Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 deficiency reduces leukocyte infiltration into adipose tissue and favors fat deposition

Abstract

Obesity is associated with low-grade inflammation and leukocyte infiltration in white adipose tissue (WAT) and is linked to diabetic complications. Semicarbazide-sensitive amine oxidase, also known as vascular adhesion protein-1 (SSAO/VAP-1), is a membrane protein that is highly expressed in adipocytes and is also present on the endothelial cell surface where it is involved in leukocyte extravasation. We studied fat deposition and leukocyte infiltration in WAT of mice with a null mutation in the amine oxidase copper-containing-3 (AOC3) gene encoding SSAO/VAP-1. Both epididymal and inguinal WATs were larger in 6-month-old AOC3-KO males than in age-matched wild-type controls. However, WAT from AOC3-KO mice contained lower CD45 mRNA levels and fewer CD45(+) leukocytes. Subpopulation analyses revealed a diminished infiltration of WAT by T cells, macrophages, natural killer, and natural killer T cells. A decrease in leukocyte content in WAT was also detected in female AOC3-KO mice as early as 2 months of age, whereas increased fat mass was evident by 6 months of age. Reduced CD45(+) populations in WAT of AOC3-KO mice was not rescued by human SSAO/VAP-1 expression on adipocytes under the control of aP2, suggesting the importance of vascular AOC3 in leukocyte entrance into fat. Our results indicate that SSAO/VAP-1 is instrumental for the presence of leukocytes in WAT. Therefore, AOC3-KO mice present a unique model of mild obesity, characterized by increased WAT devoid of low-grade inflammation.

Figures

Figure 1

Flow cytometric analysis of SVF and LN lymphocytes from adipose tissue of wild-type (WT) or AOC3-KO mice. A: Representative profiles of CD45+ cell populations according to cell size and granulosity criteria in SVF from epididymal (EPI) and inguinal (ING) adipose tissues, or LNs contained in inguinal adipose tissue (LN) from 28-week-old wild-type or AOC3-KO mice. B: Percentage of CD45+ cells contained in total SVF or LNs. Means ± SD of nine wild-type and four AOC3-KO mice. Different from control (wild-type) group at *P < 0.05, **P < 0.01. C: Representative flow cytograms of cells labeled with anti-CD45 antibody in EPI SVF from wild-type and AOC3-KO mice. Vertical dotted line separates nonspecific signal from higher intensity labeling. Bold curve represents fluorescence intensity in a preparation from a wild-type mouse, thin curve from an AOC3-KO mouse, determined under identical conditions. R1 delimits the lymphocyte gate and R2 the macrophage gate taken into account for further analyses. Similar flow cytograms were observed on ING SVF.

Figure 2

Decreased CD45 and MCP-1 expression and increased leptin mRNA abundance in ING WAT from AOC3-KO mice. mRNA levels were compared between control (wild-type) and AOC3-KO mice by real-time PCR in subcutaneous adipose tissue. A: CD34. B: MCP-1. C: Leptin. Means ± SEM of arbitrary units obtained from five to seven wild-type (WT) and six to eight AOC3-KO mice as detailed in the Materials and Methods. *P < 0.05 and **P < 0.01 versus WT mice.

Figure 3

aP2hVAP-1TG/KO mice express human AOC3 but lack endogenous murine AOC3 in adipose tissue. Murine and human AOC3 are detected by species-specific antibodies recognizing mouse (7-106) and human VAP-1 (JG2.10). Stainings of wild-type (WT) adipose tissue are shown as controls. Scale bar = 50 μm.

Figure 4

Endothelial AOC3 rather than adipose AOC3 is responsible for reduced leukocyte number in adipose tissue of AOC3-KO mice. Examples of the scarce expression of CD45-positive cells (arrows) in AOC3-KO mice (A) and aP2hVAP-1TG/KO mice (B), stainings with negative control antibodies are shown in four fold reduced insets. Original magnifications × 400. C: Number of adipocytes and CD45+ cells in sections of paraffin-embedded and zinc-fixed adipose tissue from aP2hVAP-1TG/KO and AOC3-KO mice. Mean ± SEM of counts in high-power field (n = 5 in each group).

Figure 5

Flow cytometric analysis of different lymphocyte and macrophage populations in EPI (A) and ING (B) adipose tissues of wild-type (WT) and AOC3-KO mice. Subclasses of lymphocytes represented in the left panel were determined by staining with a combination of antibodies to differentiate B (CD19+), T non-NKT (CD3+, NK1.1−), NK (CD3−, NK1.1+), and NKT (CD3+, NK1.1+) cells. Results are expressed as percentage of lymphocyte population (CD45+of R1 gate). Means ± SD of nine wild-type and four AOC3-KO mice. Subclasses of macrophages represented in the right panel were determined by the presence of antigens recognized by anti-CD11b or anti-CD115 antibodies. Results as percentage of macrophage population (CD45+ of R2 gate). Means ± SD of four wild-type and four AOC3-KO mice. Different from wild-type group at *P < 0.05, ***P < 0.001.