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J Eckel

Publications and source records attributed to J Eckel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Insulin-dependent regulation of Glut4 gene expression in ventricular cardiomyocytes: evidence for a direct effect on Glut4 transcription.

The present study examined the effect of insulin on Glut4 transcription in isolated ventricular cardiomyocytes. Upon exposure to the hormone (2 x 10(-7) M) the cellular Glut4 mRNA content increased to 224 +/- 46% of control within 3.5 hours. Nuclear run-on analysis indicated a parallel increase of transcription of the Glut4 gene to 208 +/- 49% of control. Direct incubation of cardiac nuclei with insulin resulted in a comparably significant increase of Glut4 transcription. Cross-linkage experiments with 125I-labelled insulin demonstrated the absence of soluble nuclear insulin receptors, which were readily detected in plasma and microsomal membranes. These findings suggest that expression of the cardiac Glut4 gene is subject to regulation by insulin at the transcriptional level, a process possibly involving nuclear association of the hormone.

Animals↗
PubMed1995

Insulin action on cardiac glucose transport: studies on the role of protein kinase C.

Isolated ventricular cardiomyocytes from adult rat have been used to elucidate a possible relationship between protein kinase C (PKC) and the stimulatory action of insulin on cardiac glucose transport. Cells were incubated in the presence of either insulin or phospholipase C from Clostridium perfringens (PLC-Cp) and intracellular sn-1,2-diacylglycerol (DAG) levels and initial rates of 3-O-methylglucose transport were determined. Insulin had no effect on the DAG mass level, whereas it was elevated by PLC-Cp to 200% of control. Under these conditions the hormone produced a 2.7-fold stimulation of glucose transport with no significant effect of PLC-Cp. Insulin was unable to produce a redistribution of PKC, whereas phorbol 12-myristate 13-acetate (PMA) increased membrane associated PKC twofold. The PKC inhibitors tamoxifen and staurosporine did not interfere with glucose transport stimulation by insulin. Furthermore, cells treated with PMA exhibited unaltered basal and maximally insulin stimulated rates of glucose transport. In contrast, at physiological concentrations of insulin the stimulatory action of the hormone was significantly reduced. We conclude from our data that PKC is not involved in insulin action on cardiac glucose transport. However, activation of this enzyme may lead to a modified insulin sensitivity of the cardiac cell.

Animals↗
PubMed1995

Effects of tumour necrosis factor alpha (TNF alpha) on glucose transport and lipid metabolism of newly-differentiated human fat cells in cell culture.

Tumour necrosis factor alpha (TNF alpha) has been found to cause a delipidation of fat cells and a decrease of the adipose tissue mass. In the present study, we tried to elucidate some of the mechanisms responsible for this phenomenon by investigating the action of TNF alpha on specific pathways which are involved in lipid storage. Cultured stromal cells from human adipose tissue were induced to differentiate into adipose cells by exposure to adipogenic factors and subsequently used for studying the effects of TNF alpha on fat cell metabolism. Presence of 5 nmol/l TNF alpha for 24 h resulted in a complete loss of the stimulatory effect of insulin on 2-deoxy-glucose transport. This inhibitory action was paralleled by a decrease of GLUT4 protein and mRNA levels. The amount of cellular GLUT4 protein was reduced by 49 +/- 3% after a 24-h exposure and by 82 +/- 18% after a 72-h exposure to 5 nmol/l TNF alpha. GLUT4 mRNA was almost undetectable after a 24-h incubation with 5 nmol/l TNF alpha. In a similar time-dependent manner, TNF alpha dramatically reduced the lipoprotein lipase mRNA content of the cells. Furthermore, incubation of cultured human fat cells with TNF alpha resulted in a marked dose-dependent stimulation of lipolysis, assessed by glycerol release, by up to 400% above controls, which became apparent after a 6-h exposure at the earliest.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1995

Direct stimulation of myocardial glucose transport and glucose transporter-1 (GLUT1) and GLUT4 protein expression by the sulfonylurea glimepiride.

Freshly isolated and primary cultured cardiac myocytes from adult rats were used to elucidate acute and chronic effects of the sulfonylurea drug glimepiride on basal and insulin-stimulated glucose transport and on expression of the transporter isoforms glucose transporter-1 (GLUT1) and GLUT4. A 30-min incubation with glimepiride (100 microM) was unable to modify the initial rates of 3-O-methylglucose transport in freshly isolated cardiocytes, both in the absence or presence of insulin (10(-7) M). Cells were then kept in serum-free culture for 20 h in the presence of glimepiride (10 microM) and a physiological insulin dose. Under these conditions, the sulfonylurea induced an increase in 2-deoxyglucose uptake to 186% of control. This drug effect was dose dependent and could also be demonstrated in the absence of insulin during the culture period. The acute action of insulin on glucose transport was additive to the effect of glimepiride, and the insulin responsiveness of glucose transport remained unaltered in sulfonylurea-treated cultures. Western blot analysis of crude membrane fractions obtained from cultured cardiocytes showed that glimepiride increased the expression of both GLUT1 and GLUT4 to 164% +/- 21% and 148% +/- 5% of control, respectively. We concluded that glimepiride increases cardiac glucose uptake by an insulin-independent pathway, probably involving an increased protein expression of GLUT1 and GLUT4. The increased expression of GLUT4 may have a therapeutic impact on the treatment of insulin-resistant states.

3-O-Methylglucose↗
PubMed1994

Insulin-induced translocation of the glucose transporter GLUT4 in cardiac muscle: studies on the role of small-molecular-mass GTP-binding proteins.

Subcellular fractions obtained from rat cardiac ventricular tissue were used to elucidate a possible functional relationship between small-molecular-mass G-proteins and the insulin-responsive glucose transporter GLUT4. Proteins were separated by SDS/PAGE and transferred to nitrocellulose membranes. Incubation with [alpha-32P]GTP revealed the presence of two major distinct GTP-binding protein bands of 24 and 26 kDa in both plasma and microsomal membranes. Immunoadsorption of microsomal membranes to anti-GLUT4 antibodies was used to isolate GLUT4-enriched membrane vesicles. This material was found to contain a much decreased amount of small G-proteins, with the exclusive presence of the 24 kDa species. Insulin treatment in vivo had no effect on the microsomal membrane content of small GTP-binding proteins, but significantly decreased the 24 kDa species in GLUT4-enriched vesicles by 36 +/- 5% (n = 3). This correlated with a decreased (30-40%) recovery of GLUT4-enriched vesicles from insulin-treated animals. Western-blot analysis of microsomal membranes with a panel of antisera against rab GTP-binding proteins indicated the presence of rab4A, with a molecular mass of 24 kDa, whereas rab1A, rab2 and rab6 were not observed. rab4A was barely detectable in GLUT4-enriched vesicles; however, insulin produced an extensive shift of rab4A from the cytosol and the microsomal fraction to the plasma membrane with a parallel increase in GLUT4. These data show that a small GTP-binding protein is co-localized with GLUT4 in an insulin-responsive intracellular compartment, and strongly suggest that this protein is involved in the exocytosis of GLUT4 in cardiac muscle. Furthermore, the observed translocation of rab4A is compatible with insulin-induced endosome recycling processes, possibly including the glucose transporters.

Animals↗
PubMed1994

Photoaffinity labelling of cardiac membrane GTP-binding proteins in response to insulin.

Plasma membranes from rat cardiac ventricular tissue and insulin receptors partially purified by wheat-germ-agglutinin chromatography were subjected to direct photoaffinity labelling with [alpha-32P]GTP in order to elucidate the presence of insulin-receptor-coupled GTP-binding proteins. In plasma membranes three proteins have been identified that exhibit an enhanced photolabelling with the nucleotide in response to insulin. The apparent molecular masses of these proteins were found to be 56, 60 and 74 kDa. Photolabelling of partially purified insulin receptors showed the copurification of the 60-kDa species, whereas the 56-kDa and 74-kDa proteins could not be detected. Furthermore, the 60-kDa G-protein was found to be specifically co-immunoprecipitated with the insulin receptor. Incubation of insulin receptors with insulin increased the labelling of the 60-kDa band to 205 +/- 27% (n = 5) of control. Immuno- and ligand-blotting experiments revealed the additional presence of a 39-kDa G(o)-like protein and two G-proteins with molecular masses of 24 and 26 kDa in the receptor preparation. Under basal conditions the insulin receptor and the 60-kDa G-protein exhibited an apparent inverse distribution between plasma and microsomal membranes with the G-protein being extensively labelled in the microsomal fraction. In conclusion, our data show that, in its native environment, the cardiac insulin receptor couples to at least three GTP-binding proteins. Out of these, a 60-kDa species of microsomal origin, copurifies with the insulin receptor. It is suggested that this G-protein is associated with the insulin receptor and may be involved in insulin receptor signalling in target cells.

Affinity Labels↗
PubMed1994

G protein expression and adenylate cyclase regulation in ventricular cardiomyocytes from STZ-diabetic rats.

Isolated adult ventricular cardiomyocytes have been used to study the effects of insulin-deficient diabetes on the expression of cardiac G protein alpha-subunits. Immunoblot analysis of plasma membranes revealed the presence of three different Gs proteins with molecular masses of 45, 47, and 52 kDa. Furthermore, cardiomyocytes were found to contain Gi-2 (41 kDa) and G(o) (39 kDa). Heart cells from streptozotocin-diabetic rats exhibited an unaltered expression of the Gs proteins, whereas Gi-2 and G(o) were reduced by 58 +/- 2 and 27 +/- 11%, respectively. In cells from diabetic rats, adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in response to isoproterenol decreased by approximately 30% at agonist concentrations of 10(-7) to 10(-5) M, with an unaltered maximum stimulation by forskolin. Treatment of cardiomyocytes with pertussis toxin resulted in an incremental increase of isoproterenol-stimulated cAMP formation, which was significantly lower in cardiac myocytes from streptozotocin-diabetic animals (19.2 +/- 1.7 vs. 11.5 +/- 2.4 pmol cAMP.5 x 10(4) cells-1 times 10 min-1). The inhibition of the isoproterenol-induced cAMP accumulation by carbachol in the intact cell was not altered in streptozotocin-diabetes. In conclusion, our data show that insulin-deficient diabetes is associated with a reduced expression and concomitant functional loss of Gi in ventricular cardiomyocytes. Receptor-mediated inhibition of adenylate cyclase remains unaffected by this process, whereas the beta-adrenergic stimulatory pathway involves an additional defect upstream of the adenylate cyclase/G protein system.

Adenylyl Cyclases↗
PubMed1993

Regulation of cardiac insulin receptor function by guanosine nucleotides.

The present study examined the effect of GTP-gamma-S on the function of insulin receptors partially purified from adult rat cardiomyocytes by WGA chromatography. GTP-gamma-S increased receptor autophosphorylation about two times and fully mimicked the stimulatory action of insulin on poly(Glu:Tyr) phosphorylation with no additional effect of the hormone. The effect of GTP-gamma-S was specific, dose-dependent, and due to an increase in the Vmax of the kinase. In the presence of ATP or AMP-PNP, insulin significantly enhanced the binding of [35S]GTP-gamma-S to the partially purified insulin receptor. The findings suggest coupling of the insulin receptor to a G-protein which may be involved in the regulation of tyrosine kinase activity.

Animals↗
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↗