Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation - PubMed (original) (raw)
Insulin induces phosphatidylinositol-3-phosphate formation through TC10 activation
Tania Maffucci et al. EMBO J. 2003.
Abstract
Phosphatidylinositol-3-phosphate (PtdIns-3-P) is considered as a lipid constitutively present on endosomes; it does not seem to have a dynamic role in signalling. In contrast, phosphatidylinositol-3,4,5-trisphosphate (PtdIns-3,4,5-P(3)) plays a crucial role in different signalling pathways including translocation of the glucose transporter protein GLUT4 to the plasma membrane upon insulin receptor activation. GLUT4 translocation requires activation of two distinct pathways involving phosphatidylinositol 3-kinase (PI 3-K) and the small GTP-binding protein TC10, respectively. The contribution of each pathway remains to be elucidated. Here we show that insulin specifically induces the formation of PtdIns-3-P in insulin- responsive cells. The insulin-mediated formation of PtdIns-3-P occurs through the activation of TC10 at the lipid rafts subdomain of the plasma membrane. Exogenous PtdIns-3-P induces the plasma membrane translocation of both overexpressed and endogenous GLUT4. These data indicate that PtdIns-3-P is specifically produced downstream from insulin-mediated activation of TC10 to promote the plasma membrane translocation of GLUT4. These results give a new insight into the intracellular role of PtdIns-3-P and shed light on some aspects of insulin signalling so far not completely understood.
Figures
Fig. 1. Insulin induces PtdIns-3-P formation in L6 cells and 3T3-L1 adipocytes. (A) L6 cells and (B) 3T3-L1 adipocytes were labelled with _myo_-[3H]inositol for 24 h and then stimulated with 300 nM insulin or 20 ng/ml PDGF. Phospholipids were extracted at different times of stimulation, deacylated and analysed by HPLC as described in Materials and methods. Data are mean ± SEM (A, n = 6; B, n = 2).
Fig. 2. Insulin induces the translocation of the PtdIns-3-P probe 2XFYVEHrs to the plasma membrane in L6 cells. (A) GFP–2XFYVEHrs-transfected L6 cells were serum deprived for 6 h and then left untreated (0 min) or stimulated with 300 nM insulin. At the indicated times of stimulation, cells were fixed and analysed by confocal microscopy. Arrows indicate the plasma membrane localization. Bar, 10 µm. Data on plot are mean ± SEM (n = 3–5). (B) L6 cells were co-transfected with cDNAs encoding YFP–2XFYVEHrs and the plasma membrane marker CFP–PH PLCδ1, respectively. After 24 h, cells were serum deprived for 6 h and stimulated with 300 nM insulin for 3 min before fixing for confocal microscopy analysis. Bar, 10 µm.
Fig. 3. Insulin recruits the GFP–2XFYVEHrs fusion protein to the plasma membrane of 3T3-L1 adipocytes. (A) Fully differentiated 3T3-L1 adipocytes or (B) 3T3-L1 pre-adipocytes were electroporated with the cDNA encoding GFP–2XFYVEHrs. After 24 h, cells were serum deprived for 3 h and then left untreated (0 min) or stimulated with 300 nM insulin for different times before fixing for confocal microscopy analysis. Arrows mark the plasma membrane localization. Bar, 10 µm. Data on plot are mean ± SEM (n = 2–3). Phase-contrast images show 3T3-L1 pre-adipocytes (day 0) and adipocytes at 9 days after initiation of differentiation.
Fig. 4. PtdIns-3-P formation at the plasma membrane is relatively resistant to PI 3-K inhibitors. (A and B) GFP–2XFYVEHrs-transfected L6 cells were pre-treated with the indicated concentrations of LY294002 (A) or wortmannin (B) for 15 min at 37°C and then stimulated with 300 nM insulin for 3 min. GFP–2XFYVEHrs intracellular localization was then assessed by confocal microscopy. Bar, 10 µm. Data on plot are mean ± SEM (n = 2). (C) L6 cells were transfected with GFP–PH PKB/Akt, serum deprived for 6 h and then stimulated with 300 nM insulin for 3 min. When indicated, cells were pre-treated with 100 nM wortmannin for 15 min before insulin stimulation. (D) _myo_-[3H]inositol-labelled L6 cells were left untreated or pre-treated with 100 nM wortmannin before stimulation with 300 nM insulin for different times. Deacylated phospholipids were analysed by HPLC as described in Materials and methods. Data are from one experiment representative of two independent experiments.
Fig. 5. The insulin-mediated formation of PtdIns-3-P occurs through the activation of TC10. (A) L6 cells were transfected with myc-TC10 Q75L, myc-Cdc42 L61 or the empty vector. After 24 h, cells were labelled with _myo_-[3H]inositol and HPLC analysis of PtdIns-3-P levels was performed as described in Materials and methods. Data are mean ± SEM of two independent experiments performed in duplicate. (B) L6 cells were co-transfected with GFP–2XFYVEHrs and either myc-TC10 Q75L, myc-Cdc42 L61 or HA-TC10 T31N. After 24 h, cells were serum starved for 6 h and analysed by confocal microscopy. HA-TC10 T31N-co-transfected cells were stimulated with 300 nM insulin for 3 min before confocal analysis. Arrows indicate the plasma membrane localization. Arrowheads mark the filopodia induced by overexpression of myc-Cdc42 L61. Bar, 10 µm. (C) L6 cells were transfected with myc-TC10 wt, myc-TC10 Q75L or myc-Cdc42 L61. Serum-deprived cells were left untreated or stimulated with insulin for 3 min. Lysates were incubated with GST-Pak1 PBD and pull-down assay was performed as described in Materials and methods. Association of the overexpressed proteins with the GST-Pak1 PBD was assessed in western blot analyses by using an anti-myc antibody (upper panels). Equal amount of the overexpressed proteins in lysates was confirmed by loading an aliquot of each lysate (bottom panel). Blot is representative of three independent experiments.
Fig. 6. The insulin/TC10-dependent pool of PtdIns-3-P is generated at the lipid rafts subdomain of the plasma membrane. (A) GFP–2XFYVEHrs-transfected L6 cells (a–d) were serum deprived for 6 h and then left untreated (a and d) or stimulated with 300 nM insulin for 3 min (b) or with 20 ng/ml PDGF (c). Cells were then homogenized and fractionated as described in Materials and methods. GFP–2XFYVEHrs localization in the different fractions was then assessed in western blot analyses by using an anti-GFP antibody (a–c). Flotillin was used as a marker of rafts fractions (d). Alternatively, L6 cells were transfected with both GFP–2XFYVEHrs and myc-TC10 Q75L (e and f). After 24 h, cells were serum deprived for 6 h and sucrose gradient fractions were prepared. The fusion proteins were revealed by using an anti-GFP (e) and an anti-myc (f) antibody, respectively. Alternatively, L6 cells were transfected with both GFP–2XFYVEHrs and HA-TC10 T31N (g and h). After 24 h, cells were serum deprived for 6 h, stimulated with 300 nM insulin for 3 min and then fractionated as above. The fusion proteins were revealed by using an anti-GFP (g) and an anti-HA (h) antibody, respectively. Blot is representative of at least three independent experiments. (B) GFP–2XFYVEHrs-transfected L6 cells were serum deprived for 6 h and then left untreated or incubated with 5 mM MβCD for 50 min at 37°C before stimulation with 300 nM insulin for 3 min. After fixation, cells were permeabilized, incubated with an anti-flotillin antibody and analysed by confocal microscopy. Bar, 10 µm. (C) myc-TC10 Q75L-transfected L6 cells were incubated with 5 mM MβCD for 50 min at 37°C. After fixation, cells were permeabilized, incubated with an anti-myc antibody and Alexa 594–phalloidin and analysed by confocal microscopy. Bar, 10 µm.
Fig. 7. (A–C) Exogenous PtdIns-3-P induces plasma membrane translocation of exogenous GLUT4. (A) L6 cells were transfected with GFP–GLUT4. After 24 h, cells were serum deprived for 6 h and then stimulated with 300 nM insulin or with the indicated phosphoinositides at a final concentration of 50 µM. After incubation at 37°C for 10, 20 or 30 min, cells were fixed for quantitative analyses. (B) GFP–GLUT4-transfected L6 cells were incubated for 10 min with 300 nM insulin, or the complex carrier/PtdIns-3-P at the indicated lipid final concentrations or carrier alone at a final concentration of 50 µM and then fixed for quantitative analyses. Data are mean ± SEM (n = 3–10). Significantly different from control: *P < 0.001; **P < 0.01; ***P < 0.05. (C) 3T3-L1 adipocytes were electroporated with 500 µg of a cDNA encoding GFP–GLUT4. After 24 h, cells were serum deprived for 3 h and stimulated with 300 nM insulin or incubated with the complex carrier/phosphoinositide at a lipid final concentration of 50 µM for 30 min at 37°C. Data are mean ± SEM (n = 2). (D) Exogenous PtdIns-3-P induces plasma membrane translocation of endogenous GLUT4. 3T3-L1 adipocytes were serum deprived overnight and then stimulated with insulin or with each complex carrier/phosphoinositide for 10 min. After fixing and permeabilization, the localization of endogenous GLUT4 was assessed by using an anti-GLUT4 antibody followed by a FITC-conjugated secondary antibody and analysed by confocal microscopy. (E) 3T3-L1 adipocytes were serum deprived overnight and then stimulated with insulin or incubated with either a complex carrier/phosphoinositide or carrier alone. Glucose uptake was performed as described in Materials and methods. Data are mean ± SEM (n = 3–5). (F) GFP–GLUT4-transfected L6 cells were serum deprived for 6 h and then left untreated or incubated with 100 nM wortmannin at 37°C. After 15 min, cells were stimulated with 300 nM insulin or incubated with the complex carrier/PtdIns-3-P for an additional 10 min. Data are mean ± SEM (n = 3–5). Significantly different from PtdIns-3-P: *P < 0.05.
References
- Baumann C.A., Ribon,V., Kanzaki,M., Thurmond,D.C., Mora,S., Shigematsu,S., Bickel,P.E., Pessin,J.E. and Saltiel,A.R. (2000) CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature, 407, 202–207. - PubMed
- Brown R.A., Domin,J., Arcaro,A., Waterfield,M.D. and Shepherd,P.R. (1999) Insulin activates the α isoform of class II phosphoinositide 3-kinase. J. Biol. Chem., 274, 14529–14532. - PubMed
- Chiang S.H., Baumann,C.A., Kanzaki,M., Thurmond,D.C., Watson,R.T., Neudauer,C.L., Macara,I.G., Pessin,J.E. and Saltiel,A.R. (2001) Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10. Nature, 410, 944–948. - PubMed
- Czech M.P. and Corvera,S. (1999) Signaling mechanisms that regulate glucose transport. J. Biol. Chem., 274, 1865–1868. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Research Materials
Miscellaneous