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

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.

Figure 1

Generation and characterization of immortalized human brown and white fat progenitors. (a) Light microscopic images of immortalized human WAT progenitors (hWAT-SVF) and human BAT progenitors (hBAT-SVF) at day 0 and 18 (stained with Oil Red O) from 4 subjects. Scale bar, 100 μm. (b,d) Q-RT-PCR analysis for UCP1 and LEP mRNA expression in differentiated adipocytes from hWAT-SVF and hBAT-SVF of 4 subjects. Data are presented as fold changes relative to Sub1 hWA (mean ± s.e.m., n=3). (c) Western blot analysis of UCP1 protein level in hWA and hBA differentiated from progenitors of Sub1 and Sub2. α-Tubulin serves as a loading control. (e) Oxygen consumption rate (OCR) was measured in the absence (Basal respiration, Basal Res.) and presence of oligomycin (Proton Leak) or FCCP (Maximal respiration, Max. Res.) in hWA and hBA from Sub1 (Left) and Sub2 (Right). Data are presented as mean ± s.e.m. (n=10; hWA vs hBA). (f) Glucose uptake was measured using ³H-2-deoxy-glucose in hWA and hBA stimulated with (Ins100) or without (Ins0) 100 nM insulin from Sub1 (Left) and Sub2 (Right). Data are presented as mean ± s.e.m. (n=3). (g) Fatty acid uptake (FAU) and fatty acid oxidation (FAO) were measured using ¹⁴C-palmitic acid in hWA and hBA from Sub1 (Left) and Sub2 (Right). Data are presented as a fold change compared to hWA (mean ± s.e.m., n=3). (h) Q-RT-PCR analysis for UCP1 and PPARG mRNA expression in hWA and hBA from Sub1 (Left) and Sub2 (Right). Data are presented as fold changes compared to vehicle-hWA for each subject (mean ± s.e.m., n=3; NS, not significant; Veh vs BMP7). Two-tailed Student’s t-test was used to determine P values (* P < 0.05, ** P < 0.01, *** P < 0.001).

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).

Figure 3

Clonal analysis of human brown and white fat progenitors. The strategy of clonal analysis of hWAT-SVF and hBAT-SVF progenitors is shown as a dendrogram. 152 clones from hWAT-SVF and 128 clones from hBAT-SVF were derived by limiting dilution from 4 subjects. Adipogenic capacity was determined by Nile red staining and UCP1 level was determined by luciferase activity on day 18. Detailed selection criteria are described in Supplementary Figures 6 and 7. Selected highly adipogenic clones (adipogenic ++) were pre-treated with 3.3 nM BMP7 for 6 days and then differentiated into mature adipocytes in a 96-well plate. Luciferase activity was measured on day 18 and divided into different levels (negative, Neg; low; medium, Med; high) after normalized to protein content. The positive response (+) to BMP7 pretreatment was defined by more than 1.5-fold increase of luciferase activity between BMP7-pretreated and vehicle groups.

Figure 4

Gene expression profiles in adipose progenitors predict the thermogenic capacity of mature adipocytes. (a) A schematic presentation outlining the strategy utilized to identify the genes in preadipocytes with positive or negative correlation with UCP1 levels in mature adipocytes. Microarray analyses were done in 41 selected highly adipogenic clones from 4 subjects (8 clones from hWAT-SVF and 33 clones from hBAT-SVF). (b) Histogram showing the distribution of genes that are positively and negatively correlated with UCP1 levels (determined by luciferase activities). We used P-value < 0.001 as the cutoff to prioritize candidate genes (two-tailed alternative with function cor.test). The correlation coefficient (R) is shown in the X-axis and gene frequency is shown in the Y-axis. (c) Log₂ gene expression data from 50 genes most associated with UCP1 were centered to have mean zero and restricted to the interval [−2,2] and are shown in a heatmap, along with a color bar representing UCP1 at top, where darker indicates higher UCP1. UCP1 levels were determined by luciferase activities in the reporter clones. (d) Scatter plots showing the positive and negative correlations between the UCP1-luciferase levels and expression levels of candidate genes from microarrays. The log₂ gene expression level of progenitor (day 0) is shown in the X-axis. The Y-axis represents the log₂ of UCP1 luciferase level of mature adipocyte (day 18).

Figure 5

PREX1 and EDNRB are required for determining thermogenic competency. (a) A Heatmap displaying correlations between UCP1 mRNA levels on day 18 (top row) and expression levels of candidate genes on day 0. Data were obtained from 10 independent hWAT-SVF and hBAT-SVF clones derived from the same 4 subjects that were not included in microarray analyses. Values were normalized within each row using a linear color scale. (b) Levels of PREX1 and EDNRB mRNA were measured by Q-RT-PCR in PREX1 (PREX1 KO) and EDNRB (EDNRB KO) knockout hBAT-SVF clone using CRISPR/Cas9. The experiments were verified in another progenitor clone. (c) Microscopic views of differentiated PREX1 KO and EDNRB KO hBAT-SVF cells. Scale bar, 100 μm. (d) Q-RT-PCR analysis for PPARG and brown-fat-specific markers (UCP1, DIO2 and PPARGC1A) in differentiated PREX1 KO and EDNRB KO hBAT-SVF cells. (e) SSTR1 level was detected by Q-RT-PCR in a SSTR1 knockout (SSTR1 KO) hWAT-SVF clone using CRISPR/Cas9. The experiments were verified in another progenitor clone. (f) Microscope views of differentiated SSTR1 KO hWAT-SVF clone. Scale bar, 100 μm. (g) Q-RT-PCR analysis for PPARG and brown-fat-specific markers (UCP1 and DIO2) in differentiated SSTR1 KO clone. Q-RT-PCR data are presented as fold changes compared to control vector transfected cells (Ctl) (mean ± s.e.m., n=3; two-tailed Student’s t-test; * P < 0.05, ** P < 0.01, *** P < 0.001). The Ct values (Ct) are indicated to reflect the actual levels of gene expression.

Figure 6

Isolation of progenitors possessing thermogenic potential using a cell surface marker. (a) Scatter plots showing positive correlation between the UCP1-luciferase levels (shown as log₂ level in the Y-axis) on day 18 and expression levels of ITGA10 and ITGB1 (shown as log₂ level in the X-axis) on day 0 from microarray analyses. (b) Correlation between the mRNA levels of ITGA10 and ITGB1 (shown as log2 level in the X-axis) on day 0 and UCP1 mRNA levels (shown as log2 level in the Y-axis) on day 18, in 10 independent hWAT-SVF and hBAT-SVF clones as described in Figure 5a. (c) Histogram displaying subpopulations with differential levels of CD29 from pooled hWAT-SVF (Blue) and hBAT-SVF (Red) using fluorescence-activated cell sorting. Gray line represents unstained cells. (d) Microscopic views of sorted subpopulation with different level of CD29 (CD29^low, CD29^med and CD29^high) on day 0 and 18 are shown. Note that we couldn’t sort enough numbers of CD29^low from pooled hBAT-SVF, and thus results from this subpopulation are not shown. Scale bar, 100 μm. A representative experiment from two independent studies is shown. (e) Q-RT-PCR analysis for the adipocyte markers (FASN, PPARG and FABP4) and brown-fat-specific markers (UCP1, PPARGC1A and DIO2) on the differentiated indicated populations. (f) To correct for the different degrees of adipogenesis shown in (e), expression levels of UCP1 and DIO2 were normalized to the level of mature adipocyte marker, FASN. Data are presented as mean ± s.e.m. (n=3; two-tailed Student’s t-test; * P < 0.05, ** P < 0.01, *** P < 0.001).