Insulin Regulates Adrenal Steroidogenesis by Stabilizing SF-1 Activity

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.

The activity of the hypothalamic-pituitary-adrenal (HPA) axis is dysregulated in obese and diabetic patients as well as in different animal models predisposed to metabolic syndrome including ob/ob mice and Zucker fatty rat [1][2][3] . Emerging evidence in the last few decades has shown a close relationship between disorders of the HPA axis and the development of metabolic syndrome as demonstrated by similarities in clinical features of Cushing syndrome and obesity. Cushing syndrome results from the overproduction of adrenocorticotropic hormone (ACTH) by the pituitary glands with the concomitant hypersecretion of cortisol by adrenal glands and is associated with glucose intolerance, hyperlipidemia and hypertension 4 . On the other hand, obesity is associated with an increase in the secreted levels of the adrenal gland hormones including cortisol [5][6][7] which is known to increase abdominal fat mass by stimulating the differentiation of preadipocytes to mature adipocytes 8 . The adrenal gland is therefore considered a key player in the development of metabolic syndrome owing to the role of the adrenal hormones in energy and metabolic homeostasis.
Steroidogenic factor 1 (SF-1) is a transcriptional factor belonging to the nuclear receptor superfamily and is predominantly expressed in the adrenal glands, gonads, pituitary gland and ventromedial hypothalamus (VMH). SF-1 plays an important role in steroid hormones synthesis by regulating the transcription of steroidogenic genes including StAR, Cyp11a1, Cyp17, Cyp11b1, Cyp11b2 and 3β-Hsd 9, 10 . In addition, SF-1 functions as a metabolic regulator in the brain particularly in the VMH where SF-1 expressing neurons are widespread 11,12 .
The pancreatic hormone insulin is well known for its role in energy metabolism regulation among other physiological functions 13,14 . Previous studies have suggested the involvement of insulin in gonadal steroidogenesis 15,16 . However, the molecular mechanism mediating the insulin effect on steroidogenesis as well as the physiological implication remains to be elucidated. In addition, the role of insulin in regulating the production of adrenal hormones, and the underlying molecular mechanism, has not been clearly demonstrated. In this study we therefore sought to address the role of insulin in steroidogenesis by focusing on adrenal steroidogenesis and metabolic regulation. Our data indicates that insulin regulates adrenal steroidogenesis by up regulating the transcriptional activity of SF-1 both in vitro and in vivo. Further, we show that overexpression of FoxO1, a downstream transcription factor regulated by insulin, inhibits the expression and transcriptional activity of SF-1 suggesting that insulin might increase the activity of SF-1 by phosphorylating and inactivating FoxO1. Therefore, our findings propose the insulin-FoxO1 pathway as a possible mechanism regulating adrenal hormones production and metabolic homeostasis that is distinct from the canonical cAMP/PKA pathway.

Results
Insulin upregulates SF-1 and steroidogenic target genes. To investigate the role of insulin in adrenal steroidogenesis, we treated Y1 cells with insulin for 24 h and examined the expression of genes involved in steroidogenesis. Interestingly, insulin markedly up-regulated SF-1 as well as the SF-1 target genes including StAR, Cyp11a1, Cyp11b1, Cyp11b2, and Hsd3b2 all of which are important for adrenal steroidogenic pathway (Fig. 1a). As SF-1 is a known master regulator of steroidogenesis 17 , we monitored the SF-1 protein expression after treating Y1 cells with insulin for 24 h in a dose-dependent manner and also treating 100 nM insulin time dependently. Intriguingly, insulin markedly increased SF-1 protein level dose dependently ( Fig. 1b and c). In addition, insulin significantly increased SF-1 time dependently with the strongest effect observed from 24-48 h ( Fig. 1d and e). The effect of insulin was accompanied by the phosphorylation of AKT and FoxO1, direct downstream effectors of insulin signaling ( Fig. 1b and d). We then tested the effect of insulin on DAX-1 which is a known SF-1 inhibitor 16,18 . We however did not observe any changes on DAX-1 protein by insulin treatment suggesting that the effect of DAX-1 on insulin-mediated SF-1 regulation might be minimal. In addition, treatment of 8Br-cAMP led to an increase in SF-1 protein level accompanied by phosphorylation of CREB while 24 h insulin treatment stimulated the expression of SF-1 in a manner independent of the canonical steroidogenic cAMP/PKA/pCREB pathway 19 (Fig. 1f). These results highly suggest that insulin might positively regulate the expression of steroidogenic genes by enhancing PKA-independent SF-1 function.
Insulin stimulates steroidogenic genes expression in SF-1 dependent manner. The insulin mediated increase in SF-1 protein and mRNA levels led us to investigate the effect of insulin on the transcriptional activity of SF-1 by performing luciferase assays. Insulin markedly increased SF-1 luciferase activity in a dose-dependent manner (Fig. 2a). Cyp11b1, an enzyme involved in the synthesis of corticosterone was also significantly increased in the presence of SF-1 and insulin ( Fig. 2b and Supplementary Fig. S1a). Consistent with the exogenous SF-1 overexpression, Cyp11b1 activation was up-regulated by insulin treatment in Y1 cells in which SF-1 is expressed endogenously (Fig. 2c). To investigate whether the increased expression of steroidogenic genes by insulin was dependent on SF-1, we knocked down SF-1 and then treated insulin ( Fig. 2d and e). Up-regulation of steroidogenic genes including StAR, Cyp11a1, Cyp11b1, Cyp11b2, and Hsd3b2 by insulin was markedly blunted by SF-1 knockdown indicating that SF-1 is required for the effect of insulin on adrenal steroidogenic genes (Fig. 2e).

Insulin-mediated FoxO1 suppression is critical for steroidogenic genes expression.
To gain further molecular insight into how insulin positively regulates steroidogenesis, we pre-treated cells with 1 uM of MK2206, an AKT inhibitor, for 1 h and measured the luciferase activity of Cyp11b1 with or without insulin treatment. The insulin-mediated increase in Cyp11b1 activity was significantly blunted by the AKT inhibitor, implying that the insulin/AKT pathway might be important for regulation of steroidogenic genes activities (Fig. 3a). FoxO1 is phosphorylated by AKT upon activation of insulin signaling and its transcriptional activity is inhibited through cytoplasmic localization by insulin. In addition, previous studies have shown that FoxO1 negatively regulates SF-1 activity in the ventromedial hypothalamus (VMH) 20 . Therefore, we postulated that the upregulation of SF-1 and its steroidogenic target genes by insulin might be as a result of phosphorylation and inactivation of FoxO1 by insulin. To investigate the effect of FoxO1 on SF-1 and steroidogenic target genes, we introduced the constitutively active form of FoxO1 (FoxO1-CA), in which the phosphorylation sites are mutated and is therefore always localized in the nucleus 21 and examined the luciferase activity of SF-1. FoxO1-CA markedly blunted the transcriptional activity of SF-1 from a low dose of 50 ng (Fig. 3b) and exhibited continuous suppressive effect up to 1 ug. We next overexpressed FoxO1-WT or FoxO1-CA with or without insulin and examined transcriptional activity of Cyp11b1. FoxO1-WT significantly inhibited the promoter activity of Cyp11b1 and further repressive effect was shown by FoxO1-CA ( Fig. 3c and d). Additionally, the repressive effect of FoxO1-WT and FoxO1-CA resulted in significant suppression of SF-1 mRNA levels as well as steroidogenic target genes including StAR, Cyp11a1, Cyp11b1, Cyp11b2, and Hsd3b2 ( Fig. 3e-g). Taken together, these data highly suggests that FoxO1 negatively regulates the expression of SF-1 and steroidogenic genes and that suppression of FoxO1 by insulin treatment might stimulate steroidogenesis, at least in part, through activation of SF-1. Stimulation of adrenal steroidogenesis by activation of insulin signaling. To confirm our findings that insulin stimulates steroidogenesis and to examine the effect of insulin on adrenal steroidogenesis in vivo, we generated a mouse model mimicking increased insulin activity by feeding mice on high fat diet (HFD) for 8 weeks. HFD led to a pronounced increase in plasma insulin levels ( Fig. 4a and d) and this was accompanied by a marked hyperactivation of insulin signaling pathway specifically in the adrenal gland, soleus muscle, and liver as shown by the significant increase in phosphorylation of AKT and FoxO1 in both male and female mice (Fig. 4b,c,e and f and Supplementary Fig. S2). The hyperactivation of insulin pathway by short-term HFD exposure clearly showed inactivation of FoxO1 and was accompanied by a significant increase of SF-1 in the adrenal gland (Fig. 4b,c,e and f). Corresponding with the elevated SF-1 protein levels in the adrenal gland, the mRNA levels of steroidogenic genes were also significantly elevated ( Fig. 4g and Supplementary Fig. S3). Together with the increase in expression of steroidogenic genes, plasma aldosterone and corticosterone levels were highly elevated in male and female mice ( Fig. 4h and i and Supplementary Fig. S3). Next, we generated a mouse model mimicking blunted insulin signaling by injecting mice with streptozotocin (STZ) for 5 days. STZ injection led to a marked reduction in serum insulin levels (Fig. 4j). Importantly, SF-1 protein and mRNA levels in the adrenal gland were markedly reduced in the STZ-injected mice (Fig. 4k-o). The blunted insulin signaling in adrenal gland was confirmed by the decreased levels of pAKT and pFoxO1 (Fig. 4k-n). Therefore, our results highly suggest that insulin positively regulates the expression of SF-1, and the phosphorylation and inactivation of FoxO1 through the insulin signaling might be an underlying mechanism mediating the expression and activity of SF-1.

Discussion
The synthesis and secretion of adrenal steroid hormones is coordinated by three organs, the hypothalamus, anterior pituitary and adrenal glands referred to as the HPA axis. The hypothalamus secretes corticotrophin releasing hormone (CRH) which stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary gland. ACTH in turn acts through the melanocortin 2 receptors (MC2R) expressed on the adrenal glands leading to an increase in cAMP and activation of protein kinase A followed by the transcription of steroidogenic genes and subsequent synthesis of steroid hormones 22 . Impaired adrenal steroidogenesis has detrimental effects on glucose and energy homeostasis 4 . On the other hand, dysregulation of metabolic homeostasis is associated with increased secretion of adrenal hormones showing a correlation between dysregulation of HPA axis and metabolic syndrome development [5][6][7] .
In addition to the canonical steroidogenic pathway, various factors have been shown to contribute to steroid hormones synthesis [23][24][25][26][27] . Although these studies focused on the factors involved in steroidogenesis, the direct molecular mechanisms linking metabolic hormones to adrenal steroidogenesis has not been conclusively shown. As metabolic diseases including diabetes and obesity are associated with dysregulation of adrenal steroids, we postulated that metabolic hormones including insulin might directly influence adrenal steroidogenesis. In the current study, we show that insulin directly regulates adrenal steroidogenesis by activating SF-1 and its steroidogenic target genes through a mechanism that might be independent of the canonical CRH/ACTH/MC2R/PKA pathway. To address the role of insulin on adrenal steroidogenesis, we focused on SF-1 which plays an important role in steroid hormones production as it regulates the genes involved in the steroidogenesis 17 . Insulin treatment markedly increased SF-1 and steroidogenic target genes and this increase was dependent on SF-1 as knockdown of SF-1 failed to upregulate the expression of steroidogenic target genes by insulin (Figs 1 and 2). Together with the previous finding in which deletion of the insulin and insulin-like growth factor 1 receptor was associated with adrenal agenesis 28 , our findings suggests that insulin signaling is not only essential for the adrenal glands development, but also required for adrenal steroidogenesis.
Previous studies have shown that FoxO1 negatively regulates the transcription of ovarian steroidogenic genes including luteinizing hormone β-subunit (LHβ) and the follicle stimulating hormone β (FSH β) 29,30 . In addition, FoxO1 was shown to suppress the expression of SF-1 in the ventromedial hypothalamus (VMH) 20 . This information led to our hypothesis that insulin might regulate the function of FoxO1, a direct downstream effector of insulin signaling, and thereby affect steroidogenic genes in adrenal glands. Therefore, we focused on the role of transcriptional factor FoxO1 in regulation of SF-1 expression and adrenal steroidogenesis. To address the role of FoxO1 on adrenal steroidogenesis, we overexpressed the wild-type form of FoxO1 (FoxO1-WT) and the constitutively active form of FoxO1 (FoxO1-CA) and measured the expression and activity of SF-1 and SF-1 target genes. Overexpression of FoxO1 significantly blunted the expression of SF-1 as well as steroidogenic SF-1 target genes. Similarly, FoxO1-WT and FoxO1-CA blunted the luciferase activity of SF-1 and Cyp11b1 (Fig. 3). Further, overexpression of FoxO1-CA significantly suppressed the elevation of SF-1 and its steroidogenic targets induced by insulin treatment (Fig. 3). These data suggest that the suppressive effect of FoxO1 on SF-1 and steroidogenic genes is blunted by insulin treatment. However, whether the effect of FoxO1 on steroidogenic genes is solely mediated through disinhibition of SF-1 activity requires to be further explored.
To understand the insulin-induced increase in adrenal steroids production observed in vitro, we set up a mouse model mimicking hyper-activation of insulin signaling pathway by challenging mice with HFD for 8 weeks. HFD exposure was accompanied by a significant increase in insulin levels and exhibited significant activation of insulin pathway illustrated by marked phosphorylation of AKT and FoxO1 in the adrenal gland. Having confirmed the activation of insulin signaling by HFD exposure, we then monitored the expression of SF-1 and steroidogenic target genes in the adrenal gland. As shown in Fig. 4, SF-1 and other genes involved in steroidogenesis including StAR, Cyp11a1, Cyp11b1, Cyp11b2, and Hsd3b2 were significantly increased ( Fig. 4 and Supplementary Fig. S3). In addition, the serum levels of adrenal steroid hormones such as aldosterone and corticosterone were significantly elevated in the HFD fed mice ( Fig. 4 and Supplementary Fig. S3). Consistently, low insulin condition induced by STZ administration showed a marked reduction in SF-1 levels (Fig. 4j-o). Therefore, our results highly imply that activation of insulin signaling might be directly involved, at least in part, in the regulation of steroidogenesis in the adrenal gland. In addition, it is important to note that there is possibility that insulin could be indirectly involved in steroidogenesis through insulin-induced hypoglycemia since hypoglycemia activates the HPA axis with the concomitant increase in corticosterone levels 31,32 . Therefore further studies to establish the effect of insulin on the HPA axis would provide further insight into the regulation of steroidogenesis regulated by insulin.
This study shows that FoxO1 negatively regulates adrenal steroidogenesis partly through suppression of SF-1. This finding warrants further studies to investigate the direct effects of FoxO1 on the transcription of steroidogenic genes as well as the interaction between steroidogenic genes and other members of the FoxO family in the adrenal gland. Further, clinical analysis on the expression pattern of FoxO1 and SF-1 in patients with obesity and type 2 diabetes could shed more light on the relationship between HPA axis dysregulation and obesity development.
In summary, our data suggest that insulin contributes to the dysregulation of the HPA axis observed in metabolic syndrome by directly increasing the production of adrenal gland hormones through upregulation of SF-1 and the steroidogenic genes important for steroid hormone synthesis. We suggest that the insulin mediated increase in SF-1 and steroidogenic targets might be a result of phosphorylation and inactivation of FoxO1 through a mechanism that is independent of the canonical steroidogenic cAMP/PKA pathway.
Hormone measurement. For corticosterone and aldosterone measurements, mice were housed in individual cages overnight and blood samples were collected the following day at 2 pm. Corticosterone and aldosterone and levels were measured using ELISA kit obtained from Abcam (ab136933 and ab108821 respectively Cambridge, UK) according to the manufacturer's instructions. Blood samples for insulin measurement were collected at 10 am following decapitation and insulin levels were measured using ELISA kit obtained from Morinaga Institute of Biological Science (Yokohama, Japan) following the manufacturer's protocol.
Statistical analysis. All results are expressed as mean ± SEM following analysis using the GraphPad Prism 5.0 software. Statistical comparisons were made by the Student's t-test or ANOVA and results with a p-value < 0.05 were considered to be statistically significant.