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 6·0
2·9, 3·3
0·4 and 3·1
1·3 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. McKinney & Parkinson (1987). Three antibody prep- 5-TCAGCACCCGGGACTGAGG-3. These primers arations were used in this study, #JMCK4 raised against

Journal of Endocrinology (2000) 166, 127–135 www.endocrinology.org

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access Ovine leptin ELISA · K KAUTER and others 129 the carboxyl terminus, #JMCK13 raised against the Leptin assays amino terminus and #JMCK16 raised against bovine A competitive ELISA was developed using #JMCK16 recombinant leptin (brLeptin). which was biotinylated using NHS-LC-Biotin (Pierce). Antisera and purified IgY were screened using an The ELISA plate was coated with 6 ng brLeptin in 50 µl indirect enzyme-linked immunoabsorbent assay (ELISA) 0·1 M bicarbonate buffer, pH 9·0, (binding buffer) over- technique as described below. Approximately equivalent night at 37 C. The plate was blocked with 200 µl 5% molar concentrations of peptides (9 ng/well) and brLeptin skim milk in ELISA buffer for 1 h at 37 C. The sample (100 ng/well) were coated onto the bottom of a flat bot- was added to the well in a volume of 100 µl and the tomed ELISA plate (Greiner Labortechnik, Frickenhausen, ff antiserum in a volume of 50 µl 10% Triton-X 100, 0·5% Germany) in 50 µl 0·1 M bicarbonate, pH 9·0, bu er SDS and 5% sodium deoxycholate, and the plate was (binding buffer). Excess binding sites were blocked with   incubated overnight at 37 C. Streptavidin conjugated to 200 µl 5% skim milk in PBS for 1 h at 37 C. The plate was alkaline phosphatase (Amrad Biotech) was incubated for washed 5 times with 0·9% saline and 0·05% Triton-X 100, 1 h and the plate developed with NPP. The plate was using a Titertek Microplate washer (Labsystems, Helsinki, washed 5 times between each step with a washing buffer Finland). Serial dilutions of antisera or purified IgY in PBS containing 0·9% saline and 0·05% Triton-X 100, using a containing 3·3% Triton-X 100, 1·7% sodium deoxycholate Titertek Microplate washer. and 0·17% sodium dodecyl sulphate (SDS) were added to The cross-reactivity of recombinant human (R&D the ELISA plate in a volume of 100 µl and incubated  Systems, Minneapolis, MN, USA) and mouse (Insight overnight at 37 C. The plate was washed and biotinylated Biotechnology, Wembley, Middlesex, UK) leptin was rabbit anti-chicken IgY (Chemicon, Temecula, CA, USA) ff determined in the assay using doses of up to 100 ng/ml. at a dilution of 1:10 000 in phosphate bu er, pH 8·0, The specificity of the assay was assessed using the following containing 0·5 M sodium chloride, 0·5% Tween-20 and non-specific proteins serially diluted from 10 µg/ml, ovine 0·1% BSA (ELISA buffer) was added to each well  luteinising hormone (NIDDK, Bethesda, MD, USA), and incubated at 37 C for 1 h. The plate was washed and ovine albumin (Sigma), human recombinant follistatin streptavidin conjugated to alkaline phosphatase (Amrad (NIDDK), insulin (Aza Research, West Ryde, NSW, Biotech, Boronia, Vic, Australia) at a dilution of 1:5000  Australia) and equine chorionic gonadotrophin (Horizon was incubated for 1 h at 37 C. The plate was developed Pty Ltd, North Ryde, NSW, Australia). with p-nitrophenylphosphate disodium salt hexahydrate Recoveries of leptin from plasma were determined by (NPP) and read at 405 nm using a Titertek Multiskan Plus adding 5, 10 and 20 ng brLeptin per ml of pooled ewe or microplate reader (Labsystems). wether plasma. All samples were assayed in duplicate with 3 dilutions (neat, ½, ¼) using ELISA buffer as diluent. Western blotting Pools of plasma from adult ewes, rams and castrate males (wethers) were used to determine the degree of parallelism Bovine recombinant leptin (1250 ng and 2500 ng), sub- in the assay. Individual plasma samples from 4 ewes, 5 cutaneous sheep fat extract and biotinylated broad range wethers and 4 rams were assayed to determine the mean molecular size standards (Bio-Rad), were run on 12·5% leptin concentrations in plasma from different sexes. SDS polyacrylamide reducing gels. Separated proteins were transferred to nitrocellulose membranes (0·22 µm, Schleicher and Schuell, Dassel, Germany) at 60 volts with Experimental treatments cooling, in transfer buffer containing 20 mmol l-1 Tris base, 150 mmol l-1 glycine, 20% v/v methanol. At the Adult merino wethers were housed individually in small conclusion of the transfer, the membranes were then pens for 24 h before treatment and starved overnight. blocked for 1 h with 3% non-fat dried milk in 20 mmol l-1 Insulin (1 iu/kg), adrenaline (65 µg/kg) and glucagon Tris–HCl, 0·15 mol l-1 NaCl buffer (TBS), pH 8. The (1 mg/kg) were administered intravenously to groups of membranes were then incubated with primary antiserum 3–4 sheep and blood samples were taken from a jugular #JMCK16 or preimmune serum at a dilution of 1:250 in catheter at 0, 1, 3, 5, 10, 20, 30, 60, 90 and 120 min after 1% non-fat dried milk in 20 mmol l-1 TBS for 24 h, then the injection of hormone. A single bolus dose of glucose washed several times with 1% milk/TBS/0·1% Nonidet (240 mg/kg) was given intravenously and blood samples P-40 detergent (NP-40, Sigma). The second antiserum taken at 0, 15, 30, 45, 60, 120 min post injection. Blood used for both membranes was a biotinylated rabbit anti- was collected into heparinised tubes and plasma stored at  chicken IgY (Chemicon) at a dilution of 1:4000 in 1% –20 C until assayed. milk/TBS/0·1% NP-40 for 24 h, followed by several washes as above and enhancement by avidin alkaline phosphatase conjugate (Pierce, Rockford, IL, USA) at a Data analysis dilution of 1:1000. The membranes were then developed Parallel-line statistics (Finney 1978) and one-way analy- with NBT/BCIP One-Step (Pierce). sis of variance followed by a Student-Newman-Keuls www.endocrinology.org Journal of Endocrinology (2000) 166, 127–135

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access 130 K KAUTER and others · Ovine leptin ELISA

Figure 2 Western blot analysis of brLeptin (lanes 1, 2, 4 and 5) and a subcutaneous fat extract (lanes 3 and 6) using immune (A) and preimmune (B) antibodies from chicken JMCK#16. STD=mw standards

Figure 1 The relative cross-reactivity of antibodies raised against Western blot analysis with a long period of development equivalent molar concentrations of the carboxyl (C-Terminus) and amino (N-Terminus) terminal leptin peptides and rbLeptin in an showed that the brLeptin migrated at 16·7 kDa (Fig. 2A, indirect ELISA to the 3 different antigens. lanes 1 and 2) and that a specific equivalent band (Fig. 2A, lane 3) could be observed in the fat extract. The pre- immune antibodies did not recognise the recombinant multiple range test were performed using the SAS com- leptin (Fig. 2B, lanes 4 and 5) or the putative native leptin puter software package (SAS Institute Inc., Cary, NC, (Fig. 2B, lane 6) recognised by the immune serum. Two USA). Concentration of leptin in samples was determined other bands were observed at 14 kDa and 32 kDa (Fig. 2A, using a four parameter logistic curve fitting algorithm lane 3) which were not present on the preimmune blot option in the Genesis software package (Life Sciences (Fig. 2B, lane 6). International UK, Basingstoke, Hants, UK). The competitive ELISA had a standard curve ranging These experiments were approved by the University of from 100 ng to 0·2 ng/ml with a sensitivity of approxi- New England Animal Ethics Committee and conform to mately 0·5 ng/ml. The inter- and intra-assay variation is the NHMRC/CSIRO/AAC Code of Practice for the 15% and 7% respectively. There was no interference or Care and Use of Animals for Experimental Purposes. cross-reactivity in the assay by any of the non-specific proteins at doses up to 10 µg/ml. The dilution curve from Results a pool of ewe plasma was linear and parallel to the standard curve, as shown in Fig. 3A, as were the pools of ram and Antibodies were successfully raised against recombinant wether plasma. The aqueous extract from subcutaneous fat leptin and the peptides as shown in Fig. 1. Interestingly, also diluted in a linear fashion parallel to the brLeptin the antibodies to the terminal peptides showed very little standard curve (Fig. 3A). Immunoactive leptin was de- cross-reactivity with the brLeptin, while antibodies to tected in extracts from internal fat and muscle tissue, with brLeptin had a poor cross-reactivity to either of the concentrations of 103 pg/mg and 2·4 pg/mg of protein terminal peptides. These data indicate that although respectively. This was much less than that detected in the antibodies were successfully raised against the amino extract from subcutaneous fat (623 pg/mg). The cross- and carboxyl terminus of the bovine leptin molecule, reactivity of recombinant human leptin (Fig. 3B) and these antibodies had a poor cross-reactivity with the mouse leptin was 36·5% and >1% respectively. Recovery intact molecule. Consequently, JMCK#16 was used to of brLeptin from spiked samples of pooled ewe plasma was develop the leptin ELISA as described below. The epitope 95%
3%. The mean (
..) leptin concentrations in recognised by this antibody remains to be determined. plasma samples were 6·0
2·9 ng/ml from ewes (n=4),

Journal of Endocrinology (2000) 166, 127–135 www.endocrinology.org

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access Ovine leptin ELISA · K KAUTER and others 131

Figure 4 The mean
S.E. (n=3) concentration of plasma leptin and glucose after a single injection of insulin (1 iu/kg).

to 167 mg/dl within 10 min (Fig. 5), which remained elevated until near the end of the experiment when it began to decline. Leptin levels (at approximately 4 ng/ml) Figure 3 (A) Competitive ELISA logit dose–response curves of did not change significantly (P=0·95) throughout the brLeptin, ewe plasma pool and an extract from subcutaneous fat experimental period. from a ewe. (B) The dose–response curves of human (hrLeptin) Glucagon stimulated a significant (P<0·001) rise in and mouse (mrLeptin) leptin in the leptin ELISA. blood glucose after the intravenous injection (Fig. 6). Blood glucose levels rose from 63 mg/dl to a maximum of 3·3
0·4 ng/ml from wethers (n=5) and 3·1
1·3 ng/ml 184 mg/dl at 30 min following the injection and began to from rams (n=4). decline by 120 min. No significant change (P=0·75) in The mean leptin and glucose levels from the exper- the concentration of leptin was observed during the imental sheep receiving a bolus intravenous dose of 1 sampling period. iu/kg insulin are shown in Fig. 4. The single injection of In the last experiment, a bolus intravenous injection of insulin induced a significant (P<0·05) decrease in plasma glucose significantly (P<0·001) raised blood glucose from glucose, from 63 mg/dl to 24 mg/dl at 50 min following 72 mg/dl (data not shown) to 207 mg/dl at 15 min post injection. Glucose levels remained depressed at the con- injection followed by a steady decline to normal levels by clusion of blood sampling at 120 min. As shown in Fig. 4, 180 min as shown in Fig. 7. Leptin levels remained no significant change (P=0·5) in leptin levels occurred for unchanged (P=0·92) throughout the experimental period the duration of the experiment. at approximately 3·5 ng/ml. An intravenous injection of adrenaline stimulated a The glucose levels and leptin levels in the control sheep significant (P<0·01) rise in blood glucose from 67 mg/dl which were given an injection of vehicle (saline) showed www.endocrinology.org Journal of Endocrinology (2000) 166, 127–135

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access 132 K KAUTER and others · Ovine leptin ELISA

Figure 5 The mean S.E. (n=4) concentration of plasma leptin and Figure 6 The mean
S.E. (n=4) concentration of plasma leptin glucose after a single intravenous injection of adrenaline and glucose after a single intravenous injection of glucagon (65
g/kg). (1 mg/kg).

no significant change (P=0·9, P=0·9 respectively) throughout the period of blood sampling (data not shown). recombinant molecule. This was an unexpected result since we had used peptide sequences very similar to those used by others (Horn et al. 1996, McGregor et al. 1996). Discussion These data suggest that these peptides are not readily accessible in the intact molecule and that other regions of A number of immunoassays for leptin have been described the molecule should be explored for suitable epitopes to since its recent discovery and the cloning of the gene raise specific antisera to leptin of other species. (Zhang et al. 1994). These assays have used antiserum Using the antisera raised against recombinant bovine raised against the amino (McGregor et al. 1996) or leptin, we successfully developed a specific competitive carboxyl (Horn et al. 1996) terminus of the hormone or the ELISA for bovine leptin. Ovine leptin has a 99% homol- whole protein (Considine et al. 1996). In this study, we ogy to bovine leptin (Dyer et al. 1997) so it is likely that used similar peptides derived from the bovine leptin ovine leptin would have close to 100% cross-reactivity in cDNA sequence (Genbank U50365) and brLeptin to raise the assay. Western blot analysis detects a specific im- antibodies. We were able successfully to raise antibodies munoactive band at 16 kDa which strongly suggests that with suitable titres to both peptides and to the recom- the antibodies recognise native ovine leptin. Two other binant protein; however, there was very little cross- bands were also detected which may represent degraded reactivity with the antisera raised against peptides to the and aggregated molecules or alternatively they may be

Journal of Endocrinology (2000) 166, 127–135 www.endocrinology.org

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access Ovine leptin ELISA · K KAUTER and others 133

interfering in leptin assays (Gavrilova et al. 1997). The leptin assay reported here has included reagents previously shown to dissociate follistatin (an activin binding protein) from activin, allowing the assay of activin in the presence of follistatin (McFarlane et al. 1996). The presence of these reagents in the assay would suggest that binding proteins do not interfere with this assay, although we have yet formally to demonstrate this. The plasma leptin levels in ewes were significantly higher (P<0·05) than those in ram or wether plasma, as has been reported previously in humans (Maffei et al. 1995) and in rodents (Watanobe & Suda 1999). However, no significant difference in leptin concentrations was observed between ram and wether plasma. This would suggest that the lower plasma leptin concentrations seen in ram plasma are not due to testosterone as has been suggested in humans (Rosenbaum et al. 1996, Jockenhovel et al. 1997, Roemmich et al. 1998). There is some evidence that oestradiol increases leptin production in adipose tissue (Casabiell et al. 1998) and in the plasma of transsexual men treated with oestradiol together with an androgen antagonist (Elbers et al. 1997). These studies, together with the data presented here, suggest that the higher leptin levels in ewe plasma may be due to oestrogen rather than that the lower levels seen in rams are due to testosterone. However, further work is required to con- firm this hypothesis and we are currently investigating this possibility. Using this assay, we investigated the effect of blood glucose and various regulators of blood glucose on leptin concentrations in the sheep. These preliminary results indicate that glucose is not an important short-term regulator of plasma leptin levels in sheep and consequently
Figure 7 The mean S.E. (n=4) concentration of plasma leptin and hormones that affect blood glucose do not influence leptin glucose after a bolus intravenous dose of glucose (240 mg/kg). levels. In this respect, sheep and humans are similar and contrast with mice where insulin directly influences adipocytes to secrete leptin both in vitro (Leroy et al. 1996) related molecules. Further studies are required to deter- and in vivo (Saladin et al. 1995). The fact that leptin levels mine the nature of these other proteins. Dose–response in sheep do not seem to respond to changes in blood curves of immunoactive leptin in plasma and fat extracts glucose concentration is not surprising as ruminants derive were linear and parallel to the brLeptin standard curve, the majority of their energy from bacterial fermentation and the quantitative recovery of recombinant leptin from products in the form of fatty acids. Therefore, it remains spiked plasma samples suggest that the assay reported here possible that plasma leptin levels may be modulated by is measuring ovine leptin. Our data indicate that sub- fatty acids. cutaneous fat extracts contain more leptin protein than In the sheep, plasma leptin levels may be involved internal fat, supporting results from several human studies in signalling body fat/nutrient stores rather than in modu- showing that the expression of leptin mRNA is higher in lating short-term changes in blood glucose levels, when subcutaneous than omental fat (Montague et al. 1997). leptin concentration appears to be static. Ball et al. (1998) Several different molecular weight species of leptin- observed differences between sheep in the pattern of binding proteins have been identified in both human and conservation of body tissue during feed restriction, with mouse serum (Houseknecht et al. 1996, Sinha et al. 1996, mobilization of fat being the most variable. If leptin is Gavrilova et al. 1997). One of these has been isolated, and involved in changes of energy expenditure in the sheep, after amino acid sequencing it was shown to be a form of variation in leptin concentration may be associated with the leptin receptor (Gavrilova et al. 1997). The plasma differences in an animal’s ability to cope with nutritional concentration of this soluble form of the receptor, which stress. This possible link between leptin concentration and increases during pregnancy in mice, was reported as nutritional stress in sheep remains to be explored. www.endocrinology.org Journal of Endocrinology (2000) 166, 127–135

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access 134 K KAUTER and others · Ovine leptin ELISA

References Leroy P, Dessolin S, Villageois P, Moon BC, Friedman JM, Ailhaud G & Dani C 1996 Expression of ob gene in adipose cells – regulation by insulin. Journal of Biological Chemistry 271 2365–2368. Ball AJ, Thompson JM, Alston CL, Blakely AR & Hinch GN 1998 Changes in maintenance energy requirements of mature sheep fed McFarlane JR, Foulds LM, Pisciotta A, Robertson DM & de Kretser at different levels of feed intake at maintenance, during weight loss DM 1996 Measurement of activin in biological fluids by and realimentation. Livestock Production Science 53 191–204. radioimmunoassay, utilizing dissociating agents to remove the interference of follistatin. European Journal of Endocrinology 134 Bauman WA, Spungen AM, Zhong YG & Mobbs CV 1996 Plasma 481–489. leptin is directly related to body adiposity in subjects with spinal McGregor GP, Desaga JF, Ehlenz C, Fischer A, Heese F, Hegele A, cord injury. Hormone and Metabolic Research 28 732–736. Laemmer C, PeiserC&LangRE1996Radioimmunological Bidwell CA, Ji S, Frank GR, Cornelius SG, Willis GM & Spurlock measurement of leptin in plasma of obese and diabetic human ME 1997 Cloning and expression of the porcine obese gene. subjects. Endocrinology 137 1501–1504. Animal Biotechnology 8 191–206. McKinney MM & Parkinson A 1987 A simple, non-chromatographic Casabiell X, Pineiro V, Peino R, Lage M, Camina J, Gallego R, ff procedure to purify immunoglobulins from serum and ascites fluid. Vallejo LG, DieguezC&Casanueva FF 1998 Gender di erences Journal of Immunological Methods 96 271–278. in both spontaneous and stimulated leptin secretion by human Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei omental adipose tissue in vitro: dexamethasone and estradiol H, Kim S, Lallone R, Ranganathan S, Kern PA & Friedman JM stimulate leptin release in women, but not in men. Journal of 1995 Leptin levels in human and rodent: measurement of plasma Clinical Endocrinology and Metabolism 83 2149–2155. leptin and ob RNA in obese and weight-reduced subjects. Nature Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens Medicine 1 1155–1161. TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL Mizuno TM, Bergen H, Funabashi T, Kleopoulos SP, Zhong YG, & Caro JF 1996 Serum immunoreactive-leptin concentrations in Bauman WA & Mobbs CV 1996 Obese gene expression – normal-weight and obese humans. New England Journal of Medicine reduction by fasting and stimulation by insulin and glucose in lean 334 292–295. mice, and persistent elevation in acquired (diet-induced) and Dyer CJ, Simmons JM, Matteri RL & Keisler DH 1997 cDNA genetic (yellow agouti) obesity. PNAS 93 3434–3438. cloning and tissue-specific gene expression of ovine leptin, Montague CT, Prins JB, Sanders L, Digby JE & O’Rahilly S 1997 NPY-Y1 receptor, and NPY-Y2 receptor. Domestic Animal ff Endocrinology 14 295–303. Depot- and sex-specificdi erences in human leptin mRNA expression: implications for the control of regional fat distribution. Elbers JM, Asscheman H, Seidell JC, Frolich M, Meinders AE & Diabetes 46 342–347. Gooren LJ 1997 Reversal of the sex difference in serum leptin levels upon cross-sex hormone administration in transsexuals. Journal RamsayTG,YanX&Morrison C 1998 The obesity gene in swine: of Clinical Endocrinology and Metabolism 82 3267–3270. sequence and expression of porcine leptin. Journal of Animal Science 76 484–490. Finney DJ 1978 Statistical Methods in Biological Assay, edn 3. London: Griffin. Roemmich JN, Clark PA, Berr SS, Mai V, Mantzoros CS, Flier JS, ff Frederich RC, Hamann A, Anderson S, Lollmann B, Lowell BB & WeltmanA&RogolAD1998Genderdi erences in leptin levels Flier JS 1995 Leptin levels reflect body lipid content in mice – during puberty are related to the subcutaneous fat depot and sex evidence for diet-induced resistance to leptin action. Nature steroids. American Journal of Physiology 275 E543–E551. Medicine 1 1311–1314. Rosenbaum M, Nicolson M, Hirsch J, Heymsfield SB, Gallagher D, ff Friedman JM, Leibel RL, Siegel DS, WalshJ&BaharyN1991 ChuF&Leibel RL 1996 E ects of gender, body composition, and Molecular mapping of the mouse ob mutation. Genomics 11 menopause on plasma concentrations of leptin. Journal of Clinical 1054–1062. Endocrinology and Metabolism 81 3424–3427. Gavrilova O, Barr V, Marcus-SamuelsB&Reitman M 1997 Saladin R, Devos P, Guerremillo M, Leturque A, Girard J, Staels B Hyperleptinemia of pregnancy associated with the appearance of a & Auwerx J 1995 Transient increase in obese gene expression after circulating form of the leptin receptor. Journal of Biological Chemistry food intake or insulin administration. Nature 377 527–529. 272 30546–30551. Sinha MK, Opentanova I, Ohannesian JP, Kolaczynski JW, Heiman Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz ML, Hale J, Becker GW, Bowsher RR, Stephens TW & Caro JF D, Lallone RL, Burley SK & Friedman JM 1995 Weight-reducing 1996 Evidence of free and bound leptin in human circulation – effects of the plasma protein encoded by the obese gene (see studies in lean and obese subjects and during short-term fasting. comments). Science 269 543–546. Journal of Clinical Investigation 98 1277–1282. HardieLJ,RaynerDV,HolmesS&Trayhurn P 1996 Circulating Tartaglia LA, Dembski M, Weng X, Deng NH, Culpepper J, Devos leptin levels are modulated by fasting, cold exposure and insulin R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, administration in lean but not Zucker (fa/fa) rats as measured by Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, ELISA. Biochemical and Biophysical Research Communications 223 Monroe CA & Tepper RI 1995 Identification and expression 660–665. cloning of a leptin receptor, ob-r. Cell 83 1263–1271. Horn R, Geldszus R, Potter E, von zur MuhlenA&BrabantG1996 WatanobeH&SudaT1999Adetailed study on the role of sex Radioimmunoassay for the detection of leptin in human serum. steroid milieu in determining plasma leptin concentrations in adult Experimental and Clinical Endocrinology and Diabetes 104 454–458. male and female rats. Biochemical and Biophysical Research Houseknecht KL, Mantzoros CS, Kuliawat R, Hadro E, Flier JS & Communications 259 56–59. Kahn BB 1996 Evidence for leptin binding to proteins in serum of Widjaja A, Stratton IM, Horn R, Holman RR, TurnerR&Brabant rodents and humans: modulation with obesity. Diabetes 45 G 1997 UKPDS 20: plasma leptin, obesity, and plasma insulin in 1638–1643. type 2 diabetic subjects. Journal of Clinical Endocrinology and Ji S, Willis GM, Scott RR & Spurlock ME 1998 Partial cloning and Metabolism 82 654–657. expression of the bovine leptin gene. Animal Biotechnology 9 1–14. Zhang YY, Proenca R, MaffeiM,BaroneM,LeopoldL&Friedman Jockenhovel F, Blum WF, Vogel E, Englaro P, Muller-Wieland D, JM 1994 Positional cloning of the mouse obese gene and its human Reinwein D, Rascher W & Krone W 1997 Testosterone sub- homologue. Nature 372 425–432. stitution normalizes elevated serum leptin levels in hypogonadal Zhang F, Basinski MB, Beals JM, Briggs SL, Churgay LM, Clawson men. Journal of Clinical Endocrinology and Metabolism 82 2510–2513. DK, DiMarchi RD, Furman TC, Hale JE, Hsiung HM, Schoner

Journal of Endocrinology (2000) 166, 127–135 www.endocrinology.org

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access Ovine leptin ELISA · K KAUTER and others 135

BE, Smith DP, Zhang XY, Wery JP & Schevitz RW 1997 Crystal Received 10 September 1999 structure of the obese protein leptin-E100. Nature 387 206–209. Revised manuscript received 21 February 2000 Zimmet PZ & Collier GR 1996 Of mice and (wo)men - the obesity (ob) gene, its product, leptin, and obesity. Medical Journal of Australia Accepted 14 March 2000 164 393–394.

www.endocrinology.org Journal of Endocrinology (2000) 166, 127–135

Downloaded from Bioscientifica.com at 09/27/2021 09:47:54AM via free access