Glut4 Is Sorted from a Rab10 GTPase-independent Constitutive Recycling Pathway into a Highly Insulin-responsive Rab10 GTPase-dependent Sequestration Pathway after Adipocyte Differentiation

Abstract

The RabGAP AS160/TBC1D4 controls exocytosis of the insulin-sensitive glucose transporter Glut4 in adipocytes. Glut4 is internalized and recycled through a highly regulated secretory pathway in these cells. Glut4 also cycles through a slow constitutive endosomal pathway distinct from the fast transferrin (Tf) receptor recycling pathway. This slow constitutive pathway is the only Glut4 cycling pathway in undifferentiated fibroblasts. The α2-macroglobulin receptor LRP1 cycles with Glut4 and the Tf receptor through all three exocytic pathways. To further characterize these pathways, the effects of knockdown of AS160 substrates on the trafficking kinetics of Glut4, LRP1, and the Tf receptor were measured in adipocytes and fibroblasts. Rab10 knockdown decreased cell surface Glut4 in insulin-stimulated adipocytes by 65%, but not in basal adipocytes or in fibroblasts. This decrease was due primarily to a 62% decrease in the rate constant of Glut4 exocytosis (kex), although Rab10 knockdown also caused a 1.4-fold increase in the rate constant of Glut4 endocytosis (ken). Rab10 knockdown in adipocytes also decreased cell surface LRP1 by 30% by decreasing kex 30-40%. There was no effect on LRP1 trafficking in fibroblasts or on Tf receptor trafficking in either cell type. These data confirm that Rab10 is an AS160 substrate that limits exocytosis through the highly insulin-responsive specialized secretory pathway in adipocytes. They further show that the slow constitutive endosomal (fibroblast) recycling pathway is Rab10-independent. Thus, Rab10 is a marker for the specialized pathway in adipocytes. Interestingly, mathematical modeling shows that Glut4 traffics predominantly through the specialized Rab10-dependent pathway both before and after insulin stimulation.

Keywords: LRP1; Rab10; adipocyte; as160/TBC1D4; endocytosis; exocytosis; glucose; glucose transporter type 4 (GLUT4); insulin.

FIGURE 1.

Knockdown of Rab10 decreases cell surface Glut4 in adipocytes. 3T3-L1 adipocytes expressing a Glut4 reporter construct (HA-Glut4/GFP) and either a scrambled (control; white) or Rab-targeting shRNA (Rab10 KD; gray) were incubated with 100 nm insulin for increasing amounts of time and placed on ice. Surface-exposed HA-Glut4/GFP was labeled with AlexaFluor647-conjugated α-HA antibody (AF647-α-HA). A, surface Glut4 (AF647-α-HA). B, total HA-Glut4/GFP expression (GFP). C, basal to insulin transition, MFR (mean AF647/mean GFP). Lines, single exponential fits of the data. All data are standardized to control samples as indicated and are the mean ± S.D. (A) or the average MFR ± S.D. (C) of n = 7–15 independent experiments (Table 1) or mean ± S.D. (B) of n = 96 samples from three independent experiments. ***, p ≤ 0.0001 that values are the same in control and knockdown cells.

FIGURE 2.

Knockdown of Rab10 inhibits exocytosis of Glut4 in adipocytes. A, 3T3-L1 adipocytes expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were pretreated ± 100 nm insulin for 45 min and then incubated at 37 °C for increasing times with AF647-α-HA ± insulin. Circles, basal; squares, insulin. Data are standardized to Y_max control insulin and are the average MFR ± S.D. (uptake) of n = 4–9 experiments (Table 1). Lines, single-exponential fits of the data (k_obs = k_ex; Y_max = total cycling pool size). B, k_ex ± S.D. determined from single exponential fits of the combined data. ***, p ≤ 0.0001, that control and Rab10 knockdown cells are best fit by the same function.

FIGURE 3.

Knockdown of Rab10 accelerates endocytosis of Glut4 in adipocytes. A, 3T3-L1 adipocytes expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were pretreated with 100 nm insulin for 45 min and then incubated at 37 °C for increasing times with LY294002 (LYi). Cells were then placed on ice, and surface HA-Glut4/GFP was labeled. Lines, single-exponential decay fits of the data (k_obs ≈ k_en + 0.004)_. Data are standardized to control insulin at t = 0, and are the average MFR ± S.D. (transition) of n = 6–12 independent experiments (Table 1). B, k_en± S.D. determined from single exponential fits of the combined data. **, p ≤ 0.001, that control and knockdown cells are best fit by the same function.

FIGURE 4.

Knockdown of Rab10 has no effect on Glut4 trafficking in fibroblasts. Fibroblasts expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were treated as described in Figs. 1–3, and surface HA-Glut4/GFP was labeled. A, basal to insulin transition, average MFR ± S.D. of n = 4–10 independent experiments. Lines, simulations (Table 3). B, total HA-Glut4/GFP expression (GFP), mean ± S.D. of n = 64 samples from two independent experiments. ***, p ≤ 0.0001, and values are the same in control and knockdown cells. C, k_en ± S.D. determined from single-exponential decay fits of D. Insulin + LYi transition data average MFR ± S.D. of n = 5–6 independent experiments. Lines, single exponential fits of the data. All data are standardized to control samples as indicated.

FIGURE 5.

Knockdown of Rab10 inhibits LRP1 exocytosis in adipocytes. Adipocytes expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were incubated ± 100 nm insulin for 30 min (circle, basal; squares, insulin), then incubated at 37 °C for increasing times with AF647, α₂-M ± insulin (4 μg/ml), and placed on ice. Additional cells on the same plate were then labeled for 90 min on ice to label cell surface receptors. A, surface/total LRP1; data are standardized to Y_max from cells incubated with chloroquine (data not shown (9)). B, AF647, α₂-M uptake; arbitrary units, data are standardized to control basal uptake at t = 15 min in fibroblasts (Fig. 6). Lines, linear fits of the data. C, k_en determined from the slope of the In/Sur versus time plot (In/Sur = uptake/surface; data not shown). D, k_ex calculated using surface/total Glut4 (PM) and k_en (k_{ex calc} = (PM/(1 − PM)) k_en). Data are mean ± S.D. (A and C) or mean ± S.E. (B and D) of n = 5–7 independent experiments. **, p ≤ 0.001, or *, p ≤ 0.015, that values are the same in control and knockdown cells.

FIGURE 6.

Knockdown of Rab10 has no effect on LRP1 exocytosis in fibroblasts. Fibroblasts expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were treated as described in Fig. 5. A, surface/total LRP1. B, AF647, α₂-M uptake. Lines, linear fits of the data. C, k_en determined from the slope of the In/Sur versus time plot (data not shown). D, k_ex calculated using surface/total Glut4 (PM) and k_en (k_{ex calc} = (PM/(1 − PM)) k_en). Data are mean ± S.D. (A and C) or mean ± S.E. (B and D) of n = 4 independent experiments.

FIGURE 7.

Knockdown of Rab10 has no effect on Tf receptor exocytosis in adipocytes. Adipocytes expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were incubated at 37 °C for 30 min with AF647-holo-Tf, then for increasing time in media with excess unlabeled holo-Tf and placed on ice. To label cell surface receptors, additional cells on the same plate were incubated ± insulin at 37 °C and then labeled with biotinylated α-Tf receptor and AF647-streptavidin on ice. A, AF647-Tf efflux; data are mean ± S.D. from n = 3 independent experiments standardized to Y₀ from control cells. Lines, single-exponential decay fits of the data (k_obs ≈ k_ex). There was no significant difference in k_ex in the two cell types (k_ex = 0.09 ± 0.008 min⁻¹; p = 0.94), and that k is the same for both data sets. B, AF647, Tf uptake (Y₀); mean ± S.D. from n = 9 samples from three independent experiments, data are standardized to control basal. C, surface TfR; mean ± S.D. of n = 6 samples from three independent experiments, data are standardized to control basal. ***, p ≤ 0.0001, that values are the same in control and knockdown cells.

FIGURE 8.

Knockdown of Rab10 has no effect on Tf receptor exocytosis in fibroblasts. Fibroblasts expressing HA-Glut4/GFP and shRNA (control, white; Rab10, gray) were treated as described in Fig. 7. A, AF647-Tf efflux (mean ± S.D. from n = 2 independent experiments). There was no significant difference in k_ex in the two cell types (k_ex = 0.12 ± 0.01 min⁻¹; p = 0.092, that k is the same for both data sets). B, AF647, Tf uptake (Y₀); mean ± S.D. from n = 6 samples from two independent experiments. C, surface Tf receptor; mean ± S.D. of n = 12 samples from two independent experiments. ***, p ≤ 0.0001, or **, p ≤ 0.001, that values are the same in control and knockdown cells.

FIGURE 9.

Modeling and simulations. A, Glut4 trafficking itinerary in fibroblasts and adipocytes. 1) Glut4 is internalized from the PM via a slow pathway distinct from the fast TfR pathway and delivered to SE. Proteins in the SE are sorted into different exocytic pathways, including the fast recycling pathway followed by the TfR (thin dotted lines). 2 and 3) The slow constitutive recycling pathway through endosomal recycling intermediate compartments (ERC; thin solid lines). AS160 regulates 4) trafficking from the SE into GSVs, and 5) release/priming of sequestered GSVs for fusion to the PM through its Rab substrates. 6) An AS160-independent Akt and PI3K-dependent step regulates the tethering/docking/fusion of primed GSVs to the PM. Rab10 is rate-limiting for exocytosis from the GSV, whereas an unidentified Rab (Rab-?) is required for recycling from the ERC. B, differential equations describing the transfer of Glut4 between four compartments, with a single rate constant for each of six steps: k_en, endocytosis (PM to SE); k_sort, sorting (SE to ERC); k_fuseE, endosomal fusion (ERC to PM); k_seq, sequestration (SE to GSV); k_fuseG, GSV fusion (GSV to PM); and k_rec, recycling (SE to PM; LRP and TfR). To determine the values of the rate constants to use in simulations (Fig. 10), the experimental data in Figs. 1–6 were fitted with a system of coupled ordinary differential equations derived from this model that explicitly represents the output of these experiments (10).

FIGURE 10.

Modeling and simulations, Rab10 knockdown inhibits exocytosis from GSVs and Glut4 continues to cycle through the Rab10-dependent GSVs after insulin stimulation in adipocytes. A and C, basal to insulin transitions, surface/total Glut4 (data from Fig. 1C; up triangles) and insulin + LYi transitions (data from Fig. 3; down triangles). B, α₂-M uptake (data from Fig. 5B; circles, basal; squares, insulin). D, anti-HA uptake (data from Fig. 2; circles, basal; squares, insulin). Two hypotheses were compared for Rab10 knockdown. Rab10 knockdown affects the following: 1) k_en and k_fuseG (green lines), and 2) k_en and k_seq (red lines). The control experiments were simulated with the best overall fit (black lines). We also tested the hypothesis that 3) Glut4 and LRP1 redistribute from the GSVs into the endosomal recycling pathway after insulin simulation (k_{seq, basal} = k_{seq, insulin} = 0.002 min⁻¹; control, gray lines; Rab10 knockdown, purple lines). The only hypothesis tested that accurately simulates the effects of Rab10 knockdown on Glut4 and LRP1 trafficking is hypothesis 1, Rab10 knockdown affects k_en and k_fuseG.