The contribution of arachidonate 15-lipoxygenase in tissue macrophages to adipose tissue remodeling

Abstract

Cellular plasticity in adipose tissue involves adipocyte death, its clearance, and de novo adipogenesis, enabling homeostatic turnover and adaptation to metabolic challenges; however, mechanisms regulating these serial events are not fully understood. The present study investigated the roles of arachidonate 15-lipoxygenase (Alox15) in the clearance of dying adipocytes by adipose tissue macrophages. First, upregulation of Alox15 expression and apoptotic adipocyte death in gonadal white adipose tissue (gWAT) were characterized during adipose tissue remodeling induced by β3-adrenergic receptor stimulation. Next, an in vitro reconstruction of adipose tissue macrophages and apoptotic adipocytes recapitulated adipocyte clearance by macrophages and demonstrated that macrophages co-cultured with apoptotic adipocytes increased the expression of efferocytosis-related genes. Genetic deletion and pharmacological inhibition of Alox15 diminished the levels of adipocyte clearance by macrophages in a co-culture system. Gene expression profiling of macrophages isolated from gWAT of Alox15 knockout (KO) mice demonstrated distinct phenotypes, especially downregulation of genes involved in lipid uptake and metabolism compared to wild-type mice. Finally, in vivo β3-adrenergic stimulation in Alox15 KO mice failed to recruit crown-like structures, a macrophage network clearing dying adipocytes in gWAT. Consequently, in Alox15 KO mice, proliferation/differentiation of adipocyte progenitors and β3-adrenergic remodeling of gWAT were impaired compared to wild-type control mice. Collectively, our data established a pivotal role of Alox15 in the resolution of adipocyte death and in adipose tissue remodeling.

Figure 1

β3-adrenergic stimulation induces apoptosis of adipocytes in gonadal adipose tissue. (a) Immunoblot analysis and quantification of caspase-3, cleaved caspase-3 and Alox15 expression in gWAT from mice treated with CL up to 5 days. β-actin was used as a loading control. Error bars indicated S.E.M. of four individual experiments. (*P<0.05, ***P<0.001). (b) qPCR analysis of Alox15 expression in F4/80+ macrophages and adipocytes from of mice treated with CL for 3 days and untreated controls. Error bars indicated S.E.M. of three individual experiments (***P<0.001). (c) Immunohistochemistry of cleaved caspase-3 and F4/80 in paraffin sections of gWAT of mice treated with CL for 3 days and untreated controls. Nuclei were counterstained with DAPI. Bars=20 μm. (d) Analysis of Alox15-dependent lipid metabolites in isolated F4/80+ macrophages from gWAT of mice treated with CL for 3 days and untreated controls . Error bars indicated S.E.M. of three individual experiments per condition. Three biological replicates of pooled tissues from four mice were analyzed (*P<0.05, N.S. = non-significant)

Figure 2

In vitro co-culture recapitulates adipocyte clearance by M2-like adipose tissue macrophages. (a) Immunoblot analysis of caspase-3 activation and perilipin1 expression in differentiated adipocytes from C3H10T1/2 cultures treated with Brefeldin A (5 μM) up to 24 h. β-actin was used as a loading control. (b). Analysis of ability of adipose tissue macrophages to clear apoptotic adipocytes in in vitro co-culture system. Representative images and quantitative analysis of fluorescence intensity of green (DiO-macrophages) and red (BODIPY-lipid) are shown. (c) Quantitative analysis of fluorescence intensity of red (BODIPY-lipid) and green (DiO-macrophages) to compare adipocyte clearance rates between groups. Quantifications are representative of three individual experiments. (d) qPCR analysis of efferocytosis-related genes in adipose tissue macrophages co-cultured with dying or live adipocytes, and macrophages cultured without adipocytes (no AC) . Error bars indicated S.E.M. of three individual experiments (in comparison to GM + dying adipocytes controls, *P<0.05, **P<0.01, ***P<0.001)

Figure 3

Effects of genetic deletion and chemical inhibition of Alox15 on adipocyte clearance by macrophages. Comparison of phagocytic ability of adipose tissue macrophages to clear dying adipocytes in in vitro co-culture system. (a,b) Adipose tissue macrophages were obtained from gWAT of Alox15 KO or WT mice (control, baicalein). A total of 10 μM of baicalein (Bai), or vehicle were treated during long-term imaging. Representative images (a) and quantitative analysis (b) of fluorescence intensity of green (DiO-macrophages) and red (BODIPY-lipid) are shown. (c,d) Adipose tissue macrophages were obtained from gWAT of WT mice, and 10 μM of PD146176 or 68 μM of 13-HODE were treated during long-term imaging. Representative images (c) and quantitative anlaysis (d) of fluorescence intensity of green (DiO-macrophages) and red (BODIPY-lipid) are shown. Error bars indicated S.E.M. of three individual experiments per condition

Figure 4

Alox15 is required for macrophage recruitment and cell proliferation in remodeling of gWAT induced by β3-adrenergic receptor stimulation. (a) Immunoblot analysis and quantification of Alox15 expression in gWAT from WT mice and Alox15 KO mice treated with CL for 3 days and untreated controls. β-actin was used as a loading control. (b) Representative images of paraffin sections of gWAT of mice treated with CL for 3 days and untreated controls, stained with BrdU and F4/80. Nuclei were counterstained with DAPI. Bars=20 μm. (c). Quantification of BrdU+ cells. (d). qPCR analysis of genes related to proliferation and macrophage markers in gWAT from WT mice and Alox15 KO mice treated with CL for 3 days and untreated controls. Error bars indicated S.E.M. of 3 individual experiments per condition. *P<0.05, **P<0.01, ***P<0.001

Figure 5

Analysis of markers of adipocyte progenitors and endothelial cells in gWAT of Alox15 KO mice. (a) Representative flow profiles and quantification of CD31+, PDGFRα+ and F4/80+ cells in SVC fraction from gWAT of WT and Alox15 KO mice. Error bars indicated S.E.M. of four individual experiments per condition (*P<0.05). (b) qPCR analyses of endothelial cell (PECAM), stem cell (CD34), and adipocyte progenitor (Pdgfra) markers in gWAT of WT and Alox15 KO mice. Error bars indicated S.E.M. of three individual experiments per condition (*P<0.05)

Figure 6

Alox15 is required for efferocytosis in remodeling of gWAT induced by β3-adrenergic receptor stimulation. (a) Flow cytometric analysis of CD300LF and lipid accumulation in macrophages isolated from WT and Alox15 KO mice treated with CL for 3 days and untreated controls. Representative flow profiles of two individual experiments per condition are shown. (b) Quantification of SSC-A, FSC-A, and mean fluorescence intensity of CD300LF-PE and LipidTox Deep Red. Error bars indicated ranges of two individual experiments as biological replicates of pooled tissue from three mice. (c) qPCR in macrophages of gWAT of WT and Alox15 KO mice treated with CL for 3 days and untreated controls. Error bars indicated S.E.M. of three individual experiments per condition. *P<0.05, **P<0.01, ***P<0.001

Figure 7

Alox15 is required for de novo adipogenesis in remodeling of gWAT induced by β3-adrenergic receptor stimulation. Representative images of H/E staining of paraffin sections of gWAT from WT and Alox15 KO mice treated with CL for 5 days or untreated controls. (b,c) Analyses of adipocyte size (b) and tissue weight (c) of gWAT from WT and Alox15 Ko mice treated with CL for 5 days or untreated control. Error bars indicated S.E.M. of four individual experiments per condition. (d) Representative images and quantification of BrdU and Perilipin 1 (PLIN1) staining in paraffin sections of gWAT from WT and Alox15 KO mice treated with CL for 5 days. Arrows indicate BrdU+Plin1+ adipocytes. Nuclei were counterstained with DAPI. Bars=20 μm