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

Jeffery E, Church CD, Holtrup B, Colman L, Rodeheffer MS - Nat. Cell Biol. (2015)

Bottom Line: WAT mass is composed primarily of mature adipocytes, which are generated through the proliferation and differentiation of adipocyte precursors (APs).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University, Yale University School of Medicine, 375 Congress Ave New Haven, Connecticut 06520, USA.

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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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.
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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.

Mentions: Next, we determined the molecular pathways involved in HFD-induced AP activation. To further characterize the dynamics of AP proliferation in response to HFD, we treated groups of mice with BrdU for individual days at the onset of HFD-feeding. We found that AP proliferation in VWAT was highest at day 3 of HFD feeding and returned to SD levels by day 5 (Figure 4A). This surprisingly rapid activation of APs upon switching diets suggests that WAT cellular homeostasis is closely linked to nutrient sensing. One pathway that is involved in nutrient sensing mechanisms is the phosphoinositide 3-kinase (PI3K)-AKT pathway26. To determine whether the central kinase in this pathway, AKT, is activated in VWAT at the height of the proliferation response, we analyzed AKT phosphorylation at two critical activation sites27 in the VWAT of mice after 3 days of HFD feeding. There is no significant increase in AKT phosphorylation in protein lysates from whole adipose tissue (Supplementary Figure 3A–B), which contains many cell types28. However, when we analyze APs by flow cytometry on day 3 of HFD feeding (Supplementary Figure 3C–D) we find that AKT phosphorylation at both sites is significantly elevated within APs in VWAT (Figure 4B–C). Furthermore, when we combine AKT phosphorylation analysis with BrdU labeling on the third day of diet, we find a significant positive correlation between AP proliferation rate and the level AKT phosphorylation in APs within VWAT (Figure 4D–E). When we perform the same analysis on day 14 of diet, when AP proliferation rates have returned to background levels, we observe no difference in AKT phosphorylation in APs between SD and HFD-fed groups (Figure 4F). These data indicate that AKT signaling within APs is correlated with AP proliferation and therefore may play a role in the activation of APs at the onset of obesity.


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

Jeffery E, Church CD, Holtrup B, Colman L, Rodeheffer MS - Nat. Cell Biol. (2015)

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.
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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.
Mentions: Next, we determined the molecular pathways involved in HFD-induced AP activation. To further characterize the dynamics of AP proliferation in response to HFD, we treated groups of mice with BrdU for individual days at the onset of HFD-feeding. We found that AP proliferation in VWAT was highest at day 3 of HFD feeding and returned to SD levels by day 5 (Figure 4A). This surprisingly rapid activation of APs upon switching diets suggests that WAT cellular homeostasis is closely linked to nutrient sensing. One pathway that is involved in nutrient sensing mechanisms is the phosphoinositide 3-kinase (PI3K)-AKT pathway26. To determine whether the central kinase in this pathway, AKT, is activated in VWAT at the height of the proliferation response, we analyzed AKT phosphorylation at two critical activation sites27 in the VWAT of mice after 3 days of HFD feeding. There is no significant increase in AKT phosphorylation in protein lysates from whole adipose tissue (Supplementary Figure 3A–B), which contains many cell types28. However, when we analyze APs by flow cytometry on day 3 of HFD feeding (Supplementary Figure 3C–D) we find that AKT phosphorylation at both sites is significantly elevated within APs in VWAT (Figure 4B–C). Furthermore, when we combine AKT phosphorylation analysis with BrdU labeling on the third day of diet, we find a significant positive correlation between AP proliferation rate and the level AKT phosphorylation in APs within VWAT (Figure 4D–E). When we perform the same analysis on day 14 of diet, when AP proliferation rates have returned to background levels, we observe no difference in AKT phosphorylation in APs between SD and HFD-fed groups (Figure 4F). These data indicate that AKT signaling within APs is correlated with AP proliferation and therefore may play a role in the activation of APs at the onset of obesity.

Bottom Line: WAT mass is composed primarily of mature adipocytes, which are generated through the proliferation and differentiation of adipocyte precursors (APs).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University, Yale University School of Medicine, 375 Congress Ave New Haven, Connecticut 06520, USA.

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.

Show MeSH
Related in: MedlinePlus