bluuebunny/crossref_metadata_2025 huggingface dataset:
Sphingosine 1-Phosphate Mobilizes Sequestered Calcium, Activates Calcium Entry, and Stimulates Deoxyribonucleic Acid Synthesis in Thyroid FRTL-5 Cells*
Authors: Törnquist Kid , Saarinen Pia , Vainio Minna , Ahlström Mikael
Published: January 2014

Sphingosine 1-phosphate (SPP) potently mobilizes sequestered calcium and is a mitogen in several cell types. In the present investigation, we have evaluated the effect of SPP on intracellular free calcium concentration ([Ca2+]i) and synthesis of DNA in thyroid FRTL-5 cells. SPP rapidly and transiently mobilized sequestered calcium and stimulated entry of extracellular calcium. The entry of calcium, but not the mobilization, was in part inhibited by pretreatment with pertussis toxin (Ptx), and by activation of protein kinase C. SPP did not stimulate the production of inositol 1,4,5-trisphosphate. SPP stimulated the incorporation of 3H-thymidine in a time- and dose-dependent manner. The effect was not inhibited by Ptx. Furthermore, SPP stimulated the activation of the proto-oncogene c-fos. SPP rapidly tyrosine-phosphorylated an approximately 66 kDa protein. This phosphorylation persisted for at least 1 h. Pretreatment of the cells with genistein abolished the SPP-evoked tyrosine phosphorylation, and attenuated the SPP-evoked increase in[ Ca2+]i. Furthermore, the SPP-evoked activation of Na+-H+ exchange was inhibited by genistein. The phosphorylation was not attenuated by pretreatment of the cells with Ptx. SPP per se did not affect cellular cAMP levels but attenuated the TSH-evoked increase in cAMP. As the effect of SPP might be due to activation of phospholipase D, we tested whether phosphatidic acid (PA) mobilized calcium or stimulated the incorporation of 3H-thymidine. PA mobilized sequestered calcium but did not stimulate calcium entry. PA very modestly enhanced the incorporation of 3H-thymidine. Our results suggest, that SPP stimulates DNA synthesis and activates entry of calcium in FRTL-5 cells. The effect on calcium entry appears to be dependent, at least in part, on one or several tyrosine kinases.
Hepatic Expression of ErbB3 Is Repressed by Insulin in a Pathway Sensitive to PI-3 Kinase Inhibitors*
Authors: Carver Robert S. , Mathew Puthenpurackal M. , Russell William E.
Published: January 2014

ErbB3 is an epidermal growth factor receptor-related type I tyrosine kinase receptor capable, in conjunction with ErbB2 or epidermal growth factor receptor, of transmitting proliferative and differentiative signals in a variety of cell types. We previously showed that ErbB3 messenger RNA and protein increase in cultured hepatocytes during the first 12 h in culture, as does the binding of heregulin β1, a ligand for ErbB3. Insulin inhibits the increase in heregulin β1 binding, as well as the increase in ErbB3 messenger RNA and protein. Two models of insulin deficiency in vivo (diabetes and fasting) demonstrated elevated levels of hepatic ErbB3 protein, strengthening the relevance of our observations in vitro. Using chemical activators or antagonists, we sought to identify the signaling pathways that link insulin to ErbB3 expression. The PI-3 kinase inhibitors, wortmannin and LY294002, completely blocked the inhibition of ErbB3 protein expression by insulin, suggesting a role for PI-3 kinase in the regulation of this growth factor receptor. Rapamycin, an inhibitor of p70 S6 kinase, an enzyme downstream of PI-3 kinase, failed to block the effect of insulin on ErbB3 expression. These results suggest a complex regulatory paradigm for ErbB3 that includes PI-3 kinase and may be linked, via insulin, to the metabolic status of the animal.
Changes in Facilitative Glucose Transporter Messenger Ribonucleic Acid Levels in the Diabetic Rat Kidney*
Authors: Chin Edward , Zamah A. Musa , Landau Daniel , Gronboek Henning , Flyvbjerg Allan , LeRoith Derek , Bondy Carolyn A.
Published: January 2014

Facilitative glucose transporter (GLUTs 1, 2, 4, and 5) messenger RNAs (mRNAs) are differentially distributed in the rat nephron: GLUT1 is widely expressed, GLUT4 is selectively concentrated in thick ascending limbs, and GLUT2 and 5 are exclusively localized in proximal tubules, consistent with differential roles for these transporters in renal glucose handling. In the present study, quantitative in situ hybridization was used to evaluate changes in these mRNA levels during acute (2 and 7 days) and chronic (30, 90, and 180 days) streptozotocin-induced diabetes mellitus (STZ-DM). Medullary GLUT1 and GLUT4 mRNA levels were significantly increased during the acute phase but returned to normal after 1 week. Cortical GLUT1 mRNA levels, however, were decreased significantly from 7 days through 6 months of STZ-DM. Cortical GLUT2 mRNA was slightly increased acutely and increased 5-fold in chronic STZ-DM, with the largest increase focally concentrated in the convoluted portion of the proximal tubule. Proximal tubule GLUT5 mRNA levels also were increased significantly during chronic STZ-DM.

In summary, medullary GLUT1 and GLUT4 mRNA levels are acutely increased in STZ-DM, paralleling the increased renal epithelial metabolic activity accompanying early diabetes. Proximal tubular GLUT2 and 5 mRNA levels were increased in chronic STZ-DM, possibly adapting to the increased need for glucose transport out of these epithelial cells, whereas the concomitant decrease in cortical GLUT1 expression may reflect the decreased requirement for basolateral import of glucose into these same cells. Thus, renal GLUTs demonstrate complex, nephron segment-specific and duration-dependent responses to the effects of STZ-DM.
Purification and Characterization of the Insulin-Like Growth Factor-Binding Protein-1 Phosphoform Found in Normal Plasma*
Authors: Westwood Melissa , Gibson J Martin , White Anne
Published: January 2014

Our previous work has shown that, in the normal circulation, insulin-like growth factor-binding protein-1 (IGFBP-1) is present as a single highly phosphorylated species. In this study, we have purified this previously uncharacterized isoform of IGFBP-1 to determine its ligand-binding affinity and the potential significance of highly phosphorylated IGFBP-1. Immunoaffinity chromatography was used to isolate IGFBP-1 from normal human plasma and from human hepatoma (Hep G2) cell medium as an alternative source of the IGFBP-1 phosphoform in the circulation. The affinity of this highly phosphorylated IGFBP-1 was compared with that of nonphosphorylated IGFBP-1 and recombinant human (rh) IGFBP-3 by equilibrium binding to IGF-I and IGF-II.

Anion-exchange (IEX) HPLC, nondenaturing electrophoresis, alkaline phosphatase treatment, and ligand-binding studies indicated that the highly phosphorylated IGFBP-1 from HepG2 cells was comparable with IGFBP-1 from plasma. In binding to IGF-I, the plasma phosphoform of IGFBP-1 was found to have a higher affinity (2.3 ± 1.1 × 1010m−1) than nonphosphorylated IGFBP-1 (2.5 ± 1.7 × 109m−1, P < 0.002). However, when binding to IGF-II, phosphorylation had no affect on the affinity of IGFBP-1 (3.6 ± 2 × 109m−1vs. 1.8 ± 3 × 109m−1, P not significant). Therefore, in the circulation, IGF-I has a considerably higher affinity than IGF-II for IGFBP-1 (P < 0.02). The affinity of phosphorylated IGFBP-1 from plasma (2.3 ± 1.1 × 1010m−1) also was significantly higher than the affinity of IGFBP-3 for IGF-I (5.6 ± 4.2 × 109m−1, P < 0.005).

These data suggest that the highly phosphorylated IGFBP-1 in the normal circulation will preferentially bind IGF-I rather than IGF-II, whereas in pregnancy, the affinity of IGFBP-1 for IGF-I will be reduced because of the appearance of non- and lesser-phosphorylated forms. This lends support to the theory that changes in IGFBP-1 phosphorylation may influence the modulatory effects of IGFBP-1 on IGF bioavailability.
Liver and Kidney Growth Hormone (GH) Receptors Are Regulated Differently in Diabetic GH and GH Antagonist Transgenic Mice*
Authors: Chen Nian-yi , Chen Wen Y. , Kopchick John J.
Published: January 2014

Elevated GH levels are frequently seen in poorly controlled type I diabetics and have been implicated in diabetic complications. Studies of GH and GH antagonist (GHA) transgenic mice with streptozotocin (STZ)-induced diabetes have revealed that GH has a permissive effect for diabetic nephropathy, and that expression of a GHA gene protected mice against diabetic kidney lesions. To investigate whether kidney GH receptor (GHR) and/or GH-binding protein may play a role in diabetic nephropathy, we evaluated GH-specific binding and messenger RNA levels for GHR/GH-binding protein in mouse livers and kidneys from bovine (b) GH or bGHA transgenic (Tg) mice and their nontransgenic (NTg) littermates with or without STZ-induced diabetes. We found that liver-specific GH binding is significantly higher in both bGH- and bGHA-Tg mice compared to that in their NTg controls. In contrast, kidney GH binding is significantly lower in bGH-Tg mice compared to that in NTg littermates. These results indicate that regulation of mouse GHR expression is tissue specific. STZ-induced diabetes decreased GH-specific binding in both liver and kidney of NTg and GHA-Tg mice, but not in bGH-Tg mice. The lowered GHR binding in diabetic NTg and GHA-Tg mice suggests the involvement of insulin in the regulation of GHR expression. The down-regulation of kidney GHR in GHA-Tg mice in combination with the presence of GHA may partially explain the protective mechanism of GHA against diabetic kidney lesions.
Correlation of the Activation of Ca2+/Calmodulin-Dependent Protein Kinase II with the Initiation of Insulin Secretion from Perifused Pancreatic Islets*
Authors: Easom Richard A. , Filler Natalie R. , Ings Emma M. , Tarpley Jim , Landt Michael
Published: January 2014

An experimental procedure has been designed to permit the simultaneous assessment of the activation status of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) with insulin secretion in perifused islets. By this procedure, the activation of CaM kinase II by glucose correlated closely with the initial and sustained phases of insulin secretion within a 30-min test period. By contrast, islets (160–200/tube) in static incubations neither supported second-phase insulin secretion nor CaM kinase II activation beyond 10–15 min. This was not the result of the accumulation of insulin, because the introduction of insulin (40–160 ng/ml) into the perifusion medium failed to mimic the suppression of glucose-induced insulin secretion or CaM kinase II activation. A similar addition of SRIF (0.01–1 μm) or epinephrine (1μ m) profoundly suppressed insulin secretion although failing to significantly influence CaM kinase II activation. Finally, on withdrawal of glucose from perifused islets, insulin secretion rapidly returned to basal rates, but CaM kinase II deactivation was significantly delayed. The correlation of kinase activation with the initiation of insulin secretion suggests that CaM kinase II may be important in the regulation of glucose-induced insulin secretion. The observed dissociation of these parameters in the presence of inhibitory hormones or after the withdrawal of a glucose stimulus, however, suggests that the kinase is not directly involved in the final steps of insulin exocytosis.
Cytokines Induce Deoxyribonucleic Acid Strand Breaks and Apoptosis in Human Pancreatic Islet Cells*
Authors: Delaney Carol A. , Pavlovic Dejan , Hoorens Anne , Pipeleers Daniel G. , Eizirik Décio L.
Published: January 2014

We have previously observed that a 6-day exposure of human pancreatic islets to a combination of cytokines (interleukin-1β 50 U/ml + tumour necrosis factor-α 1000 U/ml + interferon-γ 1000 U/ml) severely impairs β-cell functions. In the present study, we examined whether this condition affects DNA integrity and viability of human islet cells. Cells were studied after 3, 6, and 9 days of cytokine treatment by both single cell gel electrophoresis (the “comet assay,” a sensitive method for detection of DNA strand breaks) and by a cytotoxicity assay using the DNA binding dyes Hoechst 33342 and propidium iodide as indices for the number of viable, necrotic, and apoptotic cells. Cytokine treatment for 6 and 9 days resulted in a 50% increase in comet length (P < 0.01 vs. controls), indicating DNA strand breaks, as well as in a significant increase in the number of apoptotic cells (P < 0.02 vs. controls), but not in the number of necrotic cells. The arginine analogs NG-nitro-l-arginine and NG-monomethyl-l-arginine prevented nitric oxide formation by the cytokines but did not interfere with cytokine-induced DNA strand breaks and apoptosis. The present data suggest that prolonged (6–9 days) exposure of human pancreatic islets to a mixture of cytokines induces DNA strand breaks and cell death by apoptosis. These deleterious effects of cytokines appear to be independent of nitric oxide generation.
Telomerase Activity in Normal Adult Brown Norway Rat Seminal Vesicle: Regional Distribution and Age-Dependent Changes*
Authors: Banerjee Partha P. , Banerjee Subhadra , Zirkin Barry R. , Brown Terry R.
Published: January 2014

Telomerase activity is essential for protection of cells against the telomere erosion that occurs with each round of cell replication, and thus appears to play a role in the indefinite replication potential of some, but not all, eukaryotic cells. In this regard, some tissues contain stem cells that have a long proliferative life-span and are capable of regenerating or renewing the somatic epithelial cell population within the tissue. Because the adult seminal vesicle exhibits the ability to regenerate during androgen-replacement after castration, we hypothesized that a pool of cells with regenerating potential is present in the adult seminal vesicle, which expresses telomerase activity. In this study, we used a highly sensitive PCR-based telomerase assay [the telomeric repeat amplification protocol (TRAP) assay] to detect telomerase activity in rat seminal vesicle. Our results show that telomerase activity is, indeed, present in the normal adult rat seminal vesicle, but that, in the presence of seminal vesicle fluid, telomerase activity cannot be detected. In fact, seminal vesicle fluid was found to contain some factor(s) that is inhibitory for the TRAP assay. In addition, we found that telomerase activity in the seminal vesicle changes with age and is regionally distributed within the distal, intermediate, and proximal segments of the duct. These results suggest that as is the case for the rat prostate, a population of telomerase-positive cells is present within the adult rat seminal vesicle, and thereby, this organ retains throughout life the potential to regenerate in response to androgen replacement following castration-induced apoptotic cell death.
Identification of cis-Acting Deoxyribonucleic Acid Elements That Mediate Gonadotropin-Releasing Hormone Stimulation of the Rat Luteinizing Hormone β-Subunit Gene*
Authors: Kaiser Ursula B. , Sabbagh Elena , Saunders Brian D. , Chin William W.
Published: January 2014

GnRH plays a critical role in reproductive development and function by regulating the biosynthesis and secretion of the pituitary gonadotropins, LH and FSH. Although it is known that GnRH induces gonadotropin subunit gene transcription, the mechanism by which this occurs has not been elucidated. Studies have been hindered by the lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have transfected the rat pituitary GH3 cell line with the rat GnRH receptor complementary DNA. These cells, when cotransfected with regulatory regions of the LH or FSH subunit genes fused to a luciferase reporter gene, respond to GnRH with an increase in promoter activity comparable to that seen in primary rat pituitary cells. In this study, we have used this cell model to identify cis-acting elements of the LHβ gene that mediate stimulation by GnRH. Analysis of a series of 5′-deletion and internal deletion constructs has revealed two regions of the rat LHβ gene promoter involved in mediating the response to GnRH, region A (−490/−352) and region B (−207/−82). Fusion of region A upstream of a heterologous minimal promoter linked to the luciferase gene conferred GnRH responsiveness to the promoter, whereas region B did not. However, the presence of both regions A and B conferred a greater GnRH response than region A alone. Electrophoretic mobility shift assay revealed the presence of a protein(s) binding to region A using GH3 as well as αT3–1 nuclear extracts. Thus, region A (−490/−352) confers GnRH responsiveness to the LHβ subunit gene and binds to a protein(s) present in pituitary cell lines. DNA sequences in region B (−207/−82) also contribute to GnRH responsiveness. The identification of putative GnRH response elements in the rat LHβ gene promoter will aid in elucidation of the mechanisms of regulation of gene expression by GnRH.
Induction of Oxytocin Receptor Gene Expression in Rabbit Amnion Cells*
Authors: Jeng Yow-Jiun , Lolait Stephen J. , Soloff Melvyn S.
Published: January 2014

Oxytocin (OT)-stimulated PGE2 release by rabbit amnion is enhanced by the up-regulation of oxytocin receptors (OTR), which increase about 200-fold at the end of pregnancy. As recent studies have shown that PGs are essential for parturition, the rise in amnion OTR and associated PGE2 synthesis are probably essential for labor initiation. The present work was directed toward understanding the mechanisms of OTR up-regulation. Levels of agents that stimulate adenylyl cyclase activity and cortisol are increased in amniotic fluid at the end of pregnancy. Addition of either forskolin or cortisol to cultured amnion cells caused an increase in OTR ligand-binding sites and steady state OTR messenger RNA (mRNA) levels. Forskolin treatment elevated OTR mRNA levels rapidly, but transiently, whereas cortisol’s effects were slower and sustained. Actinomycin or cycloheximide, added 3 h after forskolin, led to a sustained elevation in OTR mRNA levels, suggesting that forskolin increases the activities of OTR mRNA-destabilizing factors along with increasing OTR mRNA concentration. Cortisol did not appear to affect OTR mRNA stability. Measurement of OTR mRNA transcription rates showed that forskolin’s effects were maximal within 1 h of treatment. In contrast, cortisol-induced transcription was not apparent until 8 h. The effects of forskolin and cortisol on OTR gene transcription were synergistic. Thus, the increase in OTR mRNA levels occurring after either forskolin or cortisol treatments is the result of induction of OTR gene expression, but the effects of the two agents appear to occur at separate sites.
Effect of Epalrestat, an Aldose Reductase Inhibitor, on the Generation of Oxygen-Derived Free Radicals in Neutrophils from Streptozotocin-Induced Diabetic Rats*
Authors: Kashima Koji , Sato Noriyuki , Sato Ken , Shimizu Hiroyuki , Mori Masatomo
Published: January 2014

Neutrophil function is impaired by a known mechanism in diabetic patients, thus increasing susceptibility to infections. We studied the effect of epalrestat, an aldose reductase inhibitor, on the generation of oxygen-derived free radicals and cytosolic sorbitol concentration in neutrophils from streptozotocin-induced diabetic rats. There were four groups: treated and untreated control and diabetic rats. Treated groups were given 0.075% epalrestat in their diet for 4 weeks from the induction of diabetes and were untreated for the subsequent 4 weeks. Oxygen radicals were measured as chemiluminescence amplified by a luciferin analog [Cypridina luciferin analog-dependent chemiluminescence (CLA-DCL), which is dependent on O2− generation] and luminol (L)-DCL, which is highly dependent on OCl− generation) in response to formyl-methonyl-leucyl-phenylalanine. Diabetes resulted in a significant decrease in CLA/L-DCL and a significant increase in sorbitol (P < 0.01); there was a negative correlation between sorbitol and CLA-DCL (P < 0.05) in diabetic groups. The 4-week treatment with epalrestat in the diabetic group completely prevented the increase in sorbitol and partially improved the CLA-DCL, although L-DCL was not significantly affected. After 4 weeks off treatment, CLA-DCL decreased and sorbitol increased. Treatment had no effect on serum insulin or glucose concentration. We conclude that an increase in sorbitol in neutrophils causes, in part, an impaired generation of O2−. Epalrestat improves the impaired O2− generation by preventing the sorbitol increase in streptozotocin-induced diabetic rats.
Repeated Intracerebroventricular Administration of Glucagon-Like Peptide-1-(7–36) Amide or Exendin-(9–39) Alters Body Weight in the Rat**This work was supported by the United Kingdom Medical Research Council.
Authors: Meeran Karim , O’Shea Donal , Edwards C. Mark B. , Turton Mandy D. , Heath Melanie M. , Gunn Irene , Abusnana Salahedeen , Rossi Michela , Small Caroline J. , Goldstone Anthony P. , Taylor Gillian M. , Sunter David , Steere Joanna , Choi Sang Jeon , Ghatei Mohammad A. , Bloom Stephen R.
Published: January 2014

Central nervous system glucagon-like peptide-1-(7–36) amide (GLP-1) administration has been reported to acutely reduce food intake in the rat. We here report that repeated intracerebroventricular (icv) injection of GLP-1 or the GLP-1 receptor antagonist, exendin-(9–39), affects food intake and body weight. Daily icv injection of 3 nmol GLP-1 to schedule-fed rats for 6 days caused a reduction in food intake and a decrease in body weight of 16 ± 5 g (P < 0.02 compared with saline-injected controls). Daily icv administration of 30 nmol exendin-(9–39) to schedule-fed rats for 3 days caused an increase in food intake and increased body weight by 7 ± 2 g (P < 0.02 compared with saline-injected controls). Twice daily icv injections of 30 nmol exendin-(9–39) with 2.4 nmol neuropeptide Y to ad libitum-fed rats for 8 days increased food intake and increased body weight by 28 ± 4 g compared with 14 ± 3 g in neuropeptide Y-injected controls (P < 0.02). There was no evidence of tachyphylaxis in response to icv GLP-1 or exendin-(9–39). GLP-1 may thus be involved in the regulation of body weight in the rat.
Altered Expression of Agouti-Related Protein and Its Colocalization with Neuropeptide Y in the Arcuate Nucleus of the Hypothalamus during Lactation*
Authors: Chen Peilin , Li Chien , Haskell-Luevano Carrie , Cone Roger D. , Smith M. Susan
Published: January 2014

During lactation, the levels of neuropeptide Y (NPY), which plays an important role in mediating food intake, are significantly elevated in a number of hypothalamic areas, including the arcuate nucleus (ARH). To identify additional hypothalamic systems that might be important in mediating the increase in food intake and alterations in energy homeostasis during lactation, the present studies examined the expression of agouti-related protein (AGRP), a recently described homologue of the skin agouti protein. AGRP is found in the hypothalamus and has been suggested to play an important role in the regulation of food intake. In the first experiment, animals were studied during diestrus of the estrous cycle, a stage of the cycle when estrogen levels are basal and similar to lactation, or during days 12–13 postpartum. Lactating animals had their litters adjusted to eight pups on day 2 postpartum. Brain tissue sections were used to measure AGRP messenger RNA (mRNA) levels by in situ hybridization. AGRP mRNA signal was found mostly in the ventromedial portion of the ARH, which has been shown to contain a high density of NPY neurons. A significant increase in AGRP mRNA content was observed in the mid- to caudal portion of the ARH of lactating animals compared with diestrous females. No difference was found in the rostral portion of the ARH. In the second experiment, double-label in situ hybridization for AGRP and NPY was performed in lactating animals to determine the extent of colocalization of the two peptides in the ARH, using 35S-labeled and digoxigenin-labeled antisense complementary RNA probes. It was found that almost all of the NPY-positive neurons throughout the ARH also expressed AGRP mRNA signal. Furthermore, AGRP expression was confined almost exclusively to NPY-positive neurons. Thus, the present study showed that during lactation, AGRP gene expression was significantly elevated in a subset of the AGRP neurons in the ARH. The high degree of colocalization of AGRP and NPY, coupled with previous reports from our laboratory demonstrating increased NPY expression in the ARH in response to suckling, suggests that AGRP and NPY are coordinately regulated and may be involved in the increase in food intake during lactation.
FKHR Binds the Insulin Response Element in the Insulin-Like Growth Factor Binding Protein-1 Promoter*
Authors: Durham Susan K. , Suwanichkul Adisak , Scheimann Ann O. , Yee Douglas , Jackson James G. , Barr Frederic G. , Powell David R.
Published: January 2014

The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription. Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif. Immunoblots using antiserum to the synthetic peptide FKHR413–430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells. Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3β, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants. These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription.
Glycosylation-Dependent Cell Adhesion Molecule 1 (GlyCAM 1) Is Induced by Prolactin and Suppressed by Progesterone in Mammary Epithelium**This work was supported by National Institutes of Health.
Authors: Hou Zhaoyuan , Bailey Jason P. , Vomachka Archie J. , Matsuda Manabu , Lockefeer Jason A. , Horseman Nelson D.
Published: January 2014

Glycosylation-dependent cell adhesion molecule 1 (GlyCAM 1), a mucin-like endothelial glycoprotein, was induced by PRL and suppressed by progesterone in the mammary gland of mice, and in HC11 mouse mammary epithelial cells. Complementary DNA microarray analysis revealed that expression of GlyCAM 1 was reduced in the mammary gland of PRL-gene disrupted mice (PRL−/−) compared with control (PRL+/−) littermates. This result was confirmed by in situ hybridization and immunostaining. The messenger RNA (mRNA) encoding GlyCAM 1 was present in mammary epithelia of PRL-stimulated mice. Immunohistochemistry indicated that GlyCAM 1 protein was detectable both in mammary epithelia and in the ductal lumen in PRL+/− virgin mice, but not in PRL−/− mice. GlyCAM 1 mRNA was highly induced by grafting pituitary glands from normal littermates. Trace amounts of mRNA for GlyCAM 1 were detected by RT-PCR in mammary tissue of PRL−/− mice. Progesterone inhibited both basal and PRL-stimulated GlyCAM 1 transcription. In HC11 cells, GlyCAM 1 mRNA was induced in cells treated with insulin, dexamethasone, and PRL. Similar to the in vivo studies, progesterone inhibited the induction of GlyCAM 1 transcription. In CHO cells, PRL stimulated transcription of a luciferase reporter gene containing an 800-bp promoter fragment of GlyCAM 1, and progesterone partially suppressed the PRL effect. These data demonstrate that expression of GlyCAM 1 in mammary gland is under the control of both PRL and progesterone.
A1 Adenosine Receptor Activation Increases Adipocyte Leptin Secretion*
Authors: Rice Alan M. , Fain John N. , Rivkees Scott A.
Published: January 2014

A1 adenosine receptors (A1ARs) are heavily expressed in adipocytes and influence fat cell metabolism. Because increasing evidence suggests a role for leptin in mediating appetite and fat cell metabolism, we tested whether A1ARs regulate leptin production. Rats were treated with the A1AR agonist N6-cyclopentyladenosine (CPA), and changes in circulating levels of leptin and leptin gene expression were examined. Serum leptin levels rose 2- to 10-fold, with peak increases seen 8–16 h after injection of CPA (P < 0.05). In contrast, CPA did not alter steady state levels of adipose tissue leptin mRNA. To assess the influence of endogenous adenosine on circulating leptin levels, rats were also injected with dipyridamole (DPY), an adenosine reuptake blocker. DPY induced 80% increases in serum levels at 8 h after injections (P < 0.05). Supporting the idea that stimulation of leptin production is A1AR mediated, pretreatment with the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine completely blocked increases in leptin levels after DPY treatment. To complement in vivo studies, the effect of A1AR activation on leptin secretion was also studied in epididymal fat pad cultures. In cultures, CPA treatment increased leptin secretion by 37% (P < 0.05). Collectively, these data show that the adenosinergic system can increase leptin secretion by directly activating A1ARs in fat tissue.
The in Vivo and in Vitro Effects of Exogenous Leptin on Ovulation in the Rat*
Authors: Duggal Priya S. , Van der Hoek Kylie H. , Milner Clyde R. , Ryan Natalie K. , Armstrong David T. , Magoffin Denis A. , Norman Robert J.
Published: January 2014

Leptin, a hormonal product of the Lep gene, is expressed by adipocytes and is thought to play a role in regulating food intake and reproduction. The leptin protein has been localized in many reproductive tissues, including the ovary. Several publications indicate that the ovary is directly affected by leptin and that leptin may be a factor linking obesity and reproductive dysfunction. In this study, the effect of systemic leptin administration on ovulation in the rat ovary, both in vivo and in vitro, was investigated. Ip administration of leptin (30 μg at 3 hourly intervals for 15 h) to immature gonadotropin-primed rats caused a decline in ovulation in vivo, from 15.9 ± 2.0 oocytes in the control animals to 5.3 ± 1.6 oocytes in the leptin-treated animals (P < 0.001). Plasma progesterone and estradiol levels were analyzed immediately before ovulation, and neither was altered significantly in animals receiving the leptin treatment. Food consumption and body weight decreased following leptin treatment; however, a loss in body weight alone (pair-fed controls) was insufficient to explain the decrease in ovulation observed in the leptin-treated animals. In vitro perfusion of FSH-primed whole ovaries showed that treatment with leptin in combination with LH significantly decreased ovulations from 5.7 ± 1.6 per ovary perfused with LH alone to 1.3 ± 0.6 in those with LH and 1 μg/ml leptin (P < 0.05). Progesterone and estradiol levels in the samples taken during the perfusion period were unaffected by leptin treatment. In summary, leptin administration resulted in fewer ovulations, both in vivo and in vitro, but did not influence steroid levels. Systemic leptin administration at these doses can therefore inhibit ovulation, a process that occurs through a direct effect on the ovary.
Interactions between the Prohormone Convertase 2 Promoter and the Thyroid Hormone Receptor*
Authors: Li Qiao-Ling , Jansen Erik , Brent Gregory A. , Naqvi Syed , Wilber John F. , Friedman Theodore C.
Published: January 2014

The majority of prohormones are cleaved at paired basic residues to generate bioactive hormones by prohormone convertases (PCs). As PC1 and PC2, two neuroendocrine-specific PCs, appear to be the key enzymes capable of processing a variety of prohormones, alterations of PC2 and/or PC1 levels will probably have a profound effect on hormonal homeostasis. We investigated the regulation of PC2 messenger RNA (mRNA) by thyroid hormone using GH3 cells to demonstrate that T3 negatively regulated PC2 mRNA levels in a dose- and time-dependent fashion. Functional analysis of progressive 5′-deletions of the human (h) PC2 promoter luciferase constructs in GH3 cells demonstrated that the regulation probably occurs at the transcriptional level, and that putative negative thyroid hormone response elements were located within the region from −44 to +137 bp relative to the transcriptional start site. Transient transfections in JEG-3 cells and COS-1 cells showed that the suppressive effect of T3 was equally mediated by the thyroid hormone receptor (TR) isoforms TRα1 and TRβ1. Electrophoretic mobility shift assays using purified TRα1 and retinoid X receptor-β protein as well as GH3 nuclear extracts showed that regions from +51 to +71 bp and from +118 to +137 bp of the hPC2 promoter bind to TRα1 as both a monomer and a homodimer and with TRα1/retinoid X receptor-β as a heterodimer. Finally, the in vivo regulation of pituitary PC2 mRNA by thyroid status was demonstrated in rats. These results demonstrate that T3 negatively regulates PC2 expression at the transcriptional level and that functional negative thyroid hormone response elements exist in the hPC2 promoter. We postulate that the alterations of PC2 activity may mediate some of the pathophysiological consequences of hypo- or hyperthyroidism.
Coexpression of ERβ with ERα and Progestin Receptor Proteins in the Female Rat Forebrain: Effects of Estradiol Treatment
Authors: Gréco Béatrice , Allegretto E. A. , Tetel M. J. , Blaustein J. D.
Published: January 2014

Estrogen and progestin receptors (ER, PgR) play a critical role in the regulation of neuroendocrine functions in females. The neuroanatomical distribution of the recently cloned, ERβ, overlaps with both ERα and PgR. To determine whether ERβ is found within ERα- or PgR-containing neurons in female rat, we used dual label immunocytochemistry. ERβ-immunoreactivity (ERβ-ir) was primarily detected in the nuclei of cells in the periventricular preoptic area (PvPO), the bed nucleus of the stria terminalis (BNSTpr), the paraventricular nucleus, the supraoptic nucleus, and the medial amygdala (MEApd). Coexpression of ERβ-ir with ERα-ir or PgR-ir was observed in the PvPO, BNSTpr, and MEApd in ovariectomized rats. E2 treatment decreased the number of ERβ-ir cells in the PvPO and BNSTpr and the number of ERα-ir cells in the MEApd and paraventricular nucleus, and therefore decreased the number of cells coexpressing ERβ-ir and ERα-ir in the PvPO, BNSTpr, and MEApd. E2 treatment increased the amount of PgR-ir in cells of the PvPO, BNSTpr, and MEApd, a portion of which also contained ERβ. These results demonstrate that ERβ is expressed in ERα- or PgR-containing cells, and they suggest that E can modulate the ratios of these steroid receptors in a brain region-specific manner.
Distribution of Chimeric IGF Binding Protein (IGFBP)-3 and IGFBP-4 in the Rat Heart: Importance of C-Terminal Basic Region
Authors: Knudtson K. L. , Boes M. , Sandra A. , Dake B. L. , Booth B. A. , Bar R. S.
Published: January 2014

IGF binding proteins-3 and -4, whether given in the perfused rat heart or given iv in the intact animal, cross the microvascular endothelium of the heart and distribute in subendothelial tissues. IGF binding protein-3, like IGF-I/II, localizes in cardiac muscle, with lesser concentrations in CT elements. In contrast, IGFBP-4 preferentially localizes in CT. In this study, chimeric IGF binding proteins were prepared in which a basic 20-amino-acid C-terminal region of IGF binding protein-3 was switched with the homologous region of IGF binding protein-4, and vice-versa, to create IGF binding protein-34 and IGF binding protein-43. Perfused IGF binding protein-34 behaved like IGF binding protein-4, localizing in connective tissue elements, whereas IGF binding protein-43 now localized in cardiac muscle at concentrations identical to perfused IGF binding protein-3. To determine whether these small mutations altered the affinity of the chimera for cells, the ability of 125I-IGF binding protein-34 and 125I-IGF binding protein-43 to bind to microvascular endothelial cells was determined and compared with IGF binding protein-3. IGF binding protein-34 retained 15% of the binding capacity of IGF binding protein-3, whereas IGF binding protein-43 bound to microvessel endothelial cells with higher affinity and greater total binding than that of IGF binding protein-3. We conclude that small changes in the C-terminal basic domain of IGF binding protein-3 and the corresponding region of IGF binding protein-4 can alter their affinity for cultured cells and influence their tissue distribution in the rat heart.
Suppression of the Basic Transcription Element-Binding Protein in Brain Neuronal Cultures Inhibits Thyroid Hormone-Induced Neurite Branching
Authors: Cayrou Christelle , Denver Robert J. , Puymirat Jack
Published: January 2014

The molecular mechanisms underlying the effect of thyroid hormone (T3) on neurite outgrowth are unknown. We recently identified the small GC-box binding protein BTEB (basic transcription element-binding protein) as a T3-regulated gene in the developing rat brain. BTEB mRNAs are rapidly (by 1 h) up-regulated by T3 in primary rat embryonic neuronal cultures. Antisense oligodeoxynucleotides (ODNs), added to the cultures, reduced by 60% the level of BTEB mRNA. Addition of BTEB antisense ODNs to the cultures, before the onset of neurite polarity, had no effect on neurite elaboration but significantly decreased, in a dose-dependent manner, the effect of T3 on neurite branching. We then examined the effects of antisense ODNs on a thyroid hormone target neuronal population, i.e. the acetylcholinesterase-positive neurons after the onset of neurite polarity. Exposure to BTEB antisense ODNs completely abolished the effects of T3 on neurite branching and on the elaboration of neuritic filopodia-like structures in acetylcholinesterase cells. By contrast, antisense ODNs did not alter the effect of T3 on neurite length. Our results show that titration of BTEB levels by T3 regulates the degree of neurite branching and that the T3-induced neurite elongation and the T3-induced neurite branching are regulated by distinct mechanisms.
Gastric Parietal Cells: Potent Endocrine Role in Secreting Estrogen as a Possible Regulator of Gastro-Hepatic Axis
Authors: Ueyama Takashi , Shirasawa Nobuyuki , Numazawa Mitsuteru , Yamada Keiko , Shelangouski Momoko , Ito Takao , Tsuruo Yoshihiro
Published: January 2014

Estrogen, if it is produced in the gastrointestinal tract, may overflow into the systemic circulation in the case of increased portal-systemic shunting. This idea is in accord with a significant step-up in serum estradiol (E2) concentration in the portal vein of rats, compared with that in the artery. Gene expression of aromatase, estrogen synthetase, was demonstrated by RT-PCR in the gastric mucosa of male and female adult rats, equivalent to that in the ovary. Aromatase activity and production of E2 in the gastric mucosa were demonstrated by 3H2O assay and gas chromatography-mass spectrometry, and they were inhibited by aromatase inhibitor, 4-hydroxyandrostenedione. Conversion of 14C-androstenedione to 14C-E2 through 14C-testosterone in cultured gastric mucosa was also demonstrated. Parietal cells exhibited strong signals for aromatase mRNA and immunoreactive protein by in situ hybridization histochemistry and immunohistochemistry. Estrogen receptor α mRNA and immunoreactive protein were demonstrated in hepatocytes by RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Total gastrectomy reduced portal venous E2 concentration, without changing systemic E2 concentration, together with down-regulation of estrogen receptor α mRNA level in the liver. These findings indicate that gastric parietal cells play a potent endocrine role in secreting estrogen that may function as a regulator of the gastro-hepatic axis.
Phosphodiesterase 8B Gene Polymorphism Is Associated with Subclinical Hypothyroidism in Pregnancy
Authors: Shields Beverley M. , Freathy Rachel M. , Knight Bridget A. , Hill Anita , Weedon Michael N. , Frayling Timothy M. , Hattersley Andrew T. , Vaidya Bijay
Published: January 2014

Background:

Maternal subclinical hypothyroidism is associated with a number of adverse outcomes in pregnancy. The Endocrine Society’s recent consensus guidelines have recommended treatment with T4 for this condition in pregnancy. The single nucleotide polymorphism rs4704397 in the phosphodiesterase 8B (PDE8B) gene has been found to be associated with altered serum TSH concentrations in the general population. We aimed to assess whether genetic variation in TSH due to the rs4704397 genotype affects the number of individuals classified as having subclinical hypothyroidism in pregnancy.

Methods:

Serum TSH, FT4, FT3, and thyroid peroxidase antibodies (TPOAbs) were measured in 970 pregnant women at 28 wk gestation. rs4704397 genotype was available on 877 subjects. Reference range calculations were based on the TPOAb-negative women.

Results:

TSH, but not FT4, FT3, or TPOAbs, varied with genotype and was highest in those with the AA genotype (median, 2.16, 1.84, and 1.73 mIU/liter for AA, AG, and GG genotypes, respectively; P = 0.0004). A greater proportion of women with the AA genotype had TSH concentrations above 4.21 mIU/liter, the upper limit of the reference range, compared with the AG and GG genotypes (9.6 vs. 3.5%, respectively; P = 0.004). Maternal PDE8B genotype was not associated with offspring birthweight or gestational age at delivery.

Conclusion:

Genetic variation in TSH levels in pregnancy associated with the PDE8B rs4704397 genotype has implications for the number of women treated for subclinical hypothyroidism under current guidelines. Consideration should be made to individualization of normal ranges, potential effects on pregnancy outcome, and intention to treat for subclinical hypothyroidism in pregnancy.
Cinacalcet HCl Reduces Hypercalcemia in Primary Hyperparathyroidism across a Wide Spectrum of Disease Severity
Authors: Peacock Munro , Bilezikian J. P. , Bolognese M. A. , Borofsky Michael , Scumpia Simona , Sterling Lulu Ren , Cheng Sunfa , Shoback Dolores
Published: January 2014

J Clin Endocrinol Metab, 10.1210/jc.2010-1221
Worsening of Obesity and Metabolic Status Yields Similar Molecular Adaptations in Human Subcutaneous and Visceral Adipose Tissue: Decreased Metabolism and Increased Immune Response
Authors: Klimčáková Eva , Roussel Balbine , Márquez-Quiñones Adriana , Kováčová Zuzana , Kováčiková Michaela , Combes Marion , Šiklová-Vítková Michaela , Hejnová Jindra , Šrámková Petra , Bouloumié Anne , Viguerie Nathalie , Štich Vladimir , Langin Dominique
Published: January 2014

Context: It is not known whether biological differences reported between sc adipose tissue (SAT) and visceral adipose tissue (VAT) depots underlie the pathogenicity of visceral fat.

Objective: We compared SAT and VAT gene expression according to obesity, visceral fat accumulation, insulin resistance, and presence of the metabolic syndrome.

Design: Subjects were assigned into four groups (lean, overweight, obese, and obese with metabolic syndrome).

Setting: Subjects were recruited at a university hospital.

Patients: Thirty-two women were included.

Main Outcome Measures: Anthropometric measurements, euglycemic-hyperinsulinemic clamps, blood analyses, and computed tomography scans were performed, and paired samples of SAT and VAT were obtained for DNA microarray-based gene expression profiling.

Results: Considering the two fat depots together, 1125 genes were more and 1025 genes were less expressed in lean compared with metabolic syndrome subjects. Functional annotation clustering showed, from lean to metabolic syndrome subjects, progressive down-regulation of metabolic pathways including branched-chain amino acid, fatty acid, carbohydrate, and mitochondrial energy metabolism and up-regulation of immune response genes involved in toll-like receptor, TNF, nuclear factor-κB, and apoptosis pathways. Metabolism and immune response genes showed an opposite correlation with fat mass, fat distribution, or insulin resistance indices. These associations were similar in SAT and VAT, although about 1000 genes showed differential expression between SAT and VAT.

Conclusions: The increase in adiposity and the worsening of metabolic status are associated with a coordinated down-regulation of metabolism-related and up-regulation of immune response-related gene expression. Molecular adaptations in SAT prove as discriminating as those in VAT.
GH Response to Oral Glucose Tolerance Test: A Comparison between Patients with Acromegaly and Other Pituitary Disorders
Authors: Verrua E. , Filopanti M. , Ronchi C. L. , Olgiati L. , Ferrante E. , Giavoli C. , Sala E. , Mantovani G. , Arosio M. , Beck-Peccoz P. , Lania A. G. , Spada A.
Published: January 2014

Context: The cutoff value of nadir GH after an oral glucose tolerance test (OGTT) used to define disease remission in acromegaly is higher than that observed in healthy subjects. However, it is uncertain whether the impaired GH inhibition might be related to subtle abnormalities of GH secretion or to functional and/or anatomical hypothalamic-pituitary disconnection due to tumor per se or treatments.

Objective: The objective of the study was to evaluate the impact of pituitary disorders other than acromegaly on GH response to OGTT.

Design, Subjects, and Methods: Thirty-three patients (24 females and nine males, aged 50.1 ± 12.3 yr, 13 operated and two irradiated) with various hypothalamic-pituitary disorders (HPDs), 45 healthy subjects (controls), and 42 cured acromegalic patients matched for sex, age. and body mass index were investigated. All subjects were studied for IGF-I levels and GH levels before and during the OGTT.

Results: In HPD patients mean postglucose nadir GH levels were 0.11 ± 0.08 μg/liter without any difference between patients treated with neurosurgery and/or radiotherapy and untreated and between patients with and without pituitary stalk alterations and/or hyperprolactinemia. Mean nadir GH values were similar in HPD patients and controls (0.11 ± 0.08 vs. 0.08 ± 0.08 μg/liter, P = 0.23) and lower than those found in cured acromegalic patients (0.18 ± 0.13 μg/liter, P = 0.02), although there was an overlapping in about half of patients.

Conclusions: Hypothalamic control of glucose-mediated GH suppression is not perturbed in patients with HPD. These data indicate that defective GH suppression to glucose that is found in acromegaly is unlikely to reflect a lack of integrity of hypothalamic function.