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. 2015 Apr;17(4):376-85.
doi: 10.1038/ncb3122. Epub 2015 Mar 2.

Rapid Depot-Specific Activation of Adipocyte Precursor Cells at the Onset of Obesity

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Free PMC article

Rapid Depot-Specific Activation of Adipocyte Precursor Cells at the Onset of Obesity

Elise Jeffery et al. Nat Cell Biol. .
Free PMC article

Abstract

Excessive accumulation of white adipose tissue (WAT) is the defining characteristic of obesity. WAT mass is composed primarily of mature adipocytes, which are generated through the proliferation and differentiation of adipocyte precursors (APs). Although the production of new adipocytes contributes to WAT growth in obesity, little is known about the cellular and molecular mechanisms underlying adipogenesis in vivo. Here, we show that high-fat diet feeding in mice rapidly and transiently induces proliferation of APs within WAT to produce new adipocytes. Importantly, the activation of adipogenesis is specific to the perigonadal visceral depot in male mice, consistent with the patterns of obesogenic WAT growth observed in humans. Furthermore, we find that in multiple models of obesity, the activation of APs is dependent on the phosphoinositide 3-kinase (PI3K)-AKT2 pathway; however, the development of WAT does not require AKT2. These data indicate that developmental and obesogenic adipogenesis are regulated through distinct molecular mechanisms.

Conflict of interest statement

Competing Financial Interests Statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. High-fat diet feeding induces depot-specific adipocyte hyperplasia
(A) Experimental scheme for analysis of in vivo adipogenesis with the Adiponectin-creER; mT/mG mouse model. (B–C) Representative images (B) and quantification (C) of adipocyte tracing in Adiponectin-creER; mT/mG mice after tamoxifen treatment followed by 8 weeks on the indicated diets (n = 3 mice for baseline and HFD, n = 4 mice for SD). Note that the vasculature remains mTomato-positive in the images in (B). Significance between the indicated groups in (C) was calculated using a two-tailed student’s t-test. Exact p-values are listed in Supplementary Table 1. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01). Scale bar in (B) is 100μm. HFD: high-fat diet, SD: standard diet, mT/mG: membrane tomato/ membrane GFP.
Figure 2
Figure 2. High-fat diet feeding induces adipocyte precursor activation
(A) Representative flow cytometry histogram showing gating for BrdU-positive AP cell populations in control (no BrdU treatment) and experimental samples. (B) Quantification of BrdU incorporation into APs from VWAT (left) or SWAT (right) of male mice at the indicated time points of diet treatment (see Supplementary Figure 1A). (n = 4 mice for wild-type male SD week 1, n = 5 mice for all other groups). (C–D) Quantification of BrdU incorporation into CD24+ and CD24 subpopulations of APs in VWAT (C) or SWAT (D) after one week (left panel) or 12 hours (right panel) of BrdU treatment at the onset of HFD feeding compared to SD controls. (For 1 week VWAT n = 4 mice for SD and n = 5 mice for HFD. For 12-hour VWAT n = 10 mice for SD and 7 mice for HFD. For 1 week SWAT n = 4 mice for SD and n = 5 mice for HFD. For 12-hour SWAT n = 8 mice for SD and n = 9 mice for HFD.) (E) 3D representation of confocal z-stack from VWAT of PdgfRα-H2B-GFP mice showing GFP-positive nuclei. Hash marks represent 50 μm increments. (F) Total number of GFP+ nuclei in the VWAT of PdgfRα-H2B-GFP mice after 1 week of HFD compared to SD controls was estimated using confocal microscopy (see methods) (n = 5 mice per group). Significance in (B) was calculated using 2-way ANOVA with Bonferroni’s post-test for multiple comparisons. Significance between the indicated groups in (C) and (D) was calculated using a two-tailed student’s t-test. Significance in (F) was calculated using a one-tailed student’s t-test. Exact p-values are listed in Supplementary Table 1. Statistics source data for 2B can be found in Supplementary Table 2. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01), *** (P<0.001), **** (P<0.0001). AP: adipocyte precursor, HFD: high-fat diet, SD: standard diet, mT/mG: membrane tomato/ membrane GFP; BrdU: bromodeoxyuridine.
Figure 3
Figure 3. Activated adipocyte precursors undergo adipogenesis in vivo
(A) Schematic depicting the time points for BrdU pulse-chase experiments. (B) BrdU incorporation into adipocyte nuclei after pulse-chase from the first week of HFD feeding (n = 5 mice for each group) (C) Quantification of BrdU incorporation into APs from VWAT of male mice at the indicated time points after BrdU treatment during the first week of diet (n = 5 mice for each group). (D–E) Representative images (D) and quantification (E) of immunofluorescence staining for BrdU in adipocyte nuclei of VWAT from mice after 1 week of BrdU treatment and 8 weeks on the indicated diet. Tissue is also stained for caveolin-1 to visualize adipocyte plasma membranes, as well as DAPI to visualize nuclei. Adipocyte nuclei are indicated with arrows (yellow indicates BrdU-positive and white indicates BrdU-negative) are identified by their location inside the adipocyte plasma membrane. Multiple sections were analyzed throughout the VWAT depot. (n = 5 mice for SD, n = 4 mice for HFD) Significance in (B) was calculated using 2-way ANOVA with Bonferroni’s post-test for multiple comparisons. Significance between the indicated groups in (C) and (E) was calculated using a two-tailed student’s t-test. Exact p-values are listed in Supplementary Table 1. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01), *** (P<0.001). Scale bars in (D) are 25μm. AP: adipocyte precursor, HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine.
Figure 4
Figure 4. Diet-induced proliferation of adipocyte precursors correlates with cell-intrinsic Akt phosphorylation
(A) Quantification of BrdU incorporation into APs from male VWAT after 24-hour pulses of BrdU for each day at the beginning of HFD-treatment. (n = 5 mice for each group) (B) Representative flow cytometry histograms of AP stained for phosphorylated AKT (T308) on day 3 of HFD feeding compared to SD and fluorescence-minus one control. (C) Quantification of mean fluorescence intensity (MFI) of pAKT staining by flow cytometry in AP cells on day 3 of HFD feeding and SD controls. (n = 5 mice for each group) (D–E) Correlation between pAKT T308 (D) or S473 (E) MFI and AP proliferation in VWAT of wild-type mice on day 3 of HFD or SD feeding. (n = 10 mice for each group). (F) Quantification of mean fluorescence intensity (MFI) of pAKT staining by flow cytometry in AP cells on day 14 of HFD feeding compared to SD controls. (n = 5 mice for each group) Significance of each HFD group compared to SD in (A) was calculated using a two-tailed student’s t-test. Significance in (C) was calculated using a two-tailed student’s t-test. Significance in (D) and (E) was calculated using two-tailed correlation analysis. Exact p-values are listed in Supplementary Table 1. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01), *** (P<0.001), **** (P<0.0001). AP: adipocyte precursor, HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine, pAkt: phosphorylated Akt, MFI: mean fluorescence intensity.
Figure 5
Figure 5. Diet-induced adipocyte precursor activation and adipogenesis requires Akt2 in adipocyte lineage cells
(A) Western blot for AKT1 and AKT2 in lysates from SVF or FACS-sorted APs from the SWAT (S) or VWAT (V) depot pooled from 12 wild-type animals. Uncropped blots are shown in Supplementary Figure 7A–B. (B) Western blot of lysates from APs enriched from SVF via Sca-1 bead pull down (see methods) after 3 days of HFD or SD. Each lane represents pooled cells from 2 mice. Uncropped blots are shown in Supplementary Figure 7C–H. (C) Quantification of western blots in (B) showing normalization of pAKT1 and pAKT2 to total AKT1 and AKT2, respectively. (D) Quantification of BrdU incorporation into VWAT APs after 1 week of HFD or SD with daily injection of wortmannin (Wort), or vehicle (Veh) (n = 5 mice per group). (E) BrdU incorporation into VWAT APs of the indicated groups during the first week of HFD feeding compared to SD controls. (n = 7 for Akt2fl/fl SD, n = 4 for PdgfRα-cre; Akt2fl/fl SD, n = 14 for PdgfRα-cre; Akt2fl/fl HFD, n = 8 for Akt2fl/fl HFD, n = 5 for wild-type and PdgfRα-cre HFD) (F) Quantification of immunofluorescence staining for BrdU in adipocyte nuclei of VWAT from the indicated groups of mice after 1 week of BrdU treatment and 8 weeks on the indicated diet. (n = 8 mice for PdgfRα-cre; Akt2fl/fl HFD, n = 5 mice for wild-type groups). Significance between the indicated groups in (C), (D), and (F) was calculated using a two-tailed student’s t-test. Significance in (E) was calculated using one-way ANOVA with Tukey’s test for multiple comparisons. Exact p-values are listed in Supplementary Table 1. Statistics source data for 5C can be found in Supplementary Table 2. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01), *** (P<0.001), **** (P<0.0001). SVF: stromal-vascular fraction, AP: adipocyte precursor, HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine, Veh: vehicle, Wort: wortmannin.
Figure 6
Figure 6. AKT2 is required for activation of adipocyte precursors in multiple models of obesity
(A–B) BrdU incorporation into VWAT (A) or SWAT (B) APs of the indicated groups after 1 week of treatment. (n = 5 mice for wild-type SD, n = 7 mice for wild-type HFD, n = 6 mice for Akt1−/− SD, n = 7 mice for Akt1−/− HFD, n = 17 mice for Akt2−/− SD, n = 15 mice for Akt2−/− HFD.) (C) Quantification of immunofluorescence staining for BrdU in adipocyte nuclei of VWAT from Akt2−/− mice after 1 week of BrdU treatment and 8 weeks on the indicated diet. (n = 4 mice for SD, n = 5 mice for HFD). (D–E) Body weight (D) and fat mass (E) in young mice after weaning at 3 weeks of age. (n = 7–8 mice for ob/ob; Akt2−/−, n = 4 mice for ob/ob). Some error bars are obstructed by symbols. (F) BrdU incorporation into VWAT APs in the indicated groups after BrdU treatment from P35–40. (n = 6 mice for wild-type, n = 4 mice for ob/ob and n = 3 mice for ob/ob; Akt2−/−). (G) VWAT weights of the indicated groups at 12 weeks of age. (n = 5 mice for wild-type, n = 8 mice for ob/ob; Akt2−/−, n = 3 mice for ob/ob). Significance between indicated groups in (A–C) was calculated using a two-tailed student’s t-test. Significance between indicated groups in (D–E) was calculated using 2-way ANOVA with Bonferroni’s post-test for multiple comparisons (p-values are adjusted p-values). Significance in (F–G) was calculated using a two-tailed student’s t-test. Exact p-values are listed in Supplementary Table 1. Statistics source data for 6D–E can be found in Supplementary Table 2. Error bars represent mean ± s.e.m. * (P<0.05), ** (p<0.01), *** (P<0.001), **** (P<0.0001). AP: adipocyte precursor, HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine.
Figure 7
Figure 7. AKT2 is not required for normal development of white adipose tissue
(A) Schematic depicting the time points when the indicated experiments were performed during WAT development. (B–C) Quantification of BrdU incorporation into APs of SWAT (A) and VWAT (B) after 24-hour BrdU treatment at the indicated days of embryonic (E) and postnatal (P) development in wild-type and Akt2−/− mice. (n = 6 mice for wild-type E18.5, n = 5 mice for wild-type P7, n = 3 mice for wild-type P16, n = 4 mice for Akt2−/− groups). Each group represents a single litter. All mice are male except mice in the E18.5 group, for which male and female data are combined (D–E) Confocal images (D) of developing wild-type and Akt2−/− VWAT stained with LipidTOX (a lipid stain) and Isolectin IB4 (an endothelial cell stain) and (E) corresponding lipid droplet size quantification based on LipidTOX staining (n = 3 mice for P8 and P15, n = 4 mice for P11). Each group includes mice from 2 different litters, except Akt2−/− P11, which contains mice from 3 litters. (F) Echo MRI analysis of fat mass throughout development in wild-type and Akt2−/− mice. n = 13 mice except for wild-type week 5 (n = 18 mice), wild-type week 4 (n = 14 mice), Akt2−/− week 5 (n = 11 mice), and Akt2−/− week 6 (n = 10 mice). wild-type mice are from 4 different litters. Akt2−/− mice are from 6 different litters. Error bars represent mean ± s.e.m. Scale bars are 50μm in (D), AP: adipocyte precursor, BrdU: bromodeoxyuridine.

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