Macrophage LRP1 Promotes Diet-Induced Hepatic Inflammation and Metabolic Dysfunction by Modulating Wnt Signaling

Hepatic inflammation is associated with the development of insulin resistance, which can perpetuate the disease state and may increase the risk of metabolic syndrome and diabetes. Despite recent advances, mechanisms linking hepatic inflammation and insulin resistance are still unclear. The low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is highly expressed in macrophages, adipocytes, hepatocytes, and vascular smooth muscle cells. To investigate the potential role of macrophage LRP1 in hepatic inflammation and insulin resistance, we conducted experiments using macrophage-specific LRP1-deficient mice (macLRP1−/−) generated on a low-density lipoprotein receptor knockout (LDLR−/−) background and fed a Western diet. LDLR−/−; macLRP1−/− mice gained less body weight and had improved glucose tolerance compared to LDLR−/− mice. Livers from LDLR−/−; macLRP1−/− mice displayed lower levels of gene expression for several inflammatory cytokines, including Ccl3, Ccl4, Ccl8, Ccr1, Ccr2, Cxcl9, and Tnf, and reduced phosphorylation of GSK3α and p38 MAPK proteins. Furthermore, LRP1-deficient peritoneal macrophages displayed altered cholesterol metabolism. Finally, circulating levels of sFRP-5, a potent anti-inflammatory adipokine that functions as a decoy receptor for Wnt5a, were elevated in LDLR−/−; macLRP1−/− mice. Surface plasmon resonance experiments revealed that sFRP-5 is a novel high affinity ligand for LRP1, revealing that LRP1 regulates levels of this inhibitor of Wnt5a-mediated signaling. Collectively, our results suggest that LRP1 expression in macrophages promotes hepatic inflammation and the development of glucose intolerance and insulin resistance by modulating Wnt signaling

Correction: LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages.

Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis

Postprandial serum lipids remain elevated in macLRP1-/- mice.

<p><b>(a</b>) Mice (macLRP1-/-, n = 11; LRP1+/+, n = 10) were fed Western diet for 3 weeks, and were then injected with Triton WR-1339 (500 mg/kg body weight), and triglyceride levels in the plasma were determined at 0, 30, 60 and 90 min following injection. The rate of triglyceride synthesis is shown. Error bars show the SEM. (<b>b</b>) LRP1+/+ mice (closed symbols, n = 5) or macLRP1-/- mice (open symbols, n = 5) fed a Chow diet were fasted and then received an intragastric olive oil load. Blood samples were collected at the indicated times, and plasma triglyceride levels measured. Error bars show the SEM. (Data were analyzed for statistical significance employing a two way ANOVA which confirmed significant effects for Genotype (p = 0.0012) and Time (p = 0.0001) with no significant Genotype X Time interaction).</p

No changes in ABCA1 and ApoE protein and mRNA levels in macLRP1-/- macrophages.

<p>(<b>a</b>) Cell extracts from thioglycollate-elicited peritoneal macrophages isolated from mice fed a Western diet for 11 weeks were subjected to immunoblot analysis for ABCA1 or ApoE (n = 4). (<b>b</b>) ABCA1 protein levels and (<b>c</b>) ApoE protein levels normalized to GAPDH were quantified using NIH ImageJ software. (<b>d,e</b>) Quantitative RT-PCR was employed to measure levels of <i>Abca1</i> (<b>d</b>) and <i>apoE</i> (<b>e</b>) and <i>Abcg1</i> (<b>f</b>) mRNA (n = 3). (<b>g</b>) Lipase activity was measured following heparin elution from LRP1+/+ and macLRP1-/- macrophages. (*p<0.001, n = 4 Student’s t-test comparing LRP1+/+ with macLRP1-/-).</p

Effective deletion of the <i>Lrp1</i> gene in elicited macrophages, bone marrow-derived macrophages, and Kupffer cells.

<p>(<b>a</b>) Quantitative real-time PCR was performed on bone marrow derived macrophages to determine <i>Lrp1</i> levels (**p = 0.005, Student’s t-test comparing LRP1+/+ with macLRP1-/-, n = 3 independent experiments). (<b>b</b>) Elicited peritoneal-derived macrophages from LRP1+/+ and macLRP1-/- mice were measured for their ability to internalize activated forms of ¹²⁵I-labeled α₂-macroglobulin (*p = 0.003, Student’s t-test comparing LRP1+/+ with macLRP1-/-, n = 2 independent experiments). (<b>c</b>) macLRP1-/- (open symbols) and LRP1+/+ (closed symbols) mice were injected with ¹²⁵I-α₂M* alone or with excess RAP. Plasma was sampled over time and counted. Counts were normalized to the amount of radioactivity detected at 1 min following injection. (¹²⁵I-α₂M*, n = 6; ¹²⁵I-α₂M* + RAP, n = 3). Error bars denote SEM. (<b>d-i</b>) Formalin-fixed paraffin-embedded liver sections from Western-fed LRP1+/+ (<b>d-f</b>) and macLRP1-/- mice (<b>g-i</b>) were stained with Alexa 488-anti-Mac-2 (<b>d,g</b>) and Alexa 546-anti-LRP1 (<b>e,h</b>) antibodies. Fluorescent channels merged with phase contrast images of the tissue (<b>f,i</b>) show yellow co-staining of LRP1 and Mac-2 in LRP1+/+ livers (<b>f</b>). No co-staining was observed in the macLRP1-/- livers (<b>i</b>). Livers from three sibling pairs of mice were stained. Representative fields from one pair are shown.</p

Normal lipase activity in macLRP1-/- mice.

<p>Adult mice were fed either Western or normal chow diet for 3 weeks. Following fasting, the epididymal fat (<b>a</b>), livers (<b>b</b>), soleus muscles (<b>c</b>) and gastrocnemius muscles (<b>d</b>) were harvested. Lipase was eluted from cell surfaces with heparin, and quantified. (Chow diet: macLRP1-/-, n = 4; LRP1+/+, n = 3; Western diet: macLRP1-/-, n = 6; LRP1+/+, n = 6) (ns, not significant as determined by Student’s t test comparing LRP1+/+ with macLRP1-/-).</p

Peritoneal macrophages from macLRP1-/- mice are defective in cholesterol ester accumulation.

<p>(<b>a</b>) LRP1+/+ (<i>upper panel</i>) or macLRP1-/- mice (<i>lower panel</i>) were fed a Western diet for 11 weeks. After this period, thioglycollate-elicited peritoneal macrophages were analyzed by Oil red O staining. (<b>b</b>). Quantification of the percentage of cells containing Oil red O positive staining. Macrophages from LRP1+/+ (n = 3) and macLRP1-/- (n = 3) were pooled and stained with Oil red O. Four fields for each genotype were counted. (*p = 0.0016, Student’s t-test comparing LRP1+/+ with macLRP1-/-). (<b>c,d</b>) Thioglycollate-elicited peritoneal macrophages from LRP1+/+ and macLRP1-/- (n = 3 for both) were isolated from mice following 11 weeks (<b>c</b>) or 3 weeks (<b>d</b>) on a Western diet, and cholesterol levels were measured. (* p<0.05, **p<0.004, Student’s t-test comparing LRP1+/+ with macLRP1-/-, n = 7). TC, total cholesterol; FC, free cholesterol; CE, cholesterol ester.</p

Macrophages from macLRP1-/- mice are defective in internalizing aggregated LDL.

<p><b>(a)</b> Thioglycollate-elicited peritoneal macrophages from LRP1+/+ and macLRP1-/- mice were cultured in DMEM supplemented with 5% delipidated fetal calf serum containing 80 μg/ml LDL, oxidized LDL, VLDL, or chlylomicrons for 3 days at 37°C. Lipids were then extracted and total cholesterol was measured and normalized to total cell protein. (<b>b,c</b>) Thioglycollate-elicited peritoneal macrophages from LRP1+/+ and macLRP1-/- mice were cultured in DMEM supplemented with 5% delipidated fetal calf serum. DiI-labeled aggregated LDL was then incubated with LRP1+/+ (<b>b</b>) or macLRP1-/- macrophages (<b>c</b>) for 24 h at 37°C and analyzed for lipoprotein internalization by fluorescence microscopy. (<b>d</b>) The extent of internalized fluorescence was quantified using Velocity Software (*p<0.001, Student’s t-test comparing LRP1+/+ with macLRP1-/-) (<b>e</b>) Cholesterol efflux of peritoneal macrophages from LRP1+/+ and macLRP1-/- mice (n = 3). (<b>f</b>) Cholesterol efflux measured at 6 h in the presence or absence of cAMP (n = 3).</p

Macrophage LRP1 deficiency leads to accumulation of triglyceride-rich lipoproteins in the plasma of LDLR-deficient mice.

<p>Mice were fed either a normal chow (<b>a</b>, <i>left</i>; <b>b</b>, <i>left</i>) or Western diet (<b>a</b>, <i>right</i>; <b>b</b>, <i>right</i>; <b>c,d</b>) for six weeks. Aliquots of serum from fasted mice were assayed for cholesterol (<b>a</b>) or triglyceride (<b>b</b>) (* p = 0.0004, **p = 0.003, n = 19 per group, Student’s t-test comparing LRP1+/+ with macLRP1-/-). (<b>c,d</b>) 100 μl of pooled serum (n = 19/group) from macLRP1-/- and LRP1+/+ mice was fractionated over a Superose 6 FPLC column. Plasma cholesterol (<b>c</b>) and triglyceride (<b>d</b>) levels were quantified. Three independent experiments were performed, each with a unique cohort of mice. Data from one representative experiment is shown.</p