bluuebunny/crossref_metadata_2025 huggingface dataset:
Fenofibrate increases the expression of high mobility group AT-hook 2 (HMGA2) gene and induces adipocyte differentiation of orbital fibroblasts from Graves' ophthalmopathy
Authors: Pasquali D , Pierantoni GM , Fusco A , Staibano S , Colantuoni V , De Bellis A , Bellastella A , Sinisi AA
Published: December 2004

Expansion of adipose tissue in the orbits is a key feature of Graves' ophthalmopathy. Recent evidence shows that orbital fibroblasts are committed to differentiate into adipocytes under appropriate stimuli. Rosiglitazone, an agonist of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma) is able to induce both differentiation of orbital fibroblasts into mature adipocytes and expression of the TSH receptor (TSHr) gene. Several studies have suggested an important role of the high mobility group AT-hook 2 (HMGA2) gene in adipocytic cell growth and development. To investigate further the association between adipogenesis-related genes and orbital fibroblasts, we treated fibroblasts from Graves' ophthalmopathy (FGOs) and from normal orbital tissues with fenofibrate, a specific agonist for PPARalpha. We then evaluated the expression of the PPARalpha, PPARgamma2, HMGA2, leptin and TSHr genes before and after 24 h of fenofibrate treatment, using semiquantitative and real-time PCR. For up to 96 h after exposure to fenofibrate, FGOs differentiated into adipocytes. PPARalpha and PPARgamma2 were expressed more in FGOs than in normal cultures, whereas TSHr mRNA was detected only in FGOs. Expression of HMGA2 mRNA and protein was significantly increased in FGOs from 6 to 24 h after fenofibrate, confirming its role in the early phase of adipocyte differentiation. Treatment with fenofibrate for 24 h significantly increased the expression of leptin and TSHr genes. Moreover, TSH treatment significantly increased the accumulation of cAMP, demonstrating that FGOs express functional TSHr. The high level of expression of PPARalpha other than PPARgamma2 transcripts and the stimulating effect of fenofibrate on adipogenesis and on HMGA2, leptin and TSHr genes also indicate that the PPARalpha pathway plays an important part in the adipocyte differentiation of FGOs. These findings suggest that novel drugs to antagonize PPARalpha, other than the PPARgamma signalling system, may also need to be considered in the treatment or prevention of Graves' ophthalmopathy.
Effect of 17beta-estradiol on hypothalamic GnRH-II gene expression in the female rhesus macaque
Authors: Densmore VS , Urbanski HF
Published: December 2004

The hypothalamus of rhesus macaques expresses two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II). However, it is unclear whether these two neuropeptides play similar roles in the control of reproductive neuroendocrine function, especially in the context of positive and negative estrogen feedback. To address this issue, in situ hybridization histochemistry was used to compare the effect of 17beta-estradiol (E) on the expression of GnRH-I and GnRH-II mRNA in the medial basal hypothalamus (MBH) of adult female macaques. GnRH-I mRNA expression was found to be significantly (P<0.01) more abundant in ovariectomized (ovx) animals compared with ovariectomized E-treated (ovx+E) animals. In marked contrast, GnRH-II mRNA expression was found to be significantly (P<0.05) more abundant in ovx+E animals than in the ovx animals. To help elucidate how E exerts this stimulatory action on GnRH-II gene expression, hypothalamic sections were subsequently double labeled using a combination of immunohistochemisty for estrogen receptor (ER) -alpha or -beta and in situ hybridization histochemistry for GnRH-II. Approximately 50% of the GnRH-II positive cells in the MBH were found to express ERbeta, but none expressed ERalpha. Taken together, these data give credence to a novel pathway by which E may control the primate neuroendocrine reproductive axis, one that involves stimulation of GnRH-II release via an ERbeta-mediated mechanism.
A quantitative RT-PCR study of the mRNA expression profile of the IGF axis during mammary gland development
Authors: Boutinaud M , Shand JH , Park MA , Phillips K , Beattie J , Flint DJ , Allan GJ
Published: December 2004

We have used quantitative RT-PCR to analyse the mRNA expression profile of the major components of the IGF axis in different stages of murine mammary gland development, including late pregnancy, lactation and involution. We have shown that all the genes studied, IGF-I, IGF-II, IGF receptor (IGFR) and IGF-binding protein (IGFBP)-1 to -6, were expressed in every stage, albeit at greatly differing levels and displaying unique expression profiles between developmental stages. IGF-I was always expressed at significantly higher levels than either IGF-II or IGFR. This suggests that IGF-I may be the more important IGF during mammary morphogenesis. Overall, IGFBP-3 demonstrated the highest level of expression of any of the IGFBP genes throughout all the developmental stages studied. However, within developmental stages, by far the highest level of expression of any of the IGFBPs was that of IGFBP-5 at day 2 of involution; this was almost an order of magnitude higher than any of the other IGFBP levels recorded. This corroborated our previous findings that the levels of IGFBP-5 protein are highly elevated in the involuting mammary gland, and demonstrated that this up-regulation of IGFBP-5 operates at the level of transcriptional control or message stability. Comparison of the expression profile for these different genes would strongly suggest that they are likely to have differential functions throughout mammary gland development, and also highlights potential interactions and co-regulation between different members of this axis. In addition, our results have identified some similarities and differences in the expression of IGFBPs between the mouse mammary epithelial cell line, HC11, and the normal mammary gland which are worthy of study, most notably the differential regulation of IGFBP-2 and the site of expression of IGFBP-4 and -6. Overall, this study has demonstrated the importance and complexity of the IGF axis during mammary gland development and provides a valuable resource for future research in this area.
Insulin-mediated activation of activator protein-1 through the mitogen-activated protein kinase pathway stimulates collagenase-1 gene transcription in the MES 13 mesangial cell line
Authors: Ayala JE , Boustead JN , Chapman SC , Svitek CA , Oeser JK , Robey RB , O'Brien RM
Published: December 2004

The initial stages of diabetic nephropathy are characterized, in part, by expansion of the mesangial matrix and thickening of the glomerular basement membrane which are caused by increased extracellular matrix (ECM) protein synthesis and reduced degradation, a consequence of decreased matrix metalloproteinase (MMP) activity. These changes have been largely attributed to the effects of hyperglycemia such that the potential contribution of impaired insulin action to alterations in the ECM have not been studied in detail. We have shown here that insulin stimulates collagenase-1 fusion gene transcription in the MES 13 mesangial-derived cell line. Multiple collagenase-1 promoter elements are required for the full stimulatory effect of insulin but the action of insulin appears to be mediated through an activator protein-1 (AP-1) motif. Thus, mutation of this AP-1 motif abolishes insulin-stimulated collagenase fusion gene transcription and, in isolation, this AP-1 motif can mediate a stimulatory effect of insulin on the expression of a heterologous fusion gene. This suggested that the other collagenase-1 promoter elements that are required for the full stimulatory effect of insulin probably bind accessory factors that enhance the effect of insulin mediated through the AP-1 motif. In MES 13 cells, the AP-1 motif is bound by Fra-1, Fra-2, Jun B and Jun D. Stimulation of collagenase-1 fusion gene transcription by insulin requires activation of the mitogen-activated protein kinase (MEK) pathway since inhibition of MEK-1 and -2 blocks this effect. The potential significance of these observations with respect to a role for insulin in the pathophysiology of diabetic glomerulosclerosis is discussed.
Expression of epiregulin and amphiregulin in the rat ovary
Authors: Sekiguchi T , Mizutani T , Yamada K , Kajitani T , Yazawa T , Yoshino M , Miyamoto K
Published: December 2004

We have previously reported that the epidermal growth factor (EGF) family growth factor, epiregulin, is expressed in rat ovarian granulosa cells by induction with pregnant mare serum gonadotropin (PMSG). In this study, we report that amphiregulin, another member of the EGF family, was also induced in the rat ovary by gonadotropin treatment. Northern blot analysis revealed that PMSG treatment induced the expression of both epiregulin and amphiregulin mRNA after 24 h, but the expression then decreased 48 h after treatment. Further treatment with human chorionic gonadotropin (hCG) rapidly induced the expression of both epiregulin and amphiregulin genes and maximal levels were reached 4 h after hCG treatment. A marginal increase in amphiregulin mRNA levels was also observed 6 h after PMSG treatment. In situ hybridization revealed that epiregulin and amphiregulin mRNAs were localized in the granulosa cells of large antral follicles. These spatio-temporal expression patterns were similar to those of cyclo-oxygenase-2 (COX-2) and progesterone receptor (PR). In adult cycling rats, epiregulin and amphiregulin were strongly induced at 1800 and 2000 h on proestrus coinciding with the preovulatory LH surge. An in situ hybridization study also showed that epiregulin and amphiregulin mRNAs were detectable in the granulosa cells of preovulatory ovarian follicles at 2000 h on proestrus, where transcripts of COX-2 and PR were co-localized with those of epiregulin and amphiregulin. These observations suggested that the EGF family members, epiregulin and amphiregulin, may play a role in the ovulatory process of cycling rats as well as in the induction of ovulation in immature rats.
Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin and mesotocin receptors
Authors: Acharjee S , Do-Rego JL , Oh DY , Moon JS , Ahn RS , Lee K , Bai DG , Vaudry H , Kwon HB , Seong JY
Published: December 2004

The neurohypophysial nonapeptides vasotocin (VT) and mesotocin (MT) are the amphibian counterparts of arginine vasopressin (AVP) and oxytocin (OT). We have here reported the cloning and functional characterization of the receptors for vasotocin (VTR) and mesotocin (MTR) in two species of frog, Rana catesbeiana and Rana esculenta. The frog VTR and MTR cDNAs encode proteins of 419 and 384 amino acids respectively. Frog VTR exhibits a high degree of sequence identity with the mammalian AVP-1a (V1a) receptor while the frog MTR possesses a high degree of sequence identity with the mammalian OT receptor. Activation of VTR induced both c-fos promoter- and cAMP-responsive element (CRE)-driven transcriptional activities, while activation of MTR induced c-fos promoter-driven transcriptional activity but failed to evoke CRE-driven transcriptional activity, suggesting differential G protein coupling between VTR and MTR. The VTR exhibited the highest sensitivity for VT followed by OT>AVP approximately MT, whereas the MTR showed preferential ligand sensitivity for MT>OT>VT>AVP. A V1a agonist but not V2 and OT agonists substantially activated both VTR and MTR with a similar sensitivity. V1a, V2 and OT antagonists inhibited MT-induced MTR activation but not VT-induced VTR activation. In the frog brain, VTR and MTR mRNAs were found to be widely expressed in the telencephalon, diencephalon and mesencephalon, and exhibited very similar regional distribution. In the pituitary, VTR and MTR were expressed in the distal and intermediate lobes but were virtually absent in the neural lobe. Taken together, these data indicated that, although the distribution of VTR and MTR largely overlaps in the frog brain and pituitary, VT and MT may play distinct activities owing to the ligand selectivity and different signaling pathways activated by their receptors.
Suppression of plasma free fatty acids upregulates peroxisome proliferator-activated receptor (PPAR) α and δ and PPAR coactivator 1α in human skeletal muscle, but not lipid regulatory genes
Authors: Watt M J , Southgate R J , Holmes A G , Febbraio M A
Published: December 2004

Fatty acids are an important ligand for peroxisome proliferator-activated receptor (PPAR) activation and transcriptional regulation of metabolic genes. To examine whether reduced plasma free fatty acid (FFA) availability affects the mRNA content of proteins involved in fuel metabolism in vivo, the skeletal muscle mRNA content of various transcription factors, transcriptional coactivators and genes encoding for lipid regulatory proteins were examined before and after 3 h of cycle exercise with (NA) and without (CON) pre-exercise ingestion of nicotinic acid (NA). NA resulted in a marked (3- to 6-fold) increase (P<0.05) in PPARα, PPARδ and PPAR coactivator 1α (PGC1α) mRNA, but was without effect on nuclear respiratory factor-1 and Forkhead transcription factor, fatty acid transcolase/CD36, carnitine palmitoyl transferase 1, hormone sensitive lipase (HSL) and pyruvate dehydrogenase kinase 4. Exercise in CON was associated with increased (P<0.05) PPARα, PPARδ and PGC1α mRNA, which was similar in magnitude to levels observed with NA at rest. Exercise was generally without effect on the mRNA content of lipid regulatory proteins in CON and did not affect the mRNA content of the measured subset of transcription factors, transcriptional co-activators and lipid regulatory proteins during NA. To determine the possible mechanisms by which NA might affect PGC1α expression, we measured p38 MAP kinase (MAPK) and plasma epinephrine. Phosphorylation of p38 MAPK was increased (P<0.05) by NA treatment at rest, and this correlated (r2=0.84, P<0.01) with increased PGC1α. Despite this close relationship, increasing p38 MAPK in human primary myotubes was without effect on PGC1α mRNA content. Plasma epinephrine was elevated (P<0.05) by NA at rest (CON: 0.27±0.06, NA: 0.72±0.11 nM) and throughout exercise. Incubating human primary myotubes with epinephrine increased PGC1α independently of changes in p38 MAPK phosphorylation. Hence, despite the fact that NA ingestion decreased FFA availability, it promoted the induction of PPARα/δ and PGC1α gene expression to a similar degree as prolonged exercise. We suggest that the increase in PGC1α may be due to the elevated plasma epinephrine levels. Despite these changes in transcription factors/coactivators, the mRNA content of lipid regulatory proteins was generally unaffected by plasma FFA availability.
Regulation of fibronectin by thyroid hormone receptors
Authors: Lin Kwang-Huei , Chen Chia-yu , Chen Shen-Liang , Yen Chun-Che , Huang Ya-Hui , Shih Chung-hsuan , Shen Jiann-Jong , Yang Rong-Chi , Wang Chia-Siu
Published: December 2004

Thyroid hormones regulate growth, development, differentiation, and metabolic processes by interacting with and activating thyroid hormone receptors and associated pathways. We investigated the triiodothyronine (T3) modulation of gene expression, in human hepatocellular carcinoma cell lines, via a PCR-based cDNA subtraction method. Here we present further data on one of the T3-upregulated genes, fibronectin (FN). We demonstrate that the induction of FN protein expression by T3 in TRα1 and TRβ1 over-expressing cells was time and dose-dependent at the mRNA and protein levels. Blockade of protein synthesis by cycloheximide almost completely inhibited the concomitant induction of FN mRNA by T3, indicating that T3 indirectly regulates FN. Furthermore, nuclear-run on and FN promoter assay clearly can specifically increase the number of FN transcriptional demonstrated that the presence of T3 initiations. In addition, we further confirmed that the up-regulation of FN by T3 was mediated, at least in part, by transforming growth factor-β (TGF-β), because the induction of FN was blocked in a dose-dependent manner by the addition of TGF-β neutralizing antibody. In an effort to elucidate the we demonstrated the involvement of the signaling pathways involved in the activation of FN by T3, mitogen activated protein kinase/c-Jun N-terminal kinase/p38 MAPK (MAPK/JNK/p38) pathway. Although T3 induces the expression of TGF-β, neither wild-type nor dominant-negative Smad3 or Smad4 over-expression affected the activation of FN by T3. Thus, we demonstrate that T3 regulates FN gene expression indirectly at the transcriptional level, with the participation of the MAPK/JNK/p38 pathway and the TGF-β signaling pathway but independent of Smad3/4.
Nuclear oxysterol receptors, LXRs, are involved in the maintenance of mouse caput epididymidis structure and functions
Authors: Frenoux J-M , Vernet P , Volle D H , Britan A , Saez F , Kocer A , Henry-Berger J , Mangelsdorf D J , Lobaccaro J-M A , Drevet J R
Published: December 2004

In this study we looked at the epididymides and spermatozoa of mice knocked-out for nuclear oxysterol receptors (LXR). We have shown that LXR-deficient mice exhibited upon ageing a severe disruption of their caput epididymides associated with abnormal accumulation of neutral lipids. The epididymis defaults were correlated with sperm head fragility and infertility. In agreement with the observed caput defect in transgenic animals in which both LXRα and LXRβ isoforms were disrupted, we have shown here that both receptors are expressed in caput and cauda epididymides regions. LXRβ was predominantly expressed throughout the mouse epididymis while the expression of LXRα was weaker. In addition, the expression of selected genes that can be considered as markers of adult epididymis function was monitored via Northern blots in the different single and double LXR-deficient backgrounds. Altogether, the data presented here suggest that LXR receptors are important actors in epididymis function.
Expression of tissue inhibitor of metalloproteinases-1 in the human corpus luteum after luteal rescue
Authors: Duncan W C , McNeilly A S , Illingworth P J
Published: December 2004

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a specific inhibitor of a group of proteolytic enzymes known as matrix metalloproteinases. These enzymes have been widely implicated in the process of tissue remodelling.

Extensive remodelling occurs in the corpus luteum during luteolysis unless human chorionic gonadotrophin (hCG) is produced by the early conceptus. This study aimed to investigate the expression and localisation of TIMP-1 in human corpora lutea during the luteal phase of the cycle and after luteal rescue with exogenous hCG to mimic the changes of early pregnancy. Human corpora lutea from the early (n = 4), mid- (n=4) and late (n=4) luteal phases and after luteal rescue by hCG (n=4) were obtained at the time of hysterectomy. Expression of TIMP-1 was investigated in these tissues by Western blotting, immunohistochemistry, Northern blotting and in situ hybridisation. Luteal cells of thecal origin were distinguished from those of granulosa origin by immunostaining for 17α-hydroxylase. A 30 kDa protein consistent with TIMP-1 was detected in human corpora lutea. This protein was localised to the granulosa lutein cells in all tissues examined. TIMP-1 mRNA was found in large quantities in all glands examined and this again localised to the granulosa lutein cells. The expression and localisation of TIMP-1 did not change throughout the luteal phase and was not altered by luteal rescue. The function of this uniform expression of TIMP-1 in the corpus luteum is not clear but these data suggest that the inhibition of structural luteolysis during maternal recognition of pregnancy is not mediated by regulation of TIMP-1 expression.

Journal of Endocrinology (1996) 148, 59–67
Evidence for androgen receptor gene expression in human thyroid cells and tumours
Authors: Rossi R , Franceschetti P , Maestri I , Magri E , Cavazzini L , degli Uberti E C , del Senno L
Published: December 2004

Androgen-binding activity has been identified in normal and pathological thyroids, but evidence for the expression of the canonic androgen receptor (AR) in the thyroid has not been provided so far. In this study we have used reverse transcription (RT)-PCR to examine RNA expression of the canonic AR gene in human thyroid tissues, in primary cultures of human thyrocytes and in a variety of neoplastic thyroid cell lines (NPA, TPC and WRO). An AR cDNA fragment with the expected size of 262 bp was detected in normal tissues and cultured thyrocytes as well as in neoplastic cell lines, demonstrating that the gene for AR is indeed expressed in thyroid follicular cells. Immunocytochemical analysis revealed the presence of the AR protein in cancer cell lines and androgen treatment increased nuclear positivity to AR. In a survey of 35 thyroid tissues AR cDNA was detected in all the non-neoplastic samples (6 normal and 3 goitrous) and in 19 of 26 neoplastic samples. AR cDNA was not detected in 4 of the 9 follicular adenomas and in 3 of the 12 papillary carcinomas. AR was revealed by immunohistochemistry in 1 of 2 normal thyroids, in 1 goiter and in 1 of 2 neoplastic thyroids. These findings show the presence of the canonic AR in the human thyroid.

Journal of Endocrinology (1996) 148, 77–85
Opioid receptor activation resets the hypothalamic clock generating growth hormone secretory bursts in the rat
Authors: Willoughby J O , Medvedev A
Published: December 2004

Plasma growth hormone (GH) concentrations were measured serially every 20 min for 6 h in unrestrained chronically-catheterised male rats to define physiological GH pulsatile secretory patterns. Bursts of GH secretion lasted 69 ± 5 min and occurred every 177 ± 4 min. Intravenous administration of the opioid receptor agonist morphine (200 μg/kg) caused an immediate GH burst of normal duration (63 ± 3 min) in all animals. This burst of secretion occurred whatever the phase of the background GH cycle and was followed by normal trough GH levels; a second GH burst occurred 177 ±6 min later, an inter-burst period not different from controls. Opioid receptor blockade with naloxone (5 mg/kg) administered i.v. every 20 min during spontaneous GH bursts significantly lengthened the interburst interval from 177 ± 4 to 200±9 min (P=0·015). Naloxone did not affect synchronisation of the GH rhythm induced by morphine but lengthened the duration of GH secretory bursts from 69 ± to 94 ± 9 min (P=0·017).

The findings indicate that opioid receptor activation resets the hypothalamic mechanism generating pulsatile GH secretion and that both the period of the GH rhythm and duration of the GH burst is normally shortened by opioid mechanisms.

Journal of Endocrinology (1996) 148, 149–155
Phospholipase C and adenylate cyclase signalling systems in the action of hCG on porcine myometrial smooth muscle cells
Authors: Kisielewska J , Flint A P F , Ziecik A J
Published: December 2004

Although the uterus is a target tissue for LH and its homologue hCG the second messenger system responding to LH/hCG in myometrial cells is not established. In this study we investigated the involvement of protein kinase A and protein kinase C in the action of hCG on porcine myometrial smooth muscle cells in vitro. Myometrium was obtained from ovariectomized gilts given 2·5 mg oestradiol benzoate plus 50 mg progesterone for five consecutive days. Myometrial cells were cultured for 48 h and different doses of hCG were then added. Increasing doses of hCG stimulated concentration-dependent increases in [3H]inositol phosphates (IPs) accumulation in incubations lasting 24 h. The highest dose of hCG (1000 mU/ml) increased turnover of IPs by 2·4-fold as reflected in elevations in IP1, IP2 and IP3, and similar effects were observed with noradrenaline. The time- and concentration-dependent effects of hCG on IPs accumulation occurred between 16 and 24 h of incubation. Incubation of myocytes with the lowest doses of hCG (0·1 and 1 mU/ml) caused a significant increase in cAMP accumulation but the highest doses (10–1000 mU/ml) had no effect on cAMP concentrations. This is the first demonstration that LH/hCG receptor signalling leads to increased inositol phosphate turnover in myometrial cells as well as cAMP generation and it leads to the conclusion that both protein kinase A and protein kinase C signalling mechanisms are involved in gonadotrophin action in porcine myometrial smooth muscle cells.

Journal of Endocrinology (1996) 148, 175–180
Effects of TRH, prolactin and TSH on cell proliferation in the intermediate lobe of the rat pituitary gland
Authors: Pawełczyk T , Pawlikowski M , Kunert-Radek J
Published: December 2004

The effect of TRH on cell proliferation in the anterior lobe of the pituitary is well known and documented. On the other hand, there are no data on the effects of TRH on the intermediate lobe of the pituitary gland. The aim of this study was to investigate the effect of TRH and its analogues (pGlu-His-Gly, pGlu-His-Gly-NH2) on cell proliferation in the intermediate pituitary lobe. The bromodeoxyuridine technique was used to detect the proliferating cells. It was found that TRH stimulated cell proliferation 24 h after a single injection at a dose of 100 μg/kg body weight. The TRH analogues did not exert any significant stimulatory effect either 12 h or 24 h after the injection.

The second experiment was carried out to distinguish the probable mechanism of the action of TRH. The effects of TSH and prolactin (PRL) on intermediate lobe cell proliferation were examined. It was found that both PRL and TSH exerted a significant stimulatory effect 24 h after a single s.c. injection of PRL at a dose of 150 IU/kg body weight or TSH at a dose 20 IU/kg body weight.

It therefore appears that the stimulatory effect of TRH on intermediate pituitary lobe cell proliferation is mediated by PRL and TSH.

Journal of Endocrinology (1996) 148, 193–196
Relationship between calbindin-D28K levels in the A and B cells of the rat endocrine pancreas and the secretion of insulin and glucagon: influence of vitamin D3 deficiency and 1,25-dihydroxyvitamin D3
Authors: Bourlon P-M , Faure-Dussert A , Billaudel B , Sutter B Ch J , Tramu G , Thomasset M
Published: December 2004

The pancreatic B cell is equipped with specific receptors for 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and contains vitamin D-dependent calcium binding proteins (calbindin-D). Insulin secretion is impaired by vitamin D deficiency and is restored by 1,25-(OH)2D3 (concomitantly with an improved calcium handling within B cells) but the effect of 1,25-(OH)2D3 on the pancreatic B cell via calbindin-D is unclear. Therefore we examined the relationship between calbindin-D28K or calbindin-D9K and the activity of the endocrine pancreas in normal (N), four week vitamin D-deficient (−D) and one week 1,25-(OH)2D3-replete (+D) rats. Calbindin-D9K was not found in the pancreas, neither in the islets nor in the exocrine part, of any of the groups of rats (N, −D, or +D). Surprisingly, total islet calbindin-D28K content was increased by vitamin D deficiency and partly restored by 1,25-(OH)2D3. Calbindin-D28K immunostaining was observed only on A and B cells in the endocrine part of the pancreas, the greatest staining being found in A cells. This difference in staining density was increased by vitamin D deficiency and decreased by 1,25-(OH)2D3 treatment. In vitro, 1,25-(OH)2D3 also produced a negative influence on calbindin-D28K staining in A cells, as demonstrated using pieces of pancreas incubated with the steroid for 2 h. No significant influence on labeling intensity of B cell calbindin-D28K could be shown. Plasma insulin and islet insulin release in response to 10 mm arginine stimulation were decreased in −D rats and enhanced in +D rats towards N values. In contrast, plasma glucagon and the amount of glucagon secretion, stimulated in vitro by 10 mm arginine or by low (1·7 mm) glucose concentration, was increased in −D rats and attenuated by 1,25-(OH)2D3.

Thus there appears to be no relationship between the steady state level of B cell calbindin-D28K and the regulation of insulin secretion by 1,25-(OH)2D3 in vitamin D-deficient rats. However there is a correlation between A cell calbindin-D28K and glucagon secretion, which are both negatively regulated by 1,25-(OH)2D3. The predominance of calbindin-D28K in A cells raises the question as to how A and B cells interact and the role of calbindin-D28K in calcium handling.

Journal of Endocrinology (1996) 148, 223–232
Ontogeny of GH receptor and GH-binding protein in the pig
Authors: Schnoebelen-Combes S , Louveau I , Postel-Vinay M-C , Bonneau M
Published: December 2004

The present study was undertaken to examine the developmental pattern of GH receptor (GHR) and GHR gene expression in skeletal muscle (longissimus dorsi and trapezius (TR)) and liver from the last third of gestation until 1 year of age in male Large White (LW) and Meishan (MS) pigs. Plasma GH-binding protein (GHBP) levels were also measured. 125I-Labelled bovine GH (bGH) specific binding (not determined in foetal TR) and GHR mRNA were detected in skeletal muscle from 75 days of gestation until the adult stage with no clear age-related changes. By contrast, 125I-labelled bGH specific binding and GHR mRNA were undetectable or barely detectable in foetal liver. After birth, 125I-labelled bGH specific binding (P<0·001) and GHR mRNA in liver increased with age. The level of bGH binding to liver membranes was higher in MS than in LW pigs at 1, 45, 80 and 120 days of age and did not differ between breeds at the other ages. Specific binding of 125I-labelled human GH (hGH) to plasma GHBP was easily detected as early as 75 days of gestation and increased with age (P<0·001). The level of hGH binding to plasma GHBP was higher in MS than in LW pigs at 1, 80 and 120 days of age. It can be concluded that (1) the developmental expression of the GHR is tissue-specific, (2) the presence of GHBP in foetuses despite the absence of GHR in liver suggests that other tissues such as skeletal muscle could contribute to the generation of GHBP and (3) the presence of GHR in skeletal muscle as early as 75 days of gestation suggests that GH may play a role in foetal muscle growth.

Journal of Endocrinology (1996) 148, 249–255
Vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct
Authors: Ninomiya Y , Arao Y , Kometani T , Hiwatashi S , Yamasaki T , Erikawa T , Yamaguchi H , Hasegawa T , Masushige S , Kato S
Published: December 2004

We examined vitamin A-deficient chicks to determine whether vitamin A affects the estrogen-induced development of the chick oviduct. When oviduct development was stimulated for 5 days with the synthetic estrogen, diethylstilbestrol, the wet weight of the oviduct in vitamin A-deficient chicks was only half that in control chicks. The DNA content in this tissue showed that the decreased oviduct weight in the vitamin A-deficient chicks was caused by the decreased proliferation of oviduct cells. However, the estrogen-induced expression of the ovalbumin gene was not affected by the vitamin A deficiency, suggesting that estrogen-induced cytodifferentiation is not affected by vitamin A. To clarify the vitamin A action on estrogen-induced development in the oviduct, transcripts of nuclear estrogen receptor (ER) and all-trans-retinoic acid (RARα, β and γ) receptors, which exert the effects of estrogen and vitamin A, were measured. The ER, RARα and RARβ genes, but not that of RARγ, were expressed during oviduct development, indicating that estrogen and vitamin A may control the expression of target genes through their cognate receptors. Thus, we have shown that vitamin A is involved in estrogen-induced cell proliferation but not in cytodifferentiation of the chicken oviduct.

Journal of Endocrinology (1996) 148, 257–265
Development and application of a two-site enzyme immunoassay for the determination of 'total' activin-A concentrations in serum and follicular fluid
Authors: Knight P G , Muttukrishna S , Groome N P
Published: December 2004

The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and α2 macroglobulin (α2M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit ∼10 pg/well), precise (mean within- and between-plate coefficients of variation 4·9 and 9·1% respectively) and accurate (activin-A recovery values of 102 ± 3 and 96 ± 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and α2M (100 μg/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all <0·5%), bovine pro-αC and follistatin (both <0·1%). Response curves parallel to the activin-A standard curve were obtained for a variety of test samples including bovine, human, ovine and porcine FF, human sera and conditioned medium from cultured bovine and human granulosa cells. Fractionation of bovine FF by SDS-PAGE confirmed assay specificity since only one peak of activin-A immunoreactivity was detected (Mr ∼25 k) in eluted gel slices. However, gel-permeation chromatography showed that under physiological conditions all of the detectable activin-A in bovine FF eluted with apparent Mr values of >700 and 60–200 k reflecting its association with binding protein(s). Analysis of bovine FF samples (n=76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r=+0·54; P<0·001) and total β subunit immunoreactivity (r=+0·32; P<0·005) but not with total α subunit immunoreactivity (r= −0·09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total β subunit levels were highest in oestrogen-inactive follicles (P<0·01) whereas total α subunit levels were lowest in these follicles (P<0·001). Activin-A levels were measurable in all human serum samples analysed, ranging from 128 pg/ml during the normal menstrual cycle, 210 pg/ml in women undergoing ovarian hyperstimulation and ∼ 500 pg/ml in postmenopausal women to over 4000 pg/ml during pregnancy.

In conclusion, the present assay provides a reliable method for quantitating total (i.e. bound+free) activin-A concentrations in a variety of biological samples and should prove useful for further in vivo and in vitro studies in a range of species including man.

Journal of Endocrinology (1996) 148, 267–279
Circulating levels of IGF-I and IGF-binding protein-1 throughout pregnancy: relation to birthweight and maternal weight
Authors: Hills F A , English J , Chard T
Published: December 2004

Serum levels of IGF-I and IGF-binding protein (IGFBP-1) have been determined in the maternal circulation between 11 and 42 weeks of gestation in women not in labour (n=335) and in the maternal and fetal circulations at the time of delivery between 37 and 42 weeks (n=55). Maternal serum (MS) IGF-I levels increased during pregnancy and showed a significant positive correlation with maternal weight (P=0·0033) but no correlation with birthweight. The MS IGFBP-1 levels did not change during the second and third trimesters and showed a negative correlation with birthweight, maternal weight, placental weight and MS glucose (P=0·0002, P<0·0001, P=0·047, P=0·024 respectively). MS IGFBP-1 levels were higher in small-for-gestational age babies than in average-for-gestational weight babies (P=0·026) and lower in the large-for-gestational weight group (P=0·048). There was a significant rise in mean MS IGFBP-1 levels during labour (P=0·0005). These findings suggest that IGFBP-1 may be an important factor in pathological growth retardation.

Journal of Endocrinology (1996) 148, 303–309
Pressor doses of angiotensin II increase hepatic glucose output and decrease insulin sensitivity in rats
Authors: Rao R H
Published: December 2004

The metabolic effects of angiotensin II (AII) were studied under steady-state conditions of euglycaemic hyperinsulinaemia in anaesthetized rats. Pressor doses of AII (50 and 400 ng/kg per min) had dose-dependent hypertensive and hyperglycaemic effects during glucose clamp studies. Glucose turnover measurements showed that hepatic glucose output (HGO) increased equally at both pressor doses compared with either saline infusion or AII infusion at a dose without a pressor effect (20 ng/kg per min); however, glucose disposal increased significantly only at 50 ng/kg per min. Infusion of the AII receptor antagonist, saralasin, did not itself alter glucose output or disposal significantly, but it abolished the effects of a simultaneous infusion of All. It is concluded that pressor doses of AII increase HGO by a receptor-mediated mechanism that is not related to the pressor response to the hormone. The hyperglycaemic reaction to this metabolic effect of AII is partially offset by increased glucose disposal at lower doses. The physiological significance of these metabolic actions of AII remains to be established, but they raise the possibility that AII could potentially play a role in glucose homeostasis in vivo.

Journal of Endocrinology (1996) 148, 311–318
Identification of epidermal growth factor-secreting cells in the anterior pituitary of lactating female rats
Authors: Mouihate A , Verrier D , Lestage J
Published: December 2004

Epidermal growth factor (EGF) is synthesized and secreted by mammalian anterior pituitary cells. It stimulates GH and prolactin (PRL) secretion, but the cellular origin of EGF is relatively unexplored. The objective of this study was to characterize the cells that secrete EGF in the anterior pituitary of lactating rats. An EGF reverse haemolytic plaque assay (RHPA) was used to identify EGF-secreting cells and this RHPA was combined with immunofluorescence using antibodies to the six major adenohypophysial hormones (i.e. PRL, GH, LH, FSH, TSH and ACTH).

Approximately 20% (20·33 ± 2·96%) of the cells in the pituitary of lactating rats secrete EGF. The EGF-secreting cell population was composed of the following labelled cells: PRL (27%), GH (20%), LH (18%), FSH (14%), TSH (14%) and ACTH (5%).

The present study showed that EGF is released by a subpopulation of anterior pituitary cells composed of all the classic hormone-containing cells.

Journal of Endocrinology (1996) 148, 319–324
Regulation of type 1 insulin-like growth factor (IGF) receptors and IGF-I mRNA by age and nutrition in ovine skeletal muscles
Authors: Oldham J M , Martyn J A K , Kirk S P , Napier J R , Bass J J
Published: December 2004

The relative abundance and location of type 1 IGF receptors in sheep muscles have been measured to determine whether changes occur during post-natal growth and nutritional stress. Using the technique of histological autoradiography, specific binding of 125I-IGF-I in muscle fibre and connective tissue of M. biceps femoris and M. gastrocnemius was demonstrated, as was specific binding to the tendon of M. gastrocnemius and the surrounding connective tissue. The binding site in both muscles was characterised as the type 1 IGF receptor in membrane preparations using competitive binding assay and SDS-PAGE.

Type 1 receptors were more abundant in connective tissue than muscle fibre or tendon (P≤0·001). Levels changed significantly with age in all tissues (P=0·054 to P≤ 0·001), while change as a result of fasting was limited to a receptor increase in the connective tissue of M. gastrocnemius (P=0·034). IGF-I mRNA in M. bicepsfemoris, as assessed by in situ hybridisation, showed changes in expression with increasing age (P≤ 0·025) but no change with fasting.

These data indicate that the distribution, relative abundance and nutritional sensitivity of type 1 receptors are related to cell type in vivo. The overall decline of receptors with increasing age may be a feature of transition from linear animal growth to cell maintenance in adult animals. Connective tissue appears to be more sensitive than muscle fibre to nutrition, possibly allowing the reduction of non-essential metabolism during fasting.

Journal of Endocrinology (1996) 148, 337–346
Characterization of the hypothalamo–pituitary–IGF-I axis in rats made obese by overfeeding
Authors: Cattaneo L , De Gennaro Colonna V , Zoli M , Müller E E , Cocchi D
Published: December 2004

Obesity is coupled to several disturbances of the endocrine axes. It has previously been shown that genetically obese Zucker male rats have an impaired secretion of growth hormone (GH), probably originating from a primary reduction of hypothalamic GH-releasing hormone (GHRH) function and resulting in a decrease of GH gene expression and release. We sought to evaluate the somatotropic function in another model of experimental obesity. Normal male Sprague-Dawley rats were fed an energy-rich highly palatable diet for 7 months until they reached body weights overlapping those reported for obese Zucker rats. They were then evaluated for different indices of the hypothalamo–pituitary–somatomedin-C (IGF-I) axis. At the end of the overfeeding period, rats were divided into overtly obese (obese group) and overweight (overweight group) rats according to the degree of overweight and the Obesity Lee Index, while rats fed ad libitum with the standard pellet chow served as controls. Acute administration of a supramaximal dose of GHRH (2 μg/rat i.v.) elicited a significantly (at least P<0·05) lower plasma GH rise in the overweight and obese groups compared with the controls although no difference was seen in the pituitary GH content and gene expression and plasma concentrations of free IGF-I in the two experimental groups vs the controls. In addition, evaluation of hypothalamic GHRH and somatostatin mRNAs (slot-blot hybridization) did not show any significant differences between the three groups. Of the different metabolic indices investigated, plasma glucose and insulin concentrations were significantly (P<0·01) higher in the obese than in the overweight and control groups. A sharp decrease in plasma testosterone levels, together with a reduction in testis weight, was seen in both groups of rats fed the palatable diet compared with the controls. These findings underline the 'peripheral' feature of the hyposomatotropinism of rats chronically fed an energy-rich diet, and may account for the reversibility of the GH impairment in many obese subjects once a normal body weight has been restored. Moreover, the peripherally-driven hyposomatotropinism of these rats is in sharp contrast with the hypothalamic-driven GH secretory impairment of the obese Zucker rats.

Journal of Endocrinology (1996) 148, 347–353
Comparison of cellular distribution of LH receptors and steroidogenic enzymes in the porcine ovary
Authors: Meduri G , Vu Hai M T , Jolivet A , Takemori S , Kominami S , Driancourt M A , Milgrom E
Published: December 2004

Previous studies have shown a heterogeneous expression of LH receptors in various structures of the porcine ovary. Specially striking was the existence in the preovulatory follicle of inner layers of theca interna cells devoid of LH receptor and the confinement in the corpus luteum of the LH receptor to the external cellular layers. In the present study, we have compared the steroidogenic capabilities of LH receptor-positive and -negative cells using immunocytochemistry for side-chain cleavage P450, 3β-hydroxysteroid-dehydrogenase, 17α-hydroxylase P450 and aromatase P450. We have also examined, using the same methods, the evolution of the various cell types after ovulation and during the development of the corpus luteum.

In preovulatory follicles the inner layers of theca cells which were not labelled with anti-LH receptor antibodies appeared to express the steroidogenic enzymes in a way similar to that of the outer LH receptor-positive cell layers. Ovulation per se did not change the distribution of LH receptors (present in the outer luteal cells and in the granulosa) or of steroidogenic enzymes. However, 48 h after follicular rupture there was a marked decrease in overall labelling with anti-LH receptor antibody, and especially a disappearance of immunostaining in the luteal cells of granulosa origin. In the mid-luteal phase (6 days after ovulation), the receptor content seemed to increase in the peripheral luteal cells derived from the theca but the receptor did not reappear in the granulosa-derived luteal cells. Thus the down-regulation of LH receptor appeared to be reversible in the external thecal layers but irreversible in the granulosa cells. Furthermore, the distribution of the various steroidogenic enzymes in the corpora lutea delineated granulosa-derived from theca-derived cells and showed that only the external layers of the latter expressed the LH receptor.

These results showed the existence in the preovulatory follicle of two theca interna regions expressing the same steroidogenic enzymes but possibly submitted to a different hormonal control. Furthermore, the cells derived from these two regions as well as the cells of granulosa origin showed a distinct pattern of variation of LH receptivity during the development of the corpus luteum.

During these studies we also observed that, in the interstitial tissue, only a minority of cells which derived from remnants of atretic follicles expressed both the LH receptor and the steroidogenic enzymes.

Journal of Endocrinology (1996) 148, 435–446
In vivo and in vitro effect of 1,25-dihydroxyvitamin D3 and 1,25-dihydroxy-16-ene-23-yne-vitamin D3 on the proliferation and differentiation of avian chondrocytes: their role in tibial dyschondroplasia
Authors: Farquharson C , Rennie J S , Loveridge N , Whitehead C C
Published: December 2004

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is regarded as the most biologically active metabolite of cholecalciferol. It prevents tibial dyschondroplasia (TD) in chicks where inhibition of chondrocyte differentiation within the growth plate occurs. However, it is unclear whether its mode of action is through direct interaction with its chondrocyte receptor and its known regulatory role in cell differentiation or is mediated by increased calcium absorption and mobilisation. Synthetic analogues of 1,25(OH)2D3 such as 1,25-dihydroxy-16-ene-23-yne cholecalciferol (RO 23–7553) with increased differentiation properties but reduced calcaemic activity have been synthesised. In this study, the in vitro and in vivo effects of 1,25(OH)2D3 and RO 23–7553 on chick chondrocyte growth and differentiation were examined. In addition, the in vivo effectiveness of these steroids in preventing TD in chicks was assessed. 1,25(OH)2D3 and RO 23–7553 (10−12-10−7 m) displayed biphasic concentration effects and had similar potencies in vitro in regulating chondrocyte proliferation and differentiation. However, while the incidence of TD in birds dosed with 1,25(OH)2D3 was lower (10%) than in control chicks (55%), RO 23–7553 was ineffective (50%). This may be the result of its reduced affinity (1000 times less) for the plasma vitamin D binding protein (DBP) and the chondrocyte receptor in comparison to that of 1,25(OH)2D3. A reduction in calcium supply to the chondrocyte may also result in decreased chondrocyte differentiation but blood ionised and plasma total calcium were normal in birds dosed with RO 23–7553. These data suggest that RO 23–7553 and 1,25(OH)2D3 regulate chondrocyte proliferation and differentiation similarly in vitro but not in vivo. This may be caused by differences in DBP binding and clearance rates of the two steroids in vivo.

Journal of Endocrinology (1996) 148, 465–474