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

Activated alpha-2 Macroglobulin (α2M*) is specifically recognized by the cluster I/II of LRP1 (Low-density lipoprotein Receptor-related Protein-1). LRP1 is a scaffold protein for insulin receptor involved in the insulin-induced glucose transporter type 4 (GLUT4) translocation to plasma membrane and glucose uptake in different types of cells. Moreover, the cluster II of LRP1 plays a critical role in the internalization of atherogenic lipoproteins, such as aggregated Low-density Lipoproteins (aggLDL), promoting intracellular cholesteryl ester (CE) accumulation mainly in arterial intima and myocardium. The aggLDL uptake by LRP1 impairs GLUT4 traffic and the insulin response in cardiomyocytes. However, the link between CE accumulation, insulin action, and cardiac dysfunction are largely unknown. Here, we found that α2M* increased GLUT4 expression on cell surface by Rab4, Rab8A, and Rab10-mediated recycling through PI3K/Akt and MAPK/ERK signaling activation. Moreover, α2M* enhanced the insulin response increasing insulin-induced glucose uptake rate in the myocardium under normal conditions. On the other hand, α2M* blocked the intracellular CE accumulation, improved the insulin response and reduced cardiac damage in HL-1 cardiomyocytes exposed to aggLDL. In conclusion, α2M* by its agonist action on LRP1, counteracts the deleterious effects of aggLDL in cardiomyocytes, which may have therapeutic implications in cardiovascular diseases associated with hypercholesterolemia.


Introduction
Cardiovascular disease is frequently associated with metabolic syndrome, type 2diabetes mellitus (T2DM) and obesity, and impaired insulin response is one of the main features in these patients [1][2][3]. The heart requires a large amount of energy for its contractile function and it is able to use different substrates [4]. The glucose uptake by the myocardium represents approximately 30% of the energy source available for this tissue [5][6][7]. In this way, insulin resistance has a marked influence on cardiac metabolism because the contribution of substrates is altered and there are drawbacks in the metabolic adaptations in the myocardium when the action of insulin is affected [8,9].
The alpha-macroglobulin family represents a well-defined group of proteinase inhibitors expressed in several vertebrate species, including human and mouse [10,11]. In human, two forms are identified, α 2 -macroglobulin (α 2 M) and pregnancy zone protein (PZP), which are analogues to tetrameric α 2 M and single chain murinoglobulin (MUG) in mouse, respectively [10,12]. Human and mouse α 2 M are structured by four identical 180-kDa subunits and can inhibit a broad spectrum of proteinases (serine-, metal-, aspartic-, and cysteine-proteinases). After α 2 M/proteinase interaction, a peptide bond localized within the bait region of the inhibitor molecule is cleaved. This event leads to a conformational change in the α 2 M, together with the trapping of the proteinase, activation, and cleavage of internal ß-cysteinyl-γ-glutamyl thiol esters, and exposure of It was identified a cardiac isoform of α 2 M (c-α 2 M) in humans and rats, which resulted to be identical to liver α 2 M [16]. Thus, we evaluate whether cardiomyocytes may express the murine c-α 2 M in cells cultured under control conditions and in the presence of aggLDL. Previously we found that aggLDL produced a marked CE accumulation in HL-1 cardiomyocytes, which impair the insulin-induced response in these cells [20]. Through reduced SDS-PAGE and subsequent Western blot using a specific antibody against tetrameric α 2 M, we detected a 180 kDa subunit in cell lysates of HL-1 cardiomyocytes cultured in control conditions, indicative of c-α 2 M (Figure 1a,b). In the same figures it is shown that after 24 h of aggLDL treatment a significant increase of murine c-α 2 M was found. Thus, these data indicate that HL-1 cardiomyocytes expressed c-α 2 M and increased its expression under CE accumulation conditions by aggLDL.
reduced SDS-PAGE and subsequent Western blot using a specific antibody against tetrameric α2M, we detected a 180 kDa subunit in cell lysates of HL-1 cardiomyocytes cultured in control conditions, indicative of c-α2M (Figure 1a,b). In the same figures it is shown that after 24 h of aggLDL treatment a significant increase of murine c-α2M was found. Thus, these data indicate that HL-1 cardiomyocytes expressed c-α2M and increased its expression under CE accumulation conditions by aggLDL.
In previous works we showed that α2M* via LRP1 activates PI3K/Akt or MAPK/ERK or both intracellular pathways depending on the cell types [21,35,36]. Herein, we evaluate the α2M* intracellular signaling activation in HL-1 cardiomyocytes by Western blot assays. Figure 1c-f shows that α2M* induced a significant phosphorylation of Akt (p-Akt) and ERK (p-ERK) at 5 min but not at 15 min of stimulus, indicating that α2M* promotes a fast and short activation of both intracellular pathways in this cell type.  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 α 2 M* 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).
In previous works we showed that α 2 M* via LRP1 activates PI 3 K/Akt or MAPK/ERK or both intracellular pathways depending on the cell types [21,35,36]. Herein, we evaluate the α 2 M* intracellular signaling activation in HL-1 cardiomyocytes by Western blot assays. Figure 1c-f shows that α 2 M* induced a significant phosphorylation of Akt (p-Akt) and ERK (p-ERK) at 5 min but not at 15 min of stimulus, indicating that α 2 M* promotes a fast and short activation of both intracellular pathways in this cell type.
In a previous work, we found that α 2 M* promotes LRP1 traffic to PM in the Müller glial-derived cell line, MIO-M1 cells [21]. Moreover, in this cell type we demonstrated that insulin increases the LRP1 expression on cell surface by a regulated exocytic pathway, involving the sorting of GLUT4-storage vesicles (GSVs) to the PM [37]. In myocardium, the insulin-induced intracellular signaling activation leads to GLUT4 translocation to the PM [20]. Considering these data, we analyze the α 2 M* effects on the GLUT4 traffic to the cell surface in HL-1 cardiomyocytes by biotin-labeling protein assays. Figure 2a,b show increased levels of GLUT4 on cell surface by α 2 M* at 5 min of stimulation with respect to the control condition. By contrast, α 2 M* stimulus for 15 min did not show effect of GLUT4 expression on PM compared to control. This evidence was corroborated by confocal microscopy in cells treated with α 2 M* for 5 min and then incubated with anti-GLUT4 in non-permeabilizing conditions (Figure 2c). Similar results were found for LRP1 and sortilin, other constitutive proteins of GSVs [29,37] (Figure 2d,e). Next, we evaluate whether GLUT4 traffic to the PM was dependent on PI 3 K/Akt and MAPK/ERK intracellular signaling activation by α 2 M*. For this, we performed a cell surface protein detection assay in which HL-1 cardiomyocytes were pre-incubated 30 min with wortmannin (PI 3 K inhibitor) or PD98059 (MAPK inhibitor) and then treated with α 2 M* for 5 min. Figure 2f,g shows that α 2 M*-induced GLUT4 increase on PM was significantly reduced through the blocking PI 3 K/Akt and MAPK/ERK intracellular signaling activation. Thus, our data shows that α 2 M* can activate PI 3 K/Akt and MAPK/ERK pathways and increase GLUT4 expression at the cell surface of HL-1 cardiomyocytes.

α2M* Promotes GLUT4 Endocytic Recycling through Rab4, Rab8A, and Rab10 GTPases
In previous work we have shown that α 2 M* promotes the exocytic route of LRP1 to PM by Rab10 GTPase activation [21]. Moreover, several reports found that GLUT4 traffic to PM induced by insulin is also dependent of Rab10 activation together with Rab8A [37][38][39][40]. Thus, our interest was to evaluate whether α 2 M* can activates the exocytic routes of GLUT4 to PM. For this, HL-1 cardiomyocytes were treated with α 2 M* for 5 min and then we analyzed the colocalization between GLUT4 and different markers of subcellular compartments, such as EEA1 for early endosomes, Rab4 for short loop endocytic recycling compartments, Rab11 for long loop endocytic recycling compartments, and Rab8A and Rab10 mainly associated with exocytic compartments. Figure 3 shows that α 2 M* increased the colocalization of GLUT4 in Rab4 + , Rab8A + , and Rab10 + subcellular compartments in comparison with control conditions. By contrast, α 2 M* did not produce significant change in GLUT4 localization in early endocytic endosome (EEA1 + ) nor long loop endocytic recycling compartments (Rab11 + ) (Supplementary Figure S1a,b). Thus, these data suggest that α 2 M* promotes the GLUT4 traffic to PM by Rab4-dependent endocytic recycling and Rab8A and Rab10-exocytic route.

α2M* Enhances Insulin-Induced 2-NBDG Uptake
Considering the α 2 M* effect on the GLUT4 traffic to the PM, we evaluated the glucose uptake in HL-1 cardiomyocytes. Thus, cells were treated with α 2 M* for 2 to 30 min together with 2-NBDG, a glucose fluorescent analogue. Figure 4a,b shows that α 2 M* did not produce significant changes in 2-NBDG uptake with respect to the non-stimulated control. Thus, although α 2 M* was able to induce the GLUT4 traffic to PM, it had no effect on the glucose control. Considering that insulin induces GLUT4 traffic and the glucose uptake into different cell types, our interest was to evaluate if α 2 M* may have effect on the insulin response in HL-1 cardiomyocytes. For this, cells were treated with α 2 M* and insulin for 2 to 30 min together with 2-NBDG. Figure 4a shows that the combined treatment of α 2 M* and insulin at the first 5 min promptly enhanced the GLUT4 expression on the PM compared with the individual effect of α 2 M* or insulin. After this time, the combined treatment of α 2 M* and insulin produced a similar level of GLUT4 expression on the cell surface than insulin and major than α 2 M* alone. In the same way, α 2 M* + insulin induced a fast 2-NBDG uptake at the first 5 min of stimulus, which was similar with insulin alone after this time ( Figure 4b). The area under the curve analysis shows that α 2 M* + insulin significantly increased the 2-NBDG uptake both at the first 5 min as well as at 30 min of stimulus compared to insulin alone (Figure 4c,d). Using simple linear regression analysis, we found that α 2 M* + insulin increased the uptake rate respect to insulin stimulus (Figure 4e,f). These results suggest that α 2 M* enhances the insulin response, increasing GLUT4 traffic to PM and insulin-induced glucose uptake in HL-1 cardiomyocytes.   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, arbitrary unit. Values are expressed as mean ± SEM. * p < 0.05 vs. indicated conditions. ** p < 0.01 vs. indicated conditions. (e,f) Analysis of 2-NBDG uptake rate in cells treated with insulin (100 nM) or (α2M* 60 nM + insulin 100 nM) for 5 min at 37 °C. Simple linear regression analysis was performed by Infostat. * p < 0.05 indicates significant differences between insulin and α2M* + insulin.

α2M* Blocks aggLDL Intracellular Accumulation by LRP1
In a previous work we found that LRP1 is the main receptor responsible for the binding and endocytosis of aggLDL in HL-1 cardiomyocytes [20]. These lipoproteins bind to cluster II of α-subunit of LRP1, and this domain is also involved in the interaction with α2M* [13,14,20,34]. Other studies showed that anti-P3 antibodies reduced foam cell Figure 4. α 2 M* improves 2-NBDG uptake in HL-1 cardiomyocytes. (a) Cell surface protein detection assay to measure GLUT4 in cells treated with α 2 M* (60 nM), insulin (100 nM) or combined (α 2 M* + 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 α 2 M* and control (p < 0.05). # indicates significant differences between insulin and control (p < 0.05). * indicates significant differences between α 2 M* + 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 α 2 M* (60 nM), insulin (100 nM) or combined (α 2 M* + 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 α 2 M* + 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, arbitrary unit. Values are expressed as mean ± SEM. * p < 0.05 vs. indicated conditions. ** p < 0.01 vs. indicated conditions. (e,f) Analysis of 2-NBDG uptake rate in cells treated with insulin (100 nM) or (α 2 M* 60 nM + insulin 100 nM) for 5 min at 37 • C. Simple linear regression analysis was performed by Infostat. * p < 0.05 indicates significant differences between insulin and α 2 M* + insulin.

α 2 M* Blocks aggLDL Intracellular Accumulation by LRP1
In a previous work we found that LRP1 is the main receptor responsible for the binding and endocytosis of aggLDL in HL-1 cardiomyocytes [20]. These lipoproteins bind to cluster II of α-subunit of LRP1, and this domain is also involved in the interaction with α 2 M* [13,14,20,34]. Other studies showed that anti-P3 antibodies reduced foam cell formation through the blockade of LRP1 interaction with atherogenic lipoproteins in cluster II [27,28]. Here, we evaluated whether α 2 M* blocks aggLDL accumulation. HL-1 cardiomyocytes were incubated with α 2 M* together with DiI-labeled aggLDL (aggLDL-DiI) for 8 h at 37 • C. Figure 5a,b shows that aggLDL-DiI was accumulated in HL-1 cardiomyocytes, but this accumulation was inhibited by α 2 M*. In normal conditions, CE is stored at a low proportion in lipid droplets [41]. However, excessive aggLDL uptake leads to CE accumulation in lysosomes [42]. In previous studies, we showed that LRP1 did not localize in lysosomes in different cell types [21,37,43]. Lysosomal degradation of α 2 M* leads to an endocytic recycling of LRP1 to the cell surface [21]. Here, we analyzed the intracellular localization of LRP1 after aggLDL stimulation by confocal microscopy. Figure 5c shows that aggLDL generated a localization of LRP1 in the degradation compartments, similar to late endosomes/lysosomes [LAMP + ], with respect to α 2 M* stimulus and control condition. This effect of aggLDL on the distribution of LRP1 in degradation compartments was fully blocked by α 2 M*. These data indicate that α 2 M* inhibits the internalization of the aggLDL by LRP1 and the receptor transport into degradation compartments. formation through the blockade of LRP1 interaction with atherogenic lipoproteins in cluster II [27,28]. Here, we evaluated whether α2M* blocks aggLDL accumulation. HL-1 cardiomyocytes were incubated with α2M* together with DiI-labeled aggLDL (aggLDL-DiI) for 8 h at 37 °C. Figure 5a,b shows that aggLDL-DiI was accumulated in HL-1 cardiomyocytes, but this accumulation was inhibited by α2M*. In normal conditions, CE is stored at a low proportion in lipid droplets [41]. However, excessive aggLDL uptake leads to CE accumulation in lysosomes [42]. In previous studies, we showed that LRP1 did not localize in lysosomes in different cell types [21,37,43]. Lysosomal degradation of α2M* leads to an endocytic recycling of LRP1 to the cell surface [21]. Here, we analyzed the intracellular localization of LRP1 after aggLDL stimulation by confocal microscopy. Figure 5c shows that aggLDL generated a localization of LRP1 in the degradation compartments, similar to late endosomes/lysosomes [LAMP + ], with respect to α2M* stimulus and control condition. This effect of aggLDL on the distribution of LRP1 in degradation compartments was fully blocked by α2M*. These data indicate that α2M* inhibits the internalization of the aggLDL by LRP1 and the receptor transport into degradation compartments.  2.5. α2M* Counteracts the Impairment of GLUT4 Traffic to PM and Glucose Uptake Induced by aggLDL Previously, we found that aggLDL/LRP1 interaction leads to intracellular CE accumulation and impaired insulin response in HL-1 cardiomyocytes [20]. Here, we evaluated whether α 2 M* restores GLUT4 traffic to PM and glucose uptake impaired by aggLDL in these cells. For this, cells were treated with aggLDL, α 2 M* or both combined ligands for 8 h, then stimulated with insulin for 30 min, and the expression levels of GLUT4 on the cell surface were analyzed by biotin-labeling cell surface protein assays. As we previously found, in Figure 6a,b it is shown that aggLDL affected insulin-induced GLUT4 expression on the PM (Line 4), whereas α 2 M* did not modify the insulin effect at this time (Line 8). Moreover, α 2 M* alone induces the GLUT4 traffic to PM (Line 7) while aggLDL did not promote this effect (Line 3). Although the combination of α 2 M* and aggLDL did not produce significant changes in the GLUT4 levels in the cell surface (Line 5), α 2 M* counteracted the aggLDL deleterious effect, since it helped to promote GLUT4 translocation to cell surface by insulin (Line 6). The presence of α 2 M*, aggLDL, and both combined ligands induced the LRP1 expression on PM, which are in agreement with previous works [20,21,37]. Finally, we evaluated insulin-induced glucose uptake by HL-1 cardiomyocytes exposed to aggLDL and α 2 M*. Figure 6c

α2M* Prevented aggLDL-Induced Cardiac Damage
Galectin-1 and 3 (Gal-1 and Gal-3) are the strongest predictors of cardiac damage and heart failure [44,45]. Our interest was to study whether aggLDL, α 2 M*, and both combined ligands may affect galectins expression in HL-1 cardiomyocytes. Figure 7a,b show that aggLDL increased the Gal-1 and Gal-3 mRNA expression in HL-1 cardiomyocytes, while this effect was counteracted by α 2 M*. These results suggest that α 2 M* also prevents aggLDL-induced cardiac damage associated with myocardial dysfunctions.

α2M* Prevented aggLDL-Induced Cardiac Damage
Galectin-1 and 3 (Gal-1 and Gal-3) are the strongest predictors of cardiac damage and heart failure [44,45]. Our interest was to study whether aggLDL, α2M*, and both combined ligands may affect galectins expression in HL-1 cardiomyocytes. Figure 7a,b show that aggLDL increased the Gal-1 and Gal-3 mRNA expression in HL-1 cardiomyocytes, while this effect was counteracted by α2M*. These results suggest that α2M* also prevents aggLDL-induced cardiac damage associated with myocardial dysfunctions.

Discussion
It is well established that LRP1 acts as a scaffold protein for the insulin receptor (IR), which is considered critical for the intracellular insulin signaling activation in cardiomyocytes, neurons, and hepatocytes [20,32,33]. In the present study we showed that α2M*, mediated by its interaction with LRP1, enhanced the insulin response, increasing insulin-induced GLUT4 traffic to PM and the glucose uptake in HL-1 cardiomyocytes. By contrast, it has also been demonstrated that aggLDL, through its binding to LRP1, induces CE accumulation and impairs insulin-induced IR activation in this cell type [20]. Here, we found that α2M* counteracted the antagonist effect of aggLDL on LRP1, blocking the lipid loading induced by this modified lipoprotein and improved the insulin response in HL-1 cardiomyocytes. In addition, α2M* was able to prevent aggLDL-induced cardiac damage characterized by a decrease in Gal-1 and Gal-3 expression in HL-1 cardiomyocytes.
In previous works, an increased level of a cardiac form of α2M (c-α2M) in patients with insulin resistance was found [46,47], which was expressed in human and rat hearts [16,17]. Through its interaction with LRP1, c-α2M improves cardiac cell function and induces hypertrophic in ventricular cardiomyocytes via ERK1/2 and PI3K/Akt activation [16,18,19]. Here, we show that mouse c-α2M, the analogue form of human α2M, is also expressed in HL-1 cardiomyocytes, which is significantly increased in cells treated with aggLDL. This c-α2M could be secreted as a native form and activated by extracellular

Discussion
It is well established that LRP1 acts as a scaffold protein for the insulin receptor (IR), which is considered critical for the intracellular insulin signaling activation in cardiomyocytes, neurons, and hepatocytes [20,32,33]. In the present study we showed that α 2 M*, mediated by its interaction with LRP1, enhanced the insulin response, increasing insulininduced GLUT4 traffic to PM and the glucose uptake in HL-1 cardiomyocytes. By contrast, it has also been demonstrated that aggLDL, through its binding to LRP1, induces CE accumulation and impairs insulin-induced IR activation in this cell type [20]. Here, we found that α 2 M* counteracted the antagonist effect of aggLDL on LRP1, blocking the lipid loading induced by this modified lipoprotein and improved the insulin response in HL-1 cardiomyocytes. In addition, α 2 M* was able to prevent aggLDL-induced cardiac damage characterized by a decrease in Gal-1 and Gal-3 expression in HL-1 cardiomyocytes.
In previous works, an increased level of a cardiac form of α 2 M (c-α 2 M) in patients with insulin resistance was found [46,47], which was expressed in human and rat hearts [16,17].
Through its interaction with LRP1, c-α 2 M improves cardiac cell function and induces hypertrophic in ventricular cardiomyocytes via ERK1/2 and PI 3 K/Akt activation [16,18,19]. Here, we show that mouse c-α 2 M, the analogue form of human α 2 M, is also expressed in HL-1 cardiomyocytes, which is significantly increased in cells treated with aggLDL. This cα 2 M could be secreted as a native form and activated by extracellular proteinases in cardiac tissue, which is then recognized and internalized by LRP1. However, it would also be interesting to know if c-α 2 M may be a product of extravasation from systemic α 2 M or only by local production at the cardiac level, and if its expression can be inducible by the aggLDL itself. In this way, α 2 M* could have an agonist effect on the LRP1 function counteracted the impairment in the insulin response induced by lipid accumulation in cardiomyocytes.
In previous studies, we found that α 2 M* activates PI 3 K/Akt or MAPK/ERK or both intracellular pathways depending on cell types [21,36]. It is known that these intracellular pathways mediate the LRP1 intracellular traffic to PM in MIO-M1 cells induced by α 2 M* or insulin [21,37]. In addition, PI 3 K/Akt and MAPK/ERK activation by insulin also leads to GLUT4 translocation to the PM [20,31]. Our present results showed that α 2 M* induced phosphorylation of Akt and ERK at short times of stimulus, suggesting that α 2 M* promotes a quick activation of both intracellular pathways in HL-1 cardiomyocytes. Then, we found that α 2 M* increased GLUT4 levels on the cell surface mediated by PI 3 K/Akt and MAPK/ERK intracellular signaling activation. Moreover, α 2 M* also increased sortilin levels at the PM, which is a constitutive protein of GSVs together with GLUT4 and LRP1 [37]. The Rab small GTPases play a critical role in the GLUT4 traffic to PM induced by insulin [31,37]. Here, we found that the α 2 M*-induced GLUT4 traffic involved the participation of Rab4, Rab8A and Rab10 GTPases from short loop endocytic route and exocytic pathway. Increased expression of GLUT4 on PM by α 2 M* significantly enhance insulin-induced glucose uptake, which was higher than insulin alone in the first 5 min of stimulation. However, α 2 M* alone did not produce significant glucose input respect to the non-stimulated control. This differential effect of α 2 M* with respect to insulin on the glucose uptake may be related to the capability of insulin to activate not only intracellular pathways such as PI 3 K/Akt and MAPK/ERK but also different proteins involved in GSVs retention inside the cell, tethering and fusion of GSVs to PM and activity of GLUT4, which α 2 M* would not activate [31,48]. In this way, it has been demonstrated that insulin stimulates endocytosis and exocytosis of GLUT4 through the participation of several molecular mediators such as ESYT1 and TC10 phosphorylated by Cdk5 [49,50]; MYO1C and MYO5 phosphorylated by CamKII [51,52]; and RIP140 and SEC5 phosphorylated by PKC [53,54]. Further studies are needed to explore if α 2 M* plays some function during the endocytosis and exocytosis of GLUT4. Nevertheless, the combined stimulation of α 2 M* and insulin showed a significant enhancement in the insulin response, characterized by an increased GLUT4 traffic to PM and insulin-induced 2-NBDG uptake in HL-1 cardiomyocytes. This improvement of the insulin response may be mediated through the enrichment of GLUT4 in the cell surface induced by α 2 M*, enabling a major number of glucose transporters available in the cells stimulated with insulin. Another possibility is that α 2 M* increases the endocytic recycling of LRP1, which could facilitate the molecular association between LRP1 and IR [20] and potentiate the IR activation induced by insulin. This evidence suggest that α 2 M* would have an important role as an insulin sensitizing agent in the heart, as proposed by other studies about patients with metabolic disturbances and heart failure [55].
In addition to α 2 M*, LRP1 can interact with different ligands with more or less affinity [15]. One of these ligands is aggLDL, which generates mainly CE uptake in cardiomyocytes, hypercholesterolemia being a promoting factor for cardiac dysfunction [20,56,57]. The aggLDL is recognized by a sequence located on the CR8/CR9 domain in the cluster II of the extracellular alpha chain of LRP1, and this interaction is mediated by heparin sulfate proteoglycans (HSPG) [15,27,28]. While α 2 M* interacts directly with LRP1 by clusters I/II with high affinity, which could represent a steric impediment to the binding of aggLDL [13,14,20,34]. In previous studies, we found that aggLDL promotes the molecular dissociation between LRP1 and IR with impairment of insulin-induced IR activation [20]. In this sense, our results showed that α 2 M* prevented aggLDL intracellular accumulation by LRP1 and the anomalous aggLDL-induced LRP1 localization in lysosomes. Moreover, α 2 M* counteracted the GLUT4 trafficking to PM and glucose uptake affected by aggLDL. In contrast to the agonist effect of α 2 M*/LRP1 on insulin response, aggLDL would be an antagonist ligand of LRP1 in this function in HL-1 cardiomyocytes. These results could underlie the link between intracellular CE accumulation in cardiomyocytes and cardiac insulin signaling abnormalities.
Finally, α 2 M* also prevented aggLDL-induced cardiac damage evidenced by a decreased level of Gal-1 and Gal-3, key mediators of cardiac lipotoxicity and heart failure [44,45]. Recent works have proposed LRP1 as an ideal target to prevent myocardial dysfunction [25][26][27][28]. In this way, it has been demonstrated that a short peptide, termed SP16, derived from serine-protease inhibitors (SERPINs), interacts with LRP1 and produces an agonist effect on the reduction of myocardial injury and preservation of cardiac systolic function in experimental acute myocardial infarction (AMI) [25,26]. On the other hand, by the use of antibodies against the CR8/CR9 domain in the cluster II of LRP1, foam cell formation and atherosclerosis development was prevented [27,28]. Further studies are needed to know if the use of α 2 M* may alter other processes in the heart such as inflammation or cardiac remodeling [25,26].
In conclusion, α 2 M* is an agonist ligand of LRP1 improving the insulin response in lipid-loaded HL-1 cardiomyocytes. Figure 8 is a schematic representation of α 2 M* counteracting the antagonist effect of aggLDL on LRP1, improving the insulin response characterized by PI 3 K/Akt and MAPK/ERK signaling activation as well as GLUT4 translocation to PM and glucose uptake in cardiomyocytes. All our findings may have important therapeutic implications in relation to the role of α 2 M* in the cardiac insulin response associated with hypercholesterolemia. cardiac systolic function in experimental acute myocardial infarction (AMI) [25,26]. On the other hand, by the use of antibodies against the CR8/CR9 domain in the cluster II of LRP1, foam cell formation and atherosclerosis development was prevented [27,28]. Further studies are needed to know if the use of α2M* may alter other processes in the heart such as inflammation or cardiac remodeling [25,26].
In conclusion, α2M* is an agonist ligand of LRP1 improving the insulin response in lipid-loaded HL-1 cardiomyocytes. Figure 8 is a schematic representation of α2M* counteracting the antagonist effect of aggLDL on LRP1, improving the insulin response characterized by PI3K/Akt and MAPK/ERK signaling activation as well as GLUT4 translocation to PM and glucose uptake in cardiomyocytes. All our findings may have important therapeutic implications in relation to the role of α2M* in the cardiac insulin response associated with hypercholesterolemia.

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). These cells showed cardiac characteristics similar of adult cardiomyocytes [58]. HL-1 cardiomyocytes were maintained in Claycomb Medium (Sigma-Aldrich, St. Louis, MO,

LDL Isolation and Modification
LDL (d 1.019-d 1.063 g/mL) was isolated by ultracentrifugation using KBr gradients, in the density range 1.019-1.063 g/mL, from pools of plasma of normocholesterolemic volunteers. A Pierce kit (#23225, ThermoFisher Scientific (Rockford, IL, USA) was used for the protein quantification. Apolipoprotein B protein integrity was tested by SDS-PAGE in 10% acrylamide gels. Aggregated LDL was obtained by vortexing LDL in PBS 1X for 5 min at room temperature [20]. AggLDL was suspended in PBS 1X to a protein concentration of 100 µg/mL.

DiI-Staining of LDL
DiI (1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, Invitrogen) is a lipophilic dialkylcarbocyanine that binds to lipoproteins and emits fluorescence at 565 nm. The LDL (100 µg/mL) was incubated with DiI in a proportion of 3 µL per 1 mg of lipoprotein in PBS 1X at 37 • C for 12 h and was then exhaustively dialyzed in PBS 1X for 24 h to eliminate the free DiI and filtered through a 0.22 µm filter [20]. Finally, DiI-LDL was aggregated mechanically by vortexing.

Western Blot Analysis
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 protein extracts were separated by electrophoresis on 10% SDSpolyacrylamide gels [61] and transferred to a nitrocellulose membrane [62] (GE Healthcare Life Science, Amsterdam, The Netherlands). Nonspecific binding was blocked with 5% non-fat dry milk in a Tris-HCl-0.01% Tween 20 (TBS-T) buffer for 60 min at room temperature. The nitrocellulose membranes were incubated overnight at 4 • C with primary antibodies, and secondary antibodies raised in goat anti-mouse IgG 680CW and goat anti-rabbit IgG 800CW (LI-COR Biosciences, Lincoln, NE, USA) diluted 1/10,000 for 1 h at room temperature. The specific bands were developed using Odyssey CLx near-infrared fluorescence imaging system (LI-COR) and were quantified by densitometric analysis using Image Studio Software (LI-COR).

Confocal Microscopy
The procedures were followed, as previously described [21,22]. Briefly, HL-1 cardiomyocytes were cultured on cover glass. After different stimulus the cells were washed with PBS 1X, fixed with 4% paraformaldehyde (PFA), quenched with 50 mM NH 4 Cl, permeabilized for 30 min with 0.5% (v/v) saponin and blocked with 2% bovine serum albumin (BSA) [20]. Primary antibodies (diluted from 1/100 to 1/250) were used for 1 h, and then secondary antibodies conjugated with Alexa Fluor (1/800) and Hoechst 33,258 colorant (1/2000) were used for 1 h. Finally, cells were mounted on glass slides with Mowiol 4-88 reagent from Calbiochem (Merck, Darmstadt, Germany). Fluorescent images were obtained with an Olympus FluoView FV1200 confocal microscope (Olympus, New York, NY, USA). Optical sections of the cells were obtained in 0.25-µm steps perpendicular to the z-axis. Images being processed using the FV10-ASW Viewer 3.1 (Olympus, New York, NY, USA) and quantified by ImageJ software. For microscope quantification of the colocalization level, a JACoP plug-in from ImageJ software (National Institutes of Health, New York, NY, USA) was used [63].

Biotin-Labeling Cell Surface Protein Assay
To determine the level of proteins at the cell surface we used a biotin-labeling protein assay (EZ-Link™ Sulfo-NHS-SS-Biotin [cat: 21331], Thermo Scientific, Rockford, IL, USA) as was previously described [20]. After stimulus, cells were incubated with a 0.12 mg/mL of biotin solution for 2 h at 4 • C, and then with 0.1 mM glycine solution for 30 min. The biotinylated proteins were pulled down using streptavidin-conjugated agarose beads (Pierce™ Streptavidin Agarose [cat: 20353], Thermo Scientific) for 2 h at room temperature. The biotinylated-plasma membrane proteins were eluted, then treated for Western blot and the nitrocellulose membranes were incubated with primary antibodies overnight at 4 • C and secondary antibodies were raised in goat anti-mouse IgG 680CW and goat anti-rabbit IgG 800CW (LI-COR) diluted 1/10,000 for 1 h at room temperature. The specific bands were developed using Odyssey CLx fluorescence imaging system (LI-COR) and were quantified by densitometric analysis using Image Studio Software (LI-COR). As the loading control of PM protein, total protein biotinylated-ATP1A1 and β-actin were used, respectively. Each biotinylated protein in the PM was related to biotinylated-ATP1A1 protein.

Cell Surface Protein Detection Assay
The procedure was followed, as previously described [37]. Briefly, after different stimulus cells were rinsed with cold PBS 1X, fixed with 4% (v/v) PFA, washed with 0.1 mM glycine, and blocked with 5% (v/v) horse serum for 30 min on ice. Cells were incubated with anti-GLUT4 (1/1000) and anti-ATP1A1 (1/2000) for 1 h on ice, followed by incubation with goat anti-rabbit IgG 800CW and goat anti-mouse IgG 680CW (LI-COR) secondary antibodies (1/10,000) for 1 h on ice. Fluorescence intensity was measured using the Odyssey CLx fluorescence imaging system (LI-COR) and quantified by densitometry using Image Studio Software (LI-COR). The content of GLUT4 in the PM was related to ATP1A1 protein.
For other experiments, before stimulus, cells were pre-incubated for 30 min with 40 µM wortmannin or PD98059.

Real Time-PCR
The cells were exposed to the different stimuli and treated with the TRIzol ® reagent (Invitrogen, Buenos Aires, Argentina) for total RNA extraction. One µg/20 µL of RNA was reverse transcribed using random hexaprimers and reverse transcriptase. The primers listed below were used to quantify the transcripts of Gal-1, Gal-3 and β-actin. The results were normalized to RT-PCR products of β-actin transcripts. For quantification we used real-time qRT-PCR (ABI 7500 Sequence Detection System, Applied Biosystems, Foster City, CA) and Sequence Detection software v1.4. Amplification conditions involved 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. Relative gene expression was calculated by the 2-Ct method. Samples was analyzed in triplicate. No amplification was observed using water or RNA samples incubated without reverse transcriptase during cDNA synthesis.

Statistical Treatment of Data
For Western blot and cell-surface protein detection assay, the data was expressed as Mean ± SEM and comparisons between groups were analyzed by one-way ANOVA followed by Dunnett's post-hoc analysis (GraphPad Prism 5.0, San Diego, CA, USA) or Student's t-test. For confocal microscopy, at least 20 cells/condition were analyzed and data was expressed as Mean ± SD. The averages of the vesicle percentages containing colocalized proteins were calculated using the Manders' coefficients. Comparisons between groups were analyzed by one-way ANOVA followed by Dunnett's post-hoc analysis (GraphPad Prism 5.0) or Student's t-test. Values of p < 0.05 were considered to be significant.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/ijms22136915/s1, Figure S1  Acknowledgments: We thank Carlos Mas and Cecilia Sampedro for excellent technical assistance with confocal microscopy in CEMINCO (Centro de Micro and Nanoscopia Cordoba).

Conflicts of Interest:
The authors declare no conflict of interest.  Low-density lipoprotein receptor-related protein-1