Very low density lipoprotein receptor (VLDLR) expression is a determinant factor in adipose tissue inflammation and adipocyte-macrophage interaction

Abstract

Obesity is associated with adipose tissue remodeling, characterized by adipocyte hypertrophy and macrophage infiltration. Previously, we have shown that very low density lipoprotein receptor (VLDLR) is virtually absent in preadipocytes but is strongly induced during adipogenesis and actively participates in adipocyte hypertrophy. In this study, we investigated the role of VLDLR in adipose tissue inflammation and adipocyte-macrophage interactions in wild type and VLDLR-deficient mice fed a high fat diet. The results show that VLDLR deficiency reduced high fat diet-induced inflammation and endoplasmic reticulum (ER) stress in adipose tissue in conjunction with reduced macrophage infiltration, especially those expressing pro-inflammatory markers. In adipocyte culture, VLDLR deficiency prevented adipocyte hypertrophy and strongly reduced VLDL-induced ER stress and inflammation. Likewise, cultures of primary peritoneal macrophages show that VLDLR deficiency reduced lipid accumulation and inflammation but did not alter chemotactic response of macrophages to adipocyte signals. Moreover, VLDLR deficiency tempered the synergistic inflammatory interactions between adipocytes and macrophages in a co-culture system. Collectively, these results show that VLDLR contributes to adipose tissue inflammation and mediates VLDL-induced lipid accumulation and induction of inflammation and ER stress in adipocytes and macrophages.

Keywords: Adipocyte; Adipose Tissue; Inflammation; Macrophages; Obesity; VLDLR.

FIGURE 1.

Effects of VLDLR deficiency on blood glucose clearance test (GTT), insulin tolerance test (ITT), and adipose tissue insulin sensitivity and morphology. A, GTT in overnight fasted wild type (WT) and very low density lipoprotein receptor-deficient (vldlr^−/−) mice (n = 7 per group) fed high a fat diet for 16 weeks. B, ITT in 4-h fasted mice. C, glucose uptake. D, representative blots. E, ratio of optical density of phosphorylated over total Akt and IRS1 tested in freshly isolated adipocytes treated with insulin (100 μm) for 20 min at 37 °C. Adipocytes were isolated from epididymal adipose tissue of mice fed a high fat diet, and glucose uptake was tested using [³H]deoxyglucose. Results are from two independent experiments conducted in triplicate for each group. Representative sections of epididymal white adipose tissues (F and G) and distribution of adipocyte diameters (H and I) from WT and vldlr^−/− mice fed a high fat diet are shown. Scale bars, 250 μm. Adipose tissues were fixed, paraffin-embedded, and stained with hematoxylin and eosin. Adipocyte diameters were measured in five random fields of adipose tissue sections from four mice per group (total adipocyte number n = 500 per group). The data are expressed as the percentage of cells found in a given diameter size range. Results are presented as mean ± S.E., and statistical differences were performed with Student's t test as **, p < 0.01, and *, p < 0.05.

FIGURE 2.

VLDLR deficiency reduces macrophage content and inflammation in adipose tissue. Mice were fed a high fat diet for 16 weeks, and adipose tissues were isolated and analyzed. A–D, representative epididymal adipose tissue sections stained with macrophage-specific antibody F4/80. Scale bars, 50 μm in A and B and 20 μm in C and D. The number of crown-like structures was determined in five different fields from four mice per group. E, adipokines and FFA released from adipose tissue cultures are presented as mean ± S.E. with n = 6 per group. Culture condition and measurement of adipokines in the medium were performed as described under “Experimental Procedures.” Expression of adipokines was assayed separately in primary adipocyte (F) and stromal vascular fraction (G) by qPCR using specific primers (Table 1). Isolation of primary adipocytes and SVF was performed by collagenase enzymatic digestion. Results are presented as mean ± S.E. with n = 8 per group. Statistical differences between vldlr^−/− and WT were performed with Student's t test as **, p < 0.01, and *, p < 0.05.

FIGURE 3.

Analysis of ATM phenotypes. A and B, representative photographs of adipose tissue sections immunostained with CD11c. Epididymal adipose tissues of WT and vldlr^−/− mice fed a high fat diet were stained with specific antibodies as described under “Experimental Procedures.” Representative dot plot analysis of CD11c (C and D) and CD206 (E and F) was determined by flow cytometry on a gated F4/80⁺ population of adipose tissue SVF. Populations with high expression of F4/80/CD11c and F4/80/CD206 ATMs are indicated in the top right quadrants of the graphs. Relative gene expression of M1 (G) and M2 (H) macrophage markers were determined by qPCR analysis of adipose tissue SVF (n = 8 per group). Data are presented as mean ± S.E., and statistical differences of vldlr^−/− and WT were performed with Student's t test as *, p < 0.05. iNOS, inducible NOS.

FIGURE 4.

Stimulation of macrophage chemotaxis by adipose tissue conditional medium. Adipose tissue isolated from WT and vldlr^−/− mice fed a high fat diet for 16 weeks were cultured as described under “Experimental Procedures.” After 24 h of culture, medium from these incubations (conditional medium) was isolated and used for the assay of the chemotaxis of PM of WT and vldlr^−/− mice. PMs were loaded in the migration chamber, and conditional medium from WT (CM^WT) and vldlr^−/− (CM^vldlrko) adipose tissue cultures was loaded in the lower chamber. Three separate experiments were performed, and the results of six wells for each experimental condition were averaged after subtracting the negative control. Results are presented as mean ± S.E., and statistical differences between cells migrating toward CM^WT and CM^vldlrko are indicated with ^a for p < 0.01.

FIGURE 5.

VLDLR deficiency reduces diet-induced inflammation and ER stress in adipocytes. Primary adipocytes were isolated from epididymal adipose tissues of WT and vldlr^−/− mice fed a high fat diet, and proteins were analyzed by Western blotting. A, representative blots; B, ratio (p/t) of optical density of phosphorylated (p) over total (t) JNK, p38 MAPK, and c-Jun. C, representative blots; D, ratio of optical density of phosphorylated (p) over total (t) of PERK, eIF2a, and ratio of CHOP over β-actin. Data of optical density are in arbitrary units and were obtained after scanning the protein bands. Results are presented as mean ± S.E. with n = 5–6 per group. E, expression of relevant genes of ER stress was quantified with qPCR relative to β-actin. Data are presented as mean ± S.E., and statistical significances between vldlr^−/− and WT were determined by Student's t test as *, p < 0.05, and **, p < 0.01.

FIGURE 6.

Effects of VLDLR deficiency on VLDL-induced adipogenesis and inflammation in adipocytes. TG content was determined enzymatically (A) and estimated by Oil Red O staining (B). C and D, representative blots (C) and optical density (D) showing the levels of total and phosphorylated forms of JNK and p38 MAPK and β-actin. Proteins were determined in adipocytes of WT and vldlr^−/− mice cultured with the indicated doses of VLDL. Relative gene expression of IL-6 (E) and MCP-1 (F) was determined in adipocytes of WT and vldlr^−/− mice cultured with 300 μg/ml VLDL. Preadipocytes were separated from epididymal adipose tissues of WT and vldlr^−/− mice, and VLDL particles were isolated from pooled plasma of WT mice. Conditions of culture, differentiation, and treatments with VLDL were as described under “Experimental Procedures.” At day 12 of differentiation, mature adipocytes were incubated with the indicated doses of VLDL for 48 h, and the cell preparations were then either tested for lipid content with Oil Red O and enzymatic assay or harvested for protein and gene expression analyses. The levels of total (t) and phosphorylated (p) JNK and p38 MAPK and β-actin were evaluated by Western blotting, and gene expression of Il6 and Ccl2 (MCP-1) was tested by qPCR. Results are presented as means ± S.E. of two independent experiments conducted in triplicate for each experimental group. Statistical differences between VLDL-treated and -untreated cells of each genotype (A–F) are indicated as *, p < 0.05, and **, p < 0.001. A, only vldlr^−/− cells treated with 300 and 400 μg/ml VLDL were significantly different (p < 0.05) from untreated vldlr^−/− cells. Statistical differences between vldlr^−/− and WT cells treated with the same dose of VLDL (C–F) are indicated with ^b for p < 0.05 and ^a for p < 0.01.

FIGURE 7.

Effects of VLDLR deficiency on VLDL-induced ER stress in adipocytes. A, representative blots of total and phosphorylated eIF-2α, CHOP, and β-actin. B, mean optical density of indicated proteins in adipocytes of WT and vldlr^−/− adipocytes cultured with VLDL (300 μg/ml, thapsigargin (THAPS) (10 μg/ml), or both for 24 h). Control nontreated cultures were incubated with an equal volume of vehicle. Data are means ± S.E. from two independent experiments performed in triplicate. Statistical differences between WT and vldlr^−/− adipocytes with the same treatment are indicated as *, p < 0.05.

FIGURE 8.

Uptakes of VLDL and palmitate complexed to albumin and palmitate induction of inflammation in adipocytes. A, uptake of ³H,¹²⁵I-labeled VLDL particles. B, distribution of VLDL-derived ³H in cellular lipids. C, uptake of [³H]palmitate complexed to albumin. Adipocytes were differentiated in vitro and cultured with double-labeled VLDL and labeled palmitate complexed to albumin for 3 h. The radioactivity and protein contents of adipocyte were analyzed to calculate uptakes of VLDL lipids and proteins and uptake of albumin-bound palmitate. Distribution of VLDL-derived ³H in adipocyte lipids was determined following extraction and separation with thin layer chromatography. Radioactivity in each lipid class was expressed as percent of total lipid incorporation. PL, polar lipids, including phospholipids and monoglycerides; DG, diglycerides. Experiments were performed in triplicate, and results are presented as mean ± S.E. Statistical differences between vldlr^−/− and WT cells are indicated as *, p < 0.05; **, p < 0.001. D, representative blots; E, ratio of optical density of phosphorylated (p) over total (t) JNK and β-actin. Expression of Il6 (F) and Ccl2 (MCP-1) (G) of adipocytes treated with various doses of palmitate complexed to albumin is shown. Data are presented as mean ± S.E. of two independent experiments performed in triplicate, and statistical significance between palmitate-treated and -untreated cells of each genotype was determined by Student's t test as *, p < 0.05, and **, p < 0.01.

FIGURE 9.

Effects of VLDLR deficiency on VLDL-induced inflammation in macrophages. A, TG content; B, representative photographs of stained lipids and nuclei of macrophages cultured with VLDL at a 200 μg/ml dose. C, relative gene expression of Tnfα and Il6. D, representative blots; E, optical density of phosphorylated (p) and total (t) JNK and p38 MAPK and β-actin protein of macrophages of WT and vldlr^−/−mice cultured with VLDL at a 200 μg/ml dose. Peritoneal macrophages of WT and vldlr^−/− mice were treated with the indicated amounts of VLDL for 24 h, and the cells were then used for enzymatic determination of triglyceride content, staining with LipidTox dye for neutral lipids and DAPI for nuclei, or collected in lysis buffer to examine protein levels by Western blotting and gene expression by qPCR. Cultures were performed in triplicate, and data are presented as means ± S.E. of two independent experiments. Statistical differences between vldlr^−/− and WT are indicated as *, p < 0.05, and **, p < 0.001.

FIGURE 10.

Effects of VLDLR deficiency on adipocyte-macrophage interactions. Secretion of IL-6 (A) and MCP-1 (B) by single cultures of AD and PM. AD + PM represent the sum of the production by adipocytes and macrophages of each genotype in single cultures. Secretion of IL-6 (C) and MCP-1 (D) by adipocyte and macrophages in co-culture systems is shown. For comparative purposes, the levels of sum (AD + PM) of chemokines in single cultures are indicated with dashed lines. For IL-6 and MCP-1, the differences between values of co-cultures and values of AD + PM of single cultures are presented by black bars (w/w cells) and open bars (vldlrko/vldlrko cells) on the right side of C and D. E, representative blot of protein levels of phosphorylated (p) and total (t) JNK; F, expression of IL-6 in adipocytes after co-cultures with WT and vldlr^−/− macrophages. Preadipocytes isolated from epididymal adipose tissue of WT and vldlr^−/− mice were differentiated in vitro to reach maturity (day 12). Peritoneal macrophages isolated from WT and vldlr^−/− mice were seeded in the transwell inserts, and cells were then co-cultured for 24 h in the presence of VLDL 300 μg/ml. Co-culture medium and adipocytes were collected for analyses. IL-6 and MCP-1 were determined by ELISA. Protein levels were examined by Western blotting, and gene expression was investigated with qPCR. Results are of two independent experiments performed in triplicate for each condition. Data are presented as mean ± S.E. and statistical differences between vldlrko cells or mixed genotype cells and WT cells are indicated as *, p < 0.05, and **, p < 0.01. Differences between WT adipocytes co-cultured with peritoneal macrophages of WT (PM^WT) or vldlr^−/− macrophages (PM^vldlrko) are indicated by ^b for p < 0.05.