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Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity

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.

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Figure 1: High-fat diet feeding induces depot-specific adipocyte hyperplasia.
Figure 2: High-fat diet feeding induces adipocyte precursor activation.
Figure 3: Activated adipocyte precursors undergo adipogenesis in vivo.
Figure 4: Diet-induced proliferation of adipocyte precursors correlates with cell-intrinsic Akt phosphorylation.
Figure 5: Diet-induced adipocyte precursor activation and adipogenesis requires Akt2 in adipocyte lineage cells.
Figure 6: Akt2 is required for activation of adipocyte precursors in multiple models of obesity.
Figure 7: Akt2 is not required for normal development of white adipose tissue.

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Acknowledgements

This work was supported by American Diabetes Association Award 7-12-JF-46, DERC pilot project grant DK045735 and NIDDK grant DK090489 to M.S.R., Lo Graduate Fellowship for Excellence in Stem Cell Research from the Yale Stem Cell Center to E.J., and EMBO long-term fellowship ALTF 132-2011 to C.D.C.

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E.J., C.D.C. and M.S.R. designed experiments. E.J., C.D.C., B.H., L.C. and M.S.R. performed experiments. E.J., C.D.C. and B.H. analysed data. E.J., C.D.C., B.H. and M.S.R. interpreted data. E.J. and M.S.R. wrote the manuscript.

Corresponding author

Correspondence to Matthew S. Rodeheffer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 3 Characterization of HFD-induced adipocyte hyperplasia.

(A) Experimental scheme for BrdU time course in Figure 2B. (B) Representative flow cytometry dot plots indicating sequential gating used to identify AP populations, including CD24+ adipocyte progenitors (CD45, CD31, CD34+, CD29+, Sca1+, CD24+), CD24 preadipocytes (CD45, CD31, CD34+, CD29+, Sca1+, CD24), and total APs (CD45, CD31, CD34+, CD29+, Sca1+). (C) Flow cytometry plots of SVF from VWAT of PdgfRα-H2B-GFP mice showing that the majority of GFP+ cells are CD29+; CD34+, of which 97.1 ± 1.5% are also Sca1+. (n = 3 mice) (D) Quantification of BrdU incorporation into GFP+ cells from VWAT of male PdgfRα-H2B-GFP mice during 1 week of SD or HFD feeding and BrdU treatment. (n = 5 mice for SD and 7 mice for HFD.) Significance between the indicated groups in (D) was calculated using a two-tailed Student’s t-test. Exact p-values are listed in Supplementary Table 1. Error bars or values represent mean ± s.e.m. (p < 0.01). HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine.

Supplementary Figure 4 Adipocyte precursor proliferation during week 2 of HFD feeding does not lead to adipogenesis.

(A) Schematic and depicting the second week BrdU pulse-chase in Supplementary Figure 2B. (B) BrdU incorporation into adipocyte nuclei after pulse-chase from the second week of HFD feeding. (n = 5 for all groups). Error bars or values represent mean ± s.e.m. HFD: high-fat diet, SD: standard diet, BrdU: bromodeoxyuridine.

Supplementary Figure 5 High-fat diet does not induce AKT phosphorylation in whole fat.

(A) Western blot analysis for phosphorylated AKT at the indicated sites and total AKT, in lysates from whole VWAT on day 3 of diet. Each lane represents one animal. Uncropped blots are shown in Supplementary Figure 7I-L. (B) Quantification of pAKT western blots in (A), normalized to total AKT. (C-D) VWAT was treated with the indicated concentrations of insulin prior to tissue digestion, and APs were isolated in the presence of wortmannin and phosphatase inhibitors to limit further changes in pathway activation (see methods). APs were then fixed and analyzed by flow cytometry for levels of phosphorylated AKT at threonine 308 (C) or serine 473 (D). The same AP cell isolation procedure was used in the experiments in Figure 4B-F and 5B-C. (E) Western blot for phosphorylated AKT1 (S473) with lysates from APs enriched from SVF via Sca-1 bead pull down (Sca1-positive) and remaining unselected cells (Sca1-negative) (see methods) after 3 days of HFD or SD. Each lane represents pooled cells from 2 mice. Uncropped blots are shown in Supplementary Figure 7M-N. Significance between the indicated groups in (B) was calculated using a two-tailed Student’s t-test. Error bars represent mean ± s.e.m. HFD: high-fat diet, SD: standard diet.

Supplementary Figure 6 Deletion of AKT2 in the adipocyte lineage does not affect body weight or food intake.

(A) Quantification of BrdU incorporation into SWAT APs after 1 week of HFD or SD with daily injection of wortmannin (Wort), or vehicle (Veh) (n = 5 mice for each group). (B) PCR analysis of DNA isolated from AP cells from the VWAT and SWAT depots of female mice show successful excision of the floxed exons 4 and 5 from Akt2 gene, while Lin+ cells (CD45+ blood cells and CD31+ endothelial cells) contain the full-length Akt2 gene. Each lane represents cells isolated from the indicated depot of individual mice. (C) Body weight of Akt2fx/fl (n = 9 mice) and PdgfRα-cre; Akt2fl/fl (n = 12 mice) at 6 weeks of age. (D) Quantification of food intake normalized to body weight in the indicated groups of mice on SD or during the first 4 days of HFD feeding. (n = 3 mice for Akt2fl/fl SD and PdgfRα-cre; Akt2fl/flSD, n = 4 mice for PdgfRα-cre HFD, n = 2 mice for Akt2fl/fl HFD, and n = 3 mice for PdgfRα-cre; Akt2fl/fl HFD. (E) BrdU incorporation into SWAT APs of the indicated groups during the first week of HFD feeding compared to SD controls. (n = 7 mice for Akt2fl/fl SD, n = 4 mice for PdgfRα-cre; Akt2fl/fl SD, n = 14 mice for PdgfRα-cre; Akt2fl/fl HFD, n = 8 mice for Akt2fl/fl HFD, n = 5 mice for wild-type and PdgfRα-cre HFD). Significance in (D) 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 4D can be found in Supplementary Table 2. Error bars represent mean ± s.e.m. (p < 0.01), (P < 0.0001).

Supplementary Figure 7 AKT2 is required for the activation of adipocyte precursors at the onset of obesity in ob/ob mice.

(A-B) Weekly intake of SD in raw kilocalories (kCal) (A) or kCal normalized to body weight (B) for the indicated groups of young mice after weaning at 3 weeks of age. Week “3” denotes food intake between the ages of 3 and 4 weeks. (n = 4 for wild-type, n = 3 for ob/ob, n = 7-8 for ob/ob; Akt2−/−) (C-D) H&E-stained sections of VWAT (C) and corresponding adipocyte size measurements (D) for the indicated groups. (n = 7 for ob/ob; Akt2−/−, n = 4 for ob/ob). Significance between indicated groups in (A-B) was calculated using a two-tailed Student’s t-test. Significance in (D) was calculated using two-way ANOVA with Bonferroni’s test for multiple comparisons. Scale bars in (C) are 50 μm. or a (P < 0.05), or b (p < 0.01), or c (P < 0.001), or d (P < 0.0001). For (A-B), indicates significance of ob/ob over wild-type, † indicates significance of ob/ob; Akt2−/− over wild-type, and ‡ indicates significance of ob/ob; Akt2−/− over ob/ob. Exact p-values are listed in Supplementary Table 1. Statistics source data for 5A-B can be found in Supplementary Table 2. Error bars represent mean ± s.e.m.

Supplementary Figure 8 AKT2 is not required for the normal development of white adipose tissue.

(A-B) Confocal images (A) of developing wild-type and Akt2−/− SWAT stained with LipidTOX (a lipid stain) and Isolectin IB4 (an endothelial cell stain) and (B) corresponding lipid droplet size quantification based on LipidTOX staining (n = 3 mice for all groups except n = 4 mice for Akt2−/−P5). Each group includes mice from 2-3 different litters. (C) Echo MRI quantification of fat mass from the indicated groups of mice at 6 weeks of age. (n = 13 mice for Akt2fl/fl and n = 14 mice for PdgfRα-cre; Akt2fl/fl). Scale bars in (A) are 50 μm. Error bars represent mean ± s.e.m. PX indicates postnatal day X.

Supplementary Table 1 Exact p-values.
Supplementary Table 2 Individual data points.

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Jeffery, E., Church, C., Holtrup, B. et al. Rapid depot-specific activation of adipocyte precursor cells at the onset of obesity. Nat Cell Biol 17, 376–385 (2015). https://doi.org/10.1038/ncb3122

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