Endocrinology

Publications in this journal

• Article: Hypothalamic inflammation without astrogliosis in response to high sucrose intake is modulated by neonatal nutrition in male rats.

  Esther Fuente-Martín, Cristina García-Cáceres, Francisca Díaz, Pilar Argente-Arizón, Miram Granado, Vicente Barrios, Jesús Argente, Julie A Chowen
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  ABSTRACT: Hypothalamic inflammation and gliosis are proposed to participate in the pathogenesis of high fat diet-induced obesity. As other factors and nutrients also induce weight gain and adiposity, we analyzed the inflammatory and glial responses to a sucrose (S)-enriched diet. Neonatal over-nutrition (NON) exacerbates weight gain in response to metabolic challenges, thus we compared the inflammatory response of male Wistar rats with NON (4 pups/litter) and controls (12 pups/litter) to increased S intake. At weaning rats received water or a 33% sucrose solution and normal chow ad libitum for 2 months. Sucrose increased serum interleukin (IL) 1β and 6 and hypothalamic IL6 mRNA levels in NON and TNFα mRNA levels in control and NON rats, while NON alone had no effect. The astrocyte marker glial fibrillary acidic protein (GFAP) was increased by NON, but decreased by S. This was associated with hypothalamic nuclei specific changes in GFAP+ cell number and morphology. Sucrose increased the number of microglia and phosphorylation of IκB and JNK in control but not NON rats, with no effect on microglia activation markers. Proteins highly expressed in astrocytes (glutamate, glucose and lactate transporters) were increased by NON but not S with no increase in vimentin expression in astrocytes, further suggesting that S-induced adiposity is not associated with hypothalamic astrogliosis. Hence, activation of hypothalamic inflammatory processes and gliosis depend not only on weight gain, but also on the diet inducing this weight gain and early nutritional status. These diverse inflammatory processes could indicate a differential disposition to obesity-induced pathologies.
  Endocrinology 05/2013;
• Article: 5α-Reductase Inhibition Suppresses Testosterone-Induced Initial Regrowth of Regressed Xenograft Prostate Tumors in Animal Models.

  Khalid Z Masoodi, Raquel Ramos Garcia, Laura E Pascal, Yujuan Wang, Hei M Ma, Katherine O'Malley, Kurtis Eisermann, Daniel H Shevrin, Holly M Nguyen, Robert L Vessella, Joel B Nelson, Rahul A Parikh, Zhou Wang
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  ABSTRACT: Androgen deprivation therapy (ADT) is the standard treatment for patients with PSA progression after treatment for localized prostate cancer. An alternative to continuous ADT is intermittent ADT (IADT), which allows recovery of testosterone during off-cycles to stimulate regrowth and differentiation of the regressed prostate tumor. IADT offers patients a reduction in side effects associated with ADT, improved quality of life, and reduced cost with no difference in overall survival. Our previous studies showed that IADT coupled with 5α-reductase inhibitor, which blocks testosterone conversion to dihydrotestosterone (DHT) could prolong survival of animals bearing androgen-sensitive prostate tumors when off-cycle duration was fixed. To further investigate this clinically relevant observation, we measured the time course of testosterone-induced regrowth of regressed LuCaP35 and LNCaP xenograft tumors in the presence or absence of a 5α-reductase inhibitor. 5α-reductase inhibitors suppressed the initial regrowth of regressed prostate tumors. However, tumors resumed growth and were no longer responsive to 5α-reductase inhibition several days after testosterone replacement. This finding was substantiated by BrdU and Ki67 staining of LuCaP35 tumors, which showed inhibition of prostate tumor cell proliferation by 5α-reductase inhibitor on day 2, but not day 14, after testosterone replacement. 5α-reductase inhibitors also suppressed testosterone-stimulated proliferation of LNCaP cells pre-cultured in androgen-free media, suggesting that blocking testosterone conversion to DHT can inhibit prostate tumor cell proliferation via an intracrine mechanism. These results suggest that short off-cycle coupled with 5α-reductase inhibition could maximize suppression of prostate tumor growth and thus, improve potential survival benefit achieved in combination with IADT.
  Endocrinology 05/2013;
• Article: Insulin and IGF-I inhibit GH synthesis and release in vitro and in vivo by separate mechanisms.

  Manuel D Gahete, José Córdoba-Chacón, Qing Lin, Jens C Brüning, C Ronald Kahn, Justo P Castano, Helen Christian, Raúl M Luque, Rhonda D Kineman
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  ABSTRACT: Insulin-like growth factor I (IGF-I) is considered a primary inhibitor of GH secretion. Insulin may also play an important role in regulating GH levels since insulin, like IGF-I, can suppress GH synthesis and release in primary pituitary cell cultures and insulin is negatively correlated with GH levels in vivo. However, understanding the relative contribution insulin and IGF-I exert on controlling GH secretion has been hampered by the fact that circulating insulin and IGF-I are regulated in parallel and insulin (INSR) and IGF-I (IGFIR) receptors are structurally/functionally related and ubiquitously expressed. In order to evaluate the separate roles of insulin and IGF-I in directly regulating GH secretion, we used the Cre/loxP system to knockdown the INSR and IGFIR in primary mouse pituitary cell cultures and found insulin-mediated suppression of GH is independent of the IGFIR. In addition, pharmacologic blockade of intracellular signals in both mouse and baboon cultures revealed insulin requires different pathways than IGF-I to exert a maximal inhibitory effect on GH expression/release. In vivo, somatotrope-specific knockout of INSR (SIRKO) or IGFIR (SIGFRKO) increased GH levels. However, comparison of the pattern of GH release, GH expression, somatotrope morphometry and pituitary explant sensitivity to acute GHRH challenge in lean SIRKO and SIGFRKO mice, strongly suggests the primary role of insulin in vivo is to suppress GH release, while IGF-I serves to regulate GH synthesis. Finally, SIRKO and/or SIGFRKO could not prevent diet-induced suppression of pituitary GH expression indicating other factors/tissues are involved in the decline of GH observed with weight gain.
  Endocrinology 05/2013;
• Article: The impact of ventral noradrenergic bundle lesions on increased IL-1 in the PVN and hormonal responses to stress in male Sprague Dawley rats.

  Peter Blandino, Cara M Hueston, Christopher J Barnum, Christopher Bishop, Terrence Deak
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  ABSTRACT: The impact of acute stress on inflammatory signaling within the CNS is of interest because these factors influence neuroendocrine function both directly and indirectly. Exposure to certain stressors increases expression of the pro-inflammatory cytokine, Interleukin-1β (IL-1), in the hypothalamus. Increased IL-1 is reciprocally regulated by norepinephrine (stimulatory) and corticosterone (inhibitory), yet neural pathways underlying increased IL-1 have not been clarified. These experiments explored the impact of bilateral lesions of the Ventral Noradrenergic Bundle (VNAB) on IL-1 expression in the PVN after footshock. Adult male Sprague Dawley rats received bilateral 6-OHDA lesions of the VNAB (VNABx) and were exposed to intermittent footshock. VNABx depleted approximately 64% of NE in the PVN and attenuated the IL-1 response produced by footshock. However, characterization of the HPA response, a crucial prerequisite for interpreting the effect of VNABx on IL-1 expression, revealed a profound dissociation between ACTH and corticosterone. Specifically, VNABx blocked the intronic CRH response in the PVN and the increase in plasma ACTH, while corticosterone was unaffected at all time points examined. Additionally, footshock led to a rapid and profound increase in COX-2 and IL-1 expression within the adrenal glands, while more subtle effects were observed in the pituitary gland. Together, these findings (a) demonstrate that exposure to acute stress increased expression of inflammatory factors more broadly throughout the HPA axis; (b) implicate a modest role for NE-containing fibers of the VNAB as an upstream regulator of PVN IL-1; and (c) suggest an ACTH-independent mechanism controlling the release of corticosterone in VNABx rats.
  Endocrinology 05/2013;
• Article: Redefining the initiation and maintenance of zebrafish interrenal steroidogenesis by characterizing the key enzyme Cyp11a2.

  Silvia Parajes, Aliesha Griffin, Angela E Taylor, Ian T Rose, Irene Miguel-Escalada, Yavor Hadzhiev, Wiebke Arlt, Cedric Shackleton, Ferenc Müller, Nils Krone
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  ABSTRACT: Zebrafish are emerging as a model to study steroid hormone action and associated disease. However, steroidogenesis in zebrafish is not well characterized. Mammalian P450 side-chain cleavage enzyme (CYP11A1) catalyzes the first step of steroidogenesis, the conversion of cholesterol to pregnenolone. Previous studies describe an essential role for zebrafish Cyp11a1 during early development. Cyp11a1 has been suggested to be the functional equivalent of mammalian CYP11A1 in the zebrafish interrenal gland (equivalent to the mammalian adrenal), gonad and brain. However, reported cyp11a1 expression is inconsistent in zebrafish larvae, after active cortisol synthesis commences. Recently, a duplicated cyp11a gene, cyp11a2, has been described, which shares an 85% identity with cyp11a1. We aimed to elucidate the specific role of the two cyp11a paralogs. cyp11a1 was expressed from 0-48 hours post-fertilization (hpf), whilst cyp11a2 expression started after development of the interrenal primordium (32 hpf), and was the only paralog in larvae. cyp11a2 is expressed in adult steroidogenic tissues, such as the interrenal, gonads, and brain. In contrast, cyp11a1 was mainly restricted to the gonads. Anti-sense morpholino knockdown studies confirmed abnormal gastrulation in cyp11a1 morphants. cyp11a2 morphants showed impaired steroidogenesis and a phenotype indicative of metabolic abnormalities. The phenotype was rescued by pregnenolone replacement in cyp11a2 morphants. Thus, we conclude that cyp11a1 is required for early development, whereas cyp11a2 is essential for initiation and maintenance of zebrafish interrenal steroidogenesis. Importantly, this study highlights the need for a comprehensive characterization of steroidogenesis in zebrafish prior its implementation as a model organism in translational research of adrenal disease.
  Endocrinology 05/2013;
• Article: Dipeptidylpeptidase Inhibition is Associated with Improvement in Blood Pressure and Diastolic Function in Insulin Resistant Male Zucker Obese Rats.

  Annayya R Aroor, James R Sowers, Shawn B Bender, Ravi Nistala, Mona Garro, Irina Mugerfeld, Melvin R Hayden, Megan S Johnson, Muhammad Salam, Adam Whaley-Connell, Vincent G Demarco
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  ABSTRACT: Diastolic dysfunction is a prognosticator for future cardiovascular events that demonstrates a strong correlation with obesity. Pharmacological inhibition of dipeptidylpeptidase-4 (DPP-4) to increase the bioavailability of glucagon like peptide-1 (GLP-1) is an emerging therapy for control of glycemia in type 2 diabetes patients. Accumulating evidence suggests GLP-1 has insulin-independent actions in cardiovascular tissue; however it is not known whether DPP-4 inhibition improves obesity-related diastolic dysfunction. Eight week old Zucker Obese (ZO) and Zucker Lean (ZL) rats were fed normal chow diet or diet containing the DPP-4 inhibitor, linagliptin (LGT), for 8 weeks. Plasma DPP-4 activity was 3.3-fold higher in ZO compared to ZL rats and was reduced by 95% with LGT treatment. LGT improved echocardiographic and pressure volume-derived indices of diastolic function that were impaired in ZO control rats, without altering food intake or body wt gain during the study period. LGT also blunted elevated blood pressure progression in ZO rats involving improved skeletal muscle arteriolar function, without reducing left ventricular hypertrophy (LVH), fibrosis, or oxidative stress in ZO hearts. Expression of phosphorylated-eNOS(ser1177), total eNOS and SERCA2a protein was elevated in the LGT-treated ZO heart suggesting improved Ca(2+) handling. The ZO myocardium had an abnormal mitochondrial sarcomeric arrangement and cristae structure that were normalized by LGT. These studies suggest that LGT reduces blood pressure and improves intracellular Cai(2+) mishandling and cardiomyocyte ultrastructure which collectively result in improvements in diastolic function in the absence of reductions in LVH, fibrosis, or oxidative stress in insulin resistant ZO rats.
  Endocrinology 05/2013;
• Article: Acute hypernatremia exerts an inhibitory oxytocinergic tone that is associated with anxiolytic mood in male rats.

  Charles J Frazier, Dipanwita Pati, Helmut Hiller, Dan Nguyen, Lei Wang, Justin A Smith, Kaley Macfadyen, Annette D de Kloet, Eric G Krause
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  ABSTRACT: Anxiety disorders are the most common psychiatric illness and are associated with heightened stress responsiveness. The neuropeptide oxytocin (OT) has garnered significant attention for its potential as a treatment for anxiety disorders; however, the mechanism mediating its effects on stress-responding and anxiety is not well-understood. Here we use acute hypernatremia, a stimulus that elevates brain levels of OT, to discern the central oxytocinergic pathways mediating stress responsiveness and anxiety-like behavior. Rats were rendered hypernatremic by acute administration of 2.0 M NaCl and had increased plasma sodium concentration, plasma osmolality and Fos induction in OT-containing neurons relative to 0.15 M NaCl treated controls. Acute hypernatremia decreased restraint-induced elevations in corticosterone (CORT) and created an inhibitory oxytocinergic tone on parvocellular neurosecretory neurons within the paraventricular nucleus of the hypothalamus. In contrast, evaluation of Fos immunohistochemistry determined that acute hypernatremia followed by restraint increased neuronal activation in brain regions receiving OT afferents that are also implicated in the expression of anxiety-like behavior. To determine whether these effects were predictive of altered anxiety-like behavior, rats were subjected to acute hypernatremia and then tested in the elevated plus maze (EPM). Relative to controls given 0.15 M NaCl, rats given 2.0 M NaCl spent more time in the open arms of the EPM, suggesting that acute hypernatremia is anxiolytic. Collectively, the results suggest that acute elevations in the pNa(+) increase central levels of OT, which decreases anxiety by altering neuronal activity in hypothalamic and limbic nuclei.
  Endocrinology 05/2013;
• Article: Progesterone Antagonism of Neurite Outgrowth Depends on Microglial Activation via Pgrmc1/S2R.

  N Bali, J M Arimoto, T E Morgan, C E Finch
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  ABSTRACT: Neuronal plasticity is regulated by the ovarian steroids estradiol (E2) and progesterone (P4) in many normal brain functions, as well as in acute response to injury and chronic neurodegenerative disease. In a female rat model of axotomy, the E2-dependent compensatory neuronal sprouting is antagonized by P4. To resolve complex glial-neuronal cell interactions, we used the "wounding-in-a-dish" model of neurons cocultured with astrocytes or mixed glia (microglia to astrocytes, 1:3). Although both astrocytes and mixed glia supported E2-enhanced neurite outgrowth, P4 antagonized E2-induced neurite outgrowth only with mixed glia, but not astrocytes alone. We now show that P4-E2 antagonism of neurite outgrowth is mediated by microglial expression of Pgrmc1/S2R, a putative nonclassical progesterone receptor mediator with multiple functions. The P4-E2 antagonism of neurite outgrowth was restored by add-back of microglia to astrocyte-neuron cocultures. Because microglia do not express the classical progesterone receptor (Pgr), we examined the role of Pgrmc1, which is expressed in microglia in vitro and in vivo. Knockdown by siRNA-Pgrmc1 in microglia before add-back to astrocyte-neuron cocultures suppressed the P4-E2 antagonism of neurite outgrowth. Conditioned media from microglia restored the P4-E2 activity, but only if microglia were activated by lipopolysaccharide (LPS) or by wounding. Moreover, the microglial activation was blocked by Pgmrc1-siRNA knockdown. These findings explain why nonwounded cultures without microglial activation lack P4 antagonism of E2-induced neurite outgrowth. We suggest that microglial activation may influence brain responses to exogenous P4, which is a prospective therapy in traumatic brain injury.
  Endocrinology 05/2013;
• Article: Alternative Splice Variants of the Rainbow Trout Leptin Receptor Encode Multiple Circulating Leptin-Binding Proteins.

  Ningping Gong, Ingibjörg E Einarsdottir, Marcus Johansson, Björn Thrandur Björnsson
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  ABSTRACT: In mammals, leptin (Lep) binding proteins (LepBPs) derived from Lep receptor (LepR) gene or protein bind most of the circulating Lep, but to date, information on LepBPs in nonmammalian vertebrate classes is lacking. This study details the characterization of multiple LepBPs in rainbow trout (Oncorhynchus mykiss), an early poikilothermic vertebrate, and presents the complete coding sequences for 3 of them. Size-exclusion chromatography and cross-linking assay identified plasma proteins bound to Lep ranging from 70 to 100 kDa. LepBPs were isolated from plasma by affinity chromatography, and their binding specificity was assessed by a competitive binding assay. A RIA for LepBPs indicates that plasma LepBP levels decline after fasting for 3 weeks. Immunoblotting of LepBPs using antibodies against different LepR epitopes shows that the LepBPs are indeed LepR isoforms. The alternatively spliced LepR transcripts (LepRS1-3) that include only the extracellular segment transcribe the 90-kDa LepBP1, the 80-kDa LepBP2, and the 70-kDa LepBP3, respectively. LepRS1 generally has lower expression than the long-form LepR in most tissues. LepRS2 is primarily expressed in adipose tissue, whereas LepRS3 is expressed abundantly in brain and spleen, and moderately in liver and gills. The mRNA levels of hypothalamic LepRS1 and hepatic LepRS3 increase after 2 weeks of fasting, but decrease after 3 weeks. This study demonstrates a mechanism in fish for the generation of LepBPs that differs from that seen in mammals and indicates that the physiologic action of Lep in these poikilothermic vertebrates can be modulated, both centrally and peripherally, by the differentiated, tissue-specific expression of multiple LepBPs.
  Endocrinology 05/2013;
• Article: The goldilocks principle and developmental androgens in males, what is "just right"?

  Paul A Fowler, Peter J O'Shaughnessy
  Endocrinology 05/2013; 154(5):1669-71.
• Article: Regulation of Lipid Metabolism by Glucocorticoids and 11β-HSD1 in Skeletal Muscle.

  Stuart A Morgan, Laura L Gathercole, Claire Simonet, Zaki K Hassan-Smith, Iwona Bujalska, Phil Guest, Lianne Abrahams, Dave M Smith, Paul M Stewart, Gareth G Lavery, Jeremy W Tomlinson
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  ABSTRACT: The prevalence of insulin resistance and type 2 diabetes mellitus are rising dramatically and as a consequence there is an urgent need to understand the pathogenesis underpinning these conditions to develop new and more efficacious treatments.We have tested the hypothesis that glucocorticoid-mediated changes in insulin sensitivity may be associated with changes in lipid flux. Furthermore, pre-receptor modulation of glucocorticoid availability by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) may represent a critical regulatory step.Dexamethasone decreased lipogenesis in both murine C2C12 and human LHC-NM2 myotubes. Inactivating p-Ser-79/218 of acetyl-CoA carboxylase 1/2 (ACC1/2) and activating p-Thr-172 of AMP-activated protein kinase (AMPK) were both increased following dexamethasone treatment in C2C12 myotubes. By contrast, dexamethasone increased β-oxidation. Selective 11β-HSD1 inhibition blocked the 11-dehydrocorticosterone (11DHC)-mediated decrease in lipogenic, and increase in lipolytic gene expression. Lipogenic gene expression was decreased, whilst lipolytic and β-oxidative genes expression increased in corticosterone (CORT) and 11DHC treated wild-type mice, and CORT (but not 11DHC) treated 11β-HSD1(-/-) mice. Furthermore, CORT and 11DHC treated wild-type mice, and CORT (but not 11DHC) treated 11β-HSD1(-/-) mice had increased p-Ser-79/218 ACC1/2, p-Thr-172 AMPK and intramyocellular diacylglyderide content.In summary, we have shown that glucocorticoids have potent actions upon intramyocellular lipid homeostasis by decreasing lipid storage, increasing lipid mobilisation and utilisation and increasing diacylglyderide content. It is plausible that dysregulated intramyocellular lipid metabolism may underpin GC-induced insulin resistance of skeletal muscle.
  Endocrinology 04/2013;
• Article: Prior history of chronic stress changes the transcriptional response to glucocorticoid challenge in the dentate gyrus region of the male rat hippocampus.

  Nicole A Datson, Jessica M E van den Oever, Oksana B Korobko, Ana Maria Magarinos, E Ronald de Kloet, Bruce McEwen
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  ABSTRACT: Chronic stress is a risk factor for several neuropsychiatric diseases such as depression and psychosis. In response to stress glucocorticoids (GCs) are secreted that bind to the glucocorticoid receptor (GR), a ligand-activated transcription factor that regulates the transcription of gene networks in the brain necessary for coping with stress, recovery and adaptation. Chronic stress particularly affects the dentate gyrus (DG) subregion of the hippocampus, causing several functional and morphological changes with consequences for learning and memory, that are likely adaptive, but at the same time make DG neurons more vulnerable to subsequent challenges.The aim of this study was to investigate the transcriptional response of DG neurons to a GC-challenge in male rats previously exposed to chronic restraint stress (CRS). An intriguing finding of the current study was that having a history of CRS had profound consequences for the subsequent response to acute GC-challenge, differentially affecting the expression of several hundreds of genes in the DG compared to challenged non-stressed control animals. This enduring effect of prior stress exposure suggests that epigenetic processes may be involved. In line with this, CRS indeed affected the expression of several genes involved in chromatin structure and epigenetic processes, including Asf1, Ash1l, Hist1h3f and Tp63. The data presented here indicate that CRS alters the transcriptional response to a subsequent GC-injection. We propose that this altered transcriptional potential forms part of the molecular mechanism underlying the enhanced vulnerability for stress-related disorders like depression caused by chronic stress.
  Endocrinology 04/2013;
• Article: Up-regulation of the Fetal Baboon Hypothalamo-Pituitary-Adrenal Axis in Intrauterine Growth Restriction: Coincidence with Hypothalamic Glucocorticoid Receptor Insensitivity and Leptin Receptor Down-regulation.

  Cun Li, Emma Ramahi, Mark J Nijland, Jaeyhek Choi, Dean A Myers, Peter W Nathanielsz, Thomas J McDonald
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  ABSTRACT: Intrauterine growth restriction (IUGR) is an important fetal developmental problem resulting from two broad causes: maternal under-nutrition and/or decreased fetal nutrient delivery to the fetus via placental insufficiency. IUGR is often accompanied by up-regulation of the hypothalamo-pituitary-adrenal axis (HPAA). Sheep studies show fetal HPAA autonomy in late gestation. We hypothesized that IUGR, resulting from poor fetal nutrient delivery, up-regulates the fetal baboon HPAA in late gestation driven by hypothalamo-pituitary glucocorticoid receptor (GR) insensitivity and decreased fetal leptin in peripheral plasma. Maternal baboons were fed as ad lib controls (CTR) or nutrient restricted to produce IUGR (fed 70% CTR diet) from 0.16 - 0.9 gestation. Peripheral ACTH, cortisol and leptin were measured by immunoassays. Corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptor (GR), leptin receptor (ObRb) and proopiomelanocortin (POMC) peptide expression were determined immunohistochemically (IHC). IUGR fetal peripheral cortisol and ACTH, but not leptin, were increased (p< 0.05). IUGR increased CRH peptide expression in the fetal hypothalamic paraventricular nucleus (PVN) and median eminence (p< 0.05), but not AVP. PVN ObRb peptide expression was decreased (p< 0.05) with IUGR, but not GR. ObRb and POMC were robustly expressed in anterior pituitary, but ∼ 1% of cells showed co-localization. We conclude that 1) CRH, but not AVP, is the major releasing hormone driving ACTH and cortisol secretion during primate IUGR, 2) fetal HPAA activation was aided by GR insensitivity and decreased ObRb expression in the PVN and 3) the AP is not a site for ObRb effects on the HPAA.
  Endocrinology 04/2013;
• Article: 17β-Estradiol rapidly attenuates P2X3 receptor-mediated peripheral pain signal transduction via ERα and GPR30.

  Yi Lu, Qian Jiang, Li-Hua Yu, Zhan-Ying Lu, Shuang-Ping Meng, Ding-Feng Su, Geoffrey Burnstock, Bei Ma
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  ABSTRACT: Estrogen has been reported to affect pain perception, although the underlying mechanisms remain unclear. In this investigation, pain behavior testing, patch clamp recording and immunohistochemistry were used on rats and transgenic mice to determine which estrogen receptors and the related signaling pathway are involved in the rapid modulation of estrogen on P2X3 receptor-mediated events. The results showed that 17β-estradiol rapidly inhibited pain induced by α,β-methylene ATP (α,β-me-ATP), a P2X1 and P2X3 receptor agonist in ovariectomized rats and normal rats in diestrus. The estrogen receptor-α (ERα) agonist 4,49,499-(4-propyl-[1H]-pyrazole-1,3,5-triyl) trisphenol (PPT) and GPR30 receptor agonist G-1 mimicked the estrogen effect, while the ERβ agonist diarylpropionitrile (DPN) had no effect. In cultured rat dorsal root ganglion (DRG) neurons, PPT and G-1 but not DPN significantly attenuated α,β-meATP-mediated currents, with the dose-response curve of these currents shifted to the right. The inhibitory effect of 17β-estradiol on P2X3 currents was blocked by G-15, a selective antagonist to the GPR30 estrogen receptor. 17β-estradiol lacked this effect in DRG neurons from ERα knockout mice while partly remained in those from ERβ knockout mice. The P2X3 and GPR30 receptors were co-expressed in the rat DRG neurons. Further, the ERK1/2 inhibitor U0126 reversed the inhibitory effect of 17β-estradiol on α,β-me-ATP-induced pain and of PPT or G-1 on P2X3 receptor-mediated currents. The cyclic AMP-protein kinase A (PKA) agonist forskolin, but not the protein kinase C agonist phorbol-12-myristate-13-acetate (PMA) mimicked the estrogen inhibitory effect on P2X3 receptor currents, which was blocked by another ERK1/2 inhibitor, PD98059. These results suggest that estrogen regulates P2X3-mediated peripheral pain by acting on ERα and GPR30 receptors expressed in primary afferent neurons, which probably involves the intracellular cAMP-PKA-ERK1/2 pathway.
  Endocrinology 04/2013;
• Article: The Neuregulin system of ligands and their receptors in rat islets of Langerhans.

  J C M South, E Blackburn, I R Brown, W J Gullick
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  ABSTRACT: Islet cell growth and function are affected by ligands from the Epidermal Growth Factor family. We describe here the expression, regional distribution and effect on growth and secretion of insulin of a subset of these, the Neuregulin family. The expression of NRG1 alpha, NRG1 beta, NRG2 alpha, NRG2 beta, NRG3 and NRG4 in rat islets was determined using immunohistochemical and double immunofluorescent staining. We also report the expression of the four receptors and the remaining seven ligands using immunohistochemistry. The NRG1 alpha splice variant was expressed in beta cells and the NRG1 beta variant mainly in alpha cells. NRG3 was also predominantly present in alpha cells. Most of the members of the EGF family of ligands were also expressed, with Epigen being present at the highest levels. The rat islet derived cell line CRI-G1 was used to study the effect of addition of EGF, NRG1 beta, NRG3 and NRG4 on cell growth and insulin secretion. Synthetic refolded NRG3 strongly stimulated the growth of the CRI-G1 cells and NRG4 gave the greatest stimulation of insulin release. Different members of the Neuregulin family are therefore potentially potent stimuli for islet cell growth and insulin release and differ in expression in alpha and beta cells.
  Endocrinology 04/2013;
• Article: Importance of His192 in the human thyroid hormone transporter MCT8 for substrate recognition.

  Stefan Groeneweg, Elaine C Lima de Souza, W Edward Visser, Robin P Peeters, Theo J Visser
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  ABSTRACT: Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). So far, functional domains within MCT8 are not well defined. Mutations in MCT8 result in severe psychomotor retardation due to impaired neuronal differentiation. One such mutation concerns His192 (H192R), located at the border of transmembrane domain (TMD) 1 and extracellular loop (ECL) 1, suggesting that this His residues is important for efficient TH transport. Here, we studied the role of different His residues, predicted within TMDs or ECLs of MCT8, in substrate recognition and translocation. Therefore, we analyzed the effects of the His-modifying reagent diethylpyrocarbonate (DEPC) and of site-directed mutagenesis of several His residues on TH transport by MCT8. Reaction of MCT8 with DEPC inhibited subsequent uptake of T3 and T4, whereas T3 and T4 efflux were not inhibited. The inhibitory effect of DEPC on TH uptake was prevented in the presence of T3 or T4, suggesting that TH blocks access to DEPC sensitive residues. Three putative DEPC target His residues were replaced by Ala: H192A, H260A, and H450A. The H260A and H450A mutants showed similar TH transport and DEPC sensitivity as wild-type MCT8. However, the H192A mutant showed a significant reduction in TH uptake, and was insensitive to DEPC. Taken together, these results indicate that His192 is sensitive to modification by DEPC, and may be located close to a putative substrate recognition site within the MCT8 protein, important for efficient TH uptake.
  Endocrinology 04/2013;
• Article: Restoration of cardiac tissue thyroid hormone status in experimental hypothyroidism: a dose-response study in female rats.

  Nathan Y Weltman, Kaie Ojamaa, Olga V Savinova, Yue-Feng Chen, Evelyn H Schlenker, Riccardo Zucchi, Alessandro Saba, Daria Colligiani, Christine J Pol, A Martin Gerdes
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  ABSTRACT: Thyroid hormones (THs) play a pivotal role in regulating cardiovascular homeostasis. To provide a better understanding of the coordinated processes that govern cardiac TH bioavailability, this study investigated the influence of serum and cardiac TH status on the expression of TH transporters and cytosolic binding proteins in the myocardium. In addition, we sought to determine if administration of T3 (instead of T4) improves the relationship between THs in serum and cardiac tissue, and cardiac function over a short-term treatment period. Adult female SD rats were made hypothyroid by seven weeks treatment with the anti-thyroid drug PTU (6-n-propyl-2-thiouracil). After establishing hypothyroidism, rats were assigned to one of five graded T3 (triiodothyronine) dosages plus PTU for a two week dose-response experiment. Untreated, age matched rats served as euthyroid controls. PTU was associated with depressed serum and cardiac tissue T3 and T4 levels, arteriolar atrophy, altered TH transporter and cytosolic TH binding protein expression, fetal gene re-expression, and cardiac dysfunction. Short-term administration of T3 led to a mismatch between serum and cardiac tissue TH levels. Normalization of serum T3 levels was not associated with restoration of cardiac tissue T3 levels or cardiac function. In fact, a 3 fold higher T3 dosage was necessary to normalize cardiac tissue T3 levels and cardiac function. Importantly, this study provides the first comprehensive data on the relationship between altered TH status (serum and cardiac tissue), cardiac function, and the coordinated in vivo changes in cardiac TH membrane transporters and cytosolic TH binding proteins in altered TH states.
  Endocrinology 04/2013;
• Article: A Surgical Model in Male Obese Rats Uncovers Protective Effects of Bile Acids Post-Bariatric Surgery.

  Rohit Kohli, Kenneth Dr Setchell, Michelle Kirby, Andriy Myronovych, Karen K Ryan, Samar H Ibrahim, Jose Berger, Kathi Smith, Mouhamadoul Toure, Stephen C Woods, Randy J Seeley
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  ABSTRACT: Bariatric surgery elevates serum bile acids. Conjugated bile acid administration, such as tauroursodeoxycholic acid (TUDCA), improves insulin sensitivity, while short-circuiting bile acid circulation through ileal interposition surgery in rats raises TUDCA levels. We hypothesized that bariatric surgery outcomes could be recapitulated by short-circuiting the normal entero-hepatic bile circulation. We established a model wherein male obese rats underwent either bile diversion (BD) or Sham (SH) surgery. The BD group had a catheter inserted into the common bile duct and its distal end anchored into the mid-distal jejunum for 4-5 weeks. Glucose tolerance, insulin and glucagon-like peptide-1 (GLP-1) response, hepatic steatosis and endoplasmic reticulum (ER) stress were measured. Rats' post-BD lost significantly more weight than the SH-rats. BD rats gained less fat mass post-surgery. BD rats had improved glucose tolerance, increased higher post-prandial GLP-1 response and serum bile acids but less liver steatosis. Serum bile acid levels including TUDCA concentrations were higher in BD compared to SH pair-fed rats. Fecal bile acid levels were not different. Liver ER stress (CHOP mRNA and pJNK protein) was decreased in BD rats. Bile acid gavage (TUDCA/UDCA) in diet-induced obese rats, elevated serum TUDCA and concomitantly reduced hepatic steatosis and ER stress (CHOP mRNA). These data demonstrate the ability of alterations in bile acids to recapitulate important metabolic improvements seen after bariatric surgery. Further, our work establishes a model for focused study of bile acids in the context of bariatric surgery that may lead to the identification of therapeutics for metabolic disease.
  Endocrinology 04/2013;
• Article: Histidines in Potential Substrate Recognition Sites Affect Thyroid Hormone Transport by Monocarboxylate Transporter 8 (MCT8).

  Doreen Braun, Iva Lelios, Gerd Krause, Ulrich Schweizer
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  ABSTRACT: Mutations in monocarboxylate transporter 8 (MCT8, SLC16A2) cause the Allan-Herndon-Dudley syndrome (AHDS), a severe X-linked psychomotor retardation syndrome. MCT8 belongs to the major facilitator superfamily (MFS) of 12 transmembrane spanning proteins and transports thyroid hormones across the blood-brain-barrier and into neurons. How MCT8 distinguishes thyroid hormone substrates from structurally closely related compounds is not known. The goal of this study was to identify critical amino acids along the transport channel cavity which participate in thyroid hormone recognition. The fact that triiodothyronine (T3)is bound between a "His-Arg clamp" in the crystal structure of the T3-receptor/T3 complex prompted us to investigate whether such a motif might potentially be relevant for T3 recognition in MCT8. We therefore replaced candidate histidines and arginines by site-directed mutagenesis and performed activity assays in MDCK-1 cells and Xenopus oocytes. Histidines were replaced by alanine, phenylalanine, and glutamine to probe for molecular properties like aromatic ring structure and H-bonding properties. It was found that some mutations in His192 and His415 significantly changed substrate transport kinetics. Arg301 at the intracellular end of the substrate channel is at an ideal distance to His415 to participate in a "His-Arg clamp" and mutation to alanine abrogated hormone transport. Molecular modeling demonstrates a perfect fit of T3 poised into the substrate channel between His415 and Arg301, while observing the same geometry as in the T3-receptor.
  Endocrinology 04/2013;