bluuebunny/crossref_metadata_2025 huggingface dataset:
Large-scale preparation and characterization of recombinant ovine placental lactogen
Authors: Sakal E , Bignon C , Grosclaude J , Kantor A , Shapira R , Leibovitch H , Helman D , Nespoulous C , Shamay A , Rowlinson S W , Djiane J , Gertler A
Published: December 2004

To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5′ and 3′ primers containing, respectively, Ncol and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(−1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into Ncol/PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-β-d-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30–40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate β-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.

Journal of Endocrinology (1997) 152, 317–327
Correlation between impairment of glucocorticoid feedback and the size of the pituitary gland in dogs with pituitary-dependent hyperadrenocorticism
Authors: Kooistra H S , Voorhout G , Mol J A , Rijnberk A
Published: December 2004

Under the assumption that the impaired inhibitory effect of glucocorticoids on cell division is an important determinant in the progression of corticotrophic adenomas, it is postulated that the magnitude of proliferation and the resistance to glucocorticoids are correlated. To test this hypothesis, 67 dogs with pituitary-dependent hyperadrenocorticism were studied to determine whether a correlation could be demonstrated between the effect of dexamethasone administration on the activity of the pituitary–adrenocortical axis and the size of the pituitary gland as estimated by computed tomography.

The volumes of the pituitary glands as calculated from summations of subsequent images of pituitary areas, ranged from 11·8 to 3238·6 mm3. Among the three dimensions, the height of the pituitary was the most sensitive indicator of enlargement. Calculation of the pituitary height/brain area ratio (P/B ratio) allowed correction for the size of the dog. The P/B ratio had the highest discriminatory power in distinguishing enlarged (n=41) from non-enlarged (n=26) pituitaries.

The effects of dexamethasone (0·1 mg/kg) on the plasma concentrations of cortisol and ACTH and on the urinary corticoid/creatinine (C/C) ratios were expressed as percentage changes from the initial values. For ACTH, cortisol and C/C ratios these figures for resistance to dexamethasone were significantly correlated with the dimensions of the pituitary, particularly the height, volume and P/B ratio.

It is concluded that the magnitude of the expansion of pituitary corticotrophic adenomas is dependent upon the loss of restraint by glucocorticoids, i.e. the degree of insensitivity to glucocorticoid feedback.

Journal of Endocrinology (1997) 152, 387–394
Dexamethasone and 11-dehydrodexamethasone as tools to investigate the isozymes of 11β-hydroxysteroid dehydrogenase in vitro and in vivo
Authors: Best R , Nelson S M , Walker B R
Published: December 2004

Dexamethasone is used in the clinic to test the sensitivity of the hypothalamic–pituitary–adrenal axis to negative feedback. It has also been proposed that metabolism of dexamethasone might differentiate between the activities of the two isozymes of 11β-hydroxysteroid dehydrogenase (11 βHSD1 and 11βHSD2). We have developed a gas chromatographic mass spectrometric assay for dexamethasone and 11-dehydrodexamethasone and have confirmed in vitro that dexamethasone is a substrate for 11 β-HSD2 but not 11 β-HSD1 (conversion to 11-dehydrodexamethasone 0·6 ± 0·3% in homogenates of rat liver with NADP+ for 11β-HSD1, and 29·4 ±10·3% and 40·0 ± 2·0% in homogenates of rat and human kidney respectively with NAD+ for 11β-HSD2). However, we have also made the novel observation that 11-dehydrodexamethasone is a substrate for both isozymes (conversion to dexamethasone 65·0±20·4% for 11βHSD1 and 53·5 ± 20·8% and 69·0 ± 4·5% for 11βHSD2, rat and human respectively). In healthy humans, the concentrations of 11-dehydrodexamethasone in plasma after an intravenous bolus of dexamethasone were less than 10% of those of dexamethasone, and 11-dehydrodexamethasone was detected (at 0·8–65·0 nm) in plasma from only 11 of 20 subjects at 0900 h on the morning after oral dexamethasone (0·1–1 mg taken at 2400 h). Concentrations of 11-dehydrodexamethasone did not correlate with the degree of suppression of plasma cortisol. Thus dexamethasone is not useful in differentiating the activities of the isozymes of 11β-HSD in vivo and variations in 11β-HSD activity do not explain the interindividual variability in suppression of plasma cortisol by low doses of dexamethasone.

Journal of Endocrinology (1997) 153, 41–48
In vitro response to interleukin-1β and streptozotocin in pancreatic islets isolated from male and female nonobese diabetic mice
Authors: Strandell E , Sandler S
Published: December 2004

The aim of the present study was to examine if the islet donor gender influences the β-cell sensitivity to possible mediators of β-cell destruction in insulin-dependent diabetes mellitus (IDDM). We have currently addressed this issue by comparing the action of the cytokine interleukin-1β (IL-1β) and the alkylating agent streptozotocin (STZ), on isolated pancreatic islets derived from prediabetic female and male nonobese diabetic (NOD) mice. In this mouse strain the females have a much higher incidence of spontaneous IDDM than the males. Pancreatic islets were isolated from female and male mice of the same litter, cultured for 6 days and thereafter either 5–50 U/ml IL-1β were added for 48 h or 1·8 mm STZ for 30 min. The cytokine exposure caused a strong increase in the medium insulin accumulation in the cultures of islets obtained from both males and females. The insulin and DNA contents were similar when comparing female and male islets before and after cytokine exposure. The male pancreatic islets exhibited a higher rate of islet glucose oxidation than islets obtained from females after IL-1β addition. Non-cytokine treated islets from males showed a higher insulin content compared with corresponding female islets. After IL-1β addition male and female islets had a similar inhibition of insulin secretion following stimulation by glucose. On the other hand, islets of both genders were equally sensitive to the inhibitory action of STZ, when studied 6 days after STZ incubation, as assessed by glucose oxidation and insulin release experiments. In conclusion the data of the present study did not demonstrate a clear gender difference in the islet β-cell sensitivity to immune mediated suppression by IL-1β or alkylation damage in IDDM-prone NOD mice. However, it cannot be excluded that in vivo, under the influence of sex hormones, a decisive alteration in β-cell sensitivity to damage might develop.

Journal of Endocrinology (1997) 153, 81–86
ATP and [3H]noradrenaline release and the presence of ecto-Ca2+-ATPases in the capsule-glomerulosa fraction of the rat adrenal gland
Authors: Jurányi Z , Orsó E , Jánossy A , Sz Szalay K , Sperlágh B , Windisch K , Vinson G P , Vizi E S
Published: December 2004

Both [3H]noradrenaline ([3H]NA) and ATP were released in response to supramaximal electric field stimulation in superfused rat adrenal capsule-glomerulosa preparations. The voltage-dependent potassium channel blocker 4-aminopyridine enhanced, while the ATP-sensitive potassium channel blocker glibenclamide failed to affect the stimulation-evoked release of [3H]NA. The selective α2-adrenoceptor antagonist CH-38083 enhanced the evoked release of [3H]NA while the P2 receptor agonist ATP and α,β-methylene-ATP failed to affect it. Neither the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) nor the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) influenced the stimulation-evoked [3H]NA release. The data showed that ATP was released from capsule-glomerulosa preparations in response to field stimulation together with but independently from [3H]NA, and that the local noradrenergic varicose axon terminals are not equipped with purinoceptors sensitive to ATP and/or adenosine. High concentrations of ATP also stimulated steroid hormone secretion in vitro, and thus may have a physiological role in this tissue. The presence of ecto-Ca2+-ATPases, enzymes able to terminate the effect of ATP, was demonstrated around the nerve profiles at the border of the capsule and zona glomerulosa tissue.

Journal of Endocrinology (1997) 153, 105–114
Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization
Authors: Fine M , Amuly R , Sandowski Y , Marchant T A , Chan S J , Gertler A , Funkenstein B
Published: December 2004

Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside. The expressed protein contained within the inclusion-body pellet was solubilized in 4·5 m urea, refolded for 24 h at pH 11·3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was ∼200-fold lower than that of hIGF-I. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active.

Journal of Endocrinology (1997) 153, 139–150
Intensive venous sampling of adrenocorticotropic hormone in rats with sham or paraventricular nucleus lesions
Authors: Boyle L L , Brownfield M S , Lent S J , Goodman B , Vo-Hill H , Litwin J , Carnes M
Published: December 2004

Adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary is predominantly regulated by corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) synthesized in neurons of the paraventricular nucleus (PVN) of the hypothalamus. Secretion of ACTH occurs in pulsatile bursts. To explore the relationship between hypothalamic control and the pulsatile pattern of ACTH secretion, we measured ACTH in 2 min blood samples over 4 h in rats with intact and lesioned PVN during hypovolemic-stress or control conditions and also measured median eminence (ME) levels of CRH, AVP, and oxytocin (OT). Mean plasma ACTH was highest in the sham lesioned-hypovolemic group, lowest in the sham lesioned-control group and intermediate in the two PVN-lesioned groups. CRH in the ME was negligible in the lesioned animals and correlated with OT and AVP. Pulsatile secretion was observed despite PVN ablation. Visual inspection of composite time series suggested different temporal patterns of ACTH secretion. Principal components analysis of the individual ACTH time series revealed three significant eigenvectors which correlated differentially with the three treatment groups. Neither lesioned group had the steep rise over 10 min seen in plasma ACTH in the non-lesioned groups. Delayed ACTH rise after 30–60 min occurred in all but the sham control group. Our data suggest that CRH is responsible for immediate secretion of ACTH in response to hypovolemic stress and that regulators from non-PVN sites may be responsible for more delayed secretion of ACTH in this setting. The persistence of ME AVP and OT levels in the face of >90% reduction in ME CRH levels leaves open the question of a role for one or both of these peptides in the delayed ACTH response following stress onset and in the generation of pulsatile ACTH secretory bursts.

Journal of Endocrinology (1997) 153, 159–167
Microinjection of rat GH but not human IGF-I into a defined area of the hypothalamus inhibits endogenous GH secretion in rats
Authors: Minami S , Suzuki N , Sugihara H , Tamura H , Emoto N , Wakabayashi I
Published: December 2004

It has been surmised that GH exerts feedback action on the hypothalamus and thereby regulates its own secretion. Our previous studies suggested that GH acts on somatostatin neurons in the hypothalamic periventricular nucleus (PeV) and neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC). However, there remains uncertainty whether GH acts directly or indirectly through the generation of IGFs on the hypothalamus to regulate its own secretion. To examine this, rat GH (rGH) or human IGF-I was injected directly into a defined area of the hypothalamus, and the blood GH profile was observed in conscious male rats. In the rats given 0·5 μg rGH into the ARC or PeV bilaterally, GH secretion was inhibited, and the inhibition lasted for 12 h. During the period of inhibition, the duration and amplitude of GH pulses were significantly decreased and the episodic secretion of GH appeared irregularly compared with the vehicle-injected control rats. In control rats given the vehicle or those given rGH into the lateral hypothalamus, the blood GH profile did not change and pulsatile GH secretion was produced every 3 h. When 0·1 μg IGF-I was injected into the ARC or PeV bilaterally, the blood GH secretory pattern was not affected. Together with the results of our previous studies showing that c-fos gene expression was induced by systemic administration of GH and that GH receptor mRNA was contained in somatostatin neurons in the PeV and NPY neurons in the ARC, the data of the present study indicate that GH, but not IGF-I, acts on the cells in the ARC and the PeV or in their vicinity to inhibit its own secretion, presumably by activating the somatostatin and NPY neurons.

Journal of Endocrinology (1997) 153, 283–290
Evidence for isoforms of 11β-hydroxysteroid dehydrogenase in the liver and kidney of the guinea pig
Authors: Quinkler M , Kosmale B , Bähr V , Oelkers W , Diederich S
Published: December 2004

In the human and in rodents like the rat and mouse, the liver enzyme 11β-hydroxysteroid dehydrogenase type I (11β-HSD-I) is a functional oxidoreductase preferring NADP+/NADPH as cosubstrate, while the renal isoenzyme (11β-HSD-II) prefers NAD+ as cosubstrate, and seems to be a pure oxidase and protects the tubular mineralocorticoid (MC) receptor from occupancy by cortisol and corticosterone. We studied the enzyme kinetics of 11β-HSDs in kidney and liver microsomes of the guinea pig, a species whose zoological classification is still a matter of debate. With a fixed concentration of 10−6 mol/l cortisol, liver and kidney microsomes preferred NAD+ to NADP+ (10−3 mol/l) for the conversion to cortisone. Kidney microsomes converted cortisol to cortisone with Km values of 0·64 μmol/l and 9·8 μmol/l with NAD+ and NADP+ as cosubstrates respectively. The reduction of cortisone to cortisol was slow with kidney microsomes, but could be markedly enhanced by adding an NADH/NADPH regenerating system: with NADPH as preferred cosubstrate, the approximate Km was 7·2 μmol/l. This indicated the existence of both isoenzymes in the guinea pig kidney. Liver microsomes oxidized cortisol to cortisone with similar Km and Vmax values for NAD+ to NADP+ as cosubstrates (Km of 4·3 μmol/l and 5·0 μmol/l respectively). The NAD+ preference for the oxidation of 10−6 mol/l cortisol described above may be due to a second, NAD+-preferring 11β-HSD with a Km of 1·4 μmol/l. In contrast to the kidney, liver microsomes actively converted cortisone to cortisol with a preference for NADPH (Km: 1·2 μmol/l; Vmax: 467 nmol/min per mg protein). Thus, the main liver enzyme is similar to the oxidoreductase of other species (11β-HSD-I) and is also present in the kidney, while the main kidney enzyme is clearly NAD+-preferring. This kidney enzyme (analogous to 11β-HSD-II of other species) seems to be suitable for the protection of the MC receptor from the high free plasma cortisol levels of the guinea pig.

Journal of Endocrinology (1997) 153, 291–298
Normal pancreastatin-like and increased post-glucose insulin levels in young offspring of insulin-resistant non-obese essential hypertensive patients
Authors: Sánchez-Margalet V , Ramos E , Mateo J , Oliván J , Pérez-Cano R , Goberna R
Published: December 2004

Pancreastatin is a regulatory peptide known to inhibit insulin secretion and insulin action with a glycogenolytic effect in the liver. This peptide is present in and secreted by many endocrine and chromaffin cells. Abnormalities of glucose, insulin and lipoprotein metabolism are common in patients with hypertension, as well as their first-degree relatives. We have recently studied a group of non-obese hypertensive subjects in which pancreastatin-like levels were increased compared with controls, and correlated with norepinephrine levels. We hypothesized that pancreastatin alongside the sympathoadrenal system might have a part in the insulin resistance of these patients, and this metabolic syndrome could play a role in the pathogenesis and complications of hypertension. In this article, we studied the normotensive offspring of these non-obese hypertensive patients and looked for metabolic abnormalities as well as plasma pancreastatin, glucagon and catecholamine levels. The subjects were separated into two groups: (1) offspring from non-insulin-resistant patients and (2) offspring from insulin-resistant patients. We found that after an intravenous glucose load, offspring from insulin-resistant patients were already hyperinsulinemic, although glucose clearance was normal, suggesting an early alteration in insulin sensitivity, whereas pancreastatin and catecholamine levels were normal compared with matched controls. However, offspring from non-insulin-resistant patients had no differences with controls. These results suggest that pancreastatin and catecholamines may not play an important role in triggering insulin resistance, although they may be important once the syndrome is established.

Journal of Endocrinology (1997) 153, 313–318
Rabbit sex hormone binding globulin: primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli
Authors: Lee W M , Wong A S T , Tu A W K , Cheung C-H , Li J C H , Hammond G L
Published: December 2004

Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability. The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract. We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79·0, 68·1 and 63·2% amino acid identity with the corresponding human, rat and mouse proteins respectively. Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1·6 kilobase mRNA in the liver of both sexes and in the testis. The rabbit SHBG cDNA was inserted into pGEX-1λT for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli. The bacterial product bound 5α-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum. The dissociation constants (Kd) for rabbit and human SHBGs produced in E. coli were 11·1 ± 1·1 nm and 2·1 ± 0·6 nm respectively, and rabbit SHBG formed a less stable protein-steroid complex (t½=5 min) than human SHBG (t½>60 min). Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity. To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5′-terminal half of SHBG from one species and 3′-terminal half of SHBG from the other species were constructed and expressed. It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species. Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex. This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.

Journal of Endocrinology (1997) 153, 373–384
Transcriptional and post-transcriptional regulation of FSH receptor in rat granulosa cells by cyclic AMP and activin
Authors: Tano M , Minegishi T , Nakamura K , Karino S , Ibuki Y , Miyamoto K
Published: December 2004

The effect of FSH on the induction of FSH receptors in granulosa cells is believed to be mediated, at least in part, by the cAMP second messenger system. We examined the effect of activin and cAMP on FSH receptor expression in this culture system. Steady-state levels of FSH receptor mRNA, analyzed by Northern blot hybridization, increased 3·5-fold in response to 24-h incubation with activin and 1·7-fold with 12-h incubation with 8-bromoadenosine 3,5-cyclic monophosphate (8-Br-cAMP; 0·2 mm). We have investigated whether 8-Br-cAMP- and/or activin-induced increases in FSH receptor mRNA levels are the result of increased transcription and/or altered mRNA stability. The rates of FSH receptor mRNA gene transcription, assessed by nuclear run-on transcription assay, increased 3-fold in cells treated with activin and 1·5-fold in cells treated with 8-Br-cAMP for 2 h. To examine the degradation rates of FSH receptor mRNA transcripts, granulosa cells were preincubated with 8-Br-cAMP, activin, or medium alone for 6 h. After the preincubation period, 5 μm actinomycin-D or 200 μm 5,6-dichloro-1-β-ribofuranosyl benzimidazole were added to arrest new RNA synthesis. The decay curves for the 2·4 kb FSH receptor mRNA transcript in granulosa cells were not significantly different in the absence or presence of 8-Br-cAMP. Activin, on the other hand, significantly altered the slope of the FSH receptor mRNA decay curve and increased the half-life of the 2·4 kb FSH receptor mRNA transcript. These data provide evidence that cAMP induces FSH receptor mRNA levels by stimulating the transcription rate and that activin increases FSH receptor mRNA levels both by stimulating transcription rates and by stabilizing the FSH receptor mRNA transcripts.

Journal of Endocrinology (1997) 153, 465–473
Involvement of SNAP-25 in TRH-induced exocytosis in pituitary GH4C1 cells
Authors: Masumoto N , Ikebuchi Y , Matsuoka T , Tasaka K , Miyake A , Murata Y
Published: December 2004

The synaptic membrane protein synaptosomal-associated protein (SNAP-25) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion in neurons. However, the role of SNAP-25 in pituitary hormone release is not known. In this study, we determined that SNAP-25 is involved in regulated exocytosis in the clonal pituitary cell line GH4C1. SNAP-25 messenger RNA and protein were detected in GH4C1 cells by RT-PCR and immunoblot analysis, respectively. Immunofluorescence analysis indicated that SNAP-25 protein was localized in the plasma membrane. Next, to determine the function of SNAP-25 in GH4C1 cells, specific inhibitors of SNAP-25, botulinum neurotoxin (BoNT) /A or /E, and antisense SNAP-25 oligonucleotide were used. Neither BoNT/A nor BoNT/E affected thyrotropin-releasing hormone (TRH)-induced cytosolic Ca2+ increase, but both inhibited TRH-induced exocytosis. Moreover, they dose-dependently inhibited TRH-induced prolactin release. The introduction of antisense oligonucleotide into the cells also inhibited TRH-induced prolactin release. These results suggest that SNAP-25 is involved in regulated exocytosis in GH4C1 cells.
Early diabetes-induced changes in rat jejunal glucose transport and the response to insulin
Authors: Sharp P A , Boyer S , Srai S K S , Baldwin S A , Debnam E S
Published: December 2004

The effects of 1 day of streptozotocin-induced diabetes in rats on glucose transport across the brush border membrane (BBM) and basolateral membrane (BLM) prepared from jejunal enterocytes has been studied. The effects on glucose transport of treatment of diabetic animals with insulin to reduce to normal the elevated blood glucose levels has also been assessed. The maximum capacity (Vmax) for SGLT1-mediated glucose uptake by BBM vesicles was unaffected by diabetes or insulin treatment of diabetic rats. In contrast, Vmax for BLM glucose uptake was increased by 206% in diabetes, a response that could not be reversed by treatment with insulin. Western blotting of BBM for SGLT1 protein revealed a single band with a molecular weight of 73 kDa and the intensity of this band was unaffected by diabetes. However, an increased level of GLUT2 was noted in diabetic BLM and this was not a consequence of changes in glycaemic or insulin status. Diabetes hyperpolarised the BBM, implying an increased driving force for Na+-sugar co-transport but insulin treatment only partially reversed this enhanced potential difference. Benzamil (2 μm), an epithelial Na+ channel blocker, hyperpolarised the BBM of control but not diabetic enterocytes, implying that a reduced Na+ permeability was responsible for the diabetic hyperpolarisation. It was concluded that in early diabetes, before the onset of hyperphagia, a greater driving force for Na+-dependent BBM sugar transport together with increased GLUT2 activity at the BLM promotes sugar movement across the enterocyte. Possible triggers for the transport responses are discussed.

Journal of Endocrinology (1997) 154, 19–25
Effect of monensin on 2-deoxyglucose uptake, the insulin receptor and phosphatidylinositol 3-kinase activity in rat muscle
Authors: Turinsky J , Damrau-Abney A , Elmendorf J S , Smith T R
Published: December 2004

Preincubation of rat soleus muscle with 1 and 10 μm monensin for 2 h increased the subsequent basal 2-deoxyglucose uptake by muscle 76 and 121% respectively. Under the same conditions, monensin decreased the insulin-stimulated (1 mU/ml) 2-deoxyglucose uptake by 29 and 37% respectively. The monensin-induced augmentation of basal 2-deoxyglucose uptake was inhibited 92% by cytochalasin B suggesting that the uptake is mediated by glucose transporters. Monensin did not increase the cellular accumulation of l-glucose in muscle indicating that it does not affect the cell membrane integrity. Neither the stimulatory effect of monensin on basal 2-deoxyglucose uptake nor the opposite, inhibitory action of monensin on the insulin-stimulated 2-deoxyglucose uptake were influenced by the removal of Ca2+ from the medium or by dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, suggesting that the actions of monensin are not mediated by calcium. Monensin had no effect on muscle ATP concentration. The monensin-induced augmentation of basal 2-deoxyglucose uptake was neither associated with stimulation of muscle phosphatidylinositol 3-kinase activity nor inhibited by wortmannin, demonstrating that the increase in basal 2-deoxyglucose uptake is not mediated by activation of phosphatidylinositol 3-kinase. The inhibition of insulin-stimulated 2-deoxyglucose uptake by monensin was associated with a 31% decrease in the abundance of insulin receptors in muscles, a 64% decrease in the insulin-induced autophosphorylation of the insulin receptor β-subunit, and a 44% reduction of the insulin-stimulated phosphatidylinositol 3-kinase activity. Addition of monensin into the phosphatidylinositol 3-kinase reaction had no effect on the activity of the enzyme, demonstrating that the inhibition in monensin-treated muscles is indirect and occurs upstream of phosphatidylinositol 3-kinase. It is concluded that monensin has a dual effect on 2-deoxyglucose uptake by skeletal muscle: it stimulates basal uptake but inhibits the insulin-stimulated uptake. The primary cause of the latter, inhibitory effect of monensin is at the level of the insulin receptor.

Journal of Endocrinology (1997) 154, 85–93
Blockade of the neonatal increase in testosterone by a GnRH antagonist: the free androgen index, reproductive capacity and postmortem findings in the male marmoset monkey
Authors: Lunn S F , Cowen G M , Fraser H M
Published: December 2004

Male marmoset monkeys which had received gonadotrophin-releasing hormone (GnRH) antagonist treatment as neonates to block the postnatal increase in testosterone were studied, with the object of determining potential long-term effects of treatment on the reproductive system, including tests of fertilising capacity. To obtain information on the nature of the circulating testosterone during this neonatal period, sequential blood samples were collected from a further control group of ten neonates, aged between birth and 3 months, and from 11 adult, normally fertile males, to examine the relative proportions of free, sex-hormone-binding globulin (SHBG)-bound, and non-SHBG-bound testosterone. In control neonates, 11% of the circulating testosterone was free, and a further 19% non-SHBG-bound, and therefore presumed to be biologically available. The remaining 70% was SHBG-bound and considered to be biologically inert. This indicates that the neonatal increase in marmoset testosterone has a biological function. After pairing with females, time to first positive vaginal lavage and first delivery was similar for females, whether they were with control or treated male partners. Pregnancy outcome, in terms of number of young delivered and sex ratio, did not differ. This indicates that there appear to be no long-term sequelae in terms of procreative ability in male marmosets treated neonatally with a GnRH antagonist. Autopsy revealed no gross changes, except in the thymus, which was significantly heavier in the treated group. These results indicate that, although the circulating testosterone is in a biologically active form during the neonatal period, inhibition of testicular function in the neonate is without major effect on the adult male reproductive system. Treatment with a GnRH antagonist may have long-term effects on the immune system.

Journal of Endocrinology (1997) 154, 125–131
Immunolocalisation of angiotensin II receptors in icefish (Chionodraco hamatus) tissues
Authors: Marsigliante S , Acierno R , Maffia M , Muscella A , Vinson G P , Storelli C
Published: December 2004

The monoclonal antibody 6313/G2 raised against the mammalian type I (AT1) angiotensin II (Ang II) receptor (Ang II-R) also recognises a component in teleost (eel) tissue preparations that binds radiolabelled Ang II, and has an isoelectric point (pI) of 6·5 and molecular mass of 75 kDa. Immunohistochemical analysis using this antibody showed specific binding sites in eel intestine, kidney, gill and liver sections.

The same antibody was used here to evaluate the presence and distribution of Ang II-R in target tissues of the Antarctic teleost icefish (Chionodraco hamatus).

Immunocytochemistry of intestine and gill sections showed that the antibody bound to uniformly distributed intracellular sites and cell surface membranes in absorptive cells in the intestine and chloride and pavement cells in the gills. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. In the kidney, only the tubules in the trunk stained positively while the head (atubular part of the kidney) was negative. In kidney tubules, in contrast with other tissues, the receptor was most concentrated in the cytoplasm underlying the basolateral membranes, with somewhat weaker staining beneath the apical cell membrane. Immunoblotting identified a single component from trunk kidney preparations that focused at pI 5·9 in isoelectric focusing gels and showed a molecular mass of 75 kDa in SDS–polyacrylamide gels.

The data suggest that, as in other teleosts, Ang II has a physiological role in the icefish.

Journal of Endocrinology (1997) 154, 193–200
Luteotrophic factors in hyperstimulated pseudopregnant rabbit: II – High sensitivity to hCG of luteal tissue and small luteal cells
Authors: Arioua R K , Benhaïm A , Féral C , Leymarie P
Published: December 2004

Previous studies on rabbit corpus luteum (CL) led to the conclusion that the luteotrophic complex, in rabbit, may include LH as well as oestradiol for normal luteal function. However, the requirement for LH is controversial. We have recently demonstrated the existence of a human chorionic gonadotrophin (hCG)-stimulated aromatase activity in cultured corpora lutea from a hyperstimulated pseudopregnant rabbit model, which develops a large number of corpora lutea, with only a few or no follicles in the ovaries. The present study was undertaken to investigate the in vitro responsiveness to hCG, dibutyryl cAMP (dbcAMP) and oestradiol of those corpora lutea.

Pseudopregnancy (PP) was induced in rabbits by i.m. injection of 200 IU equine chorionic gonadotrophin daily for 2 days followed on day 4 by i.m. injection of 200 IU hCG (day 0 of PP). Luteal tissue and small and large luteal cells obtained at days 5 and 9 of PP were cultured for 24 h or during several days respectively with or without hCG, dbcAMP or oestradiol. Basal progesterone secretion was 3·6- and 22-fold higher in large cells compared with small ones at day 5 and 9 of PP respectively. When stimulated by small doses of hCG, luteal tissue responded by a 5-fold increase in progesterone secretion. Small cells produced four times higher amounts of progesterone than controls in the presence of 1 mIU/ml hCG and more than ten times in the presence of 0·1 IU/ml hCG, whereas large cells were insensitive to hCG stimulation. dbcAMP mimicked the effect of hCG on progesterone secretion by luteal tissue and luteal cells and oestradiol stimulated basal progesterone secretion in both small and large luteal cells. Given the large contribution of non stimulated large cells to luteal progesterone production and the remarkably high sensitivity of luteal tissue to gonadotrophin in vitro it seems that interactions between the two types of cells might occur during LH stimulation. Our results suggest that LH could participate in the luteotrophic complex at least in part through the stimulation of endogenous oestradiol production by luteal cells.

Journal of Endocrinology (1997) 154, 259–265
Differential regulation of tissue insulin-like growth factor-binding protein (IGFBP)-3, IGF-I and IGF type 1 receptor mRNA levels, and serum IGF-I and IGFBP concentrations by growth hormone and IGF-I
Authors: Lemmey A B , Glassford J , Flick-Smith H C , Holly J M P , Pell J M
Published: December 2004

The aims of this investigation were (1) to examine IGF-binding protein-3 (IGFBP-3) mRNA levels in candidate tissues which might be important sources for blood IGFBP-3 (liver and skin) and in a target tissue for IGF-I action (skeletal muscle), and (2) to examine the effects of a single dose (500 μg) of GH or IGF-I on IGFBP-3 message levels in these tissues since temporal responses (4, 8 and 24 h after the single subcutaneous dose of peptide to GH-deficient dwarf rats) would indicate which peptide is the primary modulator of IGFBP-3 synthesis. Circulating IGF-I and IGFBP-3 concentrations were significantly increased (P<0·05) by IGF-I and GH. GH treatment increased liver IGFBP-3 mRNA levels by 4 h (P<0·001 over the 24 h) whereas IGF-I had no effect. Similarly, GH, but not IGF-I, increased muscle IGFBP-3 mRNA levels (P<0·001 for the 24 h study period). However, both IGF-I and GH induced increases in skin IGFBP-3 mRNA abundance throughout the 24 h period (P<0·001 and P<0·01 respectively) and skin IGFBP-3 message abundance was greater that in the liver. Liver IGF-I mRNA levels were, as expected, increased after GH and tended to decrease after IGF-I treatment; muscle IGF-I mRNA was increased by GH (P<0·001) and, interestingly, progressively increased by IGF-I (P<0·05 for the 24 h period); skin IGF-I mRNA levels were unchanged by both peptides. The IGF-I induced increase in serum IGFBP-3 concentrations in the absence of an increase in hepatic IGFBP-3 mRNA levels and a paucity of liver IGF-I type 1 receptor mRNA imply that other sources of IGFBP-3 protein or synthesis must exist. The response of skin IGFBP-3 mRNA levels to both GH and IGF-I suggests that other cell types, such as fibroblast-derived cells, could be more important than the liver in the regulation of circulating reservoir IGFBP-3 in certain circumstances. In contrast to some current suggestions, the rapid and consistent GH-induced increase in IGFBP-3 message levels in all tissues studied implies that GH might have a direct function in the regulation of IGFBP-3 synthesis.

Journal of Endocrinology (1997) 154, 319–328
Renal responses to oxytocin during the 4 days of the oestrous cycle in the rat
Authors: Windle R J , Forsling M L
Published: December 2004

Oxytocin was administered to virgin female rats at doses of 25–200 pmol/min during 0·077 mol NaCl/l infusion at 150 μl/min on each day of the oestrous cycle. The resultant rates of urine flow, glomerular filtration (GFR) and electrolyte excretion were determined. Oxytocin caused significant increases in urine flow (P<0·001) and sodium excretion (P<0·001); both responses being dose-dependent (P<0·02 and P<0·01 respectively). Significant variations in the renal responsiveness to the hormone occurred over the 4 days of the oestrous cycle. On oestrus the lowest dose of 25 pmol oxytocin/min produced a significant increase in urine flow (from 139·5 ± 4·3 to 165·6 ± 7·1 μl/min, P<0·005) and a dose of 50 pmol/min produced a significant increase in sodium excretion (from 10·6 ± 0·1 to 14·5 ± 0·7 μmol/min, P<0·005). Significant increases in urine flow and sodium excretion were seen on pro-oestrus with hormone administration rates of 50 and 100 pmol/min respectively and on dioestrus day 2 with a rate of 100 pmol/min. On dioestrus day 1 no increase in urine flow or sodium excretion was seen over the dose range of oxytocin administration. A dose of 100 pmol oxytocin/min significantly increased GFR on pro-oestrus and dioestrus day 2, but not on the other 2 days of the cycle. The circulating hormone concentrations produced by oxytocin infusion were similar on each day of the cycle and so could not account for the differences seen. Therefore, these results suggest varying renal responsiveness to oxytocin during the reproductive cycle of the female rat.

Journal of Endocrinology (1997) 154, 347–353
Growth hormone (GH) regulation of submandibular gland structure and function in the GH-deficient rat: upregulation of haptocorrin
Authors: Lobie PE , Waters MJ
Published: December 2004

Based on localization studies of the GH receptor/binding protein (BP) in the gastrointestinal tract, we have recently demonstrated growth hormone regulation of gastric intrinsic factor. In order to define the role of GH in the submandibular gland (SMG) we have investigated the effect of GH on SMG structure and function with particular reference to haptocorrin. Bovine GH (65 micrograms/100 g body weight) was administered twice daily to adult male dwarf rats for 6 days (DW+) while control animals received vehicle (DW-). Administration of GH produced a significant increase in body weight (P < 0.001) and allometric increase in SMG weight (P = 0). There was no change in RNA or protein content per g SMG and GH administration produced a small decrease in DNA content normalized to SMG weight. Morphometric analysis of the SMG revealed a significant increase in the percentage area of the gland occupied by tubular (GH receptor/BP expressing) structures and a significant increase in the diameter of both the intralobular striated and granular convoluted tubules. The effect of GH on cellular proliferation in the ductular and acinar components was determined by the immunohistochemical detection of nuclear 5'-bromo-2'-deoxyuridine (BrdU) incorporated during a 2-h pulse of BrdU. GH treatment induced a 5.5-fold increase in the labelling index of tubular cells whereas the acinar cell labelling index increased only 3.3-fold. Soluble extracts of SMG were prepared for estimation of 57Co-cyanocobalamin (vitamin B12) binding. GH administration resulted in an increase in total 57Co-cyanocobalamin (CBL) binding per mg SMG protein. To determine the contribution of haptocorrin (R-protein) the amount of cobinamide dicyanide (CD) displaceable binding was calculated. GH administration produced a 70% increase in CD displaceable CBL binding per mg SMG indicating GH regulation of haptocorrin. A comparison of total SMG CBL binding and CD displaceable CBL binding between male and female rats detected no sex difference. Therefore sex-specific GH secretory profiles are unlikely to be of importance in the regulation of haptocorrin. In conclusion we have demonstrated that GH stimulates hypertrophy and hyperplasia of components of the SMG in the dwarf rat. The observed upregulation of haptocorrin may synergize with the GH-stimulated increase in intrinsic factor to facilitate absorption of CBL during the anabolic state.
In vitro fertilizing capacity of sperm from FSH-treated photoinhibited Djungarian hamsters (Phodopus sungorus)
Authors: Niklowitz P , Lerchl A , Nieschlag E
Published: December 2004

In hypogonadal male Djungarian hamsters FSH alone can induce normal spermatogenesis. However, for the induction of mating behavior, supplementation with testosterone is necessary. We have here investigated, by in vitro fertilization, whether sperm produced by photoinhibited hamsters treated with FSH alone can fertilize without testosterone. Photoinhibited hypogonadal male Djungarian hamsters were injected daily with human FSH (10 IU; Fertinorm) for 5-7 weeks. The hormone stimulated regrowth of the testes. Neither body weight nor the weights of the androgen-dependent organs-epididymides, prostates, accessory glands-showed significant differences from photoinhibited controls; furthermore FSH treatment did not raise intratesticular or serum testosterone levels. In eleven out of the twelve FSH-treated photoinhibited hamsters, elongated spermatids were found in the testes; in five out of nine epididymides, sperm was found by histological examination. In two out of the twelve FSH-treated hamsters, the amount of sperm in the caudal part of the epididymis was sufficient for in vitro fertilization with oocytes collected from superstimulated females. These spermatozoa fertilized 16-29% of the oocytes. Spermatozoa from photostimulated controls produced similar levels of fertilization.
Intracellular calcium concentration and hormone secretion are controlled differently by TRH in rat neonatal lactotrophs and somatotrophs
Authors: Lorsignol A , Taupignon A , Horvath G , Dufy B
Published: December 2004

We studied the effects of TRH on the cytosolic free calcium concentration ([Ca2+]i) of female rat pituitary prolactin-secreting (lactotroph) and GH-secreting (somatotroph) cells in the early postnatal period, i.e. at postnatal days 5 and 10. [Ca2+]i of single identified lactotrophs and somatotrophs was recorded by dual-emission microspectrofluorimetry using the intracellular fluorescent calcium probe indo 1. An application of TRH (100 nM, 10 s) induced a marked [Ca2+]i increase in 65% of neonatal lactotrophs and 34% of neonatal somatotrophs while the remaining cells were unaffected. Most of the responsive cells, both lactotrophs and somatotrophs, exhibited a similar biphasic Ca2+ response, made up of an initial rapid large increase in [Ca2+]i followed by sustained [Ca2+]i fluctuations. In both cell types, removal of Ca2+ from the extracellular medium or addition of the Ca2+ channel blocker, cadmium chloride (500 microM), inhibited the second phase whereas the first phase persisted. Furthermore, in both cell types, protein kinase C (PKC) depletion by incubation in phorbol myristate acetate (1 microM) for 24 h abolished the second phase but did not inhibit the first phase. Conversely, when cells were pretreated with the Ca(2+)-ATPase inhibitor, thapsigargin (100 nM), all TRH-induced [Ca2+]i changes in both cell types disappeared. TRH therefore induces a biphasic increase in [Ca2+]i involving intra- and extracellular Ca2+ in neonatal lactotrophs and somatotrophs as it does in adult lactotrophs. The first phase is presumably due to mobilization of Ca2+ from intracellular stores whereas the second phase presumably results from a PKC-sensitive influx of Ca2+. TRH action on membrane potential was then investigated using the patch-clamp technique in the whole-cell mode. TRH-induced changes in membrane potential consisted of an initial hyperpolarization followed by depolarization and action potential firing. We also investigated TRH action on prolactin and GH secretion by neonatal pituitary cells using RIA. Surprisingly, static assays of prolactin and GH revealed only stimulation of prolactin release by TRH but no effect on GH secretion, although, as expected, GH-releasing factor was a potent agonist of GH secretion. Our results suggest that TRH regulates neonatal lactotrophs and somatotrophs differently, in that the [Ca2+]i changes do not correlate with stimulation of exocytosis in the latter cell type.
Dimerization properties of mutant thyroid hormone beta-receptors with auxiliary proteins
Authors: Tagami T , Nakamura H , Sasaki S , Miyoshi Y , Nakao K
Published: December 2004

Hormonal responsiveness in peripheral tissues is variable in patients with resistance to thyroid hormone (RTH). One cause of this may be differential interaction of RTH mutants of thyroid hormone receptor beta (TR beta) with TR auxiliary proteins (TRAPs). We used gel shift mobility assays to examine the interaction of wild-type and mutant TR beta s with retinoid X receptors (RXRs) and endogenous TRAPs. Some mutants showed reduced homodimerization but retained heterodimerization with recombinant RXRs. Wild-type TR beta formed heterodimeric complexes with multiple TRAPs in nuclear extracts of rat tissues, but RTH mutants showed variably altered heterodimerization with each TRAP. With liver nuclear extract, all mutants with impaired homodimerization also showed impaired TR beta-TRAP heterodimerization. Thus heterodimerizations with RXRs and TRAPs are differently affected by RTH mutations. Our results suggest that multiple TRAPs are expressed in tissue-specific patterns. The variability of TR beta heterodimerization with TRAPs may account, in part, for the variable tissue responsiveness in RTH.