scholarly journals Both insulin and epidermal growth factor stimulate lipogenesis and acetyl-CoA carboxylase activity in isolated adipocytes. Importance of homogenization procedure in avoiding artefacts in acetyl-CoA carboxylase assay

1986 ◽  
Vol 234 (2) ◽  
pp. 279-284 ◽  
Author(s):  
T A Haystead ◽  
D G Hardie
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Maximal Effect ◽  
Acetyl Coa Carboxylase ◽  
Homogenization Procedure ◽  
Acetyl Coa ◽  
Carboxylase Activity ◽  
Epidermal Growth ◽  
Isolated Adipocytes ◽  
Significant Interference

Epidermal growth factor (EGF) stimulates lipogenesis by 3-4-fold in isolated adipocytes, with a half-maximal effect at 10 nM-EGF. In the same batches of cells insulin stimulated lipogenesis by 15-fold. Freezing and prolonged homogenization of adipocytes results in release of large quantities of pyruvate carboxylase from broken mitochondria, and sufficient pyruvate can be carried through into assays for this enzyme to cause significant interference with assays of acetyl-CoA carboxylase in crude adipocyte extracts. This may account for the high amount of citrate-independent acetyl-CoA carboxylase activity reported to be present in adipocyte extracts in some previous publications. This problem may be eliminated by homogenizing very briefly without freezing. By using the modified homogenization procedure, EGF treatment of adipocytes was shown to produce an effect on acetyl-CoA carboxylase activity almost identical with that of insulin. Both messengers increase Vmax. without significant effect on the Ka for the allosteric activator, citrate.

Epidermal growth factor stimulates the prolactin synthesis and secretion in rat pituitary cells in culture (GH4C1 cells) by increasing the intracellular concentration of free calcium

1993 ◽  
Vol 128 (4) ◽  
pp. 361-366 ◽  
Author(s):  
Marie Aanestad ◽  
J Sigurd Røtnes ◽  
Peter A Torjesen ◽  
Egil Haug ◽  
Olav Sand ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Intracellular Concentration ◽  
Free Calcium ◽  
Pituitary Cells ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Epidermal Growth

Epidermal growth factor (EGF) stimulated the prolactin (PRL) synthesis and release from the GH4C1 cells in a dose-dependent manner. The ED50 was between 10−11 and 10−10 mol/l. The maximal effect was obtained at 10−9 mol/l EGF for the release, and 10−8 mol/l EGF for the synthesis. EGF stimulated the release of PRL from cell perfusion columns after a lag period of about 30 s. The maximal secretion of PRL occurred about 60 s after the start of stimulation. The PRL secretion declined to basal levels within 2 min. The EGF-stimulated PRL release was additive to the secretion evoked by thyrotropin-releasing hormone (TRH) and vasoactive intestinal peptide (VIP). An instantaneous increase in the intracellular concentration of free calcium, [Ca2+]i, of the GH4C1 cells was observed after the administration of EGF. EGF modified neither the basal nor the TRH-stimulated inositoltrisphosphate production in the GH4C1 cells, and EGF did not show any effect on the cyclic adenosine monophosphate production of these cells.

scholarly journals Functional receptors for epidermal growth factor in an epithelial-cell line derived from the rat small intestine

1985 ◽  
Vol 225 (1) ◽  
pp. 85-94 ◽  
Author(s):  
J Blay ◽  
K D Brown
Keyword(s):  
Growth Factor ◽  
Epithelial Cell ◽  
Epidermal Growth Factor ◽  
Cell Line ◽  
Intestinal Epithelial Cell ◽  
Epithelial Cell Line ◽  
Maximal Effect ◽  
Intestinal Epithelial ◽  
Egf Receptors ◽  
Epidermal Growth

Epidermal growth factor (EGF) regulates the proliferation of cells of a rat intestinal epithelial-cell line (RIE-1) in culture. Confluent RIE-1 cells were stimulated to proliferate by EGF with a half-maximal effect at 1-2 ng/ml. In contrast, the growth of sparse RIE-1 cells was inhibited by the growth factor. Binding studies at 4 degrees C with 125I-EGF identified two classes of binding sites for EGF on RIE-1 cells, one of high affinity (KD = 1.8 × 10(-10)M; 1.8 × 10(4) receptors/cell) and one of lower affinity (KD = 5.2 × 10(-9)M; 6.3 × 10(4) receptors/cell). After binding to the cells at 37 degrees C, 125I-EGF was rapidly internalized and subsequently degraded. Degradation products were released into the medium after a lag of 15-30 min. The degradation of 125I-EGF did not occur at 4 degrees C and was inhibited at 37 degrees C by chloroquine, methylamine or NH4Cl, but not by colchicine. Exposure of RIE-1 cells to EGF caused a time- and dose-dependent loss of EGF receptors from the cell surface. The recovery of receptors after the removal of EGF was retarded in the absence of serum and prevented by the presence of cycloheximide or actinomycin D. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis separation of the 125I-EGF-receptor complex from RIE-1 cells after covalently cross-linking with disuccinimidyl suberate indicated a receptor of Mr congruent to 160 000. The demonstration of functional EGF receptors in this cell line provides further evidence that EGF may regulate intestinal-epithelial-cell physiology.

Epidermal growth factor (EGF) inhibits the secretomotor response of the thyroid: effects of EGF on radioiodine turnover and fluid transport in cultured porcine thyroid cells

1991 ◽  
Vol 128 (2) ◽  
pp. 213-218 ◽  
Author(s):  
J. R. Bourke ◽  
S. Murdoch ◽  
S. W. Manley ◽  
T. Matainaho ◽  
G. J. Huxham ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cyclic Amp ◽  
Fluid Transport ◽  
Prostaglandin E ◽  
Thyroid Cell ◽  
Ionophore A23187 ◽  
Maximal Effect ◽  
Thyroid Cells ◽  
Epidermal Growth

ABSTRACT Thyrotrophin (4-256 μU/ml) promoted an increase in the rate of release of radioiodine from the organic iodine pool of cultured porcine thyroid cells in follicular formations. This action of TSH was antagonized by low concentrations of epidermal growth factor (EGF; 0·1–5 nmol/l). The maximal effect of EGF was reached by 0·5 nmol/l. EGF (0·5–5 nmol/l) also inhibited the stimulatory effect of 8-chloro cyclic AMP (0·06–1·0 nmol/l) on radioiodine turnover. Exposure of thyroid cultures to media with a calcium concentration of 17·7 μmol/l (1% of normal) resulted in a very marked increase in the rate of release of radioiodine. The effect of TSH in low-calcium media was to inhibit the increased release of radioiodine, and EGF (0·5 nmol/l) antagonized this inhibitory effect of TSH. The calcium ionophore, A23187, stimulated radioiodine release in a dose-dependent fashion, and EGF (1·7 nmol/l) inhibited this response. Fluid transport in thyroid monolayers was stimulated by prostaglandin E2 (PGE2; 1 μmol/l). EGF (5 nmol/l) also stimulated fluid transport, but antagonized the effect of PGE2 added subsequently. It was concluded that EGF exerted acute antagonistic effects on thyroid cell responses in vitro to cyclic AMP and agents promoting accumulation of cyclic AMP in time-frames too short for these inhibitory effects to be attributable to the dedifferentiative effect of the growth factor. Journal of Endocrinology (1991) 128, 213–218

Epidermal growth factor inhibits Na-Pi cotransport and mRNA in OK cells

1995 ◽  
Vol 268 (2) ◽  
pp. F309-F314 ◽  
Author(s):  
M. Arar ◽  
M. Baum ◽  
J. Biber ◽  
H. Murer ◽  
M. Levi
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase C ◽  
Mrna Abundance ◽  
Epithelial Cell Line ◽  
Maximal Effect ◽  
Maximal Velocity ◽  
Opossum Kidney ◽  
Ok Cells ◽  
Epidermal Growth

The present study examined the effect of epidermal growth factor (EGF) on Na-Pi cotransport in a tubular epithelial cell line derived from the opossum kidney (OKP cells). EGF caused a time- and dose-dependent decrease in Na-Pi cotransport. The inhibition of Na-Pi cotransport by 10(-8) M EGF was first demonstrable after 18 h with maximal effect seen at 24 h. EGF inhibited Na-Pi cotransport by decreasing the maximal velocity (10.8 +/- 0.9 in control vs. 4.9 +/- 0.8 nmol 32Pi.4 min-1.mg protein-1 in EGF, P < 0.001). Northern blot analysis indicated that EGF caused a significant decrease in NaPi-4 mRNA abundance. The abundance of NaPi-4 mRNA relative to beta-actin and/or glyceraldehyde-3-phosphate dehydrogenase mRNA was decreased by twofold in OK cells treated with EGF for 4 h and threefold in OKP cells treated with EGF for 24 h. Thus the decrease in NaPi-4 mRNA abundance preceded the decrease in Na-Pi cotransport activity. Inhibition of transcription with actinomycin D and protein synthesis with cycloheximide prevented the inhibition of Na-Pi cotransport. Furthermore, inhibition of phospholipase C activity with U-73,122 also significantly blocked the inhibitory effect of EGF on Na-Pi cotransport. The results indicate that EGF-induced decrease in OKP Na-Pi cotransport is mediated through a decrease in NaPi-4 mRNA and activation of the phospholipase C signaling pathway.

scholarly journals Epidermal growth factor administration decreases liver glycogen and causes mild hyperglycaemia in mice

1996 ◽  
Vol 315 (1) ◽  
pp. 289-293 ◽  
Author(s):  
Montserrat GRAU ◽  
Francesc TEBAR ◽  
Ignasi RAMÍREZ ◽  
Maria SOLEY
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Rat Hepatocytes ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Isolated Cells ◽  
Experimental Conditions ◽  
Epidermal Growth ◽  
Mouse Hepatocytes

Several laboratories report different effects of epidermal growth factor (EGF) on glycogen metabolism in hepatocytes. The discrepancies may be attributed to differences in the experimental conditions. It is therefore important to establish the actual effect of EGF in vivo. Because large physiological variations of EGF concentration in plasma occur in mice, we used this species to address this question. In freshly isolated mouse hepatocytes, EGF increased glycogen degradation in a dose-dependent manner. The maximal effect (36% increase over basal glycogenolysis) was smaller than maximal effects of classical glycogenolytic hormones like adrenaline or glucagon (more than 150% increase over basal). This is in keeping with the smaller effect of EGF on phosphorylase a activity. In contrast with these hormones, EGF did not inhibit glycolysis. Thus these effects of EGF in mouse hepatocytes are similar to those recently described by us in rat hepatocytes [Quintana, Grau, Moreno, Soler, Ramírez and Soley (1995) Biochem. J. 308, 889–894]. When administered to whole animals, EGF increased phosphorylase a activity, decreased the glycogen content in the liver and caused mild hyperglycaemia. Taking together the results obtained for isolated cells and for whole animals, we suggest that the glucosyl residues released from glycogen are used mostly by the liver rather than released to the circulation. This would be different from the action of the classical glycogenolytic hormones, adrenaline and glucagon.

Human growth hormone augmentation of epidermal growth factor binding sites on rat granulosa cells

1994 ◽  
Vol 142 (1) ◽  
pp. 69-75 ◽  
Author(s):  
M-A Hattori ◽  
Y Shinohara ◽  
E Yoshino ◽  
M Kanzaki ◽  
I Kojima ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Binding Sites ◽  
Egf Receptor ◽  
Scatchard Analysis ◽  
Maximal Effect ◽  
Intact Cells ◽  
Epidermal Growth

Abstract The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [125I]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 μg hGH/l and a maximal stimulatory concentration of 100 μg/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17 200 sites/cell after treatment with FSH; 31 700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites. Journal of Endocrinology (1994) 142, 69–75

Quantitative aspects of the effects of insulin, epidermal growth factor and dexamethasone on DNA synthesis in cultured adult rat hepatocytes

1985 ◽  
Vol 109 (3) ◽  
pp. 369-377 ◽  
Author(s):  
Tor-Erik Sand ◽  
Gunnar Brønstad ◽  
Vemund Digernes ◽  
Anne Killi ◽  
Wasfiyeh Amara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dna Synthesis ◽  
Time Course ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Maximal Effect ◽  
Adult Rat ◽  
Adult Rat Hepatocytes ◽  
Epidermal Growth

Abstract. Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dosedependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24–30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nm and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 μm insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5–15 nm. When added in combination with 1.7 nm EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nm. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.

scholarly journals Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells.

1988 ◽  
Vol 106 (3) ◽  
pp. 797-803 ◽  
Author(s):  
Y C Wang ◽  
P A Rubenstein
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Platelet Derived Growth Factor ◽  
Northern Analysis ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Differentiated Cells ◽  
Epidermal Growth ◽  
Dose Dependent

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.

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