Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is expressed in retinal Müller glial cells (MGCs) and regulates intracellular translocation to the plasma membrane (PM) of the membrane proteins involved in cellular motility and activity. Different functions of MGCs may be influenced by insulin, including the removal of extracellular glutamate in the retina. In the present work, we investigated whether insulin promotes LRP1 translocation to the PM in the Müller glial-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line). We demonstrated that LRP1 is stored in small vesicles containing an approximate size of 100 nm (mean diameter range of 100–120 nm), which were positive for sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. Next, we observed that LRP1 translocation to the PM was promoted by insulin-regulated exocytosis through intracellular activation of the IR/PI3K/Akt axis and Rab-GTPase proteins such as Rab8A and Rab10. In addition, these Rab-GTPases regulated both the constitutive and insulin-induced LRP1 translocation to the PM. Finally, we found that dominant-negative Rab8A and Rab10 mutants impaired insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that these GTPase proteins as well as the LRP1 level at the cell surface are involved in insulin-induced IR activation.

Introduction

Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is a member of the LDL receptor gene family and is expressed in different types of cells, including macrophages, adipocytes, muscle cells, neurons, and retinal Müller glial cells (MGCs) [13]. LRP1 is a type I transmembrane protein formed as a non-covalently associated heterodimer containing an extracellular α-subunit of 515 kDa and a transmembrane and intracytoplasmic β-subunit of 85 kDa [4]. This receptor can bind and internalize by clathrin-mediated endocytosis more than 40 ligands such as the α2-macroglobulin-protease complex (α2M*), triglyceride-rich lipoprotein-derived Apolipoprotein E, and tissue-type plasminogen activator [5]. In addition to the endocytic activity of these ligands, LRP1 can induce intracellular signaling activation, which downstream mediates cellular proliferation and migration, cell differentiation, production of pro-inflammatory factors, and extracellular matrix (ECM) remodeling [610].

In the retina, MGCs stabilize the complex retinal architecture, providing structural and functional support to retinal neurons and blood vessels [11]. It is well recognized that MGCs undergo functional and phenotypic changes in proliferative diabetic retinopathy, sickle cell retinopathy, and proliferative vitreo-retinopathy, which involve cellular hypertrophy, process extension, migration, and proliferation, followed by ECM remodeling [3,1214]. Different functions of MGCs may be influenced by insulin, which is present in the retinal microenvironment [1517]. In addition, the insulin receptor (IR) is expressed in MGCs and the PI3K (phosphatidylinositol-4,5-bisphosphate 3-kinase)–Akt axis is activated in an insulin-dependent manner [15,18]. Although the insulin–IR function in MGCs is not clearly established, it plays a key role in the regulation of activity and content of glutamine synthetase, the principal enzyme involved in the conversion of the glutamate taken up from the extracellular space to glutamine [19].

LRP1 is functionally associated with the proteolytic activity of matrix metalloproteinase 2 (MMP-2) and MMP-9 in retinas during ischemia-induced neovascularization [2,20]. In addition, LRP1 mediates α2M*-induced cell motility of the MG-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line) by regulating matrix type-1 MMP (MT1-MMP) activity at the cell surface [14]. Several studies have demonstrated that LRP1 regulates intracellular translocation to the plasma membrane (PM) of different membrane proteins and receptors, including the urokinase receptor uPAR [21], β1-integrin [7,22], and MT1-MMP [14]. This generally involves endocytosis followed by recycling processes of LRP1 toward the cell surface in order to re-establish its level and function. However, it is not known how LRP1 is returned to the PM. This receptor is mostly stored in intracellular membrane vesicles and compartments (≈80%), with a minor proportion present in the PM (≈20%) [23,24]. Previous results suggest that LRP1 is recycled to the PM by the Rab4- and Rab11-dependent pathways in non-stimulated MIO-M1 cells. However, in the presence of α2M* LRP1 might be returned to the PM by other routes that are independent of Rab4 or Rab11 [14].

Many studies have demonstrated that insulin induces glucose transporter GLUT4 translocation to the PM in adipocytes and muscle cells through regulated exocytosis [2527]. In these cells, a significant proportion of GLUT4 is accumulated in specific membrane vesicles termed GSVs (GLUT4 storage vesicles), which also contain LRP1 and other membrane proteins such as IRAP, sortilin, and VAMP2 (vesicle-associated membrane protein 2) [28,29]. The traffic and fusion of GSVs to the PM involve the activation of the insulin-mediated IR/PI3K/Akt signaling pathway and a set of diverse Rab-GPTase proteins, mainly Rab8A, Rab10, and Rab13 [26,3032]. In addition, it has also been observed that insulin can stimulate LRP1 translocation to the cell surface of hepatocytes, although the mechanism involved with this translocation has not yet been characterized [33]. In contrast with adipocytes and muscle cells, GLUT4 is not expressed in hepatocytes, so we wondered if the regulated exocytosis is also involved during hepatic insulin-stimulated LRP1 translocation to the PM. Related to this, GLUT4 is not expressed in MGCs either (unpublished data; [34]), and considering that insulin and LRP1 are involved in physiological and pathological processes in the retina, in the present study we investigated whether insulin induces regulated exocytosis of LRP1 in MIO-M1 cells. Our results demonstrated that insulin promoted LRP1 translocation to the PM from small vesicles (≤100 nm) that stored LRP1. These LRP1 storage vesicles (LSVs) were composed of sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. In addition, we found that insulin-induced LRP1 translocation to the PM in MIO-M1 cells was mediated by intracellular activation of the IR/PI3K/Akt axis, Rab8A, and Rab10. These Rab-GTPases also mediated the constitutive return of LRP1 to the PM in non-stimulated MIO-M1 cells. Finally, dominant-negative Rab8A and Rab10 mutants impaired the insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that the LRP1 level at the cell surface is fundamental for insulin-induced IR activation.

Experimental

Cell cultures and reagents

A spontaneously immortalized human Müller cell line (MIO-M1) was kindly provided by Dr G. Astrid Limb (University College London, Institute of Ophthalmology and Moorfields Eye Hospital, London, U.K.). Cells were maintained in DMEM-high glucose (4.5 mg/ml) stabilized with 2 mM l-glutamine (GlutaMAX; Invitrogen, Buenos Aires, Argentina) and supplemented with 110 mg/ml sodium pyruvate, 10% (v/v) fetal calf serum (FCS) and 100 U/ml penicillin/streptomycin (Invitrogen) at 37°C with 5% CO2. Human insulin was from Apidra® Solostar® 100 U/ml (Sanofi-Aventis, Germany). Wortmannin, LY-294002 and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma–Aldrich. Rabbit anti-IR (cs4B8), rabbit anti-pIR (cs84B2), and rabbit anti-Akt (9272) antibodies were from Cell Signaling Technology (Beverly, MA) and rabbit anti-pAkt (07-789) antibody was from Merck KGaA (Darmstadt, Germany). Mouse monoclonal anti-β-actin (A2228), and rabbit anti-hemagglutinin (HA) (SAB1306169) antibodies were from Sigma–Aldrich (St. Louis, MO), with rabbit anti-LRP1 (ab92544), mouse monoclonal anti-LRP1 (ab28320), rabbit anti-sortilin (ab16640), rabbit anti-GLUT4 (ab654), rabbit anti-VAMP2 (ab3347), rabbit anti-EEA1 (ab2900), rabbit anti-Rab4 (ab13252), rabbit anti-Rab11 (ab65200), rabbit anti-TGN46 (ab50595), mouse monoclonal anti-Rab10 (ab104859), and mouse monoclonal anti-Rab8A (ab128022) antibodies being purchased from Abcam (Cambridge, MA). Mouse monoclonal anti-transferrin receptor (TfR) (B6780) antibody was obtained from Abnova (Walnut, CA). Immunofluorescences were performed with the secondary antibodies raised in goat anti-rabbit IgG conjugated with Alexa Fluor 647, 594, or 488, and anti-mouse IgG conjugated with Alexa Fluor 594 or 488 (diluted 1/800) (Invitrogen, Buenos Aires, Argentina).

Transfection procedures

The green fluorescent protein (GFP)-GLUT4 construct as well as GFP-wild-type Rab8A (wt-Rab8A), GFP-dominant-negative (GDP-bound) Rab8AT22N (dn-Rab8A), GFP-wild-type Rab10 (wt-Rab10), and GFP-dominant-negative (GDP-bound) Rab10T23N (dn-Rab10) were kindly provided by Dr Amira Klip (The Hospital for Sick Children, Toronto, Canada). GFP-wild-type Rab11 (wt-Rab11) and GFP-dominant-negative (GDP-bound) Rab11S25N (dn-Rab11) were kindly donated by Dr María Isabel Colombo (Instituto de Histología y Embriología, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, Mendoza, Argentina). The construction of the membrane-containing mini-receptor of LRP1 (mLRP4) by PCR was performed essentially as described previously [35]. The final construction cloned in a pcDNA3.0 plasmid was composed of the LRP1 signal peptide (SP), HA epitope and GFP in their N-termini followed by the fourth ligand binding, transmembrane and cytoplasmic domains of LRP1 (SP-HA-GFP-mLRP4; Figure 3A). MIO-M1 cells (4 × 105 cells/well) were cultured in 6-well plates and transiently transfected with 2 µg/well of plasmids for 4 h, using Lipofectamine 2000 (Invitrogen) and Opti-MEM 1X (Gibco®, Thermo Fischer Scientific, Buenos Aires, Argentina) following the manufacturer's instructions. Then, these cells were washed and incubated for 18 h in DMEM-high glucose at 37°C with 5% CO2. siRNA-Rab8A was purchased from Ambion (Austin, TX, U.S.A.), while siRNA-Rab10 was obtained from Sigma–Aldrich. siRNA-Rab8A (4390824) was a 21-mer (5′-GCAAGAGAAUUAAACUGCA [dT][dT]-3′) and siRNA-Rab10 (Sigma # SASI _Hs02_000348924) was a 21-mer (5′-GCAAAUGGCUUAGAAACAU[dT][dT]-3′). For the silencing of these GTPase proteins, MIO-M1 cells (4 × 105 cells/well) were cultured in 96-well plates and transiently transfected with 5 pmol/well of siRNA for 24 h, using Lipofectamine RNAiMAX reagent (Invitrogen) and Opti-MEM 1X (Gibco®, Thermo Fischer Scientific, Buenos Aires, Argentina) following the manufacturer's instructions. To control the effect of the silencing, the Silencer™ select negative control siRNA (4390846) was used.

Western blot analysis

MIO-M1 cells were cultured in 6-well plates at 37°C for 24 h in DMEM-high glucose containing 10% FCS and 2 mM l-glutamine with 5% CO2. After fasting for 30 min, cells were treated with 100 nM insulin or 1 µM PMA for different time periods, and cell protein extracts were prepared using RIPA lysis buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Tritón X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mM sodium ortho-vanadate and protease inhibitor cocktails (Sigma–Aldrich)]. Forty micrograms of lysate were diluted in sample buffer 5× with DTT (dithiothreitol) and then heated for 5 min at 95°C. Electrophoresis on 10% SDS–polyacrylamide gels was applied and the proteins were electrotransferred to a nitrocellulose membrane (GE Healthcare Life Science, Amsterdam). Non-specific binding was blocked with 5% non-fat dry milk in a Tris–HCl saline buffer containing 0.01% Tween 20 (TBS-T) for 60 min at room temperature. The membranes were incubated overnight at 4°C with diluted primary antibodies and secondary antibodies raised in goat anti-mouse IgG IRDye® 680CW and goat anti-rabbit IgG IRDye® 800CW (LI-COR Biosciences, Lincoln, NE) diluted 1/10 000 for 1 h at room temperature. The specific bands were revealed by the Odyssey CLx near-infrared fluorescence imaging system (LI-COR) and were quantified by densitometric analysis using Image Studio Software (LI-COR).

Immunoprecipitation assays

MIO-M1 cells were cultured as above and treated with 100 nM insulin for 10 min, and cell protein extracts were prepared using RIPA lysis buffer. Protein extracts were incubated for 2 h at 4°C with rabbit anti-IR antibody or rabbit non-immune IgG as IP control (2 µg/200 µg of total proteins). Then, these were incubated overnight at 4°C with protein A-conjugated agarose beads following the manufacturer's procedure (sc-2001; Santa Cruz Biotechnology, CA), and the proteins were separated and treated for Western blot analysis using rabbit anti-IR (1/1000) and rabbit anti-p-IR (1/500) antibodies.

Biotin-labeling cell surface protein assay

MIO-M1 cells were cultured as above and treated with 100 nM insulin for different time periods. To determine the level of LRP1 at the cell surface, a biotin-labeling protein assay (EZ-Link™ Sulfo-NHS-SS-Biotin [cat: 21331], Thermo Scientific, Rockford, IL) was used following the manufacturer's procedure. Briefly, cells were incubated for 1 h at 4°C with a 0.12 mg/ml biotin solution, followed by incubation with 0.1 mM glycine solution for 30 min and washing with PBS three times to remove the excess biotin. Then, the biotin LRP1 was pulled down using streptavidin-conjugated agarose beads (Pierce™ Streptavidin Agarose [cat: 20353], Thermo Scientific) for 2 h at room temperature, and the proteins were treated for Western blot analysis using rabbit anti-LRP1 (1/10 000), rabbit anti-sortilin (1/2000), mouse monoclonal anti-TfR (1/1500) or mouse monoclonal anti-β-actin (1/5000) antibodies.

Cell surface protein detection assay

MIO-M1 cells were cultured in 96-well plates (6 × 103 cells/well) at 37°C for 24 h in DMEM-high glucose containing 10% FCS and 2 mM l-glutamine with 5% CO2. After fasting for 30 min, cells were treated with 100 nM insulin for different time periods, rinsed with cold PBS, fixed with 4% (v/v) paraformaldehyde (PFA), washed with 0.1 mM glycine and blocked with 5% (v/v) horse serum for 30 min on ice. Cells were incubated with polyclonal rabbit anti-ectodomain LRP1 β-subunit antibody (1/1000) for 1 h on ice, followed by three washes of 5 min each with ice-cold PBS before being incubated again with goat anti-rabbit IgG IRDye® 800CW (LI-COR) secondary antibody (1/10 000) for 1 h on ice. After three washes with ice-cold PBS, the resulting fluorescence was measured using the Odyssey CLx near-infrared fluorescence imaging system and quantified by densitometric analysis using Image Studio Software. For some assays, the cells were pre-incubated 30 min with 40 µM wortmannin or LY-294002. Similar experiments were carried out with MIO-M1 cells transfected with the mLRP4-GFP-HA plasmid and polyclonal rabbit anti-HA-tag antibody (1/1000).

Confocal microscopy

MIO-M1 cells were cultured as above on glass coverslips and treated with or without 100 nM insulin for 30 min. Cells were washed with PBS, fixed with 4% PFA, quenched with 50 mM NH4Cl, permeabilized for 30 min with 0.5% (v/v) saponin, blocked with 2% BSA, incubated with the respective primary antibody (diluted from 1/100 to 1/250) for 1 h, and revealed with a secondary antibody conjugated with Alexa Fluor (1/800) for 1 h. Finally, the cells were mounted on glass slides with Mowiol 4–88 reagent from Calbiochem (Merck KGaA, Darmstadt, Germany). For co-localization analyses between different cell proteins and LRP1, fluorescent images were obtained with an Olympus FluoView FV1200 confocal laser scanning biological microscope (Olympus, New York, NY). Whole cells were scanned and optical sections were obtained in 0.25-µm steps perpendicular to the z-axis, with images being processed using the FV10-ASW Viewer 3.1 (Olympus) and quantified by the ImageJ software. For mLRP4-GFP-HA detection at the cell surface, a rabbit anti-HA tag (1/100) antibody was used and revealed with Alexa 594-conjugated anti-rabbit secondary antibody (1/800). All fluorescent images were processed and quantified as described.

Immunoelectron microscopy

MIO-M1 cells overexpressing GFP-GLUT4 were incubated for 30 min in the presence or absence of 100 nM insulin. Cells were fixed in 4% PFA in 0.2 M phosphate buffer, pH 7.4 (PB) for 2 h at 4°C. After washing with PB alone and PB containing 50 mM glycine, cells were embedded in 10% gelatin and small blocks were infiltrated with 2.3 M sucrose at 4°C for 2 h and then frozen in liquid nitrogen. Ultrathin cryosections were prepared using a Leica Ultracut R and retrieved with a mixture of 2% methylcellulose and 2.3 M sucrose (vol/vol). Sections of 60 nm were labeled with rabbit anti-LRP1, followed by labeling with 15 nm gold particles coupled with protein A to detect rabbit antibodies. Subsequently, cells were incubated with a rabbit anti-sortilin, which was detected with 5 nm gold particles coupled with protein A. Finally, the samples were incubated with rabbit anti-GLUT4, which was detected with 10 nm gold particles coupled with protein A [different sizes of gold particles coupled with protein A were kindly provided by Dr Gareth Griffith, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany]. To fix the immunocomplex and to avoid a possible cross-reaction, 1% glutaraldehyde was added at the end of each labeling procedure. The sections were then contrasted and embedded in a mixture of methylcellulose and uranyl acetate, and viewed under a Zeiss 900 electron microscope.

Quantitative analysis of immunolabeled vesicle sizes

The size of GLUT4/LRP1/sortilin or LRP1/sortilin/VAMP2 positive vesicles in GLUT4-transfected or non-transfected cells, obtained by immunoelectron microscopy, was quantified using the Gatan Inc. Digital Micrograph 2.31.734 software. At least 50 triple-labeled vesicles were analyzed and the average of the major and minor diameter of each vesicle was calculated. Then, vesicles were grouped by size to construct the plot.

Statistical treatment of data

For microscope quantifications of the co-localization level, a JACoP plug-in from the ImageJ software was used [36]. At least 50 cells/condition were analyzed, and the averages of the vesicle percentages containing both proteins were calculated using the Manders' coefficients and compared by Student's t-test. Values of P < 0.05 were considered to be significant. For the Western blot and the cell surface protein detection assay, the data were expressed as mean ± SEM, and a one-way ANOVA test and Student's t-test were used for statistical analysis (GraphPad Prism 5.0). Values of P < 0.05 were considered significant.

Results

Insulin increased the LRP1 level on the cell surface in MIO-M1 cells

The insulin effect on the intracellular signaling activation in MIO-M1 cells was analyzed by Western blot assays. These cells constitutively expressed both IR and LRP1 (Supplementary Figure S1). Increased levels of phosphorylated IR (p-IR) and Akt (p-Akt) were observed in cells stimulated with insulin for different time periods (Figure 1). To investigate whether insulin would increase the LRP1 level on the PM of the MIO-M1 cells, biotin-labeling cell surface protein assays were carried out. Figure 2A,B shows that insulin significantly increased the biotin-labeled LRP1 at 10, 30, and 60 min of stimulation. Similar results were obtained when the LRP1 level on the PM was also analyzed by a cell surface protein detection assay in non-permeabilized MIO-M1 cells (Figure 2C). Thus, our data demonstrate that insulin effectively increased the LRP1 level at the cell surface of MIO-M1 cells.

Insulin induces intracellular signaling activation in MIO-M1 cells.

Figure 1.
Insulin induces intracellular signaling activation in MIO-M1 cells.

(A) Immunoprecipitation assay for IR followed by immunoblotting for phosphorylated IR (p-IR) and total IR. Cells were cultured in the presence or absence of insulin (100 nM) for 10 min at 37°C. Ig denotes the isotype immunoglobulin control. (B) Western blot assay for the analysis of phosphorylated Akt (p-Akt) in cells treated with insulin for 5 and 10 min, and with PMA (1 µM) for 10 min at 37°C. β-actin was used as loading control. (C) Densitometric quantification of the Western blot data (n = 4–5 for each group). Values are expressed as mean ± SEM. **P < 0.01 vs. non-stimulated control at the time indicated, ***P < 0.0001 vs. non-stimulated control at the time indicated. Details of experiments are indicated in Materials and Methods.

Figure 1.
Insulin induces intracellular signaling activation in MIO-M1 cells.

(A) Immunoprecipitation assay for IR followed by immunoblotting for phosphorylated IR (p-IR) and total IR. Cells were cultured in the presence or absence of insulin (100 nM) for 10 min at 37°C. Ig denotes the isotype immunoglobulin control. (B) Western blot assay for the analysis of phosphorylated Akt (p-Akt) in cells treated with insulin for 5 and 10 min, and with PMA (1 µM) for 10 min at 37°C. β-actin was used as loading control. (C) Densitometric quantification of the Western blot data (n = 4–5 for each group). Values are expressed as mean ± SEM. **P < 0.01 vs. non-stimulated control at the time indicated, ***P < 0.0001 vs. non-stimulated control at the time indicated. Details of experiments are indicated in Materials and Methods.

Insulin increases the LRP1 level at the cell surface of MIO-M1 cells.

Figure 2.
Insulin increases the LRP1 level at the cell surface of MIO-M1 cells.

(A) Biotin-labeling cell surface assays to measure the LRP1 level in the PM of cells stimulated with insulin (100 nM) for different time periods at 37°C. Biotin-labeled proteins were isolated with streptavidin-conjugated beads and then analyzed by Western blot for LRP1 and β-actin (protein loading control). Extract proteins from cell lysates were also analyzed by Western blot for LRP1 and β-actin. (B) Densitometric quantification of Western blot data for surface biotin-LRP1 related to biotin-β-actin expressed as the fold change with respect to non-stimulated control (t = 0 min; white bar). Three independent experiments were performed in triplicate. Values are expressed as mean ± SEM. *P < 0.05 vs. non-stimulated control; **P < 0.01 vs. non-stimulated control. (C) Cell surface protein detection assay to measure LRP1 levels in MIO-M1 cells stimulated with insulin (100 nM) for different time periods at 37°C. The cell surface level of LRP1 was analyzed in non-permeabilized cells using anti-LRP1. Results are expressed as the fold change of the signal under an insulin-stimulated condition with respect to non-stimulated control. Four independent experiments were performed in triplicate. Values are expressed as mean ± SEM. *P < 0.05 vs. non-stimulated control. All details of experiments are indicated in the Experimental section.

Figure 2.
Insulin increases the LRP1 level at the cell surface of MIO-M1 cells.

(A) Biotin-labeling cell surface assays to measure the LRP1 level in the PM of cells stimulated with insulin (100 nM) for different time periods at 37°C. Biotin-labeled proteins were isolated with streptavidin-conjugated beads and then analyzed by Western blot for LRP1 and β-actin (protein loading control). Extract proteins from cell lysates were also analyzed by Western blot for LRP1 and β-actin. (B) Densitometric quantification of Western blot data for surface biotin-LRP1 related to biotin-β-actin expressed as the fold change with respect to non-stimulated control (t = 0 min; white bar). Three independent experiments were performed in triplicate. Values are expressed as mean ± SEM. *P < 0.05 vs. non-stimulated control; **P < 0.01 vs. non-stimulated control. (C) Cell surface protein detection assay to measure LRP1 levels in MIO-M1 cells stimulated with insulin (100 nM) for different time periods at 37°C. The cell surface level of LRP1 was analyzed in non-permeabilized cells using anti-LRP1. Results are expressed as the fold change of the signal under an insulin-stimulated condition with respect to non-stimulated control. Four independent experiments were performed in triplicate. Values are expressed as mean ± SEM. *P < 0.05 vs. non-stimulated control. All details of experiments are indicated in the Experimental section.

Insulin-induced LRP1 translocation to the PM through IR/PI3K/Akt axis activation

To demonstrate whether LRP1 is translocated to the PM from intracellular compartments by an insulin effect, MIO-M1 cells were transiently transfected with an expression vector encoding for a mini-receptor version of LRP1, termed mLRP4-GFP-HA (Figure 3A). This mini-receptor contains the fourth ligand-binding domain of LRP1 and is capable of binding several LRP1 ligands with endocytic properties identical with full-length LRP1 [37]. In addition, it has been previously demonstrated that mLRP4 is expressed at the cell surface after its synthesis [38] and contains cytoplasmic sequences responsible for its targeting and sorting to dendritic regions in neurons [39,40]. In our study, representative images of confocal microscopy revealed that mLRP4-GFP-HA was perinuclearly localized in punctate vesicles in MIO-M1 cells (Figure 3B), thereby representing a similar subcellular distribution to constitutive LRP1 in this type of cells [14]. To examine the cell surface level of mLRP4-GFP-HA, the ectodomain HA-tag was detected using a monoclonal anti-HA antibody in non-permeabilized cells and visualized by confocal microscopy. Figure 3C shows that insulin increased the mLRP4-GFP-HA level in the PM of MIO-M1 cells after 30 min compared with non-stimulated cells. In addition, similar results were obtained when the HA level on the PM was quantified by the cell surface protein detection assay in mLRP4-GFP-HA-transfected cells, showing that the mini-receptor levels were significantly increased in the PM after 15 min of insulin stimulus (Figure 3D). Finally, this insulin effect was blocked by wortmannin (Figure 3D) or LY-294002 (Supplementary Figure S1), both PI3K inhibitors, indicating that insulin induces LRP1 translocation to the PM through IR/PI3K/Akt axis activation.

Insulin increases the LRP1 mini-receptor level at the cell surface of MIO-M1 cells.

Figure 3.
Insulin increases the LRP1 mini-receptor level at the cell surface of MIO-M1 cells.

(A) MIO-M1 cells were transfected with a pcDNA3.0 plasmid encoding for mLRP4-GFP-HA, as described in the Experimental section. SP, signal peptide; HA, hemagglutinin tag; GFP, green fluorescence protein; IV, fourth ligand-binding domain of α-subunit; and NPXY, [Asn, Pro, X residue, and Tyr] motif. (B) Confocal microscopy of mLRP4-GFP-HA-transfected cells cultured in the absence (control) or presence of insulin (100 nM) for 30 min at 37°C. Representative images of GFP signal for mLRP4-GFP-HA (Total) and immunostaining for extracellular HA in non-permeabilized cells (Surface). The Merge image shows extracellular HA-tag in red and total mLRP4-GFP-HA in green. The Mask image represents the binary images of total mLRP4-GFP-HA and extracellular HA tag co-localizations. Scale bars = 10 µm. (C) Quantification of HA-tag levels of mLRP4-GFP-HA expressed at the PM of MIO-M1 cells treated with insulin (100 nM) for 30 min at 37°C with respect to non-stimulated cells (control). Three independent experiments were performed in triplicate from the analysis of 20 cells per experiment. Values are expressed as mean ± SEM. **P < 0.01 vs. control. (D) Cell surface protein detection assay to measure HA-tag cell surface (white bars) in HA-GFP-mLRP4-transfected MIO-M1 cells cultured in the presence of insulin (100 nM) for different time periods at 37°C or pretreated with wortmannin (40 µM) for 30 min (black bars) and then stimulated with insulin for different time periods. The cell surface level of the HA-tag was analyzed in non-permeabilized cells using anti-HA antibody. Results are expressed as the fold change of the HA-tag signal under the insulin-stimulated condition with respect to control. Four independent experiments were performed in triplicate. Values are expressed as mean ± SEM. *P < 0.05 vs. control; #statistical significance (P < 0.05) between wortmannin-treated and untreated cells for the times indicated. All details of experiments are indicated in the Experimental section.

Figure 3.
Insulin increases the LRP1 mini-receptor level at the cell surface of MIO-M1 cells.

(A) MIO-M1 cells were transfected with a pcDNA3.0 plasmid encoding for mLRP4-GFP-HA, as described in the Experimental section. SP, signal peptide; HA, hemagglutinin tag; GFP, green fluorescence protein; IV, fourth ligand-binding domain of α-subunit; and NPXY, [Asn, Pro, X residue, and Tyr] motif. (B) Confocal microscopy of mLRP4-GFP-HA-transfected cells cultured in the absence (control) or presence of insulin (100 nM) for 30 min at 37°C. Representative images of GFP signal for mLRP4-GFP-HA (Total) and immunostaining for extracellular HA in non-permeabilized cells (Surface). The Merge image shows extracellular HA-tag in red and total mLRP4-GFP-HA in green. The Mask image represents the binary images of total mLRP4-GFP-HA and extracellular HA tag co-localizations. Scale bars = 10 µm. (C) Quantification of HA-tag levels of mLRP4-GFP-HA expressed at the PM of MIO-M1 cells treated with insulin (100 nM) for 30 min at 37°C with respect to non-stimulated cells (control). Three independent experiments were performed in triplicate from the analysis of 20 cells per experiment. Values are expressed as mean ± SEM. **P < 0.01 vs. control. (D) Cell surface protein detection assay to measure HA-tag cell surface (white bars) in HA-GFP-mLRP4-transfected MIO-M1 cells cultured in the presence of insulin (100 nM) for different time periods at 37°C or pretreated with wortmannin (40 µM) for 30 min (black bars) and then stimulated with insulin for different time periods. The cell surface level of the HA-tag was analyzed in non-permeabilized cells using anti-HA antibody. Results are expressed as the fold change of the HA-tag signal under the insulin-stimulated condition with respect to control. Four independent experiments were performed in triplicate. Values are expressed as mean ± SEM. *P < 0.05 vs. control; #statistical significance (P < 0.05) between wortmannin-treated and untreated cells for the times indicated. All details of experiments are indicated in the Experimental section.

LRP1 is stored in small insulin-responsive vesicles

In adipocytes and muscle cells, LRP1 and GLUT4 are co-localized in GSVs, which also store other protein membranes such as sortilin and VAMP2 [28]. However, GLUT4 is not expressed in the MGCs of the mammalian retina [34] nor in MIO-M1 cells (Supplementary Figure S1). Our hypothesis is that the MIO-M1 cells contain vesicle structures similar to GSVs, which, due to the ectopical expression of GLUT4, may be localized in these vesicles together with LRP1, sortilin and VAMP2. To evaluate this possibility, MIO-M1 cells were transiently transfected with an expression vector encoding for GLUT4-GFP, and the intracellular distribution of this protein together with LRP1 in different compartments was analyzed by confocal microscopy. In non-stimulated cells, LRP1 and GLUT4-GFP were co-localized in early endosomes (EEA1+), recycling compartments (Rab4+ and Rab11+) and the trans-Golgi network (TGN46+) (Figure 4A), which were not significantly modified in cells stimulated with insulin for 30 min (data not shown). Then, we investigated the co-localization of LRP1 with GLUT4-GFP and sortilin, and found that these three proteins were perinuclearly co-localized in punctate vesicles in non-stimulated cells (Figure 4B). In MIO-M1 cells stimulated with insulin for 30 min, the intracellular distribution of LRP1, GLUT4 and sortilin revealed a peripheral localization with an increased co-localization compared with the non-stimulated condition (≈21% vs. ≈12%; Figure 4B). Similar results were obtained for the distribution analysis of LRP1, GLUT4, and VAMP2 (Supplementary Figure S2). To evaluate the cellular ultrastructure of vesicles storing LRP1, cryoimmunoelectron microscopy was used in MIO-M1 cells transiently transfected with GLUT4 (Figure 5 and Supplementary Figure S3). Figure 5A shows small vesicles containing both LRP1 and sortilin, which were identified at the intracellular level in non-stimulated cells. Gold-labeled immunostaining for LRP1 was detected in the vesicle lumen, whereas sortilin was immunodetected on the outside of these small vesicles, which is in agreement with the LRP1 β-subunit ectodomain and sortilin C-termini epitopes recognized for each specific primary antibody. These small vesicles also showed a surrounding electron-dense zone compatible with clathrin covers and were positive for GLUT4, which was also localized in the vesicle lumen as in the case of LRP1. In insulin-stimulated cells, these small vesicles were positive for LRP1, sortilin and GLUT4, and preferentially identified near to the PM (Figure 5B), with their number per cell being significantly higher than in non-stimulated cells (Figure 5C). Under this condition, LRP1 storage small vesicles were clearly differentiated from the endocytic processes for this receptor, which occurred in the proximity of the cellular periphery (Supplementary Figure S3). Quantitative analysis of vesicle sizes determined that ∼80% had a mean diameter range of 100–120 nm (Figure 5D). Moreover, these small vesicles positive for LRP1, VAMP2, and sortilin were also characterized by specific gold-labeled immunodetection in GLUT4 non-transfected cells (Supplementary Figure S4). These data indicate that LRP1 is stored in small vesicles in MIO-M1 cells with GSV-like characteristics.

Subcellular analysis of LRP1 in MIO-M1 cells stimulated with insulin.

Figure 4.
Subcellular analysis of LRP1 in MIO-M1 cells stimulated with insulin.

(A) Confocal microscopy of GFP-GLUT4-transfected cells without insulin stimulation and immunodetected for LRP1 and EEA1 (first row), Rab4 (second row), Rab11 (third row), and TGN46 (fourth row). Mask is the binary image of the triple co-localization between all proteins mentioned and their respective quantitative analysis expressed as mean ± SEM. (B) Confocal microscopy of GFP-GLUT4-transfected cells (green) cultured in the absence or presence of insulin (100 nM) followed by immunostaining for LRP1 (red) and sortilin (blue). Mask is the binary images of the triple co-localization of GFP-GLUT4/LRP1/sortilin with their respective quantifications expressed as mean ± SEM. Scale bars = 10 µm. All details of experiments are indicated in the Experimental section.

Figure 4.
Subcellular analysis of LRP1 in MIO-M1 cells stimulated with insulin.

(A) Confocal microscopy of GFP-GLUT4-transfected cells without insulin stimulation and immunodetected for LRP1 and EEA1 (first row), Rab4 (second row), Rab11 (third row), and TGN46 (fourth row). Mask is the binary image of the triple co-localization between all proteins mentioned and their respective quantitative analysis expressed as mean ± SEM. (B) Confocal microscopy of GFP-GLUT4-transfected cells (green) cultured in the absence or presence of insulin (100 nM) followed by immunostaining for LRP1 (red) and sortilin (blue). Mask is the binary images of the triple co-localization of GFP-GLUT4/LRP1/sortilin with their respective quantifications expressed as mean ± SEM. Scale bars = 10 µm. All details of experiments are indicated in the Experimental section.

Insulin increases the number of LRP1, sortilin, and GLUT4 positive vesicles at the cell periphery in MIO-M1 cells.

Figure 5.
Insulin increases the number of LRP1, sortilin, and GLUT4 positive vesicles at the cell periphery in MIO-M1 cells.

MIO-M1 cells overexpressing GFP-GLUT4 were incubated in the absence (control) (A) or in the presence of 100 nM insulin (B) for 30 min and analyzed by cryoimmunoelectron microscopy. Ultrathin sections were triple-labeled with rabbit anti-sortilin, rabbit anti-GLUT4 and rabbit anti-LRP1 antibodies. These rabbit antibodies were detected with protein A coupled to 5, 10, or 15 nm gold particles, respectively (see Experimental section). Arrows: gold particles (protein A gold, 5 nm) detecting sortilin. Black arrowheads: gold particles (protein A gold, 10 nm) detecting GFP-GLUT4. Empty arrowheads: gold particles (protein A gold, 15 nm) detecting LRP1. The boxed regions are shown at a higher magnification. Scale bars = 100 nm. (C) The number of LRP1, sortilin and GLUT4 positive vesicles at the cell periphery per cell was quantified from images such as those displayed in (A) and (B). Values are expressed as mean ± SEM. **P < 0.01 vs. control. (D) Percentages of vesicle sizes quantified as described in the Experimental section.

Figure 5.
Insulin increases the number of LRP1, sortilin, and GLUT4 positive vesicles at the cell periphery in MIO-M1 cells.

MIO-M1 cells overexpressing GFP-GLUT4 were incubated in the absence (control) (A) or in the presence of 100 nM insulin (B) for 30 min and analyzed by cryoimmunoelectron microscopy. Ultrathin sections were triple-labeled with rabbit anti-sortilin, rabbit anti-GLUT4 and rabbit anti-LRP1 antibodies. These rabbit antibodies were detected with protein A coupled to 5, 10, or 15 nm gold particles, respectively (see Experimental section). Arrows: gold particles (protein A gold, 5 nm) detecting sortilin. Black arrowheads: gold particles (protein A gold, 10 nm) detecting GFP-GLUT4. Empty arrowheads: gold particles (protein A gold, 15 nm) detecting LRP1. The boxed regions are shown at a higher magnification. Scale bars = 100 nm. (C) The number of LRP1, sortilin and GLUT4 positive vesicles at the cell periphery per cell was quantified from images such as those displayed in (A) and (B). Values are expressed as mean ± SEM. **P < 0.01 vs. control. (D) Percentages of vesicle sizes quantified as described in the Experimental section.

Insulin-induced LRP1 translocation to the PM is mediated by Rab8A and Rab10-dependent exocytosis

Several Rab-GTPases are involved in insulin-induced vesicular traffic to the PM by intracellular activation of the IR/PI3K/Akt axis [25,28]. In this way, both Rab8A and Rab10 play key roles during insulin-dependent GLUT4 translocation from GSVs to the cell surface in muscle cells and adipocytes [26,41]. In a previous report, we observed that Rab11 is not involved with the LRP1 return to the cell surface in MIO-M1 cells [14]. Hence, we evaluated whether Rab8A and Rab10 were associated with insulin-dependent LRP1 translocation to the PM in MIO-M1 cells. Figure 6A shows an immunostaining assay for LRP1 and Rab8A, where an evident perinuclear co-localization in punctate vesicles can be observed. In addition, a significant level of co-localization between LRP1 and Rab10 is also visible (Figure 6B). Quantitative analysis revealed that co-localization levels between LRP1 and Rab8A (30.5 ± 2.3%) were higher than those between LRP1 and Rab10 (17.6 ± 3.1%). To examine Rab8A and Rab10 co-localization, MIO-M1 cells were transiently transfected with GFP-wild-type Rab8A (wt-Rab8A) and immunostained for Rab10. Figure 6C shows that Rab10 co-localized with Rab8A, and quantitative analysis showed that the co-localization levels were ∼20%. Similar results were obtained when cells were transiently transfected with GFP-wild-type Rab10 (wt-Rab10) and immunostained for Rab8A (data not shown). To investigate whether both Rab-GTPases were involved in LRP1 translocation to the PM, MIO-M1 cells were transiently transfected with wt-Rab8A, dominant-negative Rab8AT22N (dn-Rab8A), wt-Rab10 and dominant-negative Rab10T23N (dn-Rab10) plasmids. Flow cytometry quantification determined that ∼65% of MIO-M1 cells were transfected with each plasmid (data not shown). Next, after 30 min of insulin stimulation the biotin-labeling cell surface protein assay was carried out. Figure 7A,C shows that the LRP1 level in the PM was significantly decreased in non-stimulated dn-Rab8A-transfected cells with respect to non-transfected cells. Under insulin stimulus, LRP1 was slightly increased in the PM in dn-Rab8A-transfected cells. To avoid off-target effects of this construct, MIO-M1 cells were treated with specific siRNA for Rab8A, which affected LRP1 translocation to the PM in both non-stimulated and insulin-stimulated cells (Supplementary Figure S5). In contrast, the wt-Rab8A-transfected cells had increased LRP1 levels in the PM compared with control cells. When the Rab10 effect was analyzed, we found that, in both non-stimulated and insulin-stimulated dn-Rab10-transfected cells, there were significant decreases of LRP1 in the PM compared with non-stimulated control cells (Figure 7B,D). Similar results were obtained when MIO-M1 cells were pretreated with specific siRNA for Rab10 (Supplementary Figure S5). Moreover, non-stimulated wt-Rab10-transfected cells had an increased LRP1 level at the cell surface. In control experiments, sortilin showed a similar pattern of expression in the PM to that of LRP1, whereas the TfR, a well-established Rab-11-dependent endocytic recycling receptor, did not show any substantial changes at the PM level in dn-Rab8A- or dn-Rab10-transfected cells (Supplementary Figure S6). These data demonstrate that Rab8A and Rab10 are involved in maintaining a steady state of LRP1 at the cell surface, as well as in the insulin-regulated exocytosis of this receptor in MIO-M1 cells.

Rab8A and Rab10 colocalize with LRP1 in MIO-M1 cells.

Figure 6.
Rab8A and Rab10 colocalize with LRP1 in MIO-M1 cells.

Representative confocal microscopic images of immunostaining for LRP1 and Rab8A (A) or Rab10 (B). Overlaid images are magnifications of co-localization between LRP1/Rab8A and LRP1/Rab10 from merge images (boxes). Mask is the binary image of LRP1/Rab8A and LRP1/Rab10 co-localizations together with the corresponding quantification expressed as mean ± SEM. Scale bars = 10 µm. (C) Confocal microscopic images of GFP-wt-Rab8A-transfected cells and immunodetected for Rab10. Overlaid images are magnifications of the co-localization between GFP-wt-Rab8A and Rab10 from merge images (boxes). Mask is the binary image of the co-localization of GFP-wt-Rab8A and Rab10 with their respective quantifications expressed as mean ± SEM. Scale bars = 10 µm. All details of experiments are indicated in the Experimental section.

Figure 6.
Rab8A and Rab10 colocalize with LRP1 in MIO-M1 cells.

Representative confocal microscopic images of immunostaining for LRP1 and Rab8A (A) or Rab10 (B). Overlaid images are magnifications of co-localization between LRP1/Rab8A and LRP1/Rab10 from merge images (boxes). Mask is the binary image of LRP1/Rab8A and LRP1/Rab10 co-localizations together with the corresponding quantification expressed as mean ± SEM. Scale bars = 10 µm. (C) Confocal microscopic images of GFP-wt-Rab8A-transfected cells and immunodetected for Rab10. Overlaid images are magnifications of the co-localization between GFP-wt-Rab8A and Rab10 from merge images (boxes). Mask is the binary image of the co-localization of GFP-wt-Rab8A and Rab10 with their respective quantifications expressed as mean ± SEM. Scale bars = 10 µm. All details of experiments are indicated in the Experimental section.

Rab 8A and Rab10 regulate the LRP1 translocation to the PM in MIO-M1 cells.

Figure 7.
Rab 8A and Rab10 regulate the LRP1 translocation to the PM in MIO-M1 cells.

Biotin-labeling cell surface protein assay in cells transiently transfected and expressing wt-Rab8A and dn-Rab8A (A) and wt-Rab10 and dn-Rab10 (B) stimulated with insulin (100 nM) for 30 min at 37°C. In both experiments, biotin-labeled proteins were isolated with streptavidin-conjugated beads and then analyzed by Western blot for LRP1 and β-actin (protein loading control). Extract proteins from cell lysates were also analyzed by Western blot for LRP1 and β-actin. (C and D) Densitometric quantification of the Western blot data for surface biotin-LRP1 related to biotin-β-actin was expressed as the fold change with respect to control (t = 0 min; white bar). Three independent experiments were performed in triplicate. Values are expressed as mean ± SEM. **P < 0.01 vs. control, ***P < 0.0001 vs. control, #P < 0.05 vs. control. (E) Quantitative analyses of phosphorylated Akt with respect to total Akt (p-Akt/t-Akt ratio) obtained through Western blot assays for these proteins in non-transfected and transfected MIO-M1 cells with dn-Rab8A, dn-Rab10 or dn-Rab11 and stimulated with insulin (100 nM) for 5 and 10 min at 37°C. Four independent experiments were performed in triplicate. Values are expressed as mean ± SEM. **P < 0.01 and ***P < 0.0001 vs. non-stimulated cells (t = 0 min). All details of experiments are indicated in the Experimental section.

Figure 7.
Rab 8A and Rab10 regulate the LRP1 translocation to the PM in MIO-M1 cells.

Biotin-labeling cell surface protein assay in cells transiently transfected and expressing wt-Rab8A and dn-Rab8A (A) and wt-Rab10 and dn-Rab10 (B) stimulated with insulin (100 nM) for 30 min at 37°C. In both experiments, biotin-labeled proteins were isolated with streptavidin-conjugated beads and then analyzed by Western blot for LRP1 and β-actin (protein loading control). Extract proteins from cell lysates were also analyzed by Western blot for LRP1 and β-actin. (C and D) Densitometric quantification of the Western blot data for surface biotin-LRP1 related to biotin-β-actin was expressed as the fold change with respect to control (t = 0 min; white bar). Three independent experiments were performed in triplicate. Values are expressed as mean ± SEM. **P < 0.01 vs. control, ***P < 0.0001 vs. control, #P < 0.05 vs. control. (E) Quantitative analyses of phosphorylated Akt with respect to total Akt (p-Akt/t-Akt ratio) obtained through Western blot assays for these proteins in non-transfected and transfected MIO-M1 cells with dn-Rab8A, dn-Rab10 or dn-Rab11 and stimulated with insulin (100 nM) for 5 and 10 min at 37°C. Four independent experiments were performed in triplicate. Values are expressed as mean ± SEM. **P < 0.01 and ***P < 0.0001 vs. non-stimulated cells (t = 0 min). All details of experiments are indicated in the Experimental section.

LRP1 translocation to the PM is involved in the insulin-induced intracellular signaling

It was demonstrated that LRP1 interacts with IR in the brain and regulates insulin signaling, because LRP1 deficiency leads to impaired Akt phosphorylation [23]. Thus, we evaluated whether blocking insulin-regulated exocytosis of LRP1 may also affect the insulin signaling in MIO-M1 cells. Figure 7E shows that dn-Rab8A and dn-Rab10-transfected cells fully blocked the insulin-induced p-Akt activation when stimulated for 5 and 10 min. Finally, dn-Rab11 did not significantly affect the insulin signaling at 5 and 10 min compared with non-stimulated cells. Thus, these results suggest that the exocytic route of LRP1 to the PM is involved in part with the regulation of insulin signaling in MGCs.

Discussion

In the retina, MGCs play key structural and metabolic roles to preserve the correct functioning of neurons and blood vessels influenced by different extracellular factors [11]. Insulin is one of these factors, but their function is not clearly established in this type of retinal cell. In contrast, LRP1 in MGCs has a relevant participation in cell motility and the ECM remodeling produced during pathological disorders such as diabetic retinopathy [3,14,42]. Several activities of LRP1 are associated with its protein level at the PM, which is dependent on the endocytosis/recycling ratio of this receptor [7,14,21,22]. However, it is not well understood how LRP1 is returned to the PM. In the present study, we demonstrated that insulin-induced LRP1 translocation to the PM by regulated exocytosis in MIO-M1 cells, which involved downstream activation of IR/PI3K/Akt and Rab-GTPases, Rab10 and Rab8A.

In adipocytes and muscle cells, insulin regulates the uptake and metabolism of the extracellular glucose through the glucose transporter GLUT4, which is translocated to the PM from specialized vesicles termed GSVs [28,29,43]. Insulin-induced GLUT4 translocation in these types of cells involves two basic mechanisms: (i) the intracellular signaling activation of the IR/PI3K/Akt axis, which mediates phosphorylation of the Rab-GTPase-activating protein AS160, thereby promoting the activation of selective Rab-GTPases such as Rab8A and Rab13 in muscle cells and Rab10 in adipocytes; and (ii) promotion of GSV traffic and fusion to the PM, allowing GLUT4 expression and the uptake of the extracellular glucose [26,44]. These vesicles contain different membrane proteins that permit these endosomes to be identified, including sortilin, VAMP2 and LRP1, which can also be sorted to the cell surface by insulin stimulus [28,29,45]. In other types of cells expressing LRP1 but not GLUT4, such as hepatocytes, it has been demonstrated that insulin can induce LRP1 translocation and increase its cell surface level, which has been associated with an enhanced ability to interact and clear different apolipoprotein E lipoproteins from circulation [46,47]. Nevertheless, the intracellular mechanism regulating this receptor translocation has not yet been established. In the present study, we have shown LRP1 to be intracellularly distributed in different membrane vesicles including early endosomes, endocytic recycling compartments and the trans-Golgi network in MIO-M1 cells, with these distributions not being altered by the presence of insulin. On the other hand, LRP1 was also stored in punctate and perinuclear membrane vesicles that were positive for sortilin and VAMP2, and incorporated GLUT4 when it was ectopically transfected. This GLUT4 was also distributed together with LRP1 in early endosomes, endocytic recycling compartments and the trans-Golgi network, but only the co-localization levels of GLUT4 and LRP1 were increased by the presence of insulin in sortilin/VAMP2 positive vesicles. By cryoimmunoelectron microscopy of non-stimulated cells, we characterized small vesicles with an approximate size of 100 nm, which contained LRP1 and sortilin/VAMP2, and also incorporated GLUT4 when it was transiently transfected. In these cells, LRP1 and sortilin/VAMP2 storage vesicles were identified at the intracellular level in the proximity of the trans-Golgi network compartments. In addition, insulin-stimulated cells revealed an increased number of these small structural vesicles containing LRP1 in peripheral zones near the PM.

Recently, it has been demonstrated that sortilin together with retromer can mediate the retrograde transport of GLUT4 in adipocytes from endosomes to the trans-Golgi network, thereby rescuing GLUT4 degradation in the lysosomes [48]. In addition, in undifferentiated 3T3-L1 preadipocytes with ectopically expressed GLUT4, this protein has a short half-life and is found principally in degradation endosomes and lysosomes [49]. Interestingly, these preadipocytes contain retromer but not sortilin, which is expressed when these cells are differentiated to adipocytes, indicating that sortilin is essential for GLUT4 protein stability and function [50]. In our study, sortilin was constitutively expressed in MIO-M1 cells, which may be a key factor for protein stability of LRP1 through its storage in small vesicles. The formation process of these vesicles, similar to GSVs, could also involve membrane donors from the trans-Golgi network [51]. Through cellular ultrastructure evaluation by cryoimmunoelectron microscopy we revealed LRP1-VAMP2-sortilin storage vesicles with evident surrounding electron-dense zones compatible with clathrin covers, which suggest that these vesicles are originated from trans-Golgi secretion [52,53]. These processes of vesicle formation could rescue LRP1 from the degradation/lysosomal pathway and can increase the vesicle number after insulin stimulation. However, further studies are needed to explain the reasons for the dynamic formation of these vesicles in cells that do not contain GLUT4, such as Muller glial cells. Our results suggest that MIO-M1 cells contain LRP1 storage vesicles, here termed LSVs, which preserve similar structural and functional properties as those of GSVs previously characterized in muscle cells and adipocytes [28]. In addition, Rab10 and Rab8A also co-localized in non-stimulated cells, which could be interpreted as a dynamic membrane trafficking from TGN to LSVs, similar to that described for GSVs in muscle cells [32]. Moreover, both Rab-GTPases were fundamental for LRP1 translocation to the PM, because GDP-bound Rab mutants affected the cell surface level of LRP1 and sortilin in the presence of insulin. In contrast, neither negative-dominant mutant affected TfR translocation, which is known to follow a Rab11-dependent endocytic recycling route [54]. Previously, we demonstrated that the LRP1 traffic to the PM is not affected by the presence of the GDP-bound Rab11 mutant [14], indicating that the endocytic recycling route is not determinant in the return of LRP1 to the PM. Thus, our results indicate that LSVs may be constitutive vesicles that regulate LRP1 translocation to the PM by an insulin-induced regulated exocytosis in MGCs. Nevertheless, further studies are needed in order to understand the mechanistic role of Rab8A and Rab10 in MIO-M1 cells, which are selective for GLUT4 translocation to the PM in muscle cells and adipocytes, respectively.

In previous studies, we demonstrated that LRP1 can regulate the traffic and function of β1-integrin [7] and MT1-MMP [14], which mainly involve the endocytic activity of LRP1 promoting an increased accumulation of these membrane proteins in EEA1-positive early endosomes. From this endosomal structure, both β1-integrin and MT1-MMP, but not LRP1, are recycled to the PM by a Rab11-dependent recycling route, whereas LRP1 might return by a different intracellular pathway toward the cell surface. In the present study, we observed that although LRP1 was localized in Rab4- and Rab11-positive recycling compartments in non-stimulated cells, the constitutive LRP1 translocation to the PM was only blocked by the presence of a GDP-bound Rab8A and Rab10 mutant, or by specific knockdown of each protein through siRNA treatment, indicating that both Rab-GTPases are involved in a constitutive exocytosis of LRP1 to the PM. However, it is known that Rab8A is involved in insulin-dependent membrane traffic between endosomes and the trans-Golgi network, whereas Rab10 (in adipocytes) and Rab13 (in muscle cells) have a role in exocytic routes [26,32]. In this study, we did not evaluate the participation of Rab13, with further work being needed to investigate the molecular mechanisms of this Rab-GTPase and others in constitutive LRP1 translocation to the PM in MIO-M1 cells.

It has previously been demonstrated that LRP1 can interact and regulate the signaling activity of IR in the brain, suggesting that LRP1 acts as a key scaffold protein in IR activation [23]. Moreover, the somatic inactivation of LRP1 in murine liver reduced IR expression on the hepatocyte surface and decreased GLUT2 translocation, leading to diet-induced insulin resistance with dyslipidemia and non-alcoholic hepatic steatosis [47]. In the present investigation, we observed that the reduction in LRP1 in the PM of MIO-M1 cells might be essential for IR activation induced by insulin, because cells transfected with dn-Rab8A and dn-Rab10 impaired insulin-induced Akt phosphorylation. In contrast, dn-Rab11 exerted a partial, although not significant, inhibition of Akt activation, which may be attributed to a partial contribution of the endocytic recycling pathway in the LRP1 and IR return to the PM. However, further studies are needed to determine the molecular relationship between LRP1 translocation and IR signaling, as well as to establish whether these Rab-GTPase proteins are involved with the IR function in MGCs.

Summing up, we propose that insulin-induced LRP1 translocation to the PM is essential for IR activity, which might be relevant for the function of MGCs during pathological disorders of the retina associated with insulin resistance. Considering that the glutamate metabolism pathway in MGCs is regulated by the insulin/IR axis [19], we conclude that LRP1 translocation to the PM may be a critical mechanism for the correct extracellular processing of glutamate in the retina. Finally, our results may be also useful in the identification of new molecular targets for therapeutic treatments against the proliferative retinopathy occurring in diabetes, obesity and metabolic syndrome, which are deleterious pathologies for the retina and vision.

Abbreviation

     
  • ECM

    extracellular matrix

  •  
  • EEA1

    early endosome antigen 1

  •  
  • FCS

    fetal calf serum

  •  
  • GFP

    green fluorescent protein

  •  
  • GLUT4

    glucose transporter type 4

  •  
  • GSVs

    GLUT4 storage vesicles

  •  
  • HA

    hemagglutinin tag

  •  
  • IR

    insulin receptor

  •  
  • LDL

    low-density lipoprotein

  •  
  • LRP1

    low-density lipoprotein receptor-related protein-1

  •  
  • LSVs

    LRP1 storage vesicles

  •  
  • MIO-M1

    human retinal Müller glial cell-derived cell line

  •  
  • MT1-MMP

    matrix type-1 MMP

  •  
  • p-Akt

    phospho-Akt

  •  
  • PFA

    paraformaldehyde

  •  
  • PI3K

    phosphatidylinositol-4,5-bisphosphate 3-kinase

  •  
  • p-IR

    phospho-insulin receptor

  •  
  • PKB

    Akt or protein kinase B

  •  
  • PM

    plasma membrane

  •  
  • PMA

    phorbol 12-myristate 13-acetate

  •  
  • SP

    signal peptide

  •  
  • TfR

    transferrin receptor

  •  
  • TGN46

    trans-Golgi network integral membrane protein 2

  •  
  • VAMP2

    vesicle-associated membrane protein 2

Author Contribution

V.A.D. and G.A.C. designed the investigation; V.A.D., R.A.G., and C.M.F. performed the experiments; M.C.S., C.M.F., and G.A.C. contributed new reagents/analytic tools; V.A.D., M.C.S., C.M.F., and G.A.C. analyzed the data; and V.A.D. and G.A.C. wrote the paper.

Funding

This work was funded by Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT-UNC) grants 2016 and 2017; Fondo para la Investigación Científica y Tecnológica (FONCyT), Préstamo BID Proyecto de Investigación en Ciencia y Tecnología (PICT) grant 2012-2607 and grant 2015-0807; Proyecto de Investigación en Ciencia y Tecnología Orientados (PICTO)-Glaxo grant 2012-0084. V.A.D. was a doctoral fellow of FONCyT; V.A.D. and R.A.G. are doctoral fellows at CONICET, and M.C.S., C.M.F. and G.A.C. are members of the Research Career of CONICET.

Acknowledgments

We are grateful to Dr Paul David Hobson, native speaker, for revising the language of this manuscript. We thank Norberto Domizio and Elisa Bocanegra for excellent technical assistance with Electron Microscopy, as well as Carlos Mas and Cecilia Sampedro with confocal microscopy.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Supplementary data