Rapid cellular turnover in adipose tissue

Abstract

It was recently shown that cellular turnover occurs within the human adipocyte population. Through three independent experimental approaches--dilution of an inducible histone 2B-green fluorescent protein (H2BGFP), labeling with the cell cycle marker Ki67 and incorporation of BrdU--we characterized the degree of cellular turnover in murine adipose tissue. We observed rapid turnover of the adipocyte population, finding that 4.8% of preadipocytes are replicating at any time and that between 1-5% of adipocytes are replaced each day. In light of these findings, we suggest that adipose tissue turnover represents a possible new avenue of therapeutic intervention against obesity.

Figure 1. Rapid adipocyte turnover in adipose tissue.

A. Schematic showing how Rosa26-rtTA; tetO-H2BGFP pulse-chase dilution can distinguish between homogeneous adipocyte turnover (all adipocytes show loss of H2BGFP florescence) and heterogeneous adipocyte turnover (some adipocytes show loss of H2BGFP florescence, others maintain H2BGFP florescence). B. Experimental schematic for Rosa26-rtTA; tetO-H2BGFP expression. C. Timeline for Rosa26-rtTA; tetO-H2BGFP pulse-chase experiments. D. H2BGFP (green) is detected in the nucleus of single murine adipocytes expressing perilipin (red) and DAPI (blue). Scale bars 50 µm. E. Uniform loss of label in adult adipocytes following pulse-chase with Rosa26-rtTA; tetO-H2BGFP following a chase period of up to 6 months. Top row, Perilipin (red), DAPI (blue). Bottom row, CEBPα (red), DAPI (blue). No adipocytes are labeled in the absence of pulse. Exposure matched images. Scale bars 100 µm. F. FACS histograms of dissociated adipocytes of Rosa26-rtTA; tetO-H2BGFP pulse-chase animals are presented as number of H2BGFP-positive cells versus fluorescence intensity. Median fluorescence intensity of GFP-positive cells versus length of chase shown in graph. G. Even after six months of chase, LRCs were detected in the post-mitotic photoreceptor cells of the retina after expression of Rosa26-rtTA; tetO-H2BGFP from E0-6weeks. H2BGFP expression (green), recoverin (red), DAPI (blue). Magnification 200x. All images are exposure matched.

Figure 2. Cellular replication in adipose tissue assayed with the cell cycle protein Ki67.

A. In mouse adipose tissue, Ki67 (green) overlaps with C/EBPα (red) and DAPI (blue) in perilipin-positive (purple) adipose tissue in situ. White arrow indicates triple positive cell. Isotype antibody control shown in right most panel. Scale bars 50 µm. B. The percentage of Ki67-positive C/EBPα-expressing cells was determined in 6 (n = 6), 14 (n = 9), and 30-week old (n = 3) mice. Throughout adult life, 4.8% of cells in adipose tissue are Ki67 and C/EBPα-positive at any time. C. FACS plots of the stromal/vascular nuclei from dissociated fat tissue of wild-type mice, stained for Ki67 and C/EBPα, as well as appropriate secondary antibody controls. The total percentage of Ki67-positive C/EBPα-positive nuclei in the stromal/vascular fat sample (above secondary alone background for all antibodies) was 3.85%; of this 0.06% represented C/EBPα-high Ki67-positive events, and 3.79% represented C/EBPα-low Ki67-positive events. Top panels represent 8-week-old mice, bottom panels represent 10-week-old mice. D. In human adipose tissue, Ki67 (green) overlaps with C/EBPα (red) and DAPI (blue) in perilipin-positive (purple) adipose tissue in situ. White arrow indicates triple positive cell. Scale bars 50 µm.

Figure 3. New adipocyte formation assayed with BrdU incorporation.

A, Top. BrdU incorporation (green) in newly formed single mature adipocyte labeled with perilipin (red) in situ. White arrow indicates triple positive cell. BrdU was pulsed for 6 hours. DAPI stained nuclei are blue. Isotype antibody control is shown in right most panel. Scale bars 100 µm. A, Bottom. Confocal z-stack images confirming that 6 hours after injection, BrdU incorporation (green) is detected in the nucleus of single murine adipocytes expressing perilipin (red). Magnification, 200x; scale bars 50 µm. B. The rate of replication was assayed following daily injection of BrdU into 6-week-old mice for 1 (n = 7), 3 (n = 7), 5 (n = 6), 7 (n = 9) or 10 (n = 7) continuous days. Perilipin staining is shown in red, BrdU staining in green, DAPI stained nuclei are blue. Scale bars 100 µm. C. Linear regression analysis of BrdU counts. Each data point on the graph represents the percentage of BrdU-positive cells in the perilipin-positive population from one animal. Rate of new adipocyte formation in 6-week old mice is 1.1% per day. D. No BrdU is detected in post-mitotic photoreceptor cells. Recoverin, a marker of photoreceptor cells, is shown in red, BrdU staining in green, DAPI stained nuclei are blue. Top panel, 100x. Bottom panel, 400x. Bottom panel represents the region within the dashed line in the top panel.

Figure 4. Increased adipose tissue replication in OB/OB mice.

A. The rate of adipose tissue replication was assayed by co-staining for the cell cycle protein Ki67 (green) and the preadipocyte/adipocyte marker C/EBPα (red) in 14-week-old wildtype and OB/OB mice. Representative images of wildtype and OB/OB adipose tissue are shown. White arrows indicate cells co-staining for Ki67 and C/EBPα. DAPI stained nuclei are blue. Magnification, 200x; scale bars 100 µm. B. The percentage of C/EBPα-expressing cells in the growth cycle was quantitated for 14-week old C57/BL6 wildtype (n = 9) and OB/OB (n = 6) mice. There is a statistically significant increase in the rate of preadipocyte replication (p<0.05) from 4.6% to 7.3%, in wildtype and OB/OB mice, respectively. C. The rate of adipose tissue replication was assayed by co-staining for BrdU incorporation (green) and the preadipocyte/adipocyte marker C/EBPα (red) in 14-week-old wildtype and OB/OB mice. Representative images of wildtype and OB/OB adipose tissue are shown. White arrows indicate cells co-staining for BrdU and C/EBPα. DAPI stained nuclei are blue. Magnification, 200x; scale bars 100 µm. D. The percentage of C/EBPα-expressing cells having incorporated BrdU following 5 days of BrdU injection was quantitated for 14-week old C57/BL6 wildtype (n = 5) and OB/OB (n = 6) mice. There is a statistically significant increase in the rate of adipose tissue replication (p<0.05) from 2.9% to 9.7%, in wildtype and OB/OB mice, respectively.