bluuebunny/crossref_metadata_2025 huggingface dataset:
Adrenaline, insulin and glucagon do not have acute effects on plasma leptin levels in sheep: development and characterisation of an ovine leptin ELISA
Authors: Kauter K , Ball M , Kearney P , Tellam R ,   McFarlane
Published: December 2004

Leptin, a recently discovered hormone secreted mainly from adipose tissue, was first described as a regulator of adiposity, food intake and energy metabolism. It is now apparent that leptin physiology is much more complex and is likely to play an important role in many other systems including reproduction, haematopoiesis and immunity. Leptin levels have been shown to be well correlated with body fat in both humans and rodents, with administration of exogenous leptin to rats and mice resulting in loss of body fat. Leptin is, therefore, likely to be an important humoral signal to the central nervous system on body composition and regulation of food consumption. Due to the limited cross-reactivity of leptin from other species in the current assays for leptin, physiological research on leptin has, to a large extent, been restricted to rodents and humans. The aim of this study was to develop a leptin immunoassay suitable for use with sheep, enabling the investigation of the basic physiology of leptin in an animal larger than rats or mice, thus allowing repeated blood sampling. Using this assay we investigated the short-term effects of insulin, adrenaline and glucagon (all modulators of blood glucose) on plasma leptin levels. Antiserum to bovine recombinant leptin (brLeptin) raised in chickens was used to develop a competitive ELISA. Using brLeptin as standard, the assay has a sensitivity of 0. 5 ng/ml with inter- and intra-assay variation of 15% and 7% respectively. The cross-reactivity of human recombinant leptin was 36.5%, while mouse leptin showed no cross-reactivity. Plasma samples from ewes, male castrate animals and rams (n=4-5) diluted in parallel to the standard with mean leptin concentrations of 6.0+/-2. 9, 3.3+/-0.4 and 3.1+/-1.3 ng/ml respectively. Leptin levels in rams were significantly lower than in ewes. The non-significant difference in leptin levels between rams and male castrate animals suggests that testosterone may not be responsible for the lower levels of leptin. Four groups of 3-4 ewes were given intravenous insulin (1 iu/kg), adrenaline (65 microg/kg), glucagon (24 iu/kg) or saline. Blood samples were taken at 1, 3, 5, 10, 20, 30, 60, 90 and 120 min after injection. As expected, glucose levels declined within 10 min of the insulin injection and rose after 3 min following both adrenaline and glucagon injections. Leptin levels, however, remained relatively unchanged for the 2 h following the treatments. Finally, a bolus intravenous dose of glucose (240 mg/kg) was given and sequential blood samples taken. Despite plasma glucose levels rising to over 200 mg/dl, leptin levels did not significantly change over the three hours following treatment. These data indicate that plasma leptin levels in sheep, in contrast to rodents, are not responsive to short-term changes in blood glucose or insulin, as has been shown in humans.
IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland
Authors: Adam CL , Gadd TS , Findlay PA , Wathes DC
Published: December 2004

Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P<0.01), and LHRH alone (200+/-16.1%, P<0.001); there was no additive or subtractive effect of LHRH and IGF-I given together. Thus, an intrapituitary IGF system exists in sheep and the present results are consistent with an endocrine role for IGF-I in nutritional modulation of LH secretion at the level of the pituitary gland.
Primary cultured hepatocytes of the bony fish, Oreochromis mossambicus, the tilapia: a valid tool for physiological studies on IGF-I expression in liver
Authors: Schmid AC , Reinecke M , Kloas W
Published: December 2004

In spite of the importance of IGF-I for growth and development, knowledge about regulation of its production in submammalian species is rather limited. In order to create a tool for investigation of direct regulatory effects on the expression of IGF-I in bony fish liver, a primary cell culture of hepatocytes from Oreochromis mossambicus, the tilapia, was established. The cells were viable for up to 3 days and IGF-I mRNA synthesis was detected by northern blot and semiquantitative reverse transcriptase (RT)-PCR. Northern blot analysis of the primary cultured hepatocytes revealed four different IGF-I transcripts, 0.5, 1.9, 3.9 and 6.0 kb in size, which were identical to those in liver tissue. However, the expression rate was weaker than that in liver. The direct effects of recombinant tilapia (rt) growth hormone (GH) and salmon (s) IGF-I on the expression of IGF-I in primary cultured hepatocytes were investigated in time-course and dose-response experiments. In untreated cultures, IGF-I mRNA decreased with time. Hepatocytes treated with 100 nM rtGH resulted in a pronounced stimulation of IGF-I mRNA expression throughout the experiment. Treatment with rtGH in concentrations ranging from 0.1 nM to 1 microM caused a clear dose-dependent increase in the amount of IGF-I mRNA. Significant stimulation was obtained even with 0.1 nM, reaching a plateau with 10 nM. Neither significant inhibitory nor stimulatory effects were detected by adding sIGF-I from 0.1 nM to 1 microM to the hepataocytes. Our results indicate that the established primary cell culture of tilapia hepatocytes is a useful system in which to study direct effects of potential regulators of bony fish liver cell function.
Effects of infantile thymectomy on ovarian functions and gonadotrophin-induced ovulation in prepubertal mice: role of thymulin
Authors: Garcia L , Hinojosa L , Dominguez R , Chavira R , Rosas P
Published: December 2004

The effects of thymectomy performed on 10-day-old (Tx-10) mice on spontaneous puberty and the ovulatory response induced by gonadotrophin treatment were analysed, together with the effects of thymulin replacement from 10 days of age. Infantile thymectomy induced a delay of puberty, a decrease in serum 17beta-oestradiol concentration and a reduced total number of follicles. Injection of thymulin (12 ng/g body weight) to Tx-10 mice resulted in an earlier onset of puberty, a decrease in the weights of ovaries and uterus, and an increase in serum 17beta-oestradiol concentrations. In control and Tx-10 mice, treatment with pregnant mare serum gonadotrophin (PMSG) (5 IU) at 25 days of age resulted in ovulation and the numbers of ova shed by ovulating animals were similar. When the animals were injected with 1 IU PMSG ovulation did not occur. In Tx-10 mice, both 1 and 5 IU PMSG increased the number of follicles to values similar to those observed in the controls. In Tx-10 mice the sequential injection of PMSG (1 IU) and human chorionic gonadotrophin (hCG) (3 IU) resulted in ovulation, but the number of ova shed was lower than in controls. When these animals were injected daily with thymulin, an increase in the number of ova shed and serum 17beta-oestradiol concentrations was observed. The uterine weight of Tx-10 mice was always significantly reduced in response to gonadotrophin treatment. Thymulin injection in PMSG-hCG-treated Tx-10 mice provoked a significant increase in uterine weight. The results suggest that the presence of the thymus after the neonatal period is necessary to normal ovarian development and function. The increase in gonadotrophin-induced ovarian response produced by thymulin replacement indicates that this peptide has a role in this process as one of the connecting signals between thymus and ovaries.
Novel cardiac peptide hormone in several teleosts
Authors: Tervonen V , Ruskoaho H , Vuolteenaho O
Published: December 2004

To find out the significance of the newest member of the natriuretic peptide family, salmon cardiac peptide (sCP), we have determined the distribution of the peptide and its mRNA as well as the tissue and plasma molecular forms in several teleosts. Using probes based on the salmon sCP cDNA in Northern blot analysis we found mRNA homologous to that of sCP to be present in the heart of 15 fish species representing nine different genera. We developed a specific RIA for the salmon 29 amino acid peptide to be able to study the distribution of the peptide in the heart and plasma of different fish species. Despite the probable interspecies differences in the peptide sequence, large quantities of immunoreactive sCP were found in the atrium, ventricle and plasma of most of the fish species studied, suggesting that a cardiac hormone homologous to sCP has an endocrine function in a large variety of teleost species. The molecular form of the hormone secreted and stored in the tissue was determined by gel filtration high pressure liquid chromatography. In salmon, as in most of the other fish species studied, the predominant immunoreactive sCP in plasma corresponded to the low molecular weight form, with a size similar to that of the biologically active 29 amino acid sCP (sCP-29), whereas the form stored in the heart corresponded to the high molecular weight pro-sCP-sized material. The form secreted by isolated perfused salmon ventricle, in the basal state as well as when mechanically loaded, was the sCP-29-sized peptide, thus ruling out the possibility that the conversion from high to low molecular weight material is caused by plasma proteases. In conclusion, sCP-like peptides are produced and secreted from the heart of a large number of different fish species. Their post-translational processing appears to be remarkably similar to that of mammalian atrial natriuretic peptide.
alpha-MSH acetylation in the pituitary gland of the sea bream (Sparus aurata L.) in response to different backgrounds, confinement and air exposure
Authors: Arends RJ , Rotllant J ,   Metz, Mancera JM , Wendelaar Bonga SE , Flik G
Published: December 2004

MSH is a pituitary hormone derived by post-translational processing from POMC and involved in stress and background adaptation. N-terminal acetylation of MSH to monoacetyl alpha-MSH or diacetyl alpha-MSH increases the bioactivity of the peptide. The aim of this study was to characterize alpha-MSH acetylation in the sea bream (Sparus aurata L.) pituitary gland in response to the stressors air exposure and confinement, as well as in fish adapted for 15 days to a white, gray or black background. Pituitary homogenates were purified by reversed-phase HPLC (RP-HPLC). The alpha-MSH content of fractions was measured by RIA. Immunoreactive RP-HPLC fractions were further analyzed by electrospray mass spectrometry and the peptide sequence determined as SYSMEHFRWGKPV-NH2. In the pituitary gland of sea bream, des-, mono- and diacetyl alpha-MSH were identified. Then plasma alpha-MSH levels were measured in sea bream adapted to different backgrounds. Surprisingly, we found the highest plasma alpha-MSH levels in white-adapted as compared with black-adapted sea bream with intermediate values for gray-adapted fish. This observation is in contrast with results that have been obtained in eel, trout or terrestrial vertebrates. Next, des-, mono- and diacetyl alpha-MSH forms were measured in homogenates of the pituitary gland and in plasma of sea bream exposed to air, to confinement, or to different backgrounds. Monoacetyl alpha-MSH was the predominant form in all control and experimental groups. The lowest content of monoacetyl alpha-MSH relative to des- and diacetyl alpha-MSH was found in white-adapted fish. Levels of des- and diacetyl alpha-MSH forms were similar under all conditions. We observed that monoacetyl alpha-MSH is the most abundant isoform in the pituitary gland after background adaptation, confinement and air exposure, in sea bream. These data indicate that the physiologically most potent isoform of alpha-MSH may vary from species to species.
Expression and regulation of connexin43 in rat Leydig cells
Authors: You S , Li W , Lin T
Published: December 2004

Gap junctions are intercellular protein channels which provide a pathway for the exchange of ions and small molecules. This exchange of materials allows metabolic coupling of cells. Gap junction channels are made up of connexins, integral membrane proteins encoded by a multigene family. Rat testes contain mRNAs for at least five different connexins: Cx26, Cx32, Cx33, Cx37 and Cx43. Immunocytochemical studies have shown that Cx43 assembles gap junctions between Leydig cells. The present study investigated the expression and regulation of the Cx43 gene in rat Leydig cells. Purified Leydig cells were obtained from 40- to 80-day-old Sprague-Dawley rats using a combination of arterial perfusion, collagenase digestion, centrifugal elutriation and Percoll gradient centrifugation. Leydig cells from 20- and 30-day-old rats were isolated without arterial perfusion or centrifugal elutriation. Cx43 mRNA was present in 20-day-old rat Leydig cells, reached a plateau at day 40, and remained at high levels in 65- and 80-day-old rat Leydig cells. To evaluate the regulation of Cx43 gene expression, Leydig cells were cultured overnight and then treated with human chorionic gonadotropin (hCG) for variable periods of time. Addition of hCG (10 ng/ml) increased cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein mRNA levels and testosterone formation. However, Cx43 mRNA levels were inhibited by hCG in a time- and dose-dependent manner. Cx43 mRNA levels decreased 27% as early as 2 h after the addition of hCG and decreased 60% by 24 h. Treatment of Leydig cells with 8-bromo-cAMP (0.1 mM) for 6 and 24 h also reduced Cx43 mRNA levels by 36 and 56% respectively. Primary cultured Leydig cells stained strongly positive with anti-Cx43 monoclonal antibody. Treatment with hCG for 24 h reduced Cx43 signals and caused Cx43 to redistribute to the periphery of the cells. To evaluate the regulation of Cx43 in vivo, rats were treated with hCG (300 ng i.p.) and testes were removed 24 h later. Frozen section of testes revealed that these interstitial cells stained positive for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by histochemical staining and were positive for Cx43 by immunofluorescence staining. The adjacent seminiferous tubules stained only weakly positive for Cx43. Twenty-four hours after hCG treatment, 3beta-HSD activity increased while Cx43 immunostaining of Leydig cells was reduced. In conclusion, gap junction channels of Leydig cells are regulated by hCG both in vivo and in vitro. hCG increased Leydig cell steroidogenesis and steroidogenic enzyme mRNA levels but caused a redistribution of Cx43.
Exclusive androgenic effect of dehydroepiandrosterone in sebaceous glands of rat skin
Authors: Sourla A , Richard V , Labrie F , Labrie C
Published: December 2004

In order to analyze the hormonal effects of dehydroepiandrosterone (DHEA) in skin sebaceous glands, the precursor steroid was administered to ovariectomized (OVX) female Sprague-Dawley rats at a dose of 30 mg applied on the dorsal skin, twice daily, for 3, 6 and 12 months. In a parallel experiment, female OVX rats were treated with DHEA at the same daily percutaneous dose of 30 mg, alone or in combination with the antiandrogen Flutamide or the pure antiestrogen EM-800, for 12 months, in order to determine the androgenic and/or estrogenic components of DHEA action. Treatment of female OVX rats with DHEA resulted in a similar mild to moderate hyperplasia of the sebaceous glands of both dorsal (site of application) and ventral skin, as illustrated by an increase in the number and size of the acini. The above-indicated effects were observed at all time intervals studied, beginning at 3 months of treatment, and they were not further increased after longer term administration of DHEA (for 6 and 12 months). The addition of Flutamide to DHEA treatment completely prevented the DHEA-induced changes in the sebaceous glands, whereas the antiestrogen EM-800 had no effect. The present data indicate an exclusive androgenic stimulatory action of DHEA on the sebaceous glands, thus pointing out the importance of local intracrine DHEA transformation into androgens for skin anatomical integrity and function, while showing that estrogens, if active in rat skin, do not originate from DHEA.
Lanosterol 14alpha-demethylase (CYP51), NADPH-cytochrome P450 reductase and squalene synthase in spermatogenesis: late spermatids of the rat express proteins needed to synthesize follicular fluid meiosis activating sterol
Authors: Majdic G , Parvinen M , Bellamine A ,   Harwood HJ, Ku WW , Waterman MR , Rozman D
Published: December 2004

Lanosterol 14alpha-demethylase (CYP51) is a cytochrome P450 enzyme involved primarily in cholesterol biosynthesis. CYP51 in the presence of NADPH-cytochrome P450 reductase converts lanosterol to follicular fluid meiosis activating sterol (FF-MAS), an intermediate of cholesterol biosynthesis which accumulates in gonads and has an additional function as oocyte meiosis-activating substance. This work shows for the first time that cholesterogenic enzymes are highly expressed only in distinct stages of spermatogenesis. CYP51, NADPH-P450 reductase (the electron transferring enzyme needed for CYP51 activity) and squalene synthase (an enzyme preceding CYP51 in the pathway) proteins have been studied. CYP51 was detected in step 3-19 spermatids, with large amounts in the cytoplasm/residual bodies of step 19 spermatids, where P450 reductase was also observed. Squalene synthase was immunodetected in step 2-15 spermatids of the rat, indicating that squalene synthase and CYP51 proteins are not equally expressed in same stages of spermatogenesis. Discordant expression of cholesterogenic genes may be a more general mechanism leading to transient accumulation of pathway intermediates in spermatogenesis. This study provides the first evidence that step 19 spermatids and residual bodies of the rat testis have the capacity to produce MAS sterols in situ.
Exogenous GH infusion to late-gestational fetal sheep does not alter fetal growth and metabolism
Authors: Bauer MK , Harding JE , Breier BH , Gluckman PD
Published: December 2004

The role of GH in the regulation of fetal growth and metabolism in late gestation is not well defined. The aim of this study was to determine the effects of exogenous GH infusion on fetal growth and feto-placental metabolism in the normally growing late-gestation fetal sheep. Eleven fetuses received pulsatile GH infusion (3.5 mg/day) for 10 days while 12 control fetuses received vehicle. The GH infusion was given as a continuous infusion (2.5 mg/day) plus an additional pulsatile component (30 pulses equivalent to 1 mg/day) designed to mimic the natural pattern of GH secretion. Fetal GH infusion raised the circulating fetal concentrations of GH threefold, but did not change fetal concentrations of IGF-I, IGF-binding protein-3, insulin or ovine placental lactogen. GH-treated fetuses had blood urea concentrations 15% lower than controls (P<0.05) and glucose uptake 18% lower per kg fetal weig! ht (P=0.06). There were no other differences attributable to fetal GH infusion in feto-placental metabolism, placental function or placental blood flow. GH-treated fetuses were larger than controls at postmortem (weight+13%, P<0.01; girth+5%, P<0.01; crown-rump length+3%, P<0.05). However, there were no differences between groups in measures of fetal growth (increment in chest girth and hindlimb length). GH-treated fetuses had heavier mothers and when maternal weight was included as a covariate in the analysis, there was no significant difference between treatment groups that could be attributed to GH treatment. GH infusion to normal fetal sheep does not appear to have a significant effect on feto-placental metabolism or fetal growth.
Evidence of a role for neuropeptide Y and monoamines in mediating the appetite-suppressive effect of GH
Authors: Wang X ,   Day, Zhou Y , Beard JL , Vasilatos-Younken R
Published: December 2004

Among the many responses to GH administration is suppression of voluntary feed intake (FI) in some species, attributed to improvement in the efficiency of nutrient utilization and, therefore, reduced need for ingested substrates. Commercial broiler chickens have been genetically selected for generations for rapid growth, realized largely via the major correlated response of increased voluntary feed consumption. Neuropeptide Y (NPY) and monoamines play very important roles in the central regulation of feeding. Preliminary studies from our laboratory suggest that the appetite-suppressive effect of GH may be independent of its actions as a repartitioning agent, and may involve alterations in NPY expression at the pre-translational level. The purpose of this investigation was to explore the dose-response nature of the appetite-suppressive effect of GH in juvenile broilers, and the possible involvement of NPY and monoamines in this process. A GH dose-response study was conducted using 8-week-old female broilers infused i.v. with GH in a pulsatile pattern for 7 days at 0, 10, 50, 100 or 200 microgram/kg body weight per day. Hypothalamic NPY and epinephrine (EP) concentrations decreased in a dose-related manner with GH. At the highest dosage, voluntary FI decreased 19% (P<0.05) and hypothalamic NPY mRNA decreased approximately 50% in the infundibular nuclei and midline region (P<0.0001). In contrast, birds pairfed to the high-GH dosage group did not differ from controls, verifying that changes in NPY and monoamines were not secondary to reduced FI. We conclude that hypothalamic NPY and EP are likely candidates to explore further as mediators of the appetite-suppressive effect of GH.
Vasoactive intestinal polypeptide- and pituitary adenylate cyclase activating polypeptide-mediated control of catecholamine release from chromaffin tissue in the rainbow trout, Oncorhynchus mykiss
Authors: Montpetit CJ , Perry SF
Published: December 2004

The aim of the present investigation was to assess the relative contributions of cholinergic (acetylcholine) and non-cholinergic vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase activating polypeptide (PACAP) neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue lining the posterior cardinal vein of the rainbow trout (Oncorhynchus mykiss). Using an in situ saline-perfused posterior cardinal vein preparation, it was demonstrated that exogenous administration of chicken VIP or human PACAP-27 caused a dose-dependent increase in adrenaline secretion; noradrenaline secretion was unaffected. Analysis of dose-response curves indicated that VIP and PACAP stimulated the secretion of adrenaline with a similar degree of potency (ED(50) for VIP=1.90x10(-11) mol/kg; ED(50) for PACAP=1.03x10(-11) mol/kg). The VIP/PACAP-elicited secretion was diminished in the presence of the VIP receptor antagonist, VIP 6-28, but was unaffected by the PACAP receptor antagonist, PACAP 6-27, or the cholinergic antagonists, hexamethonium and atropine. Thus, this is the first study to demonstrate a direct stimulatory role for VIP or PACAP in catecholamine secretion from piscine chromaffin cells. The relative contribution of cholinergic and non-cholinergic neurotransmitters in the neuronal control of catecholamine secretion from the chromaffin tissue was evaluated using an in situ nerve-stimulating technique previously validated by us in the rainbow trout. This was accomplished by comparing catecholamine secretion in the presence or absence of cholinergic and the VIP and PACAP receptor antagonists during different levels of electrical stimulation. The results demonstrated that cholinergic stimulation predominated during high frequency of electrical stimulation (20 Hz) while the non-cholinergic component prevailed at low frequency (1 Hz). Overall, the results of the present investigation demonstrate that VIP and/or PACAP may directly stimulate adrenaline secretion from trout chromaffin cells at low levels of neuronal activity. Therefore, the neuronal control of catecholamine secretion in teleosts may not be confined to cholinergic-evoked events.
Octylphenol does not mimic diethylstilbestrol-induced oestrogen receptor-alpha expression in the newborn mouse uterine epithelium after prenatal exposure
Authors: Nielsen M , Hoyer PE , Lemmen JG , van der Burg B , Byskov AG
Published: December 2004

This study examined whether the endocrine disruptor octylphenol (OP) mimics the synthetic oestrogen diethylstilbestrol (DES) in ability to induce oestrogen receptor-alpha (ER-alpha) expression in the newborn mouse uterine epithelium after prenatal exposure. Pregnant mice were given daily s.c. injections with DES (10 or 100 microgram DES/kg maternal wt) or OP (100 or 250 mg/kg maternal wt) or with vehicle alone from day 11.5 to 16.5 of pregnancy. ER-alpha expression was evaluated on histological sections by detecting ER-alpha mRNA with the in situ hybridization technique and ER-alpha protein using immunohistochemistry. The immunostaining was quantitated using a microspectrophotometer. Oestrogen-like activity of the DES and OP batches used for in vivo exposure was confirmed in an in vitro assay based on transient gene expression of an oestrogen-dependent reporter plasmid. In mice exposed prenatally to vehicle alone, the uterine epithelium did not express either ER-alpha mRNA or protein, while both were highly expressed in the stroma. Exposure to either DES dose induced the expression of both ER-alpha mRNA and protein in the epithelium, whereas it was unchanged in the stroma. In contrast, neither OP dose induced the expression of ER-alpha mRNA or protein in the epithelium and expression was unchanged in the stroma. Our data stress the importance of in vivo studies when investigating endocrine disruptors.
Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues
Authors: McKillop AM , McCluskey JT , Boyd AC , Mooney MH , Flatt PR , O'Harte FP
Published: December 2004

Previous studies have shown that glycation of insulin occurs in pancreatic beta-cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limpet haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I(125)-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED(50)) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity <19 pg/ml) was 0. 08+/-0.01 and 1.5+/-0.6 ng/ml (P<0.01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemistry studies of the pancreas of streptozotocin-treated diabetic rats using a 1:1000 dilution of guinea-pig 2 antiserum revealed clusters of fluorescent positively stained cells in islets. These studies document the successful production of polyclonal antisera specific for glycated insulin and their usefulness in radioimmunoassays and immunocytochemistry. The demonstration of glycated insulin in plasma and islets of animal models of diabetes supports the view that glycation of insulin is involved in the pathogenesis of this disease.
Increased levels of clusterin (SGP-2) mRNA and protein accompany rat ventral prostate involution following finasteride treatment
Authors: Astancolle S , Guidetti G , Pinna C , Corti A , Bettuzzi S
Published: December 2004

Finasteride is a well-known inhibitor of the prostatic enzyme 5 alpha-reductase type 2 which prevents conversion of testosterone into 5 alpha-dihydrotestosterone, the active intraprostatic androgen, which causes prostate involution through a combination of cell atrophy and cell death. The drug is widely used to improve symptoms of benign prostatic hyperplasia in man. Clusterin, a glycoprotein which is generally up-regulated under conditions inducing cell atrophy or organ involution, is produced at a high level in the regressing rat ventral prostate following androgen ablation. According to several authors, clusterin does not respond to finasteride treatment, suggesting a different action of testosterone and 5 alpha-dihydrotestosterone. We show here that, under our conditions, finasteride was capable of inducing production of both clusterin mRNA and protein in the rat ventral prostate. In fact, by using different and converging techniques, such as Northern hybridization, in situ hybridization histochemistry and immunohistochemistry, we were able to show a strong induction of the clusterin gene in the epithelial cell population of the gland. The response to finasteride, which was similar to that seen with castration, occurred with a delay of a few days. In situ and immunohistochemistry experiments indicated that both orchidectomy and finasteride administration resulted in increased transition of the epithelial cells from the columnar to the cuboidal (atrophic) shape, and this was accompanied by an increased intensity of the signal for clusterin. Thus, it appears that induction of clusterin is part of the molecular process leading to prostate involution caused by either orchidectomy or finasteride administration.
Post-surgical recovery and time-of-day mask potentiated responses of ACTH to repeated moderate hemorrhage in conscious rats
Authors: Lilly MP , Jones RO , Putney DJ , Carlson DE
Published: December 2004

We determined how changes in the responsiveness of the hypothalamo-pituitary-adrenal (HPA) system that accompany experimentation affect facilitation of HPA responses to hemorrhage. Hemorrhage (10 ml/kg over 3 min) was performed in conscious, chronically prepared rats. Blood was sampled over 1 h followed by reinfusion of shed blood. Hemorrhage was performed either once or twice separated by 24 h in different groups of animals. To test the effect of the circadian variation in responsiveness, rats were hemorrhaged on days 4 and 5 after surgery either in the morning (AM) or in the afternoon (PM). The response of ACTH to hemorrhage on day 4 was greater in the PM than in the AM (P<0.01). The ACTH response to the second hemorrhage on day 5 was greater than that to hemorrhage on day 4 only in the AM group (P<0.01). Thus, facilitation of ACTH responses by prior hemorrhage was evident only in the AM. To determine the effects of surgical recovery, additional experiments were done in the AM either early (days 3 and 4) or later (days 6 and 7) after surgery. In these experiments, hemorrhage was performed in all rats on days 4 and 7 and either hemorrhage or blood sampling alone was performed on day 3 and 6. ACTH did not increase in rats with sampling and no hemorrhage. ACTH increased more after an initial hemorrhage on day 3 than on day 6 (P<0.01). ACTH response to hemorrhage on day 4 was greater when preceded by hemorrhage vs sampling on day 3 (P<0.01). ACTH response to hemorrhage in rats bled twice did not differ on day 3 and day 4. On day 7, the response of ACTH in rats that had hemorrhage on day 6 was greater than both their own response on day 6 and the response of a control group with sampling on day 6 (P<0.01). These results demonstrate potentiation of ACTH responses to hemorrhage by an earlier similar hemorrhage, but clearly indicate that enhanced sensitivity of the HPA to hemorrhage either by circadian factors or by surgery can mask this effect.
Beneficial effects of retinoic acid on extracellular matrix degradation and attachment behaviour in follicular thyroid carcinoma cell lines
Authors: Havekes B , Schroder van der Elst JP , van der Pluijm G , Goslings BM , Romijn JA , Smit JW
Published: December 2004

The prognosis of patients with metastasised follicular thyroid carcinoma (FTC) is limited, necessitating the search for new treatment options. Beneficial effects of retinoids have been suggested in thyroid cancer and the present study was performed to investigate the effects of retinoic acid (RA) on important determinants of metastatic behaviour in FTC: the disengagement of tumour cells from the primary tumour and the degradation of extracellular matrix, focusing on the role of the plasmin activation system and the integrin and E-cadherin families of attachment molecules. Three FTC cell lines were studied: FTC-133, derived from the primary tumour; and FTC-236 and FTC-238, derived from metastases. FTC cell lines were cultured with 0.1, 1 and 10 microM 13-cis-RA or with the solvent DMSO for 1 and 5 days. Extracellular matrix degradation by these cell lines was studied by assessing the 48-h release of radioactivity from (35)S-methionine labelled extracellular matrix proteins synthesised by the MC3T3 cell line coated onto plastic. The involvement of constituents of the plasmin activation system was investigated by semi-quantitative RT-PCR and zymography. Attachment to extracellular matrix was studied by determining the number of adhering FTC cells to extracellular matrix coated onto plastic, 3 h after seeding. The involvement of attachment molecules was studied by RT-PCR with primers for integrin subclasses and E-cadherin and immunofluorescence for E-cadherin. Five days culturing with 10 microM RA reduced the degradation of extracellular matrix significantly in all cell lines: FTC-133 by 35%, FTC-236 by 74% and FTC-238 by 31%. Zymography revealed diminished activity of urokinase type plasminogen activator (uPA) in FTC-236 and FTC-238, but not in FTC-133 cultured with RA. mRNA expression of the uPA receptor was diminished in FTC-236. In the attachment assay, 10 microM RA for 5 days increased the number of adherent cells to extracellular matrix significantly by 91% in FTC-133, 64% in FTC-236 and 87% in FTC-238. No effects of RA on integrin or E-cadherin mRNA expression were observed. Immunofluorescence, however, revealed enhanced organisation of E-cadherin along the cell membrane by RA treatment. In conclusion, the present study demonstrates beneficial effects of RA on important determinants of metastatic behaviour in FTC cell lines, e.g. decreased degradation of extracellular matrix which may in part be explained by effects on the plasmin activation system and enhanced attachment to extracellular matrix. These findings may add to the explanations for beneficial effects of retinoids in thyroid cancer.
Dimethoate inhibits steroidogenesis by disrupting transcription of the steroidogenic acute regulatory (StAR) gene
Authors: Walsh LP , Webster DR , Stocco DM
Published: December 2004

Dimethoate is a widely used organophosphate insecticide that has been shown to disrupt reproductive function in animals. Although the pathogenesis of Dimethoate-induced reproductive toxicity remains to be determined, a reduction in serum testosterone levels is thought to play an important role in the development of Dimethoate-induced infertility. Since Leydig cells play a crucial role in male reproductive function by producing testosterone, the mouse MA-10 Leydig tumor cell line was used to determine if Dimethoate can directly block steroid hormone biosynthesis and to identify the site of steroidogenic inhibition. Dimethoate inhibited steroidogenesis in both a dose- and time-dependent manner without affecting total protein synthesis or protein kinase A activity. While it decreased the activity of the P450 side chain cleavage (P450 scc) enzyme, a reduction in the activity of this enzyme alone could not account for the level of Bu(2)cAMP-inhibited progesterone production. Instead, our results suggest that Dimethoate inhibited steroidogenesis primarily by blocking transcription of the steroidogenic acute regulatory (StAR) gene. This finding is significant since StAR protein mediates the rate-limiting and acutely-regulated step in steroidogenesis, the transfer of cholesterol from the outer to the inner mitochondrial membrane. This study indicates that StAR may be an important target for environmental pollutants which disrupt steroidogenesis and impair reproductive function.
Seasonal-dependent effect of temperature on the response of adenylate cyclase to FSH stimulation in the oviparous lizard, Podarcis sicula
Authors: Borrelli L , De Stasio R , Motta CM , Parisi E , Filosa S
Published: December 2004

The study of environmental factors affecting vertebrate reproduction has long interested both developmental and evolutionary biologists. Although photoperiod has been considered to be an important environmental parameter for vertebrates such as birds, temperature is probably a primary external factor responsible for reproductive cyclicity in reptiles. In spite of the progress made in the understanding of reptilian reproductive strategies and adaptations, much remains to be learned about the interplay between endocrine physiological factors, such as hormones, and environmental parameters. In this report, we have examined the effects of in vivo administered FSH on oocyte recruitment during the most significant periods of the reproductive cycle of the lizard, Podarcis sicula. The results show that when FSH is administered in proximity to the reproductive period, it stimulates oocyte growth and ovulation; when the hormone is administered at the beginning of the winter stasis it affects ovarian activity without inducing ovulation. Ovarian adenylate cyclase activity is moderately sensitive to in vitro FSH stimulation during the pre- and post-reproductive periods. The sensitivity to hormone stimulation increases significantly during the reproductive period and winter stasis. We have also tested the hypothesis that environmental temperature affects the responsiveness of ovarian adenylate cyclase to FSH stimulation. For such a purpose, we exposed animals to 28 degrees C or 4 degrees C in different periods of the ovarian cycle. The results show that, whenever the temperature applied mimics the thermal regime of the coming season, adenylate cyclase sensitivity to FSH shifts towards levels that anticipate the natural responsiveness.
Metabolic hormones and tissue concentrations of mRNA for IGF-I in lines of sheep that differ in their protein synthesis response to feed intake
Authors: Adams NR ,   Briegel, Thompson MJ , Sammels LM
Published: December 2004

The rate of protein synthesis in the skin and muscle of sheep that have been genetically selected for high wool staple strength (SS) is less dependent on the level of dietary intake than that of low SS sheep. This study examined potential hormonal mediators of this difference in responsiveness. Sheep from SS+ and SS- genotypes were fed at 0.4, 1.1 or 1.8 times maintenance. Circulating concentrations of metabolic hormones and tissue concentrations of the mRNA for IGF-I were measured and compared with rates of protein synthesis measured previously. Plasma concentrations of GH, insulin, cortisol, thyroxine and IGF-I responded similarly to dietary intake in both genotypes, but SS+ sheep had higher plasma concentrations of IGF-I at all levels of nutrition (P<0.05). There were no interactions between diet and genotype. The concentration of mRNA for IGF-I was higher in the liver of SS+ sheep (P<0.05), and tended to increase (P=0.06) with nutrient intake, but there were no significant effects of genotype or nutrition in skin, muscle or gut. Concentrations of mRNA for IGF-I were not related to the rate of protein synthesis in any tissue examined. It was concluded that IGF-I did not drive the rate of protein synthesis directly, but it may mediate the responsiveness of protein synthesis rate, or protein degradation rate, to nutrient supply.
Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation
Authors: Sugden MC , Lall HS , Harris RA , Holness MJ
Published: December 2004

The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.
Renal Na-K-ATPase hyperactivity in diabetic Psammomys obesus is related to glomerular hyperfiltration but is insulin-independent
Authors: Scherzer P , Nachliel I , Bar-On H , Popovtzer MM , Ziv E
Published: December 2004

Psammomys obesus, a desert rodent, develops diabetes when displaced from its natural environment and fed a high energy diet in the laboratory. This study was designed to examine variations in renal function in relation to the diabetic state with emphasis on changes in Na-K-ATPase activity. The following groups of Psammomys were studied: (1) Animals fed a saltbush diet; a low energy/high salt diet (natural). (2) Animals fed a low energy/low salt diet (laboratory). Both 1 and 2 were normoglycemic and normoinsulinemic and thus served as control. (3) Animals fed a high energy diet (group C) who were hyperglycemic and hyperinsulinemic; this group was divided into two subgroups: C1 presented with glomerular hyperfiltration rate and C2 with glomerular hypofiltration rate. (4) Animals fed a high energy diet presenting with hyperglycemia-hypoinsulinemia (group D). (5) Group D+I, similar to group D but treated with external insulin (2 U/24 h). Groups D and C1, whose glomerular filtration rose above normal by 30% and 70% respectively, exhibited metabolic similarity to Type I and Type II diabetes. In these groups, Na-K-ATPase activity in the cortex increased by 80-100% and in the medulla by 180% (P<0.001 vs control). In group C2 with reduced glomerular filtration rate (GFR), Na-K-ATPase activity did not differ from control. In group D+I, with normalized glomerular filtration rate, Na-K-ATPase activity was similar to control. There was a linear and significant correlation between GFR and Na-K-ATPase activity both in the cortex and in the medulla. These experiments present a well defined animal model of diabetes mellitus. Variations in glucose and in insulin did not correlate with Na-K-ATPase activity. These results clearly demonstrated that Na-K-ATPase activity in the diabetic Psammomys was determined by glomerular filtration but was independent of plasma glucose or insulin levels.
Developmental changes of the oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of the rat--an analysis by in situ hybridization
Authors: Mowa CN , Iwanaga T
Published: December 2004

This study employed an in situ hybridization technique to compare the cellular expression of oestrogen receptor (ER) subtypes, ER alpha and beta, in the female reproductive organ of the rat during prenatal and postnatal periods. Diffuse signals of ER alpha and beta mRNAs were co-expressed in the foetal ovary; they were weak and inconsistent before onset of gonadal differentiation, but increased in intensity with age. ER beta mRNA signals in the ovary sharply increased in intensity to adult levels by postnatal days 6-7, whereas those of ER alpha mRNA remained unchanged after birth. ER alpha was the sole subtype expressed during the prenatal period from the oviduct to the vagina, being localized mainly to the sub-epithelial stromal cells, and remained predominant thereafter. Signals for ER alpha mRNA in the epithelia were confined to the oviduct during prenatal and early postnatal periods; those in uterine and vaginal epithelia first appeared by postnatal days 4-5 and 6 respectively. Expressions of ER beta mRNA in the reproductive tract were absent during the prenatal period, and were weakly expressed during the postnatal period. Thus, oestrogen action in the developing ovary may be co-mediated by both ER alpha and beta, whereas ER alpha may be the primary mediator in the differentiation and growth of the female reproductive tract.