PROPERTIES OF INSULIN AND GLUCAGON RECEPTORS ON SHEEP HEPATOCYTES: A COMPARISON OF HORMONE BINDING AND PLASMA HORMONES AND METABOLITES IN LACTATING AND NON-LACTATING EWES

1980 ◽  
Vol 84 (2) ◽  
pp. 237-247 ◽  
Author(s):  
R. D. GILL ◽  
I. C. HART
Keyword(s):  
Plasma Concentrations ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Isolated Hepatocytes ◽  
Energy Deficit ◽  
Esterified Fatty Acids ◽  
Plasma Hormones ◽  
Glucagon And Insulin ◽  
Glucagon Receptors

A method is described for the isolation of viable hepatocytes from sheep liver. The characteristics of insulin and glucagon binding to the cells were investigated by the use of mono-iodinated hormone, and from these data the optimum in-vitro incubation conditions for hormone-receptor binding were established. Glucagon and insulin receptors were examined in relation to plasma concentrations of hormones and metabolites in non-mated, and 20- and 50-day-lactating ewes (six animals/group). Measurements of insulin, growth hormone and non-esterified fatty acids in the circulation, together with a fall in body weight, suggested that at peak lactation (20 days) the ewes were in energy-deficit and were mobilizing body tissue. The percentage binding of insulin was higher in hepatocytes after 50 days of lactation when compared with that in both the unmated (P < 0·05) and 20-day-lactating animals. No changes in insulin binding were found between the unmated and 20-day-lactating groups. Glucagon binding was reduced in the 20- (P < 0·02) and increased in the 50-day-lactating group (P < 0·001) when compared with the unmated control animals. The binding of glucagon was higher at 50 days as compared with 20 days of lactation (P < 0·001). The changes in insulin binding resulted primarily from altered receptor numbers whereas changes in the binding of glucagon were due to alterations in both receptor numbers and affinity. Our results indicated that the binding of insulin and glucagon to isolated hepatocytes was altered during lactation in sheep and that these changes might modulate the sensitivity of the cells to the actions of the hormones.

Hepatic receptors for insulin and glucagon in relation to plasma hormones and metabolites in pregnant and unmated ewes

1982 ◽  
Vol 93 (2) ◽  
pp. 231-238 ◽  
Author(s):  
R. D. Gill ◽  
I. C. Hart
Keyword(s):  
Statistical Significance ◽  
Plasma Concentrations ◽  
Insulin Receptors ◽  
Isolated Hepatocytes ◽  
Energy Deficit ◽  
Esterified Fatty Acids ◽  
Plasma Hormones ◽  
Glucagon And Insulin ◽  
Glucagon Receptors ◽  
Energy Surplus

Glucagon and insulin receptors were examined in relation to plasma concentrations of hormones and metabolites in unmated and in 110- and 140-day pregnant ewes (four animals per group). The concentrations of insulin, growth hormone and non-esterified fatty acids in the circulation, together with the maintenance of body weight, suggested that the animals were in energy surplus. When compared with the unmated group the binding of insulin to isolated hepatocytes increased by 110 days of pregnancy, attaining statistical significance (P < 0·02) after 140 days. Conversely, glucagon binding was reduced by 110 days of pregnancy, also attaining statistical significance (P < 0·02) after 140 days. The changes in both insulin and glucagon binding were primarily due to changes in the number of receptors on each hepatocyte, although some fluctuations in receptor affinity were also found. These observations suggested that the number of hepatic insulin and glucagon receptors are altered during the metabolic demands of pregnancy in sheep, but unlike the changes reported during lactation in the ewe and restricted energy intake in the goat, they are not related either to energy deficit or to changes in the concentration of insulin, and probably of glucagon, in the circulation.

Distribution of 125I-labelled insulin-binding sites in peripheral tissues of the normal and diabetic rat: an autoradiographic study

1991 ◽  
Vol 128 (1) ◽  
pp. 85-NP ◽  
Author(s):  
C. S. Thompson ◽  
R. M. Sykes ◽  
J. Muddle ◽  
M. R. Dashwood
Keyword(s):  
Binding Sites ◽  
Regional Distribution ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Autoradiographic Study ◽  
Diabetic Rat ◽  
Peripheral Tissues ◽  
Liver And Kidney ◽  
Impaired Insulin Action

ABSTRACT In-vitro autoradiography was used to demonstrate the regional distribution of 125I-labelled insulin-binding sites in the liver, kidney and heart of normal rats and rats made diabetic with streptozotocin. The distribution of insulin-binding sites in the liver of control rats was uniformly high, while in the kidney of control rats there was weak 125I-labelled insulin binding in the medulla and dense binding in the cortex. In the hearts of control rats a high density of 125I-labelled insulin-binding sites was evident both in the atrial and ventricular muscle. Non-ketotic diabetes mellitus caused a marked increase in 125I-labelled insulin-binding sites in both the liver and kidney with the former tissue exhibiting a time-dependent (7 to 62 days) increase. There was no apparent effect of diabetes on insulin-binding sites in the heart. Since experimental diabetes causes (1) a decrease in circulating insulin concentration and (2) impaired insulin action at many target tissues, the increase in 125I-labelled insulin-binding sites observed in the present study may represent a compensatory 'up regulation' of insulin receptors. Journal of Endocrinology (1991) 128, 85–89

Effects of continuous intravenous infusion of an ovine placental extract enriched in placental lactogen on plasma hormones, metabolites and metabolite biokinetics in non-pregnant sheep

1987 ◽  
Vol 113 (2) ◽  
pp. 277-283 ◽  
Author(s):  
G. Thordarson ◽  
G. H. McDowell ◽  
S. V. Smith ◽  
S. Iley ◽  
I. A. Forsyth
Keyword(s):  
Jugular Vein ◽  
Control Period ◽  
Plasma Concentrations ◽  
Placental Lactogen ◽  
Saline Control ◽  
Irreversible Loss ◽  
Esterified Fatty Acids ◽  
Pregnant Sheep ◽  
Plasma Hormones ◽  
Placental Extract

ABSTRACT Continuous intravenous infusions of saline or of a placental extract containing ovine placental lactogen were given to three non-pregnant, non-lactating ewes over periods of 36 h, 1 week apart. During saline infusion no placental lactogen was detected in jugular vein plasma, but infusion of the placental extract raised the placental lactogen concentration from undetectable to 40-50 μg/l, similar to concentrations in ewes with one fetus on day 90 of pregnancy. By comparison with the saline control period, infusion of the placental extract consistently increased both plasma concentrations and irreversible loss of non-esterified fatty acids. Plasma concentrations of glucose and urea, but not irreversible loss of these metabolites, were consistently increased. Although the placental extract was not subjected to extensive purification, it was enriched in placental lactogen and contained no detectable contamination with insulin, prolactin or growth hormone. The results are suggestive of a role for placental lactogen in modifying metabolism and acting during pregnancy to provide nutrients for fetal metabolism. J. Endocr. (1987) 113, 277–283

In vitro recovery of insulin binding after downregulation in cultured normal human monocytes

1985 ◽  
Vol 249 (1) ◽  
pp. E94-E98
Author(s):  
R. H. Whitson ◽  
S. A. Kaplan
Keyword(s):  
Human Peripheral Blood ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Blood Monocytes ◽  
Peripheral Blood Monocytes ◽  
Surface Receptors ◽  
Normal Human ◽  
Lysosomal Proteases ◽  
Normal Human Peripheral Blood

When normal human peripheral blood monocytes were treated with insulin in vitro, surface insulin receptors disappeared rapidly, but total insulin receptors (surface and internalized receptors), measured in detergent-solubilized extracts of total cellular membranes, decreased slowly. Surface receptors decreased to 51 +/- 4, 36 +/- 12, and 34 +/- 12%, of control levels after 2, 6, and 18 h of insulin pretreatment, respectively. Total receptors decreased to 86 +/- 12, 69 +/- 17, and 34 +/- 12% of control levels in the same periods. Chloroquine, a lysosomotropic agent, inhibited the removal of surface receptors, indicating that lysosomal proteases play a role in this process. Unlike monocytes, IM-9 lymphocytes lost surface receptors and total receptors at the same rate when incubated with insulin. Monocytes treated with insulin for 18 h, washed free of unbound insulin and recultured for 48 h regained 94 +/- 7% of control insulin binding, indicating that cultured monocytes are competent to regenerate their insulin receptors. Monocytes treated with insulin for 6 h also required 48 h to recover their insulin binding, despite the fact that substantial numbers of insulin receptors remained intact within these cells. Two-hour pretreated monocytes recovered somewhat faster, attaining control levels of receptors after 24 h of reculture. This suggests that internalized insulin receptors pass from a recyclable pool to a nonrecyclable one.

The Relation Between Glucose Tolerance and Insulin Binding to Circulating Monocytes in Obese Children

PEDIATRICS ◽  
1982 ◽  
Vol 70 (4) ◽  
pp. 633-637
Author(s):  
Kaichi Kida ◽  
Noriyoshi Watanabe ◽  
Yoshiki Fujisawa ◽  
Yoshinori Goto ◽  
Hiroshi Matsuda
Keyword(s):  
Glucose Tolerance ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Tolerance Test ◽  
Quantitative Relation ◽  
Immunoreactive Insulin ◽  
Oral Glucose ◽  
Obese Children

The quantitative relation between insulin binding to circulating monocytes in vitro and glucose tolerance in obese children in vivo is reported. Sixty-one obese children and 11 healthy control children participated in this study. The oral glucose tolerance test (OGTT) was performed by giving them glucose (1.75 gm/kg of body weight), orally in the morning, and the binding of 125I-labeled insulin to circulating monocytes in vitro was measured prior to OGTT. The glucose tolerance expressed by ΣBS (milligrams/100 ml), the sum of the plasma glucose (blood sugar [BS]) values at OGTT, was significantly correlated with the degree of overweight (r = .316, P &lt; .01) and more highly with ΣIRI (microunits per milliliter), the sum of immunoreactive insulin (IRI) values at OGTT (r = .512, P &lt; .001). Insulin binding to monocytes in vitro (picograms/106 cells) was inversely correlated with the degree of overweight (r = -. 687, P &lt; .001). Furthermore, ΣBS was inversely correlated significantly with insulin binding to monocytes in vitro (r = -.435, P &lt; .002). These data suggest that the decrease of insulin receptors might be one cause for the impairment of the glucose tolerance associated with obesity in children.

In vitro regulation of human monocyte insulin receptors by insulin and 1-methyl-3-isobutylxanthine

1983 ◽  
Vol 245 (5) ◽  
pp. E494-E501
Author(s):  
R. H. Whitson ◽  
S. A. Kaplan
Keyword(s):  
Insulin Receptor ◽  
Cultured Cells ◽  
Phosphodiesterase Inhibitor ◽  
Insulin Receptors ◽  
Human Monocyte ◽  
Insulin Binding ◽  
Receptor Downregulation ◽  
Independent Process ◽  
Dose Dependent

Monocytes separated from human blood by Ficoll-Hypaque and adherence to polystyrene flasks were maintained successfully in culture for 7 days. The cultured cells showed normal morphology and good viability. The insulin binding properties of the cultured monocytes were also identical to those of fresh monocytes. In vitro pretreatment of the monocytes with insulin decreased both the number and affinity of insulin receptors, resulting in a 72% reduction in the binding of tracer quantities of 125I-insulin. Insulin-induced receptor down regulation was dose-dependent and specific to the insulin receptor. Monocytes pretreated with insulin in the presence of the cyclic nucleotide phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) lost significantly fewer insulin receptors than monocytes treated with insulin alone. Tracer binding to these cells was 62% of control levels. MIX had no effect on basal insulin binding. The cAMP analogues N6,O2'-dibutyryl cAMP and 8-bromo-cAMP did not counteract insulin-induced receptor downregulation by themselves and did not significantly enhance the effects of MIX. These results indicate that MIX may counteract insulin receptor downregulation by a cAMP-independent process.

scholarly journals Tyrosine kinase activity of insulin receptors from human placenta. Effects of autophosphorylation and cyclic AMP-dependent protein kinase

1986 ◽  
Vol 233 (3) ◽  
pp. 677-681 ◽  
Author(s):  
H G Joost ◽  
H J Steinfelder ◽  
C Schmitz-Salue
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Insulin Receptor ◽  
Human Placenta ◽  
Kinase Activity ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The kinase activity of partially purified insulin receptor obtained from human placenta was studied. When autophosphorylation of the beta-subunit of the receptor was initiated by ATP prior to the addition of the exogenous substrate, both basal and insulin-stimulated kinase activity was increased. However, half-maximum effective insulin concentrations were unchanged. Insulin receptor autophosphorylation as stimulated by ATP and insulin failed to affect significantly 125I-insulin binding to partially purified insulin receptor from human placenta. It is concluded that autophosphorylation of the insulin receptors regulates its kinase activity but not its affinity for insulin. The catalytic subunit of cyclic AMP-dependent protein kinase failed to phosphorylate either subunit of the insulin receptor, and each kinase failed to affect the affinity of the other one. Thus no functional interaction between cyclic AMP-dependent protein kinase and insulin receptors was observed in the in vitro system.

scholarly journals Hepatic zinc in metallothionein-null mice following zinc challenge: in vivo and in vitro studies

1995 ◽  
Vol 309 (1) ◽  
pp. 25-31 ◽  
Author(s):  
P Coyle ◽  
J C Philcox ◽  
A M Rofe
Keyword(s):  
Cell Culture ◽  
Cell Surface ◽  
Plasma Concentrations ◽  
Isolated Hepatocytes ◽  
Cultured Hepatocytes ◽  
Zn Uptake ◽  
Parallel Increase ◽  
Zn Concentration

Hepatic zinc uptake and accumulation were compared in freshly isolated and cultured hepatocytes prepared from control (MT+/+) and metallothionein (MT)-null (MT-/-) mice. In freshly isolated hepatocytes, rapid (10-15 min) exchange of 65Zn was proportional to the Zn concentration in the medium and occurred to the same extent in hepatocytes from MT+/+ and MT-/- mice. In 24 h culture experiments with MT+/+ and MT-/- hepatocytes it was shown that approx. 40% of newly acquired cell-associated Zn was attached to the cell surface and not internalized. In MT+/+ and MT-/- hepatocyte cultures, internalized Zn (intZn) increased in proportion to extracellular Zn. Zn accumulation in MT-/- hepatocytes was only 60% that of MT+/+ cells. Addition of 1 microM dexamethasone (Dex) and recombinant mouse interleukin-6 (IL-6; 100 units/ml) increased MT accumulation by 8.6-fold in MT+/+ hepatocytes (at 50 microM Zn) and there was an associated parallel increase in intZn. Dex and IL-6 did not increase intZn in the MT-/- hepatocytes. At 16 h after an intraperitoneal injection of 5 micrograms/g Zn, plasma and urine Zn concentrations were 69 +/- 10 microM and 86 +/- 25 microM respectively in MT-/- mice (n = 10) and 27 +/- 1 microM and 23 +/- 4 microM respectively in MT+/+ controls (n = 9) (P < 0.001, plasma; P < 0.05, urine). Hepatic cytosolic Zn concentrations doubled in MT+/+ mice and increased by a significant 15% in MT-/- mice. There was no increase in hepatic Zn (dry wt.) concentrations or in total hepatic Zn, demonstrating that the increase in cytosolic Zn in MT-/- mice was due to hepatic water loss rather than net Zn uptake. It appears that even at extreme plasma concentrations of Zn, little if any accumulates within the liver when there is no MT available for its sequestration. That this is not fully demonstrated in vitro is probably due to nature of cell culture, where organ architecture is lost and the external protein binding milieu is less complex.

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