bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Adenylyl Cyclase by Caveolin Peptides*
Authors: Toya Yoshiyuki , Schwencke Carsten , Couet Jacques , Lisanti Michael P. , Ishikawa Yoshihiro
Published: January 2014

Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrate ATP. Studies with various truncations and point mutations of this peptide revealed that a minimum of 16 amino acid residues and intact aromatic residues are important for the inhibitory effect. The potency of inhibition was greater when adenylyl cyclase was in stimulated condition vs. basal condition. Thus, caveolin may be another cellular component that regulates adenylyl cyclase catalytic activity. Our results also suggest that the caveolin peptide may be used as an isoform-selective inhibitor of adenylyl cyclase.
Iodothyronine Deiodinase Activities in Fetal Rat Tissues at Several Levels of Iodine Deficiency: A Role for the Skin in 3,5,3′-Triiodothyronine Economy?*
Authors: Schröder-van der Elst Janny P. , van der Heide Daan , Morreale de Escobar Gabriella , Obregón María Jesús
Published: January 2014

Iodothyronine deiodinases, types I, II, and III (D1, D2, and D3) activities were measured in tissues of fetal rats, at 18 and 21 days of gestation, at several levels of iodine deficiency (ID): mild ID diet (MID) and moderately severe ID, MID + 0.005% perchlorate (MID+P). D2 was present in fetal skin, increased between days 18 and 21, and also in MID and MID+P. In skin, D3 increased during ID at day 18, whereas there was a decrease at day 21. Skin T4 decreased in MID and MID+P, showing an inverse relationship with D2. Skin T3 decreased at day 18 in MID and MID+P but increased at day 21, probably because of the increased D2 and decreased D3, maintaining T3 concentrations. No effect of ID was observed on hepatic D1. D2 increased in brain and brown adipose tissue at day 21 in MID+P. No changes were found in maternal placental D2 and D3, but D2 and D3 increased in the fetal placenta at day 18 in MID+P.

A higher level of D2 is present in fetal skin than in the brain. As the activity is increased, in even mild ID (and already at 18 days) it can be concluded that skin D2 is likely to be of considerable physiological importance, at least for fetal thyroid hormone economy, by contributing to the intracellular T3 content of the skin and, possibly, to the plasma T3.
Differential Responses of Estrogen Target Tissues in Rats Including Bone to Clomiphene, Enclomiphene, and Zuclomiphene*
Authors: Turner Russell T. , Evans Glenda L. , Sluka James P. , Adrian M. D. , Bryant Henry U. , Turner Charles H. , Sato Masahiko
Published: January 2014

The substituted triphenylethylene antiestrogen clomiphene (CLO) prevents cancellous bone loss in ovariectomized (OVX’d) rats. However, CLO is a mixture of two stereoisomers, enclomiphene (ENC) and zuclomiphene (ZUC), which have distinctly different activities on reproductive tissues and tumor cells. The purpose of the present dose response study was to determine the effects of ENC and ZUC on nonreproductive estrogen target tissues. These studies were performed in 7-month-old female rats with moderate cancellous osteopenia that was established by ovariectomizing rats 1 month before initiating treatment. OVX resulted in increases in body weight, serum cholesterol, endocortical resorption, and indices of cancellous bone turnover, as well as decreases in uterine weight, uterine epithelial cell height, bone mineral density, bone strength, and cancellous bone area. Estrogen treatment for 3 months restored body weight, uterine histology, dynamic bone measurements, and osteoblast and osteoclast surfaces in OVX’d rats to the levels found in the age-matched sham-operated rats. In contrast, estrogen only partially restored cancellous bone volume and uterine weight, and it reduced serum cholesterol to subnormal values. CLO was a weak estrogen agonist on uterine measurements and a much more potent agonist on body weight, serum cholesterol, and dynamic bone measurements. CLO increased trabecular thickness in osteopenic rats and was the most effective treatment in improving cancellous bone volume and architecture. ZUC was a potent estrogen agonist on all tissues investigated and had dose-dependent effects. In contrast, ENC had dose-dependent effects on most measurements similar to CLO and decreased the uterotrophic effects of ZUC. It is concluded that ENC antagonizes the estrogenic effects of ZUC on the uterus but that the beneficial effects of CLO on nonreproductive tissues in OVX’d rats is conferred by both isomers. Furthermore, the combined actions of the two isomers on bone volume and architecture were more beneficial than either isomer given alone.
Role of the Src Homology 2 (SH2) Domain and C-Terminus Tyrosine Phosphorylation Sites of SH2-Containing Inositol Phosphatase (SHIP) in the Regulation of Insulin-Induced Mitogenesis1
Authors: Wada Tsutomu , Sasaoka Toshiyasu , Ishiki Manabu , Hori Hiroyuki , Haruta Tetsuro , Ishihara Hajime , Kobayashi Masashi
Published: January 2014

To examine the role of SHIP in insulin-induced mitogenic signaling, we used a truncated SHIP lacking the SH2 domain (ΔSH2-SHIP) and a Y917/1020F-SHIP (2F-SHIP) in which two tyrosines contributing to Shc binding were mutated to phenylalanine. Wild-type (WT)-, ΔSH2-, and 2F-SHIP were transiently transfected into Rat1 fibroblasts overexpressing insulin receptors (HIRc). Insulin-stimulated tyrosine phosphorylation of WT-SHIP and ΔSH2-SHIP, whereas tyrosine phosphorylation of 2F-SHIP was not detectable, indicating that 917/1020-Tyr are key phosphorylation sites on SHIP. Although SHIP can bind via its 917/1020-Tyr residues and SH2 domain to Shc PTB domain and 317-Tyr residue, respectively, insulin-induced SHIP association with Shc was more greatly decreased in 2F-SHIP cells than that inΔ SH2-SHIP cells. Insulin stimulation of Shc association with Grb2, which is important for p21ras-MAP kinase activation, was decreased by overexpression of WT- and 2F-SHIP. Importantly, insulin-induced Shc·Grb2 association was not detectably reduced in ΔSH2-SHIP cells. In accordance with the extent of Shc association with Grb2, insulin-induced MAP kinase activation was relatively decreased in both WT-SHIP and 2F-SHIP cells, but not in ΔSH2-SHIP cells. To examine the functional role of SHIP in insulin’s biological action, insulin-induced mitogenesis was compared among these transfected cells. Insulin stimulation of thymidine incorporation and bromodeoxyuridine incorporation was decreased in WT-SHIP cells compared with that of control HIRc cells. Expression of 2F-SHIP also significantly reduced insulin-induced mitogenesis, whereas it was only slightly affected by overexpression of ΔSH2-SHIP. Furthermore, the reduction of insulin-induced mitogenesis in WT-SHIP cells was partly compensated by coexpression of Shc. These results indicate that SHIP plays a negative regulatory role in insulin-induced mitogenesis and that the SH2 domain of SHIP is important for its negative regulatory function.
Scavenger Receptor Class B Type I in the Rat Ovary: Possible Role in High Density Lipoprotein Cholesterol Uptake and in the Recognition of Apoptotic Granulosa Cells*
Authors: Svensson Per-Arne , Johnson Magnus S. C. , Ling Charlotte , Carlsson Lena M. S. , Billig Håkan , Carlsson Björn
Published: January 2014

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of high density lipoprotein cholesterol. SR-BI is expressed at high levels in the ovary, indicating that it plays a role in the delivery of cholesterol as substrate for steroid hormone production. However, SR-BI also binds anionic phospholipids with high affinity and could therefore be involved in the recognition of apoptotic cells. In this study we have characterized the expression of SR-BI in rat ovarian follicles undergoing atresia. Atretic follicles with cells undergoing apoptosis were identified by in situ DNA end labeling, and SR-BI expression was determined by in situ hybridization and immunohistochemistry. SR-BI was expressed in thecal cells at all stages of follicular development, including atretic follicles, and in corpus luteum. Isolated apoptotic granulosa cells (but not viable granulosa cells) bound annexin V, indicating that they display anionic phospholipids on the cell surface. Transfection of COS-7 cells with an expression vector carrying the rat SR-BI complementary DNA resulted in increased binding to apoptotic granulosa cells (46 ± 2% of the SR-BI-expressing cells bound at least one granulosa cell compared with 24 ± 3% for the mock-transfected cells; P < 0.0001), whereas the binding to viable granulosa cells was unchanged. Apoptotic granulosa cells also bound to isolated thecal shells. We conclude that thecal cells of both nonatretic and atretic follicles express SR-BI. The location of SR-BI expression in the ovary supports a role of this receptor in the uptake of high density lipoprotein cholesterol. In addition, our data suggest that SR-BI mediates the recognition of apoptotic granulosa cells by the surrounding thecal cells and that it therefore may play a role in the remodeling of atretic follicles to secondary interstitial cells.
Genetic Bases of Estrogen-Induced Pituitary Growth in an Intercross between the ACI and Copenhagen Rat Strains: Dominant Mendelian Inheritance of the ACI Phenotype*
Authors: Spady Thomas J. , Pennington Karen L. , McComb Rodney D. , Shull James D.
Published: January 2014

Estrogens stimulate cell proliferation in a variety of tissues and are widely believed to be contributing factors in the etiology of certain cancer types in humans. The molecular mechanisms through which estrogens regulate cell proliferation are currently unknown. Estrogens stimulate proliferation of the PRL-producing lactotroph of the rat anterior pituitary gland and induce development of PRL-producing pituitary tumors in several inbred rat strains. Therefore, the lactotroph provides a well defined model for identifying the mechanisms through which estrogens regulate cell proliferation and/or survival. Data from our laboratory and others indicate that the relative sensitivity to the pituitary growth-promoting actions of estrogens is highly strain specific. This allows genetics-based approaches to be used to address the molecular mechanisms through which estrogens stimulate lactotroph proliferation and induce pituitary tumor development. In the present study we have examined the ability of diethylstilbestrol (DES) to induce pituitary growth in the genetically related AxC-Irish (ACI) and Copenhagen (COP) strains and their derived F1, F2, and backcross progeny. The data presented herein indicate that the anterior pituitary gland of the ACI strain displays approximately a 2-fold greater growth response to administered DES than does the pituitary gland of the COP strain. The average pituitary weight in male ACI rats was increased from 9.2± 0.2 mg (mean ± sd) in untreated rats to 63.7± 12.6 mg in rats treated with DES for 12 weeks, whereas in male COP rats, DES increased pituitary weight from 12.7 ± 0.9 to 38.1± 8.2 mg. The ACI phenotype was inherited in the F1, F2, and backcross progeny of an ACI × COP intercross as a dominant genetic trait, and the approximately 30 mg of additional pituitary growth displayed by the DES-treated ACI rat, relative to that of the treated COP rat, appeared to result from the actions of a single locus. Moreover, in F1 progeny from an ACI × Brown Norway intercross, the ACI phenotype was inherited as a dominant or incompletely dominant genetic trait. These data, when compared with findings of previous studies using the Fischer 344 rat strain, provide the first indication that distinct genetic pathways contribute to regulation of estrogen-induced pituitary growth and induction of PRL-producing pituitary tumors in the ACI and F344 rat strains.
Tumor Necrosis Factor-α Stimulates Lactate Dehydrogenase A Expression in Porcine Cultured Sertoli Cells: Mechanisms of Action
Authors: Boussouar Fayçal , Grataroli Renée , Ji Jingwei , Benahmed Mohamed
Published: January 2014

In the present study, we investigated the regulatory action of tumor necrosis factor-α (TNFα) on lactate dehydrogenase A (LDH A), a key enzyme involved in lactate production. To this end, use was made of a primary culture system of porcine testicular Sertoli cells. TNFα stimulated LDH A messenger RNA (mRNA) expression in a dose (ED50 = 2.5 ng/ml; 0.1 nm TNFα)-dependent manner. This stimulatory effect was time dependent, with an effect detected after 6 h of TNFα treatment and maximal after 48 h of exposition (5-fold; P < 0.001). The direct effect of TNFα on LDH A mRNA could not be accounted for by an increase in mRNA stability (half-life = 9 h), but was probably due to an increase in LDH A gene transcription. Inhibitors of protein synthesis (cycloheximide), gene transcription (actinomycin D and dichlorobenzimidazole riboside), tyrosine kinase (genistein), and protein kinase C (bisindolylmaleimide) abrogated completely (actinomycin D, dichlorobenzimidazole riboside, cycloheximide, and genistein) or partially (bisindolylmaleimide) TNFα-induced LDH A mRNA expression. These observations suggest that the stimulatory effect of TNFα on LDH A mRNA expression requires protein synthesis and may involve a protein tyrosine kinase and protein kinase C. In addition, we report that LDH A mRNA levels were increased in Sertoli cells treated with FSH. However, although the cytokine enhances LDH A mRNA levels through increased gene transcription, the hormone exerts its stimulatory action through an increase in LDH A mRNA stability. The regulatory actions of the cytokine and the hormone on LDH A mRNA levels and therefore on lactate production may operate in the context of the metabolic cooperation between Sertoli and postmeiotic germ cells in the seminiferous tubules.
Estrogen Receptor-α Detected on the Plasma Membrane of Aldehyde-Fixed GH3/B6/F10 Rat Pituitary Tumor Cells by Enzyme-Linked Immunocytochemistry*
Authors: Norfleet Andrea M. , Thomas Mary L. , Gametchu Bahiru , Watson Cheryl S.
Published: January 2014

A population of estrogen receptor-α (ERα) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ERα at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ERα. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ERα Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity of staining for ERα was confirmed by three approaches: first, treatment with an antisense oligodeoxynucleotide to nuclear ERα mRNA reduced immunolabeling of both membrane and nuclear ERα; second, labeling by two Abs raised against different ERα oligopeptides was neutralized by competing peptide; third, six Abs (ER21, H226, R4, H222, MC20, and C542) that recognize unique epitopes on rodent ERα produced immunolabeling, but neither primate-specific ERα Ab nor Ab to ERβ caused staining. In addition to demonstrating the plasma membrane ERα in GH3/B6/F10 cells, this method should be applicable to other cell types that exhibit nongenomic responses to estrogen or other steroid hormones.
Time- and Dose-Related Interactions between Glucocorticoid and Cyclic Adenosine 3′,5′-Monophosphate on CCAAT/Enhancer-Binding Protein-Dependent Insulin-Like Growth Factor I Expression by Osteoblasts1
Authors: McCarthy Thomas L. , Ji Changhua , Chen Yun , Kim Kenneth , Centrella Michael
Published: January 2014

Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-δ (C/EBPδ) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPδ and C/EBPβ expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPδ and C/EBPβ on IGF-I expression by osteoblasts.
Compartmentalization and Insulin-Induced Translocations of Insulin Receptor Substrates, Phosphatidylinositol 3-Kinase, and Protein Kinase B in Rat Liver**This work was supported by grants from the Medical Research Council and from the National Cancer Institute of Canada, and by the Cleghorn Fund at McGill University and the M. Pollack Foundation of Montreal.
Authors: Balbis Alejandro , Baquiran Gerry , Bergeron John J. M. , Posner Barry I.
Published: January 2014

Physiological doses of insulin in rats resulted in a rapid redistribution of key signaling proteins between subcellular compartments in rat liver. In plasma membranes (PM) and microsomes, insulin induced a rapid decrease in insulin receptor substrate-1/2 (IRS1/2) within 30 sec and an increase in these proteins in endosomes (EN) and cytosol. The level of p85 in PM increased 2.3-fold at 30 sec after insulin stimulation followed by a decrease at 2 min. In this interval, 60–85% and 10–20% of p85 in PM was associated with IRS1 and IRS2, respectively. Thus, in PM, IRS1/2 accounts for almost all of the protein involved in phosphatidylinositol 3-kinase activation. In ENs insulin induced a maximal increase of 40% in p85 recruitment. As in PM, almost all p85 was associated with IRS1/2. The greater level of p85 recruitment to PM was associated with a higher level of insulin-induced recruitment of Akt1 to this compartment (4.0-fold in PM vs. 2.4-fold in EN). There was a close correlation between Akt1 activity and Akt1 phosphorylation at Thr308 and Ser473 in PM and cytosol. However, in ENs the level of Akt1 activity per unit of phosphorylated Akt1 was significantly greater than in PM, indicating that in addition to phosphorylation, another factor(s) modulates Akt1 activation by insulin in rat liver. Our results demonstrate that activation of the insulin receptor kinase and modulation of key components of the insulin signaling cascade occur at the cell surface and within the endosomal system. These data provide further support for the role of the endocytic process in cell signaling.
Nuclear Receptor Coactivators Facilitate Vitamin D Receptor Homodimer Action on Direct Repeat Hormone Response Elements
Authors: Takeshita Akira , Ozawa Yasunori , Chin William W.
Published: January 2014

Vitamin D receptor (VDR) is a ligand-dependent transcription factor that regulates target gene expression. Although VDR forms stable heterodimer complex with retinoid X receptors (RXRs) on vitamin D-response elements (VDREs), it is still not clear whether VDR/RXR heterodimers are the only VDR complexes responsible for vitamin D-mediated gene transcription. In this report, we analyzed the effect of nuclear receptor coactivators (SRC-1 and TRAM-1) on VDR homodimer and VDR/RXR heterodimer formation by electrophoretic mobility shift assay. We found that VDR forms stable homodimers after interaction with the coactivators on a VDRE (DR+3). Of particular note, DR+4 and DR+5 hormone-response elements (HREs) may also support such interactions. Cotransfection experiments revealed further that the coactivators enhance ligand-induced VDR transcription on these elements. Our studies suggest the important role of VDR homodimers, in addition to VDR/RXR heterodimers, in vitamin D-induced transactivation. Thus, specific coactivator-VDR interactions on HREs may determine target gene transactivation.
Overexpression of Glutamine:Fructose-6-Phosphate Amidotransferase in Rat-1 Fibroblasts Enhances Glucose-Mediated Glycogen Accumulation via Suppression of Glycogen Phosphorylase Activity*
Authors: Crook Errol D. , Crenshaw Gregory , Veerababu Geddati , Singh Lalit P.
Published: January 2014

The hexosamine biosynthesis pathway (HBP) mediates many of the adverse effects of excess glucose. We have shown previously that glucose down-regulates basal and insulin-stimulated glycogen synthase (GS) activity. Overexpression of the rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), mimics these effects of high glucose and renders the cells more sensitive to glucose. Here we examine the role of the HBP in regulating cellular glycogen content. Glycogen content and glycogen phosphorylase (GP) activity were determined in Rat-1 fibroblasts that overexpress GFA. In both GFA and controls there was a dose-dependent increase in glycogen content (∼8-fold) in cells cultured in increasing glucose concentrations (1–20 mm). There was a shift to the left in the glucose dose-response curve for glycogen content in GFA cells (ED50 for glycogen content = 5.80 ± 1.05 vs. 8.84 ± 0.87 mm glucose, GFA vs. control). Inhibition of GFA reduced glycogen content by 28.4% in controls cultured in 20 mm glucose. In a dose-dependent manner, glucose resulted in a more than 35% decrease in GP activity in controls. GP activity in GFA cells was suppressed compared with that in controls, and there was no glucose-induced down-regulation of GP activity. Glucosamine and uridine mimicked the effects of glucose on glycogen content and GP activity. However, chronic overexpression of GFA is a unique model of hexosamine excess, as culturing control cells in low dose glucosamine (0.1–0.25 mm) did not suppress GP activity and did not eliminate the glucose-mediated down-regulation of GP activity. We conclude that increased flux through the HBP results in enhanced glycogen accumulation due to suppression of GP activity. These results demonstrate that the HBP is an important regulator of cellular glucose metabolism and supports its role as a cellular glucose/satiety sensor.
The Serum- and Glucocorticoid-Induced Kinase Is a Physiological Mediator of Aldosterone Action*
Authors: Bhargava Aditi , Fullerton Meryl J. , Myles Kathy , Purdy Timothy M. , Funder John W. , Pearce David , Cole Timothy J.
Published: January 2014

Aldosterone plays a major role in regulating sodium and potassium flux in epithelial tissues such as kidney and colon. Recent evidence suggests that serum- and glucocorticoid-regulated kinase (SGK) is induced by aldosterone and acts as a key mediator of aldosterone action in epithelial tissues. Induction of SGK messenger RNA (mRNA) has previously been shown within 30 min of addition of supraphysiological doses of aldosterone to Xenopus A6 cells and within 4 h in rat kidney in vivo. In this study we determined the time course of SGK induction, at doses of aldosterone in the physiological range, in rat kidney and colon, using Northern and Western blot analyses and in situ hybridization and determined concurrent changes in urinary sodium and potassium excretion by Kagawa bioassay. On Northern blot analysis, SGK mRNA levels were significantly elevated in both kidney and colon 60 min after the injection of aldosterone. SGK protein in late distal colon was significantly elevated 2 and 4 h after aldosterone treatment. In situ hybridization showed SGK mRNA to be induced in renal collecting ducts and distal tubular elements in both cortex and medulla by doses of aldosterone of 0.1 μg/100 g BW or more within 30 min of steroid treatment. Significant changes in urinary composition were similarly seen with an aldosterone dose of 0.1 μg/100 g BW from 90 min after aldosterone injection. The early onset of SGK induction in kidney and colon and the correlation with urinary changes in terms of both time course and dose response suggest that SGK plays an important role in mediating the effects of aldosterone on sodium homeostasis in vivo.
Catabolic Effects of Continuous Human PTH (1–38) in Vivo Is Associated with Sustained Stimulation of RANKL and Inhibition of Osteoprotegerin and Gene-Associated Bone Formation
Authors: Ma Yanfei L. , Cain Rick L. , Halladay David L. , Yang Xuhao , Zeng Qingqang , Miles Rebecca R. , Chandrasekhar Srinivasan , Martin T. John , Onyia Jude E.
Published: January 2014

Continuous infusion of PTH in vivo results in active bone resorption. To investigate the molecular basis of the catabolic effect of PTH in vivo, we evaluated the role of OPG and RANKL, which are known to influence osteoclast formation and function. Weanling rats fed a calcium-free diet were parathyroidectomized and infused with PTH via an Alzet pump to examine: 1) the changes of serum-ionized calcium and osteoclast number, 2) the expression of OPG/RANKL mRNA and protein, and 3) the expression of osteoblast phenotype bone formation-associated genes such as osteoblast specific transcription factor, osteocalcin, bone sialoprotein, and type I collagen. PTH (1–38) (0.01–20 μg/100 g) continuous infusion for 1–24 h resulted in a dose-dependent increase in serum-ionized calcium in parathyroidectomized rats and a corresponding dose-dependent increase in osteoclast number, indicating an increased bone resorption. At 20 μg/100 g PTH dose level, serum-ionized calcium was 2.1-fold of the vehicle control and not different from the Sham-parathyroidectomized rats, and osteoclast number was 3-fold of the vehicle control and 1.7-fold of the Sham-parathyroidectomized rats. In the distal femur, RANKL mRNA expression was increased (27-fold) and OPG mRNA expression was decreased (4.6-fold). The changes in RANKL and OPG mRNA levels were rapid (as early as 1 h), dose dependent, and sustained over a 24-h period that was examined. Immunohistochemical evaluation of bone sections confirmed that OPG level was reduced in proximal tibial metaphysis upon PTH infusion. Circulating OPG protein level was also decreased by 32% when compared with the parathyroidectomized control. The expression of genes that mark the osteoblast phenotype was significantly decreased [osteoblast specific transcription factor (2.3-fold), osteocalcin (3-fold), bone sialoprotein (2.8-fold), and type I collagen (5-fold)]. These results suggest that the catabolic effect of PTH infusion in vivo in this well-established resorption model is associated with a reciprocal expression of OPG/RANKL and a co-ordinate decrease in the expression of bone formation-related genes. We propose that the rapid and sustained increase in RANKL and decrease in OPG initiate maintain and favor the cascade of events in the differentiation/recruitment and activation of osteoclasts.
In Vivo Evidence for Stimulation of Placental, Myometrial, and Endometrial Prostaglandin G/H Synthase 2 by Fetal Cortisol Replacement after Fetal Adrenalectomy
Authors: Wu W. X. , Ma X. H. , Unno N. , Nathanielsz P. W.
Published: January 2014

Fetal glucocorticoid-induced premature labor in sheep is an established model of premature labor. However, the pathways by which fetal cortisol triggers subsequent maternal endocrine changes, including enhanced PG synthesis, leading to labor are unclear. The current study was undertaken to determine whether cortisol administration to adrenalectomized fetuses to clamp fetal cortisol at levels present early in the late gestation rise, which are inadequate to produce labor, can stimulate placental, myometrial, and endometrial prostaglandin G/H synthase 2 mRNA and protein expression.

At 109–113 d gestation, fetal sheep adrenals were removed (n = 8), or sham surgery was performed (n = 4). From d 6 postadrenalectomy, maternal and fetal plasma cortisol were determined daily by RIA. From d 7 postadrenalectomy, cortisol (4 μg/min) was continuously infused iv to four adrenalectomized fetuses. Endometrium, myometrium, and placentome were collected from all three groups of ewes (n = 4 for each group), and total RNA and proteins were extracted from each intrauterine tissue and analyzed by Northern and Western for prostaglandin G/H synthase 2 mRNA and protein. P45017α hydroxylase mRNA was analyzed in the placentome by Northern blot. Data were analyzed by ANOVA.

Plasma cortisol levels remained low in sham-operated and adrenalectomized fetus, whereas during cortisol infusion to adrenalectomized and cortisol-treated fetuses, plasma cortisol increased to the late gestation level. After adrenalectomy, prostaglandin G/H synthase 2 did not change in any tissue studied. Fetal plasma cortisol replacement to late gestation levels increased prostaglandin G/H synthase 2 to levels similar to term levels in all three tissues. PGHS1 mRNA and protein did not change in any group studied. There was a minimal increase in P45017α hydroxylase mRNA in the placentome in the adrenalectomized and cortisol-treated group. Cortisol- induced labor further increased P45017α hydroxylase mRNA in the placentome compared with that in adrenalectomized and cortisol-treated animals.

These data provide evidence for in vivo cortisol up-regulation of prostaglandin G/H synthase 2, but not PGHS1, in late gestation in the ovine placentome, myometrium, and endometrium. As stimulation of the estrogen biosynthetic pathway was minimal in the adrenalectomized and cortisol-treated group, these data provide support for the concept that cortisol has a direct effect on prostaglandin G/H synthase 2 expression in addition to its classical indirect pathway on prostaglandin G/H synthase 2 as a result of estrogen synthesis.
Characterization of New Selective Somatostatin Receptor Subtype-2 (sst2) Antagonists, BIM-23627 and BIM-23454. Effects of BIM-23627 on GH Release in Anesthetized Male Rats after Short-Term High-Dose Dexamethasone Treatment
Authors: Tulipano G. , Soldi D. , Bagnasco M. , Culler M. D. , Taylor J. E. , Cocchi D. , Giustina A.
Published: January 2014

We here report a pharmacological characterization of two new somatostatin (SS) receptor subtype-2 (sst2) selective antagonists by evaluating their GH-releasing activity when administered, by different routes, in anesthetized adult rats and in freely moving 10-d-old rats. Moreover, we describe the effect of these SS antagonists on the GH response to GHRH after short-term high-dose dexamethasone (DEX) treatment in young male rats. BIM-23454 and BIM-23627, given iv, were able to counteract the SS-induced inhibition of GH secretion occurring after urethane anesthesia in a dose-dependent manner. In DEX-treated animals, the GH response to GHRH was partially blunted (5-min peak values, 270 ± 50 ng/ml in saline-treated vs. 160 ± 10 ng/ml in DEX-treated, P < 0.05); however, the simultaneous administration of BIM-23627 (0.2 mg/kg, iv) restored higher amplitude GH pulse, leading to a significantly higher overall mean GH response (area under the curve, 4200 ± 120 ng/ml/30 min vs. 2800 ± 100 ng/ml/30 min after GHRH alone; P < 0.05). The SS antagonists showed a reduced GH-releasing effect when administered sc or ip, likely attributable to decreased bioavailability, as compared with the iv route. SS antagonist administration also increased plasma glucagon, insulin, and glucose levels. Based on prior reports that sst2 tonically suppresses glucagon secretion, the antagonist most likely increased glucagon secretion from the pancreatic α-cells, with resultant increases in plasma glucose and then insulin.
The Glucocorticoid Receptor Represses the Positive Autoregulation of the Trout Estrogen Receptor Gene by Preventing the Enhancer Effect of a C/EBPβ-Like Protein
Authors: Lethimonier Christèle , Flouriot Gilles , Kah Olivier , Ducouret Bernadette
Published: January 2014

Stress and cortisol are known to have negative effects on vitellogenesis in oviparous species. This provides a physiological context in which to explore in more detail the molecular mechanisms involved in transcriptional interferences between two steroids receptors, the estradiol receptor (ER) and the glucocorticoid receptor (GR). We have previously shown that the cortisol inhibitory effect on rainbow trout (rt) vitellogenesis is the result of a repression of the estradiol-induced ER-positive autoregulation by activated GR. In the present study, we demonstrate that the GR repression involves a proximal region of the rtER promoter that is unable to bind GR. This inhibition is counteracted in part by the orphan receptor COUP-TF1 that has been previously shown to cooperate with ERs on the same promoter. A detailed analysis allowed us to identify a C/EBPβ-like protein that is implicated in both the maximal stimulatory effect of estradiol and the GR repression. Indeed, GR, through its DNA-binding domain, suppresses the binding of C/EBPβ on the rtER promoter by protein-protein interactions and thereby prevents the enhancer effect of this transcription factor.
Hypercortisolemia Is Associated with Severity of Bone Loss and Depression in Hypothalamic Amenorrhea and Anorexia Nervosa
Authors: Lawson Elizabeth A. , Donoho Daniel , Miller Karen K. , Misra Madhusmita , Meenaghan Erinne , Lydecker Janet , Wexler Tamara , Herzog David B. , Klibanski Anne
Published: January 2014

Context:

Anorexia nervosa (AN) and functional hypothalamic amenorrhea (HA) are associated with low bone density, anxiety, and depression. Women with AN and HA have elevated cortisol levels. Significant hypercortisolemia, as in Cushing’s disease, causes bone loss. It is unknown whether anxiety and depression and/or cortisol dysregulation contribute to low bone density in AN or HA.

Objective:

Ourobjective was to investigate whether hypercortisolemia is associated with bone loss and mood disturbance in women with HA and AN.

Design and Setting:

We conducted a cross-sectional study in a clinical research center.

Participants:

We studied 52 women [21 healthy controls (HC), 13 normal-weight women with functional HA, and 18 amenorrheic women with AN].

Outcome Measures:

Serum samples were measured every 20 min for 12 h overnight and pooled for average cortisol levels. Bone mineral density (BMD) was assessed by dual-energy x-ray absorptiometry (DXA) at anteroposterior and lateral spine and hip. Hamilton Rating Scales for Anxiety (HAM-A) and Depression (HAM-D) were administered.

Results:

BMD was lower in AN and HA than HC at all sites and lower in AN than HA at the spine. On the HAM-D and HAM-A, AN scored higher than HA, and HA scored higher than HC. Cortisol levels were highest in AN, intermediate in HA, and lowest in HC. HAM-A and HAM-D scores were associated with decreased BMD. Cortisol levels were positively associated with HAM-A and HAM-D scores and negatively associated with BMD.

Conclusions:

Hypercortisolemia is a potential mediator of bone loss and mood disturbance in these disorders.
Dipeptidyl-Peptidase-IV Inhibition Augments Postprandial Lipid Mobilization and Oxidation in Type 2 Diabetic Patients
Authors: Boschmann Michael , Engeli Stefan , Dobberstein Kerstin , Budziarek Petra , Strauss Anke , Boehnke Jana , Sweep Fred C.G.J. , Luft Friedrich C. , He YanLing , Foley James E. , Jordan Jens
Published: January 2014

Context: Dipeptidyl-peptidase-IV (DPP-4) inhibition increases endogenous GLP-1 activity resulting in improved glycemic control in patients with type 2 diabetes mellitus. The metabolic response may be explained in part by extra-pancreatic mechanisms.

Objective: We tested the hypothesis that DPP-4 inhibition with vildagliptin elicits changes in adipose tissue and skeletal muscle metabolism.

Design: Randomized, double blind, crossover study.

Setting: Academic clinical research center.

Patients: Twenty patients with type 2 diabetes, body mass index between 28 and 40 kg/m2.

Intervention: Seven days treatment with the selective DPP-4 inhibitor vildagliptin or placebo. Standardized test meal on day seven.

Main Outcome Measures: Venous DPP-4 activity, catecholamines, free fatty acids, glycerol, glucose, (pro)insulin; dialysate glucose, lactate, pyruvate, glycerol.

Results: Fasting and postprandial venous insulin, glucose, glycerol, triglycerides and free fatty acid concentrations were not different with vildagliptin and with placebo. Vildagliptin augmented the postprandial increase in plasma norepinephrine. Furthermore, vildagliptine increased dialysate glycerol and lactate concentrations in adipose tissue while suppressing dialysate lactate and pyruvate concentration in skeletal muscle. The respiratory quotient increased with meal ingestion but was consistently lower with vildagliptin.

Conclusions: Our study is the first to suggest that DPP-4 inhibition augments postprandial lipid mobilization and oxidation. The response may be explained by sympathetic activation rather than a direct effect on metabolic status.
Increased Psychopathology and Maladaptive Personality Traits, But Normal Cognitive Functioning, In Patients after Long-Term Cure of Acromegaly
Authors: Tiemensma Jitske , Biermasz Nienke R. , van der Mast Roos C. , Wassenaar Moniek J. E. , Middelkoop Huub A. M. , Pereira Alberto M. , Romijn Johannes A.
Published: January 2014

J Clin Endocrinol Metab, 10.1210/jc.2010-1253
Update on Growth Hormone Therapy of Children
Authors: Collett-Solberg Paulo Ferrez
Published: January 2014

J Clin Endocrinol Metab, 10.1210/jc.2010-1131
Altered Autophagy in Human Adipose Tissues in Obesity
Authors: Kovsan Julia , Blüher Matthias , Tarnovscki Tanya , Klöting Nora , Kirshtein Boris , Madar Liron , Shai Iris , Golan Rachel , Harman-Boehm Ilana , Schön Michael R. , Greenberg Andrew S. , Elazar Zvulun , Bashan Nava , Rudich Assaf
Published: January 2014

Context: Autophagy is a housekeeping mechanism, involved in metabolic regulation and stress response, shown recently to regulate lipid droplets biogenesis/breakdown and adipose tissue phenotype.

Objective: We hypothesized that in human obesity autophagy may be altered in adipose tissue in a fat depot and distribution-dependent manner.

Setting and Patients: Paired omental (Om) and sc adipose tissue samples were used from obese and nonobese (n = 65, cohort 1); lean, sc-obese and intraabdominally obese (n = 196, cohort 2); severely obese persons without diabetes or obesity-associated morbidity, matched for being insulin sensitive or resistant (n = 60, cohort 3).

Results: Protein and mRNA levels of the autophagy genes Atg5, LC3A, and LC3B were increased in Om compared with sc, more pronounced among obese persons, particularly if with intraabdominal fat accumulation. Both adipocytes and stromal-vascular cells contribute to the expression of autophagy genes. The increased number of autophagosomes and elevated autophagic flux assessed in fat explants incubated with lysosomal inhibitors were observed in obesity, particularly in Om. The degree of visceral adiposity and adipocyte hypertrophy accounted for approximately 50% of the variance in Atg5 mRNA levels by multivariate regression analysis, whereas age, sex, measures of insulin sensitivity, inflammation, and adipose tissue stress were excluded from the model. Moreover, in cohort 3, the autophagy marker genes were increased in those who were insulin resistant compared with insulin sensitive, particularly in Om.

Conclusions: Autophagy is up-regulated in adipose tissue of obese persons, especially in Om, correlating with the degree of obesity, visceral fat distribution, and adipocyte hypertrophy. This may precede the occurrence of obesity-associated morbidity.
Functional Consequences of Seven Novel Mutations in the CYP11B1 Gene: Four Mutations Associated with Nonclassic and Three Mutations Causing Classic 11β-Hydroxylase Deficiency
Authors: Parajes Silvia , Loidi Lourdes , Reisch Nicole , Dhir Vivek , Rose Ian T. , Hampel Rainer , Quinkler Marcus , Conway Gerard S. , Castro-Feijóo Lidia , Araujo-Vilar David , Pombo Manuel , Dominguez Fernando , Williams Emma L. , Cole Trevor R. , Kirk Jeremy M. , Kaminsky Elke , Rumsby Gill , Arlt Wiebke , Krone Nils
Published: January 2014

Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency.

Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations.

Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein.

Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively.

Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling in 11OHD.
Progesterone Increases Manganese Superoxide Dismutase Expression via a cAMP-Dependent Signaling Mediated by Noncanonical Wnt5a Pathway in Human Endometrial Stromal Cells
Authors: Matsuoka Aki , Kizuka Fumie , Lee Lifa , Tamura Isao , Taniguchi Ken , Asada Hiromi , Taketani Toshiaki , Tamura Hiroshi , Sugino Norihiro
Published: January 2014

This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0619
Relationship between Vitamin D, Parathyroid Hormone, and Bone Health
Authors: Sai A. J. , Walters R. W. , Fang X. , Gallagher J. C.
Published: January 2014

This article appears in The Journal of Clinical Endocrinology & Metabolism, published December 15, 2010, 10.1210/jc.2010-1886
Endocrine Disruptors and Polycystic Ovary Syndrome (PCOS): Elevated Serum Levels of Bisphenol A in Women with PCOS
Authors: Kandaraki Eleni , Chatzigeorgiou Antonis E. , Livadas Sarantis , Palioura Eleni , Economou Frangiscos , Koutsilieris Michael , Palimeri Sotiria , Panidis Dimitrios , Diamanti-Kandarakis Evanthia
Published: January 2014

This article appears in The Journal of Clinical Endocrinology & Metabolism, published December 30, 2010, 10.1210/jc.2010-1658
Expanding the Phenotype and Genotype of Female GnRH Deficiency
Authors: Shaw Natalie D. , Seminara Stephanie B. , Welt Corrine K. , Au Margaret G. , Plummer Lacey , Hughes Virginia A. , Dwyer Andrew A. , Martin Kathryn A. , Quinton Richard , Mericq Veronica , Merino Paulina M. , Gusella James F. , Crowley William F. , Pitteloud Nelly , Hall Janet E.
Published: January 2014

This article appears in The Journal of Clinical Endocrinology & Metabolism, published January 5, 2011, 10.1210/jc.2010-2292
Preadipocyte Factor-1 Is Associated with Metabolic Profile in Severe Obesity
Authors: O'Connell J. , Lynch L. , Hogan A. , Cawood T. J. , O'Shea D.
Published: January 2014

Context:

Dysfunctional adipose tissue has been proposed as a key pathological process linking obesity and metabolic disease. Preadipocyte factor-1 (Pref-1) has been shown to inhibit differentiation in adipocyte precursor cells and could thereby play a role in determining adipocyte size, adipose tissue functioning, and metabolic profile in obese individuals.

Objective:

We hypothesized that adipose tissue from metabolically healthy obese (MHO) and matched metabolically unhealthy obese (MUO) individuals would demonstrate distinct differences in relation to Pref-1 expression, adipocyte size, and inflammatory markers.

Design, Setting, and Patients:

This was a cross-sectional study, investigating obese patients undergoing bariatric surgery at a tertiary referral centre. Patients included 12 MHO and 17 age- and body mass index–matched MUO individuals.

Main Outcome Measures:

Pref-1, monocyte chemotactic protein-1, TNF-α, granulocyte colony-stimulating factor, IL-6, and adiponectin levels, macrophage numbers, and adipocyte size were measured in omental and subcutaneous adipose tissue.

Results:

The MHO group had a lower level of Pref-1 (per 1000 adipocytes) in both subcutaneous [160 (136–177) versus 194 (153–355); P < 0.05] and omental adipose tissue [102 (32–175) versus 194 (100–350); P < 0.005]. This was associated with lower numbers of macrophages, lower levels of TNF-α, monocyte chemotactic protein-1, and granulocyte colony-stimulating factor, and higher levels of adiponectin. Omental Pref-1 showed strong correlations with adipocyte size (r = 0.67, P < 0.0005) and metabolic and adipokine parameters, including percent fatty liver (r = 0.62, P < 0.005), fasting glucose (r = 0.68, P < 0.0005), triglyceride (r = 0.60, P < 0.005), high-density lipoprotein cholesterol (r = −0.46, P < 0.05), and adiponectin (r = −0.71, P < 0.05).

Conclusion:

Adipose tissue in MHO individuals had lower levels of Pref-1, a known inhibitor of preadipocyte differentiation, and a more favorable inflammatory profile. These factors may be key to protecting this subgroup of obese individuals from the adverse metabolic profile associated with excess adiposity.