Oleic Acid Modulates Metabolic Substrate Channeling during Glucose-Stimulated Insulin Secretion via NAD(P)H Oxidase

Positive acute effects of fatty acids (FA) on glucose-stimulated insulin secretion (GSIS) and reactive oxygen species (ROS) formation have been reported. However, those studies mainly focused on palmitic acid actions, and reports on oleic acid (OA) are scarce. In this study, the effect of physiological OA levels on beta-cell function and the mechanisms involved were investigated. Analyses of insulin secretion, FA and glucose oxidation, and ROS formation showed that, at high glucose concentration, OA treatment increases GSIS in parallel with increased ROS content. At high glucose, OA oxidation was increased, accompanied by a suppression of glucose oxidation. Using approaches for protein knockdown of FA receptor G protein-coupled receptor 40 (GPR40) and of p47(PHOX), a reduced nicotinamide adenine dinucleotide phosphate [NAD(P) H] oxidase component, we observed that GPR40 does not mediate OA effects on ROS formation and GSIS. However, in p47(PHOX) knockdown islets, OA-induced ROS formation and the inhibitory effect of OA on glucose metabolism was abolished. Similar results were obtained by pharmacological inhibition of protein kinase C, a known activator of NAD(P) H oxidase. Thus, ROS derived from OA metabolism via NAD(P) H oxidase are an inhibitor of glucose oxidation. Put together, these results indicate that OA acts as a modulator of glucose oxidation via ROS derived from its own metabolism in beta-cells. (Endocrinology 152: 3614-3621, 2011)

UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression

Bromati CR, Lellis-Santos C, Yamanaka TS, Nogueira TC, Leonelli M, Caperuto LC, Gorjao R, Leite AR, Anhe GF, Bordin S. UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression. Am J Physiol Regul Integr Comp Physiol 300: R92-R100, 2011. First published November 10, 2010; doi:10.1152/ajpregu.00169.2010.-Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in beta-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2 alpha phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in beta-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in beta-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

Augmentation of insulin secretion by leucine supplementation in malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate kinase/mammalian target protein of rapamycin pathway

A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.

Hypothalamic Actions of Tumor Necrosis Factor alpha Provide the Thermogenic Core for the Wastage Syndrome in Cachexia

TNF alpha is an important mediator of catabolism in cachexia. Most of its effects have been characterized in peripheral tissues, such as skeletal muscle and fat. However, by acting directly in the hypothalamus, TNF alpha can activate thermogenesis and modulate food intake. Here we show that high concentration TNF alpha in the hypothalamus leads to increased O(2) consumption/CO(2) production, increased body temperature, and reduced caloric intake, resulting in loss of body mass. Most of the thermogenic response is produced by beta 3-adrenergic signaling to the brown adipose tissue (BAT), leading to increased BAT relative mass, reduction in BAT lipid quantity, and increased BAT mitochondria density. The expression of proteins involved in BAT thermogenesis, such as beta 3-adrenergic receptor, peroxisomal proliferator-activated receptor-gamma coactivator-1 alpha, and uncoupling protein-1, are increased. In the hypothalamus, TNF alpha produces reductions in neuropeptide Y, agouti gene-related peptide, proopiomelanocortin, and melanin-concentrating hormone, and increases CRH and TRH. The activity of the AMP-activated protein kinase signaling pathway is also decreased in the hypothalamus of TNF alpha-treated rats. Upon intracerebroventricular infliximab treatment, tumor-bearing and septic rats present a significantly increased survival. In addition, the systemic inhibition of beta 3-adrenergic signaling results in a reduced body mass loss and increased survival in septic rats. These data suggest hypothalamic TNF alpha action to be important mediator of the wastage syndrome in cachexia. (Endocrinology 151: 683-694, 2010)

Effects of let-7 microRNA on Cell Growth and Differentiation of Papillary Thyroid Cancer

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy and RET/PTC rearrangements represent key genetic events frequently associated to this cancer, enhancing proliferation and dedifferentiation by activation of the RET/PTC-RAS-BRAF-mitogen-activated protein kinase (MAPK) pathway. Recently, let-7 microRNA was found to reduce RAS levels in lung cancer, acting as a tumor suppressor gene. Here, we report that RET/PTC3 oncogenic activation in PCCL3 rat thyroid cells markedly reduces let-7f expression. Moreover, stable transfection of let-7 microRNA in TPC-1 cells, which harbor RET/PTC1 rearrangement, inhibits MAPK activation. As a result, let-7f was capable of reducing TPC-1 cell growth, and this might be explained, at least in part, by decreased messenger RNA (mRNA) expression of cell cycle stimulators such as MYC and CCND1 (cyclin D1) and increased P21 cell cycle inhibitor mRNA. In addition, let-7 enhanced transcriptional expression of molecular markers of thyroid differentiation such as TITF1 and TG. Thus, reduced expression of let-7f might be an essential molecular event in RET/PTC malignant transformation. Moreover, let-7f effects on thyroid growth and differentiation might attenuate neoplastic process of RET/PTC papillary thyroid oncogenesis through impairment of MAPK signaling pathway activation. This is the first functional demonstration of an association of let-7 with thyroid cancer cell growth and differentiation.

Effect of antidepressants on melatonin metabolite in depressed patients

Antidepressants increase melatonin levels, but it is still unclear whether this effect is related to the improvement of depressive symptoms or to unrelated pharmacological action of antidepressants. To answer this question, the effect of antidepressants on 6-sulphatoxymelatonin (aMT6s), the main melatonin urinary metabolite, was examined in drug-free depressed patients - most of them antidepressant-naive. aMT6s was evaluated in 34 depressed patients, before and after 8 weeks of placebo (n = 12) or antidepressant (n = 22; fluoxetine, duloxetine or Hypericum perforatum). Both groups showed an improvement of depressive symptoms after treatment compared to baseline (Hamilton Depression scores): 17.0 +/- 1.4 vs. 9.0 +/- 2.8, P = 0.007 for placebo, and 18.6 +/- 1.1 vs. 11.8 +/- 1.6, P < 0.001 for antidepressants). After treatment, aMT6s levels increased after antidepressants (P < 0.01), but not after placebo (P > 0.05). As depressive symptoms improved both in patients taking antidepressant and in those taking placebo, but an effect of antidepressants could only be seen in those taking antidepressants, we suggest that melatonin changes after antidepressants are more likely due to a pharmacological action of these drugs on melatonin secretion.

The Autoimmune Regulator (AIRE), Which Is Defective in Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Patients, Is Expressed in Human Epidermal and Follicular Keratinocytes and Associates With the Intermediate Filament Protein Cytokeratin 17

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome, which is caused by mutation of the autoimmune regulator (AIRE) gene, is a highly variable disease characterized by multiple endocrine failure, chronic mucocutaneous candidiasis, and various ectodermal defects. AIRE is a transcriptional regulator classically expressed in medullary thymic epithelial cells, monocytes, macrophages, and dendritic cells. Previous studies have suggested that AIRE can shuttle between the nucleus and cytoplasm of cells, although its cytoplasmic functions are poorly characterized. Through mass spectrometry analysis of proteins co-immunoprecipitating with cytoplasmic AIRE, we identified a novel association of AIRE with the intermediate filament protein cytokeratin 17 (K17) in the THP-1 monocyte cell line. We confirmed AIRE expression in HaCaT epidermal keratinocytes, as well as its interaction with K17. Confocal microscopy of human fetal and adult scalp hair follicles demonstrated a cytoplasmic pattern of AIRE staining that moderately colocalized with K17. The cytoplasmic association of AIRE with the intermediate filament network in human epidermal and follicular keratinocytes may provide a new path to understanding the ectodermal abnormalities associated with the APECED syndrome. (Am J Pathol 2011, 178:983-988; DOI: 10.1016/j.ajpath.2010.12.007)

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21-hydroxylase deficiency

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21-hydroxylase deficiency. Fifty-four patients from 46 families (36 female, 18 male; mean age 10.8 years) with different clinical manifestations (31 salt-wasting; 23 simple-virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme-linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX monomodular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well-protected gene segment along the evolutionary process.

Modulation of Bone Morphogenetic Protein-9 Expression and Processing by Insulin, Glucose, and Glucocorticoids: Possible Candidate for Hepatic Insulin-Sensitizing Substance

Bone morphogenetic protein 9 (BMP-9), a member of the TGF-beta superfamily predominantly expressed in nonparenchymal liver cells, has been demonstrated to improve glucose homeostasis in diabetic mice. Along with this therapeutic effect, BMP-9 was proposed as a candidate for the hepatic insulin-sensitizing substance ( HISS). Whether BMP-9 plays a physiological role in glucose homeostasis is still unknown. In the present study, we show that BMP-9 expression and processing is severely reduced in the liver of insulin-resistant rats. BMP-9 expression and processing was directly stimulated by in situ exposition of the liver to the combination of glucose and insulin and oral glucose in overnight fasted rats. Additionally, prolonged fasting ( 72 h) abrogated refeeding-induced BMP-9 expression and processing. Previous exposition to dexamethasone, a known inductor of insulin resistance, reduced BMP-9 processing stimulated by the combination of insulin and glucose. Finally, we show that neutralization of BMP-9 with an anti-BMP-9 antibody induces glucose intolerance and insulin resistance in 12-h fasted rats. Collectively, the present results demonstrate that BMP-9 plays an important role in the control of glucose homeostasis of the normal rat. Additionally, BMP-9 is expressed and processed in an HISS-like fashion, which is impaired in the presence of insulin resistance. BMP-9 regulation according to the feeding status and the presence of diabetogenic factors reinforces the hypothesis that BMP-9 might exert the role of HISS in glucose homeostasis physiology. ( Endocrinology 149: 6326-6335, 2008)

Glucocorticoids in Vivo Induce Both Insulin Hypersecretion and Enhanced Glucose Sensitivity of Stimulus-Secretion Coupling in Isolated Rat Islets

Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca(2+) signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca(2+) signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally, we explored the status of Ca(2+)-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase C alpha as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the beta-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity. (Endocrinology 151: 85-95, 2010)

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. (C) 2009 Elsevier Inc. All rights reserved.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.

Analysis of Agonist and Antagonist Effects on Thyroid Hormone Receptor Conformation by Hydrogen/Deuterium Exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T(3)) or antagonist (NH(3)). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, C-terminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T(3), but not NH(3), increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T(3) but not NH(3.) We present data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011)