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J. B. BROWN
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Interest in methoxylated oestrogens has been stimulated by the discovery of 2-methoxyoestrone and 2-methoxyoestriol in human urine (Kraychy & Gallagher, 1957; Fishman & Gallagher, 1958; Loke & Marrian, 1958). Furthermore, the 3-methyl ether of 17α-ethinyloestradiol is widely used as a potent oral oestrogen. The question arises whether the oestrogen methyl ethers can be demethylated in vivo to the parent phenols. To test this possibility, the 3-methyl ethers of oestrone, oestradiol and oestriol have been administered to human subjects, and the resulting outputs of oestrone, oestradiol and oestriol and of their methyl ethers in the urine have been measured.

EXPERIMENTAL PROCEDURES

The methyl ethers were purified by chromatography on alumina and recrystallization from ethanol. Their melting points (uncorrected) were: oestrone methyl ether 172° c; oestradiol methyl ether 97–98° c, 117–118° C; oestriol methyl ether 148–154° c. The pure compounds were dissolved in olive oil (2·5 mg./ml. for oestrone and oestradiol

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A. J. KENNY
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R. R. SAY
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SUMMARY

Acid-ethanol extracts of different regions of the gut in man, dog, rabbit and pig were assayed for glucagon-like activity by the glycogenolytic response of liver slices.

Glucagon-like activity was present in the extracts of human stomach, including the pyloric region, and in the duodenum, jejunum and ileum. No activity was found in liver, spleen and blood. The stomach of the dog yielded higher activities than that of the human. Pig tissues contained little activity.

The properties of the active substance in the human gastro-intestinal tract were those of a peptide or protein in that it was non-diffusible during dialysis, precipitated by ammonium sulphate and destroyed by chymotrypsin. Glucagon behaves in a similar way in these respects.

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N. F. CUNNINGHAM
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SUMMARY

The acid—ethanol extraction procedure described by Baird & Bornstein (1957) has been shown to extract from bovine and ovine blood plasma material having an insulin-like action in vitro in the rat diaphragm assay.

The amounts of insulin activity extracted from plasma of non-pregnant cows and sheep generally indicated plasma insulin levels of from 100–500 μu./ml. plasma. Plasma insulin activity did not appear to be related to plasma glucose level in these animals.

Insulin activities of 100 μu./ml. plasma, or less, were found in two cows soon after parturition, in some pregnant sheep bearing twins round about the time of parturition, in four acetonaemic cows and in three of six sheep with pregnancy toxaemia.

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S. SHUSTER
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SUMMARY

Plasma cortisol concentrations increased after injections of nicotine. This increase was variable and was no greater than that seen after substitution of normal saline for nicotine. Plasma cortisol concentrations did not increase after nicotine in patients with hypopituitarism and after inhibition of corticotrophin release with triamcinolone. It is concluded that the increased plasma cortisol concentration after injection of nicotine was due to non-specific pituitary stimulation associated with the experimental procedure and not due to any direct effect of the nicotine. Nicotine resulted in a similar increase in plasma cortisol in four patients with diabetes insipidus. Neither rapid infusion of hypertonic mannitol nor ingestion of ethanol had a consistent effect on the plasma cortisol concentration. It is therefore concluded that the antidiuretic hormone is not the 'corticotrophin release factor' in man.

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J P McMurtry
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G L Francis
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F Z Upton
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G Rosselot
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D M Brocht
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Abstract

The development of a homologous radioimmunoassay (RIA) for chicken insulin-like growth factor-I (cIGF-I) and its use to investigate the developmental changes in IGF-I in the chicken and turkey is described. A doubleantibody RIA has been developed using recombinantly derived cIGF-I as antigen, radiolabelled tracer and standard. The resulting immunoassay has a minimum detection limit of 0·035 ng and effective dose of 2·5 ng. Dose–response curves of chicken and turkey plasma and tissue extracts were parallel with cIGF-I standard. The antiserum is specific for IGF-I as no cross-reactivity with chicken IGF-II, insulin, glucagon, gastrin or avian pancreatic polypeptide was observed. We have also established that acid/ethanol extraction of chicken and turkey plasma reduced possible interference of IGF-binding proteins (IGFBPs) in the RIA. Comparison of IGF-I immunoactivity in unextracted and acid/ethanol-extracted samples following gel filtration under acidic and neutral conditions indicates that the cIGFBPs may be acid-labile. Analyses of samples from growing chickens and turkeys using the homologous avian reagents revealed higher IGF-I concentrations than if the IGF were quantified using heterologous mammalian-derived reagents. A similar pattern was observed when tissue extracts were assayed for IGF-I content. The application of the homologous RIA to monitor blood and tissue IGF-I levels during embryonic development and posthatch growth in avian species will provide more accurate comparisons of results from studies on the role of IGF-I in growth and metabolism of domestic birds.

Journal of Endocrinology (1994) 142, 225–234

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B. A. Crawford
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J. L. Martin
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C. J. Howe
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D. J. Handelsman
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R. C. Baxter
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ABSTRACT

This study was undertaken to compare various extraction methods for insulin-like growth factor-binding proteins (IGFBPs) from insulin-like growth factor-I (IGF-I) in rat serum systematically, before measurement of IGF-I by radioimmunoassay (RIA). The values obtained in the IGF-I RIA following acid–ethanol (AE), acid-ethanol cryoprecipitation (AEC) and formic acid-acetone (FA) extraction methods were compared with the IGF-I values obtained following high-performance liquid chromatography (HPLC), which was the reference method. Radio-ligand blots were used to determine the pattern and degree of IGFBP removal by these methods.

Over a wide range of circulating IGF-I levels, AE and AEC extraction gave IGF-I levels comparable with those obtained following HPLC. FA extraction resulted in IGF-I levels that were consistently higher (P <0·01) than those obtained following HPLC and gave non-parallel displacement curves in comparison with recombinant IGF-I standards (P <0·01). Ligand blots demonstrated a similar pattern of IGFBP removal among the three methods with almost complete removal of IGFBP-3 but only 30–40% removal of the lower molecular weight IGFBPs. These lower molecular weight IGFBPs did not interfere with the RIA measurements of IGF-I from AE and AEC extracts.

Therefore the AE extraction method of Daughaday, originally validated for use in human serum, is also satisfactory for use in rat serum. The complete removal of IGF-binding activity does not appear essential for accurate measurement of IGF-I by RIA, although this may depend on the specific binding characteristics of the IGF-I antiserum.

Journal of Endocrinology (1992) 134, 169–176

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L Gonzalez
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AI Sotelo
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A Bartke
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D Turyn
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To study the effects of homologous mouse GH (mGH) on the presence and characteristics of serum GH-binding protein (GHBP) we have used transgenic mice expressing GH-releasing hormone (GHRH) as a model. Chromatographic techniques allowed the characterization of GHBP bioactivity, and immunological techniques were used to determine its concentration and molecular components. Chromatographic separation of labeled human GH or mGH cross-linked to serum GHBPs showed two GH-binding serum fractions in normal as well as in transgenic mice serum. SDS-PAGE of this material revealed a specific band of 66 kDa and another higher molecular weight broad band, which, in the presence of 2-mercapto-ethanol, is converted into the 66 kDa fraction.Since normal mice serum has an mGH concentration of 0. 40+/-0.06 nM and a GHBP concentration of 5.7+/-1.1 nM, while the high-affinity site for mGH has a K(d)</+/-27 nM, only a small percentage (2.9%) of total serum mGH is bound to GHBP in the sera of these mice. In transgenic mice serum, where the mGH concentration is 60 times higher (23+/-2.7 nM), 22.5% of total serum mGH is bound to serum GHBP. These values agree with the experimental data (4+/-2% and 17+/-4% for normal and transgenic mice serum respectively).The concentration of GHBP in GHRH transgenic mice was found to be increased four- to tenfold, depending on the technique used. This increment closely resembles the increased concentration of GHBP in the serum of transgenic bovine GH (bGH) mice, in which peripheral bGH levels are grossly elevated. Our results support the idea that the circulating levels of mGH in normal mouse serum are capable of influencing the levels of GHBP in peripheral circulation in a way similar to that of bGH, in spite of the different affinities of these two hormones. The fact that the up-regulation of GHBP occurs, even though a small percentage of mGH is bound in these animals, strongly suggests the existence of a physiological function for GHBP. These results also question some of the assigned or attributed physiological roles of GHBP, at least in the mouse, since only a negligible percentage of total mGH would be prevented from degradation and/or renal filtration by binding to GHBP. This small percentage of bound mGH also invalidates its role as a reservoir or a buffer of mGH concentration during pulses of GH release or rapid changes of mGH levels. Our results also demonstrate the presence of high molecular weight forms of GH-GHBP complexes that could be dissociated by dilution or in the presence of 2-mercapto-ethanol.

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CW Elton
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JS Pennington
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SA Lynch
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FM Carver
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SN Pennington
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Maternal diet during pregnancy has been reported to alter the offspring's ability to respond to a glucose challenge. The current studies report changes in basal and insulin-stimulated, in vitro glucose uptake in red (soleus) and white (extensor digitorum longus) muscle fiber types, as well as whole body insulin responsiveness of adult rat offspring associated with their mother's dietary fat and alcohol content during pregnancy. The offspring of Harlan-derived Sprague-Dawley female rats, dosed during pregnancy with ethanol (ETOH) via a liquid diet (35% of calories as ETOH) with either 12% or 35% of calories as fat, were compared with offspring from litters whose mothers were pair-fed an isocaloric amount of the liquid diet without ETOH. Maternal access to the liquid diets was terminated on day 20 of the pregnancies (sperm plug=day 0). The offspring were surrogate fostered within 48 h of birth to mothers which had consumed commercial chow throughout their pregnancy. Following weaning at 21 days of age, the offspring consumed only commercial rat chow and they were examined over the next 14 months for changes in glucose homeostasis as a consequence of in utero exposure to maternal dietary fat and/or alcohol. The 35% maternal fat diet resulted in both in vivo and in vitro decreases in insulin sensitivity. Thus, compared with adults whose mother's diet contained 12% fat, significant, in vitro muscle and in vivo whole body insulin resistance (measured by hyperinsulinemic-euglycemic clamping) was observed in adult rats whose mothers consumed 35% of dietary calories as fat. The addition of ethanol to the maternal 35% fat diet further reduced the offspring's red muscle tissues in vitro response to insulin, but did not affect whole body insulin sensitivity. Muscle basal and insulin-stimulated receptor tyrosine kinase activity were significantly decreased (approximately -50%) by the 35% fat maternal diet but there was no compensatory increase in serum insulin or glucose levels. Based upon both in vivo and in vitro data, these studies suggested that in utero exposure to 35% fat has a sustained effect on the adult offspring's glucose uptake/insulin sensitivity and that the effect is paralleled, at least in part, by decreased insulin receptor tyrosine kinase activity. In utero ETOH exposure resulted in the loss of basal and insulin-stimulated, in vitro glucose uptake in red muscle fibers but maternal dietary ETOH had no detectable effect on either in vivo insulin sensitivity or muscle tyrosine kinase activity.

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I. Gy. FAZEKAS
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SUMMARY

1. Fresh splenic tissue from pigs was extracted by the method of Swingle & Pfiffner for adrenocortical extracts, 1 ml. being equivalent to 100 g splenic tissue.

2. The material extracted was readily soluble in ethanol, benzene, acetone and water, but not in petroleum ether. It contained no protein, bile pigments, fat, fatty acids or cholesterol, and had marked reducing properties.

3. Administration of the extract to adrenalectomized male mice caused an increase, continuous with increasing dose of extract, in the glycogen content of liver and muscle. The increase in liver glycogen produced by 1·2 ml. extract was about the same as that caused by 2 mg cortisone.

4. In intact rats the extract caused an increase in blood sugar and in the glycogen content of liver and muscle. Cortisone had a similar effect.

5. Neither the splenic extract nor cortisone raised the glycogen content of the brain in adrenalectomized mice or intact rats.

6. Thus the spleen contains a substance which has a glucocorticoid-like effect on the carbohydrate metabolism of adrenalectomized and intact animals. In view of its chemical and biological properties, this substance is thought to be a glucocorticoid originating in the adrenal cortex. A substance with similar effects has been previously detected in liver, brain and muscle.

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P. Laurberg
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J. F. Rehfeld
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ABSTRACT

Cholecystokinin (CCK) is a heterogeneous gut hormone which is also synthetized in extra-intestinal endocrine cells and neurones. In order to examine the possibility that CCK peptides are local modulators of calcitonin secretion, we have studied the structure– activity relationship on calcitonin secretion from perfused canine thyroid lobes as well as the presence and molecular nature of CCK in the thyroid. Peptides containing the intact COOH-terminal tetrapeptide amide of CCK (CCK-4, CCK-5, pentagastrin, CCK-8 and gastrin-17) all induced dose-dependent (0·1, 3 and 100 nmol/l) increases in calcitonin release (P<0·05, n =4) with biphasic secretion during 6-min infusion periods. The deamidated tetrapeptide and the COOHterminal tripeptide were without effect. Gel chromatography of neutral water and acid–ethanol extracts of thyroid tissue, monitored by sequence-specific CCK and gastrin radioimmunoassays, disclosed a variety of CCK and gastrin peptides of which a predominant form resembled small molecular forms like CCK-4 and CCK-5. The presence in the thyroid of small CCK-like peptides and the pronounced effect of such peptides on calcitonin secretion suggest that calcitonin secretion is modulated by local release of small CCK peptides. They could originate from intrathyroidal nerves or from sub-populations of C-cells.

J. Endocr. (1987) 115,77–82

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