Sensitivity Of Glucose Uptake Research Articles

Insulin sensitivity and inhibition by forskolin, dipyridamole and pentobarbital of glucose transport in three L6 muscle cell lines

L6 skeletal muscle myoblasts stably overexpressing glucose transporter GLUT1 or GLUT4 with exofacial myc-epitope tags were characterized for their response to insulin. In clonally selected cultures, 2-deoxyglucose uptake into L6-GLUT1myc myoblasts and myotubes was linear within the time of study. In L6-GLUT1myc and L6-GLUT4myc myoblasts, 100 nmol/L insulin treatment increased the GLUT1 content of the plasma membrane by 1.58+/-0.01 fold and the GLUT4 content 1.96+/-0.11 fold, as well as the 2-deoxyglucose uptake 1.53+/-0.09 and 1.86+/-0.17 fold respectively, all by a wortmannin-inhibitable manner. The phosphorylation of Akt in these two cell lines was increased by insulin. L6-GLUT1myc myoblasts showed a dose-dependent stimulation of glucose uptake by insulin, with unaltered sensitivity and maximal responsiveness compared with wild type cells. By contrast, the improved insulin responsiveness and sensitivity of glucose uptake were observed in L6-GLUT4myc myoblasts. Earlier studies indicated that forskolin might affect insulin-stimulated GLUT4 translocation. A 65% decrease of insulin-stimulated 2-deoxyglucose uptake in GLUT4myc cells was not due to an effect on GLUT4 mobilization to the plasma membrane, but instead on direct inhibition of GLUT4. Forskolin and dipyridamole are more potent inhibitors of GLUT4 than GLUT1. Alternatively, pentobarbital inhibits GLUT1 more than GLUT4. The use of these inhibitors confirmed that the overexpressed GLUT1 or GLUT4 are the major functional glucose transporters in unstimulated and insulin-stimulated L6 myoblasts. Therefore, L6-GLUT1myc and L6-GLUT4myc cells provide a platform to screen compounds that may have differential effects on GLUT isoform activity or may influence GLUT isoform mobilization to the cell surface of muscle cells.

Insulin-Stimulated Glucose Uptake Does Not Require p38 Mitogen-Activated Protein Kinase in Adipose Tissue or Skeletal Muscle

It has been proposed that p38 mitogen-activated protein kinase (MAPK) isoforms sensitive to the pyridinylimidazole compounds SB 203580 and SB 202190 may participate in the acute insulin-dependent activation of glucose transporters recruited to the plasma membrane of adipocytes and skeletal muscle. Here, we explore whether these kinases support the insulin stimulation of glucose uptake in these tissues by investigating the effects of a genetic loss in p38beta and that of the p38 MAPK inhibitor SB 203580. Glucose uptake in adipocytes and soleus muscle was stimulated by insulin by up to fourfold irrespective of whether tissues were isolated from wild-type or p38beta-null mice. Consistent with this finding, mice lacking p38beta exhibited normal glucose tolerance, insulinemia, and glycemia compared with their wild-type counterparts. Insulin-stimulated glucose uptake was not inhibited by SB 203580 when adipocytes were preincubated with the drug at a cytocrit of 50%, but intriguingly, uptake was suppressed (by 35%) when the cytocrit was reduced by one-half. Despite the activation of glucose uptake at the higher cytocrit, insulin failed to induce any detectable activation of p38 MAPK, whereas p38 signaling was robustly activated by anisomycin in a SB 203580-sensitive manner. Although insulin also failed to induce any detectable activation of p38 MAPK in muscle, insulin-dependent glucose uptake was reduced by SB 203580 (approximately 44%) in muscle of both wild-type and p38beta-null mice. Our results indicate that p38beta is not required for insulin-stimulated glucose uptake in adipocytes or muscle. Moreover, given that insulin fails to promote any significant activation of p38 MAPK in these tissues and the finding that sensitivity of glucose uptake, but not that of the kinase, to SB 203580 can be influenced by cytocrit, we suggest that p38 signaling is unlikely to participate in any putative activation of transporters recruited to the cell surface by insulin and that SB 203580 suppresses insulin-stimulated glucose transport by a mechanism unrelated to its inhibitory effect on p38 MAPK.

The relationship between insulin binding, insulin activation of insulin-receptor tyrosine kinase, and insulin stimulation of glucose uptake in isolated rat adipocytes. Effects of isoprenaline

We have studied the relationship between insulin activation of insulin-receptor kinase and insulin stimulation of glucose uptake in isolated rat adipocytes. Glucose uptake was half-maximally or maximally stimulated, respectively, when only 4% or 14% of the maximal kinase activity had been reached. To investigate this relationship also under conditions where the insulin effect on activation of receptor kinase was decreased, the adipocytes were exposed to 10 microM-isoprenaline alone or with 5 micrograms of adenosine deaminase/ml. An approx. 30% (isoprenaline) or approx. 50% (isoprenaline + adenosine deaminase) decrease in the insulin effect on receptor kinase activity was found at insulin concentrations between 0.4 and 20 ng/ml, and this could not be explained by decreased insulin binding. The decreased insulin-effect on kinase activity was closely correlated with a loss of insulin-sensitivity of glucose uptake. Moreover, our data indicate that the relation between receptor kinase activity and glucose uptake (expressed as percentage of maximal uptake) remained unchanged. The following conclusions were drawn. (1) If activation of receptor kinase stimulates glucose uptake, only 14% of the maximal kinase activity is sufficient for maximal stimulation. (2) Isoprenaline decreases the coupling efficiency between insulin binding and receptor-kinase activation, this being accompanied by a corresponding decrease in sensitivity of glucose uptake. (3) Our data indicate that the signalling for glucose uptake is closely related to receptor-kinase activity, even when the coupling efficiency between insulin binding and kinase activation is altered. They thus support the hypothesis that receptor-kinase activity reflects the signal which originates from the receptor and which is transduced to the glucose-transport system.

Muscle response to insulin in fetuses of diabetic pregnant rats.

To elucidate the effect of nutrition during induction on peripheral muscle responsiveness to insulin, the incorporation of radiolabeled glucose to glycogen and the uptake of radiolabeled deoxyglucose were studied in isolated diaphragms from the fetuses of normal and diabetic pregnant rats in vitro. Basal- and insulin-stimulated incorporation of [1-14C]glucose into diaphragm glycogen were greater in the fetuses of diabetic mothers (IDM) than in normal fetuses, but there was no difference in the degree of stimulation by insulin of labeled glucose into glycogen between normal fetuses and IDM. Diaphragms from normal fetuses and IDM had the same basal up ake of 2-deoxy-[1-3H]glucose as well as insulin-stimulated uptake. Consequently the sensitivity of glucose uptake to insulin was similar both in normal fetuses and IDM. These data indicate that glucose utilization (incorporation of labeled glucose into glycogen) was increased in IDM, but that the response of glucose uptake and glycogenesis to insulin was not altered.

Effect of human saliva on the fluoride sensitivity of glucose uptake by Streptococcus mutans.

The fluoride (F) sensitivity of glucose uptake by whole cell suspensions of streptococcus mutans in the presence and absence of human whole salivary supernatant was studied. It was observed that dithiothreitol (DTT) and other thiols markedly reduced the F sensitivity of cells when saliva (50%, vol/vol) was present during glucose uptake. In the absence of saliva, cells were sensitive to 2 to 2.5 mM F regardless of the presence of thiols. Supplementation of cells in phosphate or tris(hydroxymethyl)aminomethane-hydrochloride buffers with physiological concentrations of calcium or phosphate had no effect on the F sensitivity of the organism. Experiments with permeabilized cells suggested that thiols themselves had no direct effect on the F sensitivity of enolase (a principal F target). Cells pretreated with DDT subsequently exhibited decreased F sensitivity when examined in the presence of saliva but not in the absence of saliva. Cells pretreated with whole salivary supernatant were found to be subsequently less sensitive to F in the absence of saliva during glucose uptake. Furthermore, in cases where cells were pretreated with saliva, subsequent additions of DDT were unnecessary to obtain maximal reduction in the F sensitivity of glucose uptake. It was concluded that the saliva-dependent reduction in F sensitivity of glucose uptake was not due to sequestration of available F by salivary constituents. The data suggest that a salivary component(s) interacts directly with the microorganism in some manner which results in reduced F sensitivity of the process under study. Possible mechanisms of saliva action are discussed.

Sensitivity of glucose uptake and lipolysis of white adipocytes of the rat to insulin and effects of some metabolites.

1. Insulin increased glucose uptake and inhibited lipolysis in white adipocytes of the rat over the same concentration range of the hormone: the half-maximal effects were observed at approx. 10 microunits of insulin/ml. Thus, contrary to previous reports, no difference in sensitivity of the two processes to insulin could be found, which suggests that both these effects of insulin are important in increasing the rate of glucose utilization after a meal. 2. Adenosine deaminase, which lowers the concentration of adenosine in the incubation medium, decreased the sensitivity of both processes (lipolysis and glucose uptake) to insulin: this suggests that adenosine increases the sensitivity of both processes. Similarly, lactate and 3-hydroxybutyrate increased the sensitivity of both processes (to the same extent) to insulin. It is suggested that this increased sensitivity will improve the response (of adipose tissue) to insulin on refeeding after a prolonged period of starvation (when the hydroxybutyrate concentration is high), and after a short burst of exercise, when the blood lactate concentration is high and when large amounts of glucose are produced from lactate via gluconeogenesis in the liver.