Endocrinology Current Issue

• Antibodies to Thyroid Peroxidase Arise Spontaneously with Age in NOD.H-2h4 Mice and Appear after Thyroglobulin Antibodies
  

  Hashimoto’s thyroiditis, a common autoimmune disease, is associated with autoantibodies to thyroglobulin (Tg) and thyroid peroxidase (TPO). TPO, unlike abundant and easily purified Tg, is rarely investigated as an autoantigen in animals. We asked whether antibodies (Abs) develop to both TPO and Tg in thyroiditis that is induced (C57BL/6 and DBA/1 mice) or arises spontaneously (NOD.H-2h4 mice). Screening for TPOAbs was performed by flow cytometry using mouse TPO-expressing eukaryotic cells. Sera were also tested for binding to purified mouse Tg and human TPO. The antibody data were compared with the extent of thyroiditis. Immunization with mouse TPO adenovirus broke self-tolerance to this protein in C57BL/6 mice, but thyroiditis was minimal and TgAbs were absent. In DBA/1 mice with extensive granulomatous thyroiditis induced by Tg immunization, TPOAbs were virtually absent despite high levels of TgAbs. In contrast, antibodies to mouse TPO, with minimal cross-reactivity with human TPO, arose spontaneously in older (7–12 months) NOD.H-2h4 mice. Unexpectedly, TgAbs preceded TPOAbs, a time course paralleled in relatives of probands with juvenile Hashimoto’s thyroiditis. These findings demonstrate a novel aspect of murine and human thyroid autoimmunity, namely breaking B cell self-tolerance occurs first for Tg and subsequently for TPO.

  
  
  
• Erratum
• Periovulatory Leukocyte Infiltration in the Rat Ovary
  

  Ovulation is preceded by intraovarian inflammatory reactions that occur in response to the preovulatory gonadotropin surge. As a main inflammatory event, leukocytes infiltrate the ovary and release proteolytic enzymes that degrade the extracellular matrix weakening the follicular wall, a required step for follicle rupture. This study aimed to quantitatively measure the infiltrating leukocytes, determine their cell types, and localize infiltration sites in the periovulatory rat ovary. Cycling adult and gonadotropin-stimulated immature rats were used as animal models. Ovaries were collected at five different stages of estrous cycle in the adult rats (diestrus, 1700 h; proestrus, 1500 h; proestrus, 2400 h; estrus, 0600 h; and metestrus, 1700 h) and at five different time points after superovulation induction in the immature rats (pregnant mare’s serum gonadotrophin, 0 h; pregnant mare’s serum gonadotrophin, 48 h; human chorionic gonadotropin, 6 h; human chorionic gonadotropin, 12 h; and human chorionic gonadotropin, 24 h). The ovaries were either dissociated into a single cell suspension for flow cytometric analysis or fixed for immunohistochemical localization of the leukocytes. Similar numbers of leukocytes were seen throughout the estrous cycle (~500,000/ovary), except proestrus 2400 when 2-fold higher numbers of leukocytes were found (~1.1 million/ovary). A similar trend of periovulatory rise of leukocyte numbers was seen in the superovulation-induced immature rat model, recapitulating a dramatic increase in leukocyte numbers upon gonadotropin stimulation. Both macrophage/granulocytes and lymphocytes were among the infiltrating leukocytes and were localized in the theca and interstitial tissues, where platelet-endothelial cell adhesion molecule-1 and intercellular adhesion molecule-1 may play roles in the transmigration of leukocytes, because their expressions correlates spatiotemporally with the infiltrating leukocytes. In addition, a strong inverse relationship between leukocyte numbers in the ovary and spleen, as well as significant reduction of leukocyte infiltration in the splenectomized rats, were seen, indicating that the spleen may serve as an immediate supplier of leukocytes to the periovulatory ovary.

  
  
  
• Implication of Oxygen-Regulated Protein 150 (ORP150) in Apoptosis Induced by Proteasome Inhibitors in Human Thyroid Cancer Cells
  

  This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-1043

  
  
  
• Systemic Delivery of Bioactive Glucagon-Like Peptide 1 after Adenoviral-Mediated Gene Transfer in the Murine Salivary Gland
  

  An adenoviral (Ad) vector that expresses bioactive glucagon-like peptide 1 (GLP-1) was generated, and its effectiveness at modulating glucose homeostasis was evaluated after transduction of murine salivary glands. The construct was engineered with the signal sequence of mouse GH to direct the peptide into the secretory pathway, followed by a furin cleavage site and the GLP-1(7–37) sequence encoding an Ala to Gly substitution at position 8 to achieve resistance to degradation. When expressed in Neuro2A and COS7 cells, an active form of GLP-1 was specifically detected by RIA in the conditioned medium of transduced cells, showed resistance to degradation by dipeptidyl-peptidase IV, and induced the secretion of insulin from NIT1 pancreatic β-cells in vitro. In vivo studies demonstrated that healthy mice transduced with Ad-GLP-1 in both submandibular glands had serum GLP-1 levels approximately 3 times higher than mice transduced with the control Ad-luciferase vector. In fasted animals, serum glucose levels were similar between Ad-GLP-1 and Ad-luciferase transduced mice in keeping with GLP-1’s glucose-dependent action. However, when challenged with glucose, Ad-GLP-1 transduced mice cleared the glucose significantly faster than control mice. In an animal model of diabetes induced by alloxan, progression of hyperglycemia was significantly attenuated in mice given the Ad-GLP-1 vector compared with control mice. These studies demonstrate that the bioactive peptide hormone, GLP-1, normally secreted from endocrine cells in the gut through the regulated secretory pathway, can be engineered for secretion into the circulatory system from exocrine cells of the salivary gland to affect glucose homeostasis.

  
  
  
• Changes in 65-Kilodalton Glutamate Decarboxylase Antibody-Specific Antiidiotypic Antibody Levels Correlate with Changes in C-Peptide Levels and Progression to Islet Cell Autoimmunity
  

  This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0785

  
  
  
• Deletion of the Isg15 Gene Results in Up-Regulation of Decidual Cell Survival Genes and Down-Regulation of Adhesion Genes: Implication for Regulation by IL-1{beta}
  

  The ubiquitin homolog interferon stimulated gene 15 (ISG15) is up-regulated in the endometrium in response to pregnancy in primates, ruminants, pigs, and mice. ISG15 covalently attaches to intracellular proteins (isgylation) and regulates numerous intracellular responses. We hypothesized that ISG15 depletion (Isg15^–/–) alters decidual tissue gene expression and that IL-1β induces ISG15 expression and isgylation in cultured murine decidual explants and human uterine fibroblasts (HuFs). After studying the reproductive phenotype, contrary to earlier reports, up to 50% of the fetuses die between 7.5 and 12.5 d post coitum (dpc) in Isg15^–/– mothers when mated to Isg15^–/– fathers. Using microarray analysis, over 500 genes are differentially regulated in 7.5 dpc deciduas from Isg15^–/– compared with Isg15^+/+ mice. The gene for interferon-inducible protein 202b, which functions in cell-survival mechanisms, was up-regulated (mRNA and protein) in deciduas from Isg15^–/– mice. Culture of Isg15^+/+ mouse decidual explants (7.5 dpc) with IL-1β decreased Isg15 mRNA but increased free and conjugated ISG15. In predecidual HuF cells, IL-1β treatment increased ISG15 mRNA and isgylation. Additionally, IL-1β up-regulated expression of enzymes (HERC5, UBCH8) that coordinate the covalent conjugation of ISG15 to target proteins, as well as the gene that encodes the deisglyation enzyme UBP43 in HuF cells. In conclusion, deletion of Isg15 gene results in 50% fetal loss after 7.5 dpc, which can be explained through differential decidual gene expression that is functionally tied to cell survival and adhesion pathways. This fetal death also might relate to impaired IL-1β signaling, because ISG15 and isgylation are induced by IL-1β in human and murine endometrial stromal cells.

  
  
  
• Approach to Assigning Gender in 46,XX Congenital Adrenal Hyperplasia with Male External Genitalia: Replacing Dogmatism with Pragmatism
  

  This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0714

  
  
  
• Absence of Thyroid Hormone Activation during Development Underlies a Permanent Defect in Adaptive Thermogenesis
  

  Type 2 deiodinase (D2), which is highly expressed in brown adipose tissue (BAT), is an enzyme that amplifies thyroid hormone signaling in individual cells. Mice with inactivation of the D2 pathway (D2KO) exhibit dramatically impaired thermogenesis in BAT, leading to hypothermia during cold exposure and a greater susceptibility to diet-induced obesity. This was interpreted as a result of defective acute activation of BAT D2. Here we report that the adult D2KO BAT has a permanent thermogenic defect that stems from impaired embryonic BAT development. D2KO embryos have normal serum T3 but due to lack of D2-generated T3 in BAT, this tissue exhibits decreased expression of genes defining BAT identity [i.e. UCP1, PGC-1 and Dio2 (nonfunctional)], which results in impaired differentiation and oxidative capacity. Coinciding with a reduction of these T3-responsive genes, there is oxidative stress that in a cell model of brown adipogenesis can be linked to decreased insulin signaling and decreased adipogenesis. This discovery highlights the importance of deiodinase-controlled thyroid hormone signaling in BAT development, where it has important metabolic repercussions for energy homeostasis in adulthood.

  
  
  
• Progesterone Increases Manganese Superoxide Dismutase Expression via a cAMP-Dependent Signaling Mediated by Noncanonical Wnt5a Pathway in Human Endometrial Stromal Cells
  

  This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0619

  
  
  
• Real-Time Monitoring of Somatostatin Receptor-cAMP Signaling in Live Pituitary
  

  Fluorescence resonance energy transfer using genetically encoded biosensors has proven to be a powerful technique to monitor the spatiotemporal dynamics of cAMP signals stimulated by G_s-coupled receptors in living cells. In contrast, real-time imaging of G_i-mediated cAMP signals under native conditions remains challenging. Here, we describe the use of transgenic mice for cAMP imaging in living pituitary slices and primary pituitary cells. This technique can be widely used to assess the contribution of various pituitary receptors, including individual G_i protein-coupled somatostatin receptors, to the regulation of cAMP levels under physiologically relevant settings.

  
  
  
• A Role for the Orphan Nuclear Receptor Estrogen-Related Receptor {alpha} in Endometrial Stromal Cell Decidualization and Expression of Genes Implicated in Energy Metabolism
  

  This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0154

  
  
  
• Subfertility Linked to Combined Luteal Insufficiency and Uterine Progesterone Resistance
  

  Early pregnancy loss is common and can be caused by a range of factors. The Brown Norway (BN) rat exhibits reproductive dysfunction characterized by small litter size and pregnancy failure and represents a model for investigating early pregnancy loss. In this study, we investigated the establishment of pregnancy in the BN rat and gained insight into mechanisms causing its subfertility. Early stages of BN uteroplacental organization are unique. The BN primordial placenta is restricted in its development and correlates with limited BN uterine decidual development. BN uterine decidua was shown to be both structurally and functionally distinct and correlated with decreased circulating progesterone (P4) levels. Ovarian anomalies were also apparent in BN rats and included decreased ovulation rates and decreased transcript levels for some steroidogenic enzymes. Attempts to rescue the BN uterine decidual phenotype with steroid hormone therapy were ineffective. BN uteri were shown to exhibit reduced responsiveness to P4 but not to 17β-estradiol. P4 resistance was associated with decreased transcript levels for the P4 receptor (Pgr), a P4 receptor chaperone (Fkbp4), and P4 receptor coactivators (Ncoa1 and Ncoa2). In summary, the BN rat exhibits luteal insufficiency and uterine P4 resistance, which profoundly affects its ability to reproduce.

  
  
  
• Neurokinin B Stimulates GnRH Release in the Male Monkey (Macaca mulatta) and Is Colocalized with Kisspeptin in the Arcuate Nucleus
  

  Human genetics indicate that kisspeptin and neurokinin B (NKB) signaling are necessary for generating pulsatile LH release and therefore for initiation of puberty and maintaining gonadal function. In the present study, male monkeys were employed to examine 1) whether activation of the NKB receptor (NK3R) is associated with GnRH release, and 2) hypothalamic localization of these peptides using immunofluorescence histochemistry. Agonadal juveniles, in which pituitary responsiveness to GnRH was heightened by GnRH priming, were employed to indirectly examine GnRH-releasing actions of NK3R and kisspeptin receptor agonists by tracking LH after their iv injection. Castrated adults were used for immunohistochemistry. Single iv injections of NKB or senktide (an NK3R agonist) elicited robust LH discharges that were abolished by GnRH receptor antagonism (acyline) confirming the ligands’ hypothalamic action. Intermittent infusion of senktide (1-min pulse every hour for 4 h), in contrast to that of kisspeptin, failed to sustain pulsatile GnRH release. Repetitive senktide injections did not compromise the GnRH-releasing action of kisspeptin. NKB and kisspeptin were colocalized in perikarya of the arcuate nucleus and in axonal projections to the median eminence, confirming earlier findings in sheep. These results are consistent with the human genetics, and indicate that although brief activation of NK3R stimulates GnRH release, repetitive stimulation of this pathway, in contrast to that of kisspeptin receptor, fails to sustain pulsatile GnRH release. In addition, the data provide a platform for future elucidation of the interactions between NKB and kisspeptin that are required for generating pulsatile GnRH release in primates.

  
  
  
• 17{beta}-Estradiol Rapidly Increases KATP Activity in GnRH via a Protein Kinase Signaling Pathway
  

  17β-Estradiol (E2) both inhibits and excites GnRH neurons via presynaptic as well as postsynaptic mechanisms. Although it has been demonstrated that E2 can alter the excitability of GnRH neurons via direct actions, the intracellular signaling cascades mediating these actions are not well understood. Previously we have shown that the activity of one of the critical ion channels needed for maintaining GnRH neurons in a hyperpolarized state, the ATP-sensitive potassium channel (K_ATP) channel, is augmented by E2 in ovariectomized females. However, the mRNA expression of the K_ATP channel subunits Kir6.2 and SUR1 are unchanged with in vivo E2 treatment. Therefore, to elucidate the cellular signaling mechanism(s) modulating the channel activity, we did whole-cell patch-clamp recording of enhanced green fluorescent protein-GnRH neurons from ovariectomized female mice to study the acute effects of E2. E2 dose-dependently (EC₅₀ = 0.6 nM) enhanced the diazoxide (channel opener)-activated K_ATP channel currents by 1.2- to 2.0-fold, which was antagonized by ICI 182,780. E2-BSA was equally as effective as E2, whereas E2 had no effect. The protein kinase A (PKA) activator forskolin mimicked the effects of E2, whereas the PKA inhibitor H89 and the protein kinase C (PKC) inhibitor bisindolylmaleimide I blocked the effects of E2. Similar to E2, STX, a membrane estrogen receptor (ER) agonist that does not bind to ER or ERβ, also potentiated the diazoxide-induced K_ATP channel current by 1.5-fold. Therefore, E2 can potentiate K_ATP channel activity in GnRH neurons through a membrane ER-activated PKC-PKA signaling pathway.

  
  
  
• Molecular Signature of Mineralocorticoid Receptor Signaling in Cardiomyocytes: From Cultured Cells to Mouse Heart
  

  Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases.

  
  
  
• Involvement of RAGE, NADPH Oxidase, and Ras/Raf-1 Pathway in Glycated LDL-Induced Expression of Heat Shock Factor-1 and Plasminogen Activator Inhibitor-1 in Vascular Endothelial Cells
  

  Atherothrombotic cardiovascular diseases are the predominant causes of mortality of diabetic patients. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor for fibrinolysis, and it is also implicated in inflammation and tissue remodeling. Increased levels of PAI-1 and glycated low-density lipoprotein (glyLDL) were detected in patients with diabetes. Previous studies in our laboratory demonstrated that heat shock factor-1 (HSF1) is involved in glyLDL-induced PAI-1 overproduction in vascular endothelial cells (EC). The present study investigated transmembrane signaling mechanisms involved in glyLDL-induced HSF1 and PAI-1 up-regulation in cultured human vascular EC and streptozotocin-induced diabetic mice. Receptor for advanced glycation end products (RAGE) antibody prevented glyLDL-induced increase in the abundance of PAI-1 in EC. GlyLDL significantly increased the translocation of V-Ha-Ras Harvey rat sarcoma viral oncogene homologue (H-Ras) from cytoplasm to membrane compared with LDL. Farnesyltransferase inhibitor-277 or small interference RNA against H-Ras inhibited glyLDL-induced increases in HSF1 and PAI-1 in EC. Treatment with diphenyleneiodonium, a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, blocked glyLDL-induced translocation of H-Ras, elevated abundances of HSF1 and PAI-1 in EC, and increased release of hydrogen peroxide from EC. Small interference RNA for p22^phox prevented glyLDL-induced expression of NOX2, HSF1, and PAI-1 in EC. GlyLDL significantly increased V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) phosphorylation. Treatment with Raf-1 inhibitor blocked glyLDL-induced increase of PAI-1 mRNA in EC. The levels of RAGE, H-Ras, NOX4, HSF1, and PAI-1 were increased in hearts of streptozotocin-diabetic mice and positively correlated with plasma glucose. The results suggest that RAGE, NOX, and H-Ras/Raf-1 are implicated in the up-regulation of HSF1 or PAI-1 in vascular EC under diabetes-associated metabolic stress.

  
  
  
• Effects of GH Secretagogues on Contractility and Ca2+ Homeostasis of Isolated Adult Rat Ventricular Myocytes
  

  Ghrelin and its synthetic analogue hexarelin are specific ligands of GH secretagogue receptor (GHS-R) and induce a variety of cardiovascular protective and cardiac positive inotropic effects. The signaling system underlying immediate effects of both GHSs in cardiomyocytes remains undefined. In the present study, we investigated the immediate effects of GHSs on isolated ventricular myocyte shortening, intracellular Ca²⁺ ([Ca²⁺]_i) transients, and the L-type Ca²⁺ current (I_Ca,L). Putative intracellular signalling cascades were studied with specific receptor and signalling blockers. In fresh isolated adult Wistar rat ventricular myocytes, GHSs produced a positive inotropic effect in a concentration-dependent manner and increased the amplitude of [Ca²⁺]_i transients and the I_Ca,L. The positive inotropic response was abolished by the GHS-R1a antagonist [d-Lys³]-GH-releasing peptide-6 (10 µm). GHS-induced increase in the I_Ca,L was abolished by [d-Lys³]-GH-releasing peptide-6 and protein kinase C inhibitor, chelerythrine chloride (5 µm), but not by protein kinase A inhibitor, KT 5720 (10 µm). We conclude that hexarelin and ghrelin increase the I_Ca,L, through GHS-R1a receptor and protein kinase C signalling cascade, which contribute to its direct positive inotropic effect on cardiomyocytes.

  
  
  
• Identification of Early Response Genes and Pathway Activated by Androgens in the Initial Segment and Caput Regions of the Regressed Rat Epididymis
  

  To identify the initial response to androgens and estrogens in the orchidectomized, regressed epididymis, we determined the gene expression changes triggered by the administration of either of two metabolites of testosterone, 5-dihydrotestosterone (DHT) or 17β-estradiol (E2), in the regressed rat epididymis. Adult rats were orchidectomized and 8 d later implanted with either empty implants (control), DHT-filled-, or E2-filled-polydioxanone implants. Rats were euthanized 12 h, 1 d, and 7 d later, and RNA was extracted and probed on Rat230–2.0 Affymetrix arrays. Probe sets that respond to DHT or E2 were identified at early time points; although the expression of some was repressed, the expression of many others was either transiently or chronically elevated. Nerve growth factor receptor (Ngfr) and S100 calcium binding protein G (S100g) were two E2 up-regulated genes detected at 12 h. Among the genes that showed a dramatic early response to DHT were endothelin 1 (Edn1), bone morphogenetic protein 4 (Bmp4), and IGF binding protein 3 (Igfbp3), which were suppressed, and IGF-I (Igf1), which was induced. Genes that were up- or down-regulated by DHT were classified based on biological function. Using PathwayStudio 4.0, we identified genes that were linked and directly influenced either the expression or regulation of one another. Epidermal growth factor and IGF-I play an important role in the pathway due to their function in regulation and expression of many other genes. These results provide novel insights into the impact of androgen action on the expression of genes that are important for epididymal function.

  
  
  
• Adrenomedullin Relaxes Rat Uterine Artery: Mechanisms and Influence of Pregnancy and Estradiol
  

  Uterine arteries play a major role in regulating uteroplacental blood flow. Failure to maintain blood flow to the uteroplacental compartment during pregnancy often results in intrauterine growth retardation. Immunohistochemical staining of adrenomedullin (AM), an endogenous vasoactive peptide, in uterine artery was intense in pregnant compared to nonpregnant rats, but it is not known whether AM directly relaxes uterine artery or not. In this study, we elucidated the mechanisms of uterine artery relaxation by AM and its regulation by pregnancy and female sex steroids. AM was able to relax uterine artery, and this relaxation was influenced positively by pregnancy and estradiol as evidenced by the increased pD₂ and E_max values of AM. Both pregnancy and estradiol treatment to ovariectomized rats amplified RAMP₃ expression in uterine arteries while progesterone had no effect. AM-induced uterine artery relaxation is predominantly endothelium-dependent. The AM receptor antagonist CGRP_8-37 is more potent than AM_22-52 in inhibiting the AM relaxation, indicating the involvement of AM₂ receptor subtype. Moreover, AM uses the classical nitric oxide-cyclic guanosine monophosphate pathway along with K_Ca channels to mediate the vasodilatory effect in uterine artery. In conclusion, sensitivity of uterine artery to AM-induced relaxation is increased with pregnancy or estradiol treatment by increasing RAMP₃ expression, suggesting an important role for AM in regulating the uterine hemodynamics, probably maintaining uterine blood flow during pregnancy and in pre- and postmenopausal cardiovascular adaptation differences.

  
  
  
• Ovarian Steroids, Mitogen-Activated Protein Kinases, and/or Aspartic Proteinases Cooperate to Control Endometrial Remodeling by Regulating Gene Expression in the Stroma and Glands
  

  Explants from nonmenstrual endometria cultured in the absence of ovarian hormones undergo tissue breakdown. Addition of estradiol and progesterone (EP) prevents proteolysis. Explants include stromal and epithelial compartments which play different but complementary roles in endometrial physiology, including tissue remodeling and hormonal response. In order to characterize the cell type-specific contribution to regulation of tissue breakdown, we characterized the transcriptomes of microdissected stromal and glandular areas from endometrial explants cultured with or without EP. The datasets were also compared to other published endometrial transcriptomes. Finally, the contribution of proteolysis, hypoxia, and MAPKs to the regulation of selected genes was further investigated in explant culture. This analysis identified distinct gene expression profiles in stroma and glands, with differential response to EP, but functional clustering underlined convergence in biological processes, further indicating that endometrial remodeling requires cooperation between the two compartments through expression of cell type-specific genes. Only partial overlaps were observed between lists of genes involved in different occurrences of endometrial breakdown, pointing to a limited number of potentially crucial regulators but also to the requirement for additional mechanisms controlling tissue remodeling. We identified a group of genes differentially regulated by EP in stroma and glands among which some were sensitive to MAPKs and/or aspartic proteinases and were not induced by hypoxia. In conclusion, MAPKs and/or aspartic proteinases likely act in concert with EP to locally and specifically control differential expression of genes between degrading and preserved areas of the human endometrium.

  
  
  
• Osteoclast Inhibitory Peptide-1 Binding to the Fc{gamma}RIIB Inhibits Osteoclast Differentiation
  

  Osteoclast inhibitory peptide-1 (OIP) is an autocrine/paracrine inhibitor of osteoclast differentiation, and mice that overexpress OIP-1 in osteoclast lineage cells develop an osteopetrosis bone phenotype. In this study, we show that OIP-1 binding to the Fc receptor IIB (FcRIIB) inhibits osteoclast differentiation. Confocal microscopy revealed colocalization of OIP-1 with FcRIIB in osteoclasts, and we observed that OIP-1 carboxy-terminal GPI-linked peptide forms a 1:1 complex with recombinant FcRIIB protein with an affinity binding of a dissociation constant of approximately 4 µm. Immunoreceptor tyrosine-based activation motif (ITAM)-bearing adapter proteins (FcR and DNAX-activating protein of molecular mass 12 kDa) are critical for osteoclast development, and OIP-1 transgenic mouse-derived preosteoclast cells demonstrated suppression (6-fold) of ITAM phosphorylation of FcR but not DNAX-activating protein of molecular mass 12 kDa. Interestingly, these preosteoclast cells demonstrated increased levels (4-fold) of immunoreceptor tyrosine-based inhibitory motif phosphorylation of FcRIIB and Src homology 2-domain-containing proteins tyrosine phosphatase 1 activation. Further, OIP-1 mouse-derived preosteoclasts cells demonstrated inhibition of spleen tyrosine kinase activation (4.5-fold), compared with wild-type mice. These results suggest that cross-regulation of immunoreceptor tyrosine-based inhibitory motif and ITAM bearing Fc receptors may play a role in OIP-1 suppression of spleen tyrosine kinase activation and inhibition of osteoclast differentiation. Thus, OIP-1 is an important physiologic regulator of osteoclast development and may have therapeutic utility for bone diseases with high bone turnover.

  
  
  
• Interleukin-1{beta} May Mediate Insulin Resistance in Liver-Derived Cells in Response to Adipocyte Inflammation
  

  Central obesity is frequently associated with adipose tissue inflammation and hepatic insulin resistance. To identify potential individual mediators in this process, we used in vitro systems and assessed if insulin resistance in liver cells could be induced by secreted products from adipocytes preexposed to an inflammatory stimulus. Conditioned medium from 3T3-L1 adipocytes pretreated without (CM) or with TNF (CM-TNF) was used to treat Fao hepatoma cells. ELISAs were used to assess the concentration of several inflammatory mediators in CM-TNF. CM-TNF-treated Fao cells exhibited about 45% diminution in insulin-stimulated phosphorylation of insulin receptor, insulin receptor substrate proteins, protein kinase B, and glycogen synthase kinase-3 as compared with CM-treated cells, without changes in the total abundance of these protein. Insulin increased glycogenesis by 2-fold in CM-treated Fao cells but not in cells exposed to CM-TNF. Expression of IL-1β mRNA was elevated 3-fold in TNF-treated adipocytes, and CM-TNF had 10-fold higher concentrations of IL-1β but not TNF or IL-1. IL-1β directly induced insulin resistance in Fao, HepG2, and in primary rat hepatocytes. Moreover, when TNF-induced secretion/production of IL-1β from adipocytes was inhibited by the IL-1 converting enzyme (ICE-1) inhibitor II (Ac-YVAD-CMK), insulin resistance was prevented. Furthermore, liver-derived cells treated with IL-1 receptor antagonist were protected against insulin resistance induced by CM-TNF. Finally, IL-1β secretion from human omental fat explants correlated with body mass index (R² = 0.639, P < 0.01), and the resulting CM induced insulin resistance in HepG2 cells, inhibitable by IL-1 receptor antagonist. Our results suggest that adipocyte-derived IL-1β may constitute a mediator in the perturbed cross talk between adipocytes and liver cells in response to adipose tissue inflammation.

  
  
  
• The Endogenous Actions of Hypothalamic Peptides on Brown Adipose Tissue Thermogenesis in the Rat
  

  Although the neuronal pathways within the hypothalamus critical in controlling feeding and energy expenditure and projecting to brown adipose tissue (BAT) have been identified and their peptidergic content characterized, endogenous action of such peptides in the control of BAT activity has not been elucidated. Here male Sprague Dawley rats received infusions of either melanin-concentrating hormone antagonist (SNAP-7941) (1 µg/µl · h), orexin A receptor antagonist (SB-334867-A; 1 µg/µl · h), combined SB-334867-A (1 µg/µl · h), and SNAP-7941 (1 µg/µl · h), or melanocortin-3/4 receptor antagonist (SHU9119) (1 µg/µl · h) via an indwelling cannula in the lateral ventricle attached to sc implanted osmotic minipump. BAT temperature, physical activity, body weight, food intake, and changes in uncoupling protein (UCP)-1 were measured. SB-334867-A and SNAP-7941 significantly increased BAT temperature and UCP1 expression and reduced food intake and body weight. Combined infusion of SB-334867-A and SNAP-7941 produced a pronounced response that was greater than the addition of the individual effects in all parameters measured. SHU9119 significantly decreased BAT temperature and UCP1 expression and increased feeding and body weight. In a second series of experiments, the effect of SB-334867-A and SNAP-7941 alone or combination on the expression of the Fos protein was determined. SB-334867-A and SNAP-7941 increased Fos expression in key hypothalamic and brainstem feeding-related regions. In combination, these antagonists produced a greater than additive elevation of Fos expression in most of the regions evaluated. These findings support a role for endogenous orexigenic and anorexigenic hypothalamic peptides acting in concert to create a thermogenic tone via BAT activity.

  
  
  
• Essential Role for p38{alpha} MAPK But Not p38{gamma} MAPK in Igf2 Expression and Myoblast Differentiation
  

  The muscle satellite cell is established as the major stem cell contributing to fiber growth and repair. p38 MAPK signaling is essential for myoblast differentiation and in particular for up-regulation of promyogenic Igf2 expression. p38 exists as four isoforms (, β, , and ), of which p38 is uniquely abundant in muscle. The aim of this study was to characterize p38 isoform expression and importance (using shRNA knockdown; demonstrated via both reduced protein and kinase activities) during myoblast differentiation. p38 and - mRNA levels were most abundant in differentiating C2 cells with low/negligible contributions from p38β and -, respectively. Increased phosphorylation of p38 and - occurred during differentiation but via different mechanisms: p38 protein levels remained constant, whereas total p38 levels increased. Following shRNA knockdown of p38, myoblast differentiation was dramatically inhibited [reduced myosin heavy chain (MHC), myogenin, pAkt protein levels]; significantly, Igf2 mRNA levels and promoter-reporter activities decreased. In contrast, knockdown of p38 induced a transient increase in both myogenin and MHC protein levels with no effect on Igf2 mRNA levels or promoter-reporter activity. Knockdown of p38/β markedly increased but that of p38 decreased caspase 3 activity, suggesting opposite actions on apoptosis. p38 was initially proposed to have a promyogenic function; however, p38 overexpression could not rescue reduced myoblast differentiation following p38/β inhibition. Therefore, p38 is essential for myoblast differentiation, and part of its action is to convert signals that indicate cell density into promyogenic gene expression in the form of the key peptide, IGF-II; p38 has a minor, yet opposing antimyogenic, function.

  
  
  
• Knockdown of GABAA Receptor Signaling in GnRH Neurons Has Minimal Effects upon Fertility
  

  The amino acid -aminobutyric acid (GABA) is thought to play a key role in shaping the activity of the GnRH neurons throughout embryonic and postnatal life. However, the physiological roles of direct GABA inputs to GnRH neurons remain unknown. Using a Cre-LoxP strategy, we generated a targeted mouse line, in which all (98 ± 1%) GnRH neurons had the 2-subunit of the GABA_A receptor deleted. Electrophysiological recordings of GABA_A-mediated postsynaptic currents from green fluorescent protein-tagged GnRH neurons with the 2-subunit knocked out (GnRH 2 KO) showed that the amplitude and frequency of GABA_A postsynaptic currents were reduced by 70% (P < 0.01) and 77% (P < 0.05), respectively, and that the response to exogenous GABA was reduced by 90% (P < 0.01). Evaluation of male and female GnRH 2 KO mice revealed completely normal fecundity, estrous cycles, and puberty onset. Further investigation with gonadectomy and different steroid replacement regimens showed normal basal levels of LH in both sexes, and a normal estradiol-evoked positive feedback mechanism in females. However, the increment in LH after gonadectomy in GnRH 2 KO female mice was double that of controls (P < 0.05) and also more potently suppressed by 17-β-estradiol (P < 0.05). A similar but nonsignificant trend was observed in GnRH 2 KO male mice. Together, these findings show that 70–90% reductions in the normal levels of GABA_A receptor activity at the GnRH neuron appear to impact upon the estrogen negative feedback mechanism but are, nevertheless, compatible with normal fertility in mice.

  
  
  
• Designing a Long-Acting Human Growth Hormone (hGH) by Fusing the Carboxyl-Terminal Peptide of Human Chorionic Gonadotropin {beta}-Subunit to the Coding Sequence of hGH
  

  Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-β-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.

  
  
  
• Basal Plasma Levels of Insulin, Leptin, Ghrelin, and Amylin Do Not Signal Adiposity in Rats Recovering from Forced Overweight
  

  This study examined how adiposity signals are related to adiposity during recovery from forced overweight (OW). Rats were rendered OW by chronic intragastric overfeeding (OW). Overfeeding was stopped when OW rats reached 126–129% of saline-infused normal-weight (NW) rats. Adipose tissue (AT) mass was estimated by computed tomography, and blood was drawn from chronic atrial cannulas throughout. Basal levels (i.e. after 2–3 h fasts late in the diurnal phase) of the hypothesized adiposity signals insulin, leptin, ghrelin, and amylin were assayed. OW rats gained approximately 130 g more body weight (BW) and approximately 100 g more AT mass during overfeeding. Plasma levels of insulin and leptin increased, whereas those of ghrelin decreased, linearly with AT mass; amylin did not change reliably. During recovery, OW rats’ BW and AT mass decreased but were still elevated vs. NW rats after 39 d. OW rats’ insulin returned to NW levels on d 1 of recovery and decreased below NW levels thereafter. Leptin was no longer elevated after d 8 of recovery. Ghrelin and amylin did not change reliably during recovery. Although AT mass decreased in OW rats during each intermeasurement interval between d 0 and d 23 of recovery, insulin and leptin did so during only the first interval (d 0–5). Insulin and leptin levels were exponentially related to AT mass during recovery. These data indicate that basal insulin, leptin, ghrelin, and amylin do not encode AT mass in rats dynamically regulating BW and adiposity during recovery from OW.

  
  
  
• Central Injection of the Stable Somatostatin Analog ODT8-SST Induces a Somatostatin2 Receptor-Mediated Orexigenic Effect: Role of Neuropeptide Y and Opioid Signaling Pathways in Rats
  

  Somatostatin and octreotide injected into the brain have been reported to modulate food intake. However, little is known regarding the underlying mechanisms. The stable oligosomatostatin analog, des-AA^{1,2,4,5,12,13}-[DTrp⁸]-somatostatin (ODT8-SST), like somatostatin, binds to all five somatostatin receptors (sst_1–5). We characterized the effects of ODT8-SST injected intracerebroventricularly (icv) on food consumption and related mechanisms of action in freely fed rats. ODT8-SST (0.3 and 1 µg per rat, icv) injected during the light or dark phase induced an early onset (within 1 h) and long-lasting (4 h) increase in food intake in nonfasted rats. By contrast, ip injection (0.3–3 mg/kg) or icv injection of selective sst₁ or sst₄ agonists (1 µg per rat) had no effect. The 2 h food intake response during the light phase was blocked by icv injection of a sst₂ antagonist, the neuropeptide Y (NPY) Y₁ receptor antagonist, BIBP-3226, and ip injection of the µ-opioid receptor antagonist, naloxone, and not associated with changes in plasma ghrelin levels. ODT8-SST (1 µg per rat, icv) stimulated gastric emptying of a solid meal which was also blocked by naloxone. The increased food intake was accompanied by a sustained increase in respiratory quotient, energy expenditure, and drinking as well as µ-opioid receptor-independent grooming behavior and hyperthermia, while ambulatory movements were not altered after ODT8-SST (1 µg per rat, icv). These data show that ODT8-SST acts primarily through brain sst₂ receptors to induce a long-lasting orexigenic effect that involves the activation of Y₁ and opiate-receptors, accompanied by enhanced gastric transit and energy expenditure suggesting a modulation of NPYergic and opioidergic orexigenic systems by brain sst₂ receptors.

  
  
  
• A Soluble Activin Receptor Type IIB Prevents the Effects of Androgen Deprivation on Body Composition and Bone Health
  

  Androgen deprivation, a consequence of hypogonadism, certain cancer treatments, or normal aging in men, leads to loss of muscle mass, increased adiposity, and osteoporosis. In the present study, using a soluble chimeric form of activin receptor type IIB (ActRIIB) we sought to offset the adverse effects of androgen deprivation on muscle, adipose tissue, and bone. Castrated (ORX) or sham-operated (SHAM) mice received either TBS [vehicle-treated (VEH)] or systemic administration of ActRIIB-mFc, a soluble fusion protein comprised of a form of the extracellular domain of ActRIIB fused to a murine IgG2aFc subunit. In vivo body composition imaging demonstrated that ActRIIB-mFc treatment results in increased lean tissue mass of 23% in SHAM mice [19.02 ± 0.42 g (VEH) versus 23.43 ± 0.35 g (ActRIIB-mFc), P < 0.00001] and 26% in ORX mice [15.59 ± 0.26 g (VEH) versus 19.78 ± 0.26 g (ActRIIB-mFc), P < 0.00001]. Treatment also caused a decrease in adiposity of 30% in SHAM mice [5.03 ± 0.48 g (VEH) versus 3.53 ± 0.19 g (ActRIIB-mFc), NS] and 36% in ORX mice [7.12 ± 0.53 g (VEH) versus 4.57 ± 0.28 g (ActRIIB-mFc), P < 0.001]. These changes were also accompanied by altered serum levels of leptin, adiponectin, and insulin, as well as by prevention of steatosis (fatty liver) in ActRIIB-mFc-treated ORX mice. Finally, ActRIIB-mFc prevented loss of bone mass in ORX mice as assessed by whole body dual x-ray absorptiometry and micro-computed tomography of proximal tibias. The data demonstrate that treatment with ActRIIB-mFc restored muscle mass, adiposity, and bone quality to normal levels in a mouse model of androgen deprivation, thereby alleviating multiple adverse consequences of such therapy.