Rosiglitazone promotes development of a novel adipocyte population from bone marrow–derived circulating progenitor cells (original) (raw)
ROSI promotes mobilization of BM-derived mesenchymal progenitor cells into the peripheral circulation. To determine whether high-fat feeding or TZDs promote mobilization of BM-derived circulating progenitor cells to adipose tissue depots, C57BL/6 mice were subjected to lethal gamma irradiation (12 Gy split dose) to deplete their endogenous BM populations. Irradiated mice were rescued by isograft transplantation of isolated whole BM cells from GFP-expressing transgenic mice, driven by the ubiquitin C gene promoter (18). The GFP+ BM cells were allowed to engraft for 8 weeks, after which time greater than 95% of circulating PBMCs exhibited GFP expression (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI28510DS1). GFP+ BMT chimera mice were then fed either a conventional rodent chow diet (6.5% fat, 3.31 kcal/g), a diet impregnated with the TZD ROSI (15 mg/kg/d), or a high-fat diet (20% fat, 4.3 kcal/g) for up to 7 weeks.
Flow cytometric analysis of BM-derived, circulating GFP+ PBMCs isolated from the peripheral blood or collagenase-digested omental or intrascapular fat pads showed no change in any of the populations following 1 week of ROSI or high-fat diet treatment (Supplemental Table 1). However, after 3 weeks of ROSI treatment, there was a 7.74-fold increase (P = 0.02) in GFP+ cells expressing a pattern of cell-surface markers characteristic of mesenchymal progenitor cells (CD45–Sca-1+) (Table 1 and Figure 1) in the peripheral blood mononuclear layer. There was also a 3.5-fold increase in the number of peripheral blood circulating cells expressing surface markers characteristic of hematopoietic progenitor cells (CD45+c-kit+), although this change was not statistically significant (P = 0.12). Minor increases were also observed in circulating granulocytes (Gr-1+) and macrophages (CD14+). After 7 weeks of treatment, circulating levels of GFP+CD45–Sca-1+ cells were increased by almost 19-fold in ROSI-treated GFP+ BMT chimera mice (Supplemental Table 1 and Figure 1). No other significant differences in peripheral blood circulating cell populations were noted between control and high-fat diet–fed animals.
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.
Effect of ROSI on cell-surface marker expression on PBMCs and omental and dorsal intrascapular fat stromal/vascular cells
Analysis of GFP+ BM–derived populations in the stromal/vascular fraction from collagenase-digested omental and intrascapular fat pads demonstrated a modest, but reproducible decrease in CD45–Sca-1+ cells in omental fat with ROSI treatment at 3 (Table 1) and 7 (Supplemental Table 1) weeks. No significant differences were noted in other cell populations at any time point with any of the conditions. The fact that there were no significant differences in GFP+ BM–derived populations in adipose tissue with ROSI treatment while circulating levels of mesenchymal and hematopoietic progenitor cells were elevated by ROSI is likely due to the loss of progenitor cell-surface markers as these progenitor cells differentiated into adipocytes or other cell types.
BM-derived progenitor cells differentiate into ML adipocytes. Fluorescence digital deconvolution microscopy revealed GFP+ adipocytes having multiple fat droplets (ML) in all treatment groups (Figure 2A). Endogenous GFP fluorescence or immunohistochemical staining for GFP indicated that these ML adipocytes arose from BM-derived progenitor cells (Figure 2B). These cells were more abundant in adipose tissue sections from ROSI-treated animals, often clustering together in large regions. ML GFP+ adipocytes were also present at higher numbers in fat tissue from high-fat diet–fed animals than corresponding adipose tissue depots from control animals, but the levels were not as high as in samples from ROSI-treated animals. GFP+ ML adipocytes were also present in intrascapular brown fat, but rather than forming clusters as observed in white fat, the GFP+ adipocytes were observed as individual cells scattered throughout the brown fat.
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.
To quantitate these differences, adipocytes were isolated from collagenase-digested omental and intrascapular fat and subjected to FACS analysis. A 100-μm sample tip was selected for the sorting of adipocytes based on morphometric analysis of fixed adipose tissue sections and collagenase-digested adipocytes. These studies indicated that the average size of unilocular white adipocytes over all depots was approximately 40 ± 15 μm in control and ROSI-treated animals and about 70 ± 10 μm in high-fat diet–fed animals. These sizes are somewhat smaller than typically reported. This is probably due to the fact that irradiated animals and/or animals maintained at Denver, Colorado, altitude (1,600 m) eat less, gain less weight, and exhibit decreased adiposity compared with unirradiated animals maintained at lower altitudes. The large size of adipocytes required the use of log scales for both forward- and side-scatter data collection.
In samples costained with propidium iodide (PI), all GFP+ particles exhibited bright PI fluorescence, indicating the presence of nuclei in the particles. This factor along with the large size (high forward scatter) indicates that the adipocytes not lysed by digestion/flotation remained intact during their passage through the sorter. However, visual examination of post-sort samples revealed the presence of a substantial number (approximately 50%–70% of GFP+ particles) of free nuclei. Thus, only 30%–50% of the adipocytes survived the sort intact. The remaining cells were probably disrupted by the high velocity with which they left the sorter and impacted the collection tube/solution.
Figure 3 shows the flow cytometric scattergram analysis on collagenase-liberated adipocytes from a nontransplanted, wild-type C57BL/6 mouse in which no GFP+ adipocytes were detected, compared with a sample from a GFP-expressing transgenic mouse in which all adipocytes expressed GFP. By comparison, samples from GFP+ BMT chimeric mice fed control diet revealed the presence of a small number (<10% of total cells) of GFP+ adipocytes in either omental or intrascapular fat. Samples from ROSI-treated animals had increased numbers of GFP+ adipocytes, ranging from approximately 15% to 30% of total cells. Samples from high-fat diet–fed animals demonstrated more GFP+ adipocytes than control samples but fewer than samples from ROSI-treated animals.
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.
Duplicate samples of saponin-permeabilized isolated adipocytes were stained with PI and analyzed by flow cytometry for GFP and DNA content to assess the nuclear ploidy as an indicator of cell fusion events (19). Flow cytometric analysis with singlet discrimination demonstrated that 0.5%–1.5% of the GFP+ adipocyte population displayed multinuclear content characteristic of increased nuclear ploidy or putative cell fusion events (Figure 4). This percentage of fused cells was too small to account for the large number of GFP+ adipocytes detected by FACS analysis, indicating that the GFP+ ML adipocytes arose from differentiation of BM-derived circulating precursors into ML adipocytes. These data also exclude the possibility that adipocytes become GFP+ due to the adherence of GFP+ circulating cells such as macrophages to ML adipocytes.
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.
Preliminary phenotypic evaluation of GFP+ ML adipocytes. Microscopic examination of isolated GFP+ adipocytes confirmed their ML appearance, which is similar to that of brown fat cells but markedly different from that of unilocular white adipocytes (Figure 5A). Figure 5B shows a phase-contrast image of a cluster of isolated adipocytes superimposed on a fluorescence image, demonstrating the substantial number of GFP+ adipocytes among the entire adipocyte population in ROSI-treated animals after 7 weeks of ROSI treatment.
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.
Semiquantitative RT-PCR analysis demonstrated that the GFP+ ML adipocytes expressed several factors associated with terminal adipogenic development at levels comparable to those in white and brown adipocytes including CCAAT/enhancer-binding protein α (C/EBPα), PPARγ, adiponectin, perilipin, and fatty acid–binding protein (FABP) (Figure 6A). ML adipocytes expressed approximately 5-fold more UCP-1 RNA than white adipocytes, but only about 5% as much as detected in brown adipocytes. Leptin RNA levels in ML adipocytes were comparable to those in white adipocytes, while levels in brown adipocytes were approximately 5-fold lower. Levels of β3–adrenergic receptor (β3-AR) RNA in ML adipocytes were intermediate between those measured in white and brown adipocytes. Immunohistochemical analysis confirmed the results of the RT-PCR analysis. GFP+ ML adipocytes expressed factors associated with terminal adipogenic development in mature white and brown adipocytes including C/EBPα, PPARγ, adiponectin, and FABP (Supplemental Figure 2A). Western blot analysis of lysates from FACS-isolated GFP+ ML adipocytes also detected expression of perilipin, leptin, and β3-AR (Supplemental Figure 2B). Fluorescence deconvolution of omental white adipose tissue from mice treated with ROSI for 7 weeks and stained with Mitotracker dye showed that ML adipocytes had higher mitochondrial content than adjacent white adipocytes (Figure 6B).
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.
Finally, Weisberg et al. (20) have reported increased numbers of macrophages in adipose tissue of obese individuals, and Cinti et al. (21) have demonstrated that macrophages can accumulate lipid from dying adipocytes. To determine whether GFP+ ML adipocytes arise from macrophages, isolated GFP+ adipocytes, stromal/vascular cells, or PBMCs were stained with antibodies to the pan-leukocyte marker CD45 and the macrophage marker CD11b and subjected to FACS analysis. Figure 7 shows that PBMCs contained a considerable number of GFP+ cells expressing CD45 and/or CD11b. Likewise, the nonbuoyant stromal/vascular cell population contained GFP+ cells with CD45 and/or CD11b surface markers. However, CD45 was undetectable in the GFP+ ML adipocyte population with or without CD11b. A small percentage (0.05%) of ML adipocytes expressed low levels of CD11b. These data suggest that ML adipocytes do not arise from macrophages.
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.