bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone Morphogenetic Proteins Stimulate Angiogenesis through Osteoblast-Derived Vascular Endothelial Growth Factor A
Authors: Deckers Martine M. L. , van Bezooijen Rutger L. , van der Horst Geertje , Hoogendam Jakomijn , van der Bent Chris , Papapoulos Socrates E. , Löwik Clemens W. G. M.
Published: January 2014

During bone formation and fracture healing there is a cross-talk between endothelial cells and osteoblasts. We previously showed that vascular endothelial growth factor A (VEGF-A) might be an important factor in this cross-talk, as osteoblast-like cells produce this angiogenic factor in a differentiation-dependent manner. Moreover, exogenously added VEGF-A enhances osteoblast differentiation. In the present study we investigated, given the coupling between angiogenesis and bone formation, whether bone morphogenetic proteins (BMPs) stimulate osteoblastogenesis and angiogenesis through the production of VEGF-A. For this we used the murine preosteoblast-like cell line KS483, which forms mineralized nodules in vitro, and an angiogenesis assay comprising 17-d-old fetal mouse bone explants that have the ability to form tube-like structures in vitro.

Treatment of KS483 cells with BMP-2, -4, and -6 enhanced nodule formation, osteocalcin mRNA expression, and subsequent mineralization after 18 d of culture. This was accompanied by a dose-dependent increase in VEGF-A protein levels throughout the culture period. BMP-induced osteoblast differentiation, however, was independent of VEGF-A, as blocking VEGF-A activity by a VEGF-A antibody or a VEGF receptor 2 tyrosine kinase inhibitor did not affect BMP-induced mineralization.

To investigate whether BMPs stimulate angiogenesis through VEGF-A, BMPs were assayed for their angiogenic activity. Treatment of bone explants with BMPs enhanced angiogenesis. This was inhibited by soluble BMP receptor 1A or noggin. In the presence of a VEGF-A antibody, both unstimulated and BMP-stimulated angiogenesis were arrested. Conditioned media of KS483 cells treated with BMPs also induced a strong angiogenic response, which was blocked by antimouse VEGF-A but not by noggin. These effects were specific for BMPs, as TGFβ inhibited osteoblast differentiation and angiogenesis while stimulating VEGF-A production.

These findings indicate that BMPs stimulate angiogenesis through the production of VEGF-A by osteoblasts. In conclusion, VEGF-A produced by osteoblasts in response to BMPs is not involved in osteoblast differentiation, but couples angiogenesis to bone formation.
Leptin-Induced Leptin Resistance Reveals Separate Roles for the Anorexic and Thermogenic Responses in Weight Maintenance
Authors: Scarpace Philip J. , Matheny Michael , Zhang Yi , Shek Eugene W. , Prima Victor , Zolotukhin Sergei , Tümer Nihal
Published: January 2014

The purpose of this study was to determine whether leptin induces leptin resistance by examining the temporal attenuation of the anorexic and energy expenditure responses to leptin. We administered recombinant adeno-associated virus encoding rat leptin cDNA or control viral vector into mildly obese rats for 138 d and compared these results with those from pair-fed rats. We measured food consumption, body weight, oxygen consumption, leptin signal transduction, and brown adipose tissue uncoupling protein 1. The anorexic response attenuated by d 25, whereas the increase in energy expenditure persisted for 83 d before attenuating. Despite attenuation of physiological responses, phosphorylated signal transducer and activator of transcription-3 remained elevated for the duration of the study. The temporal differential attenuation of the anorexic and thermogenic responses allowed us to determine the relative contributions of each response to weight maintenance. The anorexic response predominantly mediated the initial loss of body weight, but only the energy expenditure response was necessary to maintain the reduced weight. This study provides evidence that leptin induces leptin resistance. The leptin resistance was associated with persistent elevation in hypothalamic phosphorylated signal transducer and activator of transcription-3 and was characterized by a rapid attenuation of the anorexic response and slower onset for the attenuation of the energy expenditure response. We propose that both elevated leptin and obesity may be necessary for the development of leptin resistance.
A Novel Mutation in the LHX3 Gene is Responsible for Combined Pituitary Hormone Deficiency, Hearing Impairment, and Vertebral Malformations
Authors: Kriström Berit , Zdunek Anna-Maija , Rydh Anders , Jonsson Håkan , Sehlin Petra , Escher Stefan A.
Published: January 2014

Context: The LHX3 LIM-homeodomain transcription factor gene, found in both man and mouse, is required for development of the pituitary and motor neurons and is also expressed in the auditory system.

Objective: The objective of this study was to determine the cause of, and further explore, the phenotype in six patients (aged 6 months to 22 years) with combined pituitary hormone deficiency (CPHD), restricted neck rotation, scoliosis and congenital hearing impairment. Three of the patients also have mild autistic-like behaviour.

Design: As patients with CPHD and restricted neck rotation have previously been shown to have mutations in the LHX3 gene, a candidate gene approach was applied and the gene was sequenced. Neck anatomy was explored by computed tomography and magnetic resonance imaging, including three-dimensional reformatting.

Results: A novel, recessive, splice-acceptor site mutation was found. The predicted protein encoded by the mutated gene lacks the homeodomain and carboxyl terminus of the normal, functional protein. Genealogical studies revealed a common gene source for all six families dating back to the seventeenth century. Anatomical abnormalities in the occipito–atlanto–axial joints in combination with a basilar impression of the dens axis were found in all patients assessed.

Conclusions: This study extends both the mutations known to be responsible for LHX3-associated syndromes and their possible phenotypic consequences. Previously reported traits include CPHD and restricted neck rotation; patients examined in the present study also show a severe hearing defect. Additionally the existence of cervical vertebral malformations are revealed, responsible for the rigid neck and the development of scoliosis.
A Role for the Orphan Nuclear Receptor Estrogen-Related Receptor α in Endometrial Stromal Cell Decidualization and Expression of Genes Implicated in Energy Metabolism
Authors: Bombail Vincent , Gibson Douglas , Collins Frances , MacPherson Sheila , Critchley Hilary O. D. , Saunders Philippa T. K.
Published: January 2014

This article appears in The Journal of Clinical Endocrinology & Metabolism. 10.1210/jc.2010-0154
Increased Expression of the Na+/I− Symporter in Cultured Human Thyroid Cells Exposed to Thyrotropin and in Graves’ Thyroid Tissue*
Authors: Saito Tsukasa , Endo Toyoshi , Kawaguchi Akio , Ikeda Masato , Nakazato Minoru , Kogai Takahiko , Onaya Toshimasa
Published: January 2014

The Na+/I− symporter (NIS) is important in hormone synthesis in the thyroid gland. NIS activity, as reflected by I− uptake, was increased by TSH (1 mU/mL) or forskolin (10μ mol/L) in primary cultured human thyroid cells. Northern blot analysis revealed that incubation of these cells with TSH or forskolin for 24 h increased the abundance of NIS messenger ribonucleic acid (mRNA) 2.3- and 2.5-fold, respectively. Immunoblot analysis revealed 2.7- and 2.4-fold increases, respectively, in the amount of NIS protein after 48 h, suggesting that elevated levels of intracellular cAMP induced the expression of NIS in human thyrocytes. We then studied the levels of NIS mRNA and protein in Graves’ thyroid tissue and found that the amount of NIS mRNA in thyroid tissue from individuals with Graves’ disease (n = 5) was 3.8 times that in normal thyroid tissue (n = 5). The abundance of NIS mRNA was significantly correlated with that of thyroid peroxidase or thyroglobulin mRNAs, but not with that of TSH receptor mRNA, in the Graves’ and normal thyroid tissue specimens. The amount of NIS protein was also increased 3.1-fold in Graves’ thyroid tissue compared with that in normal thyroid tissue. The increased expression of NIS may thus contribute to the development of Graves’ disease.
Liposomal Thyroxine: A Noninvasive Model for Transplacental Fetal Therapy*
Authors: Bajoria Rekha , Fisk Nicholas M. , Contractor Soli F.
Published: January 2014

Drugs that cross the placenta sparingly are currently given directly to the fetus by invasive procedures. We investigated whether anionic small unilamellar (SUV) liposomes of different lipid compositions enhanced the transfer and uptake of T4 in an in vitro model of perfused human term placenta. T4-encapsulated anionic liposomes were prepared using lecithin (F-SUV) or distearoyl phosphatidylcholine (S-SUV) with cholesterol and dicetylcholine. The size distribution, encapsulation efficiency, and stability were determined in blood-based media. The transfer kinetics of free and liposomally encapsulated T4 were studied in a dually perfused isolated lobule of human term placenta, with creatinine and liposomal carboxyfluorescein as marker substances. Concentrations of T4 and rT3 were measured by RIA. T4 crossed the placenta sparingly (1.9 ± 0.5%) because it was metabolized to rT3 (9.2 ± 1.3%). Transplacental transfer of T4 was significantly increased by F-SUV (15.8 ± 2.1%; P < 0.001) and S-SUV liposomes (7.1 ± 1.2%; P < 0.001), with a concomitant decrease in fetal rT3 levels (P < 0.001). Placental uptake of F-SUV (13.5 ± 2.0%; P < 0.001) was greater than that of S-SUV liposomes (6.7 ± 0.8%; P < 0.001). Our data suggest that anionic liposomes increase transplacental transfer of T4. If confirmed in vivo, liposomes may provide an alternative noninvasive method of drug delivery to the fetus.
Both Hypothyroidism and Hyperthyroidism Enhance Low Density Lipoprotein Oxidation*
Authors: Sundaram Vidya , Hanna Atef N. , Koneru Lata , Newman H. A. I. , Falko James M.
Published: January 2014

Hypothyroidism is frequently associated with hypercholesterolemia and an increased risk for atherosclerosis, whereas hyperthyroidism is known to precipitate angina or myocardial infarction in patients with underlying coronary heart disease. We have shown previously that l-T4 functions as an antioxidant in vitro and inhibits low density lipoprotein (LDL) oxidation in a dose-dependent fashion. The present study was designed to evaluate the changes in LDL oxidation in subjects with hypothyroidism and hyperthyroidism. Fasting blood samples for LDL oxidation analyses, lipoprotein determinations, and thyroid function tests were collected at baseline and after the patients were rendered euthyroid. The lag phase (mean ± sem hours) of the Cu+2-catalyzed LDL oxidation in the hypothyroid state and the subsequent euthyroid states were 4 ± 0.0.65 and 14 ± 0.68 h, respectively (P < 0.05). The lag phase during the hyperthyroid phase was 6 ± 0.55 h, and that during the euthyroid phase was 12 ± 0.66 h (P < 0.05). The total and LDL cholesterol levels were higher in hypothyroidism than in euthyroidism and were lower in hyperthyroidism than in the euthyroid state. We conclude that LDL has more susceptibility to oxidation in both the hypothyroid and hyperthyroid states. Thus, the enhanced LDL oxidation may play a role in the cardiac disease process in both hypothyroidism and hyperthyroidism.
Expression of Somatostatin Receptor SST4 in Human Placenta and Absence of Octreotide Effect on Human Placental Growth Hormone Concentration during Pregnancy1
Authors: Caron Philippe , Buscail Louis , Beckers Albert , Estève Jean-Pierre , Igout Ahmed , Hennen Georges , Susini Christiane
Published: January 2014

In pregnancy, the human placenta GH acts as a growth-promoting hormone and appears to be the main stimulator of insulin-like growth factor I (IGF-I) secretion. In a woman with a TSH-secreting macroadenoma, successful treatment with the somatostatin analog octreotide was conducted during the first month and the second half of pregnancy without side-effects on placental and fetal development. As observed in normal pregnancy, both serum placental GH and IGF-I levels increased throughout pregnancy and dropped sharply after delivery. In placental membranes from both treated and healthy untreated patients, we demonstrated the presence of high affinity binding sites for somatostatin-14 (Kd, 4.6 and 5.3 nmol/L; binding capacity, 1.53 and 1.35 pmol/mg protein, respectively). These receptors displayed low affinity for octreotide (IC50, 1.2–2 μmol/L), suggesting the presence of SST1 and/or SST4 receptors. We found that messenger ribonucleic acids of these two subtypes were expressed in both human placental tissue and purified human cytotrophoblast cells. Finally, the SST1-selective analog, des-AA1,2,5[d-Trp8,IAmp9]S-14 had low affinity for placental somatostatin receptors. These results argue in favor of the presence of the SST4 subtype in human placenta. At the doses administered, octreotide did not bind to placental somatostatin receptors. Our results may explain the absence of changes in both human placental GH and IGF-I concentrations that we observed during octreotide treatment.
Randomized, Double Blind, Placebo-Controlled Trial of Low Dose Iodide in Endemic Goiter
Authors: Kahaly George , Dienes Hans Peter , Beyer Jürgen , Hommel Gerhard
Published: January 2014

Iodine (I) is essential for normal thyroid function, and the majority of subjects tolerate a wide range of dietary levels. However, a subset of individuals upon exposure to normal or elevated levels of I develop thyroid dysfunction and autoimmunity. In this double blind trial, we evaluated efficacy and tolerability of low dose I in adults with euthyroid, diffuse, endemic goiter. Sixty-two subjects were randomly assigned I (0.2 mg/day) or placebo for 12 months. After termination of therapy, both groups were followed for a further 6 months. Thyroid sonography and determinations of thyroid-related hormones, urinary I excretion per 24 h, and thyroid antibodies were carried out at baseline and at 3, 6, 9, 12, 15, and 18 months. Markedly elevated urinary I values were found during therapy in subjects receiving I (32 at baseline vs. 213 μg/24 h at 12 months; P = 0.0001) compared to placebo (34 and 33 μg/24 h, respectively; P < 0.0001 vs. I). I substantially reduced thyroid volume (29 vs. 18 mL at 12 months; −38%; P = 0.0001), and at 18 months, the therapeutic effect was sustained. In the placebo group, no significant changes were observed. High microsomal and thyroglobulin autoantibody titers were present in 3 of 31 (9.7%) subjects receiving I, and I-induced hypo- and hyperthyroidism developed in 2 and 1, respectively. Fine needle biopsy revealed marked lymphocytic infiltration in all 3 cases. After withdrawal of I, thyroid dysfunctions spontaneously remitted, and antibody titers as well as lymphocytic infiltration decreased markedly. Follow-up of these 3 subjects for an additional 2 yr showed normalization of antibody titers in 2. Thus, among subjects with endemic goiter, low dose I successfully normalized thyroid volume and body I supplementation; nevertheless, reversible I-induced thyroid dysfunctions and autoimmunity were observed in nearly 10% of the subjects.
Omeprazole: Calcitonin Stimulation Test for the Diagnosis Follow-Up and Family Screening in Medullary Thyroid Carcinoma
Authors: Erdoğan Murat Faik , Güllü Sevim , Başkal Nilgün , Uysal Ali Riza , Kamel Nuri , Erdoğan Gürbüz i
Published: January 2014

Medullary thyroid carcinoma (MTC) occurs sporadically but may also be inherited as part of the multiple endocrine neoplasia (MEN) type 2 syndrome. Screening of the patients and first degree relatives annually with basal and provocative tests for serum immunoreactive calcitonin (CT) levels is essential and enables potentially curative disease. Pentagastrin and calcium are the usual provocative agents used worldwide. We used endogenous gastrin (GT) release achieved by omeprazole, 20 mg b.i.d., to stimulate CT in 9 MTC, in 3 MEN 2A family members, and in 50 healthy control subjects. A steady and significant increase both in GT and CT levels was achieved in 9 MTC patients and 3 of the 14 family members tested, whereas in healthy controls the CT increase stimulated by GT was insignificant.

Preliminary results showed that this new, safe, cheap, and outpatient-basis test can be used in MTC diagnosis, follow-up, and screening.
Deuterium and Bromide Dilution, and Bioimpedance Spectrometry Independently Show That Growth Hormone-Deficient Adults Have an Enlarged Extracellular Water Compartment Related to Intracellular Water
Authors: van Marken Lichtenbelt Wouter D. , Snel Yvonne E. M. , Brummer Robert-Jan M. , Koppeschaar Hans P. F.
Published: January 2014

GH has a strong influence on body composition. However, the effects of GH deficiency in adults on water compartments are not well understood. Therefore, extracellular water (ECW) and total body water were independently determined by deuterium and bromide dilution and by bioimpedance spectrometry in GH-deficient (GHD) adults and compared to those in controls, matched for age, sex, body weight, and height.

The results show that the percent body fat was significantly (P < 0.05) higher, and total body water and intracellular water (ICW) were significantly lower in GHD adults for males, females, and both sexes combined. ECW was not significantly different between the two groups. ECW/ICW in GHD adults (0.42 ± 0.03) was significantly (P < 0.01) higher than that in controls (0.39 ± 0.02). There was a significant positive relation between the ECW/ICW ratio and the percent body fat. These results were confirmed by the bioimpedance spectrometry measurements.
Altered Expression of Fibroblast Growth Factor Receptors in Human Pituitary Adenomas
Authors: Abbass S. Ali Asghar , Asa Sylvia L. , Ezzat Shereen
Published: January 2014

We have shown that basic fibroblast growth factor (FGF) is heterogeneously expressed by human pituitary adenomas and may be implicated as a growth stimulus for these tumors. There are four mammalian FGF receptor (FGFR) genes encoding a complex family of transmembrane tyrosine kinases. The prototypic receptor is composed of three Ig-like extracellular ligand-binding domains, a transmembrane domain, and a cytoplasmic split tyrosine kinase. Multiple forms of cell-bound or secretable isoforms of FGFR-1, -2, and -3 can be generated by cell- and tissue-specific alternative splicing, resulting in tissue-specific FGF function. Shifts in isoform expression accompany tumor progression in some systems.

We examined the normal human adenohypophysis and 40 pituitary adenomas to determine the pattern of FGFR expression by reverse transcription-PCR; all tumors were characterized clinically and morphologically. Ribonucleic acid (RNA) was extracted from frozen tumor tissue and primers were used to distinguish messenger RNA of the secretable first Ig-like domain (I) and those of the transmembrane and kinase domains (K) of each FGFR subtype. The normal pituitary-expressed mRNAs for FGFR-1 I and K, FGFR-2 I and K, FGFR-3 I and K, and FGFR-4 I but not FGFR-4 K; this represents the first report of a truncated isoform of FGFR-4, indicating possible alternative polyadenylation sites in this receptor. Only 3 tumors had the same pattern of expression of the 4 FGFRs as the normal gland. Although all tumors expressed FGFR-1 I, 1 tumor did not express FGFR-1 K, suggesting the production of only a secretable form of FGFR-1 by this tumor. Four tumors were negative for FGFR-2 I and K; 6 expressed the secretable form only, and 17 expressed FGFR-2 K but not I. All tumors expressed FGFR-3 I; 14 had secretable forms only, and no tumors expressed FGFR-3 K alone. As in the normal gland, 13 tumors expressed only the secretable I form of FGFR-4. Unlike the normal pituitary, however, 22 expressed FGFR-4 I and K, indicating a possible tumor-specific transmembrane receptor. Five tumors were negative for FGFR-4 I and K. Expression of FGFR proteins was confirmed by immunohistochemical localization of the C-terminal portion of FGFR-1, -2, -3, and -4; the results correlated with the RNA data in each case. There was no correlation between tumor type, size, or aggressiveness and the expression pattern of FGFRs.

Our study suggests that pituitary adenomas have altered FGFR subtype and isoform expression, which may determine their hormonal and proliferative responses to FGFs.
Idiopathic Hypothalamic Diabetes Insipidus, Pituitary Stalk Thickening, and the Occult Intracranial Germinoma in Children and Adolescents*
Authors: Mootha Sudha L. , Barkovich Anthony J. , Grumbach Melvin M. , Edwards Michael S. , Gitelman Stephen E. , Kaplan Selna L. , Conte Felix A.
Published: January 2014

We report nine consecutive children and adolescents [five females and four males; aged 2 yr 8 months (m) to 18 yr 1 m] studied over the last 5 yr with idiopathic central diabetes insipidus. In addition to vasopressin deficiency, anterior pituitary hormone deficiencies were detected, either on evaluation at presentation or during follow-up studies over the following 3 yr. Four patients had an increased concentration of plasma PRL. One patient had multiple pituitary hormone deficiencies at diagnosis, and two others developed the same by 21 m of follow-up. Brain magnestic resonance imaging scans, performed at presentation, were originally interpreted as normal in four of nine patients, except for absence of the bright posterior pituitary signal; after retrospective review, two of nine were considered normal. All of the brain magnetic resonance imaging (MRI) scans showed positive findings by 14 m of follow-up. The first abnormal finding in all patients was isolated pituitary stalk thickening. Evaluation of cerebrospinal fluid (CSF) for hCG was positive in three of eight evaluated patients; the three positive CSF values were found at presentation and 3 and 9 m after presentation. All eight patients assessed were negative for CSF α-fetoprotein and cytology, and no patient had serum tumor markers. Transsphenoidal biopsy of the lesion in seven of nine patients showed a germinoma in six patients and inflammatory cells in one. The six patients with documented germinoma comprise 31% of the intracranial germinomas diagnosed in this age group at the University of California-San Francisco during the last 5 yr. The patient with mononuclear inflammatory cells on biopsy along with one other patient have had spontaneous resolution of their stalk thickening. So-called “idiopathic” central diabetes insipidus warrants close follow-up to determine the etiology, especially if anterior pituitary hormone deficiencies are detected. Normal brain MRI scans or scans that show isolated pituitary stalk thickening merit follow-up with serial contrast enhanced brain MRI for the early detection of an evolving occult hypothalamic-stalk lesion. CSF evaluation is recommended at presentation because elevated CSF hCG may precede MRI abnormalities.
Ex Vivo Expansion of Human Pancreatic Endocrine Cells1
Authors: Beattie Gillian M. , Cirulli Vincenzo , Lopez Ana D. , Hayek Alberto
Published: January 2014

Cell transplantation as a therapy for type 1 diabetes is facilitated by ex vivo cell expansion of pancreatic β-cells without loss of differentiative characteristics. The aim of this study was to determine the optimal conditions for in vitro growth of functional human pancreatic endocrine tissue. We examined the mitogenicity of matrixes from a variety of cell lines; proliferation was greater in cells growing on matrixes from bladder carcinoma cell lines, especially in monolayers grown on matrix from the human cell line HTB-9. After 14-day culture, there was a more than 100-fold proliferative increase, which was augmented to a more than 200-fold when hepatocyte growth factor/scatter factor was added; however, hepatocyte growth factor/scatter factor induced a rapid decrease in insulin content. Without the growth factor, fetal cell monolayers expanded 4-fold with no insulin loss; however, after 12-fold expansion, the insulin levels decreased to 40% of those in unexpanded cells. Adult islet cells expanded 3-fold without insulin loss. After 5-fold expansion, insulin levels decreased by 25% compared to those in free floating islets while retaining a normal response to secretagogues. Together, these results indicate that HTB-9 matrix provides the best stimulatory effect on replication of human endocrine cells, with little loss of in vitro function.
Serum Leptin Levels in Women with Polycystic Ovary Syndrome: The Role of Insulin Resistance/ Hyperinsulinemia1
Authors: Laughlin G. A. , Morales A. J. , Yen S. S. C.
Published: January 2014

Polycystic ovary syndrome (PCOS) is associated with chronic anovulation, hyperandrogenemia, insulin resistance (IR)/hyperinsulinemia, and a high incidence of obesity. Thus, PCOS serves as a useful model to assess the role of IR and chronic endogenous insulin excess on leptin levels. Thirty-three PCOS and 32 normally cycling (NC) women of similar body mass index (BMI) were studied. Insulin sensitivity (SI) was assessed by rapid ivGTT in a subset of 28 PCOS and 29 NC subjects; percent body fat was determined by dual-energy x-ray absorptiometry (DEXA) in 14 PCOS and 17 NC. Fasting (0800 h) and 24-h mean hourly insulin levels were 2-fold higher (P < 0.0001), and SI was 50% lower (P = 0.005) in PCOS than in NC, while serum androstenedione (A), testosterone (T), 17-α hydroxyprogesterone (17OHP), and estrone (E1) levels were elevated (P < 0.0001), and sex hormone-binding globulin (SHBG) levels were decreased (P < 0.01). Twenty-four hour LH pulse frequency, mean pulse amplitude, and mean LH levels were elevated in PCOS (P < 0.001) as compared with NC.

Serum leptin levels for PCOS (24.1 ± 2.6 ng/mL) did not differ from NC (21.5 ± 3.5 ng/mL) and were positively correlated with BMI (r = 0.81) and percent body fat (r = 0.91) for the two groups (both P < 0.0001). Leptin levels for PCOS and NC correlated positively with fasting and 24-h mean insulin levels (r = 0.81, P < 0.0001 for both PCOS and NC) and negatively with SI and SHBG levels. Leptin concentrations for PCOS, but not NC, correlated positively with 24-h mean glucose levels and inversely with 24-h mean LH levels and 24-h mean LH pulse amplitude. Leptin levels were not correlated with estrogen or androgen levels for either PCOS or NC, although leptin levels were positively related to the ratios of E1/SHBG and E2/SHBG for both PCOS and NC and to the ratio of T/SHBG for PCOS only. In stepwise multivariate regression with forward selection, only 24-h mean insulin levels contributed significantly (P < 0.01) to leptin levels independent of BMI and percent body fat for both PCOS and NC. Given this relationship and the presence of 2-fold higher 24-h mean insulin levels in PCOS, the expected elevation of leptin levels in PCOS was not found. This paradox may be explained by the presence of adipocyte IR specific to PCOS, which may negate the stimulatory impact of hyperinsulinemia on leptin secretion, a proposition requiring further study.
Embryonic Regulation of Integrins β3,α 4, and α1 in Human Endometrial Epithelial Cells in Vitro1
Authors: Simón Carlos , Gimeno MarÍa José , Mercader Amparo , O’Connor José Enrique , RemohÍ José , Polan Mary Lake , Pellicer Antonio
Published: January 2014

In the present study, we examined the embryonic regulation ofβ 3 integrin in human endometrial epithelial cells (EEC) at the protein level and analyzed putative embryonic factors responsible for this regulation. The model employed is based on a clinical in vitro fertilization program in which single human embryos were cocultured with EEC until blastocyst stage and then transferred back to the uterus. After embryo transfer, EEC wells were divided according to the embryonic status reached: EEC with embryos that achieved the blastocyst stage, EEC with arrested embryos, and EEC without embryos. Immunostaining for β3 was positive in plasma membrane of EEC. Flow cytometry showed a mean percentage ofβ 3-stained cells of 24.1 ± 5.7 in EEC cocultured with embryos that achieved the blastocyst stage (n = 13) vs. 9.5 ± 1.6 (P < 0.05) in those EEC cultured with arrested embryos (n = 12). Immunostaining for α1 and α4 integrins was negative in EEC monolayers studied, regardless of the presence or absence of embryos, and these findings were confirmed by flow cytometry. The possibility that the embryonic IL-1 system and leukemia inhibitory factor were involved in the endometrial β3 up-regulation was investigated by neutralizing experiments demonstrating a significant inhibition of β3-stained cells when EEC monolayers were cultured in the presence of EEC/blastocyst-conditioned media with (n = 4) vs. without (n = 8) antihuman interleukin (IL)-1α + IL-1β (1.65% vs. 14.6%; P < 0.05). Dose-response experiments further demonstrated an up-regulation of β3 positive cells when IL-1α + IL-1β were added to the medium at a concentration of 10 pg/mL compared with control medium without added cytokines (40% vs. 20%, n = 4). The functional relevance of the EEC β3 up-regulation was tested using a mouse blastocyst adhesion assay. More mouse blastocysts attached to EEC previously in contact with human blastocyst (72.7%) compared with those EEC previously in contact with arrested embryos (40%). Our results demonstrate the selective effect of a developing human embryo on EEC expression of β3, which is maximal when a human blastocyst instead of an arrested embryo is considered. Furthermore, the embryonic IL-1 system seems to be involved in the EECβ 3 up-regulation, reinforcing the concept of precise paracrine cross-talk between blastocyst and endometrial epithelium during embryonic implantation.
Pharmacokinetic Characteristics, Efficacy, and Safety of Buccal Testosterone in Hypogonadal Males: A Pilot Study1
Authors: Dobs Adrian S. , Hoover Donald R. , Chen Min-Chi , Allen Richard
Published: January 2014

Transbuccal administration of drugs provides an easy route of administration. To test the safety and efficacy of a novel testosterone (T) product, we performed a randomized, double blind, placebo-controlled study in a parallel design. Men with serum T levels below 250 ng/dL were administered either an active buccal tablet containing 10 mg T (n = 7) or a buccal placebo tablet (n = 6) containing 3 mg pseudoephedrine HCl for taste matching. Men were studied while taking a standard T enanthate dose, after 6 weeks of a wash-out period, and after 8 weeks of therapy. The men were matched for age (mean ± sd, 41 ± 16 vs. 47 ± 16) and type of hypogonadism (three primary testicular failures in each group, with the remainder having a central etiology). Acute pharmacokinetic testing showed peak serum hormone levels at 30 min, with a mean serum T concentration of 2688 ± 147 ng/dL (range, 1820–3770 ng/dL). Levels returned to baseline in 4–6 h, resulting in a total T area under the curve level of 3865 ng/hn·dL, less than that using other available forms of T administration. Similar pharmacokinetics were observed for the hormone’s metabolites, bioavailable T, free T, and estradiol. After 8 weeks of treatment, the results of nocturnal penile tumescence studies evaluating rigidity and circumference were significantly different from those in the placebo group (P < 0.05) and comparable to those during T enanthate therapy. In conclusion, transbuccal T therapy was sufficient to maintain normal sexual function while minimizing the total time of exposure to elevated circulating serum T levels.
Sex Differences in Circulating Human Leptin Pulse Amplitude: Clinical Implications1
Authors: Licinio Julio , Negrão André B. , Mantzoros Christos , Kaklamani Virginia , Wong Ma-Li , Bongiorno Peter B. , Negro Paula P. , Mulla Abeda , Veldhuis Johannes D. , Cearnal Laura , Flier Jeffrey S. , Gold Philip W.
Published: January 2014

Leptin, a product of fat cells, provides a signal of nutritional status to the central nervous system. Leptin concentrations have ultradian and diurnal fluctuations. We conducted this study to assess sex differences in the levels of organization of frequently sampled leptin concentrations in healthy, normal weight women and men. Leptin levels were sampled every 7 min for 24 h in 14 healthy, normal weight individuals (6 women and 8 men). The 14 leptin time series containing a total of 2898 leptin measurements were assessed by 1) algorithms that characterize statistically significant pulsatility, 2) Spectral (Fourier) analysis, 3) analysis of time intervals and variability, and 4) approximate entropy. We found that frequently sampled plasma leptin concentrations have a 24-h profile that is numerically more than twice as high in women as in men, and leptin pulse amplitude is likewise more than twice as high in women. However, healthy men and women have nearly identical concentration-independent and frequency-related 24-h and ultradian patterns. Leptin concentrations have nonrandom fluctuations over 24 h, independent of their absolute value and underlying 24-h periodicity, that are similar in men and women. Ultradian periodicities detected by Fourier time series have similar values in men and women. The strongest distinction between the sexes in the level of organization of leptin concentration is not at the level of pulse organization or oscillation frequency, but, rather, in the mass or amount of leptin released (or removed) per unit time, indicating that women might be more resistant to the effects of leptin than men. Because leptin is clinically relevant to the regulation of body weight, future studies should examine whether the relative leptin resistance exhibited by women might contribute to their increased susceptibility to disorders whose pathophysiology involves dysregulation of food intake and body weight.
MK-677, an Orally Active Growth Hormone Secretagogue, Reverses Diet-Induced Catabolism1
Authors: Murphy M. G. , Plunkett L. M. , Gertz B. J. , He W. , Wittreich J. , Polvino W. M. , Clemmons D. R.
Published: January 2014

The reversal of diet-induced negative nitrogen balance by GH suggests a possible therapeutic role for GH treatment in catabolic patients. A double-blind, randomized, placebo-controlled, two-period, cross-over study was designed to investigate whether MK-677, an orally active nonpeptide mimic of GH-releasing peptide, can reverse diet-induced protein catabolism. Eight healthy volunteers (ages 24–39 yr) were calorically restricted (18 kcal/kg·day) for two 14-day periods. During the last 7 days of each diet period, subjects received either oral MK-677 25 mg or placebo once daily. There was a 14- to 21-day washout interval between periods. During the first week of caloric restriction (i.e. diet alone), daily nitrogen losses were similar for both treatment groups (mean ± se; MK-677 group −2.67 ± 0.40 g/day vs. placebo group− 2.83 ± 0.26 g/day). During the second week (diet and study drug), mean daily nitrogen balance was 0.31 ± 0.21 g/day in the MK-677 treatment group compared with −1.48 ± 0.21 g/day in the placebo group (P < 0.01). MK-677 improved nitrogen balance integrated over the 7 days of treatment; area under the curve day 8–14 nitrogen balance response was +2.69 ± 5.0 (se) for MK-677 and −8.97 ± 5.26 g·day for placebo (P < 0.001). MK-677 produced a peak GH response of 55.9 ± 31.7 μg/L after single dose (day 1 of treatment) and 22.6 ± 9.3 μg/L after a week of dosing compared with placebo treatment peak GH values of approximately 9 (treatment day 1) and approximately 7 μg/L (treatment day 7). Following the initial 7-day caloric restriction, insulin-like growth factor-I (IGF-I) declined from 232 ± 25 to 186 ± 19 ng/mL in the MK-677 group and from 236 ± 19 to 174 ± 23 ng/mL in the placebo group. Mean IGF-I concentration increased significantly during MK-677 to 264 ± 31 ng/mL (mean for the last 5 days of treatment) compared with 188 ± 19 ng/mL with placebo (P < 0.01). No significant difference in IGF binding protein-2 was found between the MK-677 and placebo treatments. However, the mean in IGF binding protein-3 for the last 5 days of MK-677 treatment was also significantly increased to 3273 ± 330 ng/mL (mean ± se) compared with placebo 2604 ± 253 ng/mL (P < 0.01). Neither the serum cortisol nor the PRL response was significantly greater after 7 days of MK-677 dosing compared with 7 days of placebo. MK-677 (25 mg) was generally well tolerated and without clinically significant adverse experiences. In conclusion, MK-677 reverses diet-induced nitrogen wasting, suggesting that if these short-term anabolic effects are maintained in patients who are catabolic because of certain acute or chronic disease states, it may be useful in treating catabolic conditions.
Identification of Mutations of the Arginine Vasopressin-Neurophysin II Gene in Two Kindreds with Familial Central Diabetes Insipidus
Authors: Heppner Christina , Kotzka Jörg , Bullmann Catharina , Krone Wilhelm , Müller-Wieland Dirk
Published: January 2014

Familial central diabetes insipidus is transmitted as an autosomal dominant trait with almost complete penetrance. Twenty-three different mutations of the arginine vasopressin-neurophysin II gene have been reported to date, located within the signal peptide-, the arginine vasopressin-, or the neurophysin II-coding region. In the present study two kindreds with familial central diabetes insipidus were examined. The entire coding region of the arginine vasopressin-neurophysin II gene of one affected subject of each family was amplified by PCR and subcloned into a pUC 18 plasmid, and six positive clones were sequenced. After identification of the mutation, direct sequencing was performed on the respective sequence of family members and 28 healthy control subjects. In family A, a missense mutation (C→T) at nucleotide position 280 was detected, predicting the substitution of alanine by valine at position −1 of the signal peptide. All affected subjects were heterozygote for the mutation, whereas none of the unaffected family members or control subjects displayed the mutant sequence. In family B, a missense mutation within the neurophysin II-coding sequence was identified (nucleotide 1757, G→C), predicting the substitution of glycine by arginine at position 23. Again, affected family members were found to be heterozygote for the mutation, which was not observed in unaffected family members or in control subjects. Although the mutation of family A was recently described in 3 other kindreds as well, the mutation within the neurophysin II-coding region represents a novel mutation of the AVP-NP II gene.
Corticotropin Regulates Vascular Endothelial Growth Factor Expression in Human Fetal Adrenal Cortical Cells1
Authors: Shifren Jan L. , Mesiano Sam , Taylor Robert N. , Ferrara Napoleone , Jaffe Robert B.
Published: January 2014

The human adrenal cortex has a complex vasculature that is essential for growth, organ maintenance, and access of secreted hormones to the circulation. Growth and function of the adrenal cortex are regulated by corticotropin (ACTH), the actions of which are in part mediated by locally produced growth factors. As cortical growth and vascularization must increase in a coordinated manner, we hypothesized that ACTH also influences adrenal cortical angiogenesis by stimulating the local expression of specific angiogenic factors. Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific angiogenic peptide, the expression of which has been detected in adrenal cortical cells. Therefore, we examined the localization of VEGF expression in the midgestation (16–20 weeks) human fetal adrenal cortex and determined whether VEGF expression and secretion by isolated human fetal adrenal cortical cells are regulated by ACTH. By immunohistochemical analysis, strong cytoplasmic staining for VEGF was detected in scattered clusters of fetal zone (inner cortical compartment) cells. In contrast, cells in the outer, definitive zone of the cortex stained only weakly for VEGF. The predominant staining for VEGF in the fetal zone correlated with the extensive vasculature of this zone as detected by immunohistochemical staining for von Willebrand factor, which is specific for endothelial cells. In primary cultures of human fetal adrenal cortical cells, ACTH (1 nmol/L) and forskolin (10 μmol/L) increased the abundance of messenger ribonucleic acid transcripts encoding VEGF, as assessed by Northern and slot blot analyses. The stimulatory effect of ACTH and forskolin on VEGF gene expression occurred within 2 h of agonist exposure and persisted for at least 24 h. ACTH and forskolin also increased VEGF protein secretion by fetal adrenal cortical cells, as assessed by enzyme-linked immunosorbent assay for VEGF in fetal adrenal cortical cell-conditioned medium. A significant (P< 0.05) increase in VEGF secretion was detected as early as 8 h after ACTH or forskolin treatment. By 24 h after the addition of ACTH or forskolin, VEGF secreted from isolated human fetal adrenal cells was increased 5- to 6-fold. These data demonstrate that the human fetal adrenal cortex, particularly the cells of the inner fetal zone, express VEGF and that VEGF expression and secretion by these cells are directly regulated by ACTH and the activation of adenylate cyclase. Thus, VEGF may be a local regulator of adrenal cortical angiogenesis and an important mediator of the tropic action of ACTH, ensuring the coordination of ACTH-stimulated cortical growth and vascularization.
Gender Differences in Both Spontaneous and Stimulated Leptin Secretion by Human Omental Adipose Tissue in Vitro: Dexamethasone and Estradiol Stimulate Leptin Release in Women, But Not in Men1
Authors: Casabiell Xesús , Piñeiro Verónica , Peino Roberto , Lage Mary , Camiña Jesús , Gallego Rosalía , Vallejo Luis Garcia , Dieguez Carlos , Casanueva Felipe F.
Published: January 2014

Leptin is a hormone secreted by the adipocytes to serve as a signal to the central nervous system to regulate energy homeostasis. Circulating leptin mainly reflects both total fat mass and the size of constituent adipocytes, although other ancillary hormonal factors may contribute to its blood concentration. Relevant gender differences in leptin concentrations have been reported, but it is not clear whether the elevated leptin levels in women are an intrinsic property of their adipocytes or merely reflect a greater amount of fat reserves.

To clarify these points, a systematic study with organ culture from human omental adipose tissue either stimulated or not with steroid hormones was undertaken in samples obtained at surgery from 67 nonobese donors (33 women and 34 men). The assay was standardized in periods of 24 h ending at 96 h, with no apparent tissue damage. Each adipose tissue sample from a single donor was incubated in triplicate, and leptin results are expressed as the mean ± sem of the integrated secretion to the medium (area under the curve; nanograms of leptin per g tissue/48 h).

Control nonstimulated samples showed a steady leptin secretion along the 96 h studied, with the peak of secretory activity reached at 48 h; afterward, the in vitro secretion reached a plateau state. Spontaneous leptin secretion in samples from 33 women (3904 ± 347) was significantly higher (P < 0.05) than that in samples from 34 men (2940 ± 323). Coincubation of adipose tissue with 1 μmol/L dexamethasone induced a clear-cut leptin increase (P < 0.05) in samples from women (5848 ± 624; n = 12), but did not change the spontaneous release of leptin in samples from men (3353 ± 741; n = 6). Similarly, coincubation of adipose tissue with 1 μmol/L estradiol induced a notable leptin increase (P < 0.05) in samples from women (5698 ± 688; n = 9), whereas it did not alter the secretion in the male samples (3373 ± 444; n = 6). In samples from both sexes, coincubation with 1 μmol/L estrone or progesterone had no effect, whereas 1 μmol/L forskolin significantly (P < 0.05) reduced leptin release.

In conclusion, leptin secretion from omental adipose tissue in vitro 1) is significantly higher in samples from women than in samples from men, 2) is stimulated by dexamethasone and estradiol in women but not in men, 3) is not modified by progesterone or estrone in both sexes, and 4) is inhibited by forskolin in both genders. This different response to the stimulation of adipose tissue may be the biological basis for the gender differences observed in circulating levels of human leptin.
Modest Hormonal Effects of Soy Isoflavones in Postmenopausal Women*
Authors: Duncan Alison M. , Underhill Kerry E.W. , Xu Xia , LaValleur June , Phipps William R. , Kurzer Mindy S.
Published: January 2014

Soy isoflavones have been hypothesized to exert hormonal effects in postmenopausal women. To test this hypothesis, we studied the effects of three soy powders containing different levels of isoflavones in 18 postmenopausal women. Isoflavones were consumed relative to body weight[ control: 0.11 ± 0.01; low isoflavone (low-iso): 1.00 ± 0.01; high isoflavone (high-iso): 2.00 ± 0.02 mg/kg/day] for 93 days each in a randomized crossover design. Blood was collected on day 1 of the study (baseline) and days 36–38, 64–66, and 92–94 of each diet period, for analysis of estrogens, androgens, gonadotropins, sex hormone binding globulin (SHBG), prolactin, insulin, cortisol, and thyroid hormones. Vaginal cytology specimens were obtained at baseline and at the end of each diet period, and endometrial biopsies were performed at baseline and at the end of the high-iso diet period, to provide additional measures of estrogen action. Overall, compared with the control diet, the effects of the low-iso and high-iso diets were modest in degree. The high-iso diet resulted in a small but significant decrease in estrone-sulfate (E1-S), a trend toward lower estradiol (E2) and estrone (E1), and a small but significant increase in SHBG. For the other hormones, the few significant changes noted were also small and probably not of physiological importance. There were no significant effects of the low-iso or high-iso diets on vaginal cytology or endometrial biopsy results. These data suggest that effects of isoflavones on plasma hormones per se are not significant mechanisms by which soy consumption may exert estrogen-like effects in postmenopausal women. These data also show that neither isoflavones nor soy exert clinically important estrogenic effects on vaginal epithelium or endometrium.
Hormone Replacement Therapy with Estrogen or Estrogen plus Medroxyprogesterone Acetate Is Associated with Increased Epithelial Proliferation in the Normal Postmenopausal Breast1
Authors: Hofseth Lorne J. , Raafat Ahmed M. , Osuch Janet R. , Pathak Dorothy R. , Slomski Carol A. , Haslam Sandra Z.
Published: January 2014

The relative effects of postmenopausal hormone replacement therapy (HRT) with estrogen alone vs. estrogen+progestin on breast cell proliferation and on breast cancer risk are controversial. A cross-sectional observational study was carried out to examine the proliferative effects of HRT with estrogen or estrogen plus the progestin, medroxyprogesterone acetate, in breast tissue of postmenopausal women. Benign breast biopsies from 86 postmenopausal women were analyzed with antiproliferating cell nuclear antigen (anti-PCNA) and Ki67 antibodies to measure relative levels of cell proliferation. Epithelial density and estrogen and progesterone receptor status were also determined. The women were categorized either as users of: 1) estrogen (E) alone; 2) estrogen+medroxyprogesterone acetate (E+P); or 3) no HRT. Compared with no HRT, the breast epithelium of women who had received either E+P or E alone had significantly higher PCNA proliferation indices, and treatment with E+P had a significantly higher index (PCNA and Ki67) than treatment with E alone. Breast epithelial density was significantly greater in postmenopausal women treated with E and E+P, compared with no HRT. Thus, the present study shows that postmenopausal HRT with E+P was associated with greater breast epithelial cell proliferation and breast epithelial cell density than E alone or no HRT. Furthermore, with E+P, breast proliferation was localized to the terminal duct-lobular unit of the breast, which is the site of development of most breast cancers. Further studies are needed to assess the possible association between the mitogenic activity of progestins and breast cancer risk.