bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chronic Blockade of the Melanocortin 4 Receptor Subtype Leads to Obesity Independently of Neuropeptide Y Action, with No Adverse Effects on the Gonadotropic and Somatotropic Axes**This work was supported by grants from the Swiss National Research Science Foundation (31–39729-93, 31–55732-98, and 32–04912 3–97) and in part by Ferring Pharmaceuticals Ltd. Research Laboratories.
Authors: Raposinho Paula D. , Castillo Einar , D’alleves Violaine , Broqua Pierre , Pralong François P. , Aubert Michel L. 
Published: January 2014

AbstractNeuropeptide Y (NPY) is a powerful orexigenic factor, and αMSH is a melanocortin (MC) peptide that induces satiety by activating the MC4 receptor subtype. Genetic models with disruption of MC4 receptor signaling are associated with obesity. In the present study, a 7-day intracerebroventricular infusion to male rats of either the MC receptor antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly stimulate food and water intake and to markedly increase fat pad mass. Very high plasma leptin levels were found in NPY-treated rats (27.1 ± 1.8 ng/ml compared with 9.9 ± 0.9 ng/ml in SHU9119-treated animals and 2.1 ± 0.2 ng/ml in controls). As expected, NPY infusion induced hypogonadism, characterized by an impressive decrease in seminal vesicle and prostate weights. No such effects were seen with the SHU9119 infusion. Similarly, whereas the somatotropic axis of NPY-treated rats was fully inhibited, this axis was normally activated in the obese SHU9119-treated rats. Chronic infusion of SHU9119 strikingly reduced hypothalamic gene expression for NPY (65.2 ± 3.6% of controls), whereas gene expression for POMC was increased (170 ± 19%). NPY infusion decreased hypothalamic gene expression for both POMC and NPY (70 ± 9% and 75.4 ± 9.5%, respectively). In summary, blockade of the MC4 receptor subtype by SHU9119 was able to generate an obesity syndrome with no apparent side-effects on the reproductive and somatotropic axes. In this situation, it is unlikely that hyperphagia was driven by increased NPY release, because hypothalamic NPY gene expression was markedly reduced, suggesting that hyperphagia mainly resulted from loss of the satiety signal driven by MC peptides. NPY infusion produced hypogonadism and hyposomatotropism in the face of markedly elevated plasma leptin levels and an important reduction in hypothalamic POMC synthesis. In this situation NPY probably acted both by exacerbating food intake through Y receptors and by reducing the satiety signal driven by MC peptides.
Extracellular Calcium-Sensing Receptor Expression and Its Potential Role in Regulating Parathyroid Hormone-Related Peptide Secretion in Human Breast Cancer Cell Lines*
Authors: Sanders Jennifer L. , Chattopadhyay Naibedya , Kifor Olga , Yamaguchi Toru , Butters Robert R. , Brown Edward M. 
Published: January 2014

Abstract
Metastasis of breast cancer to bone occurs with advanced disease and produces substantial morbidity. Secretion of PTH-related peptide (PTHrP) from breast cancer cells is thought to play a key role in osteolytic metastases and is increased by transforming growth factor-β (TGFβ), which is released from resorbed bone. Elevated extracellular calcium (Cao2+) also stimulates PTHrP secretion from various normal and malignant cells, an action that could potentially be mediated by the Cao2+-sensing receptor (CaR) originally cloned from the parathyroid gland. Indeed, we previously showed that both normal breast ductal epithelial cells and primary breast cancers express the CaR. In this study we investigated whether the MCF-7 and MDA-MB-231 human breast cancer cell lines express the CaR and whether CaR agonists modulate PTHrP secretion. Northern blot analysis and RT-PCR revealed bona fide CaR transcripts, and immunocytochemistry and Western analysis with a specific anti-CaR antiserum demonstrated CaR protein expression in both breast cancer cell lines. Furthermore, elevated Cao2+ and the polycationic CaR agonists, neomycin and spermine, stimulated PTHrP secretion dose dependently, with maximal, 2.1- to 2.3-fold stimulation. In addition, pretreatment of MDA-MB-231 cells overnight with TGFβ1 (0.2, 1, or 5 ng/ml) augmented both basal and high Cao2+-stimulated PTHrP secretion. Thus, in PTHrP-secreting breast cancers metastatic to bone, the CaR could potentially participate in a vicious cycle in which PTHrP-induced bone resorption raises the levels of Cao2+ and TGFβ within the bony microenvironment, which then act in concert to evoke further PTHrP release and worsening osteolysis.
Intracellular Calcium and Protein Kinase C Mediate Expression of Receptor Activator of Nuclear Factor-κB Ligand and Osteoprotegerin in Osteoblasts
Authors: Takami Masamichi , Takahashi Naoyuki , Udagawa Nobuyuki , Miyaura Chisato , Suda Koji , Woo Je-Tae , Martin T. John , Nagai Kazuo , Suda Tatsuo 
Published: January 2014

Abstract
Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.
Identification of the Lipophilic Factor Produced by Macrophages That Stimulates Steroidogenesis*
Authors: Nes W. David , Lukyanenko Yevgeniya O. , Jia Zhong Hua , Quideau Stéphane , Howald William N. , Pratum Thomas K. , West Robert R. , Hutson James C. 
Published: January 2014

AbstractMacrophages are known to release a lipophilic factor that stimulates testosterone production by Leydig cells. This macrophage-derived factor (MDF) is thought to be physiologically relevant, because removal of macrophages from the testis results in altered testosterone secretion and reduced fertility. The purpose of the present study was to purify this factor, elucidate its chemical structure, and determine whether it is both present in the testis and acts when injected intratesticularly. Culture media from testicular and peritoneal macrophages were extracted with ether, and the organic phase was sequentially purified on C18, silica, and cyano-HPLC columns. MDF was detected using a rat Leydig cell bioassay, with testosterone secretion being the end point. Purified material and crude ether extracts were analyzed by gas chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy. The time of elution of MDF from both testicular and peritoneal macrophages was identical on all three HPLC columns. A single peak was observed when MDF, obtained from the final HPLC column, was analyzed by gas chromatography. The MS fragmentation pattern of purified material from both peritoneal and testicular macrophages was identical to that of a reference preparation of 25-hydroxycholesterol. Also, the nuclear magnetic resonance spectrum of MDF was similar to that of authentic 25-hydroxycholesterol. When 25-hydroxycholesterol was subjected to the identical purification scheme as MDF, it was found to elute at the same times as MDF on all three columns and elicited activity in the Leydig cell bioassay as expected. Control medium purified identically did not contain 25-hydroxycholesterol or have biological activity. Ether extracts of testis contained 25-hydroxycholesterol, indicating that this compound is present under physiological conditions. Similarly, when 25-hydroxycholesterol was injected into the testis of adult rats, testosterone production was increased within 3 h. Taken together, these data indicate that the lipophilic factor produced by macrophages that stimulates steroidogenesis is 25-hydroxycholesterol.
Effects of Insulin on Prenylation as a Mechanism of Potentially Detrimental Influence of Hyperinsulinemia*
Authors: Draznin Boris , Miles Philip , Kruszynska Yolanta , Olefsky Jerrold , Friedman Jacob , Golovchenko Inga , Stjernholm Richard , Wall Kei , Reitman Marc , Accili Domenico , Cooksey Robert , McClain Donald , Goalstone Marc 
Published: January 2014

AbstractTo investigate the cause and effect relationship between hyperinsulinemia and the increased amounts of farnesylated p21Ras, we performed hyperinsulinemic euglycemic clamps in normal weight volunteers as well as in normal mice and dogs. Insulin infusions significantly raised the amounts of farnesylated p21Ras in the white blood cells of humans, in liver samples of mice and dogs, and in aorta samples of mice. Obese hyperinsulinemic individuals and dogs (made hyperinsulinemic by surgical diversion of the pancreatic outflow from the portal vein into the vena cava) displayed increased amounts of farnesylated p21Ras before the hyperinsulinemic clamps. Infusions of insulin did not alter the already increased levels of farnesylated p21Ras in these experimental models.To further investigate the role of acquired insulin resistance in modulating insulin’s effect on p21Ras prenylation, we induced insulin resistance in rats by glucosamine infusion. Insulin-resistant glucosamine-treated animals displayed significantly increased farnesylated p21Ras in response to insulin infusion compared to that in control saline-treated animals. Transgenic models of insulin resistance (heterozygous insulin receptor substrate-1 knockout mice, A-ZIP/F-1 fatless mice, and animals overexpressing glutamine:fructose-6-phosphate amidotransferase) contained increased amounts of farnesylated p21Ras.We conclude that hyperinsulinemia, either endogenous (a prominent feature of insulin resistance) or produced by infusions of insulin, increases the amounts of farnesylated p21Ras in humans, mice, and dogs. This aspect of insulin action may represent one facet of the molecular mechanism of the potentially detrimental influence of hyperinsulinemia.
Luteinizing Hormone (LH) Drives Diverse Intracellular Calcium Second Messenger Signals in Isolated Porcine Ovarian Thecal Cells: Preferential Recruitment of Intracellular Ca2+ Oscillatory Cells by Higher Concentrations of LH*
Authors: Aguirre C. , Jayes F. C. L. , Veldhuis J. D. 
Published: January 2014

AbstractThe present study examines Ca2+ second messenger signaling driven by LH in isolated porcine thecal cells. To this end, we implemented semiquantitative fluorescent (fura-2) videomicroscopic imaging of single thecal cells in vitro. Stimulation of 388 cells with LH (5 μg/ml) elicited an intracellular Ca2+ ([Ca2+]i) signal in 85± 5.3% of individual thecal cells (n = 11 experiments). Among 337 LH-responsive cells, we identified four predominant temporal modes of [Ca2+]i signaling: 1)[ Ca2+]i oscillations with periodicities of 0.5 to 4.5 min−1 (63 ± 4.5%), 2) a[ Ca2+]i spike followed by a sustained plateau (17 ± 2.6%), 3) a [Ca2+]i spike only (5.8 ± 2.6%); and 4) a [Ca2+]i plateau only (3.8 ± 1.5%). The prevalence, but not the amplitude or frequency, of LH-induced [Ca2+]i oscillations in thecal cells was dependent on the agonist concentration. Reduced availability of extracellular Ca2+ induced by treatment with EGTA or cobaltous chloride did not block the initiation, but reversibly abolished ongoing [Ca2+]i oscillations (72% of cells) or increased the mean[ Ca2+]i interspike periodicity from 1.09± 0.16 to 0.59 ± 0.07 min−1 (P&lt; 0.05). Putative phospholipase C inhibition with U-73122 (10μ m) also abolished or frequency-damped LH-driven[ Ca2+]i oscillations in 95 ± 4.7% of cells. [Ca2+]i oscillations in thecal cells were not abrogated by overnight pretreatment with pertussis toxin. We conclude that 1) thecal cells (unlike earlier findings in granulosa cells) manifest a diverse array of [Ca2+]i signaling responses to LH at the single cell level; 2) LH can dose dependently recruit an increasing number of individually[ Ca2+]i oscillating thecal cells; 3) extracellular Ca2+ is required for LH to sustain (but not initiate) frequent and high amplitude [Ca2+] oscillations in thecal cells; and 4) these signaling actions of LH are mediated via phospholipase C, but not a pertussis-toxin sensitive mechanism. Accordingly, the present data extend the apparent complexity of LH-induced [Ca2+]i second messenger signaling and identify at the single cell level LH’s dose-responsive drive of[ Ca2+]i oscillations in gonadal cells.
Improved Glucose and Lipid Metabolism in Genetically Obese Mice Lacking aP2
Authors: Uysal K. Teoman , Scheja Ludger , Wiesbrock Sarah M. , Bonner-Weir Susan , Hotamisligil Gökhan S. 
Published: January 2014

AbstractAdipocyte fatty acid-binding protein, aP2, is a member of the intracellular fatty acid binding protein family. Previously, studies have shown increased insulin sensitivity in aP2-deficient mice with dietary obesity. Here, we asked whether aP2-related alterations in lipolytic response and insulin production are features of obesity-induced insulin resistance and investigated the effects of aP2-deficiency on glucose homeostasis and lipid metabolism in ob/ob mice, a model of extreme obesity. ob/ob mice homozygous for the aP2 null allele (ob/ob-aP2−/−) became more obese than ob/ob mice as indicated by significantly increased body weight and fat pad size but unaltered body length. However, despite their extreme adiposity, ob/ob-aP2−/− animals were more insulin-sensitive compared with ob/ob controls, as demonstrated by significantly lower plasma glucose and insulin levels and better performance in both insulin and glucose tolerance tests. These animals also showed improvements in dyslipidemia and had lower plasma triglyceride and cholesterol levels. Lipolytic response toβ -adrenergic stimulation and lipolysis-associated insulin secretion was significantly reduced in ob/ob-aP2−/− mice. Interestingly, glucose-stimulated insulin secretion, while virtually abolished in ob/ob controls, was significantly improved in ob/ob-aP2−/− animals. There were no apparent morphological differences in the structure or size of the pancreatic islets between genotypes. Taken together, the data indicate that in obesity, aP2-deficiency not only improves peripheral insulin resistance but also preserves pancreatic β cell function and has beneficial effects on lipid metabolism.
The Peroxisome Proliferator Perfluorodecanoic Acid Inhibits the Peripheral-Type Benzodiazepine Receptor (PBR) Expression and Hormone-Stimulated Mitochondrial Cholesterol Transport and Steroid Formation in Leydig Cells*
Authors: Boujrad Noureddine , Vidic Branislav , Gazouli Maria , Culty Martine , Papadopoulos Vassilios 
Published: January 2014

AbstractThe peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.
Regulation of the GABAA Receptor by Nitric Oxide in Frog Pituitary Melanotrophs*
Authors: Castel Hélène , Jégou Sylvie , Tonon Marie-Christine , Vaudry Hubert 
Published: January 2014

AbstractNitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABAA receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABAA receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS l-arginine (l-Arg, 10−4m) or the NO donor sodium nitroprusside (10−5m) provoked a long-lasting inhibition of the current evoked by GABA (5 × 10−6m). The NOS inhibitor l-nitroarginine (10−5m) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10−5m each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by l-Arg (10−4m) and by the calcium-releasing agent caffeine (10−4m), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10−5m). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10−8–10−7m) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 × 10−7–3 × 10−5m). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABAA receptor function is mediated through activation of the cGMP/PKG pathway.
Stimulation of Intracellular Free Calcium in GH3 Cells byγ 3-Melanocyte-Stimulating Hormone. Involvement of a Novel Melanocortin Receptor?
Authors: Langouche Lies , Roudbaraki Morad , Pals Katrien , Denef Carl 
Published: January 2014

AbstractThe melanocortin (MC) γ3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca2+]i) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that γ3MSH and some analogs increase [Ca2+]i in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved.A dose as low as 1 pm γ3MSH induced an oscillating[ Ca2+]i increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nm. γ2MSH,α MSH, and NDP-αMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca2+]i rise in response to γ3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of γ3MSH showed low potency in increasing [Ca2+]i in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the γ2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca2+]i in GH3 cells, but was about 50 times less potent than γ2- or γ3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present.These data suggest that the GH3 cell line does not mediate the effect of γ3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for γ3MSH.