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J Eckel

Publications and source records attributed to J Eckel.

At least 73 records · Page 4Linked to original sources
PubMed1986

Down-regulation of insulin receptors in the heart: studies on primary cultured adult cardiac myocytes.

Primary cultured cardiac myocytes from adult rats have been used to study insulin receptor regulation. Culturing of cells in the presence of insulin induced a dose-dependent down-regulation of insulin binding with a maximal effect of 35% at an insulin concentration of 1.7 X 10(-7) mol/l. The number of high-affinity sites decreased from 110 000 to 70 000 sites per cell in control and down-regulated cells, respectively, with no change in the apparent affinity constant. Down-regulation was found to be rapid (t 1/2 = 3 h) and fully reversible. Culturing of cells in the presence of cycloheximide (0.1 mmol/l) or Tris (35 mmol/l) resulted in a further time-dependent increase in insulin-induced receptor loss with no effect on insulin binding to control cells. The action of both agents was found to be additive reaching a down-regulation of 51% after a culture period of 16 h. Recovery of insulin binding activity after removal of insulin remained unaffected in the presence of cycloheximide, whereas Tris inhibited this process by 74%. In conclusion our results show that the concept of insulin-induced receptor down regulation can be extended to the adult heart muscle. Moreover the data suggest involvement of protein synthesis and receptor recycling in this process.

Animals↗
PubMed1985

The insulin receptor of adult heart muscle cells.

Isolated cardiac myocytes possess specific receptors for insulin which consist of high- and low-affinity sites and are randomly distributed at the cell surface at 37 degrees C. Low-affinity sites can mediate a biological response comparable to that of high-affinity sites. Calcium appears to modulate the high-affinity site and thus may be involved in the regulation of cardiac sensitivity towards insulin. The data suggest involvement of the insulin receptor in insulin degradation, which includes internalization and lysosomal processing of insulin.

Animals↗
PubMed1985

Adult cardiac myocytes in primary culture: cell characteristics and insulin-receptor interaction.

Calcium-tolerant adult cardiac myocytes were kept in culture under serum-free conditions in the presence of physiological concentrations of insulin. Up to 4 days, 70% of cells retained their in vivo rodshaped morphology without gross structural alterations. During that period a constant ATP-to-ADP ratio was observed with a mean value of 10.6 +/- 0.5 (n = 4). The rate of [14C]phenylalanine incorporation remained unaltered up to 63 h in culture. Insulin binding to cultured cells was found to be time-and temperature-dependent, reversible, and highly specific. Scatchard analysis of equilibrium binding data showed a curvilinear plot with a high-affinity segment yielding an apparent dissociation constant of 4.5 X 10(-10) mol/l and a receptor number of 125,000 sites/cell. Both affinity and receptor number remained unaltered between 18 and 66 h in culture. [14C]phenylalanine incorporation was stimulated by 108% in cardiocytes cultured in the presence of high concentrations of insulin (1.7 X 10(-7) mol/l) for 63 h, when compared with control cells cultured in the absence of insulin. These data demonstrate the retention of structural integrity, insulin receptors, and insulin responsiveness in primary cultured adult cardiac myocytes and provide a useful model for long-term studies on the regulation of insulin action on the heart.

Animals↗
PubMed1985

Insulin resistance in the heart: studies on isolated cardiocytes of genetically obese Zucker rats.

Isolated cardiac myocytes from lean and genetically obese (fa/fa) Zucker rats were used to study cellular alterations related to the obesity syndrome in this tissue. Scatchard analysis of insulin binding data suggested a reduction in the number of low affinity sites in cells from obese rats; in contrast, an unaltered high affinity segment with Kd values of 5.7 +/- 0.6 and 4.5 +/- 0.7 X 10(-10) mol/liter (n = 4) in lean and obese rats, respectively, has been observed. Insulin internalization, as estimated from the amount of increased cell-associated radioactivity in chloroquine-treated cells, was decreased by 70% from 12.8 fmol insulin/10(6) cells X 120 min in lean rats to 3.8 fmol/10(6) cells X 120 min in obese rats. Determinations of initial velocities of 3-O-methylglucose influx were used for assessing glucose transport activity. Basal activity of the glucose transport system was reduced in cells from obese animals. This was found to be due to a decreased maximum velocity of the carrier with corresponding values of 69.8 +/- 5.2 and 38.3 +/- 3.2 nmol/10 sec X 10(6) cells (n = 3) in cardiocytes from lean and obese rats, respectively. Glucose transport exhibited an unaltered sensitivity toward stimulation by insulin, but an impaired responsiveness in cardiocytes from obese rats. The data suggest involvement of both receptor and postreceptor defects in the development of an insulin-resistant state in cardiac muscle.

3-O-Methylglucose↗
PubMed1985

Insulin action on cardiac glucose transport. Studies on the role of the sodium pump.

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+, K+ pump. 86Rb+ uptake by cardiac cells was found to be linear up to 2 min, with a steady state reached by 40-60 min, and was used to monitor the activity of the sodium pump. Both the ouabain-sensitive and ouabain-insensitive 86Rb+ uptake by cardiac cells were found to be unaffected by insulin treatment under conditions in which a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+ uptake was markedly reduced by the presence of calcium or magnesium or both, but remained unresponsive toward insulin treatment. Inhibition of the sodium-pump activity by ouabain and a concomitant shift in the intracellular Na+/K+ ratio did not affect basal or insulin-stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+, K+ pump and the glucose-transport system.

3-O-Methylglucose↗
PubMed1985

Glucose uptake in isolated heart cells: studies on the role of insulin.

Glucose transport in cardiac myocytes is highly sensitive to stimulation by insulin. The concentration of half-maximal action (3 X 10(-10) mol/l) corresponds to the high-affinity constant of the insulin receptor. Insulin action on the glucose carrier is exclusively due to an increase in Vmax with no changes in Km. The onset of insulin action is preceded by a lag phase of about 20 s and found to be ATP-dependent. Neither the sodium pump, nor calcium, but magnesium appears to be involved in the mediation of insulin action.

3-O-Methylglucose↗
PubMed1984

Protein kinase activity of the insulin receptor from muscle.

The insulin receptor is associated with a protein kinase activity. This has been shown for the receptor of liver, fat, and some other tissues which are not primary targets of insulin action. Here kinase activity is demonstrated for the insulin receptor of rat skeletal and cardiac muscle with similar characteristics. Insulin (10(-7) mol/l) stimulates phosphorylation of the 95-kDa receptor subunit 3- to 18-fold. The effect is detectable at 10(-10) mol/l insulin; the ED50 is approx. 3 X 10(-9) mol/l. The kinase phosphorylates exogenous substrate as well, and it is recovered after immunoprecipitation of the receptor with antireceptor antibody suggesting that kinase activity is intrinsic to the muscle receptor.

Actins↗
PubMed1984

Effect of EDTA on insulin binding and insulin action in isolated cardiocytes from adult rat. Evidence for a functional role of low-affinity insulin receptors.

Calcium-tolerant myocytes from the adult rat heart were used to study the effects of EDTA on insulin binding and insulin action. Treatment of cardiocytes with EDTA resulted in a 60% inhibition of insulin binding. This effect was partially reversible by subsequent addition of calcium or magnesium. Scatchard analysis of equilibrium binding data in the presence of calcium and magnesium showed a curvilinear plot with a high-affinity segment having a Kd of 5.7 X 10(-10) mol/L. In the presence of EDTA a linear Scatchard plot was observed with a Kd of 5.6 X 10(-9) mol/L. The total number of insulin receptors remained unaltered under these conditions. In contrast to insulin binding, insulin internalization was not affected by EDTA treatment. Insulin action was studied by measuring the effect of the hormone on the transport of 3-O-methylglucose. Half-maximal action occurred at an insulin concentration of 3 X 10(-10) mol/L and 10(-8) mol/L in control and EDTA-treated cells, respectively. Maximal transport stimulation, however, was not significantly different in both groups (130% and 106%, respectively). In conclusion, low-affinity insulin receptors in cardiac myocytes mediate a biologic response comparable to that of high-affinity sites; moreover, they may be involved in the process of internalization in this tissue. The data suggest a functional role of low-affinity insulin receptors in cardiac muscle.

3-O-Methylglucose↗
PubMed1983

Insulin action on cardiac glucose transport. Studies on the role of the Na+/K+ pump.

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na+/K+ pump. 86Rb+-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40-60 min, and was used to monitor the activity of the Na+/K+ pump. Ouabain (10(-3) mol/l) inhibited the steady-state uptake of 86Rb+ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive 86Rb+-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. 86Rb+-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na+/K+ pump activity by ouabain and a concomitant shift in the intracellular Na+ :K+ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na+/K+ pump and the glucose transport system.

Animals↗
PubMed1983

Insulin action on the glucose transport system in isolated cardiocytes from adult rat.

Calcium-tolerant myocytes from the adult rat heart were used to study the effects of insulin on the kinetics of myocardial 3-0-methylglucose transport at 37 degrees C. Insulin increased the initial velocity of sugar influx without affecting the equilibrium uptake values. Maximal stimulation averaged 50-80%, with a half-maximal response at an insulin concentration of 0.1 nM and maximal stimulation occurring at 1 nM. The onset of insulin action was preceded by a lag-phase of 20 s, reaching maximal action by 60 s. The Vmax. of the glucose transport system was increased from 160 to 287 nmol/min per 10(6) cells with an unaltered affinity. Neither extracellular nor intracellular calcium was found to be involved in the stimulatory action of insulin. Removal of intracellular magnesium resulted in a loss of insulin action. This study demonstrates that activation of the cardiac glucose transporter by insulin is due exclusively to an increase in the maximal velocity representing one of the very early effects of insulin on myocardial metabolism. The data suggest involvement of magnesium in the transmission of the insulin signal.

3-O-Methylglucose↗
PubMed1983

Influence of bradykinin on glucose uptake and metabolism studied in isolated cardiac myocytes and isolated perfused rat hearts.

To study whether bradykinin influences myocardial glucose metabolism as suggested for skeletal muscle, we studied the effect of bradykinin on myocardial glucose transport using isolated cardiac cells and on glucose metabolism using the isolated perfused rat heart. In isolated cardiac myocytes bradykinin alone had no influence on the initial velocity of basal glucose transport nor on the sensitivity of the glucose carrier towards stimulation by insulin. By contrast, in isolated perfused hearts bradykinin increased the rate of glucose uptake and oxidation as well as the formation of lactate independently of the action of insulin. In diabetic hearts, neither bradykinin nor insulin alone had a significant influence on myocardial glucose oxidation, however, both hormones together act synergistically to improve glucose oxidation. These data suggest that bradykinin enhanced the nutritional flow across the capillary wall and, thereby, indirectly accelerates the glucose metabolism in the isolated perfused heart. There is no evidence for a direct effect of bradykinin on the rate of glucose transport in the heart.

Animals↗
PubMed1983

Effects of microtubule-disrupting agents on insulin binding and degradation in isolated cardiocytes from adult rat.

Isolated muscle cells from adult rat heart have been used to study the effects of microtubule disruptive drugs on the maintenance of steady-state insulin binding to cardiac insulin receptors. Vinblastine, vincristine and podophyllotoxin significantly inhibited insulin binding (25-50%) in the presence of insulin (10(-8) mol/l). The effect of vinblastine was found to be time- and temperature-dependent and to be dependent on the amount of insulin bound to the cell. In the presence of cycloheximide (0.1 mmol/l) insulin binding decreased by 30%; this effect was found to be additive to the action of vinblastine. Treatment of cells with vinblastine significantly reduced the low-molecular mass material produced by receptor-mediated degradation of insulin. This effect was not additive to that of the lysosomotropic agent chloroquine. The results suggest involvement of microtubules in the intracellular transfer of insulin receptors from and to the plasma membrane.

Animals↗
PubMed1982

The fate of insulin in cardiac muscle. Studies on isolated muscle cells from adult rat heart.

Isolated muscle cells from adult rat heart were used to study myocardial degradation of insulin and the reactions after the initial binding event. After 60 min of association at 37 degrees C, 90% of specifically bound insulin could be dissociated from the cells; this fraction remained unaltered under steady-state conditions (up to 180 min). To assess the nature of cell-associated radioactivity, cardiocytes were solubilized and filtered on Sephadex G-50. After 5 min of association only intact insulin was observed, whereas under steady-state conditions 4% of 125I-labelled insulin bound to the cells was degraded to iodotyrosine-containing fragments. The Km for insulin degradation by isolated heart cells was estimated to be 1.75 x 10(-7)M. Receptor-mediated insulin degradation was studied by examination of the nature of radioactivity released by the cells after different times of association. After 5 min 83% of dissociating material consisted of intact insulin, whereas this fraction decreased to 50% under steady-state conditions. Treatment of cells with the lysosomotropic agent chloroquine (0.1 mM) significantly decreased the fraction that was eluted at the internal column volume. This study demonstrates that insulin degradation by the heart cell occurs by a receptor-independent and a receptor-dependent mechanism. The latter may involve internalization and a lysosomal pathway.

Animals↗
PubMed1980

Characteristics of insulin receptors in the heart muscle: binding of insulin to isolated muscle cells from adult rat heart.

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of 125I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady state increased with decreasing temperatures. Above 70% of the radioactivity bound at equilibrium at 25 degrees C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of 125I-labelled insulin was degraded by isolated heart cells after 2 h at 37 degrees C and 4 h at 25 degrees C, respectively. This degrading activity was effectively inhibited by high concentrations of albumin. Equilibrium binding studies were conducted at 25 degrees C using insulin concentrations ranging from 2.5 x 10(-11) mol/l to 10(-6) mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 x 10(7) l/mol with a total receptor concentration of 3.4 x 10(6) sites per cell. The presence of site-site interactions of the negative cooperative types among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 x 10(-9) mol/l), both, under conditions of low and high fractional saturation of receptors. These studies demonstrate the presence of specific insulin receptors in isolated muscle cells from adult rat heart and provide a useful model for the study of insulin action on the heart.

Animals↗
PubMed1979

Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins.

1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie--Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis--Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100--3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2--100 pM) and L-thyroxine (100--3000 pM) did not influence uptake by either system. 11. Under identical conditions of incubation, the uptake of L-tri-iodothyronine was 3.7 times higher than binding to cytosol proteins. The binding was insensitive to metabolic inhibitors. The results suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. 12. Plasma-membrane vesicles also take up the hormone rapidly at 23 degrees C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. 13. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal curve. The Eadie--Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. 14. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.

Animals↗
PubMed1977

Factors involved in the uptake of corticosterone by rat liver cells.

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent Kt values of the saturable systems were 64 +/- 40 nM (n = 3) and 1085 +/- 313 nM (n = 12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluoro-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35--45%. In the presence of cortisone, cortisol, dexamethasone, aldosterone, testosterone, estradiol-17beta and estrone (2 muM each) the uptake decreased 30--50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.

Animals↗