scholarly journals Association of phosphatidylinositol 3-kinase with the insulin receptor: compartmentation in rat liver

2000 ◽  
Vol 279 (2) ◽  
pp. E266-E274 ◽  
Author(s):  
Paul G. Drake ◽  
Alejandro Balbis ◽  
Jiong Wu ◽  
John J. M. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Insulin Receptor ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Metabolic Effects ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) plays an important role in a variety of hormone and growth factor-mediated intracellular signaling cascades and has been implicated in the regulation of a number of metabolic effects of insulin, including glucose transport and glycogen synthase activation. In the present study we have examined 1) the association of PI 3-kinase with the insulin receptor kinase (IRK) in rat liver and 2) the subcellular distribution of PI 3-kinase-IRK interaction. Insulin treatment promoted a rapid and pronounced recruitment of PI 3-kinase to IRKs located at the plasma membrane, whereas no increase in association with endosomal IRKs was observed. In contrast to IRS-1-associated PI 3-kinase activity, association of PI 3-kinase with the plasma membrane IRK did not augment the specific activity of the lipid kinase. With use of the selective PI 3-kinase inhibitor wortmannin, our data suggest that the cell surface IRK β-subunit is not a substrate for the serine kinase activity of PI 3-kinase. The functional significance for the insulin-stimulated selective recruitment of PI 3-kinase to cell surface IRKs remains to be elucidated.

scholarly journals Phosphatidylinositol 3-kinase acts at an intracellular membrane site to enhance GLUT4 exocytosis in 3T3-L1 cells

1996 ◽  
Vol 313 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Jing YANG ◽  
James F. CLARKE ◽  
Catriona J. ESTER ◽  
Paul W. YOUNG ◽  
Masato KASUGA ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Receptor ◽  
Time Course ◽  
Glucose Transporter ◽  
Kinase Activity ◽  
Insulin Receptor Substrate ◽  
Low Density ◽  
Intracellular Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Phosphatidylinositol 3

Glucose transporters (GLUTs) are continuously recycled in 3T3-L1 cells and so insulin, through its action on phosphatidylinositol 3-kinase (PI 3-kinase), could potentially alter the distribution of these transporters by enhancing retention in the plasma membrane or acting intracellularly to increase exocytosis, either by stimulating a budding or a docking and fusion process. To examine the site of involvement of PI 3-kinase in the glucose transporter recycling pathway, we have determined the kinetics of recycling under conditions in which the PI 3-kinase activity is inhibited by wortmannin. Wortmannin addition to fully insulin-stimulated cells induces a net reduction of glucose transport activity with a time course that is consistent with a major effect on the return of internalized transporters to the plasma membrane. The exocytosis of GLUT1 and GLUT4 is reduced to very low levels in wortmannin-treated cells (≈ 0.009 min-1), but the endocytosis of these isoforms is not markedly perturbed and the rate constants are approx. 10-fold higher than for exocytosis (0.099 and 0.165 min-1, respectively). The slow reduction in basal activity following treatment with wortmannin is consistent with a wortmannin effect on constitutive recycling as well as insulin-regulated exocytosis. PI 3-kinase activity that is precipitated by anti-phosphotyrosine, anti-[insulin receptor substrate 1 (IRS1)] and anti-α-p85 antibodies show the same level of insulin-stimulated activity, ≈ 0.5 pmol/20 min per dish of 3T3-L1 cells. Since the activities precipitated by all three antibodies are similar, it seems unlikely that a second insulin receptor substrate, IRS2, contributes significantly to the insulin signalling observed in 3T3-L1 cells. To examine whether insulin targets PI 3-kinase to intracellular membranes we have carried out subcellular fractionation studies. These suggest that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular, low-density, membranes. In addition, the association of PI 3-kinase with IRS1 appears to partially deplete the cytoplasm of α-p85-precipitatable activity, suggesting that IRS1 may redistribute PI 3-kinase from the cytoplasm to the low-density microsome membranes. Taken together, the trafficking kinetic and PI 3-kinase distribution studies suggest an intracellular membrane site of action of the enzyme in enhancing glucose transporter exocytosis.

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 Effect of nutritional state on the formation of a complex involving insulin receptor IRS-1, the 52 kDa Src homology/collagen protein (Shc) isoform and phosphatidylinositol 3′-kinase activity

1998 ◽  
Vol 335 (2) ◽  
pp. 293-300 ◽  
Author(s):  
Joëlle DUPONT ◽  
Michel DEROUET ◽  
Jean SIMON ◽  
Mohammed TAOUIS
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Residual Activity ◽  
Nutritional State ◽  
Phosphatidylinositol 3 Kinase ◽  
Regulatory Subunits ◽  
Normal Tissues ◽  
Collagen Protein ◽  
Src Homology ◽  
Phosphatidylinositol 3

The Src homology and collagen protein (Shc) is tyrosine phosphorylated in response to insulin; however, evidence for its interaction with insulin receptor (IR) in normal tissues is missing. Interactions between IR, Shc and regulatory subunits of the phosphatidylinositol 3´-kinase (PI 3´-kinase) were characterized in the present study in liver and muscles of chickens submitted to various nutritional states. A chicken liver Shc cDNA fragment encoding a 198 amino acid long fragment, including the phosphotyrosine binding domain was sequenced. It shows 89% homology with the corresponding human homologue. The amounts of the three Shc isoforms (66, 52 and 46 kDa) and Shc messenger were not altered by the nutritional state. Shc tyrosine phosphorylation was decreased by fasting in both liver and muscle. Importantly, Shc was immunoprecipitated by IR antibody (mostly the 52 kDa isoform) or by αIRS-1(mostly the 46 kDa isoform). IR–Shc association was decreased by fasting and restored by refeeding. In liver, αShc immunoprecipitated the three forms of regulatory subunits of PI 3´-kinase and a PI 3´-kinase activity which was decreased by fasting. In muscle, αShc immunoprecipitated only the p85 isoform; the associated PI 3´-kinase activity was not altered by the nutritional state. Conversely, in both tissues anti-p85 antibody precipitated only the 52 kDa Shc isoform. In liver, antibodies to insulin receptor substrate-1 (αIRS-1), Shc or IR immunoprecipitated the three regulatory subunits of PI 3´-kinase and an equal PI 3´-kinase activity, without any residual activity left in the supernatants, suggesting the presence of a large complex involving IR, IRS-1, Shc (mainly the 52 kDa isoform) and PI 3´-kinase activity. The presence of another complex containing IRS-1 and the 46 kDa Shc isoform, but no PI 3´-kinase activity, is suggested.

scholarly journals Compartmentalization and Insulin-Induced Translocations of Insulin Receptor Substrates, Phosphatidylinositol 3-Kinase, and Protein Kinase B in Rat Liver**This work was supported by grants from the Medical Research Council and from the National Cancer Institute of Canada, and by the Cleghorn Fund at McGill University and the M. Pollack Foundation of Montreal.

Endocrinology ◽  
2000 ◽  
Vol 141 (11) ◽  
pp. 4041-4049 ◽  
Author(s):  
Alejandro Balbis ◽  
Gerry Baquiran ◽  
John J. M. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Insulin Receptor ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Close Correlation ◽  
Phosphatidylinositol 3 Kinase ◽  
Maximal Increase ◽  
Kinase Activation ◽  
Almost All ◽  
Insulin Receptor Kinase ◽  
Phosphatidylinositol 3

Abstract Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60–85% and 10–20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulin-induced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr308 and Ser473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling.

Increased insulin receptor signaling and glycogen synthase activity contribute to the synergistic effect of exercise on insulin action

2003 ◽  
Vol 95 (6) ◽  
pp. 2519-2529 ◽  
Author(s):  
Christine Y. Christ-Roberts ◽  
Thongchai Pratipanawatr ◽  
Wilailak Pratipanawatr ◽  
Rachele Berria ◽  
Renata Belfort ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Insulin Infusion ◽  
Glycogen Synthase ◽  
Whole Body ◽  
Glucose Disposal ◽  
Phosphatidylinositol 3 Kinase ◽  
Insulin Stimulation ◽  
Glucose Storage ◽  
Exercise Induced ◽  
Phosphatidylinositol 3

The purpose of this study was to determine the factors contributing to the ability of exercise to enhance insulin-stimulated glucose disposal. Sixteen insulin-resistant nondiabetic and seven Type 2 diabetic subjects underwent two hyperinsulinemic (40 mU · m-2 · min-1) clamps, once without and once with concomitant exercise at 70% peak O2 consumption. Exercise was begun at the start of insulin infusion and was performed for 30 min. Biopsies of the vastus lateralis were performed before and after 30 min of insulin infusion (immediately after cessation of exercise). Exercise synergistically increased insulin-stimulated glucose disposal in nondiabetic [from 4.6 ± 0.4 to 9.5 ± 0.8 mg · kg fat-free mass (FFM)-1 · min-1] and diabetic subjects (from 4.3 ± 1.0 to 7.9 ± 0.7 mg · kg FFM-1 · min-1) subjects. The rate of glucose disposal also was significantly greater in each group after cessation of exercise. Exercise enhanced insulin-stimulated increases in glycogen synthase fractional velocity in control (from 0.07 ± 0.02 to 0.22 ± 0.05, P < 0.05) and diabetic (from 0.08 ± 0.03 to 0.15 ± 0.03, P < 0.01) subjects. Exercise also enhanced insulin-stimulated glucose storage (glycogen synthesis) in nondiabetic (2.9 ± 0.9 vs. 4.9 ± 1.1 mg · kg FFM-1 · min-1) and diabetic (1.7 ± 0.5 vs. 4.2 ± 0.8 mg · kg FFM-1 · min-1) subjects. Increased glucose storage accounted for the increase in whole body glucose disposal when exercise was performed during insulin stimulation in both groups; effects of exercise were correlated with enhancement of glucose disposal and glucose storage ( r = 0.93, P < 0.001). Exercise synergistically enhanced insulin-stimulated insulin receptor substrate 1-associated phosphatidylinositol 3-kinase activity ( P < 0.05) and Akt Ser473 phosphorylation ( P < 0.05) in nondiabetic subjects but had little effect in diabetic subjects. The data indicate that exercise, performed in conjunction with insulin infusion, synergistically increases insulin-stimulated glucose disposal compared with insulin alone. In nondiabetic and diabetic subjects, increased glycogen synthase activation is likely to be involved, in part, in this effect. In nondiabetic, but not diabetic, subjects, exercise-induced enhancement of insulin stimulation of the phosphatidylinositol 3-kinase pathway is also likely to be involved in the exercise-induced synergistic enhancement of glucose disposal.

Effect of monensin on 2-deoxyglucose uptake, the insulin receptor and phosphatidylinositol 3-kinase activity in rat muscle

1997 ◽  
Vol 154 (1) ◽  
pp. 85-93
Author(s):  
J Turinsky ◽  
A Damrau-Abney ◽  
J S Elmendorf ◽  
T R Smith
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Membrane Integrity ◽  
Insulin Receptors ◽  
Inhibitory Effect ◽  
Phosphatidylinositol 3 Kinase ◽  
Dual Effect ◽  
Cell Membrane Integrity ◽  
Rat Soleus Muscle ◽  
Phosphatidylinositol 3

Abstract Preincubation of rat soleus muscle with 1 and 10 μm monensin for 2 h increased the subsequent basal 2-deoxyglucose uptake by muscle 76 and 121% respectively. Under the same conditions, monensin decreased the insulin-stimulated (1 mU/ml) 2-deoxyglucose uptake by 29 and 37% respectively. The monensin-induced augmentation of basal 2-deoxyglucose uptake was inhibited 92% by cytochalasin B suggesting that the uptake is mediated by glucose transporters. Monensin did not increase the cellular accumulation of l-glucose in muscle indicating that it does not affect the cell membrane integrity. Neither the stimulatory effect of monensin on basal 2-deoxyglucose uptake nor the opposite, inhibitory action of monensin on the insulin-stimulated 2-deoxyglucose uptake were influenced by the removal of Ca2+ from the medium or by dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, suggesting that the actions of monensin are not mediated by calcium. Monensin had no effect on muscle ATP concentration. The monensin-induced augmentation of basal 2-deoxyglucose uptake was neither associated with stimulation of muscle phosphatidylinositol 3-kinase activity nor inhibited by wortmannin, demonstrating that the increase in basal 2-deoxyglucose uptake is not mediated by activation of phosphatidylinositol 3-kinase. The inhibition of insulin-stimulated 2-deoxyglucose uptake by monensin was associated with a 31% decrease in the abundance of insulin receptors in muscles, a 64% decrease in the insulin-induced autophosphorylation of the insulin receptor β-subunit, and a 44% reduction of the insulin-stimulated phosphatidylinositol 3-kinase activity. Addition of monensin into the phosphatidylinositol 3-kinase reaction had no effect on the activity of the enzyme, demonstrating that the inhibition in monensin-treated muscles is indirect and occurs upstream of phosphatidylinositol 3-kinase. It is concluded that monensin has a dual effect on 2-deoxyglucose uptake by skeletal muscle: it stimulates basal uptake but inhibits the insulin-stimulated uptake. The primary cause of the latter, inhibitory effect of monensin is at the level of the insulin receptor. Journal of Endocrinology (1997) 154, 85–93

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