In the adult, the insulin gene is expressed exclusively in the β cells of the islets of Langerhans. In order to understand the mechanisms involved in this cell-specific gene expression better, it is necessary to look at the molecular events controlling islet cell ontogeny. Although the timing of expression of each hormone during embryogenesis has been well documented, the exact cell lineage relationship among different islet cell types is not known in detail. In the developing mouse pancreas, glucagon immunoreactivity appears at day 12 (E12), insulin at E14·5 and somatostatin at E17, while pancreatic polypeptide immunoreactivity appears after birth (Teitelman & Lee, 1987). In transgenic mice, hybrid insulin genes are initially expressed in all cells of the embryonic endocrine pancreas (Alpert et al. 1988), suggesting a common pluripotent progenitor stem cell. This conclusion is supported by the observation that cell lines derived from islet cell tumours express multiple pancreatic hormones
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A. R. Clark and K. Docherty
R. G. Clark and A. T. Holder
This study was designed to test the hypothesis that oestrogens inhibit growth by reducing hepatic somatomedin generation. We have attempted to deliver oestrogen preferentially to the liver by transplanting ovarian tissue into the spleen. Four groups of rats were compared: intact, ovariectomized, ovariectomized with successful ovarian transplants, and animals where due to adhesions between the transplant and body wall and/or viscera oestrogens reached the general circulation. Plasma levels of LH and FSH, uterine weights, ovarian weights and vaginal smears supported this classification of animals. Ovariectomy increased body weight, body length, bioassayable serum somatomedin levels and 35SO4 2− uptake into costal cartilage in vivo compared with intact rats and animals with adhesions. Preferentially exposing the liver to oestrogen did not suppress the increased growth, serum somatomedin activity or uptake of 35SO4 2− in vivo observed in ovariectomized animals. The results suggest that the presence of oestrogen in the general circulation is associated with growth suppression and lowered somatomedin bioactivity while the presence of oestrogen in the hepatic portal circulation has little effect on body growth. We conclude that oestrogen does not appear to inhibit growth in the rat by influencing the release of somatomedin from the liver. It also seems that serum somatomedin concentration may not reflect liver somatomedin generation but overall production throughout the body.
J. Endocr. (1984) 103, 43–47
A. T. Holder, R. G. Clark, and M. A. Preece
This paper presents an investigation into the effects of prolonged oestrogen treatment (20 days) on basal growth and on growth stimulated by GH in hypopituitary dwarf mice. Body 35SO4 2− weight and tail length were measured during the treatment period and uptake of S04 into costal cartilage in vivo at the end of the treatment period. This study confirmed that treatment with human GH promotes a dose-dependent increase in body weight, tail length and uptake of 35SO4 2− in vivo; there was a highly significant correlation between these responses. Treatment with oestrogen alone had no significant effect on any of the parameters measured. All groups receiving combined oestrogen and human GH treatment showed a significant increase in body weight and tail length compared with animals receiving the same dose of oestrogen alone. However, the increase in body weight and tail length was significantly less in animals given the highest dose of oestrogen plus human GH than that observed in animals treated with the same dose of human GH alone. Treatment with oestrogen had no significant effect on the uptake of 35SO4 2− stimulated by human GH. Possible mechanisms for the growth-inhibiting effects of oestrogens are discussed.
WM Drake, SR Lowe, A Mirtella, TJ Bartlett, and AJ Clark
Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) are distantly related peptides. Both act through G protein-coupled receptors on vascular smooth muscle cells to increase intracellular cAMP concentrations, causing vasorelaxation. Recent evidence suggests that both peptides bind to a common heptahelical receptor, with specificity for each peptide being determined by a receptor activity modifying protein (RAMP). This hypothesis predicts that each peptide should desensitise the cellular response to subsequent stimulation by the other. We have studied the patterns of desensitisation of ADM/CGRP receptors in rat aortic vascular smooth muscle cells. Cells were incubated for 20 min in either serum free medium (SFM), alone (control) or in SFM containing vasoactive agonist (e.g. ADM 10(-8) M, CGRP 10(-7) M, angiotensin II 10(-9) M or isoproterenol 10(-6) M). Cells were then washed and incubated for a further 20 min in SFM containing a second agonist and 1 mM isobutyryl methyl xanthine. Cells were harvested and assayed for cAMP. Pre-exposure of cells to CGRP, isoproterenol, angiotensin II or ADM, decreased cAMP generation in response to subsequent stimulation with CGRP by 84% (+/-5), 66% (+/-18), 45% (+/-5) and 60% (+/-10) respectively (mean+/-s.d.). Pre-incubation of cells with 100 nM H-89, a protein kinase A (PKA) inhibitor, abolished the desensitisation of CGRP by itself, implying that this desensitisation was mediated through PKA. In contrast, there was no attenuation of the cAMP response to stimulation with ADM by pre-exposure to ADM and all other agonists tested. Identical results were seen with or without PKA inhibition by H-89. These results indicate that the ADM receptor does not desensitise over this time period in RAVSMCs, in contrast to the CGRP receptor, which is desensitised by pre-exposure to CGRP and other vaso-active agonists. These data also suggest that ADM and CGRP act through separate receptors in these cells.
K Walder, A Filippis, S Clark, P Zimmet, and GR Collier
Leptin is secreted from adipose tissue, and is thought to act as a 'lipostat', signalling the body fat levels to the hypothalamus resulting in adjustments to food intake and energy expenditure to maintain body weight homeostasis. In addition, plasma leptin concentrations have been shown to be related to insulin sensitivity independent of body fat content, suggesting that the hyperleptinemia found in obesity could contribute to the insulin resistance. We investigated the effects of leptin on insulin binding by isolated adipocytes. Adipocytes isolated from Sprague-Dawley rats exhibited a dose-dependent reduction in the uptake of 125I-labelled insulin when incubated with various concentrations of exogenous leptin. For example, addition of 50 nM leptin reduced total insulin binding in isolated adipocytes by 19% (P < 0.05). Analysis of displacement curve binding data suggested that leptin reduced maximal insulin binding in a dose-dependent manner, but had no significant effect on the affinity of insulin for its binding site. We conclude that leptin directly inhibited insulin binding by adipocytes, and the role of leptin in the development of insulin resistance in obese individuals requires further investigation.
J. Orlowski, C. E. Bird, and A. F. Clark
Androgen metabolism and the regulation of rat ventral prostate cell proliferation and secretory function were examined during sexual maturation. Changes in acid phosphatase (AP) characteristics were measured as a marker of androgen-dependent prostatic secretory function. In immature (21-day-old) rats, total AP activity per cell was low (14.2±1.3 mol p-nitrophenol phosphate hydrolysed/h per mg DNA); it increased threefold as the weight, protein and DNA contents of the prostate increased to adult (65-day) levels. This corresponded with significant (P<0.001) increases in the staining intensities of three of the four bands of secretory AP on isoelectric focusing gels. The extent of inhibition of AP by tartrate decreased at the same time. Secretory AP is known to be relatively tartrate-resistant. The changes in AP activity occurred after prostatic 5α-dihydrotestosterone (5α-DHT) levels increased from 4.6 ± 0.7 pmol/mg DNA (21 days) to reach a peak of 17.6±2.3 pmol/mg DNA at 58 days. Prostatic 5α-DHT concentrations were always higher than testosterone levels. Prostatic 5α-androstane-3α,17β-diol (3α-Adiol) levels were lower than 5α-DHT levels except on day 58 when levels peaked dramatically at 26.2±5.5 pmol/mg DNA. Changes in prostatic 5α-DHT and 3α-Adiol levels corresponded with changes in 5α-reductase and 3α-hydroxysteroid oxidoreductase (3α-HSOR) activities. The oxidative reaction of 3α-HSOR was approximately fourfold higher than the reductive reaction, indicating a preference for the formation of 5α-DHT. The plasma levels of testosterone, 5α-DHT and 3α-Adiol cannot account for their respective prostatic levels, indicating the importance of the steroid-metabolizing enzymes in regulating intracellular androgen levels. Changes in the AP characteristics could be correlated with the androgen status of the prostate.
J. Endocr. (1988) 116,81-90
R. G. Clark and I. C. A. F. Robinson
The GH responses to single i.v. injections of GH-releasing factor (GRF) in conscious male rats are highly variable. Although normal male rats show a pulsatile secretory pattern of GH with pulses occurring at intervals of 3–3·5 h, the peaks occur at different times in individual animals. We have compared the GH responses of young conscious male and female rats to multiple i.v. injections of 1 μg human (h) GRF1-29NH2. The peak GH responses occurred 3–5 min after hGRF1-29NH2 injection and were lower in female than in male rats. Both males and females responded uniformly to hGRF1-29NH2 injections given 180 min apart and the GH responses became entrained with no endogenous GH pulsing. Female rats produced consistent GH peaks in response to hGRF1-29NH2 injections at 90-min intervals, whereas male rats responded only to alternate injections, so that GH peaks occurred only every 180 min despite giving GRF every 90 min. When the frequency of hGRF1-29NH2 administration was increased to once every 40 min female rats again responded consistently to each injection. Male rats responded intermittently, being able to respond to two injections 40 min apart, after which they became refractory to hGRF1-29NH2. This cycle of varying sensitivity to GRF in male rats probably underlies their 3-hourly endogenous GH secretory rhythm. Female rats can respond uniformly to repeated GRF injections, consistent with their more continuous pattern of endogenous GH secretion. Introducing a pulse of 10 μg rat GH into a series of hGRF1-29NH2 injections did not induce refractoriness to hGRF1-29NH2, suggesting that GH does not itself desensitize the pituitary to GRF. Whether the different patterns of GH secretion in males and females result from different patterns of GRF and/or somatostatin secretion remains to be determined.
J. Endocr. (1985) 106, 281–289
R. G. Clark and I. C. A. F. Robinson
The 'Little' mouse is characterized by a body growth rate 60% of normal due to a defect in the synthesis and storage of GH in the anterior pituitary gland. We have now investigated the effects of GH releasing factor (GRF) in these mice and in normal animals. The pituitary GH content in Little mice was only 4% of that in normal C57: +/+ mice, and was not affected by twice daily i.p. injections of human (h) GRF1-29NH2 (0·2−2 μg) for 14 days. This treatment also had no effect on body growth. In anaesthetized normal mice, single i.v. injections of 0·1 or 2 μg hGRF1-29NH2 released large amounts of GH into the plasma, whereas this peptide was ineffective in Little mice, whether or not they had been pretreated with GRF. Therefore, although pituitaries of Little mice contain significant amounts of GH, this pool is not releasable by GRF. This suggests that the dwarfism in Little mice may be partly due to a pituitary defect in GRF receptors or their stimulus-secretion coupling, rather than a deficiency in hypothalamic GRF.
J. Endocr. (1985) 106, 1–5
J. Orlowski, C. E. Bird, and A. F. Clark
To study androgen-mediated differentiation in the rat ventral prostate, we separated the two principal cell types (epithelial and stromal) derived from prostates of immature and mature rats on two continuous Percoll gradients. Cells were immediately placed in culture medium. Testosterone metabolism by the two prostatic cell types was evaluated using [3H]testosterone and quantifying the formation of 5α-[3H]dihydrotestosterone (5α-DHT) and 5α-[3H]androstane-(3α or 3β), 17β-diols. In epithelial cells from both immature and mature rat prostates the major testosterone metabolites were 5α-DHT and 5α-androstane-3α, 17β-diol. Stromal cells metabolized less testosterone than did the epithelial cells. Differences in the relative levels of the various metabolites were observed for the two age groups.
To examine in more detail the changes in testosterone metabolism observed in vitro both types of cells and unfractionated cells from immature and mature rat prostates were assayed for testosterone 5α-reductase (using testosterone as substrate) and 3α-hydroxysteroid dehydrogenase (using 5α-DHT as substrate) activities (expressed as pmol substrate reduced/min per 106 cells). In immature rats both 5α-reductase and 3α-hydroxysteroid dehydrogenase activities were localized in the epithelial cell fraction (17 and 52 respectively); stromal cells showed lower 5α-reductase and 3α-hydroxysteroid dehydrogenase activity (4 and 4). Relative to epithelial cells from immature rats epithelial cells from mature rats showed a decrease in 5α-reductase (7) and an increase in 3α-hydroxysteroid dehydrogenase (160) activity while stromal 5α-reductase showed little change (3) and 3α-hydroxysteroid dehydrogenase increased to 22. Because there are more epithelial than stromal cells in the rat prostate, the former can be considered important sites for 5α-reductase and 3α-hydroxysteroid dehydrogenase activities. This contrasts with the human prostate where there is more 5α-reductase activity in the stroma than in the epithelium.
A. J. THODY, R. J. PENNY, DEL CLARK, and CHRISTINE TAYLOR
A sensitive and specific radioimmunoassay for α-melanocyte-stimulating hormone (α-MSH) was developed.
Extracts of the neurointermediate lobe of the rat produced displacement curves which were parallel to those obtained with synthetic α-MSH. The mean immunoreactive a-MSH concentration in neurointermediate lobes from normal adult rats was 2768 ± 200 (s.e.m.) ng/lobe. This accounted for approximately 78% of the MSH activity of the neurointermediate lobe as measured by bioassay. Much lower levels of immunoreactive α-MSH were found in the anterior lobe of the rat. Extracts of rat serum and plasma also contained immunoreactive α-MSH and the mean level was found to be 237 ± 20 pg/ml. This was slightly lower than the level measured in rat plasma by bioassay.
Increased levels of α-MSH were found in plasma of rats 1 and 3 h after a single injection of trifluoperazine and after 1·5 min of ether anaesthesia. These changes were reflected by decreases in the α-MSH content of the neurointermediate lobe.