IGFBP-3 was not due to decreased gene expression in porcine liver or kidney, since neither IGF-I nor LongR(3)IGF-I treatment altered IGFBP-3 mRNA. This study infers a direct effect of the IGF analogue LongR(3)IGF-I on GH through its inhibition of plasma IGF-I concentration and class 2 IGF-I mRNA. The decrease in plasma IGFBP-3 was not accompanied by a decrease in hepatic or renal IGFBP-3 mRNA, suggesting that in this case, plasma IGFBP-3 protein levels are posttranslationally regulated or are derived from tissues other than liver or kidney.     

Post-transcriptional and post-translational regulation of insulin-like growth factor binding protein-3 and -4 by insulin-like growth factor-I in uterine myometrial cells.

Involution of the uterus induced by oestrogen depletion is associated with a decrease in uterine insulin-like growth factor (IGF)-I and an increase in IGF binding protein (IGFBP) gene expression. We examined the effects of IGF-I on primary uterine myometrial cell proliferation, and on IGFBP-3 and IGFBP-4 gene expression. IGF-I enhanced DNA synthesis in these cells. In conditioned media, IGF-I increased IGFBP-3 accumulation by release of cell associated IGFBP-3. A low dose of IGF-I increased IGFBP-4 accumulation, and a high dose caused IGFBP-4 to disappear. In cell-free conditioned media IGF-I protected IGFBP-3 and enhanced IGFBP-4 proteolysis. Co-incubation of [(125)I]-IGFBP-4 with cell-free conditioned media cleaved IGFBP-4 into 18 and 12 kDa fragments. Northern blot analysis indicated that IGF-I increased IGFBP-4 mRNA accumulation by stabilizing the mRNA while IGFBP-3 gene expression was slightly decreased. The results demonstrate that IGF-I regulates IGFBP-4 post-trancriptionally and post-translationally, whereas IGFBP-3 is only affected post-translationally. By enhancing IGFBP-4 proteolysis, increasing cell-associated IGFBP-3 and stabilizing IGFBP-3, IGF-I may initiate a mitogenic response.     

Hepatic production of insulin-like growth factors in normal and diseased liver

BACKGROUND/AIMS: IGF-I levels are reduced in cirrhotic patients. However, it is not known whether this decreased level is the result of reduced hepatic production or modified bioavailability secondary to decreased binding proteins. We determined the hepatic production of IGF-I and IGF-II and their receptors in normal and diseased liver. METHODOLOGY: Twenty-five patients included, 11 controls with normal liver and 14 with either chronic hepatitis or cirrhosis. mRNA for IGF-1, IGF-II and their receptors were measured. Immunohistochemical staining was performed to localize the IGF-producing cells. RESULTS: In 11 normal livers, the IGF-I mRNA levels were 4.95 +/- 1.8; in the 14 diseased livers, the levels were 1.22 +/- 0.69 (p < 0.001). IGF-II mRNA levels were 3.78 +/- 1.45 for the control and 5.11 +/- 2.15 in the diseased livers (NS). IGF-I receptor levels were 1.15 +/- 0.83 in the normal and 0.31 +/- 0.22 in the liver disease group (p < 0.05). There was no statistical difference between the two groups for IGF-II receptor. CONCLUSIONS: Patients with chronic liver disease have a significant reduction in their hepatic production of IGF-I, whereas IGF-II tends to be elevated. Treatment with recombinant IGF-I in patients with metabolic or endocrine complications of cirrhosis might prove useful.     

Effects of alpha-interferon on insulin-like growth factor-I, insulin-like growth factor-II and insulin-like growth factor binding protein-3 secretion by a human lung cancer cell line in vitro

The aim of our study was to evaluate the effect of alpha-interferon (alpha-IFN) on cell growth and on the different IGF system components in a human non-small cell lung cancer line (Calu-6) in vitro. Our results confirm the release of IGF-I and IGF-II by these cells. The amount of IGF-II in conditioned media (10.25 +/- 3.95 nM/10(6) cells, mean +/- SE) was more than 10-fold higher than that of IGF-I. alpha-IFN treatment reduced IGF-II levels in the media, with a maximal effect between 1 and 10 U/ml (delta% of control: -31 and -55%, respectively, p < 0.05). IGF-I was significantly reduced at 0.5 U/ml (p < 0.01). No difference, however, was observed in IGF mRNA expression between untreated and alpha-IFN treated cells. An increase in IGF-I and IGF-II intracellular levels in alpha-IFN treated cultures was observed, suggesting that alpha-IFN can regulate the transfer of these peptides into the cells. Furthermore, IGF type-I and particularly type-lI receptor expression was increased after alpha-IFN treatment. IGFBP-3 was detected only in trace amounts in the conditioned media; however, it showed an increase after alpha-IFN treatment (+110% at 1 U/ml). IGFBP-3 mRNA expression showed a slight increase after treatment with 1 and 10 U/ml. alpha-IFN (1-10 U/ml) reduced the stimulatory effect of IGF-I on cell replication (p < 0.01), inhibited (p < 0.01) cell replication in untreated and in fetal calf serum (FCS)-stimulated cells, and increased apoptosis in Calu-6 cells. Our data suggest that alpha-IFN may exert its effects at the cellular level in part through modification of the local IGF system.     

Effects of intravenous insulin-like growth factor-I and insulin administration on insulin-like growth factor-binding proteins in the ovine fetus

The insulin-like growth factors (IGF) are important anabolic hormones in the mammalian fetus; their anabolic actions are potentially modulated by alterations in the IGF-binding proteins (IGFBP). We have previously shown that the nutritional state of the fetus affects both IGF-I and the IGFBP concentrations. The present study was designed to determine the effect of alterations in insulin and IGF-I circulating concentrations on the IGFBPs. Because both insulin and IGF-I elicit decreases in glucose and amino acid concentrations, the concentrations of these substrates were clamped during the hormone infusions. Sixteen ovine fetuses were chronically catheterized at approximately 115 days of gestation, and experimental procedures performed at approximately 130 days of gestation. Insulin, IGF-I or both were infused for an 8-h period. Baseline concentrations of hormones and binding proteins were obtained, and concentrations were also obtained at the end of the infusion. Hepatic IGFBP-1 mRNA expression was also determined. Intravenous infusion of IGF-I significantly increased IGF-I concentrations in plasma in the ovine fetus. Intravenous infusion of insulin inhibited hepatic IGFBP-1 gene expression when amino acids and glucose were clamped. In contrast, intravenous infusion of recombinant human IGF-I (rhIGF-I) enhanced hepatic IGFBP-1 gene expression. Neither insulin nor rhIGF-I treatment had an effect on hepatic IGFBP-3 gene expression. Insulin did not alter plasma IGFBP-1 significantly, but it increased IGFBP-3 in plasma. rhIGF-I increased both IGFBP-1 and IGFBP-3 protein levels in plasma. The responses of IGFBP-1 and IGFBP-3 to increased plasma IGF-I and insulin may serve to protect the fetus from exaggerated anabolic effects and to blunt the hypoglycemic potential of circulating IGFs and insulin.     

Regulation of insulin-like growth factor (IGF)-binding protein expression by growth factors and cytokines alters IGF-mediated proliferation of postnatal lung fibroblasts

Postnatal day 5 is the beginning of septation and the peak of postnatal fibroblast proliferation. The author and colleagues studied fibroblasts from this developmental time period to determine factors that regulate cell proliferation. Exposure of cells to insulin-like growth factor (IGF)-I for 48 hours increased cell number whereas exposure to epithelial growth factor (EGF), platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-7, FGF-2, tumor necrosis factor-alpha (TNF-alpha), or interleukin (L)-1beta did not alter cell number. Long[R3]IGF-I (a synthetic IGF analog with reduced affinity for IGF-binding proteins [IGFBPs]) was more potent than IGF-I, with half-maximal stimulation at a dose of 0.6 nM for long[R3]IGF-I compared to 1.5 nM for IGF-I, suggesting that IGFBPs in the conditioned medium (CM) inhibit IGF activity. Addition of exogenous IGFBP-3 inhibited the IGF-stimulated increase in cell number. Addition of IGFBP-4 did not alter IGF activity because IGF-I stimulated proteolysis of IGFBP-4. The expression of mRNA for PAPP-A (a known IGFBP-4 protease) suggests that the clearance of IGFBP-4 is mediated by pregnancy-associated plasma protein (PAPP)-A. Exposure of cells to TNF-alpha or IL-1beta increased IGFBP-3 mRNA abundance and IGFBP-3 protein in CM. PDGF-BB and IL-1beta increased IGFBP-4 protein abundance and PDGF-BB and dibutyryl cAMP increased IGFBP-4 mRNA. The increase in CM IGFBP-3 following TNF-alpha exposure blocked IGF-mediated cell proliferation, suggesting that the growth factor- and cytokine-mediated changes in IGFBP abundance regulate postnatal fibroblast cell proliferation.     

Effect of mild hypoinsulinemia on renal hypertrophy: growth hormone/insulin-like growth factor I system in mild streptozotocin diabetes

The metabolic aberrations associated with diabetes mellitus profoundly alter the growth hormone/insulin-like growth factor I (GH/IGF-I) system. In severe experimental diabetes, serum IGF-I level is reduced, reflecting altered hepatic expression. On the other hand, increased levels of kidney IGF-I have been implicated in the development of diabetic kidney disease. This study aimed to examine the effect of mild experimental diabetes with hypoinsulinemia on both the systemic and renal GH/IGF-I systems in a low-dose streptozotocin (STZ)-induced diabetic rat. Diabetic animals with mild hypoinsulinemia developed renal hyperfiltration within 3 days of diabetes, whereas the renal size increased significantly only between 30 and 48 days of diabetes. Plasma GH levels were unchanged during the entire course of the study, but a decrease in serum IGF-I, IGF-binding protein 3 (IGFBP-3), and IGF-binding protein 4 (IGFBP-4) occurred after 10, 30, and 48 days. Kidney IGF-I and IGF-binding protein 1 (IGFBP-1) mRNA expression increased after 10 and 30 days of diabetes. A significant increase in kidney IGFBP-1/2, IGFBP-3, and IGFBP-4 proteins was seen after 48 days of diabetes. A positive correlations was found between renal growth and insulin/glucose ratio (r=.57), kidney IGF-I (r=.57), IGFBP-1 mRNA (r=.43), IGFBP-1/2 (r=.41), and IGFBP-4 levels (r=.40). These results demonstrate hyperfiltration within 3 days of diabetes and a similar response in the IGF-I system in mildly and severely hypoinsulinemic rats; however, renomegaly develops slower in mildly diabetic rats at least partly due to delayed changes in the renal IGF and IGF BPs.     

Hepatic regeneration in insulin-like growth factor binding protein-1 transgenic mice

BACKGROUND/AIMS: Partial hepatectomy results in activation of genes in the residual liver tissue which serve to restore glucose homeostasis and regenerate liver mass. Expression of insulin-like growth factor binding protein-1 (IGFBP-1) is up-regulated following partial hepatectomy and IGFBP-1 can modulate both the metabolic and mitogenic effects of insulin-like growth factor-1 (IGF-I). The aim of the study was to compare the effects of partial hepatectomy on blood glucose levels and hepatic regeneration in wild-type and transgenic mice which constitutively overexpress IGFBP-1. METHODS: Hepatic IGFBP-1 mRNA, blood glucose concentrations, liver mass and hepatic DNA synthesis were compared in sham-operated and partially hepatectomized transgenic and wild-type mice. RESULTS: Hepatic IGFBP-1 mRNA was higher in sham-operated transgenic than wild-type mice, but in both groups of mice, partial hepatectomy was associated with a significant rise in IGFBP-1 mRNA. The absolute decline in blood glucose levels following partial hepatectomy was greater in transgenic mice. Basal DNA synthesis and the response to IGF-I in isolated hepatocytes from both groups of mice were similar, and DNA synthesis in the regenerating liver in vivo was not significantly different in transgenic as compared to wild-type mice: 449.3 +/- 63.9 vs. 321.6 +/- 52.3 cpm/microgram DNA. Hepatic regeneration as measured by liver weight after hepatectomy was not different between transgenic and wild-type mice. CONCLUSIONS: Constitutive overexpression of IGFBP-1 does not enhance hepatic regeneration and does not prevent the decline in blood glucose following partial hepatectomy.     

Protein-caloric food restriction affects insulin-like growth factor system in fetal Wistar rat

We have previously shown that fetuses from protein-caloric undernourished pregnant rats (35% of control diet during the last week of pregnancy) at 21.5 d post coitum exhibit increased beta-cell mass. This alteration is correlated with increased insulinemia and total pancreatic insulin content, a pattern similar to that reported in infants of mild diabetic mothers. In this work, we investigated in undernourished fetuses: 1) whether availability of growth factors such as insulin, GH, and IGFs and their binding proteins (IGFBPs) could be implicated in this alteration, and 2) the beta-cell mitogenic response to IGFs in vitro. The results show that maternal undernutrition increases pancreatic IGF-I expression and islet IGF-I receptor content in undernourished fetuses, whereas hepatic IGF-I expression and serum IGF-I levels were decreased. No changes were observed in serum IGF-II, and its expression was diminished in undernourished pancreases and unchanged in the liver, compared with control fetuses. Serum levels and liver and pancreatic mRNA expression of IGFBP-1 were found to be normal in undernourished fetuses, whereas the serum concentration and abundance of IGFBP-2 mRNA in pancreas were increased. Finally, the beta-cell mitogenic response to IGFs in vitro was significantly increased in undernourished fetal islets, compared with controls. In conclusion, in undernourished fetuses the increased beta-cell mass can be related to the stimulation of replicative beta-cell response due to locally increased pancreatic IGF-I mRNA; this effect is perhaps potentiated or favored by the enhanced islet IGF-I receptor content and pancreatic IGFBP-2 gene expression.