Y1 Adrenocortical Cells Research Articles

Capsaicin inhibits lipopolysaccharide-induced adrenal steroidogenesis by raising intracellular calcium levels.

Glucocorticoid release by adrenals has been described as significant to survive sepsis. The activation of transient receptor potential vanilloid type 1 (TRPV1) inhibited ACTH-induced glucocorticoid release by adrenal glands in vitro. The aim of this study was to investigate if capsaicin, an activator of TRPV1, would prevent LPS-induced glucocorticoid production by adrenals. Male Swiss-Webster mice were treated with capsaicin intraperitoneally (0.2 or 2 mg/kg) 30 min before LPS injection. All analyses were performed 2 h after the LPS stimulation, including plasma corticosterone and peritoneal IL-1β and TNF-α levels. Furthermore, murine adrenocortical Y1 cells were used to assess the effects of capsaicin on LPS-induced corticosterone production in vitro. Capsaicin (2 mg/kg, i.p.) significantly reduced plasma corticosterone levels and adrenal hypertrophy induced by LPS without alter the levels of pro-steroidogenic cytokines IL-1β and TNF-α in peritoneal cavity of mice, while the dose of 0.2 mg/kg of capsaicin did not interfere with adrenal steroidogenesis, attested by RIA and ELISA, respectively. Y1 cells express TRPV1, measured by immunofluorescence and western blot, and capsaicin decreased LPS-induced corticosterone production by these cells in vitro. Capsaicin also induces calcium mobilization in Y1 cells in vitro. These findings suggest that capsaicin inhibits corticosterone production induced by LPS by acting directly on adrenal cells producing glucocorticoids, in a mechanism probably associated with induction of a cytoplasmic calcium increase in these cells.

Plasma membrane cholesterol trafficking in steroidogenesis.

Cholesterol is an important component of plasma membranes (PMs) and the precursor of all steroid hormones. In steroidogenic tissues, upon hormone stimulation, there is a rapid transfer of cholesterol to the mitochondria, which is the site of the initial step in steroidogenesis. In the current study, we examined PM cholesterol trafficking for steroidogenesis. In a mitochondrial reconstitution assay, adrenal PMs supported steroidogenesis in the absence of additional transport proteins. Depletion of cholesterol in PMs by 50% eliminated the membranes' ability to support steroidogenesis in vitro and reduced steroid production in intact Y1 adrenocortical cells. Syntaxin (STX)-5 and α-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) are enriched in adrenal PMs, and adrenocorticotropic hormone treatment of rats recruited STX5 and α-SNAP to adrenal PMs and mitochondria. Immunodepletion of STX5 and α-SNAP from PMs decreased steroidogenesis supported by PMs in vitro. Protease digestion of PMs decreased, whereas recombinant STX5 or α-SNAP restored, the PMs' ability to support steroidogenesis. Knockdown of either STX5 or α-SNAP in Y1 cells decreased stimulated steroidogenesis. These results indicate that STX5 and α-SNAP facilitate cholesterol trafficking from PMs to mitochondria for adrenal steroid synthesis and underscore the importance of vesicular trafficking of PM cholesterol for steroidogenesis.-Deng, B., Shen, W.-J., Dong, D., Azhar, S., Kraemer, F. B. Plasma membrane cholesterol trafficking in steroidogenesis.

An Interdisciplinary Approach for Designing Kinetic Models of the Ras/MAPK Signaling Pathway.

We present in this article a methodology for designing kinetic models of molecular signaling networks, which was exemplarily applied for modeling one of the Ras/MAPK signaling pathways in the mouse Y1 adrenocortical cell line. The methodology is interdisciplinary, that is, it was developed in a way that both dry and wet lab teams worked together along the whole modeling process.

Role of CREB on heme oxygenase-1 induction in adrenal cells: involvement of the PI3K pathway.

In addition to the well-known function of ACTH as the main regulator of adrenal steroidogenesis, we have previously demonstrated its effect on the transcriptional stimulation of HO-1 expression, a component of the cellular antioxidant defense system. In agreement, we hereby demonstrate that, in adrenocortical Y1 cells, HO-1 induction correlates with a significant prevention of the generation of reactive oxygen species induced by H2O2/Fe(2+) ACTH/cAMP-dependent activation of redox-imbalanced related factors such as NRF2 or NFκB and the participation of MAPKs in this mechanism was, however, discarded based on results with specific inhibitors and reporter plasmids. We suggest the involvement of CREB in HO-1 induction by ACTH/cAMP, as transfection of cells with a dominant-negative isoform of CREB (DN-CREB-M1) decreased, while overexpression of CREB increased HO-1 protein levels. Sequence screening of the murine HO-1 promoter revealed CRE-like sites located at -146 and -37 of the transcription start site and ChIP studies indicated that this region recruits phosphorylated CREB (pCREB) upon cAMP stimulation in Y1 cells. In agreement, H89 (PKA inhibitor) or cotransfection with DN-CREB-M1 prevented the 8Br-cAMP-dependent increase in luciferase activity in cells transfected with pHO-1[-295/+74].LUC. ACTH and cAMP treatment induced the activation of the PI3K/Akt signaling pathway in a PKA-independent mechanism. Inhibition of this pathway prevented the cAMP-dependent increase in HO-1 protein levels and luciferase activity in cells transfected with pHO-1[-295/+74].LUC. Finally, here we show a crosstalk between the cAMP/PKA and PI3K pathways that affects the binding of p-CREB to its cognate element in the murine promoter of the Hmox1 gene.

ACTH Modulates PTP-PEST Activity and Promotes Its Interaction With Paxillin.

Adrenocorticotropic hormone (ACTH) treatment has been proven to promote paxillin dephosphorylation and increase soluble protein tyrosine phosphatase (PTP) activity in rat adrenal zona fasciculata (ZF). Also, in-gel PTP assays have shown the activation of a 115-kDa PTP (PTP115) by ACTH. In this context, the current work presents evidence that PTP115 is PTP-PEST, a PTP that recognizes paxillin as substrate. PTP115 was partially purified from rat adrenal ZF and PTP-PEST was detected through Western blot in bioactive samples taken in each purification step. Immunohistochemical and RT-PCR studies revealed PTP-PEST expression in rat ZF and Y1 adrenocortical cells. Moreover, a PTP-PEST siRNA decreased the expression of this phosphatase. PKA phosphorylation of purified PTP115 isolated from non-ACTH-treated rats increased KM and VM . Finally, in-gel PTP assays of immunoprecipitated paxillin from control and ACTH-treated rats suggested a hormone-mediated increase in paxillin-PTP115 interaction, while PTP-PEST and paxillin co-localize in Y1 cells. Taken together, these data demonstrate PTP-PEST expression in adrenal ZF and its regulation by ACTH/PKA and also suggest an ACTH-induced PTP-PEST-paxillin interaction. J. Cell. Biochem. 117: 2170-2181, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Emodin Decreases Hepatic Hypoxia-Inducible Factor-1α by Inhibiting its Biosynthesis

Hypoxia-inducible factor-1 (HIF-1) is an [Formula: see text] dimeric transcription factor. Because HIF-1[Formula: see text] is instable with oxygen, HIF-1 is scarce in normal mammalian cells. However, HIF-1[Formula: see text] is expressed in pathological conditions such as cancer and obesity. Inhibiting HIF-1[Formula: see text] may be of therapeutic value for these pathologies. Here, we investigated whether emodin, derived from the herb of Rheum palmatum L, which is also known as Chinese rhubarb, and is native to China, regulates HIF-1[Formula: see text] expression. Male C57BL/6 mice without or with diet-induced obesity were treated with emodin for two weeks, while control mice were treated with vehicle. HIF-1[Formula: see text] expression was determined by Western blot. We found that emodin inhibited obesity-induced HIF-1[Formula: see text] expression in liver and skeletal muscle but did not regulate HIF-1[Formula: see text] expression in the kidneys or in intra-abdominal fat. In vitro, emodin inhibited HIF-1[Formula: see text] expression in human HepG2 hepatic cells and Y1 adrenocortical cells. Further, we investigated the mechanisms of HIF-1[Formula: see text] expression in emodin-treated HepG2 cells. First, we found that HIF-1[Formula: see text] had normal stability in the presence of emodin. Thus, emodin did not decrease HIF-1[Formula: see text] by stimulating its degradation. Importantly, emodin decreased the activity of the signaling pathways that led to HIF-1[Formula: see text] biosynthesis. Interestingly, emodin increased HIF-1[Formula: see text] mRNA in HepG2 cells. This may be a result of feedback in response to the emodin-induced decrease in the protein of HIF-1[Formula: see text]. In conclusion, emodin decreases hepatic HIF-1[Formula: see text] by inhibiting its biosynthesis.

Osthole, a coumadin analog from Cnidium monnieri (L.) Cusson, stimulates corticosterone secretion by increasing steroidogenic enzyme expression in mouse Y1 adrenocortical tumor cells

Ethnopharmacological relevanceOsthole is an O-methylated coumadin, which was isolated and purified from the seeds of Cnidium monnieri (L.) Cusson. Osthole is a commonly used traditional Chinese medicine to treat patients with Kidney-Yang deficiency patients, who exhibit clinical signs similar to those of glucocorticoid withdrawal. However, the mechanism of action of osthole is not fully understood. ObjectiveThis study was designed to reveal the effects of osthole on corticosterone production in mouse Y1 cell. Materials and methodsMouse Y1 adrenocortical cells were used to evaluate corticosterone production, which was quantified by enzyme-linked immunosorbent assay (ELISA) kits. Cell viability was tested using the MTT assay, and the mRNA and protein expression of genes encoding steroidogenic enzymes and transcription factors was monitored by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting, respectively. ResultsOsthole stimulated corticosterone secretion from mouse Y1 cells in a dose- and time-dependent manner, and osthole enhanced the effect of dibutyryl-cAMP (Bu2cAMP) on corticosterone production. Further, osthole also increased StAR and CYP11B1 mRNA expression in a dose-dependent manner and enhanced the expression of transcription factors such as HSD3B1, FDX1, POR and RXRα as well as immediate early genes such as NR4A1. Moreover, osthole significantly increased SCARB1(SRB1) mRNA and StAR protein expression in the presence or absence of Bu2cAMP; these proteins are an important for the transport of the corticosteroid precursor cholesterol transport into mitochondria. ConclusionsOur results show that the promotion of corticosterone biosynthesis and secretion is a novel effect of osthole, suggesting that this agent can be utilized for the prevention and treatment of Kidney-Yang deficiency syndrome.

StAR Protein Stability in Y1 and Kin-8 Mouse Adrenocortical Cells.

The steroidogenic acute regulatory protein (STAR) protein expression is required for cholesterol transport into mitochondria to initiate steroidogenesis in the adrenal and gonads. STAR is synthesized as a 37 kDa precursor protein which is targeted to the mitochondria and imported and processed to an intra-mitochondrial 30 kDa protein. Tropic hormone stimulation of the cAMP-dependent protein kinase A (PKA) signaling pathway is the major contributor to the transcriptional and post-transcriptional regulation of STAR synthesis. Many studies have focused on the mechanisms of cAMP-PKA mediated control of STAR synthesis while there are few reports on STAR degradation pathways. The objective of this study was to determine the effect of cAMP-PKA-dependent signaling on STAR protein stability. We have used the cAMP-PKA responsive Y1 mouse adrenocortical cells and the PKA-deficient Kin-8 cells to measure STAR phosphorylation and protein half-life. Western blot analysis and standard radiolabeled pulse-chase experiments were used to determine STAR phosphorylation status and protein half-life, respectively. Our data demonstrate that PKA-dependent STAR phosphorylation does not contribute to 30 kDa STAR protein stability in the mitochondria. We further show that inhibition of the 26S proteasome does not block precursor STAR phosphorylation or steroid production in Y1 cells. These data suggest STAR can maintain function and promote steroidogenesis under conditions of proteasome inhibition.

NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and β-catenin accumulation.

BackgroundAdrenogonadal cell growth and differentiation are controlled by nuclear receptor NR5A1 (Ad4BP/SF-1) that regulates the expression of adrenal and gonadal genes. In addition, SF-1 also resides in the centrosome and controls centrosome homeostasis by restricting the activity of centrosomal DNA-PK and CDK2/cyclin A.ResultsHere we show that SF-1 depletion resulted in centriole splitting and amplification due to aberrant activation of DNA-PK in the centrosome of mouse adrenocortical Y1 cells. In the absence of SF-1, GSK3β was aberrantly phosphorylated during G1 phase and β-catenin was accumulated in the centrosome, but not in the nucleus. DNA-PK inhibitor vanillin reversed these phenomena. SF-1 overexpression led to inhibition of centrosomal DNA-PK activation caused by SF-1 depletion. Both full-length SF-1 and truncated SF-1 devoid of its DNA-binding domain rescued the multiple centrosome phenotype caused by SF-1 depletion, indicating that the effect of SF-1 in the centrosome is not contributed by its DNA-binding domain. Furthermore, SF-1 interacted with cyclin A in the centrosome, but not in the nucleus. Depletion of SF-1 also resulted in centriole splitting, genomic instability and reduced growth of mouse testicular Leydig MA10 cells.ConclusionCentrosomal DNA-PK signaling triggers the accumulation of β-catenin, leading to centrosome over-duplication and centriole splitting. This cascade of centrosomal events results in genomic instability and reduced cell numbers.

Mathematical modeling of the Ras/MAPK and PI3K/Akt signaling networks in the K-Ras-driven mouse Y1 adrenocortical tumor cells

Mathematical modeling of the Ras/MAPK and PI3K/Akt signaling networks in the K-Ras-driven mouse Y1 adrenocortical tumor cells

Modeling the kinetics of Ras/MAPK and PI3K/AKT signaling pathways in the K-Ras-driven mouse Y1 adrenocortical tumor cells: novel insights into the mechanisms of FGF2-driven cell cycle arrest

Modeling the kinetics of Ras/MAPK and PI3K/AKT signaling pathways in the K-Ras-driven mouse Y1 adrenocortical tumor cells: novel insights into the mechanisms of FGF2-driven cell cycle arrest

Mathematical modeling of Ras/MAPK signaling network kinetics provides insights into mechanisms of FGF2-induced cell cycle arrest in the K-Ras-driven mouse Y1 adrenocortical tumor cells

Mathematical modeling of Ras/MAPK signaling network kinetics provides insights into mechanisms of FGF2-induced cell cycle arrest in the K-Ras-driven mouse Y1 adrenocortical tumor cells

Involvement of PI3K/Akt and p38 MAPK in the induction of COX-2 expression by bacterial lipopolysaccharide in murine adrenocortical cells

Previous studies from our laboratory demonstrated the involvement of COX-2 in the stimulation of steroid production by LPS in murine adrenocortical Y1 cells, as well as in the adrenal cortex of male Wistar rats. In this paper we analyzed signaling pathways involved in the induction of this key regulatory enzyme in adrenocortical cells and demonstrated that LPS triggers an increase in COX-2 mRNA levels by mechanisms involving the stimulation of reactive oxygen species (ROS) generation and the activation of p38 MAPK and Akt, in addition to the previously demonstrated increase in NFκB activity. In this sense we showed that: (1) inhibition of p38 MAPK or PI3K/Akt (pharmacological or molecular) prevented the increase in COX-2 protein levels by LPS, (2) LPS induced p38 MAPK and Akt phosphorylation, (3) antioxidant treatment blocked the effect of LPS on p38 MAPK phosphorylation and in COX-2 protein levels, (4) PI3K inhibition with LY294002 prevented p38 MAPK phosphorylation and, (5) the activity of an NFκB reporter was decreased by p38 MAPK or PI3K inhibition.These results suggest that activation of both p38 MAPK and PI3K/Akt pathways promote the stimulation of NFκB activity and that PI3K/Akt activity might regulate both p38 MAPK and NFκB signaling pathways.In summary, in this study we showed that in adrenal cells, LPS induces COX-2 expression by activating p38 MAPK and PI3K/Akt signaling pathways and that both pathways converge in the modulation of NFκB transcriptional activity.

NR5A1 prevents centriole splitting by inhibiting centrosomal DNA-PK activation and ß-catenin accumulation

Adrenogonadal cell growth and differentiation are controlled by nuclear receptor NR5A1 (Ad4BP/SF-1) that regulates the expression of adrenal and gonadal genes. In addition, SF-1 also resides in the centrosome and controls centrosome homeostasis by restricting the activity of centrosomal DNA-PK and CDK2/cyclin A. Here we show that SF-1 depletion resulted in centriole splitting and amplification due to aberrant activation of DNA-PK in the centrosome of mouse adrenocortical Y1 cells. In the absence of SF-1, GSK3β was aberrantly phosphorylated during G1 phase and β-catenin was accumulated in the centrosome, but not in the nucleus. DNA-PK inhibitor vanillin reversed these phenomena. SF-1 overexpression led to inhibition of centrosomal DNA-PK activation caused by SF-1 depletion. Both full-length SF-1 and truncated SF-1 devoid of its DNA-binding domain rescued the multiple centrosome phenotype caused by SF-1 depletion, indicating that the effect of SF-1 in the centrosome is not contributed by its DNA-binding domain. Furthermore, SF-1 interacted with cyclin A in the centrosome, but not in the nucleus. Depletion of SF-1 also resulted in centriole splitting, genomic instability and reduced growth of mouse testicular Leydig MA10 cells. Centrosomal DNA-PK signaling triggers the accumulation of β-catenin, leading to centrosome over-duplication and centriole splitting. This cascade of centrosomal events results in genomic instability and reduced cell numbers.

Crosstalk between nitric oxide synthases and cyclooxygenase 2 in the adrenal cortex of rats under lipopolysaccharide treatment

The effect of lipopolysaccharide on the modulation of steroid production by adrenal cells has been recently acknowledged. The purpose of this study was to determine the in vivo effects of LPS on adrenal cyclooxygenase 2 (COX-2) expression, analyze its crosstalk with the nitric oxide synthase (NOS) system, and assess its involvement on the modulation of glucocorticoid production. Male Wistar rats were injected with LPS and with specific inhibitors for NOS and COX activities. PGE2 and corticosterone levels were determined by RIA. Protein levels were analyzed by immunoprecipitation and western blotting. Transfection assays were performed in murine adrenocortical Y1 cells. Results show that LPS treatment increases PGE2 production and COX-2 protein levels in the rat adrenal cortex. Systemic inhibition of COX-2 blunted the glucocorticoid response to ACTH, as well as the increase in NOS activity and the NOS-2 expression levels induced by LPS. Conversely, NOS inhibition prevented the LPS-dependent increase in PGE2 production, COX-2 protein levels, and the nitrotyrosine modification of COX-2 protein. Treatment of adrenocortical cells with a NO-donor significantly potentiated the LPS-dependent increase in NFκB activity and COX-2 expression levels. In conclusion, our results show a significant crosstalk between COX-2 and NOS in the adrenal cortex upon LPS stimulation, in which each activity has a positive impact on the other. In particular, as both the activities differently affect adrenal steroid production, we hypothesize that this kind of fine modulation enables the gland to adjust steroidogenesis to prevent either an excessive or an insufficient response to the endotoxin challenge.

Acidic Residue Glu199 Increases SUMOylation Level of Nuclear Hormone Receptor NR5A1

Steroidogenic factor 1 (NR5A1/SF1) is a well-known master regulator in controlling adrenal and sexual development, as well as regulating numerous genes involved in adrenal and gonadal steroidogenesis. Several studies including ours have demonstrated that NR5A1 can be SUMOylated on lysine 194 (K194, the major site) and lysine 119 (K119, the minor site), and the cycle of SUMOylation regulates NR5A1’s transcriptional activity. An extended consensus negatively charged amino acid-dependent SUMOylation motif (NDSM) enhances the specificity of substrate modification by SUMO has been reported; however, the mechanism of NDSM for NR5A1 remains to be clarified. In this study, we investigated the functional significance of the acidic residue located downstream from the core consensus SUMO site of NR5A1. Here we report that E199A (glutamic acid was replaced with alanine) of NR5A1 reduced, but not completely abolished, its SUMOylation level. We next characterized the functional role of NR5A1 E199A on target gene expression and protein levels. We found that E199A alone, as well as combination with K194R, increased Mc2r and Cyp19a1 reporter activities. Moreover, E199A alone as well as combination with K194R enhanced NR5A1-mediated STAR protein levels in mouse adrenocortical cancer Y1 cells. We also observed that E199A increased interaction of NR5A1 with CDK7 and SRC1. Overall, we provide the evidence that the acidic residue (E199) located downstream from the core consensus SUMO site of NR5A1 is, at least in part, required for SUMOylation of NR5A1 and for its mediated target gene and protein expression.

Secretin Receptor Promotes the Proliferation of Endocrine Tumor Cells Via the PI3K/AKT Pathway

The secretin receptor (SR), a G protein-coupled receptor, mediates the effects of the gastrointestinal hormone secretin on digestion and water homeostasis. Recently, high SR expression has been observed in pancreatic ductal adenocarcinomas, cholangiocellular carcinomas, gastrinomas, and bronchopulmonary carcinoid tumors. Receptor overexpression associates with enhanced secretin-mediated signaling, but whether this molecule plays an independent role in tumorigenesis is currently unknown. We recently discovered that pheochromocytomas developing in rats affected by the MENX (multiple endocrine neoplasia-like) syndrome express at very high-level Sctr, encoding SR. We here report that SR are also highly abundant on the membranes of rat adrenal and extraadrenal pheochromocytoma, starting from early stages of tumor development, and are functional. PC12 cells, the best characterized in vitro pheochromocytoma model, also express Sctr at high level. Thus, we used them as model to study the role of SR in neoplastic transformation. Small interfering RNA-mediated knockdown of Sctr decreases PC12 cells proliferation and increases p27 levels. The proproliferative effect of SR in PC12 cells is mediated, in part, by the phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (AKT) pathway. Transfection of Sctr in Y1 adrenocortical carcinoma cells, expressing low endogenous levels of Sctr, stimulates cell proliferation also, in part, via the PI3K/AKT signaling cascade. Because of the link between SR and PI3K/AKT signaling, tumor cells expressing high levels of the receptor (MENX-associated primary pheochromocytoma and NCI-H727 human bronchopulmonary carcinoid cells) respond well and in a SR-dependent manner to PI3K inhibitors, such as NVP-BEZ235. The association between SR levels and response to PI3K inhibition might open new avenues for the treatment of tumors overexpressing this receptor.

Death-associated Protein 6 (Daxx) Mediates cAMP-dependent Stimulation of Cyp11a1 (P450scc) Transcription

SF-1 is a key transcription factor for all steroidogenic genes. It up-regulates the expression of the steroidogenic Cyp11a1 gene in the adrenal in a pathway stimulated by cAMP through HIPK3-mediated JNK/c-Jun phosphorylation. In the present study, we have investigated the factors mediating cAMP-dependent HIPK3 action to potentiate the activity of SF-1 for Cyp11a1 transcription in mouse adrenocortical Y1 cells. We found Daxx, a HIPK kinase substrate in the apoptosis pathway, was phosphorylated by HIPK3 at Ser-669 in response to cAMP stimulation. Daxx participated in SF-1-dependent Cyp11a1 expression as shown by experiments involving both overexpression and down-regulation via a dominant negative Daxx mutant. The S669A mutant of Daxx, which could not be phosphorylated by HIPK3, lost the ability to potentiate SF-1 activity for Cyp11a1 expression. The enhancement of SF-1 activity by Daxx required JNK and c-Jun phosphorylation. Thus, Daxx functioned as a signal transducer linking cAMP-stimulated HIPK3 activity with JNK/c-Jun phosphorylation and SF-1-dependent Cyp11a1 transcription for steroid synthesis.

Gipr Is Essential for Adrenocortical Steroidogenesis; However, Corticosterone Deficiency Does Not Mediate the Favorable Metabolic Phenotype of Gipr−/− Mice

Glucose-dependent insulinotropic polypeptide (GIP) promotes glucose-dependent insulin secretion. However, GIP also enhances glucocorticoid secretion and promotes adiposity. Because obesity and diabetes are glucocorticoid dependent, we examined whether the effects of GIP on energy balance and glycemia are regulated by glucocorticoids using pharmacological activation of GIP receptor (GIPR) signaling with [d-Ala2]GIP in mice and in Y1 adrenocortical cells. Genetic elimination of GIPR activity was also studied in normal- and high-fat (HF)-fed Gipr-deficient (Gipr−/−) mice. [d-Ala2]GIP increased murine corticosterone levels in a GIPR-dependent manner. Conversely, basal corticosterone levels were reduced, whereas food deprivation resulted in significantly enhanced plasma corticosterone levels in Gipr−/− mice. [d-Ala2]GIP increased cAMP levels, activated extracellular signal\\x{2013}related kinase (ERK)1/2, increased expression of steroidogenic genes, and increased neutral lipid storage in Y1GIPR cells. Gipr−/− adrenal glands demonstrated a twofold upregulation of the ACTH receptor mRNA and increased sensitivity to ACTH ex vivo. Although HF-fed Gipr−/− mice exhibited significantly lower plasma corticosterone, glucocorticoid-treated HF-fed Gipr−/− mice had similar energy balance and glycemia compared with Gipr+/+ controls. Hence, although the Gipr is essential for adrenal steroidogenesis and links HF feeding to increased levels of corticosterone, reduced glucocorticoid levels do not significantly contribute to the enhanced metabolic phenotypes in HF-fed Gipr−/− mice.

Arginine vasopressin controls p27Kip1 protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27Kip1 protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27Kip1 is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27Kip1, AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27Kip1 protein as well as phosphorylation of the p27Kip1 protein at both Ser10 and Thr187.