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. 2011 Jul 29;286(30):26287-97.
doi: 10.1074/jbc.M111.253880. Epub 2011 May 24.

Loss of AS160 Akt substrate causes Glut4 protein to accumulate in compartments that are primed for fusion in basal adipocytes

Paul Duffield Brewer  1 , Irina Romenskaia, Mark A Kanow, Cynthia Corley Mastick
Affiliations

Loss of AS160 Akt substrate causes Glut4 protein to accumulate in compartments that are primed for fusion in basal adipocytes

Paul Duffield Brewer et al. J Biol Chem. .

Abstract

The Akt substrate AS160 (TCB1D4) regulates Glut4 exocytosis; shRNA knockdown of AS160 increases surface Glut4 in basal adipocytes. AS160 knockdown is only partially insulin-mimetic; insulin further stimulates Glut4 translocation in these cells. Insulin regulates translocation as follows: 1) by releasing Glut4 from retention in a slowly cycling/noncycling storage pool, increasing the actively cycling Glut4 pool, and 2) by increasing the intrinsic rate constant for exocytosis of the actively cycling pool (k(ex)). Kinetic studies were performed in 3T3-L1 adipocytes to measure the effects of AS160 knockdown on the rate constants of exocytosis (k(ex)), endocytosis (k(en)), and release from retention into the cycling pool. AS160 knockdown released Glut4 into the actively cycling pool without affecting k(ex) or k(en). Insulin increased k(ex) in the knockdown cells, further increasing cell surface Glut4. Inhibition of phosphatidylinositol 3-kinase or Akt affected both k(ex) and release from retention in control cells but only k(ex) in AS160 knockdown cells. Glut4 vesicles accumulate in a primed pre-fusion pool in basal AS160 knockdown cells. Akt regulates the rate of exocytosis of the primed vesicles through an AS160-independent mechanism. Therefore, there is an additional Akt substrate that regulates the fusion of Glut4 vesicles that remain to be identified. Mathematical modeling was used to test the hypothesis that this substrate regulates vesicle priming (release from retention), whereas AS160 regulates the reverse step by stimulating GTP turnover of a Rab protein required for vesicle tethering/docking/fusion. Our analysis indicates that fusion of the primed vesicles with the plasma membrane is an additional non-Akt-dependent insulin-regulated step.

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Figures

FIGURE 1.
FIGURE 1.
AS160 knockdown is partially insulin-mimetic. Surface Glut4 labeling is shown. 3T3-L1 adipocytes expressing a Glut4 reporter construct (HA-Glut4/GFP) and either a scrambled shRNA (control; white) or an shRNA directed against AS160 (AS160 KD; gray) were incubated ± insulin (100 nm or as indicated) for 45 min and placed on ice, and surface-exposed Glut4 was labeled with AlexaFluor647-conjugated anti-HA antibody (AlexaFluor647-α-HA). Data are standardized to control basal (A and B) or percentage of difference between basal and fully insulin-stimulated cells (C) and are the average mean fluorescence ratio (AlexaFluor647/GFP) ± S.D. (MFR ± S.D.) of n = 9 independent experiments. Control: basal, 1 ± 0.7, and insulin, 31 ± 0.5; AS160 KD: basal, 3.8 ± 1.0, and insulin, 23 ± 0.35.
FIGURE 2.
FIGURE 2.
AS160 knockdown increases the actively cycling pool size (Ymax) but not the intrinsic rate constant for trafficking (kex). α-HA uptake kinetics are shown. Cells were pretreated ± insulin for 45 min and then incubated at 37 °C for increasing times with AlexaFluor647-α-HA ± insulin. Data are standardized to Ymax control or AS160 KD insulin for each experiment (Ymax AS160 KD was 0.93× Ymax control) and are the average MFR ± S.D. of n = 5 experiments. Lines are single exponential fits of the average data. Control: basal, Ymax = 0.33 ± 0.05 and kex = 0.01 ± 0.005 min−1; insulin, Ymax = 1.0 ± 0.01 and kex = 0.026 ± 0.003 min−1; AS160 KD: basal, Ymax = 0.83 ± 0.04 and kex = 0.01 ± 0.002 min−1; insulin, Ymax = 1.0 ± 0.02 and kex = 0.022 ± 0.003 min−1.
FIGURE 3.
FIGURE 3.
Glut4 translocation requires Akt and PI 3-kinase activity in AS160 KD cells. Inhibitor dose response and surface Glut4 labeling are shown. Cells were preincubated with the indicated concentrations of PI 3-kinase inhibitor (A and B), LY294002 (LYi) or Akt inhibitor, Akt1/2 (Akti) (C and D) for 1 h. Insulin (0 or 100 nm) was added, and incubation was continued for 45 min and then cell surface Glut4 measured. Data are standardized to control insulin for each experiment and are the average MFR ± S.E. of n = 2 (Akti) or n = 3 (LYi) experiments. Bas, basal; Ins, insulin.
FIGURE 4.
FIGURE 4.
Inhibition of PI 3-kinase affects both kex and Ymax in control cells but only kex in AS160 KD cells. α-HA uptake kinetics are shown. Cells were preincubated ± LYi (50 μm) ± insulin (100 nm) and then α-HA uptake kinetics were measured. Data are standardized to Ymax control or AS160 KD insulin for each experiment (Ymax AS160 KD was 0.95× Ymax control) and are the average MFR ± S.D. of n = 2 experiments. Lines are single exponential fits of the averages. Control: basal, Ymax = 0.21 ± 0.01 and kex = 0.01 ± 0.001 min−1; insulin, Ymax = 1.0 ± 0.03 and kex = 0.026 ± 0.004 min−1; insulin + LYi, Ymax = 0.34 ± 0.13 and kex = 0.004 ± 0.002 min−1. AS160 KD: basal, Ymax = 0.75 ± 0.03 and kex = 0.01 ± 0.001 min−1; insulin, Ymax = 1.0 ± 0.04 and kex = 0.022 ± 0.005 min−1; insulin + LYi, Ymax = 0.78 ± 0.05 and kex = 0.007 ± 0.001 min−1.
FIGURE 5.
FIGURE 5.
Glut4 vesicles accumulate in a primed, pre-fusion pool in basal AS160 KD cells. Surface Glut4 transition kinetics are shown. A, insulin (100 nm) was added to basal cells, and incubation was continued at 37 °C for increasing times. B, cells were stimulated with insulin for 45 min and then LYi (50 μm) was added, and incubation was continued at 37 °C for increasing times. C, cells were preincubated with 20 μm Akti for 1 h prior to stimulation with insulin for increasing times. After incubation, the cells were placed on ice, and cell surface Glut4 was measured. Data are standardized to control or AS160 KD insulin for each experiment and are the average MFR ± S.D. of n = 5 experiments. Lines are single exponential fits of the average data. Control: +insulin, kobs = 0.13 ± 0.01 min−1 and Ymax = 1; Akti +insulin, kobs = 0.13 ± 0.02 min−1 and Ymax = 0.24; insulin +LYi, kobs = 0.12 ± 0.01 min−1 and Ymin = 0.04. AS160 KD: +insulin, kobs = 0.24 ± 0.03 min−1 and Ymax = 1; Akti +insulin, kobs = 0.13 ± 0.02 min−1, Ymax = 0.54; insulin +LYi kobs = 0.12 ± 0.01 min−1 and Ymin = 0.24.
FIGURE 6.
FIGURE 6.
Models. A and B, Glut4 trafficking can be modeled as six kinetically distinct steps with seven rate constants. Step 1, endocytosis, removal of Glut4 from the PM and delivery to endosomes, ken. Step 2, sorting, packaging into constitutive TV, ksort. Step 3, trafficking/fusion of the constitutive vesicles, ktr. Step 4, sequestration, the Glut4 in endosomes (endo) is also packaged into specialized Glut4 storage vesicles (GSVseq), kseq. Step 5, release, in response to insulin, GSVs are released from the retention mechanism, and the vesicles become competent to fuse (in part through GTP loading of a bound Rab, krel); AS160 regulates this step by reversing the activation of the Rab (by activating the Rab-GTPase; krev). Step 6, tethering/docking/fusion, primed GSVs bind to the plasma membrane via specific tethering and docking proteins (including Munc/SMs and SNAREs) and the vesicles fuse, kfus. Effects on any of these steps will affect the levels of Glut4 at the cell surface. C, Akt regulates the release of GSVs from retention by both increasing the rate of priming (through an unknown substrate, AS-X) and through the inhibition of AS160, which reverses the priming. Insulin also regulates vesicle tethering/docking/fusion through an additional mechanism that is Akt-independent.
FIGURE 7.
FIGURE 7.
Simulations. A series of differential equations describing the model depicted in Fig. 6 (Equations 1–5) were solved numerically using computing software (Mathematica) to simulate α-HA uptake (A) or transition kinetics (B). Four hypotheses were tested: AS160 regulates krev (solid lines); AS160 regulates kfus (dashed lines); AS160 regulates ksort and ktr (alternating lines); AS160 regulates kseq (dotted lines). Symbols: measured data (white, control; gray, AS160 KD). C, simulating the effect of Akti on transition kinetics. Two hypotheses were tested: Akt regulates krev and krel (solid line); Akt regulates krev and kfus (dotted line). Symbols: measured data, standardized to final steady state levels for each cell type.
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