bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of Thyroid Hormone Activation during Development Underlies a Permanent Defect in Adaptive Thermogenesis
Authors: Hall Jessica A. , Ribich Scott , Christoffolete Marcelo A. , Simovic Gordana , Correa-Medina Mayrin , Patti Mary Elizabeth , Bianco Antonio C.
Published: January 2014

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-1α 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.
The Poly(C)-Binding Protein-1 Regulates Expression of the Androgen Receptor
Authors: Cloke Brianna , Shah Kunal , Kaneda Hiroshi , Lavery Stuart , Trew Geoffrey , Fusi Luca , Higham Jenny , Dina Roberto E. , Ghaem-Maghami Sadaf , Ellis Patricia , Brosens Jan J. , Christian Mark
Published: January 2014

The androgen receptor (AR) is a ligand-dependent transcription factor, expressed in male and female reproductive organs, and essential for normal reproduction in both sexes. The levels of AR are tightly controlled in androgen-responsive cells in which it plays a central role in the regulation of target gene expression. The AR is abundantly expressed in human endometrial stromal cells (HESCs), but levels decline markedly after differentiation into decidual cells in vivo and in primary cultures. Decidualization profoundly down-regulated AR protein levels with no discernible effect on either AR mRNA or protein stability, suggesting that loss of the receptor was a consequence of translational inhibition. Here we show that HESCs express three RNA-binding proteins, Hu antigen R and the poly(C)-binding proteins PCBP1 and PCBP2, that reportedly target the 3′-untranslated region of AR transcripts. Only PCBP1 expression was enhanced in secretory endometrium in vivo and in decidualizing HESCs. Furthermore, knockdown of PCBP1 in decidualizing cells was sufficient to restore AR protein levels, indicating that loss of the AR protein is primarily the consequence of a translational block. PCBP1 also blocked AR translation in a cell-free system, although this did not require binding to the 3′-untranslated region of the receptor mRNA. Furthermore, knockdown of PCBP1 in the prostate cancer LNCaP cell line also increased AR protein. Therefore, PCBP1 plays a major role in the dynamic expression of AR in both male and female androgen-responsive cells.
Adrenocorticotropic Hormone Regulates the Activities of the Orphan Nuclear Receptor Nur77 through Modulation of Phosphorylation*
Authors: Li Yanzhuang , Lau Lester F.
Published: January 2014

ACTH treatment of Y1 adrenocortical cells induces the synthesis of Nur77, an orphan nuclear receptor that can act as a potent trans-activator for such genes as 21-hydroxylase (CYP21). Nur77 has thus been proposed to be a mediator of ACTH action in activating the expression of genes that encode steroidogenic enzymes. Here we show that ACTH regulates the activity of Nur77 at the level of phosphorylation. ACTH induces the synthesis of transcriptionally active, DNA-binding Nur77 that is unphosphorylated at Ser354, which resides within the DNA-binding domain. By contrast, the Nur77 population that is constitutively present in Y1 cells is phosphorylated at Ser354 and does not bind DNA. Substitutions of Ser354 with negatively charged amino acids, such as Asp or Glu, dramatically decreased Nur77 DNA-binding and trans-activation activities, whereas mutation to the neutral Ala had no effect. Aside from phosphorylation within the DNA-binding domain, ACTH treatment does not induce modifications in the N- and C-terminal domains of Nur77 that significantly affect activity. Although the specific kinases that phosphorylate Nur77 in vivo are not known, the mitogen-activated protein kinase/pp90RSK pathway is not critical to Nur77 regulation. We propose that ACTH treatment of Y1 cells results in modulation of the activities of both kinases and phosphatases, which, in turn, regulate the activities of such transcription factors as Nur77.
Transforming Growth Factor-βs Inhibit Somatostatin Messenger Ribonucleic Acid Levels and Somatostatin Secretion in Hypothalamic Cells in Culture*
Authors: Quintela M. , SeñarÍs R. M. , Diéguez C.
Published: January 2014

Treatment of hypothalamic cells in monolayer culture with transforming growth factor-β1 (TGFβ1) significantly reduced both basal and cAMP-induced somatostatin messenger RNA (mRNA) levels and somatostatin secretion. This inhibitory effect was dose- and time-dependent and not mediated by glial cells, as it was also observed in glial-free hypothalamic cell cultures treated with cytosine arabinonucleoside. TGFβ2 and -β3 mimicked the actions of TGFβ1, which indicated that the three isoforms of the TGFβ family expressed in the central nervous system displayed similar effects on the somatostatinergic neurons.

The blockade of synthesis of proteins with either cycloheximide or puromycin for 24 h prevented the inhibitory effect of TGFβ1 on somatostatin mRNA. This implied that the reduction of this mRNA by TGFβ1 required de novo protein synthesis.

We next studied whether TGFβ1 acted at the transcriptional or posttranscriptional level by altering the stability of somatostatin mRNA. Examination of the rate of disappearance of somatostatin mRNA by Northern blot, after inhibition of mRNA transcription with either actinomycin D (AcD) or 5,6-dichloro-1β-ribofuranosyl benzimidazole revealed that TGFβ1 did reduce the stability of somatostatin mRNA. This effect was observed when we pretreated the cultures with TGFβ1 4 h before the addition of AcD, but not when we administered TGFβ1 simultaneously with AcD or 5,6-dichloro-1β-ribofuranosyl benzimidazole.

Altogether these results demonstrated that the treatment of hypothalamic cells in culture with TGFβ1, TGFβ2, or TGFβ3 resulted in a decrease in somatostatin mRNA levels and somatostatin secretion. TGFβ1 reduced the steady state levels of somatostatin mRNA by inducing the synthesis of a protein (s), that appears to accelerate the degradation of the mRNA of somatostatin. Whether TGFβ1 has additional effects on the transcription of the somatostatin gene will require further study.
Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*
Authors: Bobovnikova Y. , Graves P. N. , Vlase H. , Davies T. F.
Published: January 2014

To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.
In VivoEffects of Leptin-Related Synthetic Peptides on Body Weight and Food Intake in Female ob/obMice: Localization of Leptin Activity to Domains Between Amino Acid Residues 106–140*
Authors: Grasso Patricia , Leinung Matthew C. , Ingher Stacy P. , Lee Daniel W.
Published: January 2014

In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106–167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg ip injections of LEP-(106–120), LEP-(116–130), or LEP-(126–140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106–120), LEP-(116–130), or LEP-(126–140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136–150), LEP-(146–160), or LEP-(156–167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106–120) or LEP-(126–140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116–130) were 3.4% lighter. Food intake by mice receiving LEP-(106–120), LEP-(116–130), or LEP-(126–140), but not by mice receiving LEP-(136–150), LEP-(146–160), or LEP-(156–167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106–140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.
Transcriptional Regulation of Sertoli Cell Immediate Early Genes by Interleukin-6 and Interferon-γ Is Mediated through Phosphorylation of STAT-3 and STAT-1 Proteins*
Authors: Jenab Shirzad , Morris Patricia L.
Published: January 2014

The immediate early genes are regulated by a variety of extracellular signals, including pleiotropic cytokines. The effects of the testicular cytokines, interleukin-6 (IL-6) and interferon-γ (IFN-γ), on signal transducers and activators of transcription 3 and 1 (STAT-3 and STAT-1) and on c-fos gene expression in primary Sertoli cells are suggestive of their roles in differential function. Using the tyrosine phosphorylation inhibitor, genistein, and electrophoretic mobility shift assay, we show that IL-6 and IFN-γ induce nuclear factor STAT-3 and STAT-1 DNA-binding activity to the sis-inducible element of c-fos in a genistein-dependent pathway. Quantitative solution hybridization, Northern blot, and nuclear run-on analysis show that differential induction of c-fos, junB, and c-myc messenger RNA (mRNA) by these cytokines occur at transcriptional levels. IL-6 stimulates c-fos mRNA levels by 6-fold while increasing junB levels by 2-fold. IFN-γ increases c-fos message 2-fold, but has no effect on junB mRNA levels. Furthermore, genistein treatment blocks the induction of c-fos and junB gene expression, demonstrating that tyrosine phosphorylation of STAT proteins is involved in the cytokine regulation of the Sertoli immediate early genes. H7, a serine/threonine phosphorylation inhibitor, also blocks c-fos gene induction by IL-6 and IFN-γ, but does not affect the DNA-binding activities of STAT-3 and STAT-1. Finally, IL-6 treatment of Sertoli cells (3–6 h) increases the amounts of activating protein-1 binding to activating protein-1 element and c-myc transcription.
Vectorial Production of Interleukin 1 and Interleukin 6 by Rat Sertoli Cells Cultured in a Dual Culture Compartment System*
Authors: Cudicini Corinne , Kercret Henri , Touzalin Anne-Marie , Ballet François , Jégou Bernard
Published: January 2014

The bidirectional production of interleukin-1 (IL-1) and IL-6 by Sertoli cells and its regulation by inflammatory and physiological stimuli has been studied using a dual compartment culture system allowing the study of Sertoli cell apical and basal secretory activities. Another Sertoli cell activity, the vectorial transferrin production was also studied in all culture conditions. A low constitutive IL-1 production appeared equally distributed between both poles, while IL-6 and transferrin constitutive production was predominantly directed apically. Two activators of macrophages, lipopolysaccharides and zymosan, were found to induce marked increases of IL-1 in the compartment where they had been added: basal if added to the lower compartment and vice versa. In contrast, after a basal stimulation, IL-6 production was mainly increased in the upper compartment that corresponds to a Sertoli cell apical flux. In this system, IL-1 and IL-6 levels were not modified by FSH; they were not also affected by residual bodies and latex beads, probably due to the fact that, in the bicameral system, phagocytosis is restricted to the Sertoli cells situated at the surface of the inner compartment. IL-1β, but not IL-1α, induced IL-6 secretion in the compartment of stimulation. In conclusion, the present study demonstrates that vectorial secretory patterns of IL-1 and IL-6 production greatly differ and that these cytokines are also differently regulated. These results suggest that Sertoli IL-1 and IL-6 have different targets within the testis and that, in normal and pathophysiological conditions, both the tubular and the interstitial compartments may be influenced by the action of these paracrine factors.
Fetal Sheep Pituitary Proopiomelanocortin in Late Gestation: Effect of Bilateral Lesions of the Paraventricular Nucleus on Regional and Cellular Messenger Ribonucleic Acid Levels*
Authors: Bell M. E. , Myers T. R. , McDonald T. J. , Myers D. A.
Published: January 2014

Previous experiments have clearly indicated that the successful completion of ovine gestation is dependent upon fetal adrenocortical maturation and the associated preterm rise in fetal plasma cortisol. The purposes of this study were to: 1) examine pituitary POMC messenger RNA (mRNA) levels during normal fetal development; and 2) examine the effects of bilateral lesion of the fetal paraventricular nucleus (PVN) on levels and spatial distribution of pituitary POMC mRNA.

Pituitary glands were collected from intact fetal sheep of four gestational ages [100–107 days gestational age (dga), n = 8; 117–121 dga, n = 9; 126–130 dga, n = 9; 144–147 dga, n = 8]. Lesions of the PVN (PVN Lx; n = 4) or sham lesions (Sham; n = 5) were performed at 118–122 dga. Pituitary glands from PVN Lx and Sham fetuses were collected at 139–142 dga (term∼ 147 dga). POMC mRNA levels were determined by in situ hybridization. POMC transcript levels were determined by both regional analysis (20× magnification) and analysis of individual corticotropes (400× magnification).

There was no difference among gestational age groups in superior anterior pituitary (AP) POMC mRNA levels determined by regional or cellular analysis. POMC mRNA levels were significantly greater in the inferior AP at 144–147 dga, compared with other gestational ages, using regional analysis (P = 0.003) or analysis of individual corticotropes (P < 0.01). POMC mRNA levels in the neurointermediate lobe in 126- to 130-dga fetuses were significantly greater than those in younger fetuses (P = 0.005) but not those in 144- to 147-dga fetuses.

There was no difference in POMC mRNA levels in the superior AP between PVN Lx and Sham, using regional analysis or analysis of individual corticotropes. In the inferior AP, there was a significant decrease in POMC mRNA levels in PVN Lx, compared with Sham, using both regional analysis (P < 0.01) and cellular analysis (P < 0.01). There was no difference in POMC mRNA levels in the neurointermediate lobe as the result of bilateral PVN Lx.

Our findings support that basal AP POMC mRNA levels are heterogenously distributed in the ovine fetal AP, with POMC mRNA levels in the inferior AP being significantly greater than in superior AP, by 144–147 dga. We further found that the higher POMC mRNA levels in the inferior AP reflect significantly higher corticotrope POMC transcripts and not simply a greater density of corticotropes in this AP region. The increase in POMC mRNA levels at 144–147 dga in the inferior AP seems unrelated to the onset of adrenocortical maturation (at∼ 125–130 dga). Finally, we report that increase in corticotrope POMC transcripts during late gestation in the inferior AP requires an intact PVN.
Attenuation of Calcitonin Gene Expression in Pregnant Rat Uterus Leads to a Block in Embryonic Implantation*
Authors: Zhu Li-Ji , Bagchi Milan K. , Bagchi Indrani C.
Published: January 2014

The peptide hormone calcitonin plays a key role in calcium homeostasis in many tissues, such as bone and kidney. Our previous studies revealed that the expression of calcitonin is dramatically induced in the glandular epithelium of rat uterus between days 3–5 of pregnancy before the onset of blastocyst implantation on day 5. Calcitonin expression is switched off once implantation has progressed to day 6. The coincidence in timing suggested that calcitonin may function as a regulatory signal in the uterus during the early events leading to implantation. Here we report that the implantation stage-specific expression of calcitonin can be specifically attenuated by administering antisense oligodeoxynucleotides (ODNs) directed against exon IV of calcitonin messenger RNA into the uterine horns on day 2 of gestation. The loss of calcitonin messenger RNA and protein expression upon antisense ODN treatment is accompanied by a severe impairment in implantation of embryos. Based on the observations that 1) treatment with two different antisense ODNs possessing different base compositions produced similar phenotypes; and 2) treatment with the complementary sense ODNs did not affect either calcitonin expression or implantation, we conclude that the effects of antisense ODNs on calcitonin expression and implantation are specific and functionally linked. Our study strengthens the hypothesis that a transient expression of calcitonin in the preimplantation phase uterus is critical for blastocyst implantation.
Development of Adrenal Zonation in Fetal Rats Defined by Expression of Aldosterone Synthase and 11β-Hydroxylase
Authors: Wotus Cheryl , Levay-Young Brett K. , Rogers Lisa M. , Gomez-Sanchez Celso E. , Engeland William C.
Published: January 2014

The adult rat adrenal cortex is comprised of three concentric steroidogenic zones that are morphologically and functionally distinguishable: the zona glomerulosa, zona intermedia, and the zona fasciculata/reticularis. Expression of the zone-specific steroidogenic enzymes, cytochrome P450 aldosterone synthase (P450aldo), and P450 11β hydroxylase (P45011β), produced by the zona glomerulosa and zona fasciculata/reticularis, respectively, can be used to define the adrenal cortical cell phenotype of these two zones. In this study, immunohistochemistry and in situ hybridization were used to determine the ontogeny of expression of P450aldo and P45011β to monitor the pattern of development of the rat adrenal cortex. RIA was used to measure adrenal content of aldosterone and corticosterone, the resulting products of the two enzymatic pathways. Double immunofluorescent staining for both enzymes at gestational day 16 (E16) showed P45011β protein expressed in cells distributed throughout most of the adrenal intermixed with a separate, but smaller, population of cells expressing P450aldo protein. Whereas expression of P45011β protein retained a similar pattern of distribution from E16 to adulthood (ignoring distribution of SA-1 positive, presumptive medullary cells), P450aldo protein changed its pattern of distribution by E19, becoming localized in a discontinuous ring of cells adjacent to the capsule. By postnatal day 1, P450aldo protein distribution was similar to that observed in adult glands; P450aldo-positive cells formed a continuous zone underlying the capsule. In situ hybridization showed that the pattern of P45011β messenger RNA expression paralleled protein expression at all times, whereas P450aldo messenger RNA paralleled protein at E19 and after, but was undetectable before E19. However, adrenal aldosterone and corticosterone, as measured by RIA, were detected by E16, supporting the functional capacity of both phenotypes for all ages studied. These data suggest that the development of the adrenal zona glomerulosa occurs in two distinct phases; initial expression of the glomerulosa phenotype in scattered cells of the inner cortex before E17, followed by a change in distribution to the outer cortex between E17 and E19. It is hypothesized that this change in distribution occurs via cell differentiation, rather than cell migration, and that a possible regulator of these events is the fetal renin-angiotensin system.
Regulation of Insulin-Like Growth Factor-Binding Protein-5 by Insulin-Like Growth Factor I and Interleukin-1α in Ovine Articular Chondrocytes*
Authors: Sunic Damir , McNeil Julian D. , Rayner Timothy E. , Andress Dennis L. , Belford David A.
Published: January 2014

Insulin-like growth factors (IGFs) contribute to the maintenance of the cartilage matrix by stimulating proteoglycan synthesis. In contrast, interleukin-1 (IL-1), an inflammatory cytokine, suppresses the synthesis of proteoglycans. In pathological conditions the chondrocytes’ responsiveness to IGF-I is decreased, and elevated levels of IGF-binding proteins (IGFBPs) have been implicated as a possible cause. The aim of this study was to investigate the effects of IGF-I and IL-1 on IGFBP production by ovine articular chondrocytes (OAC) and the roles of these IGFBPs in the regulation of proteoglycan synthesis. As revealed by Western ligand and immunoblotting, OACs secreted IGFBP-2 and a 24-kDa IGFBP in culture medium under basal conditions. Exposure of the cells to IGF-I for 48 h resulted in the appearance of IGFBP-5 in the medium. Des(1–3)IGF-I, an IGF-I analog with reduced affinity for IGFBPs, also increased the level of IGFBP-5, but to a lesser extent than IGF-I, whereas LR3IGF-I, which has virtually no affinity for IGFBPs, had no effect on IGFBP-5. Furthermore, IGFBP-5 underwent a time-dependent limited proteolysis when incubated with OAC-conditioned medium, degrading into 22- and 16-kDa fragments. The degradation of IGFBP-5 was significantly inhibited by IGF-I, but not by des(1–3)IGF-I or LR3IGF-I. Basic fibroblast growth factor, transforming growth factor-β, and platelet-derived growth factor had no effect on OAC IGFBPs. However, IL-1α increased the IGFBP-5 level in a dose-dependent manner, showing maximum activity at 200 U/ml. Furthermore, IL-1α, but not IGF-I, induced IGFBP-5 messenger RNA expression, as assessed by Northern blot analysis. Coincubation of IGF-I with IL-1α resulted in a substantially increased IGFBP-5 protein level, suggesting a synergism between the mechanisms of action of these two factors. Des(1–3)IGF-I and LR3IGF-I were 10 times more potent than IGF-I in stimulating proteoglycan synthesis, indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1α reduced the IGF-I bioactivity, but had no effect on the activities of the IGF-I analogs, thus implying that locally produced IGFBPs, particularly IGFBP-5, which was substantially increased when IGF-I and IL-1α were coincubated, mediated the reduction of the IGF-I activity. Our results demonstrate that IGF-I and IL-1α synergistically increase the level of IGFBP-5 in OAC by inhibiting the proteolysis and stimulating the expression of IGFBP-5, respectively. Furthermore, the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1α in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that locally produced IGFBPs, in particular IGFBP-5, may play a critical role in the regulation of cartilage matrix degradation in inflammatory and degenerative arthritides.
The Intracerebroventricular Injection of Interleukin-1β Blunts the Testosterone Response to Human Chorionic Gonadotropin: Role of Prostaglandin- and Adrenergic-Dependent Pathways*
Authors: Ogilvie Kathleen , Rivier Catherine
Published: January 2014

The present work extends our previous report that the intracerebroventricular (icv) injection of interleukin-1β (IL-1β, 80 ng) significantly blunted the testosterone response to 1 U/kg human CG (hCG), an effect that we attributed to the stimulation of inhibitory pathways connecting the hypothalamus to the testes. Systemic blockade of prostaglandin-dependent pathways with ibuprofen (α-methyl-4-[2-methyl-propyl]benzeneacetic acid; sodium salt), which did not, in itself, alter the stimulatory effect of hCG on testosterone release in control rats, modestly, but significantly (P < 0.05) reversed the inhibitory influence of IL-1β. In contrast, blockade of brain receptors for CRF was unable to alter the effect of IL-1β, as were lesions of the ventromedial hypothalamic nucleus, a brain area implicated in the control of ovarian function. Blockade of β-adrenergic receptors significantly prevented the decrease in testicular responsiveness induced by the icv injection of IL-1β. Finally, the central injection of the β-adrenergic agonist isoproterenol, as well as that of norepinephrine, mimicked the ability of icv IL-1β to blunt testicular secretory activity and produced a marked (P < 0.01) decrease in the response to hCG within 5 min of their administration.

We propose that the explanation that best fits our findings is that the icv injection of IL-1β activates a neural, catecholamine-dependent pathway that connects the brain and the testes independently of the pituitary.
Daily Melatonin Administration at Middle Age Suppresses Male Rate Visceral Fat, Plasma Leptin, and Plasma Insulin to Youthful Levels
Authors: Rasmussen Dennis D. , Boldt Brian M. , Wilkinson Charles , Yellon Steven M. , Matsumoto Alvin M.
Published: January 2014

Human and rat pineal melatonin secretion decline with aging, wheras visceral fat and plasma insulin levels increase. Melatonin modulates fat metabolism in some mammalian species, so these aging-associated melatonin, fat and insulin changes could be functionally related. Accordingly, we investigated the effects of daily melatonin supplemntation to male Sprague Dawley rats, starting at middle age (10 months) and continuing into old age (22 months). Melatonin was added to the drinking water (92%) of which was consumed at night) at a dose (4μ g/ml) previously reported to attenuate the aging-associated decrease in survival rate in male rats, as well as a 10-fold lower doase. The higher dosage produced nocturnal plasma melatonin levels in middle-aged rats which were 15-fold higher than in young (4 months) rats; nocturnal plasma melatonin levels in middle-aged rats receiving the lower dosage were not significantly different from young or middle-aged controls. Relative (% of body wt) retroperitoneal and epididymal fat, as well as plasma insulin and leptin levels, were all significantly increased at middle age when compared to young rats. All were restored within 10 weeks to youthful (4 months) levels in response to both dosages of melatonin. Continued treatment until old age maintained suppression of visceral (retroperitoneal + epididymal) fat levels. Plasma corticosterone and total thyroxine (T4) levels were not significantly altered by aging or melatonin treatment. Plasma testosterone, insulin-like growth factor i (IGF-I) and total triiodothyrone (T3) decreased by middle age; these aging-associated decreases were not significantly altered by melatonin treatment. Thus, visceral fat, insulin and leptin responses to melatonin administration may be independent of marked changes in gonadal, thyroid, adrenal or somatotropin regulation. Since increased visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggest that appropriate melatonin supplementation may potentially provide prophylaxis or therapy for some prominent pathologies associated with aging.
Activation of the Mitogen-Activated Protein Kinase Cascade Is Necessary But Not Sufficient for Basic Fibroblast Growth Factor- and Epidermal Growth Factor-Stimulated Expression of Endothelial Nitric Oxide Synthase in Ovine Fetoplacental Artery Endothelial Cells*
Authors: Zheng Jing , Bird Ian M. , Melsaether Amy N. , Magness Ronald R.
Published: January 2014

Basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) may play important roles in the placental vasculature, not only by controlling cell growth and differentiation, but also by mediating production of local vasodilators such as nitric oxide. As the mitogen-activated protein kinase (MAPK) signal cascade has been widely associated with cell growth in response to growth factors, herein we investigate whether bFGF, EGF, and VEGF also stimulate expression of endothelial nitric oxide synthase (eNOS) via activation of the MAPK cascade in ovine fetoplacental artery endothelial cells. The presence of the receptors for all three growth factors was confirmed by both immunocytochemistry and a functional cell proliferation assay. All three growth factors at 10 ng/ml rapidly (<10 min) activated MAPK. This activation was inhibited by PD 98059, a specific MAPK kinase inhibitor. bFGF and EGF, but not VEGF, dose- and time-dependently increased eNOS protein levels. Maximal stimulatory effects of bFGF and EGF on eNOS protein expression were observed at 10 ng/ml for 24 h of treatment and were associated with elevated eNOS messenger RNA. PD 98059 also significantly inhibited bFGF- and EGF-induced increases in eNOS protein expression. Because treatment with all three growth factors resulted in activation of the MAPK cascade, while bFGF and EGF, but not VEGF, increased eNOS expression, we conclude that activation of the MAPK cascade is necessary, but not sufficient, for bFGF- and EGF-induced increases in eNOS protein expression in ovine fetoplacental artery endothelial cells. Thus, additional signaling pathways are implicated in the different controls of eNOS expression and mitogenesis by growth factors.
Human Vascular Endothelial Cells Express Oxytocin Receptors*
Authors: Thibonnier Marc , Conarty Doreen M. , Preston Judy A. , Plesnicher Christine L. , Dweik Raed A. , Erzurum Serpil C.
Published: January 2014

Pharmacological studies in humans and animals suggest the existence of vascular endothelial vasopressin (AVP)/oxytocin (OT) receptors that mediate a vasodilatory effect. However, the nature of the receptor subtype(s) involved in this vasodilatory response remains controversial, and its coupled intracellular pathways are unknown. Thus, we set out to determine the type and signaling pathways of the AVP/OT receptor(s) expressed in human vascular endothelial cells (ECs).

Saturation binding experiments with purified membranes of primary cultures of ECs from human umbilical vein (HUVEC), aorta (HAEC), and pulmonary artery (HPAEC) and [3H]AVP or[ 3H]OT revealed the existence of specific binding sites with a greater affinity for OT than AVP (Kd = 1.75 vs. 16.58 nm). Competition binding experiments in intact HUVECs (ECV304 cell line) with the AVP antagonist[ 125I]4-hydroxyphenacetyl-d-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2 or the OT antagonist[ 125I]d(CH2)5[O-Me-Tyr-Thr-Orn-Tyr-NH2]vasotocin, and various AVP/OT analogs confirmed the existence of a single class of surface receptors of the classical OT subtype.

RT-PCR experiments with total RNA extracted from HUVEC, HAEC, and HPAEC and specific primers for the human V1 vascular, V2 renal, V3 pituitary, and OT receptors amplified the OT receptor sequence only. No new receptor subtype could be amplified when using degenerate primers. DNA sequencing of the coding region of the human EC OT receptor revealed a nucleotide sequence 100% homologous to that of the uterine OT receptor reported previously.

Stimulation of ECs by OT produced mobilization of intracellular calcium and the release of nitric oxide that was prevented by chelation of extra- and intracellular calcium. No stimulation of cAMP or PG production was noted. Finally, OT stimulation of ECs led to a calcium- and protein kinase C-dependent cellular proliferation response.

Thus, human vascular ECs express OT receptors that are structurally identical to the uterine and mammary OT receptors. These endothelial OT receptors produce a calcium-dependent vasodilatory response via stimulation of the nitric oxide pathway and have a trophic action.
Developmental Expression and Regulation of Adrenocortical Cytochrome P4501B1 in the Rat*
Authors: Brake Paul B. , Arai Maya , As-Sanie Suzie , Jefcoate Colin R. , Widmaier Eric P.
Published: January 2014

A 57-kDa protein whose expression in rat adrenocortical microsomes is increased after weaning has been identified as cytochrome P4501B1 (CYP1B1). Levels of CYP1B1 protein were moderately expressed in late gestation fetuses and on postnatal day 1 (pd1), but were nearly undetectable on pd6 and pd10. CYP1B1 expression initially increased in the late preweaning period (pd17–19) and again immediately postweaning (pd21–24). The temporal coincidence of CYP1B1 expression and weaning was not due to transition from suckling to solid food, as neonates that were prematurely weaned showed no increase in adrenal CYP1B1 compared with normally weaned littermates. The pattern of CYP1B1 expression paralleled changes in microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), a marker of CYP1B1 activity. Twice daily injections of ACTH to rat pups (pd3–10) failed to significantly increase the expression of CYP1B1 in pd10 adrenals, although the injections weakly stimulated steroidogenesis. Adrenocortical cells from pd17 neonates and adult cells, when cultured for 3 days, responded similarly to ACTH induction, although neonates showed more than 4-fold less basal activity. It is concluded that rat adrenal CYP1B1 may be developmentally suppressed, and its expression is independent of diet or the presence of a dam. This suppression is retained in cell culture, but is not due to deficient ACTH signaling. These results may explain the reported resistance of neonatal rat adrenals to the toxic effects of polycyclic aromatic hydrocarbons, which are metabolized by CYP1B1 into mutagenic by-products.
The Effects of Estrogen and Progesterone on Corticotropin-Releasing Hormone and Arginine Vasopressin Messenger Ribonucleic Acid Levels in the Paraventricular Nucleus and Supraoptic Nucleus of the Rhesus Monkey
Authors: Roy Brenda N. , Reid Robert L. , Van Vugt Dean A.
Published: January 2014

Ovarian steroids increase hypothalamic-pituitary-adrenal (HPA) axis activity and sensitize the hypothalamic-pituitary-ovarian (HPO) axis to stress-induced inhibition. The present study investigated the effect of ovarian steroids on CRH and arginine vasopressin (AVP) messenger RNA (mRNA) levels in the rhesus monkey hypothalamus, as both neuropeptides have been shown to stimulate the HPA axis and inhibit the HPO axis in this species. This was accomplished by measuring CRH and AVP mRNA in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) by in situ hybridization histochemistry. Menstrual cycles were simulated in ovariectomized (OVX) rhesus monkeys by sequential addition and removal of SILASTIC brand (Dow Corning Corp.) tubing containing either 17β-estradiol (E2) or progesterone (P4). On the morning of day 11 of the simulated follicular phase (E2 alone) or day 21 of the luteal phase (E2 + P4), animals were anesthetized, and the brains were perfused with paraformaldehyde via the carotid artery. Coronal sections (30 μm) were cut, and mRNA for CRH and AVP in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) were semiquantified by in situ hybridization. CRH mRNA in the PVN of E2-replaced OVX animals (n = 7) was 2-fold greater than that in untreated OVX controls (n = 4), whereas CRH mRNA after E2 + P4 (n = 4) was no different from that in controls (optical density ± sem, 0.38 ± 0.06, 0.13 ± 0.08, and 0.14 ± 0.09 for OVX + E2, OVX + E2 + P4, and OVX, respectively; P = 0.02). CRH in the SON was undetectable. In contrast to CRH, AVP mRNA in the PVN and the SON was similar in the three treatment groups. We conclude that E2 and E2 + P4 replacement to OVX monkeys exert different effects on CRH and AVP gene expression, as estrogen stimulation of CRH mRNA in the PVN was abrogated by progesterone, whereas no effect of ovarian steroids on AVP mRNA in either the PVN or SON was observed. We postulate that ovarian steroid regulation of CRH synthesis and release may in part explain the central nervous system mechanisms by which ovarian steroids affect the HPA and HPO axes during basal and stress conditions.
Localization, Regulation and Possible Consequences of Apoptotic Protease-Activating Factor-1 (Apaf-1) Expression in Granulosa Cells of the Mouse Ovary
Authors: Robles Rodolfo , Tao Xiao-Jing , Trbovich Alexander M. , Maravei Daniel V. , Nahum Ravit , Perez Gloria I. , Tilly Kim I. , Tilly Jonathan L.
Published: January 2014

The recent characterization of apoptotic protease-activating factor-1 (Apaf-1) in vertebrates as a putative homolog of the Caenorhabditis elegans gene, ced-4, indicates that the third major arm of the C. elegans programmed cell death machinery has also been conserved through evolution. Although apoptosis is now known to be important for ovarian follicular atresia in vertebrates, nothing is known of the role of Apaf-1 in ovarian function. Herein we show by immunohistochemical analysis that Apaf-1 is abundant in granulosa cells of early antral follicles whereas in vivo gonadotropin priming completely suppresses Apaf-1 expression and granulosa cell apoptosis. Western blot analysis of fractionated protein extracts prepared from granulosa cells before and after in vitro culture without hormonal support to induce apoptosis indicated that mitochondrial cytochrome c release, a biochemical step required for the activation of Apaf-1, occurs in granulosa cells cultured in vitro. Moreover, Western blot analysis of procaspase-3 processing, a principal downstream event set in motion by activated Apaf-1, indicated that healthy granulosa cells possess almost exclusively the inactive (pro-) form of the enzyme whereas granulosa cells deprived of hormonal support rapidly process procaspase-3 to the active enzyme. Lastly, we show that serum-starved granulosa cells activate caspase-3-like enzymes both prior to and after nuclear pyknosis, as revealed by a single-cell fluorescent caspase activity assay. These data, combined with previous observations regarding the role of homologs of the two other C. elegans cell death regulatory genes, ced-9 (Bc1-2 family members) and ced-3 (caspases), in atresia fully support the hypothesis that granulosa cell apoptosis is precisely coordinated by all three major arms of a cell death program conserved through evolution.
Transcriptional Activation of the Decidual/Trophoblast Prolactin-Related Protein Gene1
Authors: Orwig Kyle E. , Soares Michael J.
Published: January 2014

The decidual/trophoblast PRL-related protein (d/tPRP) is dually expressed by decidual and trophoblast cells during pregnancy. We have characterized the proximal d/tPRP promoter responsible for directing d/tPRP expression in decidual and trophoblast cells. We have demonstrated that the proximal 93 bp of d/tPRP 5′-flanking DNA are sufficient to direct luciferase gene expression in primary decidual and Rcho-1 trophoblast cells, but not in fibroblast, undifferentiated uterine stromal cells or trophoblast cells of a labyrinthine lineage. The 93-bp d/tPRP promoter was also sufficient to direct differentiation-dependent expression in trophoblast giant cells. Mutational analysis demonstrated the differential importance of activating protein-1 and Ets regulatory elements (located within the proximal 93 bp of d/tPRP 5′-flanking DNA) for activation of the d/tPRP promoter in decidual vs. trophoblast cells. Disruption of the activating protein-1 regulatory element inhibited d/tPRP promoter activity by more than 95% in decidual cells, and approximately 80% trophoblast cells. Disruption of the Ets regulatory element reduced d/tPRP promoter activity by approximately 50% in decidual cells, while inactivating the d/tPRP promoter in trophoblast cells. Protein interactions with the trophoblast Ets regulatory element were shown to be cell type specific and to change during trophoblast giant cell formation. In conclusion, a 93-bp region of the d/tPRP promoter is shown to contain regulatory elements sufficient for gene activation in decidual and trophoblast cells.
Is the Early Increase in Leptinemia One of the Anorectic Signals Induced by an Essential Amino Acid-Deficient Diet in the Rat?
Authors: Feurté Sebastien , Nicolaïdis Stylianos , Gerozissis Kyriaki
Published: January 2014

Rats start decreasing their food intake as early as 70 min after the first ingestion of a food deficient in threonine. A decrease of the limiting essential amino acid (EAA) in the plasma was proposed to be the first anorectic signal. Because many hormones regulate feeding behavior, we studied the effect of a meal (46 kJ) that was either devoid of threonine or was corrected for the deficiency, on plasma leptin, insulin and glucagon levels using a radio-immunoassay, at 0 to 180 min after the meal. One hour after ingestion of the threonine-devoid meal, a larger increase in insulinemia (22±1 vs. 15±1 μU/ml) and leptinemia (7.8±0.5 vs. 4.4±0.6 ng/ml; p<0.001) was observed than after ingestion of the corrected meal. The area under the curve of the threonine-devoid meal group was 3 and 1.34 fold larger than for the corrected meal group for insulin and leptin respectively. Glucagonemia was not different between the two groups. We propose that the rise in leptinemia, perhaps in synergy with rise in plasma insulin, might serve as one early signal to brain structures, participating in the anorectic mechanism following ingestion of an EAA-deficient diet.
Existence of Galanin in Lumbosacral Sympathetic Ganglionic Neurons That Project to the Quail Uterine Oviduct*
Authors: Sakamoto Hirotaka , Ubuka Takayoshi , Kohchi Chie , Li Dan , Ukena Kazuyoshi , Tsutsui Kazuyoshi
Published: January 2014

Oviposition in birds is conducted by vigorous contractions of the uterine oviduct. We recently isolated an oviposition-inducing peptide that was identified as avian galanin from mature quail oviducts. This peptide was localized in neuronal fibers terminating in muscle layers in the uterine oviduct and evoked vigorous uterine contractions through binding to receptors located in the uterus. However, no cell bodies that express avian galanin were detected in the uterus or other oviduct regions. To understand the control mechanism of avian oviposition by galanin, we identified the neurons that synthesize galanin and project to the uterus with the combination of retrograde labeling with neurobiotin and immunocytochemistry for galanin in mature Japanese quails. Retrograde labeling with neurobiotin from the uterus revealed that lumbosacral sympathetic ganglionic neurons located in the uterine side projected their axons to the uterine muscle layer. Abundant elementary granules were observed in somata of the retrogradely labeled sympathetic ganglionic neurons, suggesting that labeled neurons may function as a neurosecretory cell. Immunocytochemical analysis with the antiserum against avian galanin showed an intense immunoreaction restricted to somata of the retrograde-labeled ganglionic neurons. Preabsorbing the antiserum with avian galanin resulted in a complete absence of the immunoreaction. Competitive enzyme-linked immunosorbent assay using antigalanin serum confirmed that avian galanin existed in the sympathetic ganglionic neurons. Expression of the avian galanin messenger RNA in the neurons was further verified by Northern blot analysis. In addition, both avian galanin and its messenger RNA in the neurons were highly expressed in mature birds, unlike in immature birds.

These results suggest that lumbosacral sympathetic ganglionic neurons innervating the uterine muscle produce avian galanin in mature birds. Because this peptide acts directly on the uterus to evoke oviposition through a mechanism of the induction of vigorous uterine contraction, galaninergic innervation of the uterine oviduct may be essential for avian oviposition.
Adrenergic Regulation of Mitogen-Activated Protein Kinase in Rat Pinealocytes: Opposing Effects of Protein Kinase A and Protein Kinase G**This work was supported by grants from the Medical Research Council of Canada.
Authors: Ho A. K. , Chik C. L.
Published: January 2014

The role of adrenergic stimulation in the regulation of mitogen-activated protein kinase (MAPK) in rat pinealocytes was investigated by measuring phosphorylated MAPK using Western blot analysis and a MAPK enzymatic assay. Stimulation with the endogenous neurotransmitter, norepinephrine (NE; a mixed α- and β-adrenergic agonist), concentration dependently increased the phosphorylation of both p44 and p42 isoforms of MAPK. This effect of NE was blocked by PD98059 and UO126 (two inhibitors of MEK). Treatment with prazosin or propranolol significantly reduced the effect of NE on MAPK phosphorylation, suggesting the involvement of both α- andβ -adrenergic receptors. Investigation into the intracellular mechanisms of NE action revealed that the increase in MAPK phosphorylation was blocked by KT5823 (a protein kinase G inhibitor), but was enhanced by H89 (a protein kinase A inhibitor). Calphostin C (a protein kinase C inhibitor) and KN93 (a Ca2+/calmodulin-dependent protein kinase inhibitor) also attenuated NE-mediated MAPK activation, but to a lesser degree. Furthermore, inhibition of MAPK phosphorylation by (Bu)2cAMP was effective in reducing MAPK activation by (Bu)2cGMP, an active phorbol ester or ionomycin. These results indicate that the effect of NE on MAPK phosphorylation represents mainly the integration of two signaling mechanisms, protein kinase A and protein kinase G, each having an opposite effect on MAPK phosphorylation.