Insulin Regulates Adrenal Steroidogenesis by Stabilizing SF-1 Activity

Abstract

Development of metabolic syndrome is associated with hyperactivity of the HPA axis characterized by elevated levels of circulating adrenal hormones including cortisol and aldosterone. However, the molecular mechanism leading to the dysregulation of the HPA axis is not well elucidated. In this study, we found that insulin regulates adrenal steroidogenesis by increasing the expression and activity of steroidogenic factor 1 (SF-1) both in vitro and in vivo and this insulin effect was partly through inhibition of FoxO1. Specifically, insulin increased the protein and RNA levels of SF-1 and steroidogenic target genes. Further, adrenal SF-1 expression was significantly increased by hyperactivation of insulin signaling in mice. Together with the elevated SF-1 expression in adrenal glands, hyperactivation of insulin signaling led to increased aldosterone and corticosterone levels. On the other hand, suppressing the insulin signaling using streptozotocin markedly reduced the expression of adrenal SF-1 in mice. In addition, overexpression of FoxO1 significantly suppressed SF-1 and its steroidogenic target genes implying that the positive effect of insulin on SF-1 activity might be through suppression of FoxO1 in the adrenal gland. Taken together, these results indicate that insulin regulates adrenal steroidogenesis through coordinated control of SF-1 and FoxO1.

Figure 1

SF-1 and steroidogenic genes are increased by insulin. Relative gene expression of SF-1 and steroidogenic SF-1 target genes after 24 h insulin treatment (a). Dose (b and c) and time (d and e) dependent effects of insulin on SF-1 and DAX1 protein levels. Note that the activation of insulin signaling was confirmed by phosphorylation of AKT and FoxO1. Effect of insulin and 8Br-cAMP (100 uM) on SF-1 protein levels (f). 100 nM of insulin was used for experiments unless otherwise specified. All experiments were performed in triplicates. Original Western blots are provided in the Supplementary information. The values are mean ± SEM (*p < 0.05, Student’s t-test).

Figure 2

Insulin increases the transcriptional activity of SF-1. Dose-dependent effect of insulin on SF-1 transcription in HEK293 cells (a). Increase in Cyp11b1 transcriptional activity by time-dependent insulin treatment after co-transfection of SF-1 (b). Relative luciferase activity of Cyp11b1 after insulin treatment in Y1 cells (c). Effect of insulin on SF-1 and steroidogenic genes after knocking down SF-1 (d and e). The values are mean ± SEM (*p < 0.05, Student’s t-test, one-way ANOVA). EV, empty vector. SF-1-Luc, SF-1 luciferase construct. Cyp11b1-Lu, Cyp11b1 luciferase construct.

Figure 3

Suppressive effects of FoxO1 on steroidogenic genes expression. Pre-treatment of MK2206 (1 uM) significantly blunted the Cyp11b1 transcriptional activity induced by insulin treatment in HEK 293 cells (a). Dose-dependent effect of FoxO1-CA on the SF-1 promoter activity in HEK 293 cells (b). Effect of FoxO1-WT on Cyp11b1 promoter activity in Y1 cells (c). FoxO1-CA significantly suppressed the Cyp11b1 promoter activity mediated by insulin in Y1 cells (d). Overexpression of FoxO1-WT suppressed the expression of steroidogenic genes including SF-1, StAR, Cyp11a1, Cyp11b1, Cyp11b2, and Hsd3b2 in Y1 cells (e). Overexpression of FoxO1-CA blunted the insulin-mediated increase of the steroidogenic genes such as SF-1, StAR, Cyp11a1, Cyp11b1, Cyp11b2, and Hsd3b2 in Y1 cells (f). Western blot for FoxO1-WT or FoxO1-CA confirming the overexpression experiments in Fig. 3e and f (g). The values are mean ± SEM (*p < 0.05 Student’s t-test, #p < 0.05 one-way ANOVA).

Figure 4

Regulation of adrenal steroidogenesis by insulin. Plasma insulin levels in male mice fed normal chow (NC) or HFD for 8 weeks (n = 4–6) (a). Protein levels of indicated proteins from the adrenal gland (b). Densitometry of the protein intensity from (b) in male mice (c). Plasma insulin levels in female mice fed NC or HFD for 8 weeks (n = 4–6) (d). Protein levels of indicated proteins from the adrenal gland (e). Densitometry of the protein intensity from (e) in female mice (f). mRNA expression of indicated steroidogenic genes from the adrenal gland (g). Plasma aldosterone (h) and corticosterone (i) levels from male mice fed on NC or HFD. Plasma insulin levels of mice injected with streptozotocin (+STZ, n = 7) and mice injected with sodium citrate as control for vehicle (−STZ, n = 5) (j). Protein levels of indicated proteins from the adrenal gland (k). Densitometry of the indicated proteins (l–n). Relative mRNA expression of SF-1 from the adrenal gland (o). All the corresponding original Western blot images are provided in the Supplementary information. The values are mean ± SEM (*p < 0.05, Student’s t-test).