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Retinoic Acid Mediates Visceral-Specific Adipogenic Defects of Human Adipose-Derived Stem Cells

View Article: PubMed Central - PubMed

ABSTRACT

Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots.

No MeSH data available.


RA inhibits the early stage of adipocyte differentiation of ASCs. Using a standard adipogenic cocktail, adipocyte differentiation was induced in ASCs from S11, with or without pretreatment/treatment with RA at various time points. A: A representative graph showing relative fluorescence units (RFUs) of AdipoRed staining on ASCs from S11. The ASCs were treated with 1 or 10 μmol/L of RA at different time points, as indicated by D0 (D−2 to D0), D3 (D0 to D3), D6 (D3 to D6), D9 (D6 to D9), and D12 (D9 to D12). *P < 0.05 denotes significant fold change (in RFUs) against respective “No RA” control samples, corresponding to the quantitation of lipid accumulation during adipocyte differentiation. B: Representative images (original magnification ×10) showing the lipid accumulation (AdipoRed, green) and the nuclei (Hoechst 33342, blue) in SC ASCs from S11 treated with 1 or 10 μmol/L of RA at different time points: no induction (i), without RA (ii), with RA treatment during D−2 to D0 (iii), D0 to D3 (iv), D3 to D6 (v), D6 to D9 (vi), or D9 to D12 (vii). The field in ii is magnified and merged with the bright-field image to show the overlap of AdipoRed staining and lipid droplet structures (top right). Similar results were obtained from experiments using cells from S12. For the purpose of presentation, the fluorescent intensities were enhanced to the same degree for all original images. Scale bar = 100 µm. See Supplementary Fig. 3 for representative images in VS ASCs.
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Figure 5: RA inhibits the early stage of adipocyte differentiation of ASCs. Using a standard adipogenic cocktail, adipocyte differentiation was induced in ASCs from S11, with or without pretreatment/treatment with RA at various time points. A: A representative graph showing relative fluorescence units (RFUs) of AdipoRed staining on ASCs from S11. The ASCs were treated with 1 or 10 μmol/L of RA at different time points, as indicated by D0 (D−2 to D0), D3 (D0 to D3), D6 (D3 to D6), D9 (D6 to D9), and D12 (D9 to D12). *P < 0.05 denotes significant fold change (in RFUs) against respective “No RA” control samples, corresponding to the quantitation of lipid accumulation during adipocyte differentiation. B: Representative images (original magnification ×10) showing the lipid accumulation (AdipoRed, green) and the nuclei (Hoechst 33342, blue) in SC ASCs from S11 treated with 1 or 10 μmol/L of RA at different time points: no induction (i), without RA (ii), with RA treatment during D−2 to D0 (iii), D0 to D3 (iv), D3 to D6 (v), D6 to D9 (vi), or D9 to D12 (vii). The field in ii is magnified and merged with the bright-field image to show the overlap of AdipoRed staining and lipid droplet structures (top right). Similar results were obtained from experiments using cells from S12. For the purpose of presentation, the fluorescent intensities were enhanced to the same degree for all original images. Scale bar = 100 µm. See Supplementary Fig. 3 for representative images in VS ASCs.

Mentions: It was previously reported that RA is a potent inhibitor of adipocyte differentiation in mice (27,28). To determine whether RA affects the adipocyte differentiation of human ASCs and, if so, at which adipogenic stage, we treated ASCs with two different concentrations of RA (1 and 10 μmol/L) at different times during adipocyte differentiation. The results showed that the early addition of RA drastically inhibited adipocyte differentiation during the predifferentiation period and during the first 6 days after differentiation (D−2 to D0, D0 to D3, or D3 to D6) and did so to a greater extent in SC ASCs (Fig. 5A and Bi–v) than VS ASCs (Fig. 5A and Supplementary Fig. 3i–v). A higher concentration (10 μmol/L) of RA corresponded with a greater reduction in adipocyte formation, especially that of SC ASCs, when compared with a lower concentration (1 μmol/L) of RA. Importantly, the adipogenic level of SC ASCs treated with 10 μmol/L RA during D0 to D3 or D3 to D6 was similar to that of nontreated VS ASCs. Also, just 2 days of pretreatment with RA itself was sufficient to significantly reduce adipocyte formation in ASCs. RA treatment at later stages of differentiation—that is, D6 to D9 and D9 to D12—either minimally inhibited or failed to block the adipocyte formation in both SC and VS ASCs (Fig. 5A and Bvi–vii; Supplementary Fig. 3vi–vii). Furthermore, as shown in Supplementary Fig. 4, treatment of SC and VS ASCs with retinol (Supplementary Fig. 4Ai and ii) or retinal (Supplementary Fig. 4Bi and ii), intermediates in the RA synthesis pathway, significantly inhibited adipocyte differentiation at various time points during adipogenic induction, as observed by AdipoRed staining.


Retinoic Acid Mediates Visceral-Specific Adipogenic Defects of Human Adipose-Derived Stem Cells
RA inhibits the early stage of adipocyte differentiation of ASCs. Using a standard adipogenic cocktail, adipocyte differentiation was induced in ASCs from S11, with or without pretreatment/treatment with RA at various time points. A: A representative graph showing relative fluorescence units (RFUs) of AdipoRed staining on ASCs from S11. The ASCs were treated with 1 or 10 μmol/L of RA at different time points, as indicated by D0 (D−2 to D0), D3 (D0 to D3), D6 (D3 to D6), D9 (D6 to D9), and D12 (D9 to D12). *P < 0.05 denotes significant fold change (in RFUs) against respective “No RA” control samples, corresponding to the quantitation of lipid accumulation during adipocyte differentiation. B: Representative images (original magnification ×10) showing the lipid accumulation (AdipoRed, green) and the nuclei (Hoechst 33342, blue) in SC ASCs from S11 treated with 1 or 10 μmol/L of RA at different time points: no induction (i), without RA (ii), with RA treatment during D−2 to D0 (iii), D0 to D3 (iv), D3 to D6 (v), D6 to D9 (vi), or D9 to D12 (vii). The field in ii is magnified and merged with the bright-field image to show the overlap of AdipoRed staining and lipid droplet structures (top right). Similar results were obtained from experiments using cells from S12. For the purpose of presentation, the fluorescent intensities were enhanced to the same degree for all original images. Scale bar = 100 µm. See Supplementary Fig. 3 for representative images in VS ASCs.
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Figure 5: RA inhibits the early stage of adipocyte differentiation of ASCs. Using a standard adipogenic cocktail, adipocyte differentiation was induced in ASCs from S11, with or without pretreatment/treatment with RA at various time points. A: A representative graph showing relative fluorescence units (RFUs) of AdipoRed staining on ASCs from S11. The ASCs were treated with 1 or 10 μmol/L of RA at different time points, as indicated by D0 (D−2 to D0), D3 (D0 to D3), D6 (D3 to D6), D9 (D6 to D9), and D12 (D9 to D12). *P < 0.05 denotes significant fold change (in RFUs) against respective “No RA” control samples, corresponding to the quantitation of lipid accumulation during adipocyte differentiation. B: Representative images (original magnification ×10) showing the lipid accumulation (AdipoRed, green) and the nuclei (Hoechst 33342, blue) in SC ASCs from S11 treated with 1 or 10 μmol/L of RA at different time points: no induction (i), without RA (ii), with RA treatment during D−2 to D0 (iii), D0 to D3 (iv), D3 to D6 (v), D6 to D9 (vi), or D9 to D12 (vii). The field in ii is magnified and merged with the bright-field image to show the overlap of AdipoRed staining and lipid droplet structures (top right). Similar results were obtained from experiments using cells from S12. For the purpose of presentation, the fluorescent intensities were enhanced to the same degree for all original images. Scale bar = 100 µm. See Supplementary Fig. 3 for representative images in VS ASCs.
Mentions: It was previously reported that RA is a potent inhibitor of adipocyte differentiation in mice (27,28). To determine whether RA affects the adipocyte differentiation of human ASCs and, if so, at which adipogenic stage, we treated ASCs with two different concentrations of RA (1 and 10 μmol/L) at different times during adipocyte differentiation. The results showed that the early addition of RA drastically inhibited adipocyte differentiation during the predifferentiation period and during the first 6 days after differentiation (D−2 to D0, D0 to D3, or D3 to D6) and did so to a greater extent in SC ASCs (Fig. 5A and Bi–v) than VS ASCs (Fig. 5A and Supplementary Fig. 3i–v). A higher concentration (10 μmol/L) of RA corresponded with a greater reduction in adipocyte formation, especially that of SC ASCs, when compared with a lower concentration (1 μmol/L) of RA. Importantly, the adipogenic level of SC ASCs treated with 10 μmol/L RA during D0 to D3 or D3 to D6 was similar to that of nontreated VS ASCs. Also, just 2 days of pretreatment with RA itself was sufficient to significantly reduce adipocyte formation in ASCs. RA treatment at later stages of differentiation—that is, D6 to D9 and D9 to D12—either minimally inhibited or failed to block the adipocyte formation in both SC and VS ASCs (Fig. 5A and Bvi–vii; Supplementary Fig. 3vi–vii). Furthermore, as shown in Supplementary Fig. 4, treatment of SC and VS ASCs with retinol (Supplementary Fig. 4Ai and ii) or retinal (Supplementary Fig. 4Bi and ii), intermediates in the RA synthesis pathway, significantly inhibited adipocyte differentiation at various time points during adipogenic induction, as observed by AdipoRed staining.

View Article: PubMed Central - PubMed

ABSTRACT

Increased visceral fat, rather than subcutaneous fat, during the onset of obesity is associated with a higher risk of developing metabolic diseases. The inherent adipogenic properties of human adipose-derived stem cells (ASCs) from visceral depots are compromised compared with those of ASCs from subcutaneous depots, but little is known about the underlying mechanisms. Using ontological analysis of global gene expression studies, we demonstrate that many genes involved in retinoic acid (RA) synthesis or regulated by RA are differentially expressed in human tissues and ASCs from subcutaneous and visceral fat. The endogenous level of RA is higher in visceral ASCs; this is associated with upregulation of the RA synthesis gene through the visceral-specific developmental factor WT1. Excessive RA-mediated activity impedes the adipogenic capability of ASCs at early but not late stages of adipogenesis, which can be reversed by antagonism of RA receptors or knockdown of WT1. Our results reveal the developmental origin of adipocytic properties and the pathophysiological contributions of visceral fat depots.

No MeSH data available.