Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes

Abstract

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.

Keywords: adipocyte; glucose transporter type 4 (GLUT4); insulin; insulin resistance; membrane trafficking; tumor suppressor candidate 5 (TUSC5).

FIGURE 1.

Proteomic analysis of GLUT4 storage vesicles. A, number of protein groups carried through each filtering step of the analytical workflow (All, all proteins identified by mass spectrometry with known contaminants removed; pHS, proteins present in GSVs isolated from the pHS starting material (n = 2); LDM, proteins present in GSVs isolated from the LDM starting material (n = 2); Overlap, proteins identified in both LDM and pHS (n = 4); Enriched, proteins enriched within GSVs from unstimulated adipocytes with an average intensity greater than the median (n = 4, integrated p value < 0.05); GSV, enriched proteins that were also insulin-responsive (n = 4, integrated p < 0.05). B, volcano plot depicting proteins enriched within GSVs (red and blue points are proteins significantly enriched (n = 4, integrated p < 0.05)). Blue points represent known GSV proteins. C, boxplot of the average log2 LFQ intensities for all proteins present in GSVs from unstimulated adipocytes. Blue points represent known GSV proteins; red points represent enriched proteins (B) with average log2 LFQ greater than the population median; gray points are proteins below the median cutoff. D, fold response of 23 proteins that were enriched in GSVs and whose abundance decreased significantly following insulin treatment (n = 4, integrated p < 0.05). Blue bars highlight known GSV proteins. IP, immunoprecipitation.

FIGURE 2.

TUSC5 is a novel GSV protein. A, TUSC5 and GLUT4 expression was assessed in a panel of mouse tissues by Western blotting (representative image of n = 2). Asterisks denote TUSC5-specific bands of different molecular mass (* = ∼20 kDa, ** = ∼30 kDa). B, GSVs were immunoisolated from primary rat adipocytes treated with and without insulin. Abundance of GLUT4 and TUSC5 within the LDM fraction obtained from the pHS starting material and precipitates was determined by immunoblotting (representative of n = 3). C, GSVs were immunoisolated from 3T3-L1 adipocytes treated with and without insulin. GLUT4, IRAP, and TUSC5 levels were determined by immunoblotting (representative of n = 3). Two exposure levels are presented for TUSC5 to permit visualization of TUSC5 in the starting material. D, 3T3-L1 adipocytes were serum-starved for 2 h prior to fixation and processing for immunofluorescent imaging by confocal microscopy. Cells were stained for nuclei (DAPI, blue), TUSC5 (red), GLUT4 (green), and EEA1 (magenta). Cells were visualized at ×63 magnification. The basal surface (middle panel) and midpoint of specified region (cross-section, lower panel) are presented to aid visualization of colocalization. Instances of colocalization between GLUT4 and TUSC5 without EEA1 are indicated by closed arrowheads and colocalization between all three proteins is indicated by open arrowheads (top panel, scale bar, 50 μm, middle and bottom panel, scale bar, 10 μm). IP, immunoprecipitation; BAT, brown adipose tissue; Epi, epididymal fat.

FIGURE 3.

TUSC5 translocates to the PM in response to insulin. A, levels of GLUT4, IRAP, and TUSC5 were determined in purified LDM and PM subcellular fractions by immunoblotting. Subcellular fractions were generated from 3T3-L1 adipocytes treated with and without insulin as indicated. Two exposure levels are presented for TUSC5 to permit visualization of higher molecular mass forms of TUSC5 (* = ∼20 kDa, and ** = ∼30 kDa). To control for loading and to assess purity of the fractions, we immunoblotted for 14-3-3 and Caveolin1 in both fractions. B, quantification of GLUT4, IRAP, and TUSC5 levels in the LDM fraction in A relative to levels in unstimulated cells (n = 3, mean ± S.E., unpaired t test, *, p < 0.05; **, p < 0.01, all comparisons with basal values; Ins, insulin). C, quantification of GLUT4, IRAP, and TUSC5 levels in the PM fraction in A relative to levels in unstimulated cells (n = 3, mean ± S.E., unpaired t test, *, p < 0.05; **, p < 0.01, all comparisons with basal values). Black and white bars denote levels under basal and insulin conditions, respectively, as in B. D, 3T3-L1 adipocytes were serum-starved for 2 h and treated with 100 nm insulin for 20 min where indicated prior to fixation and processing for imaging by bright field light microscopy and immunofluorescent imaging by TIRF microscopy. Nonpermeabilized cells were stained for nuclei (DAPI), TUSC5, and HA-GLUT4 with anti-HA antibody. Cells were visualized at ×63 magnification (scale bar, 20 μm). E, histogram showing the distribution of TIRF signal intensity for HA-GLUT4 under basal conditions (black bars) and following insulin stimulation (gray bars) (Kolmogorov-Smirnov test comparing distributions under basal and insulin conditions, p < 0.001). F, histogram showing the distribution of TIRF signal intensity for TUSC5 under basal conditions (black bars) and following insulin stimulation (gray bars) (Kolmogorov-Smirnov test comparing distributions under basal and insulin conditions, p < 0.001). G, 3T3-L1 adipocytes were serum-starved for 2 h and treated with 100 nm insulin for 20 min where indicated prior to fixation and processing for imaging by bright field light microscopy (BF) and immunofluorescent imaging by epifluorescent (EPI) and TIRF microscopy (TIRF). Permeabilized cells were stained for nuclei (DAPI), TUSC5, and HA-GLUT4 with anti-HA antibody. Cells were visualized at ×63 magnification (scale bar, 20 μm). H, average TIRF signal/epifluorescent signal ratios for GLUT4 (black bars) and TUSC5 (white bars) under basal and insulin conditions as indicated (data from 158 to 295 cells from 10 regions of interest per condition from two independent experiments, mean ± S.E., Kolmogorov-Smirnov test, ***, p < 0.001). A.U., arbitrary units.

FIGURE 4.

TUSC5 is a positive regulator of insulin-stimulated GLUT4 trafficking. A, total cell lysates from 3T3-L1 adipocytes treated with scrambled (Scr) or a panel of three different anti-Tusc5 siRNAs (#1, #2, and #3), or a pool of all three siRNAs (T5) were immunoblotted for TUSC5 to determine the extent of TUSC5 knockdown. B, TUSC5 levels (in A) were quantified by densitometry and expressed as relative to cells treated with scrambled (Scr) siRNA (n = 4, mean ± S.E. one-way ANOVA; **, p < 0.01; ***, p < 0.001). C, 3T3-L1 adipocytes were treated with scrambled or individual anti-Tusc5 siRNAs (#1, #2, and #3) or a pool of siRNAs (T5) for 96 h before 2DOG uptake assays were performed at the insulin doses indicated. 2DOG uptake expressed as a percentage of the maximum response in cells treated with scrambled siRNA (n = 4, mean ± S.E., two-way ANOVA; NS, nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001, comparisons with control (scr) cells treated with the same insulin dose). D, PM levels of HA-GLUT4 in 3T3-L1 adipocytes overexpressing HA-GLUT4 treated with scrambled (Scr) or pooled Tusc5 (T5) siRNA was measured using a fluorescence-based assay. Adipocytes were treated with insulin as indicated (n = 3, mean ± S.E., two-way ANOVA; NS, nonsignificant; *, p < 0.05, comparisons with control (scr) cells treated with the same insulin dose). E, retroviral overexpression of TUSC5 was assessed by immunoblotting whole cell lysates from cells expressing a control vector (empty vector, EV) or overexpressing TUSC5. α-Tubulin levels were determined as a loading control. F, 3T3-L1 adipocytes expressing a control vector (EV) or overexpressing TUSC5 were subjected to 2DOG uptake assays at the insulin doses indicated. 2DOG uptake expressed as a percentage of the maximum response in cells expressing control vector (EV) (n = 4, mean ± S.E., two-way ANOVA; NS, nonsignificant; **, p < 0.01, comparisons with cells expressing control vector (EV) treated with the same insulin dose). G, PM levels of HA-GLUT4 in 3T3-L1 adipocytes overexpressing HA-GLUT4 in combination with a control vector (EV) or TUSC5 were measured using a fluorescence-based assay. PM HA-GLUT4 levels in adipocyte-treated insulin doses as indicated were plotted as a dose-response curve (n = 3, mean ± S.E., two-way ANOVA; NS, nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001 comparisons with cells expressing control vector (EV) treated with the same insulin dose). ED₅₀ values for PM-GLUT4 were determined from nonlinear fitting of dose curves and graphed as an inset (n = 3, ± S.E., unpaired t test; *, p < 0.05, compared with cells expressing empty vector (EV)). H, GLUT4, IRAP, and 14-3-3 (loading control) levels were assessed by immunoblotting whole cell lysates from control (scr) and TUSC5 knockdown cells. I, GLUT4 and IRAP levels (H) were quantified by densitometry and are expressed as relative to control (scr) cells (n = 3, mean ± S.E., unpaired t test, **, p < 0.01, all comparisons with cells treated with scrambled siRNA; dotted line represents levels in cells treated with scrambled siRNA). J, mRNA expression of Glut4 was determined by qPCR in cells treated with scrambled (Scr) and pooled anti-Tusc5 (T5) siRNA. Data were expressed relative to levels in cells treated with scrambled siRNA (n = 3, mean ± S.E., unpaired t test; NS, nonsignificant, compared with cells treated with scrambled siRNA). K, levels of GLUT4, IRAP, and 14-3-3 (loading control) were assessed by immunoblotting whole cell lysates from control (EV) cells and cells overexpressing TUSC5. L, insulin signaling was monitored at the level of phosphorylation of insulin receptor (pIR), IRS1 (pIRS1), Thr(P)-308 and Ser(P)-473 AKT and Thr(P)-642 TBC1D4 in response to 0.5 and 100 nm insulin in control (scr) cells and cells treated with TUSC5 siRNA. Total levels of insulin receptor (IR), IRS1, AKT, and TBC1D4 were assessed in all conditions, and 14-3-3 was used as loading control. M, insulin signaling was monitored at pIR, pIRS, Thr(P)-308 and Ser(P)-473 AKT, and Thr(P)-642 TBC1D4 in response to 0.5 and 100 nm insulin in control (EV) cells and cells overexpressing TUSC5. Total levels of IR, IRS, AKT, and TBC1D4 were assessed in all conditions, and 14-3-3 was used as loading control. N, quantification of signaling intermediates in L. All data are expressed relative to substrate phosphorylation in response to 100 nm insulin in control (scr) cells (n = 3–5, mean ± S.E., unpaired t test; NS, nonsignificant; *, p < 0.05; **, p < 0.01, comparisons with cells expressing treated with scrambled siRNA under the same treatment conditions). O, quantification of signaling intermediates in M. All data are expressed relative to substrate phosphorylation in response to 100 nm insulin in control (EV) cells (n = 3, mean ± S.E., unpaired t test; NS, nonsignificant, comparisons with cells expressing treated with scrambled siRNA under the same treatment conditions).

FIGURE 5.

TUSC5 is necessary, but not sufficient, for the insulin-sensitizing effects of rosiglitazone in insulin resistance. A, Tusc5 mRNA levels were determined by qPCR in cells treated with TNFα (2 ng/ml, 96 h) and/or rosiglitazone (10 μm, 48 h) as indicated. Data were expressed as relative to Tusc5 levels in control cells (n = 3, mean ± S.E., unpaired t test; *, p < 0.05; ***, p < 0.001 for comparisons with untreated cells; ##, p < 0.05 for comparisons with TNFα-treated cells). B, TUSC5 levels were determined by immunoblotting in cells treated with TNFα (2 ng/ml, 96 h) and/or rosiglitazone (10 μm, 48 h) as indicated. α-Tubulin levels were determined as a loading control. C, TUSC5 abundance (B) was quantified by densitometry and expressed as relative to untreated cells (n = 4, mean ± S.E., unpaired t test; *, p < 0.05 for comparisons with untreated cells; #, p < 0.05 for comparisons with TNFα-treated cells). D, Tusc5 mRNA levels were determined by qPCR in cells expressing an empty vector control (EV) or overexpressing TUSC5 (TUSC5) with and without TNFα treatment. Data were expressed relative to TUSC5 levels in untreated EV adipocytes (n = 3, mean ± S.E., unpaired t test; NS, nonsignificant; *, p < 0.05 for comparisons between untreated and TNFα-treated cells as indicated). E, TUSC5, GLUT4, and 14-3-3 (loading control) levels were assessed by immunoblotting whole cell lysates from cells expressing a control vector (EV) or overexpressing TUSC5 and treated with TNFα as indicated (representative of n = 4). F, 3T3-L1 adipocytes expressing a control vector (EV) or overexpressing TUSC5 were treated with TNFα (2 ng/ml, 96 h) as indicated before 2DOG uptake assays were performed. 2DOG uptake was expressed as a percentage of the maximum response (n = 4, mean ± S.E., two-way ANOVA; NS, nonsignificant; ***, p < 0.001; comparisons with cells expressing control vector (EV) under the same treatment conditions). G, 3T3-L1 adipocytes expressing a control vector (EV) or overexpressing TUSC5 were treated with TNFα (2 ng/ml, 96 h) as indicated before insulin signaling at the level of Thr(P)-308 AKT was monitored in response to 0.5 or 100 nm insulin (representative of n = 4). H, 3T3-L1 adipocytes were treated with scrambled or anti-Tusc5 siRNA for 96 h and treated with TNFα (2 ng/ml, 96 h) and/or rosiglitazone (10 μm, 48 h) as indicated before 2DOG uptake assays were performed in unstimulated cells or cells stimulated with 100 nm insulin. 2DOG uptake was expressed as a percentage of the maximum response (n = 5, mean ± S.E., two-way ANOVA; NS, nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001, comparisons with control (scr) cells under the same treatment conditions unless otherwise indicated). I, GLUT4, TUSC5, and 14-3-3 (loading control) levels were assessed by immunoblotting whole cell lysates from control (scr) and TUSC5 knockdown cells treated with TNFα and/or rosiglitazone as indicated. J, quantification of the change in GLUT4 expression in response to rosiglitazone in cells treated with scrambled (Scr) or pooled anti-Tusc5 (T5) siRNA with and without TNFα treatment (n = 3, mean ± S.E., unpaired t test; *, p < 0.05, for comparisons with control (scr) cells under the same treatment conditions). K, 3T3-L1 adipocytes were treated with scrambled or anti-Tusc5 siRNA for 96 h and treated with TNFα and/or rosiglitazone as indicated before insulin signaling in response to 100 nm insulin was monitored at the level of Thr(P)-308 AKT (representative of n = 3). L, quantification of the change in insulin-stimulated phosphorylation of AKT at Thr-308 in response to rosiglitazone in cells treated with scrambled (Scr) or pooled anti-Tusc5 (T5) siRNA with and without TNFα treatment (n = 3, mean ± S.E., unpaired t test; NS, nonsignificant; *, p < 0.05 for comparisons with control (scr) cells under the same treatment conditions).