bluuebunny/crossref_metadata_2025 huggingface dataset:
A double-staining technique for detection of growth hormone and insulin-like growth factor-I binding to rat tibial epiphyseal chondrocytes
Authors: Bentham J. , Ohlsson C. , Lindahl A. , Isaksson O. , Nilsson A.
Published: December 2008

In the present study a double-staining technique was developed to investigate simultaneous GH and insulin-like growth factor-I (IGF-I) binding to chondrocytes in a monolayer cell culture.

Rat tibial epiphyseal chondrocytes were isolated by enzymatic digestion and cultured in monolayer. GH and IGF-I were labelled with biotin. The affinity of the biotin-labelled ligands was compared with unlabelled ligands in a radioreceptor assay.

To study the distribution of GH and IGF-I binding in the monolayer, chondrocytes were incubated with biotinylated ligands with or without an excess of unlabelled ligands, followed by incubation with Vectastain ABC complex, which was then reacted with diaminobenzidine (DAB). Double staining was accomplished by carrying out the first reaction with DAB in the presence of nickel ammonium sulphate to give a black precipitate, followed by incubation with the second ligand, then ABC complex and finally DAB in the absence of nickel ammonium sulphate to give a brown stain. The presence of type-II collagen was demonstrated by immunohistochemistry and used as a marker for differentiated chondrocytes.

Biotin-labelled GH and biotin-labelled IGF-I exhibited dose-dependent displacements of 125I-labelled GH and 125I-labelled IGF-I respectively from the chondrocytes in a radioreceptor assay. The displacement curves were identical to those of unlabelled ligands indicating that the affinity was unaltered. Binding of biotinylated GH to cells was seen throughout the culture in regions where there was little or no type-II collagen staining. IGF-I binding was predominantly localized to cells at high density; areas which also showed a high degree of staining for type-II collagen.

The different locations of binding suggest that epiphyseal chondrocytes in monolayer culture comprise a heterogeneous cell population and that IGF-I and GH have different target cells.

Journal of Endocrinology (1993) 137, 361–367
Regulation of adipocyte precursor DNA synthesis by acidic and basic fibroblast growth factors: interaction with heparin and other growth factors
Authors: Butterwith S. C. , Peddie C. D. , Goddard C.
Published: December 2008

The development of adipose tissue is dependent on the growth and differentiation of fibroblast-like adipocyte precursor cells. Culture of adipocyte precursor cells in vitro has provided an ideal system for identifying potential regulators of proliferation and differentiation. We have demonstrated that both acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) stimulate chicken adipocyte precursor DNA synthesis in a dose-dependent manner up to a concentration of 100 μg aFGF/l and 1 μg bFGF/l. The effect of bFGF was biphasic, so that in incubations with 25 μg bFGF/l, DNA synthesis was not significantly different from controls. In the presence of heparin, stimulation of DNA synthesis at 25 μg bFGF/l was 1·6-fold greater than at a concentration of 1 μg bFGF/l. Addition of heparin to incubations containing aFGF reduced the concentration required for maximum stimulation of DNA synthesis to 1 μg/l. Cells incubated with aFGF (1–100 μg/l) in combination with insulin-like growth factor-I (IGF-I), platelet-derived growth factor, transforming growth factor-α or transforming growth factor-β1 (TGF-β1) exhibited a marked synergistic increase in DNA synthesis. This was also the case when 1 μg bFGF/l was used, but at a concentration of 25 pg bFGF/l synergy was only seen with IGF-I and TGF-β1. These results suggest that both basic and acidic FGF are potentially important regulators of adipocyte hyperplasia and that their effect is modulated by constituents of the extracellular matrix and the presence of other growth factors.

Journal of Endocrinology (1993) 137, 369–374
β-Cell tropin- and glucose-induced hypersecretion of insulin and amylin from perfused fatty rat pancreas
Authors: Dunmore S. J. , Cawthorne M. A. , Hislop D. C. J. , Morton J. L. , Beloff-Chain A.
Published: December 2008

The neurointermediate pituitary peptide β-cell tropin (BCT) has potent insulin-releasing and lipogenic properties and is elevated in obesity and type-2 diabetes. The effects of BCT and glucose on the release of insulin and amylin from the perfused pancreas of obese 'fatty' (fa/fa) rats and lean (Fa/?) controls were measured. Pancreata were perfused, sequentially, with buffer containing: 5·6 mmol glucose/l (basal); basal glucose±0·5 nmol BCT/l; 16·7 mmol glucose/l (high). Insulin and amylin release during basal glucose treatment was eight to nine times greater from pancreata from fatty than from lean rats. BCT induced a fivefold greater monophasic insulin and amylin release from fatty compared with lean pancreata. When not preceded by BCT there was a twofold greater high glucose-induced amylin release from fatty pancreata but no difference in insulin secretion. When preceded by BCT stimulation, high glucose induced twofold greater insulin and fourfold larger amylin release from fatty compared with lean pancreata. Molar secretion ratios of insulin: amylin varied between 30:1 and 50:1. In view of the elevated levels of BCT found in the fatty rat and in the light of the above findings, it is concluded that the peptide may have a role in the development of hyperinsulinaemia, hyperamylinaemia and insulin resistance in this animal model of obesity and diabetes.

Journal of Endocrinology (1993) 137, 375–381
In-vitro effects of mammalian and amphibian prolactins on hepatic vitellogenin synthesis in Rana esculenta
Authors: Carnevali O. , Mosconi G. , Yamamoto K. , Kobayashi T. , Kikuyama S. , Polzonetti-Magni A. M.
Published: December 2008

Male and female Rana esculenta liver was induced in an in-vitro system by homologous and Rana catesbeiana pituitary to synthesize and release vitellogenin, a lipoglycophosphoprotein precursor of yolk proteins, lipovitellins and phosvitins, in oviparous vertebrates.

In the present experiments, the action of prolactin on hepatic vitellogenin synthesis and release was investigated, using ovine prolactin and Rana catesbeiana prolactin. The effects of prolactin on hepatic vitellogenin synthesis displayed different trends related to sex; male liver was found to be more responsive than female liver to both ovine and frog prolactin; moreover, the response to prolactin was dose-related (r = 0·998; P <0·05) in male but not in female liver. In both sexes, a high degree of seasonality in the responsiveness of the liver was found, since the vitellogenin levels induced by prolactin during the winter phase were significantly (P < 0·001) higher than those produced during the summer phase. Thus, there was no significant difference between the action of ovine and frog prolactin on vitellogenin synthesis; in fact, mammalian prolactins are structurally similar with regard to nucleotide and amino acid sequences.

The direct action of prolactin on hepatic vitellogenin synthesis in the frog Rana esculenta is discussed, on the basis of the role played by prolactin as an important growth modulatory hormone in fetal and adult tissues.

Journal of Endocrinology (1993) 137, 383–389
Radioimmunoassay of FSH-suppressing protein in the ewe concentrations during the oestrous cycle and following ovariectomy
Authors: Klein R. , Findlay J. K. , Clarke I. J. , de Krester D. M. , Robertson D. M.
Published: December 2008

A sensitive and specific heterologous radioimmunoassay for FSH-suppressing protein (FSP or follistatin) was applied to ovine plasma. Following a logit–log dose transformation, parallel dose–response lines were observed between purified bovine 35 kDa FSP used as standard and serial dilutions of ewe plasma. Activin-A, inhibin-A and a range of other proteins showed low (<0·5%) cross-reactivity in the assay. Daily variations in the peripheral concentrations of FSP were measured across the ovine oestrous cycle. The peripheral concentrations of plasma FSP in adult ewes revealed a significant (P <0·01) increase (33%) during the luteal phase above follicular phase levels, peaking 10 days after the LH surge. FSP concentrations were determined in arterial and venous plasma from the ovary, head, kidney and liver. A significant (P <0·05) increase across the ovary was detected with no significant differences across the head, liver and kidney.

To investigate the relationship between gonadal FSP and the pituitary, ewes underwent ovariectomy and hypophysectomy. FSP levels rose (100–110%, P <0·01) during the period of surgery for both bilateral ovariectomy and sham ovariectomy, and then decreased significantly (37–44%) at 4–6 h after surgery. A further rise in plasma FSP (180–200% increase above pretreatment levels, P < 0·001) was observed 10–12 h after ovariectomy and sham ovariectomy. FSP levels then returned to preoperative levels during the following 26 h. Plasma FSP levels in long-term ovariectomized and hypophysectomized ewes were not significantly different from preoperative levels.

To determine whether the pattern of plasma FSP seen during the ovariectomy study was due to the effect of the induction of anaesthesia, ewes were treated with sodium thiopentone and halothane or 0·9% (w/v) NaCl by procedures of similar duration to that used during surgery. Both treatments resulted in an elevation of FSP levels (33–62%) over pretreatment values only at the time of induction of anaesthesia. To examine further whether this rise in plasma FSP observed after anaesthesia was due to a stress response and therefore under the control of the pituitary-adrenal axis, ewes were treated with ACTH, dexamethasone or saline only. A further group of sheep were exposed to a barking dog for 10 min. No change in FSP levels compared with pretreatment levels or saline-treated controls were noted following any of these treatments.

It was concluded that (1) FSP is present in the peripheral circulation; (2) ovariectomy contributes to plasma FSP, but no discernible contribution to circulating levels was evident from the head, liver or kidney; (3) plasma FSP levels rose significantly during the luteal phase of the ovine oestrous cycle; and (4) FSP secretion may be associated with a stress response, perhaps related to animal handling and intensive blood sampling through mechanisms that are as yet unclear.

Journal of Endocrinology (1993) 137, 433–443
Insulin-like growth factor-I-stimulated glucose transport in myotubes derived from chicken muscle satellite cells
Authors: Duclos M. J. , Chevalier B. , Le Marchand-Brustel Y. , Tanti J. F. , Goddard C. , Simon J.
Published: December 2008

The effects of insulin and insulin-like growth factor-I (IGF-I) on glucose transport were compared in myotubes derived from chicken breast muscle satellite cells in vitro. Myotubes were incubated (for 0·5 or 4 h) with or without glucose in the presence or absence of insulin or IGF-I. Glucose uptake was subsequently measured by the incorporation of 2-[1,2-3H(N)] deoxy-d-glucose ([3H]2DG) in glucose-free medium (10 min at 20 °C). Glucose uptake was almost completely abolished by the addition of cytochalasin B or phloretin. It was increased by a decrease in glucose concentration in the incubation medium. Insulin (5 mg/l) stimulated [3H]2DG uptake to a maximum of 43 ± 10% above basal after 30-min incubation and 101 ± 15% after 4-h incubation. IGF-I and insulin at equimolar concentrations (25 μg/l and 20 μg/l respectively) were almost equipotent after 0·5 h but after 4-h incubation IGF-I was 17-fold more potent, suggesting that this 'late' effect was mediated through the IGF-I receptor. Incubation with cycloheximide suggested that the effect of IGF-I involved increased protein synthesis. The results suggest that chicken myotubes express a glucose transporter which is regulated by IGF-I and glucose concentration. However, they do not appear to express a typical insulin-responsive transport system.

Journal of Endocrinology (1993) 137, 465–472
Effect of altering endogenous gonadotrophin concentrations on the kinetics of testicular germ cell turnover in the bonnet monkey (Macaca radiata)
Authors: Aravindan G. R. , Gopalakrishnan K. , Ravindranath N. , Moudgal N. R.
Published: December 2008

The role of FSH and diurnal testosterone rhythms in specific germ cell transformations during spermatogenesis were investigated using DNA flow cytometry and morphometry of the seminiferous epithelium of the adult male bonnet monkey (Macaca radiata), the endogenous hormone levels of which were altered by two different protocols. (1) Active immunization of five monkeys for 290 days using ovine FSH adsorbed on Alhydrogel resulted in the neutralization of endogenous FSH, leaving the LH and diurnal testosterone rhythms normal. (2) Desensitization of the pituitary gonadotrophs of ten monkeys by chronically infusing gonadotrophin-releasing hormone analogue, buserelin (50 μg/day release rate), via an Alzet pump implant (s.c.) led to a 60–80% reduction in LH and FSH as well as total abolition of testosterone rhythms. The basal testosterone level (3·3±2·0 μg/l), however, was maintained in this group by way of an s.c. testosterone silicone elastomer implant. Both of the treatments caused significant (P <0·01) nearly identical reduction in testicular biopsy scores, mitotic indices and daily sperm production rates compared with respective controls. The germ cell DNA flow cytometric profiles of the two treatment groups, however, were fundamentally different from each other. The pituitary-desensitized group exhibited a significant (P <0·001) increase in 2C (spermatogonial) and decrease in 1C (round spermatid) populations while S-phase (preleptotene spermatocytes) and 4C (primary spermatocytes) populations were normal, indicating an arrest in meiosis caused presumably by the lack of increment in nocturnal serum testosterone.

In contrast, in the FSH-immunized group, at day 80 when the FSH deprivation was total, the primary block appeared to be at the conversion of spermatogonia (2C) to cells in S-phase and primary spermatocytes (4C reduced by >90%). In addition, at this time, although the round spermatid (1C) population was reduced by 65% (P <0·01) the elongate spermatid (HC) population showed an increase of 52% (P <0·05). This, taken together with the fact that sperm output in the ejaculate is reduced by 80%, suggests a blockade in spermiogenesis and spermiation. Administration of booster injections of oFSH at time-points at which the antibody titre was markedly low (at days 84 and 180) resulted in a transient resurgence in spermatogenesis (at day 180 and 228), and this again was blocked by day 290 when the FSH antibody titre increased. Although in the two treatment groups overall spermatogenesis, as indicated by 1C:2C ratios, was significantly (P <0·01) reduced, the mechanisms by which this was brought about appeared to be different, suggesting that lack of FSH and testosterone (in particular the nocturnal testosterone surge) affect germ cell transformation at different foci.

Journal of Endocrinology (1993) 137, 485–495
Levels of insulin-like growth factor-binding protein-1 increase rapidly in amniotic fluid from 1 1 to 16 weeks of pregnancy
Authors: Wathen N.C. , Egembah S. , Campbell D.J. , Farkas A. , Chard T.
Published: December 2008

Concentrations of IGF-binding protein-1 (IGFBP-1) were measured by radioimmunassay in 176 amniotic fluid samples from 9 to 20 and 36 to 42 weeks of pregnancy. Low levels of IGFBP-1 were present at 9 and 10 weeks (median values 35.0 and 45.0 μg/l respectively). After 10 weeks, the levels increased by four orders of magnitude to reach a peak at 16 weeks (median 145.2 mg/l). After 16 weeks levels fell. Near term, the levels (median 27.1 mg/l) were lower than in the second trimester. The rapid increase in IGFBP-1 shown by radioimmunoassay was confirmed by Western ligand blotting. The findings suggest that the regulatory role of IGFBP-1 in the growth or differentiation of the fetus or of its surrounding membranes may change as pregnancy advances.
The involvement of insulin-like growth factor-I in local control of steroidogenesis and DNA synthesis of Leydig and non-Leydig cells in the rat testicular interstitium
Authors: Moore A. , Morris I. D.
Published: December 2008

Insulin-like growth factor-I (IGF-I) peptide, receptors and binding proteins are present in the rodent testis, which strongly implies that IGF-I has one or more testicular functions. In the present study we provide further information to support the concept that IGF-I is an important local mediator in the testis. High concentrations of IGF-I were measurable in interstitial fluid by radioimmunoassay, and IGF-I-binding proteins (IGFBPs) were readily detectable in interstitial fluid by ligand blotting, the predominant type being IGFBP-2. In vitro, IGF-I bound to testicular interstitial cells which did not have 3β-hydroxysteroid dehydrogenase (3β-HSD) activity and which were resistant to ethane dimethanesulphonate treatment. In vitro, IGF-I receptor-mediated actions increased both steroidogenesis and DNA synthesis. Insulin stimulated DNA synthesis at concentrations appropriate to cross-react with the IGF-I receptor, and this effect was greater in a testicular interstitial Leydig cell-depleted cell population compared with a Leydig cell-enriched cell culture. Furthermore, combinations of epidermal growth factor or transforming growth factor -α together with insulin appeared to act synergistically, causing extremely large increases in [3 H]thymidine incorporation in the interstitial cells. These results support a paracrine and/or autocrine role for IGF-I in interstitial cell growth and development.

Journal of Endocrinology (1993) 138, 107–114
Plasma measurements of corticotrophin-releasing hormone-binding protein in normal and abnormal human pregnancy
Authors: Perkins A. V. , Eben F. , Wolfe C. D. A. , Schulte H. M. , Linton E. A.
Published: December 2008

Using corticotrophin-releasing hormone-binding protein (CRHBP) purified from human plasma and a 25 amino acid peptide corresponding to the C-terminus of CRHBP we have been able to produce rabbit polyclonal antisera specific for CRHBP. This has allowed the development of a radioimmunoassay which is able to detect CRHBP specifically in human plasma regardless of the presence of endogenous CRH. We have used this assay to estimate the level of CRHBP in non-pregnant human plasma to be approximately 20 nmol/l with a range of 9·1–40·6 nmol/l. We have also examined sequential plasma samples taken from 84 normal pregnant women at fortnightly intervals from 16 weeks gestation through to term. Four women were also sampled during labour and the first week postpartum. The median plasma level of CRHBP at week 16 of normal pregnancy was 21·59 nmol/l, levels rose slightly during the early part of the third trimester (26·76 nmol/l at week 30, (P < 0·01) and fell markedly towards term (19·72 nmol/l, P < 0·01) with only 8·70 nmol/l at labour. CRHBP levels returned to normal non-pregnant levels within 48 h of parturition suggesting a role for the fetoplacental unit in CRHBP production. In eight pregnancies complicated by diabetes, CRHBP levels at each gestational age were similar to those recorded for normal pregnancy. However, in pregnancies complicated by pre-term labour (n = 9) and pre-eclampsia (n = 7), plasma CRHBP levels were significantly reduced (P < 0·01).

Journal of Endocrinology (1993) 138, 149–157
Effects of consumption of milk and milk constituents on plasma concentrations of gastric inhibitory polypeptide and metabolites in preruminant goat kids
Authors: Martin P. A. , Faulkner A. , McCarthy J. P.
Published: December 2008

Plasma concentrations of gastric inhibitory polypeptide (GIP) were measured in preruminant goat kids before and after consumption of milk, skimmed milk or solutions of milk fat, lactose, glucose or casein plus lactose. GIP concentrations increased significantly within 1 h of consumption of milk or milk fat, and were elevated for the remainder of the 5-h sampling period. The integrated mean change in GIP concentration during this period did not differ between these two meals. GIP levels were slightly increased above basal values 5 h after skimmed milk consumption, probably reflecting the absorption of a small amount of fat, but overall there was no significant GIP response to this or to any of the other test meals. The marked increase in GIP concentration after a milk feed indicates a physiological role for the hormone in preruminants but, in contrast to the situation in simple-stomached animals, carbohydrate absorption does not elicit GIP secretion in the preruminant goat. The data strongly suggest that fat is the major nutrient to stimulate GIP secretion in these animals.

Journal of Endocrinology (1993) 138, 167–173
Receptors for insulin-like growth factor-II in the growing tip of the deer antler
Authors: Elliott J. L. , Oldham J. M. , Ambler G. R. , Molan P. C. , Spencer G. S. G. , Hodgkinson S. C. , Breier B. H. , Gluckman P. D. , Suttie J. M. , Bass J. J.
Published: December 2008

Insulin-like growth factor-II (IGF-II) binding in the growing tip of the deer antler was examined using autoradiographical studies, radioreceptor assays and affinity cross-linking studies. Antler tips from red deer stags were removed 60 days after the commencement of growth, and cryogenically cut into sections. Sections were incubated with radiolabelled IGF-II, with or without an excess of competing unlabelled IGF-II and analysed autoradiographically. Radiolabelled IGF-II showed high specific binding in the reserve mesenchyme and perichondrium zones, which are tissues undergoing rapid differentiation and cell division in the antler. Binding to all other structural zones was low and significantly (P<0·001) less than binding to the reserve mesenchyme/perichondrium zones. Radioreceptor assays on antler microsomal membrane preparations revealed that the IGF-II binding was to a relatively homogeneous receptor population (Kd= 1·3 × 10−10 mol/l) with characteristics that were not entirely consistent with those normally attributed to the type 2 IGF receptor. Tracer binding was partly displaceable by IGF-I and insulin at concentrations above 10 nmol/l. However, affinity cross-linking studies revealed a single band migrating at 220 kDa under non-reducing conditions, indicative of the type 2 IGF receptor. These results indicate that, in antler tip tissues, IGF-II binds to sites which have different binding patterns and properties from receptors binding IGF-I. This may have functional significance as it appears that, whilst IGF-I has a role in matrix development of cartilage, IGF-II may have a role in the most rapidly differentiating and proliferating tissues of the antler.

Journal of Endocrinology (1993) 138, 233–241
The luteotrophic actions of prostaglandins E2 and F2α on dispersed marmoset luteal cells are differentially mediated via cyclic AMP and protein kinase C
Authors: Michael A. E. , Abayasekara D. R. E. , Webley G. E.
Published: December 2008

Progesterone production by dispersed luteal cells obtained from the marmoset monkey on day 14 after ovulation can be stimulated by both prostaglandin F2α (PGF2α) and its structural analogue, cloprostenol. To establish whether these responses can be attributed to cross-reaction with the prostaglandin E2 (PGE2) receptor, this study compared the involvement of cyclic adenosine-3′,5′-monophosphate (cAMP) and protein kinase C (PKC) in the luteotrophic responses to PGE2, PGF2α and cloprostenol. While all three prostaglandins stimulated similar increases in progesterone production (239·5 ± 7·9% of control; P <0·01), only PGE2 stimulated a significant increase in cAMP accumulation (373·2 ± 28·4% of control; P <0·01). This study is the first to demonstrate PKC activity in the marmoset ovary. Following down-regulation of PKC with a tumour-promoting phorbol ester, 4β-phorbol 12-myristate 13-acetate (4β-PMA), basal progesterone production was significantly increased (150·9 ± 8·2% of control; P <0·05) and the luteotrophic effects of PGF2α and cloprostenol were no longer evident, whereas the response to PGE2 was unaffected. These observations are consistent with the differential involvement of cAMP and PKC in the luteotrophic responses to PGE2 and PGF2α/cloprostenol respectively. Hence, we conclude that the luteotrophic actions of prostaglandins E2 and F2α on dispersed marmoset luteal cells are mediated via different receptors and signal transduction pathways.

Journal of Endocrinology (1993) 138, 291–298
Islet glucan-1,4-α-glucosidase: differential influence on insulin secretion induced by glucose and isobutylmethylxanthine in mice
Authors: Salehi A. , Lundquist I.
Published: December 2008

In previous in-vivo studies we have presented indirect evidence for the involvement of islet acid glucan-1,4-α-glucosidase (acid amyloglucosidase), a lysosomal glycogen-hydrolysing enzyme, in certain insulin secretory processes. In the present combined in-vitro and in-vivo investigation, we studied whether differential changes in islet acid amyloglucosidase activity were related to the insulin secretory response induced by two mechanistically different secretagogues, glucose and isobutylmethylxanthine (IBMX). It was observed that addition of the selective α-glucosidehydrolase inhibitor emiglitate (1 mmol/l) to isolated pancreatic islets resulted in a marked reduction of glucose-induced insulin release. This was accompanied by a pronounced suppression of islet activities of acid amyloglucosidase and acid α-glucosidase, whereas other lysosomal enzyme activities, such as acid phosphatase and N-acetyl-β-d-glucosaminidase, were unaffected. Furthermore, islets first incubated with emiglitate in the presence of high (16·7 mmol/l) glucose released less insulin than untreated controls in response to glucose in a second incubation period in the absence of emiglitate. In contrast, IBMX-induced insulin release was not influenced by emiglitate although accompanied by a marked reduction of islet activities of all three α-glucosidehydrolases. Basal insulin secretion (1 mmol glucose/1) was unaffected in the presence of emiglitate. In-vivo pretreatment of mice with highly purified fungal amyloglucosidase ('enzyme replacement'), a procedure known to increase islet amyloglucosidase activity, resulted in a greatly enhanced insulin secretory response to an i.v. glucose load. The increase in insulin release was accompanied by a markedly improved glucose tolerance curve in these animals. In contrast, enzyme pretreatment did not influence the insulin response or the blood glucose levels after an i.v. injection of IBMX. The data lend further support to our hypothesis that islet acid amyloglucosidase is involved in the multifactorial insulin secretory processes induced by glucose but not in those involving direct activation of the cyclic AMP system. The results also indicate separate, or at least partially separate, pathways for insulin release induced by glucose and IBMX.

Journal of Endocrinology (1993) 138, 391–400
Effect of high-protein feed supplements on concentrations of growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 in plasma and on the amounts of GH and messenger RNA for GH in the pituitary glands of adult rams
Authors: Clarke I. J. , Fletcher T. P. , Pomares C. C. , Holmes J. H. G. , Dunshea F. , Thomas G. B. , Tilbrook A. J. , Walton P. E. , Galloway D. B.
Published: December 2008

Three groups of mature rams were maintained on diets of hay, hay+2% lupin or hay+2% cowpea for 11 weeks. Serial blood samples were taken at 15-min intervals for 12 h for the determination of GH and IGF-I content by radioimmunoassay and for IGF-binding protein-3 (IGFBP-3) levels by Western blotting. The rams were killed after 77 days of supplementary feeding and their pituitary glands analysed for content of GH and GH mRNA. Mean plasma GH and baseline GH levels were significantly (P<0·01) decreased in the rams fed lupin and cowpea compared with controls fed hay and GH pulse amplitude was significantly (P<0·001) decreased in the group fed the cowpea diet. The frequency of GH pulses was not significantly altered by either treatment. Plasma concentrations of IGF-I were elevated in rams fed lupin (P<0·001) or cowpea (P<0·05). IGFBP-3 levels were not significantly (P>0·05) altered by either treatment. There were no significant differences in pituitary content of GH mRNA but pituitary content of GH was increased in rams fed lupin (P<0·05) and cowpea (P=0·07). In conclusion, a high-protein diet decreases plasma GH levels and increases IGF-I without changing plasma IGFBP-3 levels in rams. Thus ongoing synthesis of GH, as indicated by the mRNA levels, may cause a build up of GH stores in the pituitary gland.

Journal of Endocrinology (1993) 138, 421–427
Interleukin-1β induces nitric oxide production by a mouse pituitary tumour cell line (AtT20/D16)
Authors: Ohta K. , Hirata Y. , Imai T. , Marumo F.
Published: December 2008

To elucidate whether anterior pituitary cells express the nitric oxide (NO) synthase gene, we studied the synthesis of NO and the expression of NO synthase (NOS) mRNA by a mouse pituitary tumour cell line (AtT20/D16). Interleukin-1β (IL-1β) stimulated production of NO2−/NO3− (NOx) in a time-dependent manner and both NOx and cyclic GMP formation were stimulated in a dose-dependent manner by IL-1β. IL-1β-induced NOx production and intracellular cyclic GMP formation were similarly blocked by an NO synthase inhibitor, NG-monomethyl-l-arginine (LNMMA), whose effect was reversed by l-arginine, but not by d-arginine. Dexamethasone inhibited IL-1β-induced NOx production in a dose-dependent manner. A calmodulin inhibitor (W-7) showed no effect on IL-1β-induced NOx production, whereas cycloheximide and the actinomycin D completely inhibited NOx production. Northern blot analysis using cDNA for mouse macrophage-inducible NOS as a probe revealed the expression of inducible NOS mRNA in the cells only after exposure to IL-1β. Although IL-1β stimulated ACTH release from tumour cells, LNMMA failed to affect ACTH release stimulated by IL-1β. These results demonstrate for the first time that a pituitary tumour cell line (AtT20/D16) possesses cytokine-inducible and Ca2+/calmodulin-independent NOS, although NO may not be involved in ACTH release.

Journal of Endocrinology (1993) 138, 429–435
The uterine growth-promoting action of epidermal growth factor and its function in the fertility of mice
Authors: Tsutsumi O. , Taketani Y. , Oka T.
Published: December 2008

Epidermal growth factor (EGF) levels in the submandibular glands and plasma are increased in pregnant and aged female mice. The possible role of EGF in fertility was studied in virgin and pregnant mice ranging in age from 10 to 90 weeks of age, employing sialoadenectomy, administration of EGF antibody and EGF replacement. The uterine weight in pregnant, 10-week-old, sialoadenectomized mice was significantly less than in normal mice and the administration of EGF antibody to these mice further decreased uterine weight, resulting in an increased rate of abortion. Replacement EGF treatment in the sialoadenectomized mice prevented these changes. Uterine weight was about 70 mg at 10 weeks of age, and significantly increased from 30 to 80 weeks when it reached a plateau level of 275 mg. These changes closely followed the increase in the concentration of EGF in the submandibular glands and plasma and coincided with the decline in fertility. In contrast, uterine weight in the sialoadenectomized mice decreased immediately after the operation and remained at about 50–60 mg throughout the experimental period. Pregnancy, as judged by implantation, was achieved in the sialoadenectomized mice at later ages than in the controls. These findings suggest that elevated EGF levels may have a dual function in the control of fertility via uterine growth, depending on the age of mice.

Journal of Endocrinology (1993) 138, 437–443
Isolation and characterization of human LH isoforms
Authors: Stanton P. G. , Pozvek G. , Burgon P. G. , Robertson D. M. , Hearn M. T. W.
Published: December 2008

Thirty-nine human LH (hLH) isoforms were chromatographically separated from human pituitary extracts using a mild purification procedure which consisted of preparative isoelectric focusing, high-performance ion-exchange chromatography and immobilized metal-affinity chromatography. Twenty of these hLH isoforms were characterized by LH radioreceptor assay, SDS-PAGE and amino acid analysis, and 17 were shown to be highly purified (>90% pure). The specific activities of these hLH isoforms ranged from 1980 to 38 650 IU/mg protein in terms of the 2nd IS for human pituitary LH, based on protein content as determined by amino acid analysis. hFSH and hTSH content were <0·5% and <7·8% respectively. The purity was assessed by silver staining on SDS-PAGE. Under non-reducing conditions, a single band of apparent molecular mass 23·5–24·5 kDa was observed, whereas under reducing conditions the isoforms migrated as two distinct bands, 21·1–22·4 kDa and 18·0–20·5 kDa, probably corresponding to the α and β subunits of hLH respectively. The remaining three less pure isoform preparations (70–90% pure) contained additional bands of 16 kDa and 26·3 kDa under non-reducing conditions. All isoforms showed a low molecular mass band(s) of 11–14 kDa which was <7% of stained material as assessed by densitometry. Amino acid composition of the 17 hLH isoforms was similar to the published cDNA composition of hLH. Further fractionation of one hLH isoform (hLH IIc) on reversed-phase high-performance liquid chromatography yielded four peaks identified by N-terminal sequencing as two α and two β hLH subunits identical to their cDNA-derived N-terminal sequences. No additional sequences indicative of internal clipping of hLH were observed. The two pairs of α and β subunits probably represent two separate hLH isoforms in this preparation. It was concluded that a mild purification procedure with high recoveries for the isolation of intact hLH isoforms has been developed, and 17 isoforms of high purity suitable for further biological and physicochemical characterization have been isolated. These isoform preparations are free of other contaminating proteins, but may still contain multiple hLH-related species.

Journal of Endocrinology (1993) 138, 529–543
Effect of exogenous bovine growth hormone and ovariectomy on prepubertal mammary growth, serum hormones and acute in-vitro proliferative response of mammary explants from Holstein heifers
Authors: Purup S. , Sejrsen K. , Foldager J. , Akers R. M.
Published: December 2008

Sixteen prepubertal Holstein Friesian heifers were used to study the effect of long-term administration of bovine GH (bGH) on mammary development in intact and ovariectomized heifers. Eight heifers were ovariectomized at 2·5 months of age. Four intact and four ovariectomized heifers received subcutaneous injection of bGH (15 mg/day) for 15 weeks starting at 176 ± 3 days of age (147 ± 3 kg body weight), while the remaining eight heifers received an equal volume of excipient. Blood samples were collected weekly from 2 months of age. Heifers were slaughtered on the day after the last injection of bGH or excipient. Mammary gland development was quantified by dissection, chemical analysis and computer tomographic scanning. Mammary growth response at the time of slaughter was examined in cultures with explants prepared from parenchyma. Histological and histoautoradiographic studies with explants were performed.

Treatment with bGH resulted in a significantly (P<0·05) smaller mammary gland because of a reduced amount of extraparenchymal tissue. Ovariectomy markedly reduced the amount of parenchymal tissue. Growth response in mammary explants showed no treatment differences, suggesting that the decreased amount of parenchyma in ovariectomized heifers was caused by a decrease in mammary cell proliferation occurring some time prior to slaughter. The histological composition of mammary parenchyma was not changed by bGH treatment. However, ovariectomy resulted in less epithelial tissue (P<0·001) and lumen (P<0·05) and more stroma (P< 0·001), expressed as percentage tissue area. Serum hormone concentrations of bGH and insulin-like growth factor-I (IGF-I) were increased by bGH treatment in both intact and ovariectomized heifers. However, despite the fact that mammary growth in ovariectomized heifers was eradicated, the serum concentration of oestradiol was only decreased by one-third compared with intact heifers.

The study therefore confirms the importance of ovarian secretions for mammary growth and development in prepubertal heifers. However, the results give no clear evidence of an interaction between ovarian secretions and GH on the regulation of the development of the mammary parenchyma in heifers.

Journal of Endocrinology (1993) 139, 19–26
Ruminants express a prolactin receptor of Mr 33 000—36 000 in the mammary gland throughout pregnancy and lactation
Authors: Smith J. J. , Capuco A. V. , Mather I. H. , Vonderhaar B. K.
Published: December 2008

Developmental variation in the expression of the prolactin receptor in the ruminant mammary gland was investigated. Affinity chromatography revealed that bovine prolactin and human GH each bound to the same mammary gland proteins, yielding fractions enriched in binding activity and a protein of Mr 36 000, assumed to be a bovine prolactin receptor. Affinity cross-linking of 125I-labelled human GH to mammary microsomes confirmed that the Mr 36 000 protein was a bovine prolactin receptor. Binding assays of receptors in microsomes from the mammary tissue of cows and ewes at various stages of the lactational/reproductive cycle indicated developmental regulation of receptor concentration, but not receptor type, as no other bovine prolactin receptor type was detected by affinity cross-linking. These results suggest that differences in the response to prolactin in the mammary gland at various developmental stages in ruminants are not due to the expression of different forms of the prolactin receptor, and the lack of a prolactin effect on established lactation in ruminants is not due to the absence of the Mr 36 000 form of the prolactin receptor.

Journal of Endocrinology (1993) 139, 37–49
The effect of acute pharmacological manipulation of central serotonin neurotransmission on osmoregulated secretion of arginine vasopressin in the rat
Authors: Faull C. M. , Charlton J. A. , Butler T. J. , Baylis P. H.
Published: December 2008

To explore the hypothesis that serotonin (5-HT) is important in osmoregulated arginine vasopressin (AVP) secretion, we administered (i.p.) fluoxetine (FL) a 5-HT reuptake inhibitor (10 mg/kg body weight), ritanserin (RIT), an antagonist at the 5-HT2 and 5-HT1c receptor subtypes (1 mg/kg body weight), 1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride (DOI), a 5-HT2 receptor agonist (1 mg/kg body weight) or vehicle to rats 30 min before they were given an osmotic challenge. Rats received distilled water, normotonic saline (150 mmol NaCl/l) or hypertonic saline (500 mmol NaCl/l) (20 ml/kg i.p) and were killed 30 min later. The osmotic stimulus alone produced significant (P<0·001) effects on plasma osmolality and plasma sodium but FL, RIT and DOI did not have any significant effect on this stimulus. FL had no significant effect on the osmotic threshold of AVP release but significantly (P<0·001) increased basal AVP secretion from 1·6 ± 1·0 to 3·1 ± 1·3 (s.e.m.) pmol AVP/l and significantly (P<0·001) increased the AVP response to changes in plasma osmolality: vehicle-treated, 0·7 ± 0·4; FL-treated, 1·7 ± 0·2 pmol AVP/l per mOsm per kg. Neither RIT nor DOI had any significant effect on basal or stimulated AVP secretion. In a second study, RIT was administered 60 min i.p. prior to FL i.p. (doses as above), which was followed 30 min later by a hypertonic stimulus i.p. and rats were killed 30 min after hypertonic saline treatment. RIT had no significant effect on the AVP response to plasma osmolality and did not significantly alter the FL-augmented AVP response, suggesting that neither the 5-HT2 nor the 5-HT1c receptors are involved in the response of AVP to FL. We conclude that FL modulates osmoregulated AVP secretion but that the mechanism of this is unknown and is apparently not through the 5-HT2 or 5-HT1c receptor subtypes.

Journal of Endocrinology (1993) 139, 77–87
The relationship between the physicochemical properties and the biological effects of alloxan and several N-alkyl substituted alloxan derivatives
Authors: Munday R. , Ludwig K. , Lenzen S.
Published: December 2008

Alloxan causes diabetes in experimental animals through its ability to destroy the insulin-secreting B-cells of the pancreas. Alloxan is hydrophilic and chemically unstable; it is reactive toward thiols, undergoing redox cycling in the presence of glutathione and oxidizing protein-bound thiol groups, as reflected by inhibition of the thiol enzymes, hexokinase and glucokinase. It is apparently also selectively taken up by the GLUT-2 glucose transporter in the pancreatic B-cell membrane. In order to investigate which, if any, of these physicochemical properties are important in the toxic action of alloxan, we have examined seven N-alkyl substituted alloxan derivatives of various diabetogenic activity. Hydrophilicity was identified as a factor essential for diabetogenicity. Stability, rate of redox cycling and reactivity toward thiol groups were not correlated with diabetogenicity. Selective uptake by the GLUT-2 glucose transporter is not a prerequisite for the diabetogenicity of alloxan derivatives.

Journal of Endocrinology (1993) 139, 153–163
Effects of fasting, elevated plasma glucose and plasma insulin concentrations on milk secretion in women
Authors: Neville M. C. , Sawicki V. S. , Hay W. W.
Published: December 2008

In order to determine whether short term variations in plasma glucose and/or insulin influence milk lactose secretion in women, the effects of fasting and increased blood insulin and glucose on milk volume and composition were studied with glucose clamp methodology in exclusively and partially breast-feeding women. Twenty hours of fasting had no discernable effect on the output of milk or its macronutrient composition. Four hours of increased plasma insulin, studied under conditions where plasma glucose was maintained at the fasting level, had no effect on milk volume, milk glucose concentration, total fat content or lactose secretion rate. Increased plasma glucose, maintained at twice fasting levels for 4 to 6 h, produced a threefold increase in milk glucose concentration but had no significant effect on the rate of lactose synthesis. In partially breast-feeding women producing no more than 200 ml milk per day, a similar degree of hyperglycaemia increased milk glucose more than fourfold but did not significantly increase the milk secretion rate. It is concluded that human milk production is isolated from the homeostatic mechanisms that regulate glucose metabolism in the rest of the body, in part because the lactose synthetase system has a Km for glucose lower than the concentration available in the Golgi compartment.

Journal of Endocrinology (1993) 139, 165–173
Dopamine is taken up from the circulation by, and released from, local noradrenergic varicose axon terminals in zona glomerulosa of the rat: a neurochemical and immunocytochemical study
Authors: Vizi E. S. , Tóth I. E. , Orsó E. , Szalay K. Sz. , Szabó D. , Baranyi M. , Vinson G. P.
Published: December 2008

The effect of supramaximal electric field stimulation on [3H]dopamine (DA) release by rat adrenal capsuleglomerulosa preparations was studied using a microvolume perfusion system. When the tissues were preloaded with [3H]DA, a considerable amount of [3H]DA and [3H]noradrenaline (NA) were released in response to field stimuli. Reserpinization, calcium removal or tetrodotoxin blocking of Na+ influx all completely inhibited the stimulation-evoked release of DA/NA, indicating that the radioactivity released is of neuronal and vesicular origin. In the adrenal cortex, a substantial proportion of tyrosine hydroxylase and dopamine-β-hydroxylase immunoreactive nerve fibres and varicosities were observed around the zona glomerulosa. DA-containing nerves were not seen in the adrenal cortex; however, the same immunocytochemical procedures clearly demonstrated dopaminergic nerve cells and fibres in the substantia nigra and the striatum respectively, and cells of the adrenal medulla. Like the NA release from noradrenergic varicosities in the zona glomerulosa, the DA release from noradrenergic endings is not subject to negative feedback modulation through DA2 receptors since apomorphine, a DA2-receptor agonist, and sulpiride, a selective DA2-receptor antagonist, failed to affect the release. After in-vivo i.v. administration of [3H]DA, the glomerulosa content of DA and NA and the in-vitro release of [3H]DA and [3H]NA of zona glomerulosa both increased, indicating that the local varicose axon terminals were able to accumulate DA from the circulation, convert it into NA and release it in response to neural activity. This local arrangement of noradrenergic axon terminals, able to take up DA from the circulation and release it or convert it into NA, provides the possibility of a fine tuning of local circulation and aldosterone synthesis in the zona glomerulosa.

Journal of Endocrinology (1993) 139, 213–226
The inter-relationship between insulin and chromium in hyperinsulinaemic euglycaemic clamps in healthy volunteers
Authors: Morris B. W. , MacNeil S. , Stanley K. , Gray T. A. , Fraser R.
Published: December 2008

Evidence in the literature suggests that the trace element chromium may have a role in glucose homeostasis through the regulation of insulin action. We have previously reported a significant reduction in plasma chromium levels in healthy individuals, following a 75 g oral glucose load, and after meals and glucose-dependent uptake of chromium in insulindependent tissues in vitro. However, in vivo it is unclear whether the changes in plasma chromium relate to changes in plasma glucose or insulin.

The present study describes a series of euglycaemic hyperinsulinaemic clamps designed to attempt to define the initiator of changes in plasma chromium levels in ten healthy individuals.

The data showed a significant (P<0·01) reduction in fasting plasma chromium levels following glucose infusion and an initial bolus of insulin. Significant (P<0·02) increases in post-clamp urinary chromium excretion were insufficient to explain the decrease in plasma levels. During the recovery phase of an extended two-phase clamp protocol we found plasma insulin levels decreased by 70% within 10 min, associated with an increase in plasma chromium levels of 30% and no significant change in plasma glucose level.

These data indicate that alterations in plasma glucose are unlikely to be directly related to changes in plasma chromium, whilst supporting the hypothesis that plasma insulin may influence plasma levels of this trace element. In contrast, plasma zinc was unaffected throughout these clamp studies.

Journal of Endocrinology (1993) 139, 339–345