In vivo study of the appearance and fluctuations of insulin binding sites in different tissues during rat development

The appearance and fluctuations of specific insulin binding sites in several tissues in vivo during rat development, have been determined. After intravenous administration of 125I-insulin to fetal, suckling and adult rats, changes on specific hormone uptake were observed depending on the tissues tested and on the age of animals. Thus, in liver, specific insulin uptake was much greater in 19 day-old fetuses and 10 day-old suckling animals than in adult rats. By contrast, brown fat and spleen insulin uptake was undetected in fetal animals but present in suckling rats, while lung insulin uptake was absent in the adults but present in fetal and suckling animals. Of interest were the specific insulin uptakes by three different muscle tissues. In fact, heart insulin uptake was much higher in younger animals than in adult rats, while in the diaphragm it was significantly smaller in all groups and in skeletal muscles hormone uptake was much smaller than in the other two muscle tissues and was even absent in the fetuses. In those tissues that had previously been shown to exhibit a specific insulin uptake, the iodinated hormone uptake decreased proportionally with simultaneous injection of increasing amounts of unlabelled insulin. These results indicate that insulin binding sites appear at different times and fluctuate in a different manner according to the tissues tested during rat development; this might be important in the stimulation of the functional activities of those tissues during perinatal age.     

Mouse liver specific uptake of iodinated growth hormones: evidence for the presence of somatogenic and lactogenic sites

The distribution of human growth hormone labelled with 125I (125I-hGH) was studied in normal adult female and male mice. The radioactivity was basically concentrated by the liver and kidney reading a maximum 15 minutes after the labelled hormone injection. Only the liver showed a significant reduction of radioactivity uptake when 125I-hGH was injected together with an excess of unlabelled hormone. This reduction was dose-dependent and the amount of unlabelled hormone that prevented 50% of the liver uptake (ED50) of 125I-hGH was close to 3 micrograms for both female and male mice. Similar results were obtained in studies where bovine growth hormone labelled with 125I (125I-bGH) was injected, except that the maximum uptake value was significantly lower than that observed when 125I-hGH was used. This observation could be attributed to the difference that exists between the biological properties of both hormones since hGH has growth-promoting and lactogenic effects in rodents, whereas bGH exhibits exclusively somatotropic activity. In order to examine the nature of the radioactive material which localized in the liver soluble extracts were prepared using Triton X-100 and analyzed on Sepharose CL-6B. The majority of the radioactivity appeared as an homogeneous peak with KD = 0.31 which could be attributed to a molecular species of Stokes radius of approximately 64A. This magnitude is consistent with the effective molecular size reported for various hormone-receptor complexes.     

Lack of insulin effect on its own receptors in fetal rat hepatocytes

To determine the effect of insulin on its receptor concentrations in hepatocytes of fetal and adult rats, these cells were preincubated in the presence or absence of insulin. The reduced [125I]-insulin binding observed in adult hepatocytes was dependent on the concentration of insulin and on the duration of exposure, while in fetal hepatocytes insulin did not induce any reduction in insulin binding. In contrast, glucagon receptors were unaffected by preincubation with insulin. The modifications observed in insulin binding were accounted for by changes in receptor concentrations rather than any change in receptor affinity for the hormone. Studies on the kinetic properties of the insulin receptors of fetuses and adult rats revealed that association and dissociation rates were undistinguishable. These results indicate an absence of insulin receptor down-regulation in the fetus, which could favour anabolic processes during intrauterine life.     

Differential effects of placental lactogen,growth hormone and prolactin on rat liver ornithine decarboxylase activity in the perinatal period

Ovine placental lactogen, (oPL), ovine growth hormone, (oGH), and ovine prolactin, (oPRL) are present in high concentrations in the fetal circulation late in gestation. To determine if these hormones stimulate the activity of ornithine decarboxylase (ODC), an enzyme widely implicated in the control of cellular growth, rat fetuses were injected $\text{in utero}$ with 100 μg of oPL, oGH, oPRL, rat growth hormone (rGH) or rat prolactin (rPRL) and ODC activity in the livers, hearts, and brains of the fetuses was measured 2, 4, and 6 hours after injection. OPL stimulated fetal liver ODC activity by 282 ± 45% (mean ± SEM) as compared to litter mates injected with buffer alone but oGH, oPRL, rGH and rPRL had no effect on fetal liver ODC activity. However, in neonatal rats 24–48 hours old all five hormones significantly increased liver ODC activity. ODC activities in the hearts and brains of the fetuses and neonates were unaffected by any of the five hormones. In other experiments 50 μg of oPL significantly stimulated fetal liver ODC activity while 250 μg of oGH were without effect. However 25 μg of oGH significantly stimulated liver ODC activity in rat pups 1–2 days after birth. These results suggest that oPL, by its stimulation of ODC activity, has somatotropic effects in the fetus and that rat liver ODC activity becomes responsive to growth hormone and prolactin in the perinatal period.     

Transplacental transfer of a growth hormone-releasing hormone peptide from mother to fetus in the rat

Previous studies showed that when growth hormone-releasing hormone (GHRH) was administered to either pregnant rats or pigs as a plasmid-mediated therapy, pituitary weight, somatotroph and lactotroph numbers, and postnatal growth rate of the offspring increased. To determine if these responses resulted from direct effects of GHRH on the fetus or were secondary to effects incurred in the mother, we studied in the rat the transplacental transfer of a GHRH analog (HV-GHRH) to the fetus from the maternal circulation. For the in vivo study, HV-GHRH was labeled with 125I and purified by reverse-phase high-performance liquid chromatography (HPLC). At 18 days of gestation, pregnant dams were administered a priming intravenous dose followed by a constant infusion of the labeled peptide. Approximately 2 days later, intact [125I]-HV-GHRH was isolated from the fetal liver, stomach contents, and brain. The amounts of tracer were positively correlated with those present in the corresponding dam's plasma. These data suggest that a GHRH analog of nonplacental origin, even at physiologic concentrations, can cross the placenta and, therefore, has the potential to influence fetal pituitary development directly.     

Differential effects of growth factors on [3H]thymidine incorporation and [125I]iodine uptake in FRTL-5 rat thyroid cells

We studied the effect of several growth factors on DNA synthesis and function of FRTL-5 rat thyroid cells by simultaneous measurement of [3H]thymidine incorporation and [125I]iodide uptake. Endothelial cell growth factor, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor I stimulated thymidine incorporation in a dose-dependent manner without the parallel increase of [125I]iodide uptake. These growth factors had an additive effect with thyroid-stimulating hormone (TSH) on thymidine incorporation, but they inhibited TSH-stimulated iodide uptake. Bombesin stimulated thymidine incorporation and inhibited TSH-stimulated iodide uptake; epidermal growth factor and gastrin-releasing peptide 10 had neither effect. None of the growth factors studied affected iodide uptake in the absence of TSH. Of the growth factors tested, endothelial cell growth factor, fibroblast growth factor, insulin-like growth factor bombesin, and platelet-derived growth factor all share similar differential effects on FRTL-5 cells: stimulation of DNA synthesis, potentiation of the effects of TSH on DNA synthesis, and attenuation of the effects of TSH on cell function. The data suggest that these growth factors may play important roles in regulation of thyroid function.     

Specific binding sites for growth hormone in cultured mouse thymic epithelial cells

Growth Hormones bound specifically to murine Thymic epithelial cells, which represent the major component of thymic micro-environment and can be modulated by pituitary hormones. The Kds found with human growth hormone and bovine growth hormone were 0.14 and 0.27 nM with a Bmax 0.56 and 0.35 fmol/10(6) cells respectively. Competition experiment analysis showed ED50 of 0.24 nM for hGH, 0.46 nM for rGH, 0.71 nM for bGH, 11.8 nM for hPRL and 11.2 nM for oPRL. No specific binding of [125I]-oPRL was observed under the same conditions. Both hPRL and bGH showed a negative regulatory effect on the number of the hGH binding sites when incubated with the culture for three days. The presence of GH receptors on Thymic epithelial cells provides biochemical evidence for the effect of GH on thymic function.     

Receptor autoradiographic localization of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands

We report here the first evidence of insulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands, obtained at autopsy. Sections of tissue were incubated with 0.1 nM [125I]IGF-I and analyzed using [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry. Specific binding sites of [125I]IGF-I were found to be localized in the definitive zone, fetal zone, and fetal medulla of the fetal adrenal glands. In the adult adrenal glands, the entire cortex and medulla were specifically labeled with [125I]IGF-I. Specific binding obtained at a concentration of 0.1 nM [125I]IGF-I to areas in the fetal and adult human adrenal glands was competitively displaced by unlabeled IGF-I, with an IC50 value of 0.34-2.54 nM, and 0.38-0.73 nM, respectively, whereas insulin was much less potent in displacing the binding. Acquisition of this knowledge will aid in studies on cell growth and steroid-catecholamines biosynthesis of the human adrenal gland.     

Myocardial meta-[125l]iodobenzylguanidine uptake in awake genetically hypertensive rats at different ages: an autoradiographic study.

The purpose of this work was to evaluate changes in myocardial meta-[125I]iodobenzylguanidine ([125I]MIBG) uptake and distribution with age in awake spontaneously hypertensive rats (SHR) with respect to Wistar-Kyoto (WKY) rats. Rats were randomly divided into two groups, one for measuring myocardial [125I]MIBG uptake and distribution 4 h after its injection and the second for evaluating myocardial catecholamine concentrations. Mean arterial blood pressure, cardiac hypertrophy index (heart/body weight ratio), and heart rate were significantly higher with increasing age in SHR compared with matched WKY rats. Myocardial catecholamine concentrations and turnover did not differ between the two strains and were significantly decreased with increasing age. Myocardial [125I]MIBG uptake determined by gamma counting was similar in WKY rats and SHR and did not vary significantly with age when expressed as uptake density. However, in both strains of rats, [125I]MIBG uptake determined by autoradiography was significantly greater at the base of the heart than at the apex and midventricular levels, and the uptake values of young rats were significantly higher than those of older rats. In 21-week-old WKY rats and SHR, the highest [125I]MIBG uptake values were found in the right ventricle. Thus, quantitative autoradiography allowed detection of significant changes in myocardial [125I]MIBG uptake and showed its heterogeneous distribution in the rat heart.     

Uterine decidualization in hypophysectomized-ovariectomized rats: effects of pituitary hormones

Decidualization of the endometrial stroma occurs in rats in response to implanting blastocysts or after the application of an appropriately timed artificial stimulus. It is well established that decidualization is regulated by estrogens and progesterone (P). The present study investigated the role of pituitary hormones in this response. Decidualization produced by the bilateral intrauterine injection of 100 microliter sesame oil was compared in ovariectomized (OVX) and hypophysectomized (HYPOX)-OVX rats. All animals were treated with a sequence of 17 beta-estradiol (E2) and P that in OVX rats supported decidualization. As assessed by uterine weights 5 days after uterine stimulation, decidualization was much greater in OVX than in HYPOX-OVX rats (geometric mean uterine weights of 1539 and 376 mg, respectively). To determine the ability of pituitary hormones to restore decidualization in HYPOX-OVX rats, animals were treated with ovine prolactin (oPRL, 2 x 100 micrograms daily), bovine growth hormone (bGH, 2 x 125 micrograms daily), and thyroxine (1 microgram/day, replacement for thyrotropin) in addition to E2 and P. Combined treatment with bGH + thyroxine resulted in decidualization which was not significantly different from that obtained in OVX rats; the effects of bGH and thyroxine were additive. oPRL had no significant effect. Administration of bGH + thyroxine during the prestimulation period resulted in decidualization which did not differ significantly from that obtained when the hormones were administered both pre- and poststimulation; administration during the poststimulation period only, when growth and differentiation of decidual cells occurs, resulted in much less decidualization. Because an increase in endometrial vascular permeability is a prerequiste for decidualization, [125I]-labeled bovine serum albumin was used to assess permeability 8 h after uterine stimulation. Uterine concentrations of radioactivity indicated that endometrial vascular permeability was increased to the same extent in bGH + thyroxine-treated HYPOX-OVX rats as in OVX animals; this increase was significantly reduced in vehicle-treated HYPOX-OVX rats. Because prostaglandins (PGs) are involved in decidualization, the possibility that the reduced responses in vehicle-treated HYPOX-OVX rats were a consequence of a decreased capacity of the uterus to produce PGs in response to the deciduogenic stimulus was investigated. As indicated by uterine PGE and PGF concentrations 15 min after uterine stimulation, uterine PGE and PGF production was increased by the stimulus in both vehicle-treated and bGH + thyroxine-treated HYPOX-OVX rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Binding inhibition by monoclonal antibodies of ovine placental lactogen to growth hormone receptors in human liver

In the radioreceptor assay for growth hormone (RRA-GH) using [125I]iodo-hGH, hGH and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, ovine placental lactogen (oPL) is capable of inhibiting the binding of [125I]iodo-hGH in a parallel manner with hGH and in equipotency. Similarly, in the RRA-GH by employing [125I]iodo-oPL, oPL and human liver membrane particulate fractions as tracer, hormone standard and receptors, respectively, hGH is also equipotent as oPL in inhibiting the binding of [125I]iodo-oPL in a parallel fashion. The addition of monoclonal antibodies against oPL in the assay was effective in inhibiting the binding of [125I] iodo-oPL to human liver, but could not, however, inhibit the binding of [125I]iodo-hGH to human liver. Furthermore, the addition of the monoclonal antibodies in the RRA-GH did not affect the parallelism of the oPL standard but lowered the total binding of oPL. Our studies indicate that the structure of the binding sequence in oPL which binds to the GH receptor of human liver is not identical to the equivalent sequence of hGH and that the monoclonal antibodies compete with GH receptors in human liver for the binding of oPL.     

Pituitary-thyroid function of fetuses of hypothyroid and growth hormone treated hypothyroid rats.

Maternal hypothyroidism induced by surgical thyroidectomy (Tx) of the rat resulted in significantly higher fetal serum levels of thyroid stimulating hormone (TSH) and thyroxine (T4) on day 22 of gestation. Surprisingly, administration of growth hormone (GH) to hypothyroid mothers increased further the fetal serum T4 and TSH. The in vitro uptake of 131I-T4 by erythrocytes was elevated significantly when incubated with serum from fetuses of both hypothyroid and hypothyroid GH-treated mothers. Although the plasma protein levels of hypothyroid mothers and their fetuses are decreased significantly as compared to controls this is not true of hypothyroid GH-treated mothers and their fetuses. The T4 levels of both groups of Tx mothers were significantly below that of controls. However, as in the case of their fetuses, the serum T4 of GH-treated hypothyroid mothers was elevated from that of Tx only animals. It is concluded that the pituitary-thyroid system of fetuses of hypothyroid mothers is activated excessively during late gestation, that considerable T4 can be transported from the fetus to the mother during this period and that these fetuses are in fact born in a hyperthyroid state which is aggravated by maternal treatment with GH.     

Epinephrine regulation of amino acid transport in rat hepatocytes isolated during development

The effect of epinephrine on the amino acid transport mediated by system A was investigated by determining the uptake of 2-amino [1-14C]isobutyric acid (AIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the hormone increased AIB uptake, enhancing the Vmax, while Km was unchanged. This effect was evident in cells from adult, 18- to 20-day-old fetus, and neonate rat. Actinomycin D or cycloheximide abolished the hormone dependent increase. Experiments carried out with alpha- and beta-antagonists showed that the effect of epinephrine was beta-mediated in fetal life and alpha-mediated in adult life. Membrane binding experiments showed a higher value for epinephrine and beta-agonist dihydroalprenolol in the fetus versus the adult. The calcium depletion obtained after cell incubation with EGTA or calcium ionophore A23187 reduced the hormonal stimulation in the adult, and was ineffective in the prenatal period. An involvement of cAMP was present in the epinephrine modulation of AIB transport, both in adult and in fetal life.     

Receptors for insulin-like growth factors I and II in developing embryonic mouse limb bud

Insulin-like growth factors (IGF) or somatomedins (SM) have been classically defined as promoting the actions of growth hormone in skeletal growth. IGF is divided into two groups, IGF-I and II, and are presumed to act via IGF type I (higher affinity for IGF-I and II and very low affinity for insulin) and II (higher affinity for IGF-II than I and no affinity for insulin) receptors, respectively. Recently, a switchover role of IGF-II to I during fetal to adult growth has been suggested. We have investigated the possible transitional role of IGF-II to I in a developing mouse embryonic limb bud organ culture model. In this in vitro system, limb bud develops from the blastoma stage to a well-differentiated cartilage tissue. Both IGF type I and II receptors were found to be present in limb buds at all stages of differentiation. Type I receptor decreased with differentiation while Type II receptor increased. The effect of IGF-I on [3H]thymidine and [35S]sulfate uptake by the tissue increased with differentiation while the effect of IGF-II on [3H]thymidine uptake of the undifferentiated tissue was abolished with differentiation of the tissue. The increase of the IGF-I response with decreased type I receptor may reflect an altered receptor sensitivity (occupancy) during differentiation. The decrease of the IGF-II response with increased type II receptor with differentiation may on the other hand suggest that IGF-II in differentiated tissue no longer acts as a classical growth factor. These results tend to support the hypothesis of the switchover role of IGF-I and II during fetal and adult growth, however, confirmation of the precise role of IGF-I and II in biological growth may have to wait until further studies clarifying the significance of the increased IGF type II receptor in differentiated tissue are made.     

Regulation of transferrin receptor expression at the cell surface by insulin-like growth factors, epidermal growth factor and platelet-derived growth factor.

Addition of platelet-derived growth factor (PDGF), recombinant insulin-like growth factor I (rIGF-I) or epidermal growth factor (EGF) to BALB/c 3T3 fibroblasts causes a marked increase in the binding of [125I]diferric transferrin to cell surface receptors. This effect is very rapid and is complete within 5 min. The effect of EGF is transient, with [125I]diferric transferrin binding returning to control values within 25 min. In contrast, PDGF and rIGF-I cause a prolonged stimulation of [125I]diferric transferrin binding that could be observed for up to 2 h. The increase in the binding of [125I]diferric transferrin caused by growth factors was investigated by analysis of the binding isotherm. Epidermal growth factor, PDGF and rIGF-I were found to increase the cell surface expression of transferrin receptors rather than to alter the affinity of the transferrin receptors. This result was confirmed in human fibroblasts by the demonstration that EGF, PDGF and rIGF-I could stimulate the binding of a monoclonal antibody directed against the transferrin receptor (OKT9) to the cell surface. Furthermore, PDGF and rIGF-I stimulated the sustained uptake of [59Fe]diferric transferrin by BALB/c 3T3 fibroblasts, while EGF transiently increased uptake. Thus the effect of these growth factors to increase the cell surface expression of the transferrin receptor appears to have an important physiological consequence.     

Pharmacokinetics of radioiodinated human and ovine growth hormones in transgenic mice expressing bovine growth hormone

Pharmacokinetics of radioiodinated human growth hormone (hGH) and ovine growth hormone (oGH) were studied in normal mice and in transgenic mice carrying the bovine growth hormone (bGH) gene fused to phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK-bGH). Multiexponential plasma decay curves were obtained in both normal and transgenic mice after a¹²⁵I-oGH injection and pharmacokinetic parameters were estimated by fitting blood concentration data to a three compartment model. The half-life for the rapid compartment was shorter in transgenic than in normal mice (t_1/2:1.2±0.3 vs. 2.2±0.5 min). The slow compartment had a t_1/2 of 160±23 min for transgenic and 70±8 min for normal mice while the middle compartment had a t_1/2 of approximately 10 min for both groups of mice. The mean residence times were 167±24 and 55±5 min for transgenic and normal mice, respectively. Specific liver uptake of radioactivity after injection of¹²⁵I-oGH or¹²⁵I-hGH was found in both groups of animals. Specificity studies indicated that, similarly to normal mice, livers of transgenic mice possess a mixed population of somatotropic and lactogenic receptors. Uptake of labelled hGH by the liver was dose-dependent and the doses that prevented 50% of liver uptake (ED_50%) were 8 and 165 g per 50 g body weight for normal and transgenic mice, respectively. Thesein vivo results confirm and extend previousin vitro findings that a life-long excess of bGH increases hepatic somatotropic and lactogenic receptors. Since elevation in growth hormone (GH) receptors was reported to be associated with an increase in GH binding protein (GHBP), we suspect that both the increase in the mean residence time and the reduction in specific uptake of GH in the livers of transgenic mice may be the result of an increase in GHBP levels.     

Hepatic gene transfer in animals using retroviruses containing the promoter from the gene for phosphoenolpyruvate carboxykinase

Two methods are described for directing the expression of genes to the livers of animals using retroviral vectors containing the predominantly liver-specific promoter from the gene for phosphoenolpyruvate carboxykinase (PEPCK)-linked to the structural gene for either amino 3'-glycosyl phosphotransferase (neo) or bovine growth hormone (bGH). Replication-incompetent retrovirus was used to infect the livers of fetal rats by intraperitoneal injection of animals in utero or to infect adult rats by direct injection into the portal vein after partial hepatectomy. The proviruses were integrated into the hepatic DNA, and the chimeric genes were expressed from the PEPCK promoter for as long as 8 months after infection. The expression of the PEPCK-bGH gene was regulated by diet and hormones in a manner similar to the regulation of the endogenous PEPCK gene in the liver. The potential of this method for targeting genes to the liver is discussed.     

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.