Sex difference in the mouse BAT transcriptome reveals a role of progesterone.

Brown adipose tissue (BAT) is a metabolically active organ that exhibits sex-differential features, that is, being generally more abundant and active in females than in males. Although sex steroids, particularly estrogens, have been shown to regulate BAT thermogenic function, the underlying molecular mechanisms contributing to sexual dimorphism in basal BAT activity have not been elucidated. Therefore, we assessed the transcriptome of interscapular BAT of male and female C57BL/6J mice by RNA sequencing and identified 295 genes showing ≥2-fold differential expression (adjusted P < 0.05). In silico functional annotation clustering suggested an enrichment of genes encoding proteins involved in cell-cell contact, interaction, and adhesion. Ovariectomy reduced the expression of these genes in female BAT toward a male pattern whereas orchiectomy had marginal effects on the transcriptional pattern, indicating a prominent role of female gonadal hormones in this sex-differential expression pattern. Progesterone was identified as a possible upstream regulator of the sex-differentially expressed genes. Studying the direct effects of progesterone in vitro in primary adipocytes showed that progesterone significantly altered the transcription of several of the identified genes, possibly via the glucocorticoid receptor. In conclusion, this study reveals a sexually dimorphic transcription profile in murine BAT at general housing conditions and demonstrates a role for progesterone in the regulation of the interscapular BAT transcriptome.

Sex Difference in Corticosterone-Induced Insulin Resistance in Mice.

Prolonged exposure to glucocorticoids (GCs) causes various metabolic derangements. These include obesity and insulin resistance, as inhibiting glucose utilization in adipose tissues is a major function of GCs. Although adipose tissue distribution and glucose homeostasis are sex-dependently regulated, it has not been evaluated whether GCs affect glucose metabolism and adipose tissue functions in a sex-dependent manner. In this study, high-dose corticosterone (rodent GC) treatment in C57BL/6J mice resulted in nonfasting hyperglycemia in male mice only, whereas both sexes displayed hyperinsulinemia with normal fasting glucose levels, indicative of insulin resistance. Metabolic testing using stable isotope-labeled glucose techniques revealed a sex-specific corticosterone-driven glucose intolerance. Corticosterone treatment increased adipose tissue mass in both sexes, which was reflected by elevated serum leptin levels. However, female mice showed more metabolically protective adaptations of adipose tissues than did male mice, demonstrated by higher serum total and high-molecular-weight adiponectin levels, more hyperplastic morphological changes, and a stronger increase in mRNA expression of adipogenic differentiation markers. Subsequently, in vitro studies in 3T3-L1 (white) and T37i (brown) adipocytes suggest that the increased leptin and adiponectin levels were mainly driven by the elevated insulin levels. In summary, this study demonstrates that GC-induced insulin resistance is more severe in male mice than in female mice, which can be partially explained by a sex-dependent adaptation of adipose tissues.

Multiple effects of cold exposure on livers of male mice.

Cold exposure of mice is a common method to stimulate brown adipose tissue (BAT) activity and induce browning of white adipose tissue (WAT) that has beneficial effects on whole-body lipid metabolism, including reduced plasma triglyceride (TG) concentrations. The liver is a key regulatory organ in lipid metabolism as it can take up as well as oxidize fatty acids. The liver can also synthesize, store and secrete TGs in VLDL particles. The effects of cold exposure on murine hepatic lipid metabolism have not been addressed. Here, we report the effects of 24-h exposure to 4°C on parameters of hepatic lipid metabolism of male C57BL/6J mice. Cold exposure increased hepatic TG concentrations by 2-fold (P < 0.05) but reduced hepatic lipogenic gene expression. Hepatic expression of genes encoding proteins involved in cholesterol synthesis and uptake such as the LDL receptor (LDLR) was significantly increased upon cold exposure. Hepatic expression of Cyp7a1 encoding the rate-limiting enzyme in the classical bile acid (BA) synthesis pathway was increased by 4.3-fold (P < 0.05). Hepatic BA concentrations and fecal BA excretion were increased by 2.8- and 1.3-fold, respectively (P < 0.05 for both). VLDL-TG secretion was reduced by approximately 50% after 24 h of cold exposure (P < 0.05). In conclusion, cold exposure has various, likely intertwined effects on the liver that should be taken into account when studying the effects of cold exposure on whole-body metabolism.

Sex difference in thermal preference of adult mice does not depend on presence of the gonads.

BACKGROUND: The thermoneutral zone (TNZ) is a species-specific range of ambient temperature (T a), at which mammals can maintain a constant body temperature with the lowest metabolic rate. The TNZ for an adult mouse is between 26 and 34 °C. Interestingly, female mice prefer a higher T a than male mice although the underlying mechanism for this sex difference is unknown. Here, we tested whether gonadal hormones are dominant factors controlling temperature preference in male and female mice. METHODS: We performed a temperature preference test in which 10-week-old gonadectomized and sham-operated male and female C57BL/6J mice were allowed to choose to reside at the thermoneutral cage of 29 °C or an experimental cage of 26, 29, or 32 °C. RESULTS: All mice preferred a T a higher than 26 °C, especially in the inactive phase. Choosing between 29 and 32 °C, female mice resided more at 32 °C while male mice had no preference between the temperatures. Hence, the preferred T a for female mice was significantly higher (0.9 ± 0.2 °C) than that for male mice. However, gonadectomy did not influence the T a preference. CONCLUSIONS: Female mice prefer a warmer environment than male mice, a difference not affected by gonadectomy. This suggests that thermal-sensing mechanisms may be influenced by sex-specific pathways other than gonadal factors or that the thermoregulatory set point has already been determined prior to puberty.

Protection against renal ischemia-reperfusion injury through hormesis? Dietary intervention versus cold exposure.

AIM: Dietary restriction (DR) and fasting (FA) induce robust protection against the detrimental effects of renal ischemia-reperfusion injury (I/RI). Several mechanisms of protection have been proposed, such as hormesis. Hormesis is defined as a life-supporting beneficial effect resulting from the cellular responses to single or multiple rounds of (mild) stress. The cold exposure (CE) model is a stress model similar to DR, and has been shown to have hormetic effects and has proved to increase longevity. CE is considered to be the most robust method to increase metabolism through activation of brown adipocytes. BAT has been considered important in etiology of obesity and its metabolic consequences. MATERIALS AND METHODS: Since DR, FA, and CE models are proposed to work through hormesis, we investigated physiology of adipose tissue and effect on BAT in these models and compared them to ad libitum (AL) fed mice. We also studied the differential effect of these stress models on immunological changes, and effect of CE on renal I/RI. KEY FINDINGS: We show similar physiological changes in adiposity in male C57Bl/6 mice due to DR, FA and CE, but the CE mice were not protected against renal I/RI. The immunophenotypic changes observed in the CE mice were similar to the AL animals, in contrast to FA mice, that showed major immunophenotypic changes in the B and T cell development stages in primary and secondary lymphoid organs. SIGNIFICANCE: Our findings thus demonstrate that DR, FA and CE are hormetic stress models. DR and FA protect against renal I/IR, whereas CE could not.

Cold Exposure Partially Corrects Disturbances in Lipid Metabolism in a Male Mouse Model of Glucocorticoid Excess.

High glucocorticoid concentrations are accompanied by metabolic side effects such as high plasma triglyceride (TG) concentrations. Liver, brown adipose tissue (BAT) and white adipose tissue are important regulators of plasma TG. Exposure to 4°C reduces plasma TG concentrations, and we therefore aimed to study the interaction between glucocorticoid excess and 24 hours of exposure to 4°C on lipid metabolism. For this, mice were implanted with 50-mg corticosterone or control pellets and housed for 24 hours at 23°C or 4°C 1 week later, after which various aspects of TG metabolism in liver, BAT, and white adipose tissue were studied. Corticosterone treatment resulted in a 3.8-fold increase of plasma TG concentrations. Increased TG was normalized by cold exposure, an effect still present 24 hours after cold exposure. Corticosterone treatment increased hepatic TG content by 3.5-fold and provoked secretion of large, TG-rich very low density lipoprotein particles. Cold exposure reduced very low density lipoprotein-TG secretion by approximately 50%. Corticosterone strongly decreased BAT activity: BAT weight increased by 3.5-fold, whereas uncoupling protein 1 (Ucp1) mRNA expression and Ucp1 protein content of BAT were reduced by 75% and 60%, respectively. Cold exposure partially normalized these parameters of BAT activity. The uptake of TG by BAT was not affected by corticosterone treatment but was increased 4.5-fold upon cold exposure. In conclusion, cold exposure normalizes corticosterone-induced hypertriglyceridemia, at least partly via activating BAT.

Women have more potential to induce browning of perirenal adipose tissue than men.

OBJECTIVE: Brown adipose tissue (BAT) can generate heat by burning fatty acids, a process mediated by uncoupling protein 1 (UCP1). White adipose tissue (WAT) depots can gain BAT-like properties, and various studies have suggested that females have more active BAT or BAT-like WAT. We studied sex differences in BAT-like properties of human perirenal adipose tissue. METHODS: Perirenal and subcutaneous adipose tissue was obtained from 20 male and 24 female healthy live kidney donors. Mesenchymal stem cells (MSCs), adipocyte precursor cells, were isolated from these depots to study whether intrinsic factors control BAT-like properties of the adipose tissue depots. RESULTS: When average outside temperature a week before harvesting was below 11°C, brown-like adipocytes expressing UCP1 were present in perirenal adipose tissue of women, but not of men. MSCs derived from perirenal adipose tissue expressed significantly more UCP1 when from female origin compared to male origin (P = 0.009). However, UCP1 protein content and oxygen consumption rate did not differ between adipocytes derived from male and female perirenal MSCs. CONCLUSIONS: Female perirenal adipose tissue has a higher potency to gain BAT-like properties than male perirenal adipose tissue. The degree of gaining BAT-like properties depends on sex-specific intrinsic factors and environmental triggers such as temperature.

Estrogens increase expression of bone morphogenetic protein 8b in brown adipose tissue of mice.

BACKGROUND: In mammals, white adipose tissue (WAT) stores fat and brown adipose tissue (BAT) dissipates fat to produce heat. Several studies showed that females have more active BAT. Members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families are expressed in BAT and are involved in BAT activity. We hypothesized that differential expression of BMPs and FGFs might contribute to sex differences in BAT activity. METHODS: We investigated the expression of BMPs and FGFs in BAT of male and female C57BL/6J mice upon gonadectomy, cold exposure, and exposure to sex steroids. RESULTS: Of the FGF family, BAT Fgf1, Fgf9, Fgf18, and Fgf21 expression was induced upon cold exposure, but only Fgf1 expression was obviously different between the sexes: females had 2.5-fold lower BAT Fgf1 than males. Cold exposure induced BAT Bmp4 and Bmp8b expression, but only Bmp8b differed between the sexes: females had 35-fold higher BAT Bmp8b than males. Ovariectomy almost completely blunted BAT Bmp8b expression, while orchidectomy had no effect. Male mice and ovariectomized female mice treated with diethylstilbestrol (DES) had approximately 350-fold and approximately 36-fold higher BAT Bmp8b expression, respectively. Ninety-day and 7-day treatment of female mice with dihydrotestosterone (DHT) decreased BAT Bmp8b expression by approximately fivefold and approximately fourfold, respectively. Finally, treatment of primary murine brown adipocytes with DES did not result in changes in Bmp8b expression. CONCLUSIONS: BAT Bmp8b expression in mice is positively regulated by presence of ovaries and estrogens such as DES.

Direct activating effects of adrenocorticotropic hormone (ACTH) on brown adipose tissue are attenuated by corticosterone.

Brown adipose tissue (BAT) and brown-like cells in white adipose tissue (WAT) can dissipate energy through thermogenesis, a process mediated by uncoupling protein 1 (UCP1). We investigated whether stress hormones ACTH and corticosterone contribute to BAT activation and browning of WAT. ACTH and corticosterone were studied in male mice exposed to 4 or 23°C for 24 h. Direct effects were studied in T37i mouse brown adipocytes and primary cultured murine BAT and inguinal WAT (iWAT) cells. In vivo effects were studied using (18)F-deoxyglucose positron emission tomography. Cold exposure doubled serum ACTH concentrations (P=0.03) and fecal corticosterone excretion (P=0.008). In T37i cells, ACTH dose-dependently increased Ucp1 mRNA (EC50=1.8 nM) but also induced Ucp1 protein content 88% (P=0.02), glycerol release 32% (P=0.03) and uncoupled respiration 40% (P=0.003). In cultured BAT and iWAT, ACTH elevated Ucp1 mRNA by 3-fold (P=0.03) and 3.7-fold (P=0.01), respectively. In T37i cells, corticosterone prevented induction of Ucp1 mRNA and Ucp1 protein by both ACTH and norepinephrine in a glucocorticoid receptor (GR)-dependent fashion. ACTH and GR antagonist RU486 independently doubled BAT (18)F-deoxyglucose uptake (P=0.0003 and P=0.004, respectively) in vivo. Our results show that ACTH activates BAT and browning of WAT while corticosterone counteracts this.

Inhibin alpha-subunit (INHA) expression in adrenocortical cancer is linked to genetic and epigenetic INHA promoter variation.

Adrenocortical carcinoma (ACC) is a rare, but highly malignant tumor of unknown origin. Inhibin α-subunit (Inha) knockout mice develop ACCs following gonadectomy. In man, INHA expression varies widely within ACC tissues and its circulating peptide inhibin pro-αC has been described as a novel tumor marker for ACC. We investigated whether genetic and epigenetic changes of the INHA gene in human ACC cause loss or variation of INHA expression. To this end, analyses of INHA sequence, promoter methylation and mRNA expression were performed in human adrenocortical tissues. Serum inhibin pro-αC levels were also measured in ACC patients. INHA genetic analysis in 37 unique ACCs revealed 10 novel, heterozygous rare variants. Of the 3 coding bases affected, one variant was synonymous and two were missense variants: S72F and S184F. The minor allele of rs11893842 at -124 bp was observed at a low frequency (24%) in ACC samples and was associated with decreased INHA mRNA levels: 4.7±1.9 arbitrary units for AA, compared to 26±11 for AG/GG genotypes (P = 0.034). The methylation of four proximal INHA promoter CpGs was aberrantly increased in five ACCs (47.7±3.9%), compared to normal adrenals (18.4±0.6%, P = 0.0052), whereas the other 14 ACCs studied showed diminished promoter methylation (9.8±1.1%, P = 0.020). CpG methylation was inversely correlated to INHA mRNA levels in ACCs (r = -0.701, p = 0.0036), but not associated with serum inhibin pro-αC levels. In conclusion, aberrant methylation and common genetic variation in the INHA promoter occur in human ACCs and are associated with decreased INHA expression.

ACTH-independent macronodular adrenocortical hyperplasia reveals prevalent aberrant in vivo and in vitro responses to hormonal stimuli and coupling of arginine-vasopressin type 1a receptor to 11ß-hydroxylase.

BACKGROUND: Adrenal Cushing's syndrome caused by ACTH-independent macronodular adrenocortical hyperplasia (AIMAH) can be accompanied by aberrant responses to hormonal stimuli. We investigated the prevalence of adrenocortical reactions to these stimuli in a large cohort of AIMAH patients, both in vivo and in vitro. METHODS: In vivo cortisol responses to hormonal stimuli were studied in 35 patients with ACTH-independent bilateral adrenal enlargement and (sub-)clinical hypercortisolism. In vitro, the effects of these stimuli on cortisol secretion and steroidogenic enzyme mRNA expression were evaluated in cultured AIMAH and other adrenocortical cells. Arginine-vasopressin (AVP) receptor mRNA levels were determined in the adrenal tissues. RESULTS: Positive serum cortisol responses to stimuli were detected in 27/35 AIMAH patients tested, with multiple responses within individual patients occurring for up to four stimuli. AVP and metoclopramide were the most prevalent hormonal stimuli triggering positive responses in vivo. Catecholamines induced short-term cortisol production more often in AIMAH cultures compared to other adrenal cells. Short- and long-term incubation with AVP increased cortisol secretion in cultures of AIMAH cells. AVP also increased steroidogenic enzyme mRNA expression, among which an aberrant induction of CYP11B1. AVP type 1a receptor was the only AVPR expressed and levels were high in the AIMAH tissues. AVPR1A expression was related to the AVP-induced stimulation of CYP11B1. CONCLUSIONS: Multiple hormonal signals can simultaneously induce hypercortisolism in AIMAH. AVP is the most prevalent eutopic signal and expression of its type 1a receptor was aberrantly linked to CYP11B1 expression.

Intratumoral conversion of adrenal androgen precursors drives androgen receptor-activated cell growth in prostate cancer more potently than de novo steroidogenesis.

BACKGROUND: Despite an initial response to hormonal therapy, patients with advanced prostate cancer (PC) almost always progress to castration-resistant disease (CRPC). Although serum testosterone (T) is reduced by androgen deprivation therapy, intratumoral T levels in CRPC are comparable to those in prostate tissue of eugonadal men. These levels could originate from intratumoral conversion of adrenal androgens and/or from de novo steroid synthesis. However, the relative contribution of de novo steroidogenesis to AR-driven cell growth is unknown. METHODS: The relative contribution of androgen biosynthetic pathways to activate androgen receptor (AR)-regulated cell growth and expression of PSA, FKBP5, and TMPRSS2 was studied at physiologically relevant levels of adrenal androgen precursors and intermediates of de novo androgen biosynthesis in human prostate cancer cell lines, PC346C, VCaP, and LNCaP. RESULTS: In PC346C and VCaP, responses to pregnenolone and progesterone were absent or minimal, while large effects of adrenal androgen precursors were found. VCaP CRPC clones overexpressing CYP17A1 did not acquire an increased ability to use pregnenolone or progesterone to activate AR. In contrast, all precursors stimulated growth and gene expression in LNCaP cells, presumably resulting from the mutated AR in these cells. CONCLUSIONS: Our data indicate that at physiological levels of T precursors PC cells can generally convert adrenal androgens, while de novo steroidogenesis is not generally possible in PC cells and is not able to support AR transactivation and PC growth.

Protein kinase C-induced activin A switches adrenocortical steroidogenesis to aldosterone by suppressing CYP17A1 expression.

Functional zonation of the adrenal cortex is a consequence of the zone-specific expression of P450c17 (CYP17A1) and its cofactors. Activin and inhibin peptides are differentially produced within the zones of the adrenal cortex and have been implicated in steroidogenic control. In this study, we investigated whether activin and inhibin can function as intermediates in functional zonation of the human adrenal cortex. Activin A suppressed CYP17A1 expression and P450c17 function in adrenocortical cell lines as well as in primary adrenal cell cultures. Inhibin ßA-subunit mRNA and activin A protein levels were found to be increased up to 1,900-fold and 49-fold, respectively, after protein kinase C (PKC) stimulation through PMA or angiotensin II in H295R adrenocortical carcinoma cells. This was confirmed in HAC15 cells and for PMA in primary adrenal cell cultures. Both PMA and Ang II decreased CYP17A1 expression in the adrenocortical cell lines, whereas PMA concurrently suppressed CYP17A1 levels in the primary cultures. Inhibition of activin signaling during PKC stimulation through silencing of the inhibin ßA-subunit or blocking of the activin type I receptor opposed the PMA-induced downregulation of CYP17A1 expression and P450c17 function. In contrast, PKA stimulation through adrenocorticotrophin or forskolin increased expression of the inhibin α-subunit and betaglycan, both of which are antagonists of activin action. These data indicate that activin A acts as a PKC-induced paracrine factor involved in the suppression of CYP17A1 in the zona glomerulosa and can thereby contribute to functional adrenocortical zonation.

Activin A stimulates AKR1C3 expression and growth in human prostate cancer.

Local androgen synthesis in prostate cancer (PC) may contribute to the development of castration-resistant PC (CRPC), but pathways controlling intratumoral steroidogenic enzyme expression in PC are unknown. We investigated the effects of activin, a factor involved in the regulation of PC growth and steroidogenic enzyme expression in other steroidogenic tissues, on intratumoral steroidogenesis in PC. Activin A effects and regulation of the activin-signaling pathway molecules were studied in the PC cell lines LNCaP, VCaP, and PC-3 and in 13 individual PC xenograft models. Also, expression levels of inhibin ßA- and ßB-subunits (INHBA and INHBB) and of the activin antagonist follistatin were quantitated in patient PC tissues. Activin A induced the expression and enzyme activity of 17ß-hydroxysteroid dehydrogenase enzyme AKR1C3 in LNCaP and VCaP cells. Inhibition of endogenous activin A action in the PC-3 cell line decreased AKR1C3 levels and consequently testosterone synthesis. In return, androgens suppressed INHBA expression in both VCaP cells and the PC xenograft models. The antiproliferative effects of activin A were opposed by physiological concentrations of androstenedione in LNCaP cells. In patient PC tissues, expression levels of INHBA were increased in CRPC samples and correlated with AKR1C3 levels. Moreover, a high ratio of activin subunits to follistatin was associated with a worse metastasis-free survival in patients. In conclusion, activin A is controlled by androgens in PC models and regulates local androgen production. Activin A thus seems to mediate (residual) intratumoral androgen levels and could form a novel therapeutic target in CRPC.

Melanocortin 2 receptor-associated protein (MRAP) and MRAP2 in human adrenocortical tissues: regulation of expression and association with ACTH responsiveness.

CONTEXT: ACTH stimulates adrenocortical steroid production through the melanocortin 2 receptor (MC2R). MC2R trafficking and signaling are dependent on the MC2R accessory protein (MRAP). The MRAP homolog MRAP2 also transports the MC2R to the cell surface but might prevent activation. OBJECTIVE: The objective of the investigation was to study the regulatory pathways of MRAP and MRAP2 and their contributions to ACTH responsiveness in human adrenal tissues. DESIGN AND SETTING: MRAP, MRAP2, and MC2R expression levels were studied in 32 human adrenocortical samples. Regulation of these mRNAs was investigated in 43 primary adrenal cultures, stimulated with ACTH, forskolin, angiotensin II (AngII), phorbol-12-myristate-13-acetate (PMA), or dexamethasone. The induction of cortisol, cAMP, and ACTH-responsive genes after treatment with ACTH was related to MRAP, MRAP2, and MC2R expression levels. RESULTS: MRAP and MRAP2 levels were lower in adrenocortical carcinomas (ACC) than in other adrenal tissues (P < 0.001). Patient ACTH and cortisol levels were associated with adrenal levels of MRAP and MC2R in adrenal hyperplasia samples (P < 0.05) but not in tumors. ACTH induced the expression of MRAP 11 ± 2.1-fold and MC2R 20 ± 3.8-fold in all adrenal tissue types (mean ± SEM, both P < 0.0001), whereas AngII augmented these mRNAs 4.0 ± 1.2-fold and 12.6 ± 3.2-fold (P < 0.0001) in all but the ACC. MRAP2 expression was suppressed by forskolin (-24 ± 15%, P = 0.013) and PMA (-22 ± 7%, P = 0.0007). MRAP, MRAP2, or MC2R levels were not associated with the induction of cortisol, cAMP, or gene expression by ACTH in vitro. CONCLUSION: MRAP and MC2R expression is induced by ACTH and AngII, which would facilitate cell surface receptor availability. Physiological expression levels of MRAP, MRAP2, and MC2R were not limiting for ACTH sensitivity.

Evidence of limited contributions for intratumoral steroidogenesis in prostate cancer.

Androgen-deprivation therapy for prostate cancer (PC) eventually leads to castration-resistant PC (CRPC). Intratumoral androgen production might contribute to tumor progression despite suppressed serum androgen concentrations. In the present study, we investigated whether PC or CRPC tissue may be capable of intratumoral androgen synthesis. Steroidogenic enzyme mRNAs were quantified in hormonally manipulated human PC cell lines and xenografts as well as in human samples of normal prostate, locally confined and advanced PC, local nonmetastatic CRPC, and lymph node metastases. Overall, the majority of samples showed low or absent mRNA expression of steroidogenic enzymes required for de novo steroid synthesis. Simultaneous but low expression of the enzymes CYP17A1 and HSD3B1, essential for the synthesis of androgens from pregnenolone, could be detected in 19 of 88 patient samples. Of 19 CRPC tissues examined, only 5 samples expressed both enzymes. Enzymes that convert androstenedione to testosterone (AKR1C3) and testosterone to dihydrotestosterone (DHT; SRD5A1) were abundantly expressed. AKR1C3 expression was negatively regulated by androgens in the experimental models and was increased in CRPC samples. Expression of SRD5A1 was upregulated in locally advanced cancer, CRPC, and lymph node metastases. We concluded that intratumoral steroid biosynthesis contributes less than circulating adrenal androgens, implying that blocking androgen production and its intraprostatic conversion into DHT, such as via CYP17A1 inhibition, may represent favorable therapeutic options in patients with CRPC.