Akt may associate with insulin-responsive vesicles via interaction with sortilin

Abstract

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor–IRS–PI3 kinase–Akt–TBC1D4–Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C-terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

Graphical Abstract

Insulin-responsive vesicles (IRVs) are formed by self-assembly of Glut4, sortilin, insulin-responsive amino peptidase (IRAP), and low density lipoprotein receptor-related protein 1 (LRP1). TBC1D4 in the insulin signaling pathway binds to the cytoplasmic tails of IRAP and LRP. Here, we demonstrate that the insulin signaling protein Akt interacts with the cytoplasmic tail of sortilin. We suggest that the IRVs represent a scaffold for the final steps of insulin action.

INTRODUCTION

The regulation of blood glucose levels in mammals is mediated by the glucose transporter 4, Glut4, which is expressed primarily in fat and skeletal muscle cells and is translocated to the plasma membrane in response to insulin [1]. Glut4-mediated glucose uptake represents the rate-limiting step of insulin-stimulated glucose disposal [2–5]; therefore, if we find the way to deliver more Glut4 to the plasma membrane in response to insulin, we are almost certain to significantly improve the overall metabolic control and clinical manifestations of diabetes.

As a 12-transmembrane highly hydrophobic protein, Glut4 does not travel to and from the cell surface in a free state; instead, it is translocated to the plasma membrane by specialized insulin-responsive membrane vesicles, or IRVs, that represent the real target of insulin action. The protein composition of the IRVs has been extensively studied. The major integral membrane proteins in these vesicles are: Glut4, sortilin, IRAP, LRP1, TUSC5, SCAMPs, and VAMP2 [6–9]. Although none of these proteins is specific for the IRVs, their unique combination creates a “biochemical individuality” of the vesicles that distinguishes them from other non-insulin sensitive membrane carriers inside the cell.

Since none of the integral membrane proteins of the IRVs can be directly linked to the insulin signaling pathway (insulin receptor–IRS–PI3 kinase–Akt–TBC1D4–Rab10), it seems likely that insulin responsiveness is conferred to the vesicles by peripheral membrane proteins that may be loosely associated with this compartment. It is also clear that these peripheral membrane proteins should somehow recognize the “core” transmembrane IRV components listed above, otherwise “wrong” vesicles will be translocated by insulin.

Previously, we [10] and others [11, 12] have demonstrated that Akt is associated with the IRVs in adipose cells. Moreover, the Tavare’s group has shown that compartmentalization of Akt on the IRVs is essential for their insulin-dependent translocation to the plasma membrane [12]. However, the mechanism of Akt recruitment to the IRVs remained unknown. Here, we are filling this gap by reporting that the Akt-binding protein in the vesicles is sortilin. Interestingly, Rab10 GAP, TBC1D4 (a.k.a. AS160) [13], is compartmentalized on the IRVs by binding to the cytoplasmic tail of IRAP [14, 15] and LRP1 [8], and Rab10 itself is also associated with the IRVs via an as yet unknown mechanism [14, 16–18]. These observations seem to represent an important insight into the mechanism of compartmentalization of insulin action. Specifically, we suggest that “self assembly” of the IRVs in the cell [19] creates a scaffold for the final steps of insulin action.

MATERIALS AND METHODS

Reagents -

Human insulin and goat serum were purchased from Sigma-Aldrich (St. Louis, MO); fetal bovine serum (FBS) - from Peak Serum (Wellington, CO); calf bovine serum (CBS), Pierce^™ Ni-NTA magnetic Agarose beads, ProLong^® Gold antifade reagent with DAPI, Halt^™ Protease and Phosphatase Inhibitor Cocktail, Pierce ECL Western Blotting Substrate, Bovine Serum Albumin (BSA), Dulbecco’s phosphate-buffered saline (DPBS) and Dulbecco’s Modified Eagle Medium (DMEM) - from Thermo Fisher Scientific (Waltham, MA); Coomassie brilliant Blue R-250 and nitrocellulose membranes - from Bio-Rad (Hercules, CA). Mouse monoclonal antibodies against Akt, actin, and the myc tag, rabbit monoclonal antibodies against Akt and phospho-TBC1D4 (Thr642), rabbit polyclonal antibodies against phospho-Akt (Ser473 or Thr308) and TBC1D4 were purchased from Cell Signaling Technology (Danvers, MA). Alexa Fluor 594-conjugated goat anti-mouse antibody, Alexa Fluor 488-conjugated goat anti-rabbit antibody were purchased from Jackson ImmunoResearch (West Grove, PA). His-tagged peptides corresponding to truncated isoforms of the sortilin cytoplasmic tail were synthesized (with ca. 90% purity) by LifeTein (Hillsborough, NJ). His₆ peptide was purchased from GenScript (Piscataway NJ); recombinant human Akt2 was obtained from Abcam (Waltham, MA).

Cell culture -

3T3-L1 cells were purchased from Zen-Bio (Durham, NC). 3T3-L1 cells stably transfected with mLNCX2-sortilin-myc/His (S+ cells) were described previously in [20], and 3T3-L1 cells stably transfected with SORTILINtail and with EGFP – in [21]. All cells were grown in DMEM containing 10% calf bovine serum. For adipose differentiation, cells were transferred to differentiation medium (DMEM with 10% FBS, 0.174 μM insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine) three days after confluence. Three days later, the differentiation medium was replaced with DMEM containing 10% FBS for 4 days for full differentiation.

Pull down experiments using GST-tagged cytoplasmic tail of sortilin -

the cDNA corresponding to the cytoplasmic tail of human sortilin was amplified from the full-length sortilin cDNA (purchased from Origene, Rockville, MD) by the polymerase chain reaction using forward primer 5’-CGGGGTACCAAACAGAATTCCAAGTCA-3’ and reverse primer 5’-CGCGGATCCTATTCCAAGAGGTCCTCATC-3’ with KpnI and BamHI restriction sites (underlined) and cloned into the pGEM-T vector (Promega Corp., Madison, WI). For the bacterial expression of the cytoplasmic tail of sortilin, pET-42a vector (with GST-His tag, Millipore, Billerica, MA) and pGEM-T vector with the sortilin tail were digested with KpnI and BamH1. Digested sequences were gel-purified, ligated, and transformed into the E. coli BL21-CodonPlus (DE3)-RIPL strain (Agilent Technologies, Santa Clara, CA). Individual colonies were grown overnight in 10 ml LB broth. Next day, the overnight culture was added to fresh LB (1:100 vol) containing 1 mM IPTG and incubated at 37°C for 5 hours with 250 rpm in a bacterial shaker. Cells were pelleted by centrifugation, and the pellet was resuspended in PBS with protease inhibitors and sonicated 3 X 45sec on ice. The supernatant was then incubated with Glutathione Sepharose 4B beads (GE Healthcare LifeSciences, Pittsburg, PA) at 4°C overnight on an orbital shaker. The beads were then washed three times with PBS and used for pull down experiments. For that, 3T3-L1 adipocytes were serum starved for 4 hours and treated or not treated with insulin (100 nM) for 15 min. Cells were harvested in PBS with the protease and phosphatase inhibitor cocktail and homogenized with 10 strokes of the ball-bearing cell cracker (Isobiotec, Heidelberg, Germany). Homogenates were then centrifuged at 200,000 × g for 2 h and the supernatant was incubated with the glutathione beads with pre-immobilized GST-Sortilin tail overnight at 4°C on an orbital shaker. The beads were washed with PBS three times, eluted with Laemmli sample buffer at 95°C for 5 min and analyzed by Coomassie staining and Western blotting.

Cross-linking and isolation of His-tagged complexes -

Wild-type 3T3-L1 adipocytes (WT) and 3T3-L1 adipocytes expressing Sortilin-Myc/His (S+) in 15-cm dishes were washed twice with PBS and once with Krebs-Ringer phosphate buffer (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO₄, 1.0 mM CaCl₂, 0.6 mM Na₂HPO₄, 0.4 mM NaH₂PO₄, 2.5 mM d-glucose, pH 7.4), and dithiobis[succinimidyl propionate] (DSP) was added to the final concentration 2 mM for 30 min. Cross-linking reaction was stopped with 50 mM Tris (pH 7.4), cells were incubated for 10 min in quenching buffer (50 mM Tris, 150 mM NaCl, pH 7.4) and washed once with the same buffer. Cells were lysed in lysis buffer (10 mM HEPES, 30 mM NaCl, 5% glycerol, 10 mM imidazole, 0.5% Triton X-100, pH 7.4) supplemented with protease and phosphatase inhibitor cocktail. The suspension was incubated for 10 min at 4°C and passed three times through a 21-G needle; lysates were centrifuged for 10 min at 16,000×g in a microcentrifuge. Supernatants were adjusted to equal protein concentration and volume (300 μL) and added to 40 μl of Pierce^™ Ni-NTA magnetic Agarose beads suspension pre-washed with wash buffer (50 mM Na₂HPO₄/NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 7.4). The beads were incubated on a rotator for four hours at 4°C and washed four times with wash buffer supplemented with 0.2% Triton X-100 using magnetic stand. Elution was carried out with elution buffer (50 mM Na₂HPO₄/NaH₂PO₄, 300 mM NaCl, 300 mM imidazole, pH 7.4) supplemented with 50 mM dithiothreitol at 37°C for 30 min. Eluted proteins were analyzed by Western blotting.

Pull down experiments using His-tagged cytoplasmic tail of sortilin -

His-tagged peptides corresponding to truncated isoforms of the sortilin cytoplasmic tail (Fig. 3A) were dissolved in water to the concentration of 10 mg/ml. 3T3-L1 adipocytes grown in 15 cm dishes (normally one dish per each peptide) were washed and harvested in PBS with the protease and phosphatase inhibitor cocktail. Cells were homogenized with 10 strokes of the ball-bearing cell cracker (Isobiotec, Heidelberg, Germany). Homogenates were then centrifuged at 200,000 × g for 2 h. The supernatant was collected and incubated with 10 nmols of each peptide overnight at 4°C on an orbital shaker in the total volume of 250 μl. Next day, 30 μl of Ni-NTA magnetic beads (pre-washed with PBS) were added to the lysates for two hours at 4°C. Then, the beads were washed five times with wash buffer and eluted with elution buffer. Eluted proteins were analyzed by Ponceau staining, Western blotting, and mass-spectrometry.

Pull down of recombinant Akt2 -

Peptides (10 nmols) were mixed with 250 ng recombinant Akt2 (prepared from 0.2 mg/ml Akt2 stock) in 300 μl of PBS supplemented with 0.1% BSA and incubated for 1 hour at 4°C on an orbital shaker. The mixture was transferred to 30 μl of Ni-NTA magnetic beads (pre-washed with PBS with 1% BSA) and incubated at 4°C for an hour. Beads were washed 4 times with wash buffer and eluted with elution buffer using magnetic stand. Eluates were analyzed by gel electrophoresis followed by Comassie Brilliant Blue R-250 staining.

[³H] 2-Deoxyglucose Uptake -

The assay was performed in 6-well plates as described in [22]. Briefly, cells were washed three times with serum-free DMEM, starved for 2 h, washed twice with warm (37°C) Krebs-Ringer-HEPES (KRH) buffer without glucose (121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO₄, 0.33 mM CaCl₂, 12 mM HEPES, pH 7.4) and incubated with or without 100 nM insulin at 37°C for 13 min. Radioactive 2-deoxyglucose was added to adipocytes for 5 min. The assay was terminated by aspirating the radioactive media, and the wells were washed four times with 2 ml of ice-cold KRH containing 25 mM D-glucose. Then, 400 μL of RIPA buffer (150 mM NaCl, 0.5% Sodium Deoxycholate, 0.1% SDS, 1% NP-40P, 50 mM Tris-HCl, pH 7.4) was added to each well. The lysates were transferred to Eppendorf tubes, subjected to ultra sonication, and 300 μl aliquots were removed for determination of radioactivity by liquid scintillation counting. Measurements were made in triplicates. Nonspecific diffusion assessed in the presence of 5 μM cytochalasin B, which was <5 % of the total uptake, was subtracted. The protein concentration in the lysates was determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA) and was used to normalize counts. All 2-deoxyglucose uptake experiments have been repeated twice.

Immunofluorescence -

On the day preceding the experiment differentiated 3T3-L1 adipocytes were plated on chambered coverglass plates (Cellvis, Mountain View, CA) coated with poly-lysine. Cells were fixed with 4% methanol-free formaldehyde in DPBS for 12 minutes, blocked with 5% BSA at 4°C overnight, and stained with the primary antibody for 2 h at room temperature followed by 1 h incubation with the secondary antibody. Then, cells were examined with the Axio Observer Z1 fluorescence microscope equipped with the Hamamatsu digital camera C10600/ORCA-R2 and Axiovision 4.8.1 program (Carl Zeiss Inc., Thornwood, NY). Microscopic images show a representative result of at least three independent experiments.

Gel electrophoresis and Western blotting -

Proteins were separated in SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were blocked with 3% BSA in TBST buffer with 0.1% Tween-20 for 30 min at room temperature. Blots were probed overnight with specific primary antibodies at 4°C followed by 1 h incubation at room temperature with horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected with the enhanced chemiluminescence substrate kit (ECL) using Bio-Rad ChemiDoc^™ XRS+ System (Hercules, CA). All Western blotting images are representative of at least two independent experiments.

RESULTS AND DISCUSSION

We have shown previously that formation [20] and insulin responsiveness [23] of the IRVs in cultured adipocytes depends on the presence of sortilin. In order to further study the role of sortilin in insulin-stimulated glucose uptake, we prepared the GST-tagged cytoplasmic tail of sortilin and determined, by the GST pull down assay, that it binds to Akt. Although Fig. 1A shows no apparent effect of insulin on the interaction between Akt and sortilin tail, our experiments have been performed under non-equilibrium conditions so that any such effect or lack thereof cannot be accurately measured. It is also possible that in the living cell, sortilin tail is covalently modified after insulin administration which can affect its interaction with Akt. It would be important to explore this possibility in future experiments.

Figure 1. Akt binds to the cytoplasmic C-terminus of sortilin.

Panel A: GST and GST fused with the cytoplasmic C-terminus of sortilin (CT-GST) were immobilized on Glutathione Sepharose 4B beads as described in Materials and Methods and incubated with cytosolic lysates (4 mg of total protein) prepared from 3T3-L1 adipocytes treated or not treated with insulin (100 nM) for 15 min. Samples were analyzed by Western blotting for pan-Akt and phospho-Akt, and by Coomassie staining for GST. Panel B: 3T3-L1 adipocytes stably expressing full-length sortilin-myc/His [20] (a.k.a. S+ adipocytes) were fixed, permeabilized, and stained with mouse monoclonal antibodies against the myc tag and Alexa Fluor 594-conjugated goat anti-mouse antibody and rabbit monoclonal antibodies against Akt and Alexa Fluor 488-conjugated goat anti-rabbit antibody. The bottom panel demonstrates a magnified image of a single selected cell. The space bar corresponds to 20 μm. Panel C: 3T3-L1 adipocytes stably expressing sortilin-myc/His (S+) or wild type cells were cross-linked with DSP and subjected to pull down experiments using Ni-NTA Nickel beads. Eluates and total lysates were analyzed by immunoblotting with antibodies against the myc epitope and pan-Akt. Dotted line indicates that irrelevant lanes have been spliced out. Panel D: Panel D: Quantification of results shown in panel C (right); mean values of three independent experiments ± standard deviation is shown. Student’s test was used to assess statistical difference in the amounts of pulled down Akt between WT and S+ adipocytes; p=0.05.

To confirm an interaction between Akt and sortilin tail, we used 3T3-L1 adipocytes stably transfected with full-length sortilin-myc/His [20] that we had named S+ adipocytes. As is shown in Fig. 1B by double immunofluorescence staining, the intracellular distributions of sortilin-myc/His and Akt significantly overlap. This result was further confirmed by cross-linking. In brief, wild-type 3T3-L1 and S+ adipocytes were incubated with the membrane permeable cross-linker, DSP, for 30 min, and His-tagged complexes were isolated from cell lysates using Ni-NTA magnetic Agarose beads. Figs. 1C&D demonstrate that Akt can be cross-linked to sortilin-myc/His in living cells.

Note, that previous proteomics studies of the IRVs (such as [8, 9]) failed to identify Akt in this compartment. This is not surprising since peripheral membrane proteins tend to dissociate from the IRVs in the process of immunoadsorption and thus escape identification. At the same time, alternative methods have clearly demonstrated that Akt is associated with the IRVs [10–12].

Sortilin is involved in several different membrane trafficking pathways in various cells. Correspondingly, its cytoplasmic tail interacts with multiple adaptor proteins. In particular, sortilin binds to GGA on the TGN membranes [24, 25]; trafficking of sortilin from TGN to endosomes is mediated by the clathrin adaptor AP-1 [26, 27], retrograde traffic from endosomes to TGN – by retromer [28], and endocytosis from the plasma membrane – by AP-2 [29]. Binding sites of trafficking adaptors on the C-terminus of sortilin have been identified. Thus, GGA binds to DEDLL (a.a. 820–824 in mouse sortilin) [29], AP1 and AP2 interact with YSVL (a.a. 790–793)[29]; in addition, AP1 can bind to DSDEDL (a.a. 818–823) [27]; retromer binds to FLV (a.a. 785–787) [30] and to YSVL (a.a. 790–793) [26].

In order to determine the Akt-binding site in the C-terminus of sortilin, we have chemically synthesized the peptide with the same amino acid sequence (mouse isoform, a.a. 777–825) and 3 truncated isoforms: 777–815, 777–794, and 777–789. All four peptides have His tags at their N-termini (Figs. 2A). We immobilized these peptides on Ni-NTA magnetic beads, conducted pull-down experiments with the cytosolic extract prepared from 3T3-L1 adipocytes, and analyzed bound material using Western blotting (Fig. 2B) and Ponceau Red staining (Fig. 2C). “Empty beads” and the peptide corresponding to the His tag alone served as controls. We have found that not only the full-length C-terminus of sortilin, but all the truncated peptides, including the shortest one (777–789) were fully capable of binding to Akt suggesting that the Akt-binding site is localized in the juxtamembrane region of sortilin somewhere between amino acids 777 and 789 (Figs. 2 B&D). Since one of the identified retromer-binding sites has also been mapped to this region (specifically - FLV, a.a. 785–787 [30]), it seems feasible that binding of Akt and retromer to sortilin is mutually exclusive. This may suggest that Akt specifically binds to sortilin localized in the TGN or IRVs, but not in endosomes where sortilin interacts with retromer [28]. This hypothesis requires further investigation.

Figure 2: Akt interacts with the juxtamembrane region of the sortilin cytoplasmic tail.

Panel A: Sequences of the peptide reproducing the full-length (FL) mouse sortilin cytoplasmic tail (SorC, GenBank accession number NM_019972) and truncated peptides used in this study. All peptides were tagged with 6xHis at the N-terminus. Panel B: Cytosolic lysates of differentiated 3T3-L1 adipocytes (4 mg of total protein) were used for pull-down experiments with peptides (10 nmols each) shown in panel A and immobilized on Ni-NTA Nickel beads. The eluates were analyzed by SDS electrophoresis in a 10% gel and immunoblotted with pan-Akt antibody. A representative result of 3 independent experiments is shown. Panel C: Same eluates were analyzed by electrophoresis in a 16% gel and Western blotting followed by Ponceau staining. Panel D: Quantification of results shown in panel B; mean values of three independent experiments ± standard deviation is shown. Student’s test was used to assess statistical significance as compared to SorC-FL. For SorC-789: p=0.064, SorC-794: p=0.14, SorC-815: p=0.092. Panel E: Recombinant Akt2 (250 ng) was used instead of cell lysates for pull down experiments with peptides shown in panel A immobilized on Ni-NTA Nickel beads. The eluates were analyzed by Coomassie Blue staining.

We have also analyzed the eluates from the pull-down experiments (Figs. 2B) using mass-spectrometry. Not only these results confirmed the data shown in Fig. 2B, but also showed the presence of both Akt1 and Akt2 in the eluates (Supplemental Table 1). Recently, Pallesen et al reported that PAK kinases 1–3 can also interact with the cytoplasmic tail of sortilin and their binding site partially overlaps with that of Akt [31]. Our mass-spectrometry analysis did not reveal any presence of PAK kinases in the eluates (not shown) suggesting that PAK kinases are not major sortilin-interacting proteins in adipocytes.

It is possible that Akt binds to the C-terminus of sortilin not directly but via some unidentified component(s). To exclude such a possibility, we repeated our experiments with recombinant Akt2 instead of the cytosolic extract. As is shown in Fig. 2E, recombinant Akt2 also binds to the juxtamembrane region of sortilin suggesting that the interaction is direct. Interestingly, Akt tends to better interact with truncated peptides, than with the full-length sortilin tail (Figs. 2 B,D&E). This may be explained by the 3D structure of the sortilin tail that may partially block Akt access to its juxtamembrane region.

Our observation that Akt may be recruited to the IRVs by sortilin supports the notion that sortilin plays a central role in insulin-stimulated glucose uptake [20, 23]. In agreement with this idea, it has been reported that expression of sortilin in cultured cells, experimental animals, and humans is decreased in insulin resistance and diabetes [32, 33]. Moreover, diabetogenic factors, such as saturated fatty acids, high fat diet, and obesity also decrease sortilin expression [32, 34–36]. In addition, inhibition of the PI3 kinase/Akt pathway causes degradation of the sortilin protein [37]. Importantly, several mutations in the Sort1 gene in humans have been linked strongly and specifically to type 2 diabetes, fasting glucose, etc. (https://t2d.hugeamp.org/gene.html?gene=SORT1).

We have shown previously that the IRVs are formed in the trans-Golgi network by self-assembly of Glut4, sortilin, IRAP, and LRP1 that interact with each other via luminal domains [6–8, 19–21, 28, 38, 39]. The heteromeric complex of the IRV proteins may bud from the TGN donor membranes as a single entity with the help of GGA adaptors that recognize a specific sequence DEDLL in the cytoplasmic tail of sortilin and recruit clathrin to budding vesicles [24]. Recruitment of clathrin bends the membrane eventually leading to vesicle severing [40–42]. In addition, Arf6 [43] and PIP4 (not shown) are likely to play important (but not totally clear) roles in vesicle biogenesis.

Upon formation of the IRVs, Akt and TBC1D4 bind to the cytoplasmic tails of sortilin (this report) and IRAP/LRP1 [8, 14, 15] correspondingly (Fig. 3A) which completes the formation of the insulin-responsive vesicular compartment. Under basal conditions, TBC1D4 maintains the intracellular localization of the IRVs by keeping its target Rab10 (which is also compartmentalized on the IRVs [14, 16, 18]) in the inactive GDP-bound conformation. Insulin stimulation leads to Akt-mediated phosphorylation of TBC1D4 that inhibits the GAP activity of TBC1D4 either directly [18] or by recruiting 14–3-3 [44]. In addition, phosphorylation of TBC1D4 may trigger its dissociation from the cytoplasmic tail of IRAP [14, 15, 45]. In any case, functional inactivation of TBC1D4 in the IRVs by Akt-mediated phosphorylation [13, 46] should increase the GTP load of Rab10 that allows for the recruitment of myosin Va [17] and translocation of the vesicles. Alternatively, Rab10 can activate RalA that stimulates translocation of the IRVs via the exocyst-mediated mechanism [18].

Figure 3. Self-assembly of the IRVs via luminal interactions creates a platform for insulin signaling to this compartment.

Panel A: Akt binds to the cytoplasmic tails of sortilin and, upon insulin stimulation, phosphorylates TBC1D4 associated with the cytoplasmic tails of IRAP and LRP1. See text for more details. Panel B, left panel: 3T3-L1 adipocytes stably expressing sortilin-myc/His (S+), SORTILINtail or wild type cells were treated or not treated with insulin for 15 min. Total cell lysates were analyzed by Western blotting in the same gel. Dotted lines indicate that irrelevant lanes have been spliced out. Intensity of p-TBC1D4 bands (shown in the last lane beneath insulin concentrations) was normalized by actin signals and the basal level of TBC1D4 phosphorylation in corresponding samples. The panel shows a typical result of three independent experiments. Panel B, right panel: total amounts of signaling proteins in cell lines. Panel C: Insulin-stimulated glucose uptake was measured in 3T3-L1 cells stably transfected with SORTILINtail and with EGFP as a control. Mean values of three independent experiments ± standard deviation is shown. P values were calculated by Student’s test.

How is IRV-bound Akt activated by insulin? Although the “classical” model of Akt activation postulates that it happens at the plasma membrane, recent reports have convincingly demonstrated that activation of Akt in response to insulin and growth factors takes place in the intracellular membranes as well [47, 48]. In fact, our earlier studies directly demonstrate that IRV-associated Akt is strongly activated by insulin [10]. Since the IRVs constantly communicate with intracellular membranes such as endosomes and TGN by budding and fusing [49], it is feasible that Akt activated in either endosomes or TGN is partitioned to the vesicles due to binding to sortilin.

According to our model shown in Fig. 3A, self-assembly of the IRVs creates a scaffold for insulin-stimulated phosphorylation of TBC1D4 by Akt. To test this idea, we used S+ adipocytes that stably over-express sortilin-myc/His [20]. In these cells, sortilin-myc/His is faithfully targeted to the IRVs [20, 21] thus significantly increasing insulin-stimulated translocation of Glut4 to the plasma membrane [23] and glucose uptake [20]. Interestingly, insulin-stimulated phosphorylation of TBC1D4 in these cells is elevated in spite of similar levels of Akt phosphorylation (Fig. 3B).

To confirm this result, we used cells stably transfected with SORTILINtail. In this construct, the luminal Vps10p domain of sortilin had been replaced with EGFP followed by two Flag epitopes. As we had shown previously, SORTILINtail is not targeted to the IRVs but is recovered in a different type of vesicular carriers that do not have insulin responsiveness [21]. Fig. 3B shows that insulin-stimulated phosphorylation of TBC1D4 in these cells is decreased in comparison to control adipocytes, and insulin-stimulated glucose uptake is significantly lower (Fig. 3C) while Glut4 levels stay the same (not shown). We attribute this effect to competition between the sortilin tale and the IRV-localized full-length sortilin for binding to Akt.

Last but not least, we would like to point out that activation of only 5–10% of total Akt is required for robust phosphorylation of TBC1D4 and translocation of Glut4 [50]. Correspondingly, attenuated insulin signaling may not account for reduced insulin-stimulated glucose uptake in insulin resistance and diabetes [51–53]. These results are difficult to explain if we assume that phosphorylated Akt is randomly “spread” throughout the whole cell volume. However, if Akt and TBC1D4 are specifically compartmentalized on the IRVs, insulin signal can reach this compartment in spite of a significant reduction in total Akt phosphorylation. By the same token, a decreased expression of sortilin may lead to insulin resistance [19, 20, 23, 36] not only because of impaired formation of the IRVs but also, due to reduced recruitment of Akt to this compartment. In any case, our findings may offer a new insight into the molecular nature of insulin action and insulin resistance.

List of Abbreviations:

Glut4

glucose transporter isoform 4

IRAP

insulin-responsive amino peptidase

IRVs

insulin-responsive vesicles

LRP1

low density lipoprotein receptor-related protein 1

GAP

GTPase activating protein

SCAMPs

secretory carrier associated membrane proteins

TBC1D4

Tre-2/BUB2/CDC16 domain family member 4

TUSC5

tumor suppressor candidate 5

VAMP2

vesicle associated membrane protein 2

DATA AVAILABILITY:

The data that support the findings of this study are available in Figs.1–3 and the supplementary material of this article