Bone formation in the context of growth retardation induced by hIGFBP-1 overexpression in transgenic mice.

In humans, intrauterine growth retardation (hIUGR) is correlated with an overexpression of insulin-like growth factor binding protein 1 (IGFBP-1). The affected children also present a delay in bone mineralization. In this study, transgenic 12-day-old mutant mice overexpressing human IGFBP-1 hepatospecifically showed a severe growth retardation. Alcian blue and alizarin red S staining of the skeleton revealed mineralization defects at the posterior level of the skull (delayed suture closure) and in appendicular and axial skeleton. Furthermore, microradiographic analysis showed a reduced bone density in the same areas. Thus, overexpressing of hIGFBP-1 demonstrates early postnatal life growth retardation and a delay in mineralization in transgenic mutant mice. These data show the involvement of the IGF/IGFBP system and more particularly IGFBP-1 in the biomineralization process.

Insulin-like growth factor (IGF) binding proteins modulate the glucocorticoid-dependent biological effects of IGF-II in cultured fetal rat hepatocytes.

The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1-6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.

Regulation by insulin-like growth factor (IGF) binding proteins of IGF-II-stimulated glycogenesis in cultured fetal rat hepatocytes.

We previously showed that when added to fresh medium, insulin-like growth factor (IGF)-II stimulates glycogen synthesis in cultured 18-day-old fetal rat hepatocytes. In the present study, we investigated the influence of 24-h culture-conditioned medium on IGF-II- and insulin-induced glycogenesis. The stimulatory effect of IGF-II (2.9-fold) on [14C]glucose incorporation into glycogen over 3 h was dose dependently inhibited by conditioned medium, whereas that of insulin (3.2-fold) was unaffected. Western ligand and immunoblot analysis of the conditioned media revealed IGF binding protein (IGFBP)-1, IGFBP-2, and IGFBP-4, with a predominance of IGFBP-1. IGFBP-3 was not detected. Preincubation of conditioned medium with IGF-II for 4 h at 4 C restored the glycogenic effect of newly added IGF-II. Preincubation of fresh medium with recombinant IGFBP-1 or IGFBP-3 in the presence of IGF-II suppressed IGF-II stimulation of glycogen synthesis. IGFBPs alone had no effect on glycogenesis. Des(1-6)IGF-II and R6IGF-II, structural analogs of IGF-II that have weak affinity for the IGFBPs, elicited maximal stimulation, near that of IGF-II (2.8-fold and 3.1-fold vs. 3.0-fold for IGF-II), whether tested in fresh or conditioned medium. The inhibitory effect of conditioned medium on IGF-II-induced glycogenesis is therefore mediated by the IGFBPs via sequestration of IGF-II. This suggests that the IGFBPs, particularly IGFBP-1, produced by fetal rat hepatocytes in culture may play a role in regulating glycogenesis.

Variations in the antagonistic effects of insulin and glucagon on glycogen metabolism in cultured foetal hepatocytes.

The antagonistic effects of insulin and glucagon on glycogen formation and mobilization were studied in cultured 18-day foetal rat hepatocytes with regard to different modes of exposure. Hormone combinations were achieved with a constant dose of 10 nM-insulin (maximal for the glycogenic effect of this hormone) and increasing doses of glucagon [from 0.03 to 10 nM: concn. causing half-maximal response (ED50) = 0.3 nM)]. When insulin and glucagon were added simultaneously, increasing glucagon concentrations progressively depressed the glycogenic effect of insulin and 0.3 nM-glucagon antagonized the insulin effect completely. The maximal glycogenolytic effect of glucagon was observed at concentrations greater than 1 nM. When the two hormones were introduced successively, with an interval of 4 h between additions, the effect of the second hormone was always fully expressed between 4 and 8 h. at which time the effect of the first hormone had ceased; the dominance of glucagon over insulin was also lost, due to cell desensitization to glucagon. Both continuous or intermittent (10 min on/10 min off periods) exposure to insulin and/or glucagon gave similar antagonistic effects, while in cells exposed to insulin plus glucagon alternating with exposure to insulin or glucagon alone, the glycogenic effect of insulin was less or more antagonized respectively by glucagon. Whatever the situation, the results obtained could not be related to antagonism by a glucagon-induced rise in either cyclic AMP levels (ED50 = 3 nM) or cell-surface hormone binding. Thus, depending on the hormonal state and the mode of hormone administration, regulation of glycogenesis in cultured foetal hepatocytes appears to be different from that predicted by the insulin/glucagon molar ratio, which is strikingly altered in the perinatal period.

Effects of modified insulin B21-B26 fragments on glycogenesis and on insulin-receptor complex fate in cultured fetal hepatocytes.

Biological activity and interference with insulin receptor complex fate of two modified sequences of insulin B21-B26, beta-Ala-Arg-Gly-Phe-Phe-Tyr-NH2 (DP-432) and beta-Ala-Arg-Pro-Phe-Phe-Tyr-NH2 (DP-640), were studied in cultured 18-day-old fetal rat hepatocytes known to respond to insulin by an acute stimulation of glycogenesis. The two derivatives stimulated [14C]glucose incorporation into glycogen in the absence of insulin independently of the deprivation of serum in the medium. The maximal effect of 3 mM DP-640 after 2 h, more pronounced than with 3 mM DP-432, was of the same order as that obtained with 10 nM insulin alone (stimulation index: 4.7 +/- 0.7, 2.5 +/- 0.2 and 3.6 +/- 0.9, n = 4, with DP-640, DP-432 and insulin, respectively) whereas insulin B-chain decreased glycogen labeling. Simultaneous addition of derivatives and insulin at maximal concentrations produced nearly additive effects. DP-640, as well as DP-432, increased the amount of [125I](A14) or (B26) human insulin associated with cells at 37 degrees C and inhibited intracellular insulin degradation with differences depending on the kind of insulin isomer and derivative, while the rapid insulin receptor cycle was not affected. Thus, the two derivatives specifically modified the cellular processing of insulin in cultured fetal hepatocytes, and exerted an insulin-like effect on glycogenesis clearly enhanced through modification of DP-432 by substitution of glycine for proline (DP-640).

Compared roles of glucose, galactose and fructose as glycogen precursors during the acute response to insulin in cultured rat foetal hepatocytes.

1. The efficiency of the contribution of hexoses to basal- and stimulated-glycogenesis, when studied in cultured 18 day-old rat foetal hepatocytes in the presence of glucose, was as follows: galactose greater than glucose greater than fructose. 2. Glucose deprivation had opposite effects on the contributions of [14C]galactose (decreased) and [14C]fructose (increased) to glycogenesis, which occurred independently of insulin and were reversed by glucose concentrations as low as 30-100 microM. 3. The stimulation of glycogenesis by insulin measured with [14C]glucose (3.2-fold) was superior to that obtained with either [14C]galactose or [14C]fructose (2.7-fold in both cases), which revealed a specific beneficial effect of insulin on glucose contribution.

Differences between glucose and insulin stimulation of glycogenesis in cultured fetal hepatocytes.

The glycogenic effects of a glucose load (15 mM) and/or insulin (10 nM) were studied in 18-day-old fetal rat hepatocytes after 2 days of culture when medium contained 4 mM glucose. A glucose load led to a stimulation of [14C]glucose glycogen labelling (20 min) earlier than with insulin (30-40 min); maximal stimulations were 3-fold after 1 h for the glucose load and 5-fold after 2-3 h for insulin. Simultaneous addition of the two agents produced synergic effects. When insulin was added 4 h after a glucose load (or vice versa), a second glycogenic response was elicited: a further addition of the same glycogenic agent was ineffective. The early glycogenic effects (up to 2 h) also occurred in the presence of 10 microM cycloheximide, with, however, some decrease of insulin stimulation. The contribution of medium glucose to the glycogen formed for 2 days (67% in the absence of glycogenic agent) was clearly enhanced by a glucose load and to a lesser degree by insulin after a 4-h exposure (83 and 71%, respectively). This was accompanied by a related modification of the participation of glucogenic precursors such as fructose and galactose. Thus, acute glycogenic response to glucose and insulin appeared both synergic and independent, and quite different in several aspects in cultured fetal hepatocytes.

[Expression of the glycogenic response to insulin in cultured fetal hepatocytes]. / Expression de la réponse glycogénique à l'insuline dans les hépatocytes foetaux en culture.

In the present study, we used primary cultures of fetal rat hepatocytes which are highly suitable for studying the glycogenic effect of insulin and its regulation. After a lag in the period of culture in the presence of cortisol, glycogenic response to insulin developed together with a progressive accumulation of glycogen. When insulin was added, the rate of glycogen synthesis increased, becoming maximal after 2-3 h, due to the activation of the glycogen synthase system already present. Modification of glycogen precursors in the medium did not alter the amplitude of the insulin effect. The glycogenic effect of insulin was unrelated to that of glucose load and occurred after the formation of glucose-1-phosphate. This only happened when the cyclic AMP-dependent glycogenolytic system was not stimulated, since it was suppressed by low doses of glucagon. Insulin effect, which was time-dependent, ceased after 4 h. This corresponded to a desensitization of hepatocytes without any alteration in the specific binding of insulin. These variations in the glycogenic effect of insulin were likely due to different causes; one of these could be the first events following the interaction of insulin with its receptor.

Relationship between insulin binding and glycogenesis in cultured fetal hepatocytes.

Binding of 125I-insulin and the stimulatory effect of insulin on 14C-glucose incorporation into glycogen have been studied in cultured fetal rat hepatocytes. Measurement of both variables was possible at 37 degrees C because of the slow rate of insulin degradation in the medium. 125I-insulin binding approached maximum after 10 min, thus largely preceding the insulin glycogenic effect which became significant after 45 min. Maximal effect was observed after 3 h with 10 nmol/l insulin when 16,000 specific sites per hepatocyte were occupied and half-maximal response was achieved with 0.3 nmol/l insulin (2,900 sites/hepatocyte). Dissociation of bound insulin was rapid (t 1/2 = 3 min) and accelerated by the availability of native insulin. In hepatocytes preincubated with insulin, binding was measured after 30 min incubation in the absence of hormone which allowed the liberation of most (95%) of the bound insulin. No modification of insulin binding was observed over extended periods (2-24 h) of exposure to 10 nmol/l insulin, when the glycogenic effect of insulin showed striking variations, notably a cessation of the effect between 4 and 12 h. Thus the time-dependence of the glycogenic effect of insulin cannot be related to a defect in insulin binding in cultured fetal hepatocytes.