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D. R. LANGSLOW and C. N. HALES

SUMMARY

The effects on lipolysis of various compounds have been studied in intact chicken adipose tissue and in isolated fat cells prepared from chicken adipose tissue. Glucagon stimulated lipolysis at concentrations down to 1 ng./ml. in intact pieces and 0·1 ng./ml. in isolated fat cells. The effect was enhanced by high concentrations of insulin. No anti-lipolytic effect of insulin was observed. Adrenaline, noradrenaline, porcine corticotrophin (ACTH) and long-acting ACTH were lipolytic but the effects were small and high concentrations were required. The adrenaline effect was blocked by propranolol hydrochloride. Dibutyryl 3′,5′-(cyclic)-AMP and theophylline stimulated lipolysis as did a combination of crude chicken growth hormone and hydrocortisone sodium succinate. It was concluded that the pattern of response of chicken adipose tissue was markedly different from that of the rat.

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T. W. BURNS, C. N. HALES and A. STOCKELL HARTREE

SUMMARY

Human sera and fractions derived from human pituitary tissue have been tested for lipolytic activity using intact epididymal fat pads of the rat and isolated cell suspensions prepared from rat fat pads and from human adipopose tissue.

(1) Glycerol release was enhanced when human serum was incubated with epididymal fat pads and with isolated rat adipose tissue cells. No difference in this effect was noted between fasting serum and serum obtained 30 min. after glucose administration.

(2) The three primary fractions of pituitary powder were assayed for lipolytic activity with rat cells, the one containing the gonadotrophins and thyroid-stimulating hormone (TSH) was highly potent while those containing human growth hormone (HGH) and corticotrophin respectively were inactive. The lipolytic activity was associated with TSH but not entirely due to TSH.

(3) Human cells were prepared from adipose tissue from both sexes, from ages 4 to 78 and from subcutaneous, perirenal and intra-abdominal sites.

(4) Compared with rat cells, human cells were less sensitive and less consistent in their pattern of response. Human cells reacted poorly or not at all to large doses of HGH while rat cells react to small doses.

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CAROL READHEAD, G. M. ADDISON, C. N. HALES and H. LEHMANN

SUMMARY

An immunoradiometric assay for human follicle-stimulating hormone (FSH) is described. A non-specific antiserum to human FSH was purified by incubation of the antiserum with human luteinizing hormone and thyroid-stimulating hormone before its reaction with an immunoadsorbent containing human FSH. The specific antibodies to human FSH isolated on immunoadsorbent were iodinated and used for the FSH assay. The sensitivity of the assay was improved by the extraction of plasma FSH onto antibody-coated polyethylene tubes before its reaction with iodinated FSH antibodies producing a two-site assay. Optimal conditions for the preparation and use of antibody-coated tubes in such a procedure are described. Using serial extraction a sensitivity of 11 pg/ml (0·1 mu./ml) was achieved. There was good agreement between the results obtained with this method and those obtained with radioimmunoassay. The advantages of the increased sensitivity of this new method are discussed.

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ANNE STOCKELL HARTREE, D. R. LANGSLOW and C. N. HALES

Because of a misunderstanding, the information on chicken pituitary fractions provided by one of us (A.S.H) was erroneously presented in a paper by two of us (Langslow & Hales, 1969). Figure 1 gives an accurate representation of the purification scheme that was employed together with further information on the pituitary fractions that were used.

The designation of certain fractions as 'GTN residue' and 'GTN supernate' (Langslow & Hales, 1969) is misleading since there is no evidence that either fraction contains significant amounts of glycoprotein or of gonadotrophic activity. The fraction designated GTN precipitate contains follicle-stimulating and luteinizing hormone activities (Stockell Hartree & Cunningham, 1969) and there is suggestive evidence that thyrotrophin is also present (Mitchell, 1967).

The fraction designated crude 'growth hormone' might be expected to contain growth-promoting activity by analogy with corresponding fractions from human and horse pituitaries (Stockell Hartree, 1966; Stockell Hartree, Mills, Welch & Thomas, 1968). However,

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N. C. Sturgess, M. L. J. Ashford, C. A. Carrington and C. N. Hales

ABSTRACT

Using the patch-clamp technique we observed three distinct classes of K+ channels which were spontaneously active in excised 'inside-out' membrane patches from an insulin-secreting rat pancreatic islet cell line (CRI-G1). Two of these occurred infrequently, one with a conductance of approximately 7 pS, and the other a conductance of 220 pS. The activation of the 220 pS K+ channel was dependent upon the membrane voltage and was sensitive to the concentration of calcium ions at the cytoplasmic surface of the membrane. The third, and by far the most common class of K+ channel, was characterized by its sensitivity to ATP. Application of ATP to the cytoplasmic side of the membrane reversibly inhibited this K+ channel in a dose-dependent manner, but had no effect when applied to the external side. The properties of the ATP-sensitive K+ channel appear to be indistinguishable from those of a channel found in rat neonatal β cells. Thus this insulin-secreting cell line should prove valuable in the investigation of the role of K+ channels in the regulation of insulin secretion.

J. Endocr. (1986) 109, 201–207

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G. M. ADDISON, C. N. HALES, J. S. WOODHEAD and J. L. H. O'RIORDAN

SUMMARY

An immunoradiometric assay for parathyroid hormone has been developed. Antisera to bovine parathyroid hormone (BPTH) were screened for their ability to bind BPTH and human parathyroid hormone (HPTH). A BPTH-immunoadsorbent was used to extract antibodies from an antiserum which did not discriminate between BPTH and HPTH in a standard radioimmunoassay. These antibodies were labelled with 125I for use in the immunoradiometric assay. With this system as little as 5 pg BPTH and 8 pg HPTH could be detected. The serum concentration of BPTH was shown to rise in a cow rendered hypocalcaemic by an infusion of EDTA. Sera from patients with hyperparathyroidism contained high concentrations of hormone. These sera were found to dilute-out parallel to calibration curves obtained using HPTH extracted from parathyroid adenomata. The advantages of this method over the standard radioimmunoassay are discussed.

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C. A. Carrington, E. D. Rubery, E. C. Pearson and C. N. Hales

ABSTRACT

Five cell lines have been derived from a rat transplantable islet cell tumour using two different methods. The lines differ in morphology and contain and release different amounts of insulin and glucagon (insulin content, 1–90 pmol/106 cells; insulin release, 6–250 pmol/106 cells per 24 h; glucagon content, < 0·005–35 pmol/106 cells; glucagon release, < 0·05– 10 pmol/106 cells per 24 h). All the lines responded to the presence of the secretagogues leucine (20 mmol/l) plus theophylline (5 mmol/l) by increasing the rate of release of insulin approximately twofold. A high extracellular concentration of potassium (40 mmol/l) caused a three- to tenfold calcium-dependent increase in release of insulin and a parallel release of glucagon. Increasing the concentration of glucose from 2·8 to 16·7 mmol/l did not alter the rate of insulin release by any of the cell lines.

J. Endocr. (1986) 109, 193–200

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D. R. LANGSLOW, E. J. BUTLER, C. N. HALES and A. W. PEARSON

SUMMARY

The relationships between plasma insulin, glucose, non-esterified fatty acid (NEFA) and α-amino nitrogen concentrations in the domestic fowl have been studied. During a 72-hr. fast the plasma glucose concentration fell while the NEFA concentration rose but there was no change in plasma insulin concentration. Both oral and intracardiac glucose increased the plasma insulin concentration and lowered the plasma NEFA and α-amino nitrogen concentrations. Oral amino acids increased plasma insulin and glucose concentrations but had no effect on plasma NEFA. Intracardiac ox insulin depressed plasma glucose and α-amino nitrogen and increased the plasma NEFA concentration. Intracardiac glucagon increased both plasma glucose and NEFA and depressed the plasma α-amino nitrogen concentrations but had no significant effect on plasma insulin. Intracardiac adrenaline had no effect on plasma NEFA but increased plasma glucose concentration and caused a small depression in plasma insulin concentration.

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P. L. STORRING, T. W. BURNS, BRIDGET E. FURNIVAL, C. N. HALES, P. LANGLEY and ANNE STOCKELL HARTREE

SUMMARY

Attempts to isolate, free of thyroid-stimulating hormone (TSH), the peptide(s) responsible for the lipolytic activity of a fraction of acetone-dried human pituitary powder are described. It was not found possible to separate the lipolytic and TSH activities, even by isoelectric focusing which gave fractions consisting predominantly of single protein components. In the four lipolytic fractions isolated by electrofocusing, the two activities paralleled one another. It is therefore concluded that the TSH present is responsible for the lipolytic activity of this human pituitary fraction, though some evidence suggests that luteinizing hormone (LH), which is a minor contaminant, might also be lipolytic.

The minimum effective lipolytic concentration of the most potent fraction isolated was in the range 0·1–1·0 μg/ml. This is far in excess of plasma concentrations of TSH or LH encountered physiologically. Hence the lipolytic action of these molecules is unlikely to be of physiological significance.

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P. MARY COTES, MARJORIE V. MUSSETT, I. BERRYMAN, R. EKINS, S. GLOVER, N. HALES, W. M. HUNTER, CLARA LOWY, R. W. J. NEVILLE, E. SAMOLS and PATRICIA M. WOODWARD

SUMMARY

Results of a collaborative study in which the insulin concentrations of a number of plasma samples were estimated in several laboratories are reported. They indicate that: (1) laboratories agreed on the ranking of plasma samples according to insulin content; (2) the reproducibility of replicate estimates obtained in the same assay usually overestimated the reproducibility of estimates made on different occasions; (3) values reported for the insulin concentration of the same plasma examined in each of a number of laboratories were likely to range from about one half to twice the inter-laboratory mean estimate.

Possible reasons for the variability of estimates are discussed.