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Insulin regulates Rab3-Noc2 complex dissociation to promote GLUT4 translocation in rat adipocytes.

Koumanov F, Pereira VJ, Richardson JD, Sargent SL, Fazakerley DJ, Holman GD - Diabetologia (2015)

Bottom Line: We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation.Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2.This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.

ABSTRACT

Aims/hypothesis: The glucose transporter GLUT4 is present mainly in insulin-responsive tissues of fat, heart and skeletal muscle and is translocated from intracellular membrane compartments to the plasma membrane (PM) upon insulin stimulation. The transit of GLUT4 to the PM is known to be dependent on a series of Rab proteins. However, the extent to which the activity of these Rabs is regulated by the action of insulin action is still unknown. We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation.

Methods: We developed a new photoaffinity reagent (Bio-ATB-GTP) that allows GTP-binding proteomes to be explored. Using this approach we screened for insulin-responsive GTP loading of Rabs in primary rat adipocytes.

Results: We identified Rab3B as a new candidate insulin-stimulated G-protein in adipocytes. Using constitutively active and dominant negative mutants and Rab3 knockdown we provide evidence that Rab3 isoforms are key regulators of GLUT4 translocation in adipocytes. Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2. Disruption of the Rab3-Noc2 complex leads to displacement of Noc2 from the PM. This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation.

Conclusions/interpretation: The discovery of the involvement of Rab3 and Noc2 in an insulin-regulated step in GLUT4 translocation suggests that the control of this translocation process is unexpectedly similar to regulated secretion and particularly pancreatic insulin-vesicle release.

No MeSH data available.


FLAG-Rab3B and endogenous Rab3D are activated upon insulin stimulation. (a) Distribution of expressed FLAG-Rab3B between the total-membrane and the cytoplasmic fractions of rat adipocytes. (b) Insulin stimulation (Ins) of the Bio-ATB-GTP loading state of FLAG-Rab3B. Streptavidin-precipitated proteins were detected with anti-FLAG antibody. (c) Effect of wortmannin (200 nmol/l for 10 min) (Wtm) on insulin-stimulated FLAG-Rab3B GTP loading. (d) Quantification of the data presented in (b) and (c). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. (e) Effect of insulin stimulation on endogenous Rab3D GTP loading. Total-membrane preparations (300 μg/condition) were labelled with Bio-ATB-GTP and streptavidin-precipitated proteins immunoblotted with anti-Rab3D antibody. (f) Quantification of the data shown in (e). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. SA ppt, streptavidin precipitation; TM, total-membrane loading control
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Fig2: FLAG-Rab3B and endogenous Rab3D are activated upon insulin stimulation. (a) Distribution of expressed FLAG-Rab3B between the total-membrane and the cytoplasmic fractions of rat adipocytes. (b) Insulin stimulation (Ins) of the Bio-ATB-GTP loading state of FLAG-Rab3B. Streptavidin-precipitated proteins were detected with anti-FLAG antibody. (c) Effect of wortmannin (200 nmol/l for 10 min) (Wtm) on insulin-stimulated FLAG-Rab3B GTP loading. (d) Quantification of the data presented in (b) and (c). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. (e) Effect of insulin stimulation on endogenous Rab3D GTP loading. Total-membrane preparations (300 μg/condition) were labelled with Bio-ATB-GTP and streptavidin-precipitated proteins immunoblotted with anti-Rab3D antibody. (f) Quantification of the data shown in (e). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. SA ppt, streptavidin precipitation; TM, total-membrane loading control

Mentions: As no suitable commercially available antibodies to rodent Rab3B were available, FLAG-tagged rat Rab3B was expressed in rat adipocytes and was found to be strongly associated with membrane fractions (approximately 70%, Fig. 2a). We used Bio-ATB-GTP to tag membrane preparations isolated from basal or insulin-stimulated rat adipocytes expressing FLAG-Rab3B. A time-dependent activation of Rab3B loading was observed following treatment with insulin (Fig. 2b, d). Activation of Rab3B was independent of phosphoinositide 3 (PI 3)-kinase activity as wortmannin failed to inhibit Rab3B loading (Fig. 2c, d). Rab3D was previously detected in 3T3-L1 adipocytes [20] and has been shown to be present in insulin-responsive tissues [21]. We therefore explored whether this isoform also exhibited insulin-dependent GTP loading. We observed an increase in the GTP loading state of Rab3D (Fig. 2e, f). Wortmannin treatment of the cells before membrane isolation did not inhibit insulin-dependent stimulation of GTP loading of Rab3D. These data are consistent with PI 3-kinase-independent activation of both Rab3B and Rab3D.Fig. 2


Insulin regulates Rab3-Noc2 complex dissociation to promote GLUT4 translocation in rat adipocytes.

Koumanov F, Pereira VJ, Richardson JD, Sargent SL, Fazakerley DJ, Holman GD - Diabetologia (2015)

FLAG-Rab3B and endogenous Rab3D are activated upon insulin stimulation. (a) Distribution of expressed FLAG-Rab3B between the total-membrane and the cytoplasmic fractions of rat adipocytes. (b) Insulin stimulation (Ins) of the Bio-ATB-GTP loading state of FLAG-Rab3B. Streptavidin-precipitated proteins were detected with anti-FLAG antibody. (c) Effect of wortmannin (200 nmol/l for 10 min) (Wtm) on insulin-stimulated FLAG-Rab3B GTP loading. (d) Quantification of the data presented in (b) and (c). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. (e) Effect of insulin stimulation on endogenous Rab3D GTP loading. Total-membrane preparations (300 μg/condition) were labelled with Bio-ATB-GTP and streptavidin-precipitated proteins immunoblotted with anti-Rab3D antibody. (f) Quantification of the data shown in (e). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. SA ppt, streptavidin precipitation; TM, total-membrane loading control
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Fig2: FLAG-Rab3B and endogenous Rab3D are activated upon insulin stimulation. (a) Distribution of expressed FLAG-Rab3B between the total-membrane and the cytoplasmic fractions of rat adipocytes. (b) Insulin stimulation (Ins) of the Bio-ATB-GTP loading state of FLAG-Rab3B. Streptavidin-precipitated proteins were detected with anti-FLAG antibody. (c) Effect of wortmannin (200 nmol/l for 10 min) (Wtm) on insulin-stimulated FLAG-Rab3B GTP loading. (d) Quantification of the data presented in (b) and (c). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. (e) Effect of insulin stimulation on endogenous Rab3D GTP loading. Total-membrane preparations (300 μg/condition) were labelled with Bio-ATB-GTP and streptavidin-precipitated proteins immunoblotted with anti-Rab3D antibody. (f) Quantification of the data shown in (e). Data are means ± SEM from three independent experiments. *p < 0.05 vs basal. SA ppt, streptavidin precipitation; TM, total-membrane loading control
Mentions: As no suitable commercially available antibodies to rodent Rab3B were available, FLAG-tagged rat Rab3B was expressed in rat adipocytes and was found to be strongly associated with membrane fractions (approximately 70%, Fig. 2a). We used Bio-ATB-GTP to tag membrane preparations isolated from basal or insulin-stimulated rat adipocytes expressing FLAG-Rab3B. A time-dependent activation of Rab3B loading was observed following treatment with insulin (Fig. 2b, d). Activation of Rab3B was independent of phosphoinositide 3 (PI 3)-kinase activity as wortmannin failed to inhibit Rab3B loading (Fig. 2c, d). Rab3D was previously detected in 3T3-L1 adipocytes [20] and has been shown to be present in insulin-responsive tissues [21]. We therefore explored whether this isoform also exhibited insulin-dependent GTP loading. We observed an increase in the GTP loading state of Rab3D (Fig. 2e, f). Wortmannin treatment of the cells before membrane isolation did not inhibit insulin-dependent stimulation of GTP loading of Rab3D. These data are consistent with PI 3-kinase-independent activation of both Rab3B and Rab3D.Fig. 2

Bottom Line: We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation.Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2.This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.

ABSTRACT

Aims/hypothesis: The glucose transporter GLUT4 is present mainly in insulin-responsive tissues of fat, heart and skeletal muscle and is translocated from intracellular membrane compartments to the plasma membrane (PM) upon insulin stimulation. The transit of GLUT4 to the PM is known to be dependent on a series of Rab proteins. However, the extent to which the activity of these Rabs is regulated by the action of insulin action is still unknown. We sought to identify insulin-activated Rab proteins and Rab effectors that facilitate GLUT4 translocation.

Methods: We developed a new photoaffinity reagent (Bio-ATB-GTP) that allows GTP-binding proteomes to be explored. Using this approach we screened for insulin-responsive GTP loading of Rabs in primary rat adipocytes.

Results: We identified Rab3B as a new candidate insulin-stimulated G-protein in adipocytes. Using constitutively active and dominant negative mutants and Rab3 knockdown we provide evidence that Rab3 isoforms are key regulators of GLUT4 translocation in adipocytes. Insulin-stimulated Rab3 GTP binding is associated with disruption of the interaction between Rab3 and its negative effector Noc2. Disruption of the Rab3-Noc2 complex leads to displacement of Noc2 from the PM. This relieves the inhibitory effect of Noc2, facilitating GLUT4 translocation.

Conclusions/interpretation: The discovery of the involvement of Rab3 and Noc2 in an insulin-regulated step in GLUT4 translocation suggests that the control of this translocation process is unexpectedly similar to regulated secretion and particularly pancreatic insulin-vesicle release.

No MeSH data available.