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Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes.

Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH - Nat. Med. (2015)

Bottom Line: Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes.Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29.These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

View Article: PubMed Central - PubMed

Affiliation: 1] Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA. [2] Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

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Utilization of a UCP1 reporter system for in vitro and in vivo monitoring of UCP1 expression. (a) Schematic structure of the hUCP1 promoter reporter system. 4148 bp of human UCP1 promoter drives the expression of bicistronic luciferase and GFP. T2A is the internal ribosomal entry site. (b) In hBAT-SVF and hWAT-SVF stably expressed the reporter construct, luciferase activity (Right) was strongly correlated with endogenous UCP1 gene expression (Left) during the course of differentiation (see Fig. 1a and Method). Data are presented as fold changes compared to hWAT-SVF on day 0 (mean ± s.e.m., n=3). A representative experiment from a total of two independent studies is shown. (c) Monitoring UCP1 expression by GFP in vitro using a time lapse imaging system during differentiation of hBAT-SVF from Sub1. (d) Representative IVIS images of nude mice after 22 days of transplantation of hWAT-SVF and hBAT-SVF are shown on the left panel. Quantifications of luciferase activity by total flux are shown on the right panel (mean ± s.e.m.). The experiments have been repeated twice (n=2 for hWAT-SVF group; n=3 for hBAT-SVF group). (e) Q-RT-PCR analysis for expression of FABP4, UCP1 and LEP in fat pads developed from the transplanted cells. Data are presented as fold changes compared to fat pads developed from hWAT-SVF with vehicle treatment (mean ± s.e.m.). Two-tailed Student’s t-test was used to determine P values (* P < 0.05, ** P < 0.01, *** P < 0.001).
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Figure 2: Utilization of a UCP1 reporter system for in vitro and in vivo monitoring of UCP1 expression. (a) Schematic structure of the hUCP1 promoter reporter system. 4148 bp of human UCP1 promoter drives the expression of bicistronic luciferase and GFP. T2A is the internal ribosomal entry site. (b) In hBAT-SVF and hWAT-SVF stably expressed the reporter construct, luciferase activity (Right) was strongly correlated with endogenous UCP1 gene expression (Left) during the course of differentiation (see Fig. 1a and Method). Data are presented as fold changes compared to hWAT-SVF on day 0 (mean ± s.e.m., n=3). A representative experiment from a total of two independent studies is shown. (c) Monitoring UCP1 expression by GFP in vitro using a time lapse imaging system during differentiation of hBAT-SVF from Sub1. (d) Representative IVIS images of nude mice after 22 days of transplantation of hWAT-SVF and hBAT-SVF are shown on the left panel. Quantifications of luciferase activity by total flux are shown on the right panel (mean ± s.e.m.). The experiments have been repeated twice (n=2 for hWAT-SVF group; n=3 for hBAT-SVF group). (e) Q-RT-PCR analysis for expression of FABP4, UCP1 and LEP in fat pads developed from the transplanted cells. Data are presented as fold changes compared to fat pads developed from hWAT-SVF with vehicle treatment (mean ± s.e.m.). Two-tailed Student’s t-test was used to determine P values (* P < 0.05, ** P < 0.01, *** P < 0.001).

Mentions: To allow direct assessment of the thermogenic potential of differentiated cells, we introduced a transgenic reporter construct into the white and brown fat precursors to measure UCP1 gene expression by coupling a bicistronic luciferase and green fluorescent protein (GFP) reporter system to a 4.1-Kb human UCP1 promoter fragment (Fig. 2a). In mature adipocytes that stably expressed the reporter construct, luciferase activity was strongly correlated with endogenous UCP1 gene expression and only detected in mature brown adipocytes but not in undifferentiated cells (Fig. 2b). We monitored differentiating cells using time-lapse microscopy and could detect activation of the GFP reporter as early as day 9 in differentiating BAT cells (Fig. 2c and Supplementary video). To determine if these cells were capable of differentiation in vivo, we transplanted progenitor cells into immune-deficient nude mice and used in vivo bioluminescent imaging to measure UCP1 reporter activity. Luciferase activity was high in mice implanted with hBAT progenitors, and could be further induced by BMP7 pretreatment of progenitors (Fig. 2d). Conversely, mice receiving transplanted hWAT progenitors displayed almost no detectable luciferase activity. While both grafts expressed similar level of FABP4, fat grafts from hBAT-SVF displayed at least 100-fold increase in UCP1 mRNA compared to hWAT-SVF-derived fat pads which is consistent with luciferase activity. LEP was selectively expressed in fat grafts from hWAT-SVF (Fig. 2e). These data demonstrate that the UCP1 reporter system accurately indicates differentiation into mature brown adipocytes in both in vivo and in vitro settings.


Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes.

Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH - Nat. Med. (2015)

Utilization of a UCP1 reporter system for in vitro and in vivo monitoring of UCP1 expression. (a) Schematic structure of the hUCP1 promoter reporter system. 4148 bp of human UCP1 promoter drives the expression of bicistronic luciferase and GFP. T2A is the internal ribosomal entry site. (b) In hBAT-SVF and hWAT-SVF stably expressed the reporter construct, luciferase activity (Right) was strongly correlated with endogenous UCP1 gene expression (Left) during the course of differentiation (see Fig. 1a and Method). Data are presented as fold changes compared to hWAT-SVF on day 0 (mean ± s.e.m., n=3). A representative experiment from a total of two independent studies is shown. (c) Monitoring UCP1 expression by GFP in vitro using a time lapse imaging system during differentiation of hBAT-SVF from Sub1. (d) Representative IVIS images of nude mice after 22 days of transplantation of hWAT-SVF and hBAT-SVF are shown on the left panel. Quantifications of luciferase activity by total flux are shown on the right panel (mean ± s.e.m.). The experiments have been repeated twice (n=2 for hWAT-SVF group; n=3 for hBAT-SVF group). (e) Q-RT-PCR analysis for expression of FABP4, UCP1 and LEP in fat pads developed from the transplanted cells. Data are presented as fold changes compared to fat pads developed from hWAT-SVF with vehicle treatment (mean ± s.e.m.). Two-tailed Student’s t-test was used to determine P values (* P < 0.05, ** P < 0.01, *** P < 0.001).
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Utilization of a UCP1 reporter system for in vitro and in vivo monitoring of UCP1 expression. (a) Schematic structure of the hUCP1 promoter reporter system. 4148 bp of human UCP1 promoter drives the expression of bicistronic luciferase and GFP. T2A is the internal ribosomal entry site. (b) In hBAT-SVF and hWAT-SVF stably expressed the reporter construct, luciferase activity (Right) was strongly correlated with endogenous UCP1 gene expression (Left) during the course of differentiation (see Fig. 1a and Method). Data are presented as fold changes compared to hWAT-SVF on day 0 (mean ± s.e.m., n=3). A representative experiment from a total of two independent studies is shown. (c) Monitoring UCP1 expression by GFP in vitro using a time lapse imaging system during differentiation of hBAT-SVF from Sub1. (d) Representative IVIS images of nude mice after 22 days of transplantation of hWAT-SVF and hBAT-SVF are shown on the left panel. Quantifications of luciferase activity by total flux are shown on the right panel (mean ± s.e.m.). The experiments have been repeated twice (n=2 for hWAT-SVF group; n=3 for hBAT-SVF group). (e) Q-RT-PCR analysis for expression of FABP4, UCP1 and LEP in fat pads developed from the transplanted cells. Data are presented as fold changes compared to fat pads developed from hWAT-SVF with vehicle treatment (mean ± s.e.m.). Two-tailed Student’s t-test was used to determine P values (* P < 0.05, ** P < 0.01, *** P < 0.001).
Mentions: To allow direct assessment of the thermogenic potential of differentiated cells, we introduced a transgenic reporter construct into the white and brown fat precursors to measure UCP1 gene expression by coupling a bicistronic luciferase and green fluorescent protein (GFP) reporter system to a 4.1-Kb human UCP1 promoter fragment (Fig. 2a). In mature adipocytes that stably expressed the reporter construct, luciferase activity was strongly correlated with endogenous UCP1 gene expression and only detected in mature brown adipocytes but not in undifferentiated cells (Fig. 2b). We monitored differentiating cells using time-lapse microscopy and could detect activation of the GFP reporter as early as day 9 in differentiating BAT cells (Fig. 2c and Supplementary video). To determine if these cells were capable of differentiation in vivo, we transplanted progenitor cells into immune-deficient nude mice and used in vivo bioluminescent imaging to measure UCP1 reporter activity. Luciferase activity was high in mice implanted with hBAT progenitors, and could be further induced by BMP7 pretreatment of progenitors (Fig. 2d). Conversely, mice receiving transplanted hWAT progenitors displayed almost no detectable luciferase activity. While both grafts expressed similar level of FABP4, fat grafts from hBAT-SVF displayed at least 100-fold increase in UCP1 mRNA compared to hWAT-SVF-derived fat pads which is consistent with luciferase activity. LEP was selectively expressed in fat grafts from hWAT-SVF (Fig. 2e). These data demonstrate that the UCP1 reporter system accurately indicates differentiation into mature brown adipocytes in both in vivo and in vitro settings.

Bottom Line: Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes.Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29.These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

View Article: PubMed Central - PubMed

Affiliation: 1] Section on Integrative Physiology and Metabolism, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts, USA. [2] Division of Endocrinology and Metabolism, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT
Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

Show MeSH
Related in: MedlinePlus