PubMed Health⌕ Search

Biomedical subjects

J Eckel

Publications and source records attributed to J Eckel.

At least 19 recordsLinked to original sources
PubMed1992

Contraction-induced translocation of the glucose transporter Glut4 in isolated ventricular cardiomyocytes.

Field stimulation of isolated adult ventricular cardiomyocytes was used to study the effect of contractile activity on 3-O-methylglucose transport and the subcellular distribution of Glut4. Cells contracting at a frequency of 1 Hz for 30 min exhibited unaltered basal and insulin-stimulated rates of glucose transport when compared to resting cells. However, at 5 Hz 3-O-methylglucose transport increased to 224% of control after 5 min. Under these conditions insulin was unable to produce a significant additional stimulation of glucose transport. Immunoblotting with an anti-Glut4 polyclonal antibody showed that both insulin and contraction (5 Hz) increased the amount of Glut4 in a plasma membrane fraction by about 8-fold with a parallel decrease in an intracellular membrane fraction by 60-65%. These data suggest the existence of an identical insulin- and contraction-recruitable Glut4 transporter pool in cardiomyocytes.

3-O-Methylglucose↗
PubMed1991

Diabetes-induced decrease in the mRNA coding for sarcoplasmic reticulum Ca(2+)-ATPase in adult rat cardiomyocytes.

The present study examined the level of the mRNA coding for the sarcoplasmic reticulum (SR) Ca(2+)-ATPase in isolated ventricular myocytes from streptozotocin-diabetic rats and genetically obese (fa/fa) rats using Northern blotting techniques. In both animal models one single transcript with a mean size of 4.12 kb could be detected. In insulin-deficient rats the Ca(2+)-ATPase transcript level decreased by 51% when compared to normal rats. In obese animals a modest decrease in the Ca(2+)-ATPase mRNA content to 77% of lean controls has been detected. Decreased mRNA expression of the SR Ca(2+)-ATPase may partly explain the delayed diastolic relaxation observed in the diabetic heart.

Animals↗
PubMed1991

Inverse regulation of glucose transporter Glut4 and G-protein Gs mRNA expression in cardiac myocytes from insulin resistant rats.

The present study examined the mRNA levels of glucose transporter Glut4 and G-protein Gs alpha-subunit in isolated ventricular myocytes from lean and genetically obese (fa/fa) Zucker rats and streptozotocin-diabetic rats. In obese animals the amount of transcripts coding for Glut4 increased to 122 +/- 6% of lean controls, whereas the mRNA coding for Gs alpha-subunit decreased by 42 +/- 12%. An unaltered level of Gs mRNA was observed in insulin deficient rats. When cardiomyocytes from normal rats were treated with insulin, the Glut4 transcript level increased by 48 +/- 5%, whereas the Gs mRNA level decreased by 55 +/- 8%. The findings suggest that insulin may act as a potential regulator of Glut4 and Gs mRNA expression in the cardiac cell.

Animals↗
PubMed1991

Alpha-adrenoceptor-mediated increase in cytosolic free calcium in isolated cardiac myocytes.

The effect of alpha-adrenoceptor stimulation on the concentration of cytosolic free calcium (Cai2+) was determined by measuring indo-l fluorescence in isolated ventricular cardiomyocytes from normal and streptozotocin-diabetic rat; 1.3 x 10(5) alpha 1-adrenoceptors per normal myocyte and an unaltered number of these receptors in cells from diabetic rats were detected using the alpha 1-selective ligand WB-4101. Under basal conditions, Cai2+ was found to be 154 +/- 4 nM (n = 34) reaching a value of 192 +/- 10 nM (n = 15) after stimulation of myocytes with a maximal dose of methoxamine for 5 min. Under the same conditions the leakage of dye produced a significantly smaller increase of basal values of 169 +/- 5 nM (n = 17). Indo-l loaded cells did not respond to beta-stimulation unless in the presence of KCl (50 mM), demonstrating the specificity of methoxamine action. Treatment of cells with nifedipine or chelation of extracellular calcium by EGTA did not modify the alpha-adrenergic response. Experiments with cardiomyocytes from streptozotocin-diabetic rats showed an unaltered modulation of Cai2+ by both alpha- and beta-receptor stimulation. It is concluded that signalling by alpha 1-adrenoceptors in ventricular cardiomyocytes results in mobilization of intracellular calcium stores.

Animals↗
PubMed1991

Induction of insulin resistance in primary cultured adult cardiac myocytes.

Primary cultured cardiac myocytes from adult rats were used to elucidate the role of insulin and catecholamines in the development of insulin resistance in this tissue. Cardiomyocytes exhibited a stable response toward insulin up to at least 48 h in serum-free culture, as determined by measuring the effect of the hormone on initial rates of 2-deoxyglucose uptake. Culturing of cells in the absence of insulin for 6 and 19 h, respectively, resulted in a loss of insulin sensitivity and a reduced (33%) maximal responsiveness after 19 h of insulin deficiency. This was paralleled by a decrease in [14C]phenylalanine incorporation and an unaltered level of insulin binding. Insulin action was completely lost in cells cultured in the presence of cycloheximide for 19 h. When added to the culture medium for 4 h, both isoproterenol and (Bu)2cAMP decreased insulin binding by about 50%. Under these conditions maximal insulin responsiveness was not affected by isoproterenol but was reduced by 46% by (Bu)2cAMP. Nifedipine antagonized the inhibitory action of (Bu)2cAMP, but was ineffective when the culture period was extended to 19 h. Cardiomyocytes cultured in the presence of palmitate exhibited a largely reduced (67%) insulin responsiveness, which was only partly restored by inhibition of fatty acid oxidation. From these data we conclude that: 1) insulin deficiency induces insulin resistance due to decreased protein synthesis; 2) sustained, prolonged elevation of cAMP modulates insulin action by both Ca(++)-dependent and Ca(++)-independent mechanisms; and 3) free fatty acids antagonize insulin action by both metabolic and nonmetabolic pathways.

Animals↗
PubMed1990

G-protein-mediated regulation of the insulin-responsive glucose transporter in isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the involvement of G-proteins in the regulation of the glucose transporter by insulin and isoprenaline. Efficient modification of G-protein functions was established by measuring isoprenaline-stimulated cyclic AMP production, viability and ATP content after treating the cells with cholera toxin and pertussis toxin for 2 h. Under these conditions cholera toxin decreased the stimulatory action of insulin on 3-O-methylglucose transport by 56%, but pertussis toxin had no effect. Basal transport was not affected by toxin treatment. Isoprenaline increased 3-O-methylglucose transport by 63%. This effect was not mimicked by dibutyryl cyclic AMP, but was completely blocked by cholera toxin. Streptozotocin-diabetes abolished isoprenaline action and decreased stimulation of transport by 64%. Concomitantly, cholera-toxin sensitivity of glucose transport was lost in cells from diabetic animals. This was paralleled by a large decrease (87 +/- 4%) in mRNA expression of the insulin-regulatable glucose transporter, as shown by Northern-blot analysis of RNA isolated from cardiomyocytes of diabetic rats. These data suggest a functional association between the insulin-responsive glucose transporter and a cholera-toxin-sensitive G-protein mediating stimulation by insulin and isoprenaline.

3-O-Methylglucose↗
PubMed1990

Modulation of transmembrane potential of isolated cardiac myocytes by insulin and isoproterenol.

Isolated muscle cells from adult rat heart have been used to study the effects of insulin and catecholamines on transmembrane potential by following triphenylmethylphosphonium cation uptake. Insulin was found to hyperpolarize the cells with a maximal effect of 3.2 +/- 0.7 mV (n = 4) at an insulin concentration of 3 x 10(-9) mol/l. This insulin action was fully antagonized by isoproterenol (10(-5) mol/l), which depolarized the cardiocytes in a dose-dependent fashion with a maximal effect of 9.5 +/- 2.2 mV. Treatment of cardiocytes with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or CsCl resulted in a total loss of insulin action, whereas isoproterenol action was not affected. Cardiac myocytes from streptozotocin diabetic rats exhibited an unaltered hyperpolarization by insulin within the physiological concentration range. Isoproterenol now induced a biphasic response with a significant hyperpolarization at low doses and a decreased depolarization at maximal concentrations. In conclusion, 1) hormonal modulation of cardiac myocyte membrane potentials involves hyperpolarization by insulin and depolarization by beta-agonists, 2) insulin action appears to be related to an increased potassium conductance and may be antagonized by beta-stimulation, and 3) membrane potential modulation may be profoundly altered in the diabetic state.

Animals↗
PubMed1988

Involvement of hormone processing in insulin-activated glucose transport by isolated cardiac myocytes.

Isolated muscle cells from adult rat heart were used to study the relationship between myocardial insulin processing and insulin action on 3-O-methylglucose transport at 37 degrees C. Internalization of the hormone as measured by determination of the non-dissociable fraction of cell-bound insulin increased linearly up to 10 min, reaching a plateau by 30-60 min at 3 nM-insulin. At this hormone concentration the onset of insulin action was found to be biphasic, with a rapid phase up to 8 min, followed by a much slower phase, reaching maximal insulin action by 30-60 min. Insulin internalization was totally blocked by phenylarsine oxide, whereas dansylcadaverine had no effect on this process. Initial insulin action (5 min) on glucose transport was not affected by chloroquine and dansylcadaverine, but was completely abolished by treatment of cardiocytes with phenylarsine oxide. This drug effect was partly prevented by the presence of 2,3-dimercaptopropanol. Under steady-state conditions (60 min), the stimulatory action of insulin was decreased by about 60% by both chloroquine and dansylcadaverine. This study, demonstrates that insulin action on cardiac glucose transport is mediated by processing of the hormone. The data suggest dual pathways of insulin action involving initial processing of hormone-receptor complexes and lysosomal degradation.

3-O-Methylglucose↗
PubMed1987

Insulin binding and action in isolated cardiocytes from spontaneously diabetic BB rats.

Isolated cardiac myocytes from control and insulin treated diabetic BB rats were used to study cellular alterations related to partly controlled diabetes. Scatchard analysis of equilibrium binding data showed an unaltered affinity and number of insulin receptors in cardiocytes from both groups of animals. Moreover, insulin internalization was found to be identical under these conditions. Insulin action was determined by measuring the effect of the hormone on initial velocities of 3-0-methylglucose influx. Basal activity of the glucose transporter and maximal transport stimulation by insulin remained unaffected. In contrast, the sensitivity of the carrier towards stimulation by insulin was markedly reduced in cardiocytes from diabetic rats with a half-maximal action occurring at an insulin concentration of 3 X 10(-10) mol/l and 9 X 10(-9) mol/l in control and diabetic animals, respectively. The onset of insulin action was much slower in cells from diabetic BB rats exhibiting an increase in the coupling time by 400% from 5 to 20 min, respectively. The data suggest an association of partly controlled diabetes with myocardial alterations located at the postreceptor level.

Animals↗
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↗
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↗