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Impaired LDL receptor-related protein 1 translocation correlates with improved dyslipidemia and atherosclerosis in apoE-deficient mice.

Gordts PL, Bartelt A, Nilsson SK, Annaert W, Christoffersen C, Nielsen LB, Heeren J, Roebroek AJ - PLoS ONE (2012)

Bottom Line: Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes.These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling.These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Mouse Genetics, Center for Human Genetics, KU Leuven, Leuven, Belgium.

ABSTRACT

Objective: Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1) dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE.

Methods and results: LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with apoE-deficient mice. In the absence of apoE, relative to LRP1 wild-type animals, LRP1 mutated mice showed an increased clearance of postprandial lipids despite a compromised LRP1 endocytosis rate and inefficient insulin-mediated translocation of the receptor to the plasma membrane, likely due to inefficient slow recycling of the mutated receptor. Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes. One year-old apoE-deficient mice having the dysfunctional LRP1 revealed a 3-fold decrease in spontaneous atherosclerosis development and a 2-fold reduction in LDL-cholesterol levels.

Conclusion: These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling. These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.

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Inefficient insulin-mediated LRP1 translocation and impaired slow recycling.A–B, LRP1 staining in primary hepatocytes before and after insulin stimulation (A) and immunoblot analysis (B) in liver plasma membrane (PM) extracts [ApoE−/− (□) and apoE−/−LRP1n2/n2 (▪)] before and after insulin injection (n = 3–4; bars are 20 µm). C, Cell Fractionation analysis of LRP1+/+ and LRP1n2/n2 MEFs. D–F, Steady-state internalization or binding (4°C) of FITC-α2M in mouse embryonic fibroblasts (MEFs) (D), steady-state internalization of FITC-α2M in MEFs in the absence (−) or presence (+) of either a lysosomal inhibitor, chloroquine (E), or a proteasomal inhibitor, MG123 (F) [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)].G–H, Fast (G) and slow (H) recycling kinetics of LRP1 in MEFs at the indicated time intervals [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)]. Data are mean±SEM. *P<0.05, **P<0.005, ***P<0.001.
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pone-0038330-g004: Inefficient insulin-mediated LRP1 translocation and impaired slow recycling.A–B, LRP1 staining in primary hepatocytes before and after insulin stimulation (A) and immunoblot analysis (B) in liver plasma membrane (PM) extracts [ApoE−/− (□) and apoE−/−LRP1n2/n2 (▪)] before and after insulin injection (n = 3–4; bars are 20 µm). C, Cell Fractionation analysis of LRP1+/+ and LRP1n2/n2 MEFs. D–F, Steady-state internalization or binding (4°C) of FITC-α2M in mouse embryonic fibroblasts (MEFs) (D), steady-state internalization of FITC-α2M in MEFs in the absence (−) or presence (+) of either a lysosomal inhibitor, chloroquine (E), or a proteasomal inhibitor, MG123 (F) [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)].G–H, Fast (G) and slow (H) recycling kinetics of LRP1 in MEFs at the indicated time intervals [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)]. Data are mean±SEM. *P<0.05, **P<0.005, ***P<0.001.

Mentions: We evaluated whether the mutation had an additional effect on insulin-mediated LRP1 translocation to the PM. In primary hepatocytes from apoE−/− mice, LRP1 translocated to the PM upon insulin stimulation. In apoE−/−LRP1n2/n2 hepatocytes, however, LRP1 did not translocate efficiently (Figure 4A). Additionally, a different cellular distribution of LRP1 with a predominantly juxta-nuclear staining in the apoE−/−LRP1n2/n2 hepatocytes compared to apoE−/− hepatocytes was observed. In vivo, absence of increased LRP1 at the PM after insulin injection was also found in preparations of purified PM isolated from apoE−/−LRP1n2/n2 livers (Figure 4B). Cell fractionation of wild-type LRP1 MEFs revealed that mature wild-type LRP1 (Figure 4C; LRP1) has a bimodal-distribution with the largest amounts of mature LRP1 present in the cis-Golgi to trans-golgi network (TGN) and the endosomal fractions [29]. Fractionation of MEFs with the LRP1 NPxYxxL mutation showed a unimodal distribution, as LRP1-β is not abundantly present in the endosomal fractions but the bulk of the protein is rather restricted to Golgi and recycling endosomal fractions (Figure 4C; LRP1-β). These distribution differences are suggestive for an altered cellular trafficking of LRP1 carrying the NPxYxxL inactivation. Initial binding to the plasma membrane of α2M was not different between LRP1+/+ and LRP1n2/n2 MEFs. The internalization rate, however, was significantly reduced by almost 25% (Figure 4D). Absence or presence of either lysosomal inhibitor chloroquine (Figure 4E), or the proteasomal inhibitor, MG132 (Figure 4F), did, however, not significantly improve the α2M internalization rate in LRP1n2/n2 MEFs. As LRP1 degradation was not significantly enhanced, we evaluated fast LRP1 recycling. After incubation with a fluorochrome labelled specific LRP1 antibody (Alexa488-5A6), the quantity of LRP1 recycling back to the cell surface was measured. LRP1 fast recycling kinetics in LRP1+/+ and LRP1n2/n2 MEFs were, however, almost identical (Figure 4G). Therefore, recycling from inside the cell to the plasma membrane at steady state was also investigated as described [35]. Cells were incubated with Alexa488-5A6 for 30 min, washed and chased for 2 h, to achieve a steady-state distribution. After this step, return of labelled LRP1 from perinuclear endosomal recycling compartments, slow recycling, was measured and revealed that 2 h after chase up to 75% of labelled wild-type LRP1 recycled back to surface, whereas only 50% of labelled LRP1 in the LRP1n2/n2 MEFs reached the cell surface (Figure 4H). These results indicate that the NPxYxxL motif is important for insulin-mediated translocation of LRP1 and slow perinuclear endosomal recycling.


Impaired LDL receptor-related protein 1 translocation correlates with improved dyslipidemia and atherosclerosis in apoE-deficient mice.

Gordts PL, Bartelt A, Nilsson SK, Annaert W, Christoffersen C, Nielsen LB, Heeren J, Roebroek AJ - PLoS ONE (2012)

Inefficient insulin-mediated LRP1 translocation and impaired slow recycling.A–B, LRP1 staining in primary hepatocytes before and after insulin stimulation (A) and immunoblot analysis (B) in liver plasma membrane (PM) extracts [ApoE−/− (□) and apoE−/−LRP1n2/n2 (▪)] before and after insulin injection (n = 3–4; bars are 20 µm). C, Cell Fractionation analysis of LRP1+/+ and LRP1n2/n2 MEFs. D–F, Steady-state internalization or binding (4°C) of FITC-α2M in mouse embryonic fibroblasts (MEFs) (D), steady-state internalization of FITC-α2M in MEFs in the absence (−) or presence (+) of either a lysosomal inhibitor, chloroquine (E), or a proteasomal inhibitor, MG123 (F) [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)].G–H, Fast (G) and slow (H) recycling kinetics of LRP1 in MEFs at the indicated time intervals [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)]. Data are mean±SEM. *P<0.05, **P<0.005, ***P<0.001.
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Related In: Results  -  Collection

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pone-0038330-g004: Inefficient insulin-mediated LRP1 translocation and impaired slow recycling.A–B, LRP1 staining in primary hepatocytes before and after insulin stimulation (A) and immunoblot analysis (B) in liver plasma membrane (PM) extracts [ApoE−/− (□) and apoE−/−LRP1n2/n2 (▪)] before and after insulin injection (n = 3–4; bars are 20 µm). C, Cell Fractionation analysis of LRP1+/+ and LRP1n2/n2 MEFs. D–F, Steady-state internalization or binding (4°C) of FITC-α2M in mouse embryonic fibroblasts (MEFs) (D), steady-state internalization of FITC-α2M in MEFs in the absence (−) or presence (+) of either a lysosomal inhibitor, chloroquine (E), or a proteasomal inhibitor, MG123 (F) [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)].G–H, Fast (G) and slow (H) recycling kinetics of LRP1 in MEFs at the indicated time intervals [twice in triplicate, LRP1+/+ (□) and LRP1n2/n2 (▪)]. Data are mean±SEM. *P<0.05, **P<0.005, ***P<0.001.
Mentions: We evaluated whether the mutation had an additional effect on insulin-mediated LRP1 translocation to the PM. In primary hepatocytes from apoE−/− mice, LRP1 translocated to the PM upon insulin stimulation. In apoE−/−LRP1n2/n2 hepatocytes, however, LRP1 did not translocate efficiently (Figure 4A). Additionally, a different cellular distribution of LRP1 with a predominantly juxta-nuclear staining in the apoE−/−LRP1n2/n2 hepatocytes compared to apoE−/− hepatocytes was observed. In vivo, absence of increased LRP1 at the PM after insulin injection was also found in preparations of purified PM isolated from apoE−/−LRP1n2/n2 livers (Figure 4B). Cell fractionation of wild-type LRP1 MEFs revealed that mature wild-type LRP1 (Figure 4C; LRP1) has a bimodal-distribution with the largest amounts of mature LRP1 present in the cis-Golgi to trans-golgi network (TGN) and the endosomal fractions [29]. Fractionation of MEFs with the LRP1 NPxYxxL mutation showed a unimodal distribution, as LRP1-β is not abundantly present in the endosomal fractions but the bulk of the protein is rather restricted to Golgi and recycling endosomal fractions (Figure 4C; LRP1-β). These distribution differences are suggestive for an altered cellular trafficking of LRP1 carrying the NPxYxxL inactivation. Initial binding to the plasma membrane of α2M was not different between LRP1+/+ and LRP1n2/n2 MEFs. The internalization rate, however, was significantly reduced by almost 25% (Figure 4D). Absence or presence of either lysosomal inhibitor chloroquine (Figure 4E), or the proteasomal inhibitor, MG132 (Figure 4F), did, however, not significantly improve the α2M internalization rate in LRP1n2/n2 MEFs. As LRP1 degradation was not significantly enhanced, we evaluated fast LRP1 recycling. After incubation with a fluorochrome labelled specific LRP1 antibody (Alexa488-5A6), the quantity of LRP1 recycling back to the cell surface was measured. LRP1 fast recycling kinetics in LRP1+/+ and LRP1n2/n2 MEFs were, however, almost identical (Figure 4G). Therefore, recycling from inside the cell to the plasma membrane at steady state was also investigated as described [35]. Cells were incubated with Alexa488-5A6 for 30 min, washed and chased for 2 h, to achieve a steady-state distribution. After this step, return of labelled LRP1 from perinuclear endosomal recycling compartments, slow recycling, was measured and revealed that 2 h after chase up to 75% of labelled wild-type LRP1 recycled back to surface, whereas only 50% of labelled LRP1 in the LRP1n2/n2 MEFs reached the cell surface (Figure 4H). These results indicate that the NPxYxxL motif is important for insulin-mediated translocation of LRP1 and slow perinuclear endosomal recycling.

Bottom Line: Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes.These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling.These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Mouse Genetics, Center for Human Genetics, KU Leuven, Leuven, Belgium.

ABSTRACT

Objective: Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1) dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE.

Methods and results: LRP1 knock-in mice carrying an inactivating mutation in the NPxYxxL motif were crossed with apoE-deficient mice. In the absence of apoE, relative to LRP1 wild-type animals, LRP1 mutated mice showed an increased clearance of postprandial lipids despite a compromised LRP1 endocytosis rate and inefficient insulin-mediated translocation of the receptor to the plasma membrane, likely due to inefficient slow recycling of the mutated receptor. Postprandial lipoprotein improvement was explained by increased hepatic clearance of triglyceride-rich remnant lipoproteins and accompanied by a compensatory 1.6-fold upregulation of LDLR expression in hepatocytes. One year-old apoE-deficient mice having the dysfunctional LRP1 revealed a 3-fold decrease in spontaneous atherosclerosis development and a 2-fold reduction in LDL-cholesterol levels.

Conclusion: These findings demonstrate that the NPxYxxL motif in LRP1 is important for insulin-mediated translocation and slow perinuclear endosomal recycling. These LRP1 impairments correlated with reduced atherogenesis and cholesterol levels in apoE-deficient mice, likely via compensatory LDLR upregulation.

Show MeSH
Related in: MedlinePlus