bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypothalamic-pituitary-adrenal activity and pro-opiomelanocortin mRNA levels in the hypothalamus and pituitary of the rat are differentially modulated by acute intermittent morphine with or without water restriction stress
Authors: Zhou Y , Spangler R , Maggos CE , Wang XM , Han JS , Ho A , Kreek MJ
Published: December 2004

Acute administration of morphine stimulates the secretion of hypothalamic-pituitary-adrenal (HPA) hormones, ACTH, beta-endorphin and corticosterone in the rat. In this study we investigated the effects of repeated multiple-dose morphine on HPA activity under two different conditions: without or with water restriction stress. Rats received six intermittent injections of morphine (6.25 mg/kg per injection, s.c.) every 2 h and were killed 30 min after the last injection. The results were as follows. (1) Morphine significantly elevated plasma ACTH and corticosterone levels; water restriction also significantly increased ACTH secretion, but with no significant increase of plasma corticosterone levels. In contrast, rats treated with morphine under the water restriction condition failed to show any increases of either ACTH or corticosterone levels. (2) Morphine did not change pro-opiomelanocortin (POMC) mRNA levels in the anterior pituitary; whereas water restriction significantly increased the POMC mRNA levels. The water restriction-induced increases of POMC mRNA in the anterior pituitary were absent in the rats which received morphine. (3) Morphine significantly increased POMC mRNA levels in the hypothalamus; water restriction had no effect. The morphine-induced increases in POMC mRNA in the hypothalamus were absent in the rat under the water restriction condition. These findings, that the effects of morphine on HPA activation or POMC mRNA expression depend on the presence of stress, suggest a counter-regulatory role of opiates on a stress response and opioid gene expression.
Effect of GHRH and peptides from the vasoactive intestinal peptide family on cAMP production of human cancer cell lines in vitro
Authors: Csernus V , Schally AV , Groot K
Published: December 2004

Antagonistic analogs of GHRH inhibit growth of various human cancers both in vivo and in vitro. To elucidate the mechanism of direct action of the antagonistic analogs of GHRH on tumor cells, cultured human cancer cells were exposed to GHRH, vasoactive intestinal peptide (VIP), secretin, glucagon, neuropeptide-Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), and VIP analogs in a superfusion system, and changes in cAMP and IGF-II release from the cells were measured. Various human cancer cell lines, such as mammary (MDAMB-468 and ZR-75-1), prostatic (PC-3), pancreatic (SW-1990 and Capan-2), ovarian (OV-1063), and colorectal (LoVo) responded to pulsatile stimuli with GHRH (0.5-20 nM), VIP (0.02-10 nM), and PACAP-38 (0.05-5 nM) with a rapid, transient increase in cAMP release from the cells. The VIP antagonist, PG-97-269, and the adenylate cyclase inhibitor, MDL-12330A, but not SQ-22536 or pertussis toxin, blocked the cAMP responses to these peptides. Stimulation of the cells with 100 nM secretin, glucagon or NPY did not alter the cAMP release. Our results suggest that GHRH receptors different from the type expressed in the pituitary are involved in mediating these effects. As cAMP is a potent second messenger controlling a wide variety of intracellular functions, including those required for cell growth, our results indicate that GHRH might have a direct stimulatory effect on growth of human cancers. Blockade of the autocrine/paracrine action of GHRH with its antagonistic analogs may provide a new approach to tumor control.
Desensitization and resensitization of lutropin receptors expressed in transfected Y-1 adrenal cells
Authors: Ulaner GA , Chuang J , Lin W , Woodbury D , Myers RV , Moyle WR
Published: December 2004

Stimulation of gonadal cells by lutropins such as human chorionic gonadotropin (hCG) is often transient and followed by down-regulation and/or desensitization of lutropin receptors (LHR). Here we describe desensitization/resensitization of LHR in Y-1 adrenal cell lines (termed Y-1L) expressing a rat cDNA lacking most 5' and 3' LHR untranslated regions under the control of a metallothionein promoter. Using a simple morphological assay in which stimulated cells are round and unstimulated cells are flat, we identified clones that rounded and remained round and others that became insensitive to lutropin stimulation and reverted to their flat appearance within 2-4 h. Flattened cells were insensitive to further hormonal stimulation but rounded after treatments with cholera toxin, forskolin, or cyclic AMP, showing that loss of responsiveness was associated with an early step in signal transduction, not loss of rounding potential. Removing the lutropin stimulus for at least 90-120 min reversed hormone insensitivity, even in the presence of the protein synthesis inhibitor puromycin. The number of surface bound receptors did not change during a cycle of rounding/flattening and hCG bound to rounded or flattened cells was replaced equally by radioiodinated hCG during incubations at 4 degrees C. Thus, desensitization/resensitization of LHR in Y-1L cells occurred in the absence of new receptor synthesis, receptor degradation, or receptor recycling. These observations suggest that LHR desensitization/resensitization in Y-1L cells was closely coupled to receptor occupancy and that this cell line may be useful for identifying factors that modulate the activities of occupied receptors.
GH regulates secretory activity and apoptosis in cultured bovine granulosa cells through the activation of the cAMP/protein kinase A system
Authors: Sirotkin AV , Makarevich AV
Published: December 2004

We have studied the action of GH on the production of hormones, growth factors, growth factor-binding protein and the occurrence of apoptosis in bovine ovarian granulosa cells, as well as the role of cAMP-stimulated protein kinase A (PKA) in the mediation of these effects. For this purpose we investigated the effects of exogenous bovine GH (0.001-10 microgram/ml), PKA blockers KT5720 (100 ng/ml) and adenosine-3',5'-monophosphothiodate (Rp-cAMPS) (1 micromol), alone and in combination, on IGF-I, IGF-binding protein (IGFBP)-3, oxytocin, progesterone and estradiol secretion, cAMP and PKA content and the occurrence of apoptosis.The secretion of hormones, IGF-I and IGFBP-3 into the culture medium was measured using RIA/IRMA. The presence of PKA was detected using immunocytochemistry and Western immunoblotting. The presence of cAMP in cells was demonstrated using immunocytochemistry, whilst the proportion of apoptotic cells was determined by the TUNEL method.It was found that the addition of GH to the culture medium strongly (P<0.05) stimulated IGF-I (at a concentration of 0.001-10 microgram GH/ml medium), IGFBP-3 (0.001-1 microgram GH/ml) and oxytocin (0.01-10 microgram GH/ml) secretion. Low concentrations (1-100 ng/ml) of GH stimulated, whilst a higher concentration (10 microgram/ml) inhibited estradiol output. GH slightly (P<0.05) inhibited progesterone (1-100 ng GH/ml) secretion and significantly (P<0.05) decreased the incidence of apoptosis (0.01-1 microgram GH/ml) in cultured cells. The addition of GH (100 ng/ml) caused a dramatic (P<0.05) increase in the proportion of cells possessing the immunoreactive catalytic subunit of PKA and a slight decrease in the proportion of cells containing the regulatory PKA subunit.PKA blockers KT5720 and Rp-cAMPS significantly (P<0.05) reduced the proportion of granulosa cells containing cAMP, and the catalytic and (in the case of KT5720) regulatory subunits of PKA. KT5720 given alone significantly (P<0.05) inhibited the secretion of IGFBP-3, but not that of IGF-I or progesterone. Rp-cAMPS decreased (P<0.05) the secretion of oxytocin but not that of estradiol output or the occurrence of apoptosis. KT5720 and Rp-cAMPS fully or partially prevented the GH effect on IGF-I, IGFBP-3, oxytocin, progesterone, estradiol and apoptosis.These observations suggest the involvement of GH and a cAMP/PKA-dependent intracellular cascade in the control of IGF-I, IGFBP-3, oxytocin, progesterone, estradiol, cAMP and apoptosis in bovine ovarian granulosa cells. The stimulation of PKA by GH and the prevention of GH-induced effects by PKA blockers suggest that the observed GH effects on bovine ovarian cells are probably mediated by the cAMP/PKA system.
Regulation of circulating levels of IGF-I in pregnant rats: changes in nitrogen balance correspond with changes in serum IGF-I concentrations
Authors: Nakago S , Funakoshi T , Ueda Y , Maruo T
Published: December 2004

Serum IGF-I concentrations in rats decrease significantly in late pregnancy. To determine if the reduction in serum IGF-I concentrations is attributable to circulating GH or maternal nutritional status, we investigated the effect of treatment with recombinant human GH (rhGH: 100 microgram/rat per day) on IGF-I concentrations during late pregnancy, and evaluated the relationship between maternal nitrogen balance and IGF-I concentrations. Serum IGF-I concentrations and maternal nitrogen balance ((nitrogen intake)-(nitrogen content in faeces and urine)-(nitrogen content in fetus and placenta)) were measured by RIA and the Dumas method. In non-pregnant rats treated with rhGH for 3 days, serum IGF-I concentrations (835.4+/-59.5 ng/ml; P<0.01) were significantly greater than in those animals treated with saline (319.6+/- 95.6 ng/ml). In the pregnant rats, however, there was no significant difference in serum IGF-I between those treated with rhGH (151. 1+/-43.0 ng/ml) and those treated with saline (142.0+/- 39.9 ng/ml) from day 17 to 19 of pregnancy. Maternal nitrogen balance in the pregnant rats increased significantly from day 4 to day 10 of pregnancy (169.5+/-57.4 and 196.1+/- 33.4 mg/day, respectively; P<0. 05) compared with non-pregnant controls (31.9+/-19.9 mg/day) and decreased markedly from day 12 of pregnancy (79.8+/-60.1 mg/day; P<0. 05) onwards, to 14.9+/-47.8 mg/day on day 20 of pregnancy (P<0.01), significantly different from the value on day 10 of pregnancy. The mean difference in maternal nitrogen balance between pregnant and non-pregnant rats was positively correlated (r=0.87, P<0.01) with the mean difference in maternal IGF-I concentrations, using linear regression analysis. These results support the conclusion that the circulating concentration of IGF-I in pregnant rats is associated with the change in nitrogen balance, but not with circulating GH.
Maternal hypothyroxinemia influences glucose transporter expression in fetal brain and placenta
Authors: Pickard MR , Sinha AK , Ogilvie LM , Leonard AJ , Edwards PR , Ekins RP
Published: December 2004

The influence of maternal hypothyroxinemia on the expression of the glucose transporters, GLUT1 and GLUT3, in rat fetal brain and placenta was investigated. Fetal growth was retarded in hypothyroxinemic pregnancies, but only before the onset of fetal thyroid hormone synthesis. Placental weights were normal, but placental total protein concentration was reduced at 19 days gestation (dg). Immunoblotting revealed a decreased abundance of GLUT1 in placental microsomes at 16 dg, whereas GLUT3 was increased. Fetal serum glucose levels were reduced at 16 dg. In fetal brain, the concentration of microsomal protein was deficient at 16 dg and the abundance of parenchymal forms of GLUT1 was further depressed, whereas GLUT3 was unaffected. Northern hybridization analysis demonstrated normal GLUT1 mRNA levels in placenta and fetal brain. In conclusion, maternal hypothyroxinemia results in fetal growth retardation and impaired brain development before the onset of fetal thyroid function. Glucose uptake in fetal brain parenchyma may be compromised directly, due to deficient GLUT1 expression in this tissue, and indirectly, as a result of reduced placental GLUT1 expression. Though corrected by the onset of fetal thyroid hormone synthesis, these deficits are present during the critical period of neuroblast proliferation and may contribute to long term changes in brain development and function seen in this model and in the progeny of hypothyroxinemic women.
Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells
Authors: Tetsuka M , Haines LC , Milne M , Simpson GE , Hillier SG
Published: December 2004

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.
Regulation of 11beta-hydroxysteroid dehydrogenase isoforms and glucocorticoid receptor gene expression in the rat uterus
Authors: Ho CK , Tetsuka M , Hillier SG
Published: December 2004

Glucocorticoids are known to have diverse effects on the uterus, generally believed to be mediated by the glucocorticoid receptor (GR). To date, two isoforms of the enzyme 11beta-hydroxysteroid dehydrogenase (11betaHSD) have been identified, namely 11betaHSD1 and 11betaHSD2, which interconvert active and inactive glucocorticoids and regulate local levels of hormones available to the GR in target tissues. The aim of the present study was to examine the uterine expression of 11betaHSD and GR mRNA. The interplay of these parameters is probably an important factor in determining actions of glucocorticoids on the uterus. Using Northern analysis we investigated the uterine expression of 11betaHSD1, 11betaHSD2 and GR mRNA in relation to serum levels of sex steroid hormones and uterine progesterone receptor mRNA expression in an animal model. Immature female rats were treated with 10 IU pregnant mare serum gonadotrophin (PMSG) followed by 10 IU human chorionic gonadotrophin (hCG) 48 h afterwards, and then killed at 0, 3, 6, 9, 12 and 24 h and 5 days after the hCG injection. Expression of both 11betaHSD1 and 11betaHSD2 mRNA in total uterine RNA was found to be up-regulated by more than 50% at 48 h after PMSG injection when oestradiol levels were also high. Following hCG treatment the expression of 11betaHSD1 and 11betaHSD2 further increased to reach maximal levels at 24 and 12 h respectively. GR mRNA expression was down-regulated by more than 50% by PMSG but gradually recovered after hCG injection. The results show that mRNA expression of 11betaHSD1, 11betaHSD2 and GR in the uterus is developmentally regulated, suggesting that these key determinants of glucocorticoid action may play an important role in uterine function.
Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe
Authors: Byrne B , McGregor A , Taylor PL , Sellar R , Rodger FE , Fraser HM , Eidne KA
Published: December 2004

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.
Inhibition of the secretion of LH in ovariectomised pigs by sustained but not repeated acute elevation of cortisol in the absence but not the presence of oestradiol
Authors: Turner AI , Hemsworth PH , Canny BJ , Tilbrook AJ
Published: December 2004

Prolonged stress is known to impair reproduction. It has been proposed that reproduction will also be impaired when a severe acute stress occurs during a period of elevated plasma concentrations of oestradiol, such as during the follicular phase of the oestrous cycle. In this experiment, we hypothesised that repeated acute and sustained elevation of cortisol would suppress the secretion of LH in ovariectomised pigs and that these effects would be enhanced in the presence of oestradiol negative feedback. Cortisol (or vehicle) was administered 12 hourly to ovariectomised pigs (n=6/treatment) for 8 days in the absence of oestradiol treatment and for a further 8 days during treatment with oestradiol. Vehicle was administered to 'control' pigs, 10 or 20 mg cortisol was administered i.v. to pigs to produce 'repeated acute' elevation of cortisol and 250 mg cortisol was administered i.m. to pigs to give a 'sustained' elevation of cortisol. Both before and during treatment with oestradiol, plasma concentrations of LH were monitored on the day before treatment, on the 4th and 8th days of treatment and following an i.v. injection of GnRH at the end of the 8th day of treatment. The repeated acute elevation of cortisol did not impair any parameters of LH secretion (i.e. mean plasma concentrations of LH, pulse amplitude or frequency, pre-LH pulse nadir or the LH response to GnRH) in the absence or in the presence of oestradiol. In contrast, when the elevation of cortisol was sustained, the mean plasma concentrations of LH and the pre-LH pulse nadir were significantly (P<0.05) lower on the 8th day of treatment than on the day before treatment and on the 4th day of treatment. Nevertheless, no other parameters of LH secretion were affected and these effects only occurred in the absence (not in the presence) of oestradiol. In conclusion, cortisol needed to be elevated for more than 4 days to impair the secretion of LH, and oestradiol did not enhance the impact of cortisol on LH secretion in ovariectomised pigs.
Transport of circulating IGF-I and LR3IGF-I from blood to extracellular wound fluid sites in rats
Authors: Bastian SE , Walton PE , Belford DA
Published: December 2004

Significant levels of IGF-I are found in wound fluid. The contribution that systemic IGF-I makes to the total IGF-I pool in wounds and the influence of IGF binding proteins (IGFBPs) on the delivery of systemic IGF-I to these wound sites has not been established. In the present series of experiments, we have shown that IGF-I flux across a model endothelial cell barrier is decreased in the presence of IGFBPs, whereas flux of Long R(3)IGF-I (LR(3)IGF-I, an IGF-I analogue with low affinity for IGFBPs) is unaffected. On the basis of these findings, the transport of IGF-I and LR(3)IGF-I from blood to extracellular wound fluid was assessed. Wound chambers were implanted subcutaneously in the backs of adult male rats and left in place for 14 days. A single i.v. bolus of either (125)I-IGF-I or (125)I-LR(3)IGF-I (10x10(6) c.p.m.) was administered via a jugular catheter and wound fluid and plasma samples taken at sequential time points between 5 and 240 min. (125)I-LR(3)IGF-I was removed from the circulation more rapidly than (125)I-IGF-I in both sham control and chamber implanted rats. Although implantation of the chambers did not alter the pharmacokinetic parameters of (125)I-IGF-I, significant increases in the steady state volume of distribution, clearance rate and half-life were recorded for (125)I-LR(3)IGF-I. In addition, significantly more intact (125)I-LR(3)IGF-I was recovered in wound fluid than (125)I-IGF-I at each time point, although only 0.08% of administered (125)I-LR(3)IGF-I was recovered per ml of wound fluid at 240 min. Compared with plasma, a greater proportion of wound fluid IGF-I radioactivity had distributed to the lower molecular weight IGFBPs or existed as free peptide. However, a small amount of wound fluid (125)I-IGF-I was detected in the 150 kDa region 30 min after injection. A greater proportion of (125)I-LR(3)IGF-I was associated with the lower molecular weight IGFBPs or existed as free peptide in both wound fluid and plasma. These data point to the importance of IGFBPs in determining the pharmacokinetic parameters of IGF-I in an extracellular fluid-expanded state. They also suggest only a minor role for endocrine IGF-I in surface wound repair.
Conversion of dehydroepiandrosterone to downstream steroid hormones in macrophages
Authors: Schmidt M , Kreutz M , Loffler G , Scholmerich J , Straub RH
Published: December 2004

Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal hormone with immunomodulatory effects such as inhibition of the production of monokines. Whether DHEA itself or the downstream steroids are the immunomodulatory effector hormones in target cells is not known. In this study, we investigated the conversion of DHEA to downstream steroid hormones in target macrophages. Within 1 day of culture with radiolabeled DHEA, monocyte-derived macrophages converted DHEA to significant amounts of Delta5-derivatives such as 16OH-DHEA, 3beta, 17beta-androstenediol (A'diol), and 3beta,16alpha, 17beta-androstenetriol (A'triol). However, the production of Delta4-steroids (androstenedione (A'dione), testosterone (T), and 16OH-T) and estrogens (estrone, estradiol, and estriol) was relatively low. Further cultivation of macrophages for 5 days with radiolabeled DHEA resulted in a significant (P<0.05) increase of the molar amounts of A'triol (P=0.012), 16OH-T (P=0.008), and estriol (P=0.003). In contrast to monocyte-derived macrophages, monocytes did not express aromatase mRNA, which was demonstrated by RT-PCR (P<0.01). Furthermore, DHEA in macrophages significantly inhibited one of the downstream converting enzymes, the aromatase, which was not demonstrated in the presence of the typical macrophage activator, lipopolysaccharide (LPS) (P<0.01). In conclusion, conversion of DHEA to physiologically relevant amounts of Delta5- and Delta4-steroids and estrogens was demonstrated in monocyte-derived macrophages. The conversion depends on maturation of monocytes and local factors such as the presence of LPS. The conversion of DHEA leads to an increase of downstream effector hormones in target macrophages which may be an important factor for local immunomodulation.
8-bromo-cyclicAMP stimulates glucose transporter-1 expression in a human choriocarcinoma cell line
Authors: Ogura K , Sakata M , Okamoto Y , Yasui Y , Tadokoro C , Yoshimoto Y , Yamaguchi M , Kurachi H , Maeda T , Murata Y
Published: December 2004

Facilitative glucose transporter-1 (GLUT1) is abundant in trophoblast cells and is responsible for glucose transport in the placenta. However, the change in GLUT expression in human placenta upon trophoblast differentiation remains to be clarified. Therefore, we first examined the localization of GLUT1 and GLUT3 using human first-trimester chorionic villi. We found that GLUT1 and GLUT3 were mainly localized to syncytiotrophoblast and cytotrophoblast cells respectively. We analyzed whether placental GLUT1 and GLUT3 expression changes during differentiation using a human choriocarcinoma (BeWo) cell line which is known to show functional and morphological differentiation in response to cAMP in culture. Treatment of BeWo cells with 8-bromo-cyclicAMP (8-bromo-cAMP) increased the level of hCG secretion and induced cell fusion leading to the formation of large syncytia. Treatment of BeWo cells with 8-bromo-cAMP also resulted in a significant increase in glucose uptake on days 2-3 of culture. The stimulating effect of 8-bromo-cAMP on glucose uptake was concentration dependent. Northern and immunoblot analyses revealed that the levels of mRNA and protein of GLUT1, but not of GLUT3, were significantly increased by 8-bromo-cAMP. These findings suggest that 8-bromo-cAMP stimulates GLUT1 expression with differentiation in BeWo cells.
Stimulation of glucose transport by thyroid hormone in 3T3-L1 adipocytes: increased abundance of GLUT1 and GLUT4 glucose transporter proteins
Authors: Romero R , Casanova B , Pulido N , Suarez AI , Rodriguez E , Rovira A
Published: December 2004

In 3T3-L1 adipocytes we have examined the effect of tri-iodothyronine (T(3)) on glucose transport, total protein content and subcellular distribution of GLUT1 and GLUT4 glucose transporters. Cells incubated in T(3)-depleted serum were used as controls. Cells treated with T(3) (50 nM) for three days had a 3.6-fold increase in glucose uptake (P<0.05), and also presented a higher insulin sensitivity, without changes in insulin binding. The two glucose carriers, GLUT1 and GLUT4, increased by 87% (P<0.05) and 90% (P<0. 05), respectively, in cells treated with T(3). Under non-insulin-stimulated conditions, plasma membrane fractions obtained from cells exposed to T(3) were enriched with both GLUT1 (3. 29+/-0.69 vs 1.20+/-0.29 arbitrary units (A.U.)/5 microg protein, P<0.05) and GLUT4 (3.50+/-1.16 vs 0.82+/-0.28 A.U./5 microg protein, P<0.03). The incubation of cells with insulin produced the translocation of both glucose transporters to plasma membranes, and again cells treated with T(3) presented a higher amount of GLUT1 and GLUT4 in the plasma membrane fractions (P<0.05 and P<0.03 respectively). These data indicate that T(3) has a direct stimulatory effect on glucose transport in 3T3-L1 adipocytes due to an increase in GLUT1 and GLUT4, and by favouring their partitioning to plasma membranes. The effect of T(3) on glucose uptake induced by insulin can also be explained by the high expression of both glucose transporters.
Endogenous vasopressin contributes to hypothalamic-pituitary-adrenocortical alterations in aged rats
Authors: Hatzinger M , Wotjak CT , Naruo T , Simchen R , Keck ME , Landgraf R , Holsboer F , Neumann ID
Published: December 2004

The ageing process in animals and humans is thought to be accompanied by a gradual impairment of corticosteroid receptor function, which is reflected by increased pituitary-adrenocortical hormone secretion at baseline and a number of aberrant neuroendocrine function test results. The latter include the ACTH and corticosteroid responses to a combined dexamethasone (DEX)/corticotropin-releasing hormone (CRH) challenge. The excessive hormonal response to this test among aged individuals has been taken as indirect evidence of enhanced endogenous arginine vasopressin (AVP) release, which - together with peripherally administered CRH - is capable of overriding DEX-induced ACTH suppression. The current study was designed to explore the role of endogenous AVP in mediating excessive hypothalamic-pituitary-adrenocortical (HPA) activity in ageing. The combined DEX/CRH test was administered to aged (22-24 months old) Wistar rats and the effect of the AVP type 1 (V1) receptor antagonist, d(CH(2))(5)Tyr(Me)AVP, on ACTH release was studied. Infusion of the V1 receptor antagonist after DEX pretreatment and before CRH administration prevented the CRH-induced rise in ACTH secretion in comparison with vehicle-treated aged rats (area under the concentration-time curve: 699+/-479 versus 2896+/-759; P<0.01). This difference was absent in young (3 months old) control rats. In situ hybridization showed an increased number of AVP mRNA-expressing neurons in the parvocellular but not the magnocellular, portion of the hypothalamic paraventricular nucleus in DEX-pretreated aged rats. The number and synthetic activity of parvocellular neurons expressing CRH mRNA was also increased. We have concluded that the increased HPA activity in aged rats involves enhanced synthesis and release of AVP from parvocellular neurons, possibly secondary to impaired corticosteroid receptor function.
Transforming growth factor-beta induces growth inhibition and IGF-binding protein-3 production in prostatic stromal cells: abnormalities in cells cultured from benign prostatic hyperplasia tissues
Authors: Cohen P , Nunn SE , Peehl DM
Published: December 2004

The IGF axis has been implicated in the pathogenesis of benign prostatic hyperplasia (BPH) via the paracrine action of IGFs and IGF-binding proteins (IGFBPs). In this study, we examined the regulation of cell growth and IGFBP-3 secretion by transforming growth factor-beta (TGF-beta) in prostatic stromal cell (PC-S) cultures from histologically normal tissues and tissues from BPH. PC-S cultures were treated with varying doses of TGF-beta1. Forty-eight hour conditioned media (CM) from these cultures were subjected to Western immunoblotting and ligand blotting for detection and quantification of IGFBPs. IGFBPs-2, -3 and -4 were detected in the CM from normal PC-S cultures. In CM from BPH PC-S, IGFBP-3 levels were 2-fold lower at baseline than in the normal PC-S CM, in addition to the differences in IGFBPs-2 and -5 which we have previously reported. In response to TGF-beta1, a 15-fold increase in the levels of IGFBP-3 was observed in normal PC-S CM, while a mere 2-fold increase was observed in BPH PC-S CM (P<0.001). These findings were confirmed by specific immunoblotting and immunocytochemistry. IGFBP-3 mRNA levels detected by Northern blotting of total RNA extracted from similar cultures showed the induction of IGFBP-3 expression by TGF-beta1 in normal PC-S and its lack of induction in BPH PC-S. Cell growth inhibition in response to TGF-beta1 correlated with the IGFBP-3 concentrations found in CM. Normal PC-S showed a 60% decrease in cell number after 10 days in media with 1 ng/ml TGF-beta1, compared with the untreated control. The decrease in proliferation observed in comparably treated BPH cells was only 20% (P<0.001). In conclusion, BPH PC-S had a reduced IGFBP-3 response to TGF-beta1 and demonstrated decreased TGF-beta1-induced growth inhibition relative to normal PC-S. We hypothesize that in normal PC-S, TGF-beta exerts its anti-proliferative effects by stimulating the production of IGFBP-3, which acts as an inhibitory factor, either by inhibiting IGFs or directly by interacting with cells, and that this process is altered in BPH PC-S.
High-resolution micro-computed tomography analyses of the abnormal trabecular bone structures in klotho gene mutant mice
Authors: Yamashita T , Nabeshima Y , Noda M
Published: December 2004

Inactivation mutation of the recently discovered klotho gene in mice causes a syndrome resembling aging. Manifestations include short life span, atherosclerosis, gonadal atropy, skin atropy, emphysema, ataxia and ectopic calcification. These mice also exhibit abnormally high bone density in the epiphyses of their tibiae based on X-ray and histological analyses. However, micro-structures of the trabecular bones in arbitrary two-dimensional planes or three-dimensional regions are difficult to analyze by these techniques. Therefore, we applied high resolution micro-computed tomography (microCT) to characterize the micro-structural abnormality in the trabecular bone in long bones as well as in vertebrae of four- to six-week-old klotho mutant mice. Two-dimensional microCT analyses in the mid-sagittal plane as well as three-dimensional microCT analyses indicated that the trabecular bone volume fraction measured in the proximal metaphyses of the tibiae was increased more than twofold in klotho mutant mice compared with the wild-type mice. Similarly, the trabecular bone area fraction in the mid-sagittal plane of the lumbar vertebral bodies was also increased by about 80% at the proximal and distal ends. No significant difference was observed with regard to the cortical thickness in the mid-shaft of femora between klotho mutant and wild-type mice. Three-dimensional microCT analyses also indicated that the trabecular number and thickness of the proximal metaphyses of the tibiae were increased by about 80% and 300% respectively in the klotho mutant mice, while trabecular separation was 60% less in klotho mutant mice compared with the wild-type mice. These quantitative microCT analyses indicate that the inactivation of klotho gene expression results in an increase in three-dimensional bone volume fraction, number and thickness of the trabecular bones in these mice.
The role of the StAR protein in steroidogenesis: challenges for the future
Authors: Stocco DM
Published: December 2004

The steroidogenic acute regulatory or StAR protein has been shown to be instrumental in the acute regulation of steroid hormone biosynthesis through its action in mediating cholesterol transfer to the inner mitochondrial membrane and the cholesterol side chain cleavage enzyme system. Since the time of its cloning in 1994, a number of studies have been performed which underscore the important role that this protein plays in steroidogenesis. While it is now quite apparent that StAR fulfills the criteria for the acute regulator as proposed by early studies, several crucial areas remain poorly understood. This list is topped by the so far intractable nature of the mechanism of action of StAR in transferring cholesterol to the P450scc enzyme. A second area which should prove to be of great interest is that of further understanding the regulation of the StAR gene which, like many genes, is quite complex. Lastly, with the recent demonstration of StAR being present in the brain, determining if StAR has a role in the synthesis of neurosteroids should prove to be of great importance.
ACTH treatment disrupts ovarian IGF-I and steroid hormone production
Authors: Viveiros MM , Liptrap RM
Published: December 2004

Hyper-adrenal activity and increased glucocorticoid hormone release are associated with disruptions in reproductive function and adverse effects on the ovary. The aim of this investigation was to determine whether elevated glucocorticoid hormone levels can influence ovarian IGF-I synthesis and action in vivo. To elevate endogenous glucocorticoid levels, gilts were treated with ACTH during the luteal phase of the oestrous cycle (days 9-13) while the control group received saline. The gilts were subsequently ovariectomized on either day 14 or day 18 of the oestrous cycle. Follicular fluid (FF) was collected from individual follicles; IGF-I and steroid hormone concentrations were determined by radioimmunoassay, and IGF-binding protein (IGFBP) expression was assessed by Western ligand blotting. Granulosa cells were also recovered and placed in culture to determine IGF-I, progesterone (P(4)) and oestradiol-17beta (E(2)) production levels. The cells were cultured in serum-free medium for 5 days and supplemented with: (a) media alone, (b) IGF-I, (c) FSH and androstenedione (A(4)), or (d) IGF-I with FSH and A(4). The FF from ACTH-treated gilts was characterized by elevated (P<0.05) cortisol levels on day 14 and lower (P<0.05) E(2) values on both day 14 and day 18. Lower (P<0.05) IGF-I concentrations were also measured in the FF of ACTH-treated gilts collected on day 18. This altered hormone profile in FF was associated with impaired IGF-I and steroid hormone synthesis by granulosa cells. IGF-stimulated P(4) production (P<0.01) by cells recovered from ACTH-treated gilts on day 14 was lower (P<0.05). By day 18, IGF-I, P(4) and E(2) production by cells from the ACTH group were all significantly (P<0. 05) lower. These results demonstrate that increased glucocorticoid concentrations can disrupt subsequent ovarian IGF-I synthesis and IGF action in vivo and can, potentially, impair follicle maturation.
hCGbeta core fragment is a metabolite of hCG: evidence from infusion of recombinant hCG
Authors: Norman RJ , Buchholz MM , Somogyi AA , Amato F
Published: December 2004

The availability of recombinant human chorionic gonadotrophin (r-hCG) has allowed us to measure its metabolic and renal clearance rates and to study the origin of the beta core fragment of hCG (hCGbetacf). Serum and urine samples were collected from six subjects, after an intravenous injection of 2 mg (equivalent to 44 000 IU Urinary hCG) r-hCG, and assayed for hCG and the beta subunit (hCGbeta). Urine from four of the subjects was also subjected to gel chromatography and assayed for hCGbetacf and hCG. r-hCG, administered as an intravenous dose, was distributed, initially in a volume of 3.4+/-0.7 l (mean+/-s.d.) and then in 6.5+/-1.15 l at steady-state. The disappearance of r-hCG from serum was bi-exponential, with an initial half-life of 4.5+/-0.7 h and a terminal half-life of 29.0+/-4.6 h. The mean residence time was 28. 6+/- 3.6 h and the total systemic clearance rate of r-hCG was 226+/-18 ml/h. The renal clearance rate was 28.75+/-6.2 ml/h (mean+/-s.d). hCGbetacf was detected in all urine samples collected at 6 h intervals. Over the 138 h period of urine collection, 12.9% (range 10.1-17.3% ) of r-hCG injected was recovered as the intact molecule and 1.7% (range 0.8-2.9%) was recovered as the hCGbetacf, in 4 subjects. The molar ratio of hCGbetacf to hCG in urine increased from 3.1+/-1.7%, on day 1, to 76+/-34.3% (mean+/-s.e.m.) on day 5, after r-hCG infusion, suggesting that hCGbetacf is a metabolic product of the infused r-hCG.
Stable overexpression of the glucose-6-phosphatase catalytic subunit attenuates glucose sensitivity of insulin secretion from a mouse pancreatic beta-cell line
Authors: Iizuka K , Nakajima H , Ono A , Okita K , Miyazaki J , Miyagawa J , Namba M , Hanafusa T , Matsuzawa Y
Published: December 2004

Glucose-6-phosphatase (G-6-Pase) hydrolyzes glucose-6-phosphate to glucose, reciprocal with the so-called glucose sensor, glucokinase, in pancreatic beta cells. To study the role of G-6-Pase in glucose-stimulated insulin secretion from beta cells, we have introduced rat G-6-Pase catalytic subunit cDNA and have established permanent clones with 3-, 7- and 24-fold G-6-Pase activity of the mouse beta-cell line, MIN6. In these clones, glucose usage and ATP production in the presence of 5.5 or 25 mM glucose were reduced, and glucose-stimulated insulin secretion was decreased in proportion to the increased G-6-Pase activity. In addition, insulin secretory capacity in response to d-fructose and pyruvate was unchanged; however, 25 mM glucose-stimulated insulin secretion and intracellular calcium response were completely inhibited. In the clone with 24-fold G-6-Pase activity, changes in intracellular NAD(P)H autofluorescence in response to 25 mM glucose were reduced, but the changes with 20 mM fructose and 20 mM pyruvate were not altered. Stable overexpression of G-6-Pase in beta cells resulted in attenuation of the overall glucose-stimulated metabolic responses corresponding to the degree of overexpression. This particular experimental manipulation shows that the possibility exists of modulating glucose-stimulated insulin release by thoroughly altering glucose cycling at the glucokinase/G-6-Pase step.
Transmembrane protein tyrosine phosphatase IA-2 (ICA512) is expressed in human midgut carcinoids but is not detectable in normal enterochromaffin cells
Authors: Cunningham JL ,   Lopez-Egido, Janson ET , Eriksson B , Oberg K , Gobl AE
Published: December 2004

A potential upregulation of receptor type protein tyrosine phosphatase IA-2 (ICA512) expression was detected by differential display and investigated in midgut carcinoid tumours. Normal intestine tissue and tumour tissue from 13 midgut carcinoid patients were studied by in situ hybridisation using an IA-2 ribonucleotide probe and confocal microscopy using specific IA-2 antibodies. Previously, it had been shown that IA-2 is located in the secretory granules of virtually all neuroendocrine cells. However, we found that IA-2 was not detectable in resting normal enterochromaffin (EC) cells of the small intestine, while high expression of IA-2 mRNA and protein was confirmed in both primary and metastatic carcinoid tissue. This difference in expression was not observed with chromogranin A or serotonin, two secretory granule hormones known to be expressed in EC cells, indicating that IA-2 was seemingly not necessary for the basal production and packaging of these hormones. When comparing patients receiving biotherapy before operation with untreated patients, we found expression of IA-2 to be lower in tumours from patients that had been treated with a combination of alpha-interferon and the somatostatin analogue, octreotide. There was no correlation between IA-2 expression and proliferation rates as measured by immunohistochemistry with antibodies against the Ki 67 antigen. Furthermore, we show that IA-2 is co-localised with serotonin in carcinoid tumours as well as in the pancreatic tumour cell line, BON1, which is interesting as serotonin secretion rate is presumably higher in tumour cells than in resting EC cells. Taken together, these findings may indicate a role for IA-2 in the later stages of the regulated secretory process.
Melatonin administration can entrain the free-running circadian system of blind subjects
Authors: Lockley SW , Skene DJ , James K , Thapan K , Wright J , Arendt J
Published: December 2004

Although melatonin treatment has been shown to phase shift human circadian rhythms, it still remains ambiguous as to whether exogenous melatonin can entrain a free-running circadian system. We have studied seven blind male subjects with no light perception who exhibited free-running urinary 6-sulphatoxymelatonin (aMT6s) and cortisol rhythms. In a single-blind design, five subjects received placebo or 5 mg melatonin p.o. daily at 2100 h for a full circadian cycle (35-71 days). The remaining two subjects also received melatonin (35-62 days) but not placebo. Urinary aMT6s and cortisol (n=7) and core body temperature (n=1) were used as phase markers to assess the effects of melatonin on the During melatonin treatment, four of the seven free-running subjects exhibited a shortening of their cortisol circadian period (tau). Three of these had taus which were statistically indistinguishable from entrainment. In contrast, the remaining three subjects continued to free-run during the melatonin treatment at a similar tau as prior to and following treatment. The efficacy of melatonin to entrain the free-running cortisol rhythms appeared to be dependent on the circadian phase at which the melatonin treatment commenced. These results show for the first time that daily melatonin administration can entrain free-running circadian rhythms in some blind subjects assessed using reliable physiological markers of the circadian system.
Estrogen treatment in vivo increases keratinocyte growth factor expression in the mammary gland
Authors: Pedchenko VK , Imagawa W
Published: December 2004

Proliferation and differentiation of mammary epithelia are regulated by the combined action of systemic hormones and locally derived paracrine growth factors. Keratinocyte growth factor (KGF) is a potential candidate stromal factor that may participate in the hormonal control of stromal/epithelial interactions. In this study, we have examined the in vivo effect of 17beta-estradiol (E) treatment on KGF expression in mammary glands of peripubertal (5-week-old) and mature (11-week-old) mice. Mice received subcutaneous injections of hormone after which KGF mRNA levels were assayed by ribonuclease protection analysis of mammary gland RNA. E treatment caused a dose- and time-dependent increase in KGF mRNA levels in intact mice from both age groups. Neither 17alpha-estradiol nor progesterone injection affected KGF mRNA levels. Comparison of the relative expression of KGF in parenchyma-free fat pads and in intact glands demonstrated that the basal and E-dependent KGF mRNA levels did not require the presence of mammary epithelium. ELISA assay of KGF tissue content demonstrated that concomitantly with an up-regulation of mRNA, E treatment also increased KGF protein in mammary glands from peripubertal and mature mice. These data show that E treatment stimulates both KGF mRNA and protein expression in mammary stroma in vivo and raises the possibility that KGF has a role in E-regulated mammary gland development.
Expression of ErbB receptors during pancreatic islet development and regrowth
Authors: Kritzik MR , Krahl T , Good A , Gu D , Lai C , Fox H , Sarvetnick N
Published: December 2004

We have characterized expression of the ErbB receptor family and one of its ligands, heregulin, in an effort to identify molecules associated with pancreatic development and regeneration. In addition to studying expression during fetal pancreatic development, we have also studied expression during pancreatic regeneration in the interferon-gamma (IFNgamma)-transgenic mouse, which exhibits significant duct cell proliferation and new islet formation. These studies demonstrate significant expression of the ErbB2, ErbB3, and ErbB4 receptors, in addition to heregulin isoforms, in the developing murine fetal pancreas. We also report significant ductal expression of these proteins during IFNgamma-mediated pancreatic regeneration. This striking expression was absent in 1-week-old neonates, but was clearly visible in pups by 5 weeks of age. These data therefore indicate that ErbB receptor and ligand expression decline by birth in both the IFNbeta-transgenic and non-transgenic mice, and that expression resumes early in postnatal life in the IFNbeta-transgenic mice. The expression of ErbB receptor family members at sites of islet development and regrowth suggests that these molecules might be relevant to these processes.