Serum interleukin 1 receptor antagonist as an independent marker of non-alcoholic steatohepatitis in humans.

BACKGROUND & AIMS: Mechanisms leading to non-alcoholic steatohepatitis (NASH) have remained unclear, and non-invasive diagnosis of NASH is challenging. In this study, we investigated the benefits of measuring serum interleukin 1 receptor antagonist (IL-1RA) levels. METHODS: Liver biopsies from 119 morbidly obese individuals (47.5 ± 9.0 years, BMI 44.9 ± 5.9 kg/m(2)) were used for histological and gene expression assessment. In a cross-sectional population-based cohort of 6447 men (58 ± 7 years, BMI 27.0 ± 3.9 kg/m(2)) the association of serum IL1-RA with serum alanine aminotransferase (ALT) levels was investigated. RESULTS: Serum levels of IL-1RA, and liver mRNA expression of IL1RN are associated with NASH and the degree of lobular inflammation in liver (p<0.05). The decrease in serum IL-1RA level and expression of IL1RN after obesity surgery correlated with the improvement of lobular inflammation (p<0.05). We developed a novel NAFLD Liver Inflammation Score, including serum Il-1RA concentration, which performed better to diagnose NASH than did previously published scores. Results from the population study confirmed the potential of measuring serum IL-1RA level. The strongest determinants of the ALT concentration at the population level were Matsuda insulin sensitivity index (r(2)=0.130, p=7 × 10(-197)) and serum IL-1RA concentration (r(2)=0.074, p=1 × 10(-110)). IL-1RA concentrations associated significantly with ALT levels even after adjusting for BMI, alcohol consumption and insulin sensitivity (p=2 × 10(-21)). CONCLUSIONS: IL-1RA serum levels associate with liver inflammation and serum ALT independently of obesity, alcohol consumption and insulin resistance, suggesting a potential use of IL-1RA as a non-invasive inflammatory marker for NASH.

Androgen receptor gene CAG repeat polymorphism and X-chromosome inactivation in children with premature adrenarche.

CONTEXT: There is variation in the adrenal androgen levels and clinical findings of children with premature adrenarche (PA). OBJECTIVES: We hypothesized that androgen sensitivity, indicated by the length of CAG repeat in the X-chromosomal androgen receptor (AR) gene has a role in the polygenic pathogenesis of PA. DESIGN AND PATIENTS: We performed a cross-sectional association study among 73 Finnish Caucasian children with PA (10 boys and 63 girls) and 97 age- and gender-matched healthy controls (18 boys and 79 girls). MAIN OUTCOME MEASURES: AR gene methylation-weighted CAG(n)(mwCAG(n)) via CAG(n) length and X-chromosome inactivation analysis and clinical phenotype were determined. SETTING: The study took place at a university hospital. RESULTS: PA subjects had significantly shorter mwCAG(n) than controls [mean difference (95% confidence interval); 0.76 (0.14-1.38); P = 0.017]. AR gene mwCAG(n) did not correlate with androgen or SHBG levels in either group. In children with PA, mwCAG(n) correlated positively with body mass index (BMI) (tau = 0.19; P = 0.02). The mean of mwCAG(n) was significantly shorter in PA children with lower BMI compared with PA children with higher BMI [BMI sd score < 0.79, n = 35, vs. BMI sd score > 0.79, n = 36; 1.13 (0.38-1.87), P = 0.004] and in PA children with lower BMI compared with healthy children with same BMI (P = 0.004). CONCLUSIONS: The AR gene CAG(n) polymorphism may have a significant role in the pathogenesis of PA, especially in lean children.

Androgen receptor gene CAG repeat length in women with metabolic syndrome.

OBJECTIVE: The length of the androgen receptor gene CAG repeat [AR (CAG)(n)] modulates the activity of the androgen receptor (AR), and this polymorphism has been shown to modulate body fat mass and serum concentrations of insulin in men. We hypothesized that shorter AR (CAG)(n) is associated with metabolic syndrome (MBS) or its components in women. DESIGN, PATIENTS AND MEASUREMENTS: In a cross-sectional controlled study we studied 52 Finnish women aged 34-55 years with MBS and 69 age-matched controls. All participants were recruited from a sample of women drawn from the Finnish population register. We compared the mean AR (CAG)(n) in the two groups. Furthermore, we correlated the AR (CAG)(n) with serum testosterone, androstenedione, dehydroepiandrosterone sulfate and several parameters of glucose and lipid metabolism in each group and in all 121 women. RESULTS: There was no difference in the biallelic mean AR (CAG)(n) between the MBS and the control group (21.6+/-0.2 vs. 21.8+/-0.2, not significant). The AR (CAG)(n) did not correlate significantly with any of the clinical or biochemical parameters of glucose or fat metabolism. However, it correlated negatively with serum testosterone (-0.195, p = 0.04) and androstenedione concentrations (-0.205, p = 0.03) in all studied women. CONCLUSIONS: The AR (CAG)(n) is not a major determinant of MBS in women but it contributes to ovarian androgen production.

Tumor necrosis factor-alpha regulates steroidogenesis, apoptosis, and cell viability in the human adrenocortical cell line NCI-H295R.

TNF-alpha regulates the hypothalamo-pituitary-adrenal axis at several levels. It has been shown to modify adrenal steroidogenesis in many species, and it is supposed to act as an auto/paracrine factor. However, its significance in human adrenocortical function remains unclear. Therefore, we investigated the effect of TNF-alpha on adrenal steroidogenesis, expression of the key steroidogenic genes, apoptosis, and cell viability in the human adrenocortical cell line NCI-H295R. TNF-alpha treatment (1 nM for 48 h) decreased the basal production of cortisol, androstenedione, dehydroepiandrosterone sulfate (DHEAS), and aldosterone (14, 18, 35, and 52%, respectively), and the 8-bromo-cAMP-induced production of cortisol, androstenedione, dehydroepiandrosterone (DHEA), and DHEAS (44, 66, 58, and 48%, respectively). However, when the steroid production data were normalized by the cell number, TNF-alpha increased the basal production of cortisol, androstenedione, DHEA, DHEAS, and aldosterone (137, 121, 165, 73, and 28%, respectively), and the 8-bromo-cAMP-induced production of cortisol, DHEAS, and aldosterone (122, 121, and 256%, respectively). This was accompanied by a parallel increase in the expression of the genes encoding for the steroidogenic acute regulatory protein, 3beta-hydroxysteroid dehydrogenase 2, and 17-hydroxylase/17,20-lyase (74, 200, and 50%, respectively; quantitative real-time RT-PCR analysis). TNF-alpha increased caspase 3/7 activity (an indicator of apoptosis) and decreased cell viability dose and time dependently. The effect of TNF-alpha on apoptosis was neutralized by a monoclonal TNF-alpha antibody. These findings indicate that TNF-alpha is a potent regulator of steroidogenesis and cell viability in adrenocortical cells. TNF-alpha may have physiological and/or pathophysiological significance as an endocrine and/or paracrine/autocrine regulator of adrenocortical function.

Inhibition of DNA methylation increases follistatin expression and secretion in the human adrenocortical cell line NCI-H295R.

Activin affects adrenocortical steroidogenesis and increases apoptosis, while follistatin (FS) acts as an activin antagonist by binding to activin, preventing attachment to its receptors. The regulation of FS expression in the adrenal cortex is poorly understood. Adrenocortical tumors often display aberrant methylation. In the present study, we investigated the effect of DNA methylation on FS mRNA expression and peptide secretion in adrenocortical cells. We treated human NCI-H295R adrenocortical cells with the methylation inhibitor 5-Aza-2'deoxycytidine (Azad; 0.1-100 microM for 1, 4 or 7 days) and measured FS mRNA expression by Northern blot and quantitative real time RT-PCR analyses as well as FS secretion by specific ELISA. Methylation-specific PCR showed decreased methylation in the FS promoter region after Azad treatment. A significant (P < 0.05) time- and dose-dependent increase in FS mRNA expression (up to 4.6-fold) and peptide secretion (up to 17.1-fold) was detected after Azad treatment. We conclude that FS gene expression and peptide secretion in NCI-H295R adrenocortical cells are regulated by DNA methylation. Thus, variable methylation in different adrenocortical tumors may influence activin bioactivity and its consequences in steroidogenesis and cell proliferation/apoptosis.

Transcription factors GATA-6, SF-1, and cell proliferation in human adrenocortical tumors.

Transcription factor GATA-6 is expressed in fetal and adult human adrenal cortex and has been suggested to have a role in adrenal androgen synthesis. In other tissues GATA-6 has been linked to the cell cycle regulation and the dedifferentiation of carcinoma cells. GATA-6 has been shown to be downregulated in mouse adrenocortical tumors, but has not been studied in human adrenocortical tumors in detail. We have now analyzed GATA-6 expression in 20 human adrenocortical adenomas and 16 carcinomas using Northern blot analysis and immunohistochemistry. GATA-6 mRNA and protein expression was remarkably diminished in adrenocortical carcinomas as compared to normal adrenal cortex and adenomas (p<0.05). In opposite to other tumor types GATA-6 expression was, however, high in virilizing carcinomas. Steroidogenic factor 1 (SF-1) has been functionally linked to GATA-6, and the expression of these two factors correlated in the adrenal tumors. Furthermore, GATA-6 immunoreactivity was linked to P450c17 expression. In contrast to GATA-6, we found upregulated cyclin-dependent kinase inhibitor p21 and proliferation marker Ki67 in adrenocortical carcinomas indicating that GATA-6 is not linked to cell proliferation in human adrenal tumors. Taken together, the present and earlier results link GATA-6 to adrenocortical steroidogenesis and to the benign adrenocortical phenotype.

Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells.

Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin alpha-, betaA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.

Expression of betaglycan, an inhibin coreceptor, in normal human ovaries and ovarian sex cord-stromal tumors and its regulation in cultured human granulosa-luteal cells.

Activins and inhibins are often antagonistic in the regulation of ovarian function. TGFbeta type III receptor, betaglycan, has been identified as a coreceptor to enhance the binding of inhibins to activin type II receptor and thus to prevent the binding of activins to their receptor. In this study we characterized the expression and regulation pattern of betaglycan gene in normal ovaries and sex cord-stromal tumors and in cultured human granulosa-luteal cells from women undergoing in vitro fertilization. Expression of betaglycan mRNA was detected by RT-PCR or Northern blotting in normal ovarian granulosa, thecal, and stroma cells as well as in granulosa-luteal cells. Immunohistochemical analysis revealed positive staining for betaglycan in antral and preovulatory follicular granulosa and thecal cells and in corpora lutea of normal ovaries. Furthermore, betaglycan expression was detected in the vast majority of granulosa cell tumors, thecomas, and fibromas, with weaker staining in granulosa cell tumors compared with fibrothecomas. In cultured granulosa-luteal cells, FSH and LH treatment increased dose-dependently the accumulation of betaglycan mRNA, as did the protein kinase A activator dibutyryl cAMP and the protein kinase C inhibitor staurosporine. In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol 13-acetate had no significant effect on betaglycan mRNA levels. Treatment with prostaglandin E(2) and with its receptor EP2 subtype agonist butaprost increased betaglycan mRNA accumulation and progesterone secretion dose- and time-dependently. In summary, betaglycan gene is expressed in normal human ovarian steroidogenic cells and sex cord-stromal ovarian tumors. The accumulation of its mRNA in cultured granulosa-luteal cells is up-regulated by gonadotropins and prostaglandin E(2), probably via the protein kinase A pathway. The specific expression and regulation pattern of betaglycan gene may be related to the functional antagonism of inhibins to activin signal transduction in human ovaries.

Link between GIP and osteopontin in adipose tissue and insulin resistance.

Low-grade inflammation in obesity is associated with accumulation of the macrophage-derived cytokine osteopontin (OPN) in adipose tissue and induction of local as well as systemic insulin resistance. Since glucose-dependent insulinotropic polypeptide (GIP) is a strong stimulator of adipogenesis and may play a role in the development of obesity, we explored whether GIP directly would stimulate OPN expression in adipose tissue and thereby induce insulin resistance. GIP stimulated OPN protein expression in a dose-dependent fashion in rat primary adipocytes. The level of OPN mRNA was higher in adipose tissue of obese individuals (0.13 ± 0.04 vs. 0.04 ± 0.01, P < 0.05) and correlated inversely with measures of insulin sensitivity (r = -0.24, P = 0.001). A common variant of the GIP receptor (GIPR) (rs10423928) gene was associated with a lower amount of the exon 9-containing isoform required for transmembrane activity. Carriers of the A allele with a reduced receptor function showed lower adipose tissue OPN mRNA levels and better insulin sensitivity. Together, these data suggest a role for GIP not only as an incretin hormone but also as a trigger of inflammation and insulin resistance in adipose tissue. Carriers of the GIPR rs10423928 A allele showed protective properties via reduced GIP effects. Identification of this unprecedented link between GIP and OPN in adipose tissue might open new avenues for therapeutic interventions.

Adipose tissue TCF7L2 splicing is regulated by weight loss and associates with glucose and fatty acid metabolism.

We investigated the effects of obesity surgery-induced weight loss on transcription factor 7-like 2 gene (TCF7L2) alternative splicing in adipose tissue and liver. Furthermore, we determined the association of TCF7L2 splicing with the levels of plasma glucose and serum free fatty acids (FFAs) in three independent studies (n = 216). Expression of the short mRNA variant, lacking exons 12, 13, and 13a, decreased after weight loss in subcutaneous fat (n = 46) and liver (n = 11) and was more common in subcutaneous fat of subjects with type 2 diabetes than in subjects with normal glucose tolerance in obese individuals (n = 54) and a population-based sample (n = 49). Additionally, there was a positive correlation between this variant and the level of fasting glucose in nondiabetic individuals (n = 113). This association between TCF7L2 splicing and plasma glucose was independent of the TCF7L2 genotype. Finally, this variant was associated with high levels of serum FFAs during hyperinsulinemia, suggesting impaired insulin action in adipose tissue, whereas no association with insulin secretion or insulin-stimulated whole-body glucose uptake was observed. Our study shows that the short TCF7L2 mRNA variant in subcutaneous fat is regulated by weight loss and is associated with hyperglycemia and impaired insulin action in adipose tissue.

Response to Brosch et al.

We would like to respond to Brosch et al. regarding our manuscript "Expression of the Splicing Factor Gene SFRS10 Is Reduced in Human Obesity and Contributes to Enhanced Lipogenesis" (Pihlajamäki et al., 2011b). Brosch performed RT-PCR in liver samples from 13 lean and 34 obese individuals, finding no differences in SFRS10 or LPIN1 expression. We wish to address points raised by Brosch, including experimental strategy and analysis of human SFRS10 expression.

Expression of the splicing factor gene SFRS10 is reduced in human obesity and contributes to enhanced lipogenesis.

Alternative mRNA splicing provides transcript diversity and may contribute to human disease. We demonstrate that expression of several genes regulating RNA processing is decreased in both liver and skeletal muscle of obese humans. We evaluated a representative splicing factor, SFRS10, downregulated in both obese human liver and muscle and in high-fat-fed mice, and determined metabolic impact of reduced expression. SFRS10-specific siRNA induces lipogenesis and lipid accumulation in hepatocytes. Moreover, Sfrs10 heterozygous mice have increased hepatic lipogenic gene expression, VLDL secretion, and plasma triglycerides. We demonstrate that LPIN1, a key regulator of lipid metabolism, is a splicing target of SFRS10; reduced SFRS10 favors the lipogenic ß isoform of LPIN1. Importantly, LPIN1ß-specific siRNA abolished lipogenic effects of decreased SFRS10 expression. Together, our results indicate that reduced expression of SFRS10, as observed in tissues from obese humans, alters LPIN1 splicing, induces lipogenesis, and therefore contributes to metabolic phenotypes associated with obesity.

ACTH receptor promoter polymorphism associates with severity of premature adrenarche and modulates hypothalamo-pituitary-adrenal axis in children.

The genetic mechanisms underlying the regulation of adrenarche are unknown. The aim of the study was to find out whether ACTH receptor (MC2R) promoter polymorphism associates with premature adrenarche (PA) and its characteristics. DNA samples of 74 prepubertal children with PA and their age- and gender-matched 97 healthy controls were genotyped for the -2 bp T/C diallelic MC2R promoter polymorphism (MC2R -2 T>C) All children were examined clinically, and hormonal measurements after an overnight fast and a low-dose ACTH stimulation test were performed. In controls, the baseline ACTH/cortisol ratio was significantly higher (p = 0.002) in subjects with the polymorphism than in the T/T group indicating decreased ACTH sensitivity. The frequency of the MC2R -2 T>C polymorphism was significantly higher in PA children with premature pubarche than in those with milder signs of PA or in control children (p = 0.04). In children with PA, the polymorphism associated with higher baseline serum dehydroepiandrosterone (p = 0.03), androstenedione (p = 0.02), plasma ACTH (p = 0.03) levels and with lower birth weight (p = 0.02). Our study provides evidence that the MC2R promoter polymorphism modulates the hypothalamo-pituitary-adrenal axis in children and may play a role in altered regulation of adrenarche.