bluuebunny/crossref_metadata_2025 huggingface dataset:
8-Bromo-cAMP inhibits glucose transport activity in mouse placental cells in culture
Authors: Sakata M , Yamaguchi M , Imai T , Tadokoro C , Yoshimoto Y , Oka Y , Kurachi H , Miyake A
Published: December 2004

Glucose plays an important role in fetal development and energy metabolism. Facilitative glucose transporter-1 (GLUT1) has been found in placenta. However, little is known about GLUT1 modulation in placental cells. To examine changes in mouse placental GLUT1 levels caused by 8-bromo-cAMP, we performed 2-deoxyglucose uptake experiments, Northern blot analysis and immunoblot analysis using a primary mouse placental cell culture. Immunohistochemical analysis showed that GLUT1 was localized to the ectoplacental cone and the labyrinth zone of mouse placentas on days 7 and 11 of pregnancy respectively. Treatment of mouse placental cells with 250 μmol/l 8-bromo-cAMP resulted in a significant (P<0·01) decrease in glucose uptake on days 2–5 of culture. The inhibitory effect of 8-bromo-cAMP on glucose uptake was concentration-dependent. Glucose uptake was also inhibited by 100 μg/l cholera toxin and by 0·1 mmol/l forskolin. Northern blot and immunoblot analysis revealed that both GLUT1 mRNA and protein levels were also decreased by 8-bromo-cAMP. These findings suggest that 8-bromo-cAMP inhibits glucose transport activity in mouse placental cells in culture.

Journal of Endocrinology (1996) 150, 319–327
Parathyroid hormone-related protein (PTHrP) action in rat articular chondrocytes: comparison of PTH(1–34), PTHrP(1–34), PTHrP(1–141), PTHrP(100–114) and antisense oligonucleotides against PTHrP
Authors: Tsukazaki T , Ohtsuru A , Namba H , Oda J , Motomura K , Osaki M , Kiriyama T , Iwasaki K , Yamashita S
Published: December 2004

Parathyroid hormone-related protein (PTHrP) is thought to be an important autocrine/paracrine factor for chondrocyte metabolism since mice lacking the PTHrP gene exhibit abnormal cartilage development. To determine the biological role of PTHrP in chondrocytes, we first compared the agonist potency of human (h) PTHrP(1–34) with hPTH(1–34) in cultured rat articular chondrocytes. Neither hPTHrP(1–34) nor hPTH(1–34) altered basal DNA synthesis, but attenuated the stimulatory effect of transforming growth factor β (TGF-β). Both agents suppressed the expression of α(1) type II collagen mRNA in a dose–response fashion with the same potency. In addition, the action of exogenously added hPTHrP(1–34) and hPTH(1–34) on intracellular cAMP and [Ca2+]i levels was similar. We next compared the effect of PTHrP within its entire amino acid sequence (1–141). With regard to thymidine incorporation, α(1) type II collagen gene expression and accumulation of cAMP and [Ca2+]i level, there was no significant difference between hPTHrP(1–34) and hPTHrP(1–141). PTHrP C-terminal (100–114) did not show any function. To further investigate PTHrP function, intracellular PTHrP translation was inhibited by a transgene of antisense oligonucleotides against PTHrP. Antisense oligonucleotides decreased PTHrP mRNA translation, specifically inhibited DNA synthesis in control as well as TGF-β-treated chondrocytes and enhanced α(1) type II collagen mRNA expression in TGF-β-treated chondrocytes.

These results suggest that there is no significant difference between exogenously added hPTH(1–34), hPTHrP(1–34) and PTHrP(1–141) with regard to the biological action of these agents, including cell growth, differentiation and second messenger pathway. However, the result of DNA synthesis in the antisense PTHrP-inhibition study suggests that intracellular PTHrP may have an as yet unknown biological role, in addition to a classical PTH/PTHrP receptor-mediated function in the rat articular chondrocyte.

Journal of Endocrinology (1996) 150, 359–368
Ontogeny of growth hormone, insulin-like growth factor-I, estradiol and cortisol in the growing lamb: effect of testosterone
Authors: Arnold A M , Peralta J M , Thonney M L
Published: December 2004

Exogenous sex steroids have altered growth hormone secretion in some domestic species. This study examined whether different physiological concentrations of testosterone alter muscle growth in sheep through modification of the somatotropic axis. The effects of testosterone on growth hormone (GH), insulin-like growth factor-I (IGF-I), estradiol (E2) and cortisol concentrations in growing lambs were evaluated in 20 rams, 20 wethers and 20 wethers receiving subcutaneous testosterone replacement therapy. Two animals from each of the three testosterone status groups were slaughtered at 14-day intervals from 49 to 133 days of age, and then at 28-day intervals until 217 days of age for a total of 10 slaughter ages. Animals were sampled every 10 min for an 8-h period 1 day prior to slaughter to characterize the episodic patterns of GH and testosterone. Immediately after slaughter, the semitendinosus, splenius and triceps brachii muscles were removed, trimmed of adhering fat and connective tissue, and weighed. Testosterone increased the combined muscle weight. GH concentrations decreased during the course of the experiment. However, there was no effect of testosterone on GH mean, baseline, amplitude or GH pulse frequency measured by PULSAR. IGF-I concentrations increased in response to testosterone treatment. Testosterone had no effect on cortisol levels while E2 levels were increased after 133 days. Increased muscle growth due to testosterone appeared to be caused either by a direct effect or by increased levels of IGF-I independent of circulating GH concentrations.

Journal of Endocrinology (1996) 150, 391–399
The effect of oxytocin and oestradiol on the action of conceptus secretory protein in progesterone-treated ovariectomized ewes
Authors: Mann G E , Payne J H , Lamming G E
Published: December 2004

In intact cyclic ewes intrauterine infusion of conceptus secretory proteins results in the suppression of both endometrial oxytocin receptor concentrations and oxytocin-induced prostaglandin F2α release. However, similar infusion in progesterone-treated ovariectomized ewes, while suppressing endometrial oxytocin receptors, does not fully inhibit oxytocin-induced prostaglandin F2α release. To examine whether this anomaly resulted from an inadequate simulation of the luteal phase in the ovariectomized ewe treated with progesterone alone, the effects of additional treatment with two other ovarian hormones, oestradiol-17β and oxytocin, was investigated. Rather than permitting conceptus secretory protein to successfully inhibit oxytocin-induced prostaglandin F2α release, treatment with oestradiol-17β in addition to progesterone actually resulted in an advancement in the timing of release. However, treatment with oxytocin, alone or in combination with oestradiol, permitted the full inhibition of oxytocin-induced prostaglandin F2α release. To confirm that this effect did not result from the action of oxytocin alone, independently of the action of conceptus secretory protein, a second experiment was undertaken using a similar protocol but without the infusion of conceptus secretory protein. In this situation, oxytocin-induced prostaglandin F2α release was only partially inhibited suggesting that both luteal oxytocin and conceptus secretory proteins are necessary to facilitate the full inhibition of luteolysis during early pregnancy in the ewe.

Journal of Endocrinology (1996) 150, 473–478
Changes in oxytocin receptor mRNA in the rat uterus measured by competitive reverse transcription-polymerase chain reaction
Authors: Liu C-X , Takahashi S , Murata T , Hashimoto K , Agatsuma T , Matsukawa S , Higuchi T
Published: December 2004

Large changes in the responsiveness of target organs to oxytocin are thought to originate from alteration of the number of oxytocin receptors (OTR). To elucidate the molecular mechanisms regulating the synthesis of the OTR, we developed a competitive reverse transcription-PCR protocol to measure OTR mRNA. We synthesized cRNA comprising a small stuffer introduced into the target mRNA. Using this cRNA as an internal standard, we made a quantitative estimation of OTR mRNA. Application of this method to the rat uterus revealed that the mean levels of OTR mRNA remained unchanged until 1030–1100 h on day 21 of pregnancy, increased significantly after 2200–2230 h on the same day and declined rapidly after parturition. A similar rapid increase in uterine OTR mRNA content was observed in rats given prostaglandin on day 18, inducing premature delivery on day 19 of pregnancy. All parturient rats had higher OTR mRNA levels regardless of whether parturition was spontaneous or prostaglandin induced. However, in a few rats, OTR mRNA remained as low as that observed during mid pregnancy even on day 22 of gestation, the expected day of parturition in about 70% of the rats in our colony. A similar increase in uterine OTR mRNA content to that observed at parturition was induced by oestrogen treatment for 3 days in ovariectomized virgin rats, but concomitant injection of progesterone did not influence the effect of oestrogen. The present results revealed that the large increase of uterine OTR at the peripartum period is accompanied by an increase in OTR mRNA content that may be brought about, at least in part, by increased oestrogen secretion following luteolysis.

Journal of Endocrinology (1996) 150, 479–486
Desensitisation of somatostatin, TRH and GHRH responses to glucose in the diabetic (Goto-Kakizaki) rat hypothalamus
Authors: Lewis B M , Ismail I S , Issa B , Peters J R , Scanlon M F
Published: December 2004

We have studied the effects of glucose on the release of somatostatin (SS), TRH and GHRH from incubated hypothalami of normal and genetically diabetic, Goto-Kakizaki (GK) rats. The active isomer d-glucose caused a dose-related inhibition of SS, TRH and GHRH from normal rat hypothalami over a 20-min incubation period in vitro. In contrast, in GK rats the effects of glucose on TRH and SS were significantly reduced and the effects on GHRH were abolished. These data indicate that the sensitivity of SS-, TRH- and GHRH-producing hypothalamic neurones is reduced in diabetic rats. The effect is most pronounced for GHRH release as there was no change in the release of this peptide with increasing glucose concentrations. In conclusion, it appears that the diabetic state in GK rats causes differential desensitisation (GHRH>TRH and SS) of neuronal responses to subsequent changes in glucose concentrations in vitro. This may be due to alterations in the neurotransmitter control and/or a reduction in number, affinity or function of glucose transporters on these peptidergic neurones or other intermediary neuronal pathways.

Journal of Endocrinology (1996) 151, 13–17
Stage-specific expression of the FSH receptor gene in the prepubertal and adult rat seminiferous epithelium
Authors: Rannikko A , Penttilä T-L , Zhang F-P , Toppari J , Parvinen M , Huhtaniemi I
Published: December 2004

Stage-specific expression of the FSH receptor (FSHR) gene in the rat seminiferous epithelium was studied. Using transillumination-assisted microdissection for sample preparation and Northern hybridization for analysis of total RNA, we first reassessed the stage specificity of the FSHR gene expression in the adult rat testis. Sixfold higher FSHR mRNA levels were found in stages XIII–I compared with stage VI of the seminiferous epithelial cycle, which had the lowest signal level (P<0·01). The other stages had intermediate signal levels. In situ hybridization showed distribution of grains which confirmed the data obtained by Northern analysis. Prepubertal stage-specific FSHR gene expression was studied using in situ hybridization. Stage specificity could first be demonstrated at the age of 16 days when the average grain counts in stages I–IV were threefold higher than in stages VI–VII (P<0·01). The present data are in agreement with earlier findings on stage-specific FSH binding and FSHR gene expression using both microdissected and stage-synchronized seminiferous tubules. The onset of stage-specific FSHR gene expression is concomitant with maturation of the Sertoli cell population and completion of the first generation of spermatocytes. This supports the hypothesis that spermatogonia and spermatocytes may be involved in the regulation of FSHR gene expression.

Journal of Endocrinology (1996) 151, 29–35
Regulation of intestinal cholecystokinin gene expression by glucocorticoids
Authors: Ratineau C , Roche C , Chuzel F , Cordier-Bussat M , Blanc M , Bernard C , Cuber J-C , Chayvialle J-A
Published: December 2004

The effect of glucocorticoids on the expression of intestinal cholecystokinin (CCK) was investigated both in vivo and in cell culture systems. In vivo, 2-day administration of methylprednisolone to adult male rats induced a decrease in CCK-like immunoreactivity (CCK-LI) and CCK mRNA levels in mucosal extracts. In two CCK-producing cell lines, RIN 1056E and STC-1 of pancreatic and intestinal origin respectively, dexamethasone induced dose-dependent decreases in both CCK-LI and steady-state CCK mRNA levels. The decrease in CCK mRNA was totally prevented by incubation of cells with an excess of RU 38486, a competitive inhibitor for the binding of glucocorticoids to their receptor. Actinomycin D, used to prevent RNA synthesis, did not modify CCK mRNA stability in dexamethasone-pretreated cells as compared with cells not exposed to dexamethasone. When cells were first incubated with actinomycin D, subsequent addition of dexamethasone left the steady-state CCK mRNA levels unaltered in both cell lines. Nuclear run-on assays performed in RIN 1056E cells showed that glucocorticoids decreased the rate of transcription of the CCK gene. In addition, cycloheximide, used to prevent protein synthesis, abolished the inhibitory effects of dexamethasone on steady-state CCK mRNA levels. These results demonstrate that glucocorticoids down-regulate CCK gene expression in the rat intestinal mucosa and in two CCK-producing cell lines. The effect is blocked by a glucocorticoid receptor antagonist. Inhibition of CCK gene expression may result from a decrease in the transcription rate, and probably involves one or several steps that depend on protein synthesis.

Journal of Endocrinology (1996) 151, 137–145
Interleukin-1β inhibits rat thyroid cell function in vivo and in vitro by an NO-independent mechanism and induces hypothyroidism and accelerated thyroiditis in diabetes-prone BB rats
Authors: Reimers J I , Rasmussen A K , Karlsen A E , Bjerre U , Liang H , Morin O , Andersen H U , Mandrup-Poulsen T , Burger A G , Feldt-Rasmussen U , Nerup J
Published: December 2004

Interleukin-1β has been implicated as a pathogenic factor in the development of autoimmune thyroiditis. When given for 5 days to normal non-diabetes-prone Wistar Kyoto rats, it decreased plasma concentrations of total tri-iodothyronine and thyroxine and increased plasma TSH. These effects were not prevented by co-injection of nitroarginine methyl ester or aminoguanidine, inhibitors of NO synthases. Exposure to interleukin-1β dose-dependently reduced iodine uptake in FRTL-5 cells, but had no effect on thyroglobulin secretion. Nitrite was not detected in the FRTL-5 cell culture media after exposure to interleukin-1β. However, reverse transcription PCR analysis of mRNA isolated from interleukin-1β-exposed FRTL-5 cells revealed a transitory expression of the inducible NO synthase, which was markedly lower than inducible NO synthase induction in interleukin-1β-exposed isolated rat islets of Langerhans. Co-incubation with the NO synthase inhibitor NG-monomethylarginine did not ameliorate the effect of interleukin-1β on FRTL-5 cell iodine uptake. Furthermore, we demonstrate that daily injections of interleukin-1β for 13 weeks aggravated spontaneous thyroiditis and induced severe hypothyroidism in non-diabetic diabetes-prone BB rats. The data suggest that NO does not mediate interleukin-1β-induced inhibition of rat thyroid function in vivo or in vitro in FRTL-5 cells, and the induction of hypothyroidism by interleukin-1β in diabetes-prone BB rats is speculated to be due to exacerbation of recruitment and activation of intrathyroidal mononuclear cells.

Journal of Endocrinology (1996) 151, 147–157
Transcription initiation of the rat insulin-like growth factor-I gene in hepatocyte primary culture
Authors: Krishna A Y , Pao C-I , Thulé P M , Villafuerte B C , Phillips L S
Published: December 2004

Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10−6 or 10−11 m, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (−380 bp from the 3′ end of exon 1), site 2 (−343 bp), site 3 (−242 bp), sites 1 and 2 spliced, and site 4 (−32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 8, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4–5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4–6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH.

In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms.

Journal of Endocrinology (1996) 151, 215–223
Plasma levels of chromogranin A are directly proportional to tumour burden in neuroblastoma
Authors: Wassberg E , Stridsberg M , Christofferson R
Published: December 2004

A novel animal experimental model involving the human, poorly differentiated, and adrenergic neuroblastoma cell line SH-SY5Y xenotransplanted to subcutaneous tissue of 13 nude rats (WAG rnu/rnu) was used to investigate the usefulness of six proposed neuroblastoma markers. It was shown that the plasma concentrations of human chromogranin A (CgA) as measured by RIA were directly proportional to tumour volume (r=0·83, P<0·001). To rule out possible liberation of CgA by tumour cell lysis, the CgA degradation product pancreastatin was also measured in plasma by a specific RIA, but was not detectable. Plasma neurone-specific enolase (NSE) was elevated in tumour-bearing animals (P<0·01), but did not correlate with tumour volume (r=0·49, P>0·05). Urine homovanillic acid (HVA), detected by HPLC, was elevated in tumour-bearing animals (P<0·01), but did not correlate with tumour volume (r=−0·32, P>0·05). Urine vanillyl mandelic acid was not detectable. Urine dopamine was found in low concentrations that did not correlate with tumour volume. In summary, although plasma NSE and urinary HVA were elevated in tumour-bearing animals only plasma CgA correlated with tumour burden. This makes CgA a promising biochemical marker for neuroblastomas.

Journal of Endocrinology (1996) 151, 225–230
Differential regulation of granulocyte-macrophage colony-stimulating factor mRNA and protein expression in human thyrocytes and thyroid-derived fibroblasts by interleukin-1α and tumour necrosis factor-α
Authors: Aust G , Hofmann A , Laue S , Ode-Hakim S , Scherbaum W A
Published: December 2004

In this study, we provide the first report on the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) by human thyroid epithelial cells. Primary cultures of highly purified thyrocytes and thyroid-derived fibroblasts (n=3) and three thyroid anaplastic and one largely papillary carcinoma cell lines were exposed to different potent GM-CSF stimulators, employing interleukin 1α (Il-1α) and tumour necrosis factor-α (TNF-α). Cytokine mRNA levels were monitored by semiquantitative reverse transcriptase-PCR including an internal heterologous competitor fragment after 3, 6 and 18 h of culture. Culture supernatants were assayed for GM-CSF using a highly sensitive ELISA (detection limit ≤ 0·5 pg/ml) after 24 h.

Basal GM-CSF mRNA expression was higher in fibroblasts and SW 1736 cells compared with thyrocytes, C 634, 8505 C and HTh 74 cells. GM-CSF was spontaneously secreted by fibroblasts (means ± s.e.m.; 43 ± 15 pg/ml), SW 1736 (59 ± 4 pg/ml), HTh 74 (34 ± 4 pg/ml) and C 643 cells (12 ± 1 pg/ml) but not by thyrocytes and 8505 C cells. Treatment with Il-1α (10 U/ml) resulted in a marked increase of GM-CSF mRNA within 3 h and an increase or induction of protein expression in thyrocyte (2350 ± 214 pg/ml), fibroblast (5242 ± 1400 pg/ml), SW 1736 (20016 ± 280 pg/ml) and C 643 cultures (1285 ± 79 pg/ml). Stimulation with TNF-α (10 U/ml) yielded divergent results. No significant increase of GM-CSF mRNA or protein expression was found in thyrocytes although TNF-α receptor expression in these cells is well documented. Stimulation with TNF-α resulted in an increased GM-CSF production in fibroblasts (361 ± 14 pg/ml), HTh 74 (148 ± 51 pg/ml) and SW 1736 cultures (235 ± 43 pg/ml). TSH (10 mU/ml) did not stimulate GM-CSF secretion in thyrocytes and HTh 74 cells, both expressing the TSH receptor. Phorbol 12-myristate 13-acetate (10 ng/ml) enhanced GM-CSF mRNA and protein levels in all cell types investigated.

Our data suggest that both thyrocytes and fibroblasts synthesize GM-CSF in response to Il-1α, but only fibroblasts respond to TNF-α with a significant increase in GM-CSF. Anaplastic thyroid carcinomas are potential GM-CSF producers.

Journal of Endocrinology (1996) 151, 277–285
Investigation of the effects of antisense oligodeoxynucleotides to islet amyloid polypeptide mRNA on insulin release, content and expression
Authors: Kulkarni R N , Smith D M , Ghatei M A , Jones P M , Bloom S R
Published: December 2004

We have investigated the effects of antisense oligodeoxynucleotides (oligos) to islet amyloid polypeptide (IAPP) mRNA on the expression and secretion of IAPP and insulin, in the clonal β-cell line HIT-T15. Phosphorothioate-modified oligos were cytotoxic compared with phosphodiester (D)-oligos. Of the nine oligos tested using a lipofection reagent, 03, a 30-mer D-oligo complementary to a sequence downstream of the IAPP initiation codon, showed a significant dose-dependent suppression of IAPP mRNA, with a 42% decrease at 7·5 μm, compared with a scrambled (MS03) control oligo (n=3, P<0·01). A subsequent 89% suppression of IAPP release was observed in the 4-h period following antisense treatment (1·78 ± 0·13 (MS03) vs 0·19 ± 0·14 (03) pmol/106 cells per 240 min, n=7, P<0·01). A significant increase in insulin mRNA (100 ± 10% (MS03) vs 124 ± 8% (03), n=3, P<0·05) and insulin content (13·0 ± 0·9 (MS03) vs 17·4 ± 1·4 (03) pmol/106 cells, n=7, P=0·028) was observed following treatment with 03 at 7·5 μm. 08, a 20-mer D-oligo directed to a region of IAPP mRNA further downstream than 03, also showed a decrease in IAPP mRNA and peptide release and an increase in insulin content. No significant changes were observed in the expression and release of the unrelated β-cell peptide, neuropeptide Y. We thus show a suppression of synthesis and release of IAPP in HIT-T15 cells using antisense oligos. The associated increase in insulin mRNA and content in these cells after treatment with IAPP antisense oligos is in accord with an inhibitor action of IAPP on insulin availability.

Journal of Endocrinology (1996) 151, 341–348
Repartitioning of maternal muscle protein towards the foetus induced by a polyclonal antiserum to rat GH
Authors: Palmer R M , Thom A , Flint D J
Published: December 2004

Growth and protein accretion were studied in maternal muscle and liver and in foetuses of rats on day 20 of pregnancy. In young rats, weighing 120 g at mating, muscle mass and protein content of three hind-limb muscles, soleus, plantaris and gastrocnemius, increased on average by 7% compared with non-pregnant controls although the rate of muscle protein synthesis was decreased. In mature rats, rates of muscle protein synthesis were also reduced on day 20 of pregnancy but no change in muscle mass was observed. Rates of liver protein synthesis and accretion were increased in the pregnant animals; the effect was larger in the young pregnant rat. Administration of an antibody to rat GH (anti-rGH) for 10 days to young pregnant rats reversed the effect on the same three maternal muscles and resulted in a 9–11% lower muscle mass and protein content, compared with control pregnant animals. In both young and mature dams serum IGF-I concentrations were halved on day 20 of pregnancy, a further small reduction was observed in response to anti-rGH. No significant change in serum insulin or corticosterone levels was observed. Anti-rGH treatment also reduced food intake but foetal weight at 20 days was significantly increased (14%). The effects on maternal muscle were not the result of loss of appetite associated with anti-GH administration as, in rats pair-fed to the intake of the anti-rGH group, maternal muscle and foetal weights were the same as in animals with food available ad libitum. The data suggest that the GH/IGF axis is involved in the partitioning of nutrients between the dam and the foetus.

Journal of Endocrinology (1996) 151, 395–400
The effect of epidermal growth factor on circulating levels of IGF and IGF-binding proteins in adult Goettingen minipigs
Authors: Vinter-Jensen L , Orloff Juhl C , Frystyk J , Dajani E Z , Oksbjerg N , Flyvbjerg A
Published: December 2004

It has recently been demonstrated that epidermal growth factor (EGF) administration to neonatal rodents causes growth retardation with concomitant reductions in circulation levels of IGF-I. We describe the effects of systemic EGF administration for 4 weeks on circulating levels of IGF-I and IGF-binding proteins (IGFBPs) and on thyroid hormones (tri-iodothyronine, T3; thyroxine, T4) in sexually mature pigs. Goettingen minipigs of either sex were treated with placebo (n=5) or EGF (30 μg/kg per day, n=6) s.c. for 4 weeks (in relation to an oesophageal sclerotherapy regimen). Blood samples were taken under anaesthesia before and after 1, 2, 3 and 4 weeks of treatment. Circulating levels of IGF-I, insulin, glucose, T3 and T4 were analysed every week and IGFBPs every second week. IGF-I was not reduced significantly after 1 week but significantly reduced after 2 and 3 weeks of EGF treatment. A similar decline was observed for the major IGFBP, IGFBP-3, which was reduced after 2 and 4 weeks. IGFBP-1, IGFBP-2 and IGFBP-4 increased throughout the treatment period (all significantly at week 4). EGF treatment induced increased circulating T3 after 2, 3 and 4 weeks of EGF treatment. In conclusion, we report that EGF treatment for 4 weeks in Goettingen minipigs reduces circulating IGF-I and IGFBP-3, increases circulating IGFBP-1, IGFBP-2 and IGFBP-4, and induces a slight hyperthyroidism as judged from increased circulating levels of T3.

Journal of Endocrinology (1996) 151, 401–407
Thyroxine effect on intestinal Cl−/HCO3− exchange in hypo- and hyperthyroid rats
Authors: Tenore A , Fasano A , Gasparini N , Sandomenico M L , Ferrara A , Di Carlo A , Guandalini S
Published: December 2004

Changes in transepithelial water and electrolyte transport as causative or contributing factors of the diarrhoea and constipation found associated with changes in thyroid physiology were studied. Albino Wistar rats were pharmacologically made either hypothyroid or hyperthyroid. After sacrifice, the small intestine was mounted in Ussing chambers in order to measure in vitro ion net fluxes under short-circuit conditions. Hypothyroid animals showed an increase in intestinal transit time, Cl− absorption (mainly due to an increment in its mucosal to serosal component) and residual ion flux (which is believed to represent HCO3− secretion) when compared with euthyroid animals. The hyperthyroid animals showed a decrease in Cl− mucosal to serosal transport. Furthermore, a significant correlation was found between serum l-thyroxine (T4) levels and both net Cl− transport (r= −0·74, P<0·00001) and residual ion flux (r= −0·55, P<0·005).

These results indicate that the effect of T4 is firstly to inhibit Cl−/HCO3− anion exchange thereby influencing transepithelial flux transport and secondly to affect intestinal motility. Such inhibition was not found when T4 was acutely added to rat ileum, suggesting that the effect on electrolyte transport probably requires protein synthesis. In conclusion, the phenomenon observed in vitro could explain the clinical manifestations of constipation and diarrhoea in hypo- and hyper-thyroidism respectively.

Journal of Endocrinology (1996) 151, 431–437
Hepatocyte growth factor/scatter factor and its receptor c-met: localisation and expression in the human placenta throughout pregnancy
Authors: Clark D E , Smith S K , Sharkey A M , Sowter H M , Charnock-Jones D S
Published: December 2004

Hepatocyte growth factor (HGF), also known as scatter factor, acts via the c-met receptor resulting in pronounced effects on certain epithelial cells. We hypothesised that HGF would be important in placental development where the trophoblast represents a specialised barrier of epithelial origin. In this paper we examine the expression and production of HGF and its receptor in the human placenta throughout pregnancy. In addition, RT-PCR was undertaken on human embryos to ascertain whether preimplantation embryonic or trophoblast cells were under the influence of this growth factor. In samples from the first trimester of pregnancy in situ hybridisation with a c-met antisense probe detected message expression in villous cytotrophoblast and in decidual glands but not in extravillous trophoblast. Some c-met expression was detected in cytotrophoblast from the second trimester placentae; this declined to negligible levels by term. Staining with an anti c-met antibody largely confirmed these findings but found relatively strong staining of cytotrophoblast at term. HGF was confined to the villous core throughout pregnancy when examined by both in situ hybridisation and immunohistochemistry. Trophoblast was consistently negative for HGF. Pre-implantation embryos examined by RT-PCR were negative for both c-met and HGF mRNA. These results indicate that the HGF may exert an important influence on cytotrophoblast throughout the process of placental formation and growth.

Journal of Endocrinology (1996) 151, 459–467
Effect of lactation on insulin signal transduction in sheep adipose tissue and skeletal muscle
Authors: Wilson L A , Mills S E , Finley E , Kilgour E , Buttery P J , Vernon R G
Published: December 2004

The molecular basis of the insulin resistance of adipocytes and skeletal muscle during lactation has been investigated in sheep. The number of insulin receptors per adipocyte or per unit membrane protein for skeletal muscle is unchanged by lactation. The ability of insulin to stimulate autophosphorylation of its β-subunit was enhanced in adipocytes but not in skeletal muscle during lactation. This increased autophosphorylation was due, at least in part, to enhanced tyrosine phosphorylation and was found when both solubilised, immunoprecipitated insulin receptors and intact adipocytes were incubated with insulin. The ability of the insulin receptor kinase to phosphorylate other proteins did not appear to be altered by lactation; this was shown with lectin-purified insulin receptors using the artificial substrate, polyglutamyl tyrosine, and in intact adipocytes. Lactation had no effect on the ability of insulin to activate two key downstream kinases, mitogenactivated protein kinase and phosphatidyl inositol-3-kinase in adipocytes. The study thus shows that the insulin resistance of lactation in sheep is due to changes downstream of the receptor in both adipocytes and skeletal muscle.

Journal of Endocrinology (1996) 151, 469–480
Characterization of the ovine LHβ-subunit gene: the promoter is regulated by GnRH and gonadal steroids in transgenic mice
Authors: McNeilly J R , Brown P , Mullins J , Clark A J , McNeilly A S
Published: December 2004

We have previously demonstrated that 1·9 kb of ovine LHβ promoter fused to bacterial chloramphenicol transferase (CAT) coding sequence is sufficient to target expression of the transgene specifically to the gonadotroph cells of the anterior pituitary in mice with no expression being observed in other tissues. However, it is not known if this region of the ovine LHβ promoter contains the necessary elements that confer transcriptional regulation by gonadal steroids and GnRH. Following gonadectomy, both endogenous pituitary LH and CAT activity significantly (P>0·001) increased as did plasma LH. This postgonadectomy increase in CAT, pituitary and plasma LH could be suppressed in females by treatment with oestradiol alone or oestradiol and progesterone, with an additional significant (P<0·05) reduction in CAT activity being observed in one line following the combined steroid treatment. In castrated males, testosterone suppressed CAT activity in one line. Treatment of transgenic ovariectomized females with oestradiol alone significantly suppressed plasma LH (P<0·01) with no change in pituitary LH content. There was no difference in pituitary LH between oestradiol-treated ovariectomized transgenic and non-transgenic females. Treatment of intact females from both lines with either GnRH antiserum or agonist demonstrated a decrease in pituitary CAT activity whereas similar treatment in intact males had no effect. While endogenous pituitary LH concentrations were variable, plasma LH was lower in all treated animals irrespective of line, sex or expression of the transgene. In conclusion, these results indicate that (1) the presence of CAT protein is not toxic and does not compromise either endogenous LH synthesis, storage and secretion and (2) the ovine LHβ–CAT gene is regulated in a similar but more variable manner to the endogenous LHβ gene. This may relate to the use of CAT as a reporter where its release is not necessarily related to that of the endogenous hormone whose synthesis, storage and release may differ.

Journal of Endocrinology (1996) 151, 481–489
Effects of adrenal steroids on the concentration of Na+–K+ pumps in rat skeletal muscle
Authors: Dørup I , Clausen T
Published: December 2004

Since adrenal steroids have been shown to upregulate the concentration of Na+–K+-ATPase in cardiac muscle, similar effects could be expected in skeletal muscle. Following infusion of dexamethasone (0·02–0·1 mg/kg per day) for 7 days in 10-week-old rats, the total concentration of [3H]ouabain-binding sites rose by up to 22–42% in soleus, extensor digitorum longus, gastrocnemius and diaphragm muscle. Dexamethasone produced no or minute changes in the Na+–K+ contents of skeletal muscle. In contrast, infusion with aldosterone (0·02–0·5 mg/kg per day) for 7 days produced hypokalemia and a graded reduction in the K+ content of skeletal muscle, which was closely correlated to a downregulation of the [3 H]ouabain-binding site concentration (r= 0·65–0·70; P<0·001). The results indicate that in skeletal muscle high doses of glucocorticoids upregulate the concentration of Na+–K+ pumps whereas mineralocorticoids induce a downregulation, which is secondary to the concomitant K+ deficiency. Since adrenalectomy produced no significant change in [3 H]ouabain-binding site concentration, basal levels of endogenous adrenal steroids seem to be of minor importance for the regulation of Na+–K+ pump concentration in skeletal muscle.

Journal of Endocrinology (1997) 152, 49–57
Concentrations of GnRH receptor and GnRH receptor mRNA in pituitary tissue of orchidectomized sheep: effect of oestradiol, progesterone, and progesterone withdrawal
Authors: Sakurai H , Adams B M , Adams T E
Published: December 2004

The effect of progesterone (P4) and P4 withdrawal on oestradiol (E2)-induced change in gonadotrope responsiveness (GR) and concentration of GnRH receptor and GnRH receptor mRNA in pituitary tissue of orchidectomized sheep (wethers) was determined. Thirty wethers were assigned at random to one of six treatment groups (n=5 animals/group). Wethers received E2 (2 μg/h; groups 2, 4, and 6) in 10% ethanol-saline (vehicle), or vehicle alone (groups 1, 3, and 5), as a continuous infusion for 24 h beginning 7 days after insertion (s.c.) of blank (groups 1 and 2) or P4-containing (groups 3–6) implants. The effect of P4 withdrawal was assessed by removing P4-containing implants at the beginning of vehicle (group 5) or E2 (group 6) infusion. Gonadotrope responsiveness (increase in serum LH induced by 500 ng GnRH, i.v.) was assessed at the end of infusion. In a companion study, anterior pituitary tissue was collected at the end of the 24-h infusion period. Infusion of E2 increased (P<0·05) GR relative to GR noted in control wethers receiving vehicle alone. The magnitude of E2-induced augmentation of GR was not affected by concurrent administration of P4 or P4 withdrawal. Pituitary tissue concentrations of GnRH receptor and GnRH receptor mRNA were significantly reduced in wethers implanted with P4. This P4-induced decrease in tissue concentration of GnRH receptor and GnRH receptor mRNA was not reversed during the 24-h period after P4 withdrawal. Steady-state concentrations of GnRH receptor and GnRH receptor mRNA were significantly increased by E2. However, the magnitude of oestrogen-induced increase in tissue concentrations of GnRH receptor mRNA was not significantly affected by P4 or P4 withdrawal. Conversely, concurrent progestin stimulation potentiated the E2-induced augmentation of tissue concentrations of GnRH receptor. However, this P4-induced potentiation of the oestrogenic response was not evident 24 h after removal of the P4-containing implants. Steady-state concentrations of mRNA encoding the LHβ and FSHβ subunits were reduced (P<0·05) by P4. Infusion of E2 had a similar affect. These data indicate that prolonged progestin stimulation leads to a decrease in tissue concentrations of GnRH receptor and GnRH receptor mRNA. This P4-induced suppression of GnRH receptor activity is not reversed within 24 h of P4 withdrawal. In addition, the increase in steady-state concentrations of GnRH receptor and GnRH receptor mRNA induced by E2 is not compromised by concurrent progestin stimulation.

Journal of Endocrinology (1997) 152, 91–98
Effect of the protein phosphatase inhibitor okadaic acid on FSH-induced granulosa cell steroidogenesis
Authors: Reyes J González , Santana P , Robaina I González , Oliva J Cabrera , Estévez F , Hernández I , Blanco F López , Aguiar J Quintana , Fanjul L F , de Galarreta C M Ruiz
Published: December 2004

To address a possible role of type 1 and 2A serine/threonine protein phosphatases (PP1 and PP2A) in regulating granulosa cell hormonal responses, we investigated the effects of okadaic acid (OA) on FSH- and cAMP-induced steroidogenesis in these cells. When added alone (0·01–1 nmol/l), the cell-permeant phosphatase inhibitor did not affect progesterone and 3β-hydroxysteroid dehydrogenase/Δ5–4 isomerase (3β-HSD) enzyme activity, whereas when added with FSH it dose-dependently augmented (minimal effective dose, 0·1 nmol/l) gonadotropin-stimulated steroidogenesis in cultured granulosa cells. A similar stimulatory effect of the toxin was observed in cells cultured for 48 h with the cell-permeant analogue dibutyryl cAMP (1 mmol/l), or when granulosa cells were stimulated with the cAMP-inducing agents cholera toxin (1 μg/ml), forskolin (15 μmol/l) or 1-methyl-3-isobutyl-xanthine (0·1 mmol/l). The observed effect of OA on FSH-supported granulosa cell steroidogenesis was not a consequence of increased cAMP generation, and time course experiments also revealed that a minimal time period of 12 h was necessary for OA (0·1 and 1 nmol/l) to significantly enhance FSH-induced progesterone and 3β-HSD enzyme activity. Since OA also inhibits the dephosphorylation of protein kinase C (PKC) substrates, we also compared the effect of OA and the PKC activator 12-0-tetradecanoylphorbol-13-acetate (TPA) on FSH-induced granulosa cell steroidogenic activity. While activation of the PKC pathway with the tumor promoter TPA (10 nmol/l) inhibited progesterone and cAMP accumulation in FSH-stimulated granulosa cells, treatment with OA augmented steroidogenesis and did not affect gonadotropin-induced cAMP generation. Collectively these results suggest that PP1 and PP2A may be important in regulating the phosphorylation state of proteins implicated in the cAMP-protein kinase A-stimulated steroidogenic activity of these cells.

Journal of Endocrinology (1997) 152, 131–139
IGF-binding protein-6 is involved in growth inhibition in SH-SY5Y human neuroblastoma cells: its production is both IGF- and cell density-dependent
Authors: Babajko S , Leneuve P , Loret C , Binoux M
Published: December 2004

The IGF system is involved in the growth and differentiation of neuroblastoma cells, but the precise roles played by the IGF-binding proteins (IGFBPs) remain unknown. We have examined the expression and functions of IGFBPs produced by the neuroblastoma cell line, SHSY5Y, in the presence of: insulin, IGF-I, IGF-II, des(1–3)IGF-I (an IGF-I analogue with weak affinity for IGFBPs), acidic fibroblast growth factor, basic fibroblast growth factor, or nerve growth factor. Under basal conditions, SH-SY5Y cells in serum-free medium secreted IGF-II, and traces of IGF-I, IGFBP-2 and IGFBP-4. After 24 h of culture, comparative mitogenic potencies were: des(1–3)IGF-I>IGF-1>IGF-II>insulin. After 48 h, when IGFBP-2 and IGFBP-4 concentrations in the culture media had increased, des(1–3)IGF-I remained the most active, but the activity of insulin now equalled or exceeded that of IGF-I and IGF-II. This suggests a negative feedback mechanism involving partial sequestration of IGF-I and IGF-II by IGFBP-2 and IGFBP-4. At high cell density and with high concentrations of IGF-I, des(1–3)IGF-I (40 ng/ml) or IGF-II (80 ng/ml), the mitogenic activities of the IGFs diminished concomitantly with the appearance in the culture medium of an additional IGFBP identified as IGFBP-6, whose production depended on activation of the type 1 IGF receptor. These findings suggest that IGFBP-6 contributes as an autocrine inhibitor in the regulation of growth by the IGF system in these neuroblastoma cells.

Journal of Endocrinology (1997) 152, 221–227
Mitogenic actions of endothelin and other growth factors in ovine endometrium
Authors: Salamonsen L A , Young R J , Garcia S , Findlay J K
Published: December 2004

Endothelin-1 (ET-1) is present in ovine endometrium, primarily in epithelial cells, and increases around the time of implantation. We examined the cell type expressing ET-binding sites in vitro and whether ET-1 has mitogenic actions in the endometrium, alone or in synergy with other growth factors. Purified epithelial and stromal cells were prepared from luteal-phase endometrium. Specific receptors were demonstrated by binding of 125I-ET-1 and proliferative effects of ET-1 and/or other growth factors determined by uptake of [3H]thymidine by cells in serum-free culture. 125I-ET-1 bound to both epithelial (2516 ± 820 c.p.m./well) and stromal (6368 ± 1350 c.p.m./well) cells and was displaced by ET-1 (1 μmol l−1). There were no proliferative effects of ET on epithelial cells. ET-1 (10 nmol l−1) stimulated uptake of [3H]thymidine by stromal cells under serum-free conditions in 13/20 individual cell preparations, to 149 ± 13% of control (untreated=100%) with dose-dependence between the range of 1 to 100 nmol l−1. Stimulation by fetal calf serum was to 377 ± 126% of control. The effects on proliferation by other growth factors (dose; % of control ± s.e.m., number of positives/total number of cell preparations) were: IGF-I (13 nmol l−1; 182 ± 14, 4/4), epidermal growth factor (EGF; 4·8 nmol l−1; 132 ± 5%, 7/7), platelet-derived growth factor-BB (0·4 nmol l−1; 146 ± 3, 2/2) and leukaemia inhibitory factor (0·4 nmol l−1; 110 ± 2, 3/3). All stimulations except that of EGF were significant and dose-responsive but only insulin was additive with ET (350 ± 35, 5/5). ET-1 also stimulated expression of the the AP-1 cis element c-jun, this being maximal at 60 min of exposure to mitogen. ET-1, along with other growth factors has a likely paracrine role in cellular proliferation in the endometrium, possibly in association with blastocyst implantation.

Journal of Endocrinology (1997) 152, 283–290
Moderate food restriction reduces serum IGF-I and alters circulating IGF-binding protein profiles in lactating rats
Authors: Monaco M H , Donovan S M
Published: December 2004

The role of somatogenic and lactogenic hormones in the adaptative mechanisms which occur in response to nutrient restriction during lactation is unknown. To characterize the effect of food restriction during lactation on serum IGF-I, GH and prolactin concentrations and serum IGF-binding protein (IGFBP) profiles, lactating dams had free access to food (control) or were restricted to 60% of control intake during pregnancy and lactation (RPL) or only during lactation (RL). Serum, milk and mammary gland samples were collected throughout lactation. RL dams lost body weight, control dams gained weight, while RPL dams maintained body weight during lactation. By day 20, body and mammary gland weights of RL and RPL dams did not differ and were lower than control (P<0·05). Serum IGF-I concentrations in restricted groups were lower than control (P<0·05), however, hepatic expression of IGF-I mRNA did not differ between groups in early (day 1) or mid-lactation (day 8) and was increased on day 20 in RL dams compared with RPL or control. These data suggest that serum IGF-I and hepatic IGF-I mRNA expression are not co-ordinately regulated in the food-restricted lactating rat. In early lactation, serum IGFBP-3 was lower in RPL dams than control (P<0·05), whereas IGFBP-1 and -2 were increased in RL and RPL dams in late lactation compared with control. The decrease in IGFBP-3 and increase in lower molecular weight IGFBP may have contributed to the reduction in serum IGF-I by increasing IGF-I clearance from the circulation. Serum GH and prolactin were measured in samples obtained between 0900 and 1200 h. Serum GH did not differ with the exception of an increase on day 1 in control relative to RPL dams and on day 20 in RL dams relative to RPL and control. Serum prolactin was higher in the RL dams than controls on day 4. In summary, food restriction during pregnancy and lactation or solely during lactation results in similar reductions in serum IGF-I and alterations in serum IGFBP despite differences in body weight responses to food restriction during lactation.

Journal of Endocrinology (1997) 152, 303–316