bluuebunny/crossref_metadata_2025 huggingface dataset:
Minireview: Insights into G Protein-Coupled Receptor Function Using Molecular Models**The work from our laboratories was supported by USPHS Grant DK-43036.
Authors: Gershengorn Marvin C. , Osman Roman
Published: January 2014

G protein-coupled receptors (GPCRs) represent the largest family of signal-transducing molecules known. They convey signals for light and many extracellular regulatory molecules. GPCRs have been found to be dysfunctional/dysregulated in a growing number of human diseases and have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how GPCRs function at the molecular level is an important goal of biological research. In order to understand function at this level, it is necessary to delineate the 3D structure of these receptors. Recently, the 3D structure of rhodopsin has been resolved, but in the absence of experimentally determined 3D structures of other GPCRs, a powerful approach is to construct a theoretical model for the receptor and refine it based on experimental results. Computer-generated models for many GPCRs have been constructed. In this article, we will review these studies. We will place the greatest emphasis on an iterative, bi-directional approach in which models are used to generate hypotheses that are tested by experimentation and the experimental findings are, in turn, used to refine the model. The success of this approach is due to the synergistic interaction between theory and experiment.
PPARγ/RXRα Heterodimers Are Involved in Human CGβ Synthesis and Human Trophoblast Differentiation
Authors: Tarrade Anne , Schoonjans Kristina , Guibourdenche Jean , Bidart Jean Michel , Vidaud Michel , Auwerx Johan , Rochette-Egly Cécile , Evain-Brion Danièle
Published: January 2014

Recent studies performed with null mice suggested a role of either RXRα or PPARγ in murine placental development. We report here that both PPARγ and RXRα are strongly expressed in human villous cytotrophoblasts and syncytiotrophoblasts. Moreover, specific ligands for RXRs or PPARγ (but not for PPARα or PPARδ) increase both human CGβ transcript levels and the secretion of human CG and its free β-subunit. When combined, these ligands have an additive effect on human CG secretion. Pan-RXR and PPARγ ligands also have an additive effect on the synthesis of other syncytiotrophoblast hormones such as human placental lactogen, human placental GH, and leptin. Therefore, in human placenta, PPARγ/RXRα heterodimers are functional units during cytotrophoblast differentiation into the syncytiotrophoblast in vitro. Elements located in the regulatory region of the human CGβ gene (β5) were found to bind RXRα and PPARγ from human cytotrophoblast nuclear extracts, suggesting that PPARγ/RXRα heterodimers directly regulate human CGβ transcription. Altogether, these data show that PPARγ/RXRα heterodimers play an important role in human placental development.
Parallel Declines in Fos Activation of the Medial Anteroventral Periventricular Nucleus and LHRH Neurons in Middle-Aged Rats
Authors: Le W.-W. , Wise P. M. , Murphy A. Z. , Coolen L. M. , Hoffman G. E.
Published: January 2014

The middle-age decline in reproductive function is manifested by reduced LHRH release, resulting in a decreased magnitude and delay of onset of the LH surge. Earlier studies suggested that the reductions in LHRH neural activation in middle-aged rats resulted from deficits in the afferent drive to the LHRH neurons. One critical afferent to the LHRH neurons lies in the anteroventral periventricular preoptic area (AVPv) nucleus. The neurons of the medial AVPv are synchronously activated to express Fos with LHRH neurons at the time of an LH surge in young adult animals. The present study examined whether, in middle age, reductions in the activation of AVPv neurons accompany the reduction in Fos activation in LHRH neurons.

Young (3- to 4-month-old) and middle-aged (10- to 12-month-old) spontaneously cycling and ovariectomized steroid-replaced rats were killed during peak and early descending phase of the LH surge, and their brains were examined for Fos in LHRH and AVPv neurons. Young animals had a characteristic increase in Fos expression in both LHRH and AVPv neurons. In middle-aged rats, the proportion of LHRH neurons expressing Fos at the time of an LH surge was reduced by approximately 50%, irrespective of whether surges were spontaneous or induced by exogenous steroids. A similar reduction in the number of Fos+ cells (by approximately 50%) was noted in the medial AVPv. Linear regression analysis of the relationship between the extent of Fos activation in LHRH and AVPv neurons revealed a strong positive correlation (r2 = 0.66; P < 0.01), suggesting that changes in the AVPv’s drive to LHRH neurons underlie the decrease in LHRH activity in middle age.

A second series of experiments examined whether decreased input from the AVPv could account for reduced Fos activation in LHRH neurons seen in middle-aged animals. When the medial AVPv was lesioned, LHRH neurons failed to express Fos on the side ipsilateral to the lesion. Animals with lesioned medial AVPv also had significantly lower LH values than animals with an intact medial AVPv. Taken together, these data suggest that a principal deficit in middle-aged rats is the ability of the medial AVPv to stimulate LHRH neurons.
Development of an ER Action Indicator Mouse for the Study of Estrogens, Selective ER Modulators (SERMs), and Xenobiotics
Authors: Nagel Susan C. , Hagelbarger Jennifer L. , McDonnell Donald P.
Published: January 2014

We have developed a transgenic mouse that functions as a reporter of ER activity, termed ER action indicator (ERIN), by incorporating a transgene with an estrogen-responsive promoter (three copies of the vitellogenin estrogen response element with a minimal thymidine kinase promoter) linked to the reporter gene β-galactosidase. Evaluation of ER activity in female ERIN mice demonstrated estrogen-inducible expression of the reporter gene in the uterus, pituitary, and hypothalamus; established targets of estrogen action. Importantly, we also identified ER activity in a number of nonclassical estrogen target tissues, including kidney, liver, adrenal, and thyroid gland. ERIN provides a system to measure the same end point (transgene regulation) in different target tissues, permitting separation of the contributions of cell- and promoter-specific factors in determining ER pharmacology. In this regard we observed that on this specific promoter the pituitary gland was 25-fold more sensitive than the uterus to the estrogen diethylstilbestrol, implying the existence of cell-specific factors that influence ligand sensitivity. Our studies also identified considerable difference in the efficacy and potency of ER ligands in the uterus when ER transcriptional activity was assayed vs. uterine weight gain. Specifically, we observed that the environmental estrogen bisphenol A was a potent agonist in stimulating ER transcriptional activity, whereas it exhibited little uterotropic activity. In contrast to bisphenol A, tamoxifen significantly increased uterine weight, but minimally induced ER reporter activity in this tissue. Given the results of these studies, we believe that ERIN will be a useful model to evaluate ER ligand pharmacology and will assist in defining the cellular and molecular mechanisms that determine agonist and antagonist activity.
Identification of Specific Inhibin A-Binding Proteins on Mouse Leydig (TM3) and Sertoli (TM4) Cell Lines*
Authors: Harrison Craig A. , Farnworth Paul G. , Chan Karen L. , Stanton Peter G. , Ooi Guck T. , Findlay Jock K. , Robertson David M.
Published: January 2014

The binding of human inhibin A to cell surface binding proteins of mouse Leydig (TM3) and Sertoli (TM4) cell lines was investigated. Scatchard analysis identified two classes of inhibin A-binding sites on TM3 (Kd(1) = 85 pm and 4,160 sites/cell; Kd(2) = 520 pm and 12,500 sites/cell) and TM4 (Kd(1) = 61 pm and 2,620 sites/cell; Kd(2) = 520 pm and 10,400 sites/cell) cells. Compared with inhibin A, inhibin B only partially competed[ 125I]inhibin A binding (6–8%), whereas activin A competed weakly (<0.01%). Chemical cross-linking of[ 125I]inhibin A to both cell lines identified five[ 125I]inhibin A binding complexes with apparent molecular masses of 70, 95, 145, 155, and more than 200 kDa. Inhibin A displacement of [125I]inhibin A from each of these cross-linked species (ED50 = 60–110 pm) closely resembled displacement from intact TM3 (ED50 = 97 ± 32 pm) and TM4 (ED50 = 75± 28 pm) cells, suggesting that all of these proteins are involved in the high affinity inhibin A binding complex. Immunoprecipitation of iodinated inhibin A complexed to TM3 and TM4 cells with an antibody against human betaglycan identified protein complexes of more than 200, 145, and 95 kDa. It is concluded that the high affinity binding complex for inhibin A found in these cell lines consists of betaglycan and several proteins of unknown identity and may represent the putative inhibin receptor complex.
Male Germ Cells Regulate Transcription of the Cathepsin L Gene by Rat Sertoli Cells*
Authors: Zabludoff Sonya D. , Charron Martin , DeCerbo Joshua N. , Simukova Natalia , Wright William W.
Published: January 2014

It is well known that male germ cells regulate the steady state levels of numerous transcripts expressed by Sertoli cells. To date, however, there has been no direct test of whether this regulation reflects changes in gene transcription and/or transcript stability. This study used two experimental approaches to test the hypothesis that germ cells regulate transcription of the cathepsin L gene by rat Sertoli cells. We examined this gene because, in vivo, steady state levels of cath L messenger RNA in Sertoli cells change in a stage-specific manner as the surrounding germ cells progress through the 14 stages of the cycle of the seminiferous epithelium. In the first experimental approach, seminiferous tubules at stages VI–VII and stages IX–XII were incubated for 1 h in 4-thiouridine, and the amount of metabolically labeled cath L messenger RNA was quantified. The results demonstrate that transcription of the cath L gene by Sertoli cells is 7-fold higher at stages VI–VII than at stages IX–XII. The second experimental approach examined the ability of germ cells to regulate the activity of cath L reporter constructs in mature Sertoli cells. Before these studies, we isolated a cath L genomic clone and demonstrated that this clone contains the transcription start site of the cath L gene expressed by Sertoli cells. Transient transfection analysis then demonstrated that two reporter constructs, containing 244 and about 2.1 kb of sequence upstream from the transcription start site, had similar activities in mature Sertoli cells. However, germ cells only affected the activity of the larger construct in Sertoli cells, which was reduced by 30%. We conclude that germ cells regulate transcription of the cath L gene by Sertoli cells and that repressive effects of germ cells are mediated by elements upstream from nucleotide− 244 of this gene.
Expression of the mRNAs and Proteins for the Na+/H+ Exchangers and Their Regulatory Factors in Baboon and Human Placental Syncytiotrophoblast
Authors: Pepe Gerald J. , Burch Marcia G. , Sibley Colin P. , Davies William A. , Albrecht Eugene D.
Published: January 2014

In polarized epithelial cells of several organ systems, e.g. the kidney, a family of Na+/H+ exchangers (e.g. Na+/H+ exchanger-1 and -3) and their regulatory proteins, Na+/H+ exchanger regulatory factor and Na+/H+ exchanger-3 kinase A regulatory protein play a major role in regulating Na+/H+ exchange integral to cellular homeostasis. Because the primate placenta regulates exchange of Na+ and H+ between the mother and fetus critical to fetal-placental homeostasis, the current study determined whether Na+/H+ exchanger-1 and -3 were compartmentalized and associated with expression of Na+/H+ exchanger regulatory factor and Na+/H+ exchanger-3 kinase A regulatory protein in baboon and human syncytiotrophoblast. Using RT-PCR, single 413-bp Na+/H+ exchanger-1 and 190-bp Na+/H+ exchanger-3 products were expressed by baboon and human syncytiotrophoblasts. The 104-kDa Na+/H+ exchanger-1 protein was detected by Western blot in microvillus membranes and to a much lesser extent in the basal membranes of the baboon and human syncytiotrophoblasts. In contrast, the 85-kDa Na+/H+ exchanger-3 protein was detected primarily in membranes contiguous with the basal membranes of the syncytiotrophoblast of both species. Differential localization of Na+/H+ exchanger-1 and -3 was confirmed by immunocytochemistry. The Na+/H+ exchanger-3 regulatory protein, Na+/H+ exchanger-3 kinase A regulatory protein, resided almost exclusively in the basal membranes, whereas Na+/H+ exchanger regulatory factor was localized primarily to the microvillus membranes in the baboon and human syncytiotrophoblast. Collectively, these results are the first to show that the baboon and human term placental syncytiotrophoblast expressed the mRNAs and proteins for Na+/H+ exchanger-1 and -3 and their regulatory factors and that Na+/H+ exchanger-1 and Na+/H+ exchanger regulatory factor resided primarily in the microvillus membranes, whereas Na+/H+ exchanger-3 and Na+/H+ exchanger-3 kinase A regulatory protein were localized to membranes contiguous with the basal membranes and to the basal membranes, respectively. We conclude that a complete Na+/H+ exchange system is present in the baboon and human term placental syncytiotrophoblast and suggest that the primate placenta exhibits polarity with respect to the capacity for regulation of Na+/H+ exchange between the placenta and the maternal and fetal circulations.
The C-Terminal Region of PTHrP, in Addition to the Nuclear Localization Signal, Is Essential for the Intracrine Stimulation of Proliferation in Vascular Smooth Muscle Cells
Authors: de Miguel F. , Fiaschi-Taesch N. , López-Talavera J. C. , Takane K. K. , Massfelder T. , Helwig J.-J. , Stewart A. F.
Published: January 2014

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has “intracrine” actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1–36), mid region (38–86), nuclear localization signal, C terminus (108–139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.
The Vasopressin Receptors Colocalize with Vasopressin in the Magnocellular Neurons of the Rat Supraoptic Nucleus and Are Modulated by Water Balance
Authors: Hurbin Amandine , Orcel Hélène , Alonso Gérard , Moos Françoise , Rabié Alain
Published: January 2014

Activity of the magnocellular neurons that synthesize vasopressin in the supraoptic and paraventricular nuclei of the hypothalamus is modulated by local release of the neuropeptide within the nuclei. V1a and V1b vasopressin receptor genes are expressed in these cells. The present study reports the localization of V1a and V1b receptors using multiple labeling immunocytochemistry. Both receptors are mainly located in vasopressinergic magnocellular neurons and colocalized with vasopressin in cytoplasmic vesicles dispersed throughout the cell. Possible functional modifications of the mRNA and protein levels of the V1a receptor, the major isoform, were also investigated by semiquantitative in situ hybridization and immunocytochemistry in rats submitted to reduced or increased water intake. V1a mRNA and receptor levels varied with water balance. V1a mRNA level dropped in rats submitted to high water intake. Conversely, dehydration up-regulated the V1a receptor content. These observations suggest that the pathways that regulate the expression of the genes encoding vasopressin and the V1a receptor are linked, which fits the present findings that the two partners are colocalized in cytoplasmic vesicles. Colocalization might explain how V1 autoreceptors are controlled by cell activity and/or local concentration of vasopressin (released locally by the neurons themselves), allowing fine adjustment of magnocellular neuron activity.
Distribution, Neuronal Colocalization, and 17β-E2 Modulation of Small Conductance Calcium-Activated K+ Channel (SK3) mRNA in the Guinea Pig Brain
Authors: Bosch Martha A. , Kelly Martin J. , Rønnekleiv Oline K.
Published: January 2014

Molecular cloning has revealed the existence of three distinct small conductance (SK1–3) Ca2+-activated K+ channels. Because SK channels underlie the afterhyperpolarization (AHP) that is critical for sculpturing phasic firing in hypothalamic neurons, we investigated the distribution of these channels in the female guinea pig. Both SK1 and SK3 cDNA fragments were cloned using PCR, and ribonuclease protection assay as well as in situ hybridization analysis illustrated that the SK3 channel was the predominant subtype expressed in the guinea pig hypothalamus. Combined in situ hybridization and fluorescence immunocytochemistry revealed that SK3 mRNA was expressed in GnRH, dopamine, and vasopressin neurons, and all of these neurons exhibited an AHP current. Moreover, SK3 mRNA was found in other brain areas, including the septum, bed nucleus, amygdala, thalamus, midbrain, and hippocampus. Using quantitative ribonuclease protection assay, the rank order of SK3 mRNA expression was septum ≥ midbrain > rostral thalamus ≥ rostral basal hypothalamus ≥ caudal thalamus ≥ preoptic area ≫ caudal basal hypothalamus ≥ hippocampus. Moreover, 17β-E2 treatment, which reduces plasma LH during the negative feedback phase, significantly increased SK3 mRNA levels in the rostral basal hypothalamus (P < 0.05; n = 6). Therefore, these findings suggest that estrogen increases the mRNA expression of SK3 channels, which may represent a mechanism by which estrogen regulates hypothalamic neuronal excitability during negative feedback.
Cbl, IRS-1, and IRS-2 Mediate Effects of Rosiglitazone on PI3K, PKC-λ, and Glucose Transport in 3T3/L1 Adipocytes
Authors: Standaert Mary L. , Kanoh Yoshinori , Sajan Mini P. , Bandyopadhyay Gautam , Farese Robert V.
Published: January 2014

The thiazolidenedione, rosiglitazone, increases basal and/or insulin-stimulated glucose transport in various cell types by diverse but uncertain mechanisms that may involve insulin receptor substrate (IRS)-1-dependent PI3K. Presently, in 3T3/L1 adipocytes, rosiglitazone induced sizable increases in basal glucose transport that were: dependent on PI3K, 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and PKC-λ; accompanied by increases in tyrosine phosphorylation of Cbl and Cbl-dependent increases in PI3K and PKC-λ activity; but not accompanied by increases in IRS-1/2-dependent PI3K or protein kinase B activity. Additionally, rosiglitazone increased IRS-1 and IRS-2 levels, thereby enhancing insulin effects on IRS-1- and IRS-2-dependent PI3K and downstream signaling factors PKC-λ and protein kinase B. Our findings suggest that Cbl participates in mediating effects of rosiglitazone on PI3K, PDK-1, and PKC-λ and the glucose transport system and that this Cbl-dependent pathway complements the IRS-1 and IRS-2 pathways for activating PI3K, PDK-1, and PKC-λ during combined actions of rosiglitazone and insulin in 3T3/L1 cells.
Minireview: Neuro-Immuno-Endocrine Modulation of the Hypothalamic-Pituitary-Adrenal (HPA) Axis by gp130 Signaling Molecules
Authors: Chesnokova Vera , Melmed Shlomo
Published: January 2014

The neuroendocrine and immune systems communicate bidirectionally. The neuro-immune-endocrine interface is mediated by cytokines acting as auto/paracrine or endocrine factors regulating pituitary development, cell proliferation, hormone secretion, and feedback control of the hypothalamic-pituitary-adrenal (HPA) axis. At birth or during neonatal ontogenesis, cytokines produce permanent alterations of HPA axis function and the stress response. Overexpressing IL-6 or leukemia inhibitory factor leads to significant changes in pituitary development and functions. Pituitary corticotroph POMC gene expression is regulated by CRH as well as several gp130 cytokines acting as neuro-immuno-endocrine modulators. Conversely, HPA axis functions modulate susceptibility or resistance to inflammatory disease. Cytokines (including IL-1, TNF, and members of the gp130 cytokine family) participate as mediators of a complex HPA axis response to stress and inflammation. Prolonged exposure to proinflammatory cytokines increases levels of the dominant negative glucocorticoid receptor isoform. Nonresponsiveness of the HPA axis to glucocorticoid negative feedback control provides a defense from destructive effects of cytokine excess. At the same time, gp130 cytokines stimulate pituitary suppressor of cytokine signaling (SOCS)-3, which represses cytokine signaling and abrogates cytokine-induced corticotroph POMC gene transcription and ACTH secretion.
The Estrogen-Occupied Estrogen Receptor Functions as a Negative Regulator to Inhibit Cell Proliferation Induced by Insulin/IGF-1: A Cell Context-Specific Antimitogenic Action of Estradiol on Rat Lactotrophs in Culture
Authors: Kawashima Kengo , Yamakawa Koji , Takahashi Wakaba , Takizawa Soichi , Yin Ping , Sugiyama Nobuhiro , Kanba Shigenobu , Arita Jun
Published: January 2014

Estrogens stimulate cell proliferation in typical estrogen-responsive tissues including the anterior pituitary gland. Here we report that 17-β estradiol (E2) has estrogen receptor-mediated mitogenic and antimitogenic actions on rat lactotrophs in primary culture, depending on the cell context. E2 did not affect basal proliferation at 2 d after treatment, but it increased it at 4 d. Insulin markedly increased proliferative activity, which was inhibited by simultaneous treatment with E2, even after only 2 d of treatment. This antimitogenic action on insulin-induced proliferation was also observed with other estrogens but not with nonestrogenic steroids. Treatment with antiestrogens in combination with E2 antagonized both the mitogenic and antimitogenic actions of E2. Antiestrogen treatment alone inhibited basal proliferation, and it mimicked the inhibitory action of E2 on insulin-induced proliferation with less potency. In parallel with cell proliferation, an insulin-induced increase in the cell number of cyclin D1-immunoreactive lactotrophs was inhibited by E2 treatment. Although the antimitogenic action of E2 was seen with a wide range of doses of insulin or IGF-1, proliferation was stimulated rather than inhibited by E2 when cells were treated with serum or forskolin/isobutylmethylxanthine instead of insulin, indicating a mitogen-specific, but not proliferative activity-dependent, inhibition by E2. The results of estrogen-occupied estrogen receptors as negative regulators of proliferation suggest a novel interaction between estrogen and growth factors in the regulation of proliferation in estrogen-responsive cells.
The Association of Endogenous Sex Hormones, Adiposity, and Insulin Resistance with Incident Diabetes in Postmenopausal Women
Authors: Kalyani Rita Rastogi , Franco Manuel , Dobs Adrian S. , Ouyang Pamela , Vaidya Dhananjay , Bertoni Alain , Gapstur Susan M. , Golden Sherita Hill
Published: January 2014

Context:

In postmenopausal women, endogenous bioavailable testosterone (T) and estradiol (E2) have been positively associated, and SHBG has been negatively associated, with incident type 2 diabetes (T2DM). Previous studies have not explored possible factors explaining these relationships.

Objective:

Our objective was to examine the association of endogenous sex hormones with incident T2DM in postmenopausal women and possible explanatory factors.

Design, Setting, and Participants:

The Multi-Ethnic Study of Atherosclerosis (MESA) is a prospective study that included 1612 postmenopausal women aged 45–84 yr, followed between the years 2000-2006, who were not taking hormone replacement therapy, had no prevalent cardiovascular disease or diabetes, and had complete ascertainment of sex hormones.

Main Outcome Measures:

T2DM was defined based on fasting glucose and/or treatment for diabetes.

Results:

There were 116 incident cases of diabetes during follow-up. Across higher quartiles of bioavailable T and E2 and lower quartiles of SHBG, we found significantly greater hazards of developing incident T2DM (all P for trend ≤ 0.001). After adjustment for body mass index and insulin resistance estimated by homeostasis model assessment of insulin resistance, bioavailable T was no longer associated with incident T2DM. The associations of E2 and SHBG with incident T2DM were partially explained by body mass index and insulin resistance but persisted in fully adjusted models (both P for trend < 0.02). Dehydroepiandrosterone had no relationship with incident T2DM.

Conclusions:

Adiposity and insulin resistance explained most of the association of bioavailable T but only partially explained the associations of E2 and SHBG with incident T2DM among postmenopausal women.
Identification of Adipocyte Genes Regulated by Caloric Intake
Authors: Franck Niclas , Gummesson Anders , Jernås Margareta , Glad Camilla , Svensson Per-Arne , Guillot Gilles , Rudemo Mats , Nyström Fredrik H , Carlsson Lena M. S. , Olsson Bob
Published: January 2014

Context: Changes in energy intake have marked and rapid effects on metabolic functions, and some of these effects may be due to changes in adipocyte gene expression that precede alterations in body weight.

Objective: The aim of the study was to identify adipocyte genes regulated by changes in caloric intake independent of alterations in body weight.

Research Design and Methods: Obese subjects given a very low-caloric diet followed by gradual reintroduction of ordinary food and healthy subjects subjected to overfeeding were investigated. Adipose tissue biopsies were taken at multiple time-points, and gene expression was measured by DNA microarray. Genes regulated in the obese subjects undergoing caloric restriction followed by refeeding were identified using two-way ANOVA corrected with Bonferroni. From these, genes regulated by caloric restriction and oppositely during the weight-stable refeeding phase were identified in the obese subjects. The genes that were also regulated, in the same direction as the refeeding phase, in the healthy subjects after overfeeding were defined as being regulated by caloric intake. Results were confirmed using real-time PCR or immunoassay.

Results: Using a significance level of P < 0.05 for all comparisons, 52 genes were down-regulated, and 50 were up-regulated by caloric restriction and regulated in the opposite direction by refeeding and overfeeding. Among these were genes involved in lipogenesis (ACLY, ACACA, FASN, SCD), control of protein synthesis (4EBP1, 4EBP2), β-oxidation (CPT1B), and insulin resistance (PEDF, SPARC).

Conclusions: Metabolic genes involved in lipogenesis, protein synthesis, and insulin resistance are central in the transcriptional response of adipocytes to changes in caloric intake.
Insufficient Activation of Autophagy Allows Cellular Damage to Accumulate in Critically Ill Patients
Authors: Vanhorebeek Ilse , Gunst Jan , Derde Sarah , Derese Inge , Boussemaere Magaly , Güiza Fabian , Martinet Wim , Timmermans Jean-Pierre , D'Hoore André , Wouters Pieter J. , Van den Berghe Greet
Published: January 2014

Context:

Responses to critical illness, such as excessive inflammation and hyperglycemia, may trigger detrimental chain reactions that damage cellular proteins and organelles. Such responses to illness contribute to the risk of (nonresolving) multiple organ dysfunction and adverse outcome.

Objective:

We studied autophagy as a bulk degradation pathway able to remove toxic protein aggregates and damaged organelles and how these are affected by preventing hyperglycemia with insulin during critical illness.

Design and Setting:

Patients participated in a randomized study, conducted at a university hospital surgical/medical intensive care unit.

Patients:

We studied adult prolonged critically ill patients vs. controls.

Interventions:

Tolerating excessive hyperglycemia was compared with intensive insulin therapy targeting normoglycemia.

Main Outcome Measures:

We quantified (ultra)structural abnormalities and hepatic and skeletal muscle protein levels of key players in autophagy.

Results:

Morphologically, both liver and muscle revealed an autophagy-deficiency phenotype. Proteins involved in initiation and elongation steps of autophagy were induced 1.3- to 6.5-fold by critical illness (P < 0.01), but mature autophagic vacuole formation was 62% impaired (P = 0.05) and proteins normally degraded by autophagy accumulated up to 97-fold (P < 0.03). Mitophagy markers were unaltered or down-regulated (P = 0.05). Although insulin preserved hepatocytic mitochondrial integrity (P = 0.05), it further reduced the number of autophagic vacuoles by 80% (P = 0.05).

Conclusions:

Insufficient autophagy in prolonged critical illness may cause inadequate removal of damaged proteins and mitochondria. Such incomplete clearance of cellular damage, inflicted by illness and aggravated by hyperglycemia, could explain lack of recovery from organ failure in prolonged critically ill patients. These data open perspectives for therapies that activate autophagy during critical illness.
Reversal of the Sex Difference in Serum Leptin Levels upon Cross-Sex Hormone Administration in Transsexuals*
Authors: Elbers Jolanda M. H. , Asscheman Henk , Seidell Jacob C. , Frölich Marijke , Meinders A. Edo , Gooren Louis J. G.
Published: January 2014

Women have higher circulating leptin levels than men. This sex difference is not simply explained by differences in the amount of body fat and is possibly influenced by their different sex steroid milieus. This prompted us to study prospectively the effects of cross-sex steroid hormones on serum leptin levels in 17 male to female transsexuals and 15 female to male transsexuals. Male to female transsexuals were treated with 100 μg ethinyl estradiol and 100 mg cyproterone acetate (antiandrogen) daily, and female to male transsexuals received testosterone esters (250 mg/2 weeks, im). Before and after 4 and 12 months of cross-sex hormone treatment, serum leptin levels and measures of body fatness were assessed. Before treatment, female subjects had higher serum leptin levels than male subjects independently of the amount of body fat (P < 0.01). Cross-sex hormone administration induced a reversal of the sex difference in serum leptin levels. Estrogen treatment in combination with antiandrogens in male subjects increased median serum leptin levels from 1.9 ng/mL before treatment to 4.8 ng/mL after 4 months and 5.5 ng/mL after 12 months of treatment (P < 0.0001). Testosterone administration in female subjects decreased median serum leptin levels from 5.6 to 2.6 ng/mL after 4 months and to 2.5 ng/mL after 12 months (P < 0.0001). Analysis of covariance revealed that the changes in serum leptin levels were independent of changes in body fatness in both groups (P < 0.01).

In conclusion, these results indicate that sex steroid hormones, in particular testosterone, play an important role in the regulation of serum leptin levels. The prevailing sex steroid milieu, not genetic sex, is a significant determinant of the sex difference in serum leptin levels.
Somatic Mutations in the Thyrotropin Receptor Gene and Not in the Gsα Protein Gene in 31 Toxic Thyroid Nodules
Authors: Führer Dagmar , Holzapfel Hans-Peter , Wonerow Peter , Scherbaum Werner A. , Paschke Ralf , Führer-Sakel Dagmar
Published: January 2014

Studies on frequency and distribution pattern of TSH receptor (TSHR) and Gsα protein (gsp) mutations in toxic thyroid nodules (TTNs) reported conflicting results, most likely also related to the different screening methods applied and the investigation of only part of exon 10 of the TSHR. Therefore, we screened a consecutive series of 31 TTNs for both TSHR and gsp mutations by direct sequencing of exon 9 and the entire exon 10 of the TSHR gene and exons 7–10 of the gsp gene. Somatic TSHR mutations were identified in 15 of 31 TTNs. TSHR mutations were localized in the third intracellular loop (Asp619Gly and Ala623Val), the sixth transmembrane segment (Phe631Leu and Thr632Ile, Asp633Glu) and the second extracellular loop (Ile568Thr). One mutation was found in the extracellular TSHR domain (Ser281Asn). Two new TSHR mutations were identified. One involves codon 656 in the third extracellular loop (Val656Phe). The other new mutation is a 27-bp deletion in the third intracellular loop resulting in deletion of 9 amino acids at codons 613–621. Transient expression of the new TSHR mutations in COS-7 cells demonstrated their constitutive activity. No mutation was found in exons 7–10 of the gsp gene. This finding was confirmed by an allele-specific PCR for mutations in gsp codons 201 (Arg→His, Cys) and 227 (Gln→His, Arg). Our data indicate that constitutively activating TSHR mutations can be found in 48% of TTNs and thus currently represent the most frequent molecular mechanism known in the etiopathogenesis of TTNs. Moreover, the absence of gsp mutations in our series argues for an only minor role of these mutations in TTNs. Constitutive activation of the TSHR by a deletion in a region that might be involved in G protein coupling of the TSHR offers new insights into TSHR activation.
Phenotype: Genotype Relationships in Growth Hormone Insensitivity Syndrome1
Authors: Woods Katie A. , Dastot Florence , Preece Michael A. , Clark Adrian J. L. , Postel-Vinay Marie-Catherine , Chatelain Pierre G. , Ranke Michael B. , Rosenfeld Ron G. , Amselem Serge , Savage Martin O.
Published: January 2014

GH insensitivity syndrome (GHIS) is associated with many different mutations of the GH receptor (GHR) gene. We examined the phenotypic and biochemical features in 82 GHIS patients from 23 countries, each fulfilling diagnostic criteria of GHIS. There were 45 males and 37 females [mean age, 8.25 yr; mean height, −6.09 sd score, and mean insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3),− 7.99 sd score]. Sixty-three were GH-binding protein (GHBP) negative; 19 were GHBP positive (>10% binding). The mean height in GHBP-negative subjects was −6.5 sd score, and that in GHBP-positive patients was −4.9 sd score (P = <0.001). Clinical and biochemical heterogeneity was demonstrated by the wide range of height (−2.2 to− 10.4 sd score) and IGFBP-3 (−1.4 to −14.7 sd score) values, which were positively correlated (r2 = 0.45; P = <0.001). This contrasted with the lack of correlation between mean parental height sd score and height sd score (r2 = 0.01).

Fifteen different GH receptor gene mutations were identified in 27 patients. All had homozygous defects, except 1 who had a compound heterozygous defect. The mutations were 5 nonsense, 2 frame shift, 4 splice, 4 missense, and 1 compound heterozygote. There was no relationship between mutation type or exon of the GHR gene involved and height or IGFBP-3 sd score.

In conclusion, GHIS is associated with wide variation in the severity of clinical and biochemical phenotypes. This variation cannot clearly be accounted for by defects in the GHR gene. Other genetic and/or environmental factors must, therefore, contribute to phenotype in GHIS.
Diabetes during Pregnancy Does Not Alter Whole Body Bone Mineral Content in Infants
Authors: Lapillonne Alexandre , Guerin Sophie , Braillon Pierre , Claris Olivier , Delmas Pierre D. , Salle Bernard L.
Published: January 2014

A previous study using single photon absorptiometry has reported low bone mineral density of the radius in infants of diabetic mothers. The aim of this study was to assess by dual x-ray absorptiometry the whole body bone mineral content (WbBMC) and the body composition of 40 infants of diabetic mothers at birth (mean gestational age ± sd, 37.5 ± 1.3 weeks; mean birth weight ± sd, 3815 ± 641 g).

WbBMC was not correlated with gestational age, but was well correlated with birth weight (r = 0.73; P = 0.0001) and also with fat mass (r = 0.87; P = 0.0001) and lean mass (r = 0.42; P = 0.008). The z-scores ± sd adjusted for weight for WbBMC and fat mass were significantly increased (1.3 ± 0.9 and 2.6 ± 1.3, respectively (P < 0.0001), but were not significantly influenced either by in utero growth or by the type of the diabetes mellitus of the mother.

Bone mineralization and fat mass studied by whole body dual x-ray absorptiometry are increased at birth in these infants compared with reference curves.
Calcium Absorptive Effects of Vitamin D and Its Major Metabolites1
Authors: Heaney Robert P. , Barger-Lux M. Janet , Dowell M. Susan , Chen Tai C. , Holick Michael F.
Published: January 2014

The absorptive response to graded doses of vitamin D3, 25(OH)D, and 1,25(OH)2D was measured in healthy adult men after treatment periods of eight, four, and two weeks, respectively. While no relationship was found between baseline absorption and serum vitamin D metabolite levels, all three vitamin D compounds significantly elevated 45Ca absorption from a 300 mg calcium load given as part of a standard test meal. 1,25(OH)2D was active even at the lowest dose (0.5μ g/day), and the slope was such that doubling of absorption would occur at an oral dose of approximately 3 μg/day. 25(OH)D was also active in elevating absorption and did so without raising total 1,25(OH)2D levels. On the basis of the dose response curves for 1,25(OH)2D and 25(OH)D, the two compounds exhibited a molar ratio for physiological potency of approximately 100:1. The absorptive effect of vitamin D3 was seen only at the highest dose level (1250 μg, or 50,000 IU/day) and was apparently mediated by conversion to 25(OH)D. Analysis of the pooled 25(OH)D data from both the 25(OH)D- and vitamin D3-treated groups suggests that approximately one eighth of circulating vitamin D-like absorptive activity under untreated conditions in winter may reside in 25(OH)D. This is a substantially larger share than has been predicted from studies of in vitro receptor binding.
Thyroid Nodules in the Follow-Up of Irradiated Individuals: Comparison of Thyroid Ultrasound with Scanning and Palpation1
Authors: Schneider Arthur B. , Bekerman Carlos , Leland Joel , Rosengarten Jeffrey , Hyun Hyewon , Collins Barbara , Shore-Freedman Eileen , Gierlowski Theresa C.
Published: January 2014

In 1974 we began a prospective study of a cohort of 4296 individuals exposed to therapeutic head and neck irradiation during childhood for benign conditions. To define the role of thyroid ultrasonography in following irradiated individuals, we studied a subgroup of 54 individuals. They all had been screened between 1974–1976 and had normal thyroid scans and no palpable nodules at that time. Thyroid ultrasonography, thyroid scanning, physical examination, and serum thyroglobulin measurements were performed. One or more discrete ultrasound-detected nodules were present in 47 of 54 (87%) subjects. There were a total of 157 nodules, 40 of which were 1.0 cm or larger in largest dimension. These 40 nodules occurred in 28 (52%) of the subjects. Thirty (75%) of these 1.0-cm or larger nodules matched discrete areas of diminished uptake on corresponding thyroid scans. The 10 that did not match (false negative scans for ≥1.0-cm nodules) were the only nodules of this size in 7 subjects. Of 11 nodules 1.5 cm or larger, only 5 were palpable. Serum thyroglobulin correlated to the number (P = 0.04; r2 = 0.10), but not the volume of the thyroid nodules (P = 0.07; r2 = 0.08). We conclude that thyroid nodules are continuing to occur and are exceedingly common in this irradiated cohort of individuals. The results confirm that thyroid ultrasonography is more sensitive than physical examination and scanning. However, thyroid ultrasound is so sensitive and nodules so prevalent that great caution is needed in interpreting the results.
Evidence for Production and Functional Activity of Nitric Oxide in Seminiferous Tubules and Blood Vessels of the Human Testis1
Authors: Middendorff R. , Müller D. , Wichers S. , Holstein A. F. , Davidoff M. S.
Published: January 2014

Previous studies have demonstrated that nitric oxide (NO) influences Leydig cell function. Here we provide evidence for NO production and activity in seminiferous tubules and blood vessels of the human testis. By immunohistochemistry, the soluble guanylyl cyclase (sGC), the intracellular NO receptor, and the second messenger, cyclic guanosine monophosphate (cGMP), were detected in myofibroblasts of the peritubular lamina propria in Sertoli cells, as well as in endothelial and smooth muscle cells of testicular blood vessels. Performed with isolated tubules and blood vessels, the biological activity of sGC could be proved by cGMP generation in response to treatments with the NO donor, sodium nitroprusside. The endothelial and neuronal subtypes of NO synthase (NOS) were localized immunohistochemically to the same cell types that express sGC and cGMP. In isolated tubules and vessels, the presence of endothelial NOS and neuronal NOS was confirmed by immunoblotting, and NOS activity was demonstrated by decreased cGMP production upon incubation with the NOS inhibitor l-nitro arginine methylester. These findings show that peritubular cells, Sertoli cells, and testicular blood vessels may be sites of NO production and activity, possibly involved in relaxation of seminiferous tubules and blood vessels to modulate sperm transport and testicular blood flow, respectively.
Clinical, Biochemical, and Molecular Investigations of a Genetic Isolate of Growth Hormone Insensitivity (Laron’s Syndrome)*
Authors: Baumbach L. , Schiavi A. , Bartlett R. , Perera E. , Day J. , Brown M. R. , Stein S. , Eidson M. , Parks J. S. , Cleveland W.
Published: January 2014

We have characterized the GH receptor mutation that is responsible for extreme short stature and GH insensitivity in a Bahamian genetic isolate. Heights of affected individuals ranged from −4.0 to −6.3 sd. Like others with Laron’s syndrome, they had normal to high serum GH concentrations and low serum insulin-like growth factor I concentrations. Circulating levels of GH-binding protein activity were below limits of detection. Amplification of exons 2–7 and screening with single strand conformational polymorphism analysis located an abnormality in exon 7. Sequencing identified homozygosity for a C to T transition in the third position of codon 236. Reverse transcription and PCR amplification of complementary DNA from lymphocytes showed that this same sense mutation generated a new splice donor site 63 bp 5′ to the normal exon 7 splice site. This novel site was used to the exclusion of the normal site in homozygotes. Both normal and variant messenger ribonucleic acid species were detected in heterozygotes. The predicted protein lacks 21 amino acids, including those defining the WS-like motif of the GH receptor extracellular domain. The high frequency of Laron’s syndrome in this isolated island population probably reflects the introduction of the G236 splice mutation by a settler early in the 300-yr history of English settlement.
Estimation of Tissue Hypothyroidism by a New Clinical Score: Evaluation of Patients with Various Grades of Hypothyroidism and Controls1
Authors: Zulewski Henryk , Müller Beat , Exer Pascale , Miserez André R. , Staub Jean-Jacques
Published: January 2014

The classical signs and symptoms of hypothyroidism were reevaluated in the light of the modern laboratory tests for thyroid function. We analyzed 332 female subjects: 50 overt hypothyroid patients, 93 with subclinical hypothyroidism (SCH), 67 hypothyroid patients treated with T4, and 189 euthyroid subjects. The clinical score was defined as the sum of the 2 best discriminating signs and symptoms. Beside TSH and thyroid hormones, we measured parameters known to reflect tissue manifestations of hypothyroidism, such as ankle reflex relaxation time and total cholesterol.

Classical signs of hypothyroidism were present only in patients with severe overt hypothyroidism with low T3, but were rare or absent in patients with normal T3 but low free T4 or in patients with SCH (normal thyroid hormones but elevated basal TSH; mean scores, 7.8 ± 2.7 vs. 4.4 ± 2.2 vs. 3.4 ± 2.0; P < 0.001). Assessment of euthyroid subjects and T4-treated patients revealed very similar results (mean score, 1.6 ± 1.6 vs. 2.1 ± 1.5). In overt hypothyroid patients, the new score showed an excellent correlation with ankle reflex relaxation time and total cholesterol (r = 0.76 and r = 0.60; P < 0.0001), but no correlation with TSH (r = 0.01). The correlation with free T4 was r = −0.52 (P < 0.0004), and that with T3 was r = −0.56 (P < 0.0001). In SCH, the best correlation was found between the new score and free T4 (r =− 0.41; P < 0.0001) and TSH (r = 0.35; P < 0.0005).

Evaluation of symptoms and signs of hypothyroidism with the new score in addition to thyroid function testing is very useful for the individual assessment of thyroid failure and the monitoring of treatment.
I. Adrenal Cortex and Steroid 21-Hydroxylase Autoantibodies in Adult Patients with Organ-Specific Autoimmune Diseases: Markers of Low Progression to Clinical Addison’s Disease
Authors: Betterle Corrado , Volpato Marina , Smith Bernard Rees , Furmaniak Jadwiga , Chen Shu , Greggio Nella A. , Sanzari Maria , Tedesco Francesco , Pedini Beniamino , Boscaro Marco , Presotto Fabio
Published: January 2014

Adrenal cortex antibodies (ACA) were measured by immunofluorescence in 8840 adult patients with organ-specific autoimmune diseases without overt hypoadrenalism. Sixty-seven (0.8%) patients were ACA-positive, with the highest prevalence in those with premature ovarian failure (8.9%). Forty-eight ACA-positive and 20 ACA-negative individuals were enrolled into a prospective study. Antibodies to steroid 21-hydroxylase (21-OH), steroid 17α-hydroxylase (17α-OH) and cytochrome P450 side chain cleavage enzyme (P450scc) were measured by immunoprecipitation assay. Human leucocyte antigens D-related (HLA-DR) genotyping was also carried out and adrenal function assessed by ACTH test. On enrollment, 75% of ACA-positive patients had a normal adrenal function, while 25% revealed a subclinical hypoadrenalism. 21-OH antibodies were positive in 91% of ACA-positive sera. Eleven patients were positive for steroid-cell antibodies by immunofluorescence, and 9 revealed a positivity for antibodies to 17α-OH and/or P450scc. During the prospective study, overt Addison’s disease developed in 21% and subclinical hypoadrenalism in 29% of ACA-positive patients, while 50% maintained normal adrenal function. Progression to Addison’s disease was more frequent in patients with subclinical hypoadrenalism, high titers of ACA and higher levels of 21-OH antibodies, complement-fixing ACA and HLA-DR3 status. All 20 persistently ACA-negative patients were also negative for antibodies to 21-OH, 17α-OH, and P450scc, and all maintained normal adrenal function during follow-up. In conclusion, the detection of ACA/21-OH antibodies in adults is a marker of low progression toward clinical Addison’s disease. .