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The glucocorticoid receptor, not the mineralocorticoid receptor, plays the dominant role in adipogenesis and adipokine production in human adipocytes.

Lee MJ, Fried SK - Int J Obes (Lond) (2014)

Bottom Line: Knockdown of GR by 65% decreased leptin and adiponectin while increasing IL-6 production.In contrast, although MR knockdown increased leptin, it did not affect adiponectin and IL-6 expression.Our data demonstrate that although both GR and MR have roles in regulating leptin expression, GR plays more important roles in mediating the actions of cortisol to regulate adipogenesis and adipokine production in human adipocytes.

View Article: PubMed Central - PubMed

Affiliation: Section of Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA, USA.

ABSTRACT

Background: Both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) are expressed in adipose tissue and assumed to mediate cortisol actions on adipose tissue. The relative significance of the two receptors in mediating glucocorticoid regulation of adipogenesis and adipokine expression in human adipocytes has not been addressed.

Methods: We investigated the differential roles of the GR and MR in mediating glucocorticoid actions on adipogenesis and adipokine production using RNA interference in primary cultures of human preadipocytes and adipocytes.

Results: Both types of receptors are expressed, but levels of GR were several hundred fold higher than MR in both human preadipocytes and adipocytes. As expected, cortisol added during adipogenesis increased the differentiation of human preadipocytes. Silencing of GR, but not MR, blocked these proadipogenic actions of cortisol. In differentiated human adipocytes, addition of cortisol increased leptin and adiponectin, while suppressing interleukin-6 (IL-6), messenger RNA levels and protein secretion. Knockdown of GR by 65% decreased leptin and adiponectin while increasing IL-6 production. In addition, GR silencing blocked the effects of cortisol on adipokine expression. In contrast, although MR knockdown increased leptin, it did not affect adiponectin and IL-6 expression.

Conclusion: Our data demonstrate that although both GR and MR have roles in regulating leptin expression, GR plays more important roles in mediating the actions of cortisol to regulate adipogenesis and adipokine production in human adipocytes.

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Knockdown of GR blocked the cortisol-induction of GILZ mRNA in human adipocytesDifferentiated adipocytes (d9) were transfected with siRNA and treated for 24h with cortisol (200 nM) on day 14. Knockdown of GR and MR were confirmed on d15. (A) GR mRNA levels. GR siRNA decreased GR mRNA levels (p<0.05 with 2-way ANOVA); effect of cortisol vs. control treatment, *, p<0.05. (B) MR mRNA levels. MR siRNA reduced MR mRNA levels (p<0.05 with 2-way ANOVA). (C) Immunoblotting of GR and MR protein. Band intensities were quantified and expression levels relative to the control gene, HSP90, are . (D) GILZ mRNA expression levels. Interaction of GR siRNA and cortisol with 2 way-ANOVA, p<0.01, n=4. Effect of cortisol vs. control, *, p<0.05, ** p<0.01 by paired T test.
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Figure 3: Knockdown of GR blocked the cortisol-induction of GILZ mRNA in human adipocytesDifferentiated adipocytes (d9) were transfected with siRNA and treated for 24h with cortisol (200 nM) on day 14. Knockdown of GR and MR were confirmed on d15. (A) GR mRNA levels. GR siRNA decreased GR mRNA levels (p<0.05 with 2-way ANOVA); effect of cortisol vs. control treatment, *, p<0.05. (B) MR mRNA levels. MR siRNA reduced MR mRNA levels (p<0.05 with 2-way ANOVA). (C) Immunoblotting of GR and MR protein. Band intensities were quantified and expression levels relative to the control gene, HSP90, are . (D) GILZ mRNA expression levels. Interaction of GR siRNA and cortisol with 2 way-ANOVA, p<0.01, n=4. Effect of cortisol vs. control, *, p<0.05, ** p<0.01 by paired T test.

Mentions: To test whether GC regulation of adipokine expression is mediated through GR or MR, we silenced GR and MR in adipocytes. Differentiated adipocytes were transfected with MR or GR siRNA on day 9 and cultured in the maintenance media. Gene silencing effects were effective after 4 days of transfection and maintained for at least additional 5 days (data not shown). GR or MR silenced adipocytes were deprived of dexamethasone overnight (the maintenance media contains 10 nM dexamethasone and 10 nM insulin), and then treated with cortisol (200 nM) for 24h in the presence of 10 nM insulin. siRNA mediated-knockdown in primary human adipocytes was effective, reducing GR mRNA expression by 65±7% and MR mRNA expression by 60±9% (n=5, p<0.01, Fig 3A and B). Similar levels of knockdown were achieved at the protein expression (Fig 3C).


The glucocorticoid receptor, not the mineralocorticoid receptor, plays the dominant role in adipogenesis and adipokine production in human adipocytes.

Lee MJ, Fried SK - Int J Obes (Lond) (2014)

Knockdown of GR blocked the cortisol-induction of GILZ mRNA in human adipocytesDifferentiated adipocytes (d9) were transfected with siRNA and treated for 24h with cortisol (200 nM) on day 14. Knockdown of GR and MR were confirmed on d15. (A) GR mRNA levels. GR siRNA decreased GR mRNA levels (p<0.05 with 2-way ANOVA); effect of cortisol vs. control treatment, *, p<0.05. (B) MR mRNA levels. MR siRNA reduced MR mRNA levels (p<0.05 with 2-way ANOVA). (C) Immunoblotting of GR and MR protein. Band intensities were quantified and expression levels relative to the control gene, HSP90, are . (D) GILZ mRNA expression levels. Interaction of GR siRNA and cortisol with 2 way-ANOVA, p<0.01, n=4. Effect of cortisol vs. control, *, p<0.05, ** p<0.01 by paired T test.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321810&req=5

Figure 3: Knockdown of GR blocked the cortisol-induction of GILZ mRNA in human adipocytesDifferentiated adipocytes (d9) were transfected with siRNA and treated for 24h with cortisol (200 nM) on day 14. Knockdown of GR and MR were confirmed on d15. (A) GR mRNA levels. GR siRNA decreased GR mRNA levels (p<0.05 with 2-way ANOVA); effect of cortisol vs. control treatment, *, p<0.05. (B) MR mRNA levels. MR siRNA reduced MR mRNA levels (p<0.05 with 2-way ANOVA). (C) Immunoblotting of GR and MR protein. Band intensities were quantified and expression levels relative to the control gene, HSP90, are . (D) GILZ mRNA expression levels. Interaction of GR siRNA and cortisol with 2 way-ANOVA, p<0.01, n=4. Effect of cortisol vs. control, *, p<0.05, ** p<0.01 by paired T test.
Mentions: To test whether GC regulation of adipokine expression is mediated through GR or MR, we silenced GR and MR in adipocytes. Differentiated adipocytes were transfected with MR or GR siRNA on day 9 and cultured in the maintenance media. Gene silencing effects were effective after 4 days of transfection and maintained for at least additional 5 days (data not shown). GR or MR silenced adipocytes were deprived of dexamethasone overnight (the maintenance media contains 10 nM dexamethasone and 10 nM insulin), and then treated with cortisol (200 nM) for 24h in the presence of 10 nM insulin. siRNA mediated-knockdown in primary human adipocytes was effective, reducing GR mRNA expression by 65±7% and MR mRNA expression by 60±9% (n=5, p<0.01, Fig 3A and B). Similar levels of knockdown were achieved at the protein expression (Fig 3C).

Bottom Line: Knockdown of GR by 65% decreased leptin and adiponectin while increasing IL-6 production.In contrast, although MR knockdown increased leptin, it did not affect adiponectin and IL-6 expression.Our data demonstrate that although both GR and MR have roles in regulating leptin expression, GR plays more important roles in mediating the actions of cortisol to regulate adipogenesis and adipokine production in human adipocytes.

View Article: PubMed Central - PubMed

Affiliation: Section of Endocrinology, Diabetes and Nutrition, Department of Medicine, Boston University School of Medicine, Boston, MA, USA.

ABSTRACT

Background: Both the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) are expressed in adipose tissue and assumed to mediate cortisol actions on adipose tissue. The relative significance of the two receptors in mediating glucocorticoid regulation of adipogenesis and adipokine expression in human adipocytes has not been addressed.

Methods: We investigated the differential roles of the GR and MR in mediating glucocorticoid actions on adipogenesis and adipokine production using RNA interference in primary cultures of human preadipocytes and adipocytes.

Results: Both types of receptors are expressed, but levels of GR were several hundred fold higher than MR in both human preadipocytes and adipocytes. As expected, cortisol added during adipogenesis increased the differentiation of human preadipocytes. Silencing of GR, but not MR, blocked these proadipogenic actions of cortisol. In differentiated human adipocytes, addition of cortisol increased leptin and adiponectin, while suppressing interleukin-6 (IL-6), messenger RNA levels and protein secretion. Knockdown of GR by 65% decreased leptin and adiponectin while increasing IL-6 production. In addition, GR silencing blocked the effects of cortisol on adipokine expression. In contrast, although MR knockdown increased leptin, it did not affect adiponectin and IL-6 expression.

Conclusion: Our data demonstrate that although both GR and MR have roles in regulating leptin expression, GR plays more important roles in mediating the actions of cortisol to regulate adipogenesis and adipokine production in human adipocytes.

Show MeSH
Related in: MedlinePlus