bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Pancreatic Islet Blood Flow in 48-Hour Glucose-Infused Rats: Involvement of Central and Autonomic Nervous Systems*
Authors: Atef Nadia , Laury Marie-Claude , N’Guyen Jean-Michel , Mokhtar Najad , Ktorza Alain , Penicaud Luc
Published: January 2014

The pancreatic islet blood flow of rats 24 h after a prolonged (48-h) glucose infusion was investigated using a nonradioactive microsphere technique. In the basal state, islet blood flow was significantly increased in previously hyperglycemic rats (HG) compared to that in controls (C). During an iv glucose challenge, both plasma insulin and islet blood flow were increased in the two groups, but these increases were significantly higher in HG than in C rats. Although less pronounced, the results were similar when glucose was injected into the carotid artery toward the brain at a dose that did not modify the peripheral glucose level. The effect of this intracarotid injection was abolished after bilateral subdiaphragmatic vagotomy in both C and HG rats. Furthermore, in the latter group, both plasma insulin concentration and islet blood flow returned to values similar to those observed in the basal state in C rats. After pretreatment with the α2-adrenoceptor agonist clonidine, the insulin response to the intracarotid glucose load was totally blunted in the two groups of rats. By contrast, whereas such a pretreatment lowered the glucose-induced increase in islet blood flow in C rats, it was without effect in HG rats. These data suggest that a period of hyperglycemia and/or hyperinsulinemia is sufficient to induce a perturbation of pancreatic islet blood flow, which appears to be mainly due to an increased parasympathetic activity, whereas the decrease in sympathetic tone does not play a role. These modifications in autonomic nervous system activity could be due to alterations in some brain areas involved in “glucose sensing.”
Potential Role of Protein Kinase B in Glucose Transporter 4 Translocation in Adipocytes*
Authors: Tanti Jean-François , Grillo Sophie , Grémeaux Thierry , Coffer Paul J. , Van Obberghen Emmanuel , Le Marchand-Brustel Yannick
Published: January 2014

Phosphatidylinositol 3-kinase (PI 3-kinase) activation promotes glucose transporter 4 (Glut 4) translocation in adipocytes. In this study, we demonstrate that protein kinase B, a serine/threonine kinase stimulated by PI 3-kinase, is activated by both insulin and okadaic acid in isolated adipocytes, in parallel with their effects on Glut 4 translocation. In 3T3-L1 adipocytes, platelet-derived growth factor activated PI 3-kinase as efficiently as insulin but was only half as potent as insulin in promoting protein kinase B (PKB) activation. To look for a potential role of PKB in Glut 4 translocation, adipocytes were transfected with a constitutively active PKB (Gag-PKB) together with an epitope tagged transporter (Glut 4 myc). Gag-PKB was associated with all membrane fractions, whereas the endogenous PKB was mostly cytosolic. Expression of Gag-PKB led to an increase in Glut 4 myc amount at the cell surface. Our results suggest that PKB could play a role in promoting Glut 4 appearance at the cell surface following exposure of adipocytes to insulin and okadaic acid stimulation.
Cloning and Characterization of the Vitamin D Receptor from Xenopus laevis*
Authors: Li Yan Chun , Bergwitz Clemens , Jüppner Harald , Demay Marie B.
Published: January 2014

The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species.

The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor α, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (preneurulation), increasing to maximum at stages 57–61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.
Direct Administration of Insulin-Like Growth Factor to Fetal Rhesus Monkeys (Macaca mulatta)*
Authors: Tarantal Alice F. , Hunter Maya K. , Gargosky Sharron E.
Published: January 2014

A potential treatment for the amelioration of fetal growth failure is insulin-like growth factor-I (IGF-I). To address concerns of safety and efficacy, IGF-I (80 μg/kg; GroPep Pty.) was administered ip to healthy rhesus monkey fetuses via ultrasound guidance every other day between gestational days (GD) 110–120 and 130–140 (third trimester; term = approximately GD 165 ± 10; n = 6). Pregnancies were monitored sonographically, and fetal/maternal blood samples were collected for complete blood counts, immunophenotyping, and biochemical analyses. Blood samples, external measures of the fetus and newborn, and tissue and organ weights were collected at fetal necropsy (GD 150; n = 2) or at term delivery of neonates (GD 160; n = 4). The results of these investigations have shown no evidence of hypoglycemia in the fetus or dam during the course of treatment. Circulating concentrations of fetal, but not maternal, IGF-I increased with treatment (∼80 to ∼1015 ng/ml), and there was no evidence of a change in serum IGF-II or an increase in IGF binding protein-3 compared with historical control values. Fetal lymphocytes and select red cell parameters increased, and a significant elevation in circulating B cells and CD4/CD8 ratios in fetal lymph nodes was shown. Although no changes were detected in body weights, increases in thymic, splenic, and kidney weights and small intestine lengths occurred. Thus, administration of IGF-I to the fetal monkey is safe and results in 1) transient increases in circulating IGF-I, 2) a significant effect on fetal hematopoietic and lymphoid tissues, and 3) an increase in select fetal organ weights and measures. These data suggest that IGF-I may represent a potential candidate for therapeutic treatment of growth-compromised human fetuses in utero.
Pheromone Regulated Production of Inositol-(1, 4, 5)-Trisphosphate in the Mammalian Vomeronasal Organ*
Authors: Wekesa Kennedy S. , Anholt Robert R. H.
Published: January 2014

Social behaviors of most mammals are profoundly affected by chemical signals, pheromones, exchanged between conspecifics. Pheromones interact with dendritic microvilli of bipolar neurons in the vomeronasal organ (VNO). To investigate vomeronasal signal transduction pathways, microvillar membranes from porcine VNO were prepared. Incubation of such membranes from prepubertal females with boar seminal fluid or urine results in an increase in production of inositol-(1, 4, 5)-trisphosphate (IP3). The dose response for IP3 production is biphasic with a GTP-dependent component at low stimulus concentrations and a nonspecific increase in IP3 at higher stimulus concentrations. The GTP-dependent stimulation is mimicked by GTPγS and blocked by GDPβS. Furthermore, the GTP-dependent component of the stimulation of IP3 production is sex specific and tissue dependent. Studies with monospecific antibodies reveal a Gαq/11-related protein in vomeronasal neurons, concentrated at their microvilli. Our observations indicate that pheromones in boar secretions act on vomeronasal neurons in the female VNO via a receptor mediated, G protein-dependent increase in IP3. These observations set the stage for further investigations on the regulation of stimulus-excitation coupling in vomeronasal neurons. The pheromone-induced IP3 response also provides an assay for future purification of mammalian reproductive pheromones.
Effects of Nitric Oxide on Ovulation and Ovarian Steroidogenesis and Prostaglandin Production in the Rabbit*
Authors: Yamauchi Jun , Miyazaki Toyohiko , Iwasaki Shinya , Kishi Ikuko , Kuroshima Masako , Tei Chisei , Yoshimura Yasunori
Published: January 2014

Evidence supports the involvement of nitric oxide (NO) in ovarian physiology. The present study was undertaken to investigate the role of the NO/NO synthase (NOS) systems in ovulation, oocyte maturation, ovarian steroidogenesis, and PG production using in vitro perfused rabbit ovaries. The addition of the NOS inhibitors, aminoguanidine hemisulfate salt (AG) and N-omega-nitro-l-arginine methyl ester (L-NAME), to the perfusate inhibited the ovulation induced by hCG in a dose-dependent manner, whereas D-NAME had no significant effect. Neither AG nor L-NAME affected the hCG-induced meiotic maturation of the ovulated ova. The exogenous administration of the NO generator, sodium nitroprusside (NP), induced follicle rupture in the absence of gonadotropin, but did not induce oocyte maturation. Inhibition of endogenous NOS by AG and L-NAME resulted in a significant elevation in the production of estradiol (E2), but not of progesterone, stimulated by hCG. The concomitant administration of NP significantly reduced the AG-stimulated production of E2 by ovaries perfused in the presence of hCG, which suggests that NO down-regulates ovarian E2 synthesis. Ovarian production of PGE2 and PGF2α in response to hCG was significantly blocked by L-NAME, and exogenous administration of NP stimulated the production of PGs in the absence of gonadotropin. Significant correlations were observed between the ovulatory efficiencies and the production of PGs by rabbit ovaries perfused with or without L-NAME. In conclusion, the ovarian NO/NOS system is involved in follicle rupture during the ovulatory process. NO may induce follicle rupture in rabbit ovaries at least in part by the stimulation of PG production.
Oncogene Transformation of PC Cl3 Clonal Thyroid Cell Line Induces an Autonomous Pattern of Proliferation That Correlates with a Loss of Basal and Stimulated Phosphotyrosine Phosphatase Activity*
Authors: Florio Tullio , Scorziello Antonella , Thellung Stefano , Salzano Salvatore , Berlingieri Maria Teresa , Fusco Alfredo , Schettini Gennaro
Published: January 2014

The effects of the stable expression of E1A and/or middle T oncogenes on the proliferative activity of PC Cl3 normal thyroid cells are reported. The proliferation of PC Cl3 cells is mainly regulated by insulin and TSH in a stimulatory way and by somatostatin in an inhibitory fashion. The transformed cell lines, named PC Py and PC E1A Py, show an autonomous pattern of proliferation. The blockade of phosphotyrosine phosphatase activity with vanadate increased the proliferation rate of PC Cl3 under basal and stimulated conditions and completely prevented the inhibitory activity of somatostatin, suggesting that in PC Cl3 cells, a tonic tyrosine phosphatase activity regulates basal and stimulated proliferation, and that a somatostatin-dependent increase in this activity may represent a cytostatic signal. Conversely, in both PC Py and PC E1A Py, vanadate did not modify basal and stimulated proliferation. We analyzed tyrosine phosphatase activity in the different cell lines basally and under conditions leading to the arrest of cell proliferation: confluence (contact inhibition), growth factor deprivation (starvation), and somatostatin treatment. Under basal conditions, tyrosine phosphatase activity was significantly lower in PC Py and PC E1APy cell lines than that in the normal cells. The inhibition of the proliferation induced by contact inhibition or somatostatin treatment was accompanied by an increase in tyrosine phosphatase activity only in PC Cl3 cells. The reduction in tyrosine phosphatase activity in PC E1APy cells correlated with a significant reduction in the expression of R-PTPη, a tyrosine phosphatase cloned from PC Cl3 cells. Conversely, the expression of another receptor-like PTP, PTPμ, was unchanged. Thus, PTPη may be a candidate to mediate inhibitory signals (i.e. activation of somatostatin receptors or cell to cell contact) on the proliferative activity of PC Cl3 cells, and the reduction of its expression in the transformed cell lines may lead to an alteration in the control of cell proliferation.
Desensitization of AT1 Receptor-Mediated Cellular Responses Requires Long Term Receptor Down-Regulation in Bovine Adrenal Glomerulosa Cells*
Authors: Richard Darren E. , Laporte Stéphane A. , Bernier Sylvie G. , Leduc Richard , Guillemette Gaétan
Published: January 2014

Angiotensin II (Ang II) regulates aldosterone production in bovine adrenal glomerulosa cells by interacting with the AT1 receptor. This receptor is coupled to a G protein that controls the activity of phospholipase C. With a primary culture of bovine adrenal glomerulosa cells, we evaluated the desensitization of cellular responses after pretreatment with Ang II. When cells were pretreated for 30 min with 1 μm Ang II at 37 C, we observed a 48% loss of [125I]Ang II-binding activity. Scatchard analysis revealed that this decreased binding activity corresponded to a 53% loss of the total number of binding sites. This phenomenon was time dependent, with a t1/2 of 20 min, and a maximal loss of 76% of the total binding sites was observed after 14 h. A time-dependent decrease in AT1 receptor messenger RNA levels was also observed after pretreatment with 1 μm Ang II for 12–24 h. Taken together, these results are interpreted as a down-regulation of the AT1 receptor. Desensitization of phospholipase C activity under similar conditions was, however, a slower process, with a t1/2 of 9 h and a maximal response reduction of 83% observed after 24 h. Dose-response experiments indicated that maximal phospholipase C desensitization was obtained in the presence of 1 μm Ang II, with an EC50 of 90 nm. The desensitization was of a homologous nature, as a 24-h pretreatment with Ang II did not affect bradykinin-induced inositol phosphate production. A 24-h pretreatment with 1 μm Ang II also significantly desensitized the steroidogenic effect of Ang II and the potentiating effect of Ang II on ACTH-induced cAMP production. Lower concentrations of Ang II (10 nm) did not produce any desensitizing effect on these two parameters. This study provides evidence that glomerulosa cells are functionally resistant to short term desensitization of the AT1 receptor and that long term down-regulation with high concentrations of Ang II is needed to desensitize AT1-mediated cellular responses.
Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice
Authors: Carpenter Thomas O. , Moltz Kathleen C. , Ellis Bruce , Andreoli Monica , McCarthy Thomas L. , Centrella Michael , Bryan Duane , Gundberg Caren M.
Published: January 2014

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts.

Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17–25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response.

Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.
Macroorchidism in FMR1 Knockout Mice Is Caused by Increased Sertoli Cell Proliferation during Testicular Development*
Authors: Slegtenhorst-Eegdeman Karin E. , de Rooij Dirk G. , Verhoef-Post Miriam , van de Kant Henk J. G. , Bakker Cathy E. , Oostra Ben A. , Grootegoed J. Anton , Themmen Axel P. N.
Published: January 2014

The fragile X syndrome is the most frequent hereditary form of mental retardation. This X-linked disorder is, in most cases, caused by an unstable and expanding trinucleotide CGG repeat located in the 5′-untranslated region of the gene involved, the fragile X mental retardation 1 (FMR1) gene. Expansion of the CGG repeat to a length of more than 200 trinucleotides results in silencing of the FMR1 gene promoter and, thus, in an inactive gene.

The clinical features of male fragile X patients include mental retardation, autistiform behavior, and characteristic facial features. In addition, macroorchidism is observed. To study the role of Sertoli cell proliferation and FSH signal transduction in the occurrence of macroorchidism in fragile X males, we made use of an animal model for the fragile X syndrome, an Fmr1 knockout mouse.

The results indicate that in male Fmr1 knockout mice, the rate of Sertoli cell proliferation is increased from embryonic day 12 to 15 days postnatally. The onset and length of the period of Sertoli cell proliferation were not changed compared with those in the control males. Serum levels of FSH, FSH receptor messenger RNA expression, and short term effects of FSH on Sertoli cell function, as measured by down-regulation of FSH receptor messenger RNA, were not changed.

We conclude that macroorchidism in Fmr1 knockout male mice is caused by an increased rate of Sertoli cell proliferation. This increase does not appear to be the result of a major change in FSH signal transduction in Fmr1 knockout mice.
Effects of Castration and Chronic Steroid Treatments on Hypothalamic Gonadotropin-Releasing Hormone Content and Pituitary Gonadotropins in Male Wild-Type and Estrogen Receptor-α Knockout Mice
Authors: Lindzey Jonathan , Wetsel William C. , Couse John F. , Stoker Tammy , Cooper Ralph , Korach Kenneth S.
Published: January 2014

Testicular androgens are integral components of the hormonal feedback loops that regulate circulating levels of LH and FSH. The sites of feedback include hypothalamic areas regulating GnRH neurons and pituitary gonadotropes. To better define the roles of androgen receptor (AR), estrogen receptor-α (ERα), and estrogen receptor-β (ERβ) in mediating feedback effects of sex steroids on reproductive neuroendocrine function, we have determined the effects of castration and steroid replacement therapy on hypothalamic GnRH content, pituitary LHβ and FSHβ messenger RNA (mRNA) levels, and serum gonadotropins in male wild-type (WT) and estrogen receptor-α knockout (ERKO) mice. Hypothalami from intact WT and ERKO males contained similar amounts of GnRH, whereas castration significantly reduced GnRH contents in both genotypes. Replacement therapy with estradiol (E2), testosterone (T), or dihydrotestosterone (DHT) restored hypothalamic GnRH content in castrated (CAST) WT mice; only the androgens were effective in CAST ERKOs.

Analyses of pituitary function revealed that LHβ mRNA and serum LH levels in intact ERKOs were 2-fold higher than those in intact WT males. Castration increased levels of LHβ mRNA (1.5- to 2-fold) and serum LH (4- to 5-fold) in both genotypes. Both E2 and T treatments significantly suppressed LHβ mRNA and serum LH levels in CAST WT males. However, E2 was completely ineffective, and T was only partially effective in suppressing these two indexes in the CAST ERKO males. DHT treatments stimulated a 50% increase in LHβ mRNA and serum LH levels in WT males, whereas serum LH was significantly suppressed in DHT-treated ERKO males.

Although the pituitaries from intact ERKO males contained similar amounts of FSHβ mRNA, serum FSH levels were 20% higher than those in the intact WT males. Castration increased FSHβ mRNA levels only in WT males, but significantly increased serum FSH levels in both genotypes. Both E2 and T treatments significantly suppressed serum FSH in CAST WT males, whereas only E2 suppressed FSHβ mRNA. DHT treatments of CAST WT mice stimulated a small increase in serum FSH, but failed to alter FSHβ mRNA levels. None of the steroid treatments exerted any significant effect on FSHβ mRNA or serum FSH levels in CAST ERKOs.

These data suggest that hypothalamic GnRH contents can be maintained solely through AR signaling pathways. However, normal regulation of gonadotrope function requires aromatization of T and activation of ERα signaling pathways in the gonadotrope. In addition, serum FSH levels in male ERKOs appear to be regulated largely by nonsteroidal testicular factors such as inhibin. Finally, these data suggest that hypothalamic ERβ may not be involved in mediating the negative feedback effects of T on serum LH and FSH in male mice.
The New Antidiabetic Drug MCC-555 Acutely Sensitizes Insulin Signaling in Isolated Cardiomyocytes**This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit, EU COST Action B5, and a grant from Mitsubishi Chemical (Yokohama, Japan).
Authors: Liu Li Sen , Tanaka Hideho , Ishii Shinichi , Eckel Jürgen
Published: January 2014

Freshly isolated adult rat ventricular cardiomyocytes have been used to characterize the action profile of the new thiazolidinedione antidiabetic drug MCC-555. Preincubation of cells with the compound (100 μm for 30 min or 10 μm for 2 h) did not modify basal 3-O-methylglucose transport, but produced a marked sensitizing effect (2- to 3-fold increase in insulin action at 3 × 10−11m insulin) and a further enhancement of maximum insulin action (1.8-fold). MCC-555 did not modulate autophosphorylation of the insulin receptor and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1). However, insulin action (10−10 and 10−7m) on IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was enhanced 2-fold in the presence of MCC-555. Association of the p85 adapter subunit of PI 3-kinase to IRS-1 was not modified by the drug. Immunoblotting experiments demonstrated expression of the peroxisomal proliferator-activated receptor-γ in cardiomyocytes reaching about 30% of the abundance observed in adipocytes. The insulin-sensitizing effect of MCC-555 was lost after inhibition of protein synthesis by preincubation of the cells with cycloheximide (1 mm; 30 min). Cardiomyocytes from obese Zucker rats exhibited a completely blunted response of glucose transport at 3 × 10−11m insulin. MCC-555 ameliorates this insulin resistance, producing a 2-fold stimulation of glucose transport, with maximum insulin action being 1.6-fold higher than that in control cells. This drug effect was paralleled by a significant dephosphorylation of IRS-1 on Ser/Thr. In conclusion, MCC-555 rapidly sensitizes insulin-stimulated cardiac glucose uptake by enhancing insulin signaling resulting from increased intrinsic activity of PI 3-kinase. Acute activation of protein expression leading to a modulation of the Ser/Thr phosphorylation state of signaling proteins such as IRS-1 may be underlying this process. It is suggested that MCC-555 may provide a causal therapy of insulin resistance by targeted action on the defective site in the insulin signaling cascade.
A Winged-Helix Family Member Is Involved in a Steroid Hormone-Triggered Regulatory Circuit**This work was supported by NIH Grants RO1-DK-40082 (to M.M.S.) and T32-DK-0703 (to D.M.D.).
Authors: Dean Diane M. , Berger Ryan R. , Sanders Michel M.
Published: January 2014

A common theme emerging in eukaryotic gene regulation is that maximal gene induction requires several transcription factors acting in concert to regulate the activation of critical genes. Increasingly, nuclear receptors play key roles in orchestrating this regulation, often by integrating additional signaling pathways, through complex regulatory elements known as hormone response units. The ovalbumin gene contains one such unit, known as the steroid-dependent regulatory element. The binding of the chicken ovalbumin induced regulatory protein-I (Chirp-I) to this element occurs only in response to treatment with estrogen and glucocorticoid. Evidence presented herein demonstrates that Chirp-I has many features in common with the winged-helix (W-H) family of transcription factors. The binding sites for Chirp-I and for the W-H proteins have similar sequence recognition requirements. Northern blots establish that members of the W-H family are expressed in oviduct. Most convincing, the Chirp-I complex interacts with two different antibodies specific to W-H family members. The culmination of this work supports the hypothesis that Chirp-I is a member of the W-H family, and it lends credence to the idea that W-H proteins are essential components of some steroid hormone regulatory circuits.
Interspecies Differences in Bone Composition, Density, and Quality: Potential Implications for in Vivo Bone Research*
Authors: Aerssens Jeroen , Boonen Steven , Lowet Geert , Dequeker Jan
Published: January 2014

This study compares bone composition, density, and quality in bone samples derived from seven vertebrates that are commonly used in bone research: human, dog, pig, cow, sheep, chicken, and rat. Cortical femoral bone samples were analyzed for their content of ash, collagen, extractable proteins, and insulin-like growth factor-I. These parameters were also measured in bone powder fractions that were obtained after separation of bone particles according to their density. Large interspecies differences were observed in all analyses. Of all species included in the biochemical analyses, rat bone was most different, whereas canine bone best resembled human bone. In addition, bone density and mechanical testing analyses were performed on cylindrical trabecular bone cores. Both analyses demonstrated large interspecies variations. The lowest bone density and fracture stress values were found in the human samples; porcine and canine bone best resembled these samples. The relative contribution of bone density to bone mechanical competence was largely species-dependent. Together, the data reported here suggest that interspecies differences are likely to be found in other clinical and experimental bone parameters and should therefore be considered when choosing an appropriate animal model for bone research.
Dynamic Changes in Prolactin Promoter Activation in Individual Living Lactotrophic Cells
Authors: Takasuka N. , White M. R. H. , Wood C. D. , Robertson W. R. , Davis J. R. E.
Published: January 2014

The firefly luciferase gene has become widely used as a convenient reporter for studies of gene promoter regulation. Very recently, the development of ultralow-light imaging cameras has enabled the quantitative digital imaging of light signals resulting from luciferase activation in the presence of luciferin substrate. We have applied this technology to the study of PRL promoter activation in individual pituitary tumor cells to study the temporal and spatial characteristics of the expression of a well-characterized pituitary hormone gene.

Rat pituitary GH3 cells were transfected by lipofection with a luciferase reporter gene linked to 5000 bp from the human PRL gene 5′-flanking region. A series of stably transfected cell clones were generated, and one of these was chosen for detailed study on the basis of appropriate regulation of high-level luciferase expression by a series of known stimuli including TRH, forskolin, the calcium channel agonist Bay K8644, and basic fibroblast growth factor (bFGF). These cells were subjected to direct imaging of luciferase activity using a Hamamatsu photon-counting camera linked to a Zeiss Axiovert microscope with an Argus-50 image processor. Cells were exposed to 1 mm luciferin, and images were integrated over 30-min periods for up to 72 h. The total photon count over a given field settled to steady levels within 10 h and then remained constant for over 55 h. Addition of forskolin, TRH, or bFGF increased the total photon count of fields of 20–100 cells by 2- to 4-fold consistent with previous data from transient expression assays using the human PRL promoter. Individual cells, on the other hand, showed marked marked temporal and spatial heterogeneity and variability of luciferase expression when studied at 3-h intervals. Unstimulated cells showed variable luciferase expression with up to 40-fold excursions in photon counts per single cell area within 12-h periods. Stimulation of cells with either TRH, forskolin, or bFGF resulted in smooth increases in photon output over fields of 20–100 cells, but again individual cell responses differed widely, with some cells showing slow progressive rises in photon output, others showing phasic or transient responses, and yet others showing no response.

In conclusion, we found a surprising degree of heterogeneity and temporal variability in the level of gene expression in individual living pituitary tumor cells over long periods of time, with markedly divergent responses to hormonal or intracellular stimulation. The use of stably transfected clonal cell lines with extended periods of reporter gene imaging offers a valuable insight into control of gene expression in living cells in real time.
Histomorphometric Evidence for Echistatin Inhibition of Bone Resorption in Mice with Secondary Hyperparathyroidism
Authors: Masarachia Patricia , Yamamoto Michiko , Leu Chih-Tai , Rodan Gideon , Duong Le
Published: January 2014

Echistatin, an RGD-containing peptide, was shown to inhibit the acute calcemic response to exogenous PTH or PTH-related protein (PTH-rP) in thyroparathyroidectomized rats, suggesting that echistatin inhibits bone resorption. In this study: 1) we present histological evidence for echistatin inhibition of bone resorption in mice with secondary hyperparathyroidism, and show that 2) echistatin binds to osteoclasts in vivo, 3) increases osteoclast number, and 4) does not detectably alter osteoclast morphology. Infusion of echistatin (30μ g/kg·min) for 3 days prevented the 2.6-fold increase in tibial cancellous bone turnover and the 36% loss in bone volume, produced by a low calcium diet. At the light microscopy level, echistatin immunolocalized to osteoclasts and megakaryocytes. Echistatin treatment increased osteoclast-covered bone surface by about 50%. At the ultrastructural level, these osteoclasts appeared normal, and the fraction of cells containing ruffled borders and clear zones was similar to controls. Echistatin was found on the basolateral membrane and in intracellular vesicles of actively resorbing osteoclasts. Weak labeling was found in the ruffled border, and no immunoreactivity was detected at the clear zone/bone surface interface. These findings provide histological evidence for echistatin binding to osteoclasts and for inhibition of bone resorption in vivo, through reduced osteoclast efficacy, without apparent changes in osteoclast morphology.
Nutrient Augmentation of Ca2+-Dependent and Ca2+-Independent Pathways in Stimulus-Coupling to Insulin Secretion Can Be Distinguished by Their Guanosine Triphosphate Requirements: Studies on Rat Pancreatic Islets*
Authors: Komatsu Mitsuhisa , Noda Mitsuhiko , Sharp Geoffrey W. G.
Published: January 2014

To delineate the underlying mechanisms by which glucose augments both Ca2+-dependent and Ca2+-independent insulin release, the latter induced by the simultaneous activation of protein kinases A and C, we examined the effects of GTP depletion by mycophenolic acid (MPA), an inhibitor of GTP synthesis, on the augmentation of insulin release from rat pancreatic islets. MPA treatment reduced GTP content by 30–40% and completely abolished glucose-induced augmentation of Ca2+-independent insulin release. Thus, this pathway is extremely sensitive to a decrease in cellular GTP content. Complete inhibition was also observed in islets treated with MPA plus adenine, to maintain ATP levels, under which conditions GTP is selectively depleted. Provision of guanine, which increases the activity of a salvage pathway for GTP synthesis and normalizes GTP content, completely reversed the inhibitory effect of MPA. Neither glucose utilization nor glucose oxidation was affected by MPA. The augmentation of Ca2+-independent insulin release by several other metabolizable nutrients including α-ketoisocaproic acid (KIC) was also inhibited by MPA. In sharp contrast, augmentation of Ca2+-dependent insulin release by KIC was resistant to GTP depletion, indicating that nutrient-induced augmentation of the Ca2+-dependent- and Ca2+-independent secretory pathways can be differentiated by GTP dependency. We interpret these data in accord with current knowledge concerning the two known stimuli for exocytosis, Ca2+ and GTP (independently of Ca2+). We propose that both Ca2+-dependent and Ca2+-independent augmentation occurs via one metabolic pathway acting upon Ca2+- and upon GTP-stimulated exocytosis. Activation of PKA and PKC stimulates the GTP-sensitive exocytosis.
Megalin (gp330): A Putative Endocytic Receptor for Thyroglobulin (Tg)
Authors: Zheng Gang , Marino Michele , Zhao Jun , McCluskey Robert T.
Published: January 2014

Megalin (gp330) is a large glycoprotein receptor found mainly on a group of absorptive epithelial cells, including renal proximal tubule, epididymal and thyroid cells. Megalin has been shown to bind multiple, unrelated ligands, mainly in vitro, and to mediate endocytosis of ligands in cultured cells. However, physiologic ligands of megalin are largely unknown. In the present study we have demonstrated that purified rat megalin binds rat thyroglobulin (Tg) in solid phase assays, with an estimated Kd of 9.2±0.6 nM. Binding was calcium dependent and was almost completely inhibited by excess Tg, by three megalin ligands—lactoferrin, lipoprotein lipase and apolipoprotein J- and by the receptor associated protein (RAP), which inhibits binding of all megalin ligands. Three anti-megalin antibodies partially inhibited Tg binding to megalin. 125I labeled Tg bound to megalin was released by EDTA and heparin; the released product was shown by SDS-PAGE and autoradiography to be 660 kD (dimeric) Tg. However, an immunoblotting experiment showed binding of megalin both to monomeric (330 kD) and dimeric Tg. We propose that megalin, which is known to mediate ligand endocytosis and is found on the apical surface of thyrocytes, may participate in the endocytosis of Tg from the colloid, a process that is required for hormone release from Tg.
Reduced 11β-Hydroxysteroid Dehydrogenase Activity in the Remaining Kidney Following Nephrectomy*
Authors: Escher Geneviève , Vogt Bruno , Beck Thomas , Guntern Daniel , Frey Brigitte M. , Frey Felix J.
Published: January 2014

Intracellular access of steroids to gluco- and mineralocorticoid receptors is regulated by reduced 11β-hydroxysteroid dehydrogenase (OHSD) 1 and 2. These enzymes convert active 11β-OH-steroids into inactive 11-keto-steroids. The purpose of the present study was to establish whether the 11β-OHSD1 and 11β-OHSD2 are modulated in the remnant kidney 24 h or 14 days after uninephrectomy (UNX) in rats. Overall, 11β-OHSD activity was analyzed by measuring the ratio of the exogenous 11β-OH-steroid prednisolone to its 11-keto metabolite prednisone in vivo in kidney tissue using high performance liquid chromatography. To determine which isoenzyme accounts for the changed activity 24 h after UNX, the oxidation and reduction attributable to 11β-OHSD1 and oxidation to 11β-OHSD2 were analyzed in total renal extracts and in isolated glomeruli, proximal convoluted tubules (PCT), cortical ascending limbs, and cortical convoluted tubules (CCT). The messenger RNA content of 11β-OHSD1 and 11β-OHSD2 was measured by RT-PCR in renal tissues and single segments, using glyceraldehyde-3-phosphate-dehydrogenase as an internal standard. Protein amounts of 11β-OHSD1 and 11β-OHSD2 were assessed by Western blot. The prednisolone/prednisone ratio increased 24 h after UNX in 9 out of 10 animals (P ≤ 0.0011), and was unchanged 14 days after UNX. 11β-OHSD1 oxidation (P ≤ 0.032) and reduction activity (P ≤ 0.002) declined 24 h after UNX in total extracts. 11β-OHSD1 oxidase activity was more than 3 times higher in PCT than in glomeruli, cortical ascending limbs, and CCT, and declined by 50% after UNX (P ≤ 0.001). The reductase activity did not change following UNX in PCT. 11β-OHSD2 activity was 5–15 times higher in CCT than in the other segments, and decreased significantly after UNX (P ≤ 0.008). UNX did not affect messenger RNA and protein levels of both enzymes in total renal extracts. In conclusion, 11β-OHSD1 and 11β-OHSD2 are predominantly expressed in PCT and CCT, respectively, and their corresponding oxidative activities decline after UNX. Thus, the access of 11β-glucocorticosteroids to gluco- and mineralocorticoid receptors in the remaining kidney is facilitated after UNX.
Growth Hormone and Mild Exercise in Combination Markedly Enhance Cortical Bone Formation and Strength in Old Rats*
Authors: Oxlund H. , Andersen N. B. , Ørtoft G. , Ørskov H. , Andreassen T. T.
Published: January 2014

The effects of a combination of mild exercise and GH injections on bone were studied in old female rats. Biosynthetic human GH, 2.7 mg/kg/day, was injected sc for 73 days. Exercised rats ran 8 m/min on a treadmill for 1 h/day. All rats (age 21 months old) were labeled with a tetracycline injection 56 days and a calcein injection 11 days before killing. The GH injections resulted in an 11-fold increase in femoral middiaphyseal bone formation rate and a 12% increase in cross-sectional area compared with the saline-injected group. The mild exercise doubled the mineralizing surface but did not influence the bone formation rate significantly. The combination of GH injections plus exercise, however, resulted in a further increase of 39% in bone formation rate, primarily at the anterolateral aspects, and an increase of 5% in cross-sectional area compared with the group injected with GH only. The femur ultimate breaking load was increased by 37% and the stiffness by 42% in the group injected with GH compared with the saline-injected group. Exercise alone did not influence the femur mechanical properties. The combination of GH injections plus exercise induced a 4% further increase in ultimate breaking load and 7% further increase in stiffness compared with the group injected with GH alone. The GH injections induced a 117% increase in serum insulin-like growth factor I. The GH-insulin-like growth factor I axis stimulates recruitment of osteoblast precursor cells, resulting in increased bone formation at the periosteal surface. GH injections and mild excercise in combination modulate and increase further the formation and strength of cortical bone in old female rats.
Molecular Cloning and Hormonal Regulation of a Murine Epididymal Retinoic Acid-Binding Protein Messenger Ribonucleic Acid
Authors: Lareyre Jean-Jacques , Zheng Weng-Li , Zhao Guang-Quan , Kasper Susan , Newcomer Marcia E. , Matusik Robert J. , Ong David E. , Orgebin-Crist Marie-Claire
Published: January 2014

A complementary DNA encoding the mouse epididymal secretory protein MEP 10 (mouse epididymal protein 10) was cloned and is now renamed murine epididymal retinoic acid binding protein (mE-RABP). The analysis of the predicted primary amino acid sequence showed that mE-RABP has a 75% identity with rat ESP I (epididymal secretory protein I), another epididymal retinoic acid-binding protein. The homology strongly suggests that mE-RABP is the mouse orthologue of rat ESP I. A computer analysis of the predicted three-dimensional structure confirmed that mE-RABP can accommodate retinoic acid as ligand. In the rat, ESP I messenger RNA (mRNA) is expressed in the efferent ducts and in the entire caput epididymidis. However, in the mouse, the expression of a 950-bp mE-RABP mRNA was detected only in principal cells of the mid/distal caput epididymidis, suggesting that the regulation of region-specific expression is different in rat and mouse. Northern blot analyses showed that mE-RABP gene expression is no longer detected 10 days after castration but progressively rebounds between days 15 and 60. However, mE-RABP protein could not be detected by Western blot 30 days after castration. Androgen replacement, begun 5 days after castration and continued for 4 days restored significant expression of mE-RABP mRNA. Efferent duct ligation for 10 days did not affect gene expression. Taken together, these results indicate that mE-RABP mRNA expression is regulated by androgens but not by testicular factors. The overall similarity in the primary amino acid sequence of mE-RABP with ESP I and other members of the lipocalin superfamily suggests that they are evolutionarily related.
Pregnancy Increases Soluble and Particulate Guanylate Cyclases and Decreases the Clearance Receptor of Natriuretic Peptides in Ovine Uterine, But Not Systemic, Arteries*
Authors: Itoh Hiroaki , Bird Ian M. , Nakao Kazuwa , Magness Ronald R.
Published: January 2014

Pregnancy increases uterine blood flow by 30- to 50-fold and uterine production of cGMP by 38-fold. Moreover, cGMP causes potent vasodilatation. We hypothesized that pregnancy up-regulates soluble and particulate guanylate cyclases (sGC and pGC) in ovine uterine arteries. Activities of sGC and pGC were compared by measuring cGMP production (37 C; 10 min) by uterine arteries from nonpregnant (n = 5) and pregnant (n = 4, 120 ± 2 days’ gestation; term = 145 ± 3 days; mean ± se) ewes after sodium nitroprusside (100 μm), atrial natriuretic peptide (1μ m), or C-type natriuretic peptide (CNP; 1μ m) treatment. The protein and/or messenger RNA expressions of sGC β1-subunit, pGC-A, pGC-B, the clearance receptor of natriuretic peptide (CR), and CNP were investigated in uterine and systemic (renal and/or omental) arteries from nonpregnant (n = 29) and pregnant (n = 21; 125 ± 2 days’ gestation) ewes. The potencies of uterine arterial GC activities were sGC ≫ pGC-A> pGC-B. Activities as well as protein expression of sGC, pGC-A, and pGC-B in pregnant uterine arteries were increased 48–128% above those in nonpregnant controls concomitant with a 34% down-regulation of CR protein expression; systemic arterial protein expressions were unaltered. These changes in uterine arterial GC-B and CR were confirmed using RT-PCR. Immunohistochemical staining of CNP in uterine, but not systemic, arterial endothelium from pregnant ewes was much stronger than that from nonpregnant ewes. Thus, two distinct GC pathways are present in ovine uterine artery, and both may be specifically up-regulated during pregnancy and so contribute to the tremendous local increase in cGMP production during pregnancy.
Phospholipase D- and Protein Kinase C Isoenzyme-Dependent Signal Transduction Pathways Activated by the Calcitonin Receptor*
Authors: Naro Fabio , Perez Marie , Migliaccio Silvia , Galson Deborah L. , Orcel Philippe , Teti Anna , Goldring Steven R.
Published: January 2014

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product[ 3H]choline with an EC50 = 2.5 ± 0.3 × 10−8m, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 ± 1.0 × 10−8m). The hormone failed to induce release of [3H]phosphocholine and[ 3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-Cα, but not the δ isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-Cζ, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.