SUMMARY 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, down-regulated 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 β isoform of LPIN1. Importantly, LPIN1β-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.

The variant of rs12255372 of TCF7L2 was associated with incident type 2 diabetes in the DPS and in a separate population-based cross-sectional study. Impaired insulin secretion is likely to be the main cause for our findings.

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.

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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.

Activins and inhibins are structurally related glycoprotein hormones modulating pituitary FSH secretion and gonadal steroidogenesis. Activins and inhibins are also produced in the adrenal cortex where their physiological role is poorly known. Hormonally active human adrenocortical tumors express and secrete inhibins, while in mice adrenal inhibins may function as tumor suppressors. To clarify the significance of adrenal activins and inhibins we investigated the localization of activin/inhibin signaling components in the adrenal gland, and the effects of activins and inhibins on adrenocortical steroidogenesis and apoptosis.Activin receptor type II/IIB and IB, activin signal transduction proteins Smad2/3, and inhibin receptor betaglycan were expressed throughout the adrenal cortex, whereas Smad4 expression was seen mainly in the zona reticularis and the innermost zona fasciculata as evaluated by immunohistochemistry. Treatment of cultured adrenocortical carcinoma NCI-H295R cells with activin A inhibited steroidogenic acute regulatory protein and 17 -hydroxylase/17,20-lyase mRNA accumulation as evaluated by the Northern blot technique, and decreased cortisol, androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate secretion as determined by specific enzyme immunoassays. Activin A increased apoptosis as measured by a terminal deoxynucleotidyl transferase in situ apoptosis detection method. Inhibins had no effect on steroidogenesis or apoptosis.In summary, activin/inhibin signaling components are coexpressed in the zona reticularis and the innermost zona fasciculata indicating full signaling potential for adrenal activins and inhibins in these layers. Activin inhibits steroidogenic enzyme gene expression and steroid secretion, and increases apoptosis in human adrenocortical cells. Thus, the activin-inhibin system may have a significant role in the regulation of glucocorticoid and androgen production and apoptotic cell death in the human adrenal cortex.

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.

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.