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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 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.
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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.

Mentions: The AKT kinases regulate several processes including cellular growth, survival, and metabolism29. The most prominent mammalian isoforms are AKT1 and AKT2. While AKT1 is widely expressed and promotes the growth of many tissues30,31, AKT2 regulates metabolic flux within liver, muscle, and, adipose tissue.32,33. Since AKT1 and AKT2 are both expressed in APs isolated from both VWAT and SWAT (Figure 5A), we isolated APs from VWAT after 3 days of HFD feeding and analyzed AKT1 and AKT2 phosphorylation by western blot. Using antibodies specific for phosphorylated AKT2 at S474 and AKT1 at S473, we find significant elevation of phospho-AKT2 at the height of AP activation in HFD-fed mice while levels of phospho-AKT1 are low in APs and do not increase in response to HFD (Figure 5B–C). We do, however, detect phospho-AKT1 in the non-AP cell population. (Supplementary Figure 3E). These data indicate that AKT2 is specifically activated in APs in response to HFD-feeding.


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 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.
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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.
Mentions: The AKT kinases regulate several processes including cellular growth, survival, and metabolism29. The most prominent mammalian isoforms are AKT1 and AKT2. While AKT1 is widely expressed and promotes the growth of many tissues30,31, AKT2 regulates metabolic flux within liver, muscle, and, adipose tissue.32,33. Since AKT1 and AKT2 are both expressed in APs isolated from both VWAT and SWAT (Figure 5A), we isolated APs from VWAT after 3 days of HFD feeding and analyzed AKT1 and AKT2 phosphorylation by western blot. Using antibodies specific for phosphorylated AKT2 at S474 and AKT1 at S473, we find significant elevation of phospho-AKT2 at the height of AP activation in HFD-fed mice while levels of phospho-AKT1 are low in APs and do not increase in response to HFD (Figure 5B–C). We do, however, detect phospho-AKT1 in the non-AP cell population. (Supplementary Figure 3E). These data indicate that AKT2 is specifically activated in APs in response to HFD-feeding.

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