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Adrenaline, Insulin and Glucagon Do Not Have Acute Effects on Plasma Leptin Levels in Sheep: Development and Characterisation of an Ovine Leptin ELISA

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Adrenaline, Insulin and Glucagon Do Not Have Acute Effects on Plasma Leptin Levels in Sheep: Development and Characterisation of an Ovine Leptin ELISA 127 Adrenaline, insulin and glucagon do not have acute effects on plasma leptin levels in sheep: development and characterisation of an ovine leptin ELISA K Kauter, M Ball, P Kearney, R Tellam1 and J R McFarlane Physiology, University of New England, Armidale, New South Wales 2351, Australia 1Division of Tropical Agriculture, CSIRO, Long Pocket Laboratories, Indooroopilly, Queensland 4068, Australia (Requests for offprints should be addressed to J R McFarlane; Email: [email protected]) Abstract Leptin, a recently discovered hormone secreted mainly human recombinant leptin was 36·5%, while mouse leptin from adipose tissue, was first described as a regulator of showed no cross-reactivity. Plasma samples from ewes, adiposity, food intake and energy metabolism. It is now male castrate animals and rams (n=4–5) diluted in parallel apparent that leptin physiology is much more complex and to the standard with mean leptin concentrations of is likely to play an important role in many other systems 6·02·9, 3·30·4 and 3·11·3 ng/ml respectively. including reproduction, haematopoiesis and immunity. Leptin levels in rams were significantly lower than Leptin levels have been shown to be well correlated with in ewes. The non-significant difference in leptin levels body fat in both humans and rodents, with administration between rams and male castrate animals suggests that of exogenous leptin to rats and mice resulting in loss of testosterone may not be responsible for the lower levels of body fat. Leptin is, therefore, likely to be an important leptin. humoral signal to the central nervous system on body Four groups of 3–4 ewes were given intravenous insulin composition and regulation of food consumption. (1 iu/kg), adrenaline (65 µg/kg), glucagon (24 iu/kg) or Due to the limited cross-reactivity of leptin from other saline. Blood samples were taken at 1, 3, 5, 10, 20, 30, 60, species in the current assays for leptin, physiological 90 and 120 min after injection. As expected, glucose levels research on leptin has, to a large extent, been restricted to declined within 10 min of the insulin injection and rose rodents and humans. The aim of this study was to develop after 3 min following both adrenaline and glucagon a leptin immunoassay suitable for use with sheep, enabling injections. Leptin levels, however, remained relatively the investigation of the basic physiology of leptin in an unchanged for the 2 h following the treatments. Finally, a animal larger than rats or mice, thus allowing repeated bolus intravenous dose of glucose (240 mg/kg) was given blood sampling. Using this assay we investigated the and sequential blood samples taken. Despite plasma glu- short-term effects of insulin, adrenaline and glucagon (all cose levels rising to over 200 mg/dl, leptin levels did not modulators of blood glucose) on plasma leptin levels. significantly change over the three hours following treat- Antiserum to bovine recombinant leptin (brLeptin) ment. These data indicate that plasma leptin levels in raised in chickens was used to develop a competitive sheep, in contrast to rodents, are not responsive to ELISA. Using brLeptin as standard, the assay has a short-term changes in blood glucose or insulin, as has been sensitivity of 0·5 ng/ml with inter- and intra-assay vari- shown in humans. ation of 15% and 7% respectively. The cross-reactivity of Journal of Endocrinology (2000) 166, 127–135 Introduction which appears to be expressed primarily in adipose tissue and encodes a protein of approximately 16 kDa. There is Zhang and co-workers (1994) recently reported the dis- no known close homology with leptin and other proteins covery of leptin, a hormone secreted primarily by adipose but it is structurally similar to the long chain helical tissue. This report, together with the identification of its cytokine family (Zhang et al. 1997), with the homology of receptor in the hypothalamus and other peripheral tissues the derived leptin amino acid sequence across the different (Tartaglia et al. 1995), has provided important new infor- species ranging from 95% to 67% (Zhang et al. 1997). In mation to help unravel some of the gaps in our current the ob/ob strain of mice, a non-functional leptin gene knowledge on the recognition of nutritional status. The results in the mutant mice having an increased food intake, leptin gene encodes a 4·5 kb mRNA (Zhang et al. 1994), reduced energy expenditure and elevated insulin and Journal of Endocrinology (2000) 166, 127–135 Online version via http://www.endocrinology.org 0022–0795/00/0166–0127 2000 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access 128 K KAUTER and others · Ovine leptin ELISA cortisol levels which result in obesity, sterility and non- were used to amplify DNA from bovine fat cDNA. The insulin dependent diabetes mellitus (Friedman et al. 1991). amplified DNA of approximately 440 bp corresponded to These effects can be reversed with the administration of the mature form of bovine leptin (i.e. without the region exogenous leptin (Halaas et al. 1995). coding for the signal sequence) as determined by sequenc- Although it has been shown that plasma leptin levels are ing both strands. This DNA was subcloned into the highly correlated with body fat in humans (Considine et al. expression vector pQE9 (QIAGEN Pty Ltd, Cliffton Hill, 1996, Zimmet & Collier 1996) and rodents (Frederich Vic, Australia) and recombinant leptin containing an et al. 1995, Maffei et al. 1995), the mechanisms which N-terminal hexaHis-affinity tag expressed in E. coli. The modulate leptin levels remain unclear. Insulin has been protein was affinity purified by nickel nitrilotriacetic acid shown to stimulate leptin secretion by rat adipocytes both (Ni-NTA) affinity chromatography according to the in vitro (Leroy et al. 1996) and in vivo (Hardie et al. 1996). manufacturer’s instructions (QIAGEN). The purified In humans, although correlations between leptin concen- hexaHis-leptin was dialysed against 20 mM Tris–HCl, trations and insulin concentrations have been reported 500 mM NaCl, 8 M urea, pH 8·5, and then diluted (Rosenbaum et al. 1996, Widjaja et al. 1997), short-term dropwise to a final protein concentration of 50 µg/ml into insulin infusions fail to modify leptin mRNA expression, 20 mM Tris–HCl, 500 mM NaCl, 2 M urea, pH 8·0. The whereas in mice a single injection of insulin increases protein was allowed to refold in this buffer for 18 h at 4 C mRNA (Saladin et al. 1995, Mizuno et al. 1996). A single and afterwards dialysed into 40 mM Tris–HCl, 140 mM injection of glucose also results in elevation of leptin NaCl, pH 8·0. The authenticity of the purified hexaHis- mRNA in mice (Mizuno et al. 1996), while in humans, leptin was verified by its relative size on SDS-PAGE and glucose does not appear to modulate leptin levels (Bauman amino-terminal amino acid sequencing using an Applied et al. 1996). Biosystems 471A protein sequencer (Foster City, CA, Recently, mRNA encoding leptin has been isolated in USA). pig (Bidwell et al. 1997, Ramsay et al. 1998), sheep (Dyer et al. 1997) and cattle (Ji et al. 1998) adipose tissue. These studies showed that breed, stage of growth, feed intake and Tissue extracts body fat were correlated with leptin mRNA expression. The majority of the current information on leptin, how- Fresh subcutaneous and internal visceral fat and muscle ever, is restricted to work on rodents and studies of human tissue was collected from two euthanased ewes and stored obesity, partly due to the unavailability of suitable assays at –20 C. The tissues were homogenised in phosphate for leptin from other species. buffered saline (PBS), pH 7·5, containing 10 mM benza- The aim of this study was to develop an assay capable of midine (Sigma Chemical Company, St Louis, MO, USA) measuring leptin in sheep and to investigate the effects of and 10 mM EDTA. The homogenate was then centri- glucose and glucose modulating hormones on the short- fuged at 10 000g using a Sorval SS-34 fixed angle rotor term regulation of plasma leptin concentrations in sheep. (Dupont Medical Products, Newtown, CT, USA) for 30 min at 4 C. The supernatant was retained and concen- trated 10 times using an Amicon stirred cell concentrator Materials and Methods (Amicon, Beverly, MA, USA) and a PM10 (Amicon) membrane. The protein content of the extracts was then Leptin peptides determined using the Bio-Rad DC protein kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine immuno- An amino terminus peptide 6–14, VQDDTKTLIKT(C) globulin (Ig) G as the standard. The concentrate was stored and a carboxyl terminus peptide 124–135 (C)VALSR- at –20 C until analysed. LQGSLQD were selected, based on the bovine leptin sequence (Genbank accession U50365), and synthesised by Chiron Mimotopes (Clayton, Victoria, Australia). Antibodies Antibodies were raised in female chickens, which were Recombinant bovine leptin immunised with 200 nM peptide conjugated to diphtheria Two oligonucleotide primers were synthesised based on toxoid or with 100 µg recombinant bovine leptin. Blood the known genomic sequence of the bovine leptin gene was initially collected to assess titres, followed by eggs (Genbank accession U50365). The first primer, a sense when suitable titres had been achieved. IgY was isolated primer, corresponded to the 5 end of the mature protein from the egg yolk of preimmune and immune chickens sequence i.e. 5-GTGCCCATCCGCAAGGTCC-3. using a combination of caprylic acid and ammonium The second primer was a reverse sense primer based in sulphate precipitation as previously described by the immediate 3 UTR following the stop codon i.e.
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