De novo generation of white adipocytes from the myeloid lineage via mesenchymal intermediates is age, adipose depot, and gender specific

Abstract

It is generally assumed that white adipocytes arise from resident adipose tissue mesenchymal progenitor cells. We challenge this paradigm by defining a hematopoietic origin for both the de novo development of a subset of white adipocytes in adults and a previously uncharacterized adipose tissue resident mesenchymal progenitor population. Lineage and cytogenetic analysis revealed that bone marrow progenitor (BMP)-derived adipocytes and adipocyte progenitors arise from hematopoietic cells via the myeloid lineage in the absence of cell fusion. Global gene expression analysis indicated that the BMP-derived fat cells are bona fide adipocytes but differ from conventional white or brown adipocytes in decreased expression of genes involved in mitochondrial biogenesis and lipid oxidation, and increased inflammatory gene expression. The BMP-derived adipocytes accumulate with age, occur in higher numbers in visceral than in subcutaneous fat, and in female versus male mice. BMP-derived adipocytes may, therefore, account in part for adipose depot heterogeneity and detrimental changes in adipose metabolism and inflammation with aging and adiposity.

Fig. 1.

BM hematopoietic progenitors generate adipocytes via myeloid intermediates. (A) Representative photomicrographs of LacZ-expressing adipocytes in adipose tissue sections or the adipocyte fraction from mice transplanted with BM from LacZ-expressing male mice. LacZ was detected in sections by immunostaining and in adipocytes with the fluorescent LacZ substrate FDG. (Scale bar, 50 μm.) (B) Competitive BM transplants were performed with GFP-expressing CD45+ and CD45-, and lin+ and lin− subpopulations. At 8 and 40 wk posttransplantation, adipocytes from gonadal fat were analyzed by flow cytometry. Representative scattergrams are shown for each transplant and time point and the average (n = 3) percentage of GFP+ cells is indicated. (C) Adipocytes from LysMcreROSA^flox/STOP mice were loaded with FDG and examined by phase contrast and fluorescence microscopy. Representative overlay micrographs show LacZ-expressing adipocytes adjacent to marker-negative fat cells. (D) Cytogenetic analysis of X and Y chromosome number in LacZ and GFP-expressing fat cells from transplant recipients. Y chromosome = red, X chromosome = green in DAPI-stained nuclei. (E) Myeloid BM cells were cultured in Matrigel and treated with inducers of adipogenesis. Representative brightfield micrographs before and after staining with Oil Red O.

Fig. 2.

Myeloid-derived adipocyte progenitor cells lacking hematopoietic markers are present in adipose tissue. Gonadal white adipose tissue was taken from LysMcreROSA^flox/STOP mice and digested with collagenase. Stroma was recovered by centrifugation, loaded with FDG and stained with APC-conjugated antibodies to CD45 and PE-conjugated antibodies to CD11b. Expression of Ter119 was used to exclude red blood cells from analysis. (A) Flow cytometry for FDG hydrolysis resolved myeloid (circled in red) from nonmyeloid populations. (B) Flow sorting of LacZ-expressing cells (myeloid) from (A) revealed a population of cells negative for CD45 and CD11b expression (circled in red). (C) The LacZ positive cells lacking CD45 and CD11b from B were cultured and treated with a mixture of adipogenic inducing agents. Representative fluorescence image (LacZ is red, BODIPY stained lipid droplets are green and DAPI is blue) showing LacZ-positive adipocytes (myeloid origin) adjacent to other cells.

Fig. 3.

BMP-derived adipocytes accumulate with age in a depot and gender-specific manner. (A) Gonadal adipose tissue was harvested from female recipient mice at 4, 8, and 12 wk after GFP BM transplantation. Adipocyte fraction was isolated, and GFP-expressing adipocytes were identified by flow cytometry. (B) Gonadal, perirenal, subcutaneous, and intrascapular adipose tissue was harvested 8 wk posttransplant. Representative scattergrams are shown for each depot, and GFP-positive region is indicated. (C) Gonadal adipocytes from male and female LysMcreROSAflox/STOP mice were loaded with FDG and examined by flow cytometry. Representative histograms are shown for each sex and the GFP positive region is indicated. Average (n = 3) percentage of GFP+ fat cells in each sample is indicated.

Fig. 4.

BMP-derived adipocytes are distinct from conventional white and brown adipocytes. BMP-derived adipocytes were purified from transplanted recipient mice by flow cytometry. GFP/LacZ-negative white adipocytes (two samples) were purified from gonadal fat and GFP/LacZ-negative brown adipocytes (two samples) were purified from intrascapular fat striped of white fat by flow cytometry from the same transplanted mice. GFP/LacZ-negative CD11b+ cells were purified from peripheral blood mononuclear cells or fat stroma, also from the same animals. cDNA from each sample was hybridized to Affymetrix mouse whole genome microarrays. (A) Principal component analysis of array data before normalization showing clustering of white, brown and BMP-derived adipocytes (BMP-D 1 and 2) separate from either circulating (Pb) or stromal (St) myeloid cells. (B) Supervised hierarchical cluster analysis of 26 adipocyte genes and the myeloid/dendritic markers CD11b and CD11c following normalization of all data sets. ACC α and β, acetyl-coA carboxylase α and β; C/EBP α and β, CCAAT/enhancer binding protein α and β; CD11b and ¹¹C, integrin aM and aX; CREB, cAMP-response element binding protein; CXCL9, aka monokine induced by gamma-IFN; CX3CL1, aka fractalkine/neurotactin; DGAT1, diacylglycerol-acytransferase 1; FABP4/aP2, fatty acid binding protein 4/adipocyte protein 2; FAS, fatty acid synthase; FSP 27, fat specific protein 27; Glut4, glucose transporter 4; HSL, hormone-sensitive lipase; Insulin R, insulin receptor; IRS1, insulin receptor substrate 1; LPL, lipoprotein lipase; PEPCK, phosphoenolpyruvate carboxykinase (cytosolic); PGC1α, PPARγ coactivator 1α; PPARγ, peroxisome proliferator-activated receptor γ; SCD1, stearoyl-coA desaturase 1; SCF, stem cell factor/kit ligand; SDF-1, stromal cell-derived factor-1; SMAF1, small adipocyte factor 1; SREBP1, sterol regulatory element binding protein 1; UCP-1, uncoupling protein-1. (C) Principal component analysis of only white, brown and BMP-derived adipocyte microarray data sets. Analysis reveals that BMP-derived adipocytes are distinct from conventional white and brown adipocytes. (D) Supervised hierarchical cluster analysis of select mitochondrial/peroxisomal and cytokine gene expression levels in all data sets. Data show decreased expression of mitochondrial/peroxisomal genes related to organelle biogenesis and lipid oxidation, and up-regulation of certain inflammatory cytokines.

Fig. 5.

Model showing generation of myeloid-derived progenitor cells from hematopoietic stem cells in BM. The myeloid-derived progenitors traffic to fat tissue where they loose hematopoietic marker expression and transdifferentiate into BMP-derived white adipocytes.