scholarly journals Phorbol esters stimulate phosphatidylinositol 3,4,5-trisphosphate production in 3T3-L1 adipocytes: implications for stimulation of glucose transport

1996 ◽  
Vol 318 (1) ◽  
pp. 203-205 ◽  
Author(s):  
Barbara T. NAVÉ ◽  
Kenneth SIDDLE ◽  
Peter R SHEPHERD
Keyword(s):  
Glucose Transport ◽  
Kinase Activity ◽  
Phorbol Esters ◽  
Signalling Pathways ◽  
End Point ◽  
Stimulation Of

The effects of insulin and phorbol 12-myristate 13-acetate (PMA) on the levels of cellular phosphoinositides were investigated in 3T3-L1 adipocytes. Stimulation for 4 min with PMA (1 µM) or insulin (10 nM) increased levels of PtdIns(3,4,5)P3 approx. 2-fold and 6-fold respectively. PMA also had a small effect on the cellular levels of PtdIns4P, whereas insulin had no effect on PtdIns4P levels; levels of PtdIns(4,5)P2 and PtdIns3P were not significantly affected by either agent. Insulin increased the levels of the p85α subunit of phosphoinositide (PI) 3-kinase associated with membranes, whereas PMA decreased levels of membrane-associated p85α. PMA did not increase PI 3-kinase activity in anti-phosphotyrosine or anti-p85 immunoprecipitates. The stimulation of glucose transport by insulin or PMA was blocked by 100 nM wortmannin or 10 ng/ml LY294002, indicating that PI 3-kinase is essential for stimulation by both agents. In summary, these results demonstrate: (1) that PMA and insulin stimulate PtdIns(3,4,5)P3 production by distinct mechanisms in 3T3-L1 adipocytes, and (2) that stimulation of PtdIns(3,4,5)P3 production by PMA is likely to be important in signalling pathways leading from PMA stimulation to end-point responses such as glucose transport.

scholarly journals Signalling pathways of an insulin-mimetic phosphoinositolglycan–peptide in muscle and adipose tissue

1998 ◽  
Vol 330 (1) ◽  
pp. 277-286 ◽  
Author(s):  
Alexandra KESSLER ◽  
Günter MÜLLER ◽  
Susanne WIED ◽  
Anna CRECELIUS ◽  
Jürgen ECKEL
Keyword(s):  
Adipose Tissue ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Glucose Transport ◽  
Insulin Action ◽  
Kinase Activity ◽  
Signalling Pathways ◽  
Insulin Mimetic ◽  
Isolated Adipocytes ◽  
Stimulation Of

A novel phosphoinositolglycan-peptide (PIG-P) from the yeast Saccharomyces cerevisiae potently mimicks insulin action on glucose transport and metabolism in rat muscle and adipose tissue. The aim of the present study was to elucidate the cellular signalling pathways of this insulin-mimetic compound. Rapid onset and reversibility of PIG-P action on glucose transport were observed in isolated adipocytes with a half-time of transport stimulation of 6-8 min (insulin less than 5 min). Combined treatment with PIG-P and insulin indicated additive stimulation of glucose transport at submaximal concentrations and non-additive action of both agents at maximal doses. The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was markedly increased in response to PIG-P in rat cardiomyocytes without any effect on the tyrosine phosphorylation of the insulin receptor β-subunit. PIG-P action in these cells was accompanied by phosphorylation/dephosphorylation of several proteins with molecular masses of 15-30 kDa, a response not detected with insulin. Downstream signalling of IRS-1 was then analysed by monitoring IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in cardiomyocytes. A stable (2 and 15 min incubation with PIG-P) 7-fold stimulation corresponding to about 50% of insulin action could be detected. Increased tyrosine phosphorylation of IRS-1 and enhanced PI 3-kinase activity in response to PIG-P independent of the insulin receptor was also observed in isolated adipocytes. Involvement of PI 3-kinase in PIG-P action was subsequently confirmed by the dose-dependent inhibition of PIG-P-activated glucose transport in rat diaphragm and adipocytes by the PI 3-kinase inhibitors wortmannin and LY294002. These data suggest divergent upstream signalling by insulin and PIG-P involving phosphoproteins not affected by insulin. However, PIG-P and insulin action converge at the level of IRS-1 inducing insulin-independent PI 3-kinase-mediated signalling to glucose transport.

scholarly journals Positive and Negative Regulation of Phosphoinositide 3-Kinase-Dependent Signaling Pathways by Three Different Gene Products of the p85α Regulatory Subunit

2000 ◽  
Vol 20 (21) ◽  
pp. 8035-8046 ◽  
Author(s):  
Kohjiro Ueki ◽  
Petra Algenstaedt ◽  
Franck Mauvais-Jarvis ◽  
C. Ronald Kahn
Keyword(s):  
Glucose Transport ◽  
Kinase Activity ◽  
Glycogen Synthesis ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Irs Proteins ◽  
Terminal Structure ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85α regulatory subunit yields three splicing variants, p85α, AS53/p55α, and p50α. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110α catalytic subunit. PI 3-kinase activity associated with p50α was greater than that associated with p85α or AS53. Increasing the level of p85α or AS53, but not p50α, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85α mutant lacking the p110-binding site (Δp85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70S6K), was decreased in cells expressing p85α or AS53 but not in cells expressing p50α. Similar inhibition of PI 3-kinase, Akt, and p70S6K was observed, even when p110α was coexpressed with p85α or AS53. Expression of p110α alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110α was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.

scholarly journals Stimulation of the human neutrophil respiratory burst by formyl peptides is primed by a protein kinase inhibitor, staurosporine

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2890-2898 ◽  
Author(s):  
C Combadiere ◽  
J el Benna ◽  
E Pedruzzi ◽  
J Hakim ◽  
A Perianin
Keyword(s):  
Protein Kinase ◽  
Respiratory Burst ◽  
Phorbol Esters ◽  
Kinase Inhibitor ◽  
Polymorphonuclear Neutrophils ◽  
Time Dependent ◽  
Signalling Pathways ◽  
Dependent Manner ◽  
Formyl Peptides ◽  
Stimulation Of

Abstract Stimulation of polymorphonuclear neutrophils (PMN) by phorbol esters or formyl peptides (fMLP) generates large quantities of superoxide anion, the so-called respiratory burst (RB), a phenomenon associated with intense phosphorylation of a 47-kD protein (p47 phox). Staurosporine, a potent protein kinase C (PKC) antagonist, inhibits both responses when PMN are stimulated by phorbol myristate acetate (PMA), suggesting a positive role of PKC. In this study, we reassessed these PMN responses in fMLP-stimulated cells and found that staurosporine had opposite effects depending on the duration of PMN treatment with staurosporine. Short PMN incubation (0.5 to 3 minutes) with 25 to 100 nmol/L staurosporine inhibited the fMLP-induced RB, whereas longer treatment (15 to 20 minutes) enhanced it by up to about 200% relative to controls. In contrast, the PMA-mediated RB was depressed by staurosporine in a time-dependent manner. A primed fMLP-induced RB was also observed after long (15 minutes) PMN treatment with 5 to 100 mumol/L H-7, whereas shorter treatment (5 minutes) resulted in a small decrease in RB. By contrast, the tyrosine kinase inhibitor genistein (2 to 80 mumol/L) depressed fMLP-induced RB whatever the duration of PMN treatment. Analysis of 32P-phosphorylated proteins in fMLP-stimulated cells showed that short PMN treatment (< 8 minutes) with staurosporine abolished the phosphorylation of the 47-kD protein, which was identified as p47 phox, whereas long treatment partially restored p47 phox phosphorylation up to approximately 50% of the control value. In PMA-stimulated PMN, phosphorylation was reduced in a time-dependent manner. Furthermore, the staurosporine-primed RB and the staurosporine- induced recovery of phosphorylation were inhibited by sphingosine but not by genistein. Thus, in addition to its known depressive effect, staurosporine markedly potentiated fMLP-stimulated RB as a function of the duration of PMN treatment. The restoration of p47 phox phosphorylation suggests that staurosporine may alter the interactions between different protein kinases, producing marked time-dependent changes in signalling pathways. These data emphasize the care that should be taken in interpreting data obtained using this kinase inhibitor that may, however, be helpful analyzing in signalling pathways.

scholarly journals Stimulation of the human neutrophil respiratory burst by formyl peptides is primed by a protein kinase inhibitor, staurosporine

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2890-2898 ◽  
Author(s):  
C Combadiere ◽  
J el Benna ◽  
E Pedruzzi ◽  
J Hakim ◽  
A Perianin
Keyword(s):  
Protein Kinase ◽  
Respiratory Burst ◽  
Phorbol Esters ◽  
Kinase Inhibitor ◽  
Polymorphonuclear Neutrophils ◽  
Time Dependent ◽  
Signalling Pathways ◽  
Dependent Manner ◽  
Formyl Peptides ◽  
Stimulation Of

Stimulation of polymorphonuclear neutrophils (PMN) by phorbol esters or formyl peptides (fMLP) generates large quantities of superoxide anion, the so-called respiratory burst (RB), a phenomenon associated with intense phosphorylation of a 47-kD protein (p47 phox). Staurosporine, a potent protein kinase C (PKC) antagonist, inhibits both responses when PMN are stimulated by phorbol myristate acetate (PMA), suggesting a positive role of PKC. In this study, we reassessed these PMN responses in fMLP-stimulated cells and found that staurosporine had opposite effects depending on the duration of PMN treatment with staurosporine. Short PMN incubation (0.5 to 3 minutes) with 25 to 100 nmol/L staurosporine inhibited the fMLP-induced RB, whereas longer treatment (15 to 20 minutes) enhanced it by up to about 200% relative to controls. In contrast, the PMA-mediated RB was depressed by staurosporine in a time-dependent manner. A primed fMLP-induced RB was also observed after long (15 minutes) PMN treatment with 5 to 100 mumol/L H-7, whereas shorter treatment (5 minutes) resulted in a small decrease in RB. By contrast, the tyrosine kinase inhibitor genistein (2 to 80 mumol/L) depressed fMLP-induced RB whatever the duration of PMN treatment. Analysis of 32P-phosphorylated proteins in fMLP-stimulated cells showed that short PMN treatment (< 8 minutes) with staurosporine abolished the phosphorylation of the 47-kD protein, which was identified as p47 phox, whereas long treatment partially restored p47 phox phosphorylation up to approximately 50% of the control value. In PMA-stimulated PMN, phosphorylation was reduced in a time-dependent manner. Furthermore, the staurosporine-primed RB and the staurosporine- induced recovery of phosphorylation were inhibited by sphingosine but not by genistein. Thus, in addition to its known depressive effect, staurosporine markedly potentiated fMLP-stimulated RB as a function of the duration of PMN treatment. The restoration of p47 phox phosphorylation suggests that staurosporine may alter the interactions between different protein kinases, producing marked time-dependent changes in signalling pathways. These data emphasize the care that should be taken in interpreting data obtained using this kinase inhibitor that may, however, be helpful analyzing in signalling pathways.

scholarly journals Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin

1994 ◽  
Vol 300 (3) ◽  
pp. 631-635 ◽  
Author(s):  
J F Clarke ◽  
P W Young ◽  
K Yonezawa ◽  
M Kasuga ◽  
G D Holman
Keyword(s):  
Glucose Transport ◽  
Glucose Transporter ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Insulin Signalling ◽  
Reversible Inhibitor ◽  
Transport Activity ◽  
Insulin Stimulation ◽  
Gamma S ◽  
Stimulation Of

Wortmannin is a potent and reversible inhibitor of insulin-stimulated PtdIns 3-kinase activity in 3T3-L1 cells (IC50 = 2.6 +/- 0.8 nM). Wortmannin inhibits the PtdIns 3-kinase activity which is precipitated with antibodies against insulin receptor substrate 1 and against the alpha-p85 subunit of PtdIns 3-kinase. These observations suggest that wortmannin inhibits at the p110 catalytic subunit of PtdIns 3-kinase. Insulin stimulation of glucose transport in permeabilized 3T3-L1 cells is also inhibited by wortmannin (IC50 = 6.4 +/- 1.4 nM). Wortmannin did not inhibit basal glucose transport activity. The close similarity of the IC50 values for wortmannin inhibition of insulin-stimulated PtdIns 3-kinase and glucose transport activities suggests that the PtdIns 3-kinase is a key intermediate in insulin signalling of glucose-transport stimulation. The wortmannin inhibitory effect on transport is associated with a reduction in the cell-surface, but not the total cellular, levels of both GLUT1 and GLUT4 glucose transporter isoforms that are accessible to the cell-impermeant photolabel, ATB-BMPA. These photolabelling results suggest that the glucose transporter translocation process is dependent upon PtdIns 3-kinase activity. The stimulatory effect of guanosine 5′-[gamma-thio]triphosphate (GTP gamma S) on glucose transport activity in permeabilized cells is only partially blocked by concentrations of wortmannin that completely inhibit the stimulatory effect of insulin. The residual stimulatory effect of GTP gamma S that occurs in the presence of wortmannin suggests that at least part of the GTP gamma S effect is mediated at a signalling site that is downstream of the site at which wortmannin inhibits the insulin stimulation of PtdIns 3-kinase and glucose transport activities.

Mechanisms underlying impaired GLUT-4 translocation in glycogen-supercompensated muscles of exercised rats

2000 ◽  
Vol 279 (6) ◽  
pp. E1311-E1318 ◽  
Author(s):  
Kentaro Kawanaka ◽  
Lorraine A. Nolte ◽  
Dong-Ho Han ◽  
Polly A. Hansen ◽  
John O. Holloszy
Keyword(s):  
Glucose Transport ◽  
Kinase Activity ◽  
Amp Kinase ◽  
Glut 4 ◽  
Glut 4 Translocation ◽  
Insulin Responsiveness ◽  
Muscle Glucose Transport ◽  
Pkb Phosphorylation ◽  
Fasted Rats ◽  
Stimulation Of

Exercise training induces an increase in GLUT-4 in muscle. We previously found that feeding rats a high-carbohydrate diet after exercise, with muscle glycogen supercompensation, results in a decrease in insulin responsiveness so severe that it masks the effect of a training-induced twofold increase in GLUT-4 on insulin-stimulated muscle glucose transport. One purpose of this study was to determine whether insulin signaling is impaired. Maximally insulin-stimulated phosphatidylinositol (PI) 3-kinase activity was not significantly reduced, whereas protein kinase B (PKB) phosphorylation was ∼50% lower ( P < 0.01) in muscles of chow-fed, than in those of fasted, exercise-trained rats. Our second purpose was to determine whether contraction-stimulated glucose transport is also impaired. The stimulation of glucose transport and the increase in cell surface GLUT-4 induced by contractions were both decreased by ∼65% in glycogen-supercompensated muscles of trained rats. The contraction-stimulated increase in AMP kinase activity, which has been implicated in the activation of glucose transport by contractions, was ∼80% lower in the muscles of the fed compared with the fasted rats 18 h after exercise. These results show that both the insulin- and contraction-stimulated pathways for muscle glucose transport activation are impaired in glycogen-supercompensated muscles and provide insight regarding possible mechanisms.

scholarly journals Phorbol esters imitate in rat fat-cells the full effect of insulin on glucose-carrier translocation, but not on 3-O-methylglucose-transport activity

1988 ◽  
Vol 249 (3) ◽  
pp. 865-870 ◽  
Author(s):  
C Mühlbacher ◽  
E Karnieli ◽  
P Schaff ◽  
B Obermaier ◽  
J Mushack ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Cytochalasin B ◽  
Phorbol Esters ◽  
Fat Cells ◽  
Transport Activity ◽  
Rat Adipocytes ◽  
Glucose Carrier ◽  
Stimulation Of

Tumour-promoting phorbol esters have insulin-like effects on glucose transport and lipogenesis in adipocytes and myocytes. It is believed that insulin activates the glucose-transport system through translocation of glucose transporters from subcellular membranes to the plasma membrane. The aim of the present study was to investigate if phorbol esters act through the same mechanism as insulin on glucose-transport activity of rat adipocytes. We compared the effects of the tumour-promoting phorbol ester tetradecanoylphorbol acetate (TPA) and of insulin on 3-O-methylglucose transport and on the distribution of D-glucose-inhibitable cytochalasin-B binding sites in isolated rat adipocytes. Insulin (100 mu units/ml) stimulated 3-O-methylglucose uptake 9-fold, whereas TPA (1 nM) stimulated the uptake only 3-fold (mean values of five experiments, given as percentage of equilibrium reached after 4 s: basal 7 +/- 1.3%, insulin 60 +/- 3.1%, TPA 22 +/- 2.3%). In contrast, both agents stimulated glucose-transporter translocation to the same extent [cytochalasin B-binding sites (pmol/mg of protein; n = 7): plasma membranes, basal 6.2 +/- 1.0, insulin 13.4 +/- 2.0, TPA 12.7 +/- 2.7; low-density membranes, basal 12.8 +/- 2.1, insulin 6.3 +/- 0.9, TPA 8.9 +/- 0.7; high-density membranes, 6.9 +/- 1.1; insulin 12.5 +/- 1.0, TPA 8.1 +/- 0.9]. We conclude from these data: (1) TPA stimulates glucose transport in fat-cells by stimulation of glucose-carrier translocation; (2) insulin and TPA stimulate the carrier translocation to the same extent, whereas the stimulation of glucose uptake is 3-fold higher with insulin, suggesting that the stimulatory effect of insulin on glucose-transport activity involves other mechanisms in addition to carrier translocation.

Mechanism of IGF-I-stimulated glucose transport in human adipocytes. Demonstration of specific IGF-I receptors not involved in stimulation of glucose transport

Diabetes ◽  
1989 ◽  
Vol 38 (10) ◽  
pp. 1217-1225 ◽  
Author(s):  
M. K. Sinha ◽  
C. Buchanan ◽  
N. Leggett ◽  
L. Martin ◽  
P. G. Khazanie ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Human Adipocytes ◽  
Igf I ◽  
Stimulation Of
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