scholarly journals Sphingomyelinase stimulates 2-deoxyglucose uptake by skeletal muscle

1996 ◽  
Vol 313 (1) ◽  
pp. 215-222 ◽  
Author(s):  
Jiri TURINSKY ◽  
G. William NAGEL ◽  
Jeffrey S. ELMENDORF ◽  
Alice DAMRAU-ABNEY ◽  
Terry R. SMITH
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Cellular Accumulation ◽  
C6 Ceramide ◽  
Phosphatidylinositol 3

The effects of sphingomyelinase, phosphorylcholine, N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide) and sphingosine on basal and insulin-stimulated cellular accumulation of 2-deoxy-D-glucose in rat soleus muscles were investigated. Preincubation of muscles with sphingomyelinase (100 or 200 m-units/ml) for 1 or 2 h augmented basal 2-deoxyglucose uptake by 29-91%, and that at 0.1 and 1.0 m-unit of insulin/ml by 32-82% and 19-25% respectively compared with control muscles studied at the same insulin concentrations. The sphingomyelinase-induced increase in basal and insulin-stimulated 2-deoxyglucose uptake was inhibited by 91% by 70 μM cytochalasin B, suggesting that it involves glucose transporters. Sphingomyelinase had no effect on the cellular accumulation of L-glucose, which is not transported by glucose transporters. The sphingomyelinase-induced increase in 2-deoxyglucose uptake could not be reproduced by preincubating the muscles with 50 μM phosphorylcholine, 50 μM C2-ceramide or 50 μM C6-ceramide. Preincubation of muscles with 50 μM sphingosine augmented basal 2-deoxyglucose transport by 32%, but reduced the response to 0.1 and 1.0 m-unit of insulin/ml by 17 and 27% respectively. The stimulatory effect of sphingomyelinase on basal and insulin-induced 2-deoxyglucose uptake was not influenced by either removal of Ca2+ from the incubation medium or dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum. This demonstrates that Ca2+ does not mediate the action of sphingomyelinase on 2-deoxyglucose uptake. Sphingomyelinase also had no effect on basal and insulin-stimulated activities of insulin receptor tyrosine kinase and phosphatidylinositol 3-kinase. In addition, 1 and 5 μM wortmannin, an inhibitor of phosphatidylinositol 3-kinase, failed to inhibit the sphingomyelinase-induced increase in 2-deoxyglucose uptake. These results suggest that sphingomyelinase does not increase 2-deoxyglucose uptake by stimulating the insulin receptor or the initial steps of the insulin-transduction pathway. The data suggest the possibility that sphingomyelinase increases basal and insulin-stimulated 2-deoxyglucose uptake in skeletal muscle as the result of an unknown post-receptor effect.

scholarly journals Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase

1991 ◽  
Vol 280 (3) ◽  
pp. 769-775 ◽  
Author(s):  
H Hayashi ◽  
N Miyake ◽  
F Kanai ◽  
F Shibasaki ◽  
T Takenawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Phosphorylated Form ◽  
Phosphatidylinositol 3

Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo.

scholarly journals Roles of 1-phosphatidylinositol 3-kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells.

1995 ◽  
Vol 15 (10) ◽  
pp. 5403-5411 ◽  
Author(s):  
M J Quon ◽  
H Chen ◽  
B L Ing ◽  
M L Liu ◽  
M J Zarnowski ◽  
...  
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Glucose Transport ◽  
Expression Vectors ◽  
Regulatory Subunit ◽  
Dominant Negative Effect ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Adipose Cells ◽  
Phosphatidylinositol 3

Insulin stimulates glucose transport in insulin target tissues by recruiting glucose transporters (primarily GLUT4) from an intracellular compartment to the cell surface. Previous studies have demonstrated that insulin receptor tyrosine kinase activity and subsequent phosphorylation of insulin receptor substrate 1 (IRS-1) contribute to mediating the effect of insulin on glucose transport. We have now investigated the roles of 1-phosphatidylinositol 3-kinase (PI 3-kinase) and ras, two signaling proteins located downstream from tyrosine phosphorylation. Rat adipose cells were cotransfected with expression vectors that allowed transient expression of epitope-tagged GLUT4 and the other genes of interest. Overexpression of a mutant p85 regulatory subunit of PI 3-kinase lacking the ability to bind and activate the p110 catalytic subunit exerted a dominant negative effect to inhibit insulin-stimulated translocation of epitope-tagged GLUT4 to the cell surface. In addition, treatment of control cells with wortmannin (an inhibitor of PI 3-kinase) abolished the ability of insulin to recruit epitope-tagged GLUT4 to the cell surface. Thus, our data suggest that PI 3-kinase plays an essential role in insulin-stimulated GLUT4 recruitment in insulin target tissues. In contrast, over-expression of a constitutively active mutant of ras (L61-ras) resulted in high levels of cell surface GLUT4 in the absence of insulin that were comparable to levels seen in control cells treated with a maximally stimulating dose of insulin. However, wortmannin treatment of cells overexpressing L61-ras resulted in only a small decrease in the amount of cell surface GLUT4 compared with that of the same cells in the absence of wortmannin. Therefore, while activated ras is sufficient to recruit GLUT4 to the cell surface, it does so by a different mechanism that is probably not involved in the mechanism by which insulin stimulates GLUT4 translocation in physiological target tissues.

scholarly journals Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells

1998 ◽  
Vol 274 (4) ◽  
pp. E611-E617 ◽  
Author(s):  
Rae D. Record ◽  
Larry L. Froelich ◽  
Chris J. Vlahos ◽  
Bonnie L. Blazer-Yost
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Sodium Transport ◽  
Specific Inhibitor ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
A6 Cells ◽  
Phosphatidylinositol 3

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 ≈ 6 μM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

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