Rosiglitazone promotes development of a novel adipocyte population from bone marrow-derived circulating progenitor cells

Abstract

Obesity and weight gain are characterized by increased adipose tissue mass due to an increase in the size of individual adipocytes and the generation of new adipocytes. New adipocytes are believed to arise from resident adipose tissue preadipocytes and mesenchymal progenitor cells. However, it is possible that progenitor cells from other tissues, in particular BM, could also contribute to development of new adipocytes in adipose tissue. We tested this hypothesis by transplanting whole BM cells from GFP-expressing transgenic mice into wild-type C57BL/6 mice and subjecting them to a high-fat diet or treatment with the thiazolidinedione (TZD) rosiglitazone (ROSI) for several weeks. Histological examination of adipose tissue or FACS of adipocytes revealed the presence of GFP(+) multilocular (ML) adipocytes, whose number was significantly increased by ROSI treatment or high-fat feeding. These ML adipocytes expressed adiponectin, perilipin, fatty acid-binding protein (FABP), leptin, C/EBPalpha, and PPARgamma but not uncoupling protein-1 (UCP-1), the CD45 hematopoietic lineage marker, or the CDllb monocyte marker. They also exhibited increased mitochondrial content. Appearance of GFP(+) ML adipocytes was contemporaneous with an increase in circulating levels of mesenchymal and hematopoietic progenitor cells in ROSI-treated animals. We conclude that TZDs and high-fat feeding promote the trafficking of BM-derived circulating progenitor cells to adipose tissue and their differentiation into ML adipocytes.

Figure 1. ROSI increases circulating levels of BM-derived mesenchymal and hematopoietic progenitor cells.

Flow cytometric analysis of PBMCs isolated from GFP⁺ BMT mice fed a control (Cntrl) or ROSI-impregnated (ROSI) diet for 3 (top and bottom rows) or 7 (middle row) weeks. Cells were stained with APC-conjugated anti-CD45 antibodies and either PE-labeled anti–Sca-1 (top and middle rows) or anti–c-kit (bottom row) antibodies and analyzed by flow cytometry as described in Methods. Gates were set and data corrected for results obtained with unstained cells or cells stained with APC- or PE-conjugated isotype-matched control antibodies. Representative scattergrams for each analysis are shown. Blue ovals indicate cells staining weakly for Sca-1 that were not detected in samples from control animals. The average percentage (from 3 independent experiments) of total GFP⁺ cells is indicated in the top left, top right, and bottom right quadrants.

Figure 2. Appearance and distribution of GFP⁺ ML adipocytes in adipose tissue from untreated, ROSI-treated, and high-fat diet–fed mice.

(A) Five-micrometer sections were prepared from paraffin-embedded omental (left 3 columns) and dorsal intrascapular (right column) adipose tissue from GFP⁺ BMT animals fed control, ROSI-impregnated, or high-fat diets for 3 weeks. Sections were deparaffinized, rehydrated, and mounted with aqueous mounting medium. Sections were examined by phase-contrast and fluorescence digital deconvolution microscopy. The GFP fluorescence signal was digitally overlayed on the corresponding phase-contrast image. Representative photomicrographs of both white fat (left 3 columns) and brown fat (Brn fat) are shown. Scale bar (red): 100 μm. (B) Serial sections of omental white fat from GFP⁺ BMT mice fed ROSI for 3 weeks were compared for GFP fluorescence and immunohistochemical staining for GFP (GFP Ab). Lack of staining with an isotype-matched negative control antibody (Iso match Ab) is also shown.

Figure 3. FACS analysis of GFP⁺ adipocytes.

Adipocytes were isolated from a nontransgenic, nontransplanted (WT C57BL/6) mouse (as a negative control); a UBI-GFP/BL6 transgenic (UBI-GFP Tg) donor mouse (as a positive control); and untreated GFP⁺ BMT mice (Cntrl), ROSI-treated mice, or mice fed a high-fat diet for 7 weeks. Adipocytes were isolated by collagenase digestion and flotation from omental or dorsal intrascapular depots. Shown are representative scattergrams in which green dots indicate GFP⁺ cells and black dots represent either non-GFP⁺ cells, free lipid droplets, or debris. The average percentage of particles that were GFP⁺ is indicated at the top right of each scattergram. The results demonstrate that ROSI increases the number of GFP⁺ adipocytes in the tissue samples. High-fat diet also increases GFP⁺ adipocyte numbers but to lesser extent than ROSI treatment. GFP comp, GFP compensation.

Figure 4. Nuclear DNA content analysis of GFP⁺ adipocytes.

Adipocytes were isolated from GFP⁺ BMT mice fed control, ROSI-impregnated, or high-fat diets for 7 weeks. Adipocytes were permeablized with saponin and stained with PI. Flow cytometry was conducted with singlet discrimination. GFP-negative cells were excluded from the analysis. The position of polyploid (fused or multinuclear) cells or cells in the G₀/G₁ and G₂/M regions of the cell cycle histogram are indicated. The small peak to the left of the G₀/G₁ peak in each histogram indicates apoptotic cells. The average percentage of polyploid cells in each treatment is indicated in parentheses below the label for each treatment histogram.

Figure 5. Microscopic observation of GFP⁺ ML adipocytes isolated by collagenase digestion.

(A) Omental and dorsal intrascapular adipose tissue was isolated from GFP⁺ BMT mice fed ROSI-impregnated chow for 7 weeks. The tissue was digested with collagenase, and adipocytes were isolated by flotation. Adipocytes were then subjected to flow sorting to separate GFP⁺ and GFP^– cells. Isolated cells were examined by phase-contrast and fluorescence digital deconvolution microscopy to evaluate morphology and GFP expression. Shown are representative phase-contrast and fluorescence images of GFP⁺ ML adipocytes (MLAs) compared with a GFP^– unilocular white adipocyte (from omental tissue) and a GFP^– ML brown adipocyte (from dorsal intrascapular brown fat). Digital overlay of GFP fluorescence signal and phase-contrast images in shown. Scale bar (red): 100 μm. (B) Phase-contrast, fluorescence, and digital overlay images of adipocytes isolated by collagenase digestion and flotation from GFP⁺ BMT mice fed ROSI-impregnated diet for 7 weeks. The image shows the substantial number of GFP⁺ adipocytes present in the total adipocyte population.

Figure 6. GFP⁺ ML adipocytes express C/EBPα, PPARγ, adiponectin, FABP, perilipin, leptin, and β3-AR but not UCP-1 and have high mitochondrial content.

(A) cDNA was prepared from RNA from white (from omental adipose tissue), brown (from dorsal intrascapular adipose tissue), and ML adipocytes isolated by collagenase digestion and flotation as described in Methods. Equal amounts of cDNA (1 μg) were subjected to PCR with validated primer sets for the targets indicated to the left of each gel photograph. PCR reactions were then resolved on 2% agarose gels run in the presence of ethidium bromide. Fluorescence photographs of the gels were captured to computer, and band intensities were measured using ImageJ software. Representative gel photographs are shown in the left column. Densitomentry data were averaged over 3 experiments and corrected for differences in β-actin levels. Average band intensities are shown in the corresponding bar graphs to the right of each gel photograph. (B) Mitotracker Red 580 staining was performed on minced white adipose tissue fragments from GFP⁺ BMT mice fed ROSI-impregnated chow for 7 weeks. Representative fluorescence deconvolution images for GFP and Mitotracker signals, as well as a digital overlay of GFP and Mitotracker signals, are shown (yellow: GFP plus Mitotracker; red or orange: Mitotracker plus little or no GFP). The general location of white (W) and ML adipocytes is indicated by the white ovals.

Figure 7. GFP⁺ adipocytes do not express CD45 or CD11b.

PBMCs and omental adipose tissue were isolated from GFP⁺ BMT mice fed ROSI-impregnated chow for 7 weeks. The adipose tissue was digested with collagenase and nonbuoyant stromal/vascular cells, and buoyant adipocytes were separated by differential centrifugation. All 3 cell fractions were stained with APC-conjugated anti-CD45.2 (clone 104-2) antibodies and PE-conjugated anti-CD11b (clone M1/70) antibodies. The stained cell suspensions were subjected to FACS analysis for GFP⁺ cells (GFP^– cells were excluded) expressing CD45 and/or CD11b. Representative scattergrams are shown, and the average percentage (from 3 independent sorts) of cells is indicated in the top left (CD11b⁺), top right (CD45⁺ and CD11b⁺), and bottom right (CD45⁺) quadrants. Gates were set and data were corrected using unstained cell suspensions or suspensions incubated with APC- and PE-conjugated isotype-matched control antibodies.