Activated Alpha-2 Macroglobulin Improves Insulin Response via LRP1 in Lipid-Loaded HL-1 Cardiomyocytes

Figures

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  Figure 1. α2M* activates PI3K/Akt and MAPK/ERK pathways in HL-1 cardiomyocytes. (a) Western blot assay for the analysis of 180 kDa-subunit from mouse tetrameric α2M (c-α2M) expression in protein extract of HL-1 cardiomyocytes treated with aggLDL 100 µg/mL for 24 h. Bands are shown in duplicates. β-actin was used as loading control. The electrophoresis conditions are detailed in the Materials and Methods section. (b) Densitometric quantification of Western blot data for c-α2M/β-actin expressed as fold change respect to non-stimulated control (white bar). Values are expressed as mean ± SEM. ** p < 0.01 vs. non-stimulated control. (c,e) Western blot assay for the analysis of phosphorylated Akt (p-Akt) (c) or phosphorylated ERK (p-ERK) (e) in cells treated with α2M* 60 nM for 5 and 15 min. Total Akt (Akt), total ERK (ERK) and β-actin were used as loading control (d,f). Densitometric quantification of Western blot data for p-Akt/Akt (d) or p-ERK/ERK (f) expressed as fold change respect to non-stimulated control (white bar). Values are expressed as mean ± SEM. * p < 0.05 vs. non-stimulated control. Three experiments were performed (n = 3).

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  Figure 2. α2M* increases GLUT4 on cell surface by PI3K/Akt and MAPK/ERK signaling activation in HL-1 cardiomyocytes. (a) Biotin-labeling protein assay to measure GLUT4 level in the PM of cells stimulated with α2M* (60 nM) for 5 and 15 min at 37 °C. Biotin-labeled proteins were isolated with streptavidin-conjugated beads and then analyzed by Western blot for GLUT4 expression. ATP1A1 and β-actin were used as protein loading controls. (b) Densitometric quantification of Western blot data for surface biotin-GLUT4 related to biotin-ATP1A1 expressed as fold change with respect to the non-stimulated control (white bar). Values are expressed as mean ± SEM. ** p < 0.01 vs. non-stimulated control. Three experiments were performed (n = 3). (c) Confocal microscopy of HL-1 cardiomyocytes treated with α2M* (60 nM) for 5 min at 37 °C. Representative images of immunostained GLUT4 (red) in non-permeabilized cells (Surface). Quantitative analysis of fluorescence intensity per cell area are expressed as mean ± SD (%). ** p < 0.01 vs. non-stimulated control. Scale bar = 10 µm. At least 20 cells per condition were analyzed (n = 20). (d) Biotin-labeling protein assay was used to measure LRP1 and sortilin…

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  Figure 3. α2M* promotes endocytic and exocytic recycling of GLUT4 in HL-1 cardiomyocytes. (a) Confocal microscopy in cells treated with α2M* (60 nM) for 5 min at 37 °C. Images show colocalization between GLUT4 and Rab4 (green), Rab8A (red) or Rab10 (red). INSET represents magnification 4× of framed regions in dotted lines. White arrowheads indicate colocalization sectors. Scale bar = 10 µm. (b) Quantitative analysis of colocalization between GLUT4 and different markers by Manders’ coefficients expressed as mean ± SD (%). * p < 0.05 vs. indicated conditions. At least 20 cells per condition were analyzed (n = 20).

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  Figure 4. α2M* improves 2-NBDG uptake in HL-1 cardiomyocytes. (a) Cell surface protein detection assay to measure GLUT4 in cells treated with α2M* (60 nM), insulin (100 nM) or combined (α2M* + insulin) for different times at 37 °C. The cell surface level of GLUT4 was analyzed in non-permeabilized cells using anti-GLUT4 antibody. Values are expressed as mean ± SEM. + indicates significant differences between α2M* and control (p < 0.05). # indicates significant differences between insulin and control (p < 0.05). * indicates significant differences between α2M* + insulin and control (p < 0.05). Three experiments were performed (n = 3). (b) Uptake assay to measure 2-NBDG uptake by confocal microscopy in cells treated with α2M* (60 nM), insulin (100 nM) or combined (α2M* + insulin) for different times together with 2-NBDG 80 µM at 37 °C. Graph represents mean ± SEM of the fluorescence intensity of 2-NBDG per cell area. # indicates significant differences between insulin and control (p < 0.05). * indicates significant differences between α2M* + insulin and control (p < 0.05). Three experiment were performed (n = 3). (c,d) Area under curve quantified from (b) at 5 and 30 min, respectively. AU,…

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  Figure 5. α2M* blocks the aggLDL intracellular accumulation and redistribution of LRP1 to degradation compartments in HL-1 cardiomyocytes. (a) Confocal microscopy in cells treated with α2M* (60 nM) or aggLDL-DiI (red) (100 µg/mL) or both ligands combined for 8 h at 37 °C. Hoechst was used to stain nuclei. Scale bar = 10 µm. (b) Quantitative analysis of aggLDL-DiI uptake as fluorescence intensity per cell area expressed as fold change with respect to the non-stimulated control. Values are expressed as mean ± SD. * p < 0.05 vs. indicated conditions. At least 20 cells per condition were analyzed (n = 20). (c) Confocal microscopy in cells treated with α2M* (60 nM) or aggLDL 100 µg/mL or both ligands combined for 8 h at 37 °C. Images show colocalization between LRP1 (red) and LAMP1 (green). INSET represents 4× magnification of the framed regions in dotted lines. White arrowheads indicate colocalization sectors. Scale bar = 10 µm. Quantitative analysis of colocalization between LRP1 and LAMP1 by Manders’ coefficients expressed as mean ± SD (%). * p < 0.05 vs. control conditions. At least 20 cells per condition were analyzed (n = 20).

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  Figure 6. α2M* counteracts GLUT4 trafficking to PM and glucose uptake impaired by aggLDL in HL-1 cardiomyocytes. (a) Biotin-labeling protein assay to measure GLUT4 and LRP1 levels in the PM of cells stimulated with α2M* 60 nM, or aggLDL 100 µg/mL or both for 8 h, and then stimulated with insulin 100 nM for 30 min at 37 °C. Biotin-labeled proteins were isolated with streptavidin-conjugated beads and then analyzed by Western blot for GLUT4 and LRP1 expression. ATP1A1 and β-actin were used as protein loading controls. (b) Densitometric quantification of Western blot data for surface GLUT4 and LRP1 related to biotin-ATP1A1 expressed as fold change with respect to the non-stimulated control (white bar). Values are expressed as mean ± SEM. ** p < 0.01 and *** p < 0.001 vs. non-stimulated control. Three experiments were performed (n = 3). (c) Confocal microscopy of HL-1 cardiomyocytes treated with α2M* 60 nM, aggLDL 100 µg/mL or both for 8 h and then stimulated with insulin 100 nM together with 2-NBDG 80 µM (green) for 30 min at 37 °C. Hoechst was used to stain nuclei. Scale bar = 10 µm. (d) Quantitative analysis of 2-NBDG uptake as fluorescence intensity per cell area expressed as fold change…

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  Figure 7. α2M* reduces Gal-1 and Gal-3 expression induced by aggLDL in HL-1 cardiomyocytes. (a,b) Quantitative RT-PCR assay to measure Gal-1 and Gal-3 mRNA expression of HL-1 cardiomyocytes treated with α2M* 60 nM, aggLDL 100 µg/mL or both ligands combined for 8 h at 37 °C. The bar graph shows the mean ± SEM of Gal-1 and Gal-3 mRNA levels relative to β-actin mRNA expressed as fold change respect to control. ** p < 0.01 vs. non-stimulated control (white bar).

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  Figure 8. Schematic summary of the main findings. α2M* is an agonist ligand of LRP1 that improves the insulin response characterized by PI3K/Akt and MAPK/ERK signaling activation, GLUT4 translocation to PM by Rab4, Rab8A and Rab10 GTPases activation and glucose uptake induced by insulin in cardiomyocytes. Moreover, α2M* counteracts the antagonist effect of aggLDL on LRP1 in insulin response in lipid-loaded HL-1 cardiomyocytes.