cells Article LRP1-Mediated AggLDL Endocytosis Promotes Cholesteryl Ester Accumulation and Impairs Insulin Response in HL-1 Cells Virginia Actis Dato 1,2 , Aleyda Benitez-Amaro 3,4 , David de Gonzalo-Calvo 3,4,5 , Maximiliano Vazquez 1,2 , Gustavo Bonacci 1,2 , Vicenta Llorente-Cortés 3,4,5, * and Gustavo Alberto Chiabrando 1,2, * 1 Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba 5000, Argentina; vickyactisdato@gmail.com (V.A.D.); mvazquezgc@gmail.com (M.V.); gustavobonacci@gmail.com (G.B.) 2 Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Córdoba 5000, Argentina 3 Institute of Biomedical Research of Barcelona (IIBB)-Spanish National Research Council (CSIC), 08025 Barcelona, Spain; aleyda.benitez.amaro@gmail.com (A.B.-A.); david.degonzalo@gmail.com (D.d.G.-C.) 4 Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025 Barcelona, Spain 5 CIBERCV, Institute of Health Carlos III, 28029 Madrid, Spain * Correspondence: cllorente@santpau.cat (V.L.-C.); gustavo.chiabrando@unc.edu.ar (G.A.C.); Tel.: +54-351-4334264 (ext. 3431) (G.A.C.); Fax: +54-351-4333048 (G.A.C.)  Received: 17 October 2019; Accepted: 6 January 2020; Published: 10 January 2020  Abstract: The cardiovascular disease (CVD) frequently developed during metabolic syndrome and type-2 diabetes mellitus is associated with increased levels of aggregation-prone small LDL particles. Aggregated LDL (aggLDL) internalization is mediated by low-density lipoprotein receptor-related protein-1 (LRP1) promoting intracellular cholesteryl ester (CE) accumulation. Additionally, LRP1 plays a key function in the regulation of insulin receptor (IR) and glucose transporter type 4 (GLUT4) activities. Nevertheless, the link between LRP1, CE accumulation, and insulin response has not been previously studied in cardiomyocytes. We aimed to identify mechanisms through which aggLDL, by its interaction with LRP1, produce CE accumulation and affects the insulin-induced intracellular signaling and GLUT4 trafficking in HL-1 cells. We demonstrated that LRP1 mediates the endocytosis of aggLDL and promotes CE accumulation in these cells. Moreover, aggLDL reduced the molecular association between IR and LRP1 and impaired insulin-induced intracellular signaling activation. Finally, aggLDL affected GLUT4 translocation to the plasma membrane and the 2-NBDG uptake in insulin-stimulated cells. We conclude that LRP1 is a key regulator of the insulin response, which can be altered by CE accumulation through LRP1-mediated aggLDL endocytosis. Keywords: glucose; signaling; lipid; membranes; intracellular traffic 1. Introduction Metabolic syndrome (MS) and type-2 diabetes mellitus (T2DM) are chronic metabolic disorders and prevalent diseases adversely impacting health worldwide, which are characterized by increased levels of circulating glucose, insulin, atherogenic lipoprotein subfractions, and inflammatory cytokines [1,2]. These mediators of metabolic disease modify multiple molecular pathways, such as ceramide-induced apoptosis, increased formation of reactive oxygen species, mitochondrial dysfunction, and endoplasmic reticulum stress, which drive multiple alterations in different cell types, including cardiomyocytes [1,3]. Cardiomyocytes are insulin-responsive cells and impaired insulin-signaling are associated with heart Cells 2020, 9, 182; doi:10.3390/cells9010182 www.mdpi.com/journal/cells Cells 2020, 9, 182 2 of 17 failure [4]. The excessive accumulation of neutral lipids is a phenomena associated with altered insulin signaling activation in hepatocytes, muscle cells, and myocardium [5,6]. However, the molecular mechanisms underlying the link between impaired insulin action and lipid accumulation are largely unknown. Patients with MS and T2DM usually present increased levels of aggregation-prone small LDL particles in addition to other anomalies in the lipoprotein profile [7]. These small LDL particles have a high tendency to become retained and aggregated (aggLDL) in extracellular spaces, but a small proportion of aggLDL has also been detected in circulation [7–9]. Whereas native LDL are recognized by the LDL receptor (LDLR), aggLDL are taken up by the LDL receptor-related protein 1 (LRP1) [10]. Moreover, LRP1 is involved in intracellular CE accumulation in muscle cells [11–13], which promotes a significant up-regulation of the lrp1 gene expression mediated by the sterol regulatory element-binding proteins (SREBP) down-regulation [14–16]. It is known that LRP1 regulates the intracellular traffic of insulin-responsive vesicles containing the glucose transporter GLUT4 (GSV for GLUT4 storage vesicles) in fat and muscle cells [17]. These vesicles are trafficked and fused with the plasma membrane (PM) under insulin stimulus, through a mechanism dependent on the activation of the PI3 K (phosphatidylinositol-3-kinase)/Akt pathway [18,19]. LRP1 depletion in GSV substantially reduces GLUT4 sorting to the PM promoting decreased glucose uptake [20]. In addition, it has been shown in hepatic and neuron-specific LRP1 knock-out mice that this receptor interacts with insulin receptor (IR) and regulates its intracellular signaling in neurons and hepatocytes [21,22]. Recently, we found that the blockage of LRP1 exocytosis towards the PM affected the insulin-induced intracellular signaling in retinal Müller glial cells [23]. These data suggest that LRP1 plays a key role in the insulin response in different types of cells and tissues. On the basis of these previous results, we hypothesize that LRP1 is involved both in CE accumulation and insulin response impairment in cardiomyocytes treated with aggLDL. Thus, the main objective of the present study was to evaluate the role of LRP1 in the aggLDL-mediated intracellular CE accumulation and in the impairment of insulin response evaluated through the insulin signaling activation, GLUT4 trafficking and glucose uptake in HL-1 cardiomyocytes-derived cell line. Herein, we demonstrated that LRP1 mediates the aggLDL binding and endocytosis, promoting CE accumulation in these cells. The aggLDL/LRP1 complex was accumulated in early endosomes [EEA1+ ] but not in other recycling vesicles such as TGN [TGN46+ ] or recycling endocytic compartments [Rab4+ and Rab11+ ]. Finally, aggLDL-treated HL-1 cells showed a decreased insulin response, which was evidenced by: (i) reduced molecular association between LRP1 and IR; (ii) decreased insulin-induced intracellular signaling (IR, Akt, and AS160 phosphorylation); (iii) impaired GLUT4 translocation to the PM; and (iv) reduced extracellular glucose uptake. 2. Material and Methods 2.1. HL-1 Cardiomyocyte-Derived Cell Line, Cultures, and Reagents The murine HL-1 cardiomyocyte-derived cell line was generated by Dr. W.C. Claycomb (Louisiana State University Medical Centre, New Orleans, LA, USA) [24,25]. HL-1 cells were maintained in Claycomb Medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Buenos Aires, Argentina), 100 µM nor-epinephrine (Sigma-Aldrich, St. Louis, MO, USA), 100 units/mL penicillin, 100 g/mL streptomycin (Invitrogen, Buenos Aires, Argentina), and L-glutamine 2 mM (GlutaMAX from Invitrogen, Buenos Aires, Argentina) in plastic dishes, coated with 12.5 g/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA) and 0.02% gelatin, in a 5% CO2 atmosphere at 37 ◦ C. Insulin was from Apidra® Solostar® 100 U/mL (Sanofi-Aventis, Germany). Rabbit anti-IR (cs4B8), rabbit anti-pIR (Tyr1361, cs84B2), and rabbit anti-Akt (#9272) monoclonal antibodies were from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-pAkt (Ser473, #07-789) antibody was from Merck KGaA (Darmstadt, Germany). Rabbit anti-AS160 (#ab24469) and rabbit anti-pAS160 (Thr642, #ab65753) antibodies were from Abcam (Cambridge, MA, USA). Mouse monoclonal anti-β-actin Cells 2020, 9, 182 3 of 17 (#A2228) was from Sigma-Aldrich (St. Louis, MO, USA). Mouse monoclonal anti-APT1A1 (M7-PB-E9) was from ThermoFisher Scientific (Rockford, IL, USA). Rabbit anti-LRP1 (ab92544), mouse monoclonal anti-LRP1 (#ab28320), rabbit anti-GLUT4 (#ab654), rabbit anti-EEA1 (#ab2900), rabbit anti-Rab4 (#ab13252), rabbit anti-Rab11 (#ab65200), and rabbit anti-TGN46 (#ab50595) monoclonal antibodies being purchased from Abcam (Cambridge, MA, USA). 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). GST-RAP was expressed and purified as described elsewhere [26] and used without further modification. In this work, 400 nM GST-RAP was used to inhibit the binding of aggLDL to LRP1. 2.2. LDL Isolation and Modification Total LDL (d 1.019–d 1.063 g/mL) was isolated by sequential ultracentrifugation using KBr gradients, in the density range of 1.019–1.063 g/mL, from six different pools of plasma of normocholesterolemic volunteers. For the protein quantification we used a Pierce kit (#23225, ThermoFisher Scientific (Rockford, IL, USA). Apolipoprotein B integrity was tested by SDS-PAGE in 10% acrylamide gels. Aggregated LDL was generated by vortexing LDL in PBS 1X for 5 min at room temperature [27], and then was centrifuged at 10,000 rpm for 10 min to precipitate the aggLDL. Finally, aggLDL was suspended in PBS 1X to a protein concentration of 1 mg/mL. The ultrastructure of aggLDL obtained by vortexing was similar to that of LDL modified by versican, one of the main chondroitin sulfate proteoglycans structuring the arterial intima [28]. 2.3. DiI-Labeling of LDL DiI (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, Invitrogen, Buenos Aires, Argentina) is a lipophilic long-chain dialkylcarbocyanine that binds to lipoproteins and emits fluorescence at 565 nm. The LDL (1 mg/mL) were incubated for 12 h with DiI in a proportion of 3 µL per 1 mg of lipoprotein in PBS 1X at 37 ◦ C, exhaustively dialyzed in PBS 1X for 24 h to eliminate the excess of free DiI and filtered through a 0.22 µm filter. Finally, these stained LDL were aggregated mechanically by vortexing. 2.4. Lipid Extraction and Determination of CE, TG, and FC Content HL-1 cells were serum-starved overnight and then exposed to aggLDL (100 µg/mL) for 8 h. Next, cells were exhaustively washed and harvested in NaOH 0.1 M. Lipids were extracted with dichloromethane/methanol [1:2] and cholesteryl esters (CE), free cholesterol (FC) and triglycerides (TG) content was analyzed by thin layer chromatography. The organic solvent was removed under N2 stream, the lipid extract was dissolved in dichloromethane and one aliquot was partitioned by thin layer chromatography (TLC) performed on silica G-24 plates. Different concentrations of standards (a mixture of cholesterol, cholesterol palmitate and triglycerides) were applied to each plate. The chromatographic solution was heptane/diethylether/acetic acid (74:21:4, v/v/v). The developing was carried out with a 5% solution of phosphomolybdic acid/5% sulfuric acid in ethanol and heating 7 min at 100 ◦ C. The spots corresponding to CE, TG, and FC were quantified by densitometry against the standard curve of cholesterol palmitate, triglycerides, and cholesterol, respectively, using a computing densitometer. 2.5. Western Blot Analysis HL-1 cells were cultured as above and cell protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 10 mM sodium ortho-vanadate, and protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). Forty micrograms of cell protein extracts were diluted in sample buffer 5X with DTT (dithiothreitol) and then heated for 5 min at 95 ◦ C. Electrophoresis on 10% SDS-polyacrylamide gels was applied and proteins were electrotransferred to a nitrocellulose membrane (GE Healthcare Life Science, Amsterdam, The Netherlands). Nonspecific 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 Cells 2020, 9, 182 4 of 17 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, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were revealed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR Biosciences, Lincoln, NE, USA). 2.6. Confocal Microscopy HL-1 cells were cultured as above on cover glass. After different stimulus the cells were washed with PBS 1X, fixed with 4% paraformaldehyde (PFA), quenched with 50 mM NH4 Cl, permeabilized for 30 min with 0.5% (v/v) saponin, blocked with 2% bovine serum albumin (BSA) and 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) and Hoechst 33,258 colorant (1/2000) 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, fluorescent images were obtained with an Olympus FluoView FV1200 confocal laser scanning biological microscope (Olympus, NY, USA). 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, NY, USA) and quantified by ImageJ software (NIH, Bethesda, MD, USA). 2.7. Real Time-PCR The cells were serum-starved overnight and then exposed to the different stimuli and treated with the TRIzol® reagent (Invitrogen, Buenos Aires, Argentina) for total RNA extraction. One µg of total RNA was reverse transcribed in a total volume of 20 µL using random hexaprimers and reverse transcriptase. The PCR primers listed below were used to quantify the transcripts of LRP1, and β-actin. The results were normalized to RT-PCR products of β-actin transcripts. Transcripts were quantified by real-time qRT-PCR (ABI 7500 Sequence Detection System, Applied Biosystems, Foster City, CA, USA) using Sequence Detection software v1.4. Amplification conditions included a warm start at 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 1 min. Specificity was verified by fusion curve analysis and electrophoresis in 2% agarose gel with fluorescence detection with SYBR® Safe DNA (Invitrogen, Buenos Aires, Argentina). Relative gene expression was calculated according to the 2-Ct method. Each sample was analyzed in triplicate. No amplification was observed in the PCRs using water or RNA samples incubated without reverse transcriptase during cDNA synthesis. Sequences of mouse primers: lrp1 forward 50 -TGGAGCAGATGGCAATCGACT-30 lrp1 reverse 50 -CGAGTTGGTGGCGTAGAGATAGTT-30 β-actin forward 50 -AAATCTGGCACCACACCTTC-30 β-actin reverse 50 -GGGGTGTTGAAGGTCTCAAA-30 2.8. Immunoprecipitation (IP) Assays HL-1 cells were cultured as above, they were treated with or without aggLDL (100 µg/mL) for 8 h and then stimulated or not with 100 nM of insulin for 30 min. Cell protein extracts were prepared using RIPA lysis buffer and incubated for 2 h at 4 ◦ C with rabbit anti-LRP1 monoclonal 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, Santa Cruz, CA, USA) and the proteins were separated and treated for Western blot using rabbit anti-IR (1/1000) and rabbit anti-LRP1 (1/10,000) monoclonal antibodies. Secondary antibodies raised in goat anti-mouse IgG IRDye® 680CW and goat anti-rabbit IgG IRDye® 800CW (LI-COR Biosciences, Lincoln, NE, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were revealed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR) and Cells 2020, 9, 182 5 of 17 were quantified by densitometric analysis using Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA). As loading control of total protein extracts β-actin was used. 2.9. Biotin-Labeling Cell Surface Protein Assay HL-1 cells were cultured as above and then treated with or without aggLDL (100 µg/mL) for 8 h, followed by the stimulation with 100 nM of insulin for 30 min. To determine the level of LRP1 and GLUT4 at the cell surface a biotin-labeling protein assay (EZ-Link™ Sulfo-NHS-SS-Biotin (cat: 21331), Thermo Scientific, Rockford, IL, USA) was used. Briefly, cells were incubated for 2 h at 4 ◦ C with a 0.12 mg/mL of biotin solution. Then, were incubate with 0.1 mM glycine solution for 30 min and washed with PBS 1X three times to remove the excess of biotin, after which corresponding cell lysates were generated. The biotinylated proteins were pulled down using streptavidin-conjugated agarose beads (Pierce™ Streptavidin Agarose (cat: 20353), Thermo Scientific, Rockford, IL, USA) for 2 h at room temperature. The biotinylated-plasma membrane proteins and total proteins (corresponding to 10% of proteins incubated with agarose beads) were eluted by adding sample buffer 6X, DTT 1M and heating at 100 ◦ C for 5 min, and then treated for Western blot using rabbit anti-LRP1 (1/10,000), rabbit anti-GLUT4 (1/2000), mouse monoclonal anti-ATP1A1 (1/1500), or mouse monoclonal anti-β-actin (1/5000) monoclonal antibodies. 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, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were revealed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR Biosciences, Lincoln, NE, USA) and were quantified by densitometric analysis using Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA). As loading control of PM protein and total protein extracts biotinylated-ATP1A1 and β-actin were used, respectively. The content of each biotinylated protein in the PM was analyzed by densitometry and related to biotinylated-ATP1A1 protein. 2.10. 2-NBDG Uptake Assay HL-1 cells were cultured as above, and they were treated with or without aggLDL (100 µg/mL) for 8 h and then stimulated or not with 100 nM of insulin for 30 min together with 80 µM of 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG solution; Sigma-Aldrich, St. Louis, MO, USA). The 2-NBDG is a glucose fluorescent analog that is not metabolized and displays emission maxima of 540 nm. After stimulus, cells were washed with PBS 1X, fixed with 4% of paraformaldehyde, quenched with 50 mM NH4 Cl, permeabilized for 30 min with 0.5% (v/v) saponin, blocked with 2% BSA and incubated with Hoechst 33,258 colorant (1/2000) for 1 h. Finally, cells were mounted on glass slides with Mowiol 4–88 reagent. Fluorescent images were obtained with an Olympus FluoView FV1200 confocal laser scanning biological microscope (Olympus, NY, USA). 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, NY, USA) and the total fluorescence in the whole cell area was quantified by ImageJ software. For microscope quantification of the co-localization level, a JACoP plug-in from ImageJ software (NIH, Bethesda, MD, USA) was used. 2.11. Statistical Treatment of Data 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 the Student’s t-test. For Western blot and cell-surface protein detection assay, the data was expressed as the mean ± SEM and comparisons between two groups were performed using a paired t-test. Results from more than two groups were analyzed by one-way ANOVA followed by Dunnett’s post-hoc analysis (GraphPad Prism 5.0, San Diego, CA, USA). Values of p < 0.05 were considered to be significant. Cells 2020, 9, 182 6 of 17 Cells 2020, 9, 182 3. Results 6 of 19 3. Results 3.1. LRP1 Mediates Intracellular CE Accumulation by AggLDL Endocytosis It3.1. LRP1 has Mediates been Intracellular reported CE Accumulation that LRP1 is the cellbysurface AggLDLreceptor Endocytosisresponsible for the binding and endocytosis It has been reported that LRP1 is the cell surface receptor responsible and of aggLDL and CE accumulation in VSMCs, macrophages HL-1 for the cellsand binding [11,29]. endocytosis of aggLDL and CE accumulation in VSMCs, macrophages and HL-1 cells [11,29]. Accordingly, using lipid extraction and subsequent thin layer chromatography, also we detected Accordingly, an increased using lipidofextraction accumulation CE as wellandassubsequent thin formation lipid droplet layer chromatography, in HL-1 cells also we detected treated an with aggLDL for 8 increased and 18 h,accumulation respectivelyof(Figure CE as well 1A,Basand lipidFigure dropletS1A). formation AggLDLin HL-1 cells treated treatment did with aggLDL not significantly affectfor cell8 survival and 18 h,(Figure respectively S1B).(Figures Next, to1A,B and S1A). evaluate AggLDL whether LRP1 treatment did not is mediating significantly this affect lipid accumulation, cell survival (Figure S1B). Next, to evaluate whether LRP1 is mediating this lipid HL-1 cells were preincubated with GST-RAP, which blocks the binding of all LRP1 ligands, followed accumulation, HL- by 1 cells were preincubated with GST-RAP, which blocks the binding of all LRP1 ligands, followed by treatment with DiI-labeled aggLDL (aggLDL-DiI) during 8 h and next analyzed by confocal microscopy. treatment with DiI-labeled aggLDL (aggLDL-DiI) during 8 h and next analyzed by confocal As expected, the presence of GST-RAP significantly blocked the uptake of aggLDL (Figure 1C,D). microscopy. As expected, the presence of GST-RAP significantly blocked the uptake of aggLDL These(Figure results1C,D). support theresults These concept that LRP1 support mediates the concept the aggLDL that LRP1 mediatesuptake by HL-1 the aggLDL cellsbyproducing uptake HL-1 intracellular CE accumulation. cells producing intracellular CE accumulation. FigureFigure 1. Intracellular 1. Intracellular lipid lipid content content andaggLDL and aggLDLuptake uptake ininHL-1 HL-1cells. cells.(A)(A) Thin layer Thin chromatography layer chromatography assay of lipid extracts from HL-1 cells incubated with 100 µg/mL aggLDL for 8 h. Bandscorrespond assay of lipid extracts from HL-1 cells incubated with 100 µg/mL aggLDL for 8 h. Bands correspond to to intracellular intracellular cholesteryl cholesteryl ester triglycerides ester (CE), (CE), triglycerides (TG),(TG), and and free free cholesterol cholesterol (FC). (FC). TheThe CE/TG/FC CE/TG/FC bands bands from the standards analyzed by duplicate are shown in the right panel. (B) Bar graph represents from the standards analyzed by duplicate are shown in the right panel. (B) Bar graph represents the the mean ± SEM of the optical densities of the bands corresponding to CE relative to the FC content mean ± SEM of the optical densities of the bands corresponding to CE relative to the FC content for each for each condition. The statistical comparison of means was carried out through Student’s t-test for condition. The statistical comparison of means was carried out through Student’s t-test for independent independent samples. (***) indicates significant differences (p < 0.0001) vs. control. Three independent samples. (***) indicates experiments significant were performed. (C)differences (p < 0.0001) Confocal microscopy vs. control. of HL-1 Three independent cells preincubated or not withexperiments 400 nM were performed. (C) Confocal microscopy of HL-1 cells preincubated or GST-RAP and next treated with 100 µg/mL aggLDL-DiI for 8 h. The fluorescent emission not with 400 nMofGST-RAP aggLDL- and DiI is observed next treated with 100 redµg/mL and yellow dotted line aggLDL-DiI forrepresents the cell shape. 8 h. The fluorescent The cell nuclei emission were labeled of aggLDL-DiI with is observed Hoechst. red and yellow The scale line dotted bar corresponds represents the to 10cell µm. (D) Bar shape. Thegraph cell represents nuclei were thelabeled Mean ± with SEM Hoechst. of the Dil The scale fluorescence bar correspondsintensity perµm. to 10 cell area. (D) Bar(*) indicates significantthe graph represents differences Mean ±(pSEM < 0.05) of between aggLDL the Dil fluorescence treatment with or without GST-RAP. Three independent experiments were performed intensity per cell area. (*) indicates significant differences (p < 0.05) between aggLDL treatment with or by duplicate andGST-RAP. without 20 cells were analyzed Three per experiment. independent experiments were performed by duplicate and 20 cells were analyzed per experiment. Cells 2020, 9, 182 7 of 17 3.2. Aggregated LDL Endocytosis Leads to AggLDL/LRP1 Complex Accumulation in Endosomal Compartments To evaluate the endocytic pathway that follows LRP1 after binding to aggLDL, HL-1 cells were incubated with aggLDL-DiI for 8 h at 37 ◦ C and then immunostained for detection of LRP1 at different intracellular compartments. Confocal microscopy experiments showed that LRP1 and aggLDL-DiI colocalized in vesicular structures in a high proportion (−50%) (Figure 2A). The co-localization analysis of the different subcellular compartments indicated that main of the LRP1 co-localization with aggLDL occurred in endocytic early endosome [EEA1+ ] (Figure 2B,C) while a weak co-localization occurred in [Rab11+ ] and [Rab4+ ] endocytic recycling as well as trans-Golgi [TGN46+ ] compartments (Figure 2C). These data indicate that aggLDL/LRP1 complex is internalized and accumulated in endocytic compartments in HL-1 cells. Cells 2020, 9, 182 8 of 19 Intracellular Figure 2.Figure distribution 2. Intracellular and expression distribution and expressionlevellevel of LRP1 in HL-1 of LRP1 cells.cells. in HL-1 (A) (A) Confocal Confocalmicroscopy microscopy of HL-1 cells of HL-1in incubated cells theincubated absenceinor thepresence absence or of presence of 100 µg/mL 100 µg/mL aggLDL-Dil aggLDL-Dil forfor 8 8h.h.LRP1 LRP1 in green in green and and aggLDL-DiI aggLDL-DiI in red are shown.in red Inset: are shown. Inset: 4× magnification 4× magnification of framed of framed regionregion in the in the dottedlines. The dotted lines. The MASK represents the binary image where co-localization pixels between LRP1 and aggLDL MASK represents the binary image where co-localization pixels between LRP1 and aggLDL are shown are shown in white. The corresponding quantitative analysis is shown as the mean ± SD of the in white.percentages The corresponding quantitative of co-localization. The images analysis is shownofas are representative 30 the cellsmean ± SD ofcoming per condition, the percentages from of co-localization. The images three independent are representative experiments. The scale barofcorresponds 30 cells per condition, to 10 comingmicroscopy μm. (B) Confocal from three independent in HL- 1 cells The experiments. incubated scale with 100 µg/mL aggLDL-DiI bar corresponds to 10 µm. for (B) 8 h Confocal showing the triple co-localization microscopy in HL-1analysis cells incubated with 100between µg/mLLRP1 and aggLDL-Dil aggLDL-DiI for 8 together h showing with the earlytriple endosome marker [EEA1+analysis co-localization ]. The insetbetween representsLRP1 and the 4× magnification of the framed regions in the dotted lines. The white arrowheads indicate vesicles aggLDL-Dil together with early endosome marker [EEA1+ ]. The inset represents the 4× magnification containing triple co-localization sectors. The images are representative of 30 cells per condition, coming from three independent experiments. The scale bar corresponds to 10 μm. (C) The bar graph represents the mean ± SEM of the co-localization percentages of the triple co-localization between LRP1, aggLDL-Dil and different intracellular compartment markers (EEA1, Rab4, Rab11, and TGN46), obtained from the quantification performed in 30 cells per condition. (D) Quantitative RT- PCR assay to evaluate LRP1 mRNA expression in HL-1 cells exposed to 100 μg/mL aggLDL for 8 h. Cells 2020, 9, 182 8 of 17 of the framed regions in the dotted lines. The white arrowheads indicate vesicles containing triple co-localization sectors. The images are representative of 30 cells per condition, coming from three independent experiments. The scale bar corresponds to 10 µm. (C) The bar graph represents the mean ± SEM of the co-localization percentages of the triple co-localization between LRP1, aggLDL-Dil and different intracellular compartment markers (EEA1, Rab4, Rab11, and TGN46), obtained from the quantification performed in 30 cells per condition. (D) Quantitative RT-PCR assay to evaluate LRP1 mRNA expression in HL-1 cells exposed to 100 µg/mL aggLDL for 8 h. Bar graph showing the Mean ± SEM of mRNA levels. The RT-PCR products of Actin transcripts were used as loading controls. (E) Western blot assay for the immunodetection of the LRP1 β-subunit in HL-1 cells treated with 100 µg/mL aggLDL for 8 h. The immunodetection of β-actin was used as a protein loading control. The bar graph represents the relative intensity of LRP1 band normalized to β-actin and reported as a fold change against controls from three different experiments performed in triplicate. It has been shown that aggLDL prolongs the half-life of LRP1 by inhibiting the proteasomal LRP1 degradation, which in turn favors the uptake and intracellular CE accumulation in VSMC [11]. Therefore, we evaluated the effect of HL-1 exposure to aggLDL for 8 h on LRP1 mRNA and protein levels through quantitative RT-PCR and Western blot assays, respectively. Figure 2D,E shows that aggLDL did not alter mRNA or protein levels of LRP1 at this end point. Therefore, these results demonstrate that aggLDL does not modify the LRP1 protein levels in HL-1 cells. 3.3. Aggregated LDL Reduces the Molecular Association Between LRP1 and IR It has been demonstrated that LRP1 mediates the insulin-induced IR phosphorylation in mouse brain [21]. In addition, hepatic LRP1 deficiency impaired the insulin response of mice fed with high-fat diet [22]. Considering that aggLDL/LRP1 complex is accumulated in endocytic compartments, the cellular functionality of this receptor may be affected. Therefore, we have focused to investigate whether aggLDL may alter the molecular association between LRP1 and IR. In this way, HL-1 cells were incubated with aggLDL for 8 h and then stimulated with insulin for 30 min. Next, immunoprecipitation procedures were carried out using an anti-IR antibody followed by Western blot assays for LRP1 (Figure 3A). The quantitative analysis (Figure 3B) of bands showed that under basal conditions (without insulin) a molecular association is produced between LRP1 and IR (lane 2) that was reduced by aggLDL (lane 3). Insulin increased the LRP1/IR association (lane 5) that was also impaired in the presence of aggLDL (lane 4). Similar results were obtained when immunoprecipitation assays were carried out with anti-LRP1 antibody followed by Western blot assays for IR (Figure S1C,D). To evaluate whether the inhibitory effect of aggLDL on the molecular association between IR and LRP1 involves the ligand-receptor interaction, immunoprecipitation assays were carried out in cells preincubated with GST-RAP for 30 min. Under basal conditions (without insulin), GST-RAP did not affect the molecular association between LRP1 and IR (lanes 2 and 6) but effectively blocked the aggLDL effect on LRP1/IR association (lanes 3 and 8) (Figure 3C,D). In the presence of insulin, GST-RAP restored the molecular association between LRP1 and IR in cells treated with aggLDL (lanes 4, 5, and 7) (Figure 3C,D). Thus, these results indicate that the molecular association between LRP1 and IR in HL-1 cells is reduced by aggLDL. 3.4. Aggregated LDL Impairs Insulin-Induced Intracellular Signaling Activation Our group has previously demonstrated that insulin promotes the LRP1 translocation to the PM by activation of the insulin-induced intracellular signaling pathway [23,30]. Here, we investigated whether aggLDL may affect both LRP1 and IR levels on the cell surface. For this purpose, HL-1 cells were treated with aggLDL for 8 h, stimulated or not with insulin for 30 min and the levels of both receptors in the PM were analyzed by biotinylation assay of surface proteins followed by Western blot. Figure 4A,B show that insulin as well as aggLDL significantly increased IR and LRP1 protein levels in Cells 2020, 9, 182 9 of 17 the PM respect to control. In cells treated with aggLDL and then stimulated with insulin, the levels of LRP1 protein on the PM were increased over the control while those of IR remained unaltered. Cells 2020, 9, 182 10 of 19 Figure 3.3. Molecular Figure Molecular association association between between IR IR andand LRP1LRP1 inin aggLDL-treated aggLDL-treated HL-1 HL-1cells. cells. (A) (A) Immunoprecipitation assay Immunoprecipitation assay for forIR.IR.Cell Celllysate lysatewas wasimmunoprecipitated immunoprecipitated with an anti-IR with an anti-IRantibody and antibody thenthen and subjected subjectedto SDS-PAGE to SDS-PAGE and followed and followed byby Western Westernblot blotassays assaysfor forLRP1 LRP1and and total total IR. Cells were IR. Cells were incubatedor incubated ornot notwith with100 100µg/mL µg/mLaggLDL aggLDLfor for 8h 8h and and then then stimulated stimulated or or notnot with with 100100nMnM insulin insulin for for 30 30 min. min. Lane Lane 1, immunoglobulin 1, immunoglobulin isotype isotype control control (IC).(IC). TheThe detection detection of LRP1, of LRP1, IR and IR and β-actin β-actin in total in total cell cell lysates lysates is shownis shown in thepanel in the lower lower of panel of theblot. the Western Western (B) The blot. bar (B) graphThe bar graph represents therepresents densitometric the densitometric quantification of the bands obtained in the Western blot. Values were expressed as the quantification of the bands obtained in the Western blot. Values were expressed as the mean ± SEM of mean the ± SEM relative of the of intensity relative intensity LRP1 bands with ofrespect LRP1 bands to IR and with respect shown as ato IR change fold and shownagainstas control a fold change (white against bar). (***)control indicate(white bar).differences significant (***) indicate(p < 0.0001) significant differences vs. control. Three(pindependent < 0.0001) vs. control. Three experiments were independent performed experiments in duplicate. (C)were performed in duplicate. Immunoprecipitation assays for (C)IRImmunoprecipitation followed by Western blot assaysfor for LRP1 IR followed by Western blot for LRP1 β-subunit. Cells were or not preincubated with 400 nM GST-RAP β-subunit. Cells were or not preincubated with 400 nM GST-RAP for 30 min, then incubated or not for 30100 with min, then aggLDL µg/mL incubated foror8not h inwith 100 µg/mL the presence ofaggLDL for 8 h in the 400 nM GST-RAP andpresence of 400 stimulated subsequently nM GST-RAP or andwith not subsequently stimulated 100 nM insulin for 30ormin. not with Lane100 nM insulin for 30 isotype 1, immunoglobulin min. Lane 1, immunoglobulin control (IC). The detection isotypeof control LRP1 and(IC). The detection β-actin of LRP1 in the total and β-actin cell lysates is shownin thein total cell lysates the lower panelisofshown in the lower the Western panel blot. (D) Theof the Western blot. (D) The bar graph represents the densitometric quantification of the bands obtained bar graph represents the densitometric quantification of the bands obtained in the Western blot. The in the Western values blot. The were expressed asvalues Mean were± SEM expressed as Mean of the relative ± SEM of intensity of LRP1 the relative bandsintensity of LRP1 with respect to IR,bands and with respect to IR, and reported as fold change against the corresponding control (white bar). (*) (p < reported as fold change against the corresponding control (white bar). (*) (p < 0.05) and (**) (p < 0.01) indicate 0.05) and significant (**) (p