www.elsevier.com/locate/livprodsci

Livestock Production Science 87 (2004) 189–195

Growth hormone induced alterations of leptin serum concentrations

in dairy cows as measured by a novel enzyme immunoassay

H. Sauerweina,*, U. Heintgesa, M. Henniesa, T. Selhorstb, A. Daxenbergerc,1

a Institute of Physiology, Biochemistry and Animal Hygiene, Bonn University, Katzenburgweg 7-9, 53115 Bonn, GermanybFederal Research Center for Virus Diseases, Institute of Epidemiology, Seestrasse 55, 16868 Wusterhausen, Germany

c Institute of Physiology, Technical University Munich, Weihenstephaner Berg 3, 85354 Freising, Germany

Received 6 June 2002; received in revised form 29 July 2003; accepted 7 August 2003

Abstract

The present knowledge on the interrelationship between leptin and the somatotropic axis in livestock, in particular the effects

of growth hormone (GH) treatment on leptin secretion is inconsistent. We therefore aimed to characterise GH-induced

alterations of blood leptin concentrations in dairy cows and first developed and validated a competitive enzyme immunoassay

(EIA). The assay is valid in cattle, sheep, goat, pig and in horses. Lactating cows were injected once with 500 mg depot-

formulated bovine GH and blood samples were drawn every second day 2 weeks before and 4 weeks after the GH injections

and were assayed for leptin and for insulin-like growth factor-1 (IGF-1). Leptin concentrations were decreased in response to

the GH treatment solely in pregnant cows ( p < 0.05), whereas IGF-1 concentrations were increased independently of pregnancy

status. In conclusion, the response of leptin blood concentrations towards GH treatment in lactating cows might depend from

endocrine and metabolic cues related to pregnancy.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Leptin; Enzyme immunoassay; Growth hormone; IGF-1; Ruminant; Pig; Horse

1. Introduction and energy expenditure (Houseknecht and Portocar-

Leptin, the first of several recently discovered

adipocyte proteins, putatively signals the extent of

fat energy reserves to the hypothalamus and is thus

involved in the coordination of the metabolic, endo-

crine and behavioural systems regulating food intake

0301-6226/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.livprodsci.2003.08.001

* Corresponding author. Tel.: +49-228-732804; fax: +49-228-

737938.

E-mail address: sauerwein@uni-bonn.de (H. Sauerwein).1 Present address: VITACERT GmbH, Westendstrasse 199,

80686 Munchen, Germany.

rero, 1998; Ahima and Flier, 2000; Vernon et al.,

2001; Ingvartsen and Boisclair, 2001). Besides its

central effects, leptin might also exert peripheral

effects in a number of different tissues, both acting

in an endocrine or a paracrine/autocrine manner, e.g.

in skeletal muscle (Bates et al., 2002), ovary (Spicer,

2001) or adipose tissue itself (Newby et al., 2001).

The functions demonstrated in man and in rodents for

leptin in appetite regulation, lean and fat deposition

and also reproduction are of crucial interest not only

for human medicine but also for animal science. For

dairy as well as for meat production, most of the

H. Sauerwein et al. / Livestock Production Science 87 (2004) 189–195190

research on the endocrine regulatory mechanisms

involved was focused on steroid hormones, somato-

tropic and lactogenic hormones. The exogenous ap-

plication of some of these hormones has evolved a

relatively detailed understanding of their respective

physiological role during the past decades. For live-

stock species, the position of leptin within these

regulative systems could not be investigated properly

until the first species specific assay became available

in 2000 (Delavaud et al., 2000). Since then, the

number of publications dealing with leptin in farm

animals has rapidly increased and several groups have

developed adequate assay systems by now (for review

see Chilliard et al., 2001). Most of the studies on

leptin have focused on the effects of various levels of

energy intake; less is known about the role of leptin

within the hormonal system of the somatotropic axis,

i.e. the growth hormone/insulin-like growth factor

axis. There are several in vivo as well as in vitro

studies on the effects of leptin on growth hormone

(GH) release (e.g. Chen et al., 2001; Henry et al.,

2001; Morrison et al., 2001). Vice versa, studies on

the effects of GH on leptin secretion in farm animals

are presently limited to leptin mRNA quantification in

white adipose tissue (Raymond et al., 1997; House-

knecht et al., 2000). We therefore aimed to further

characterise the hormonal regulation of leptin, in

particular the effects of growth hormone on blood

leptin concentrations in cows of different physiolog-

ical status. A specific enzyme immunoassay was

developed, validated and applied for this purpose.

2. Materials and methods

2.1. Development of a competitive enzyme immuno-

assay (EIA) for leptin in domestic animals

Specific polyclonal antisera against leptin were

raised in five crossbreed rabbits using a mixture of

200 Ag recombinant ovine leptin (roLep; Gertler et

al., 1998) and 100 Ag each of two modified syn-

thetic peptides out of the bovine leptin sequence

(SWISS-PROT protein sequence database; peptide

1: VSSKQRVTGLDFIPGLKY; peptide 2: DLENL-

RDLLHKY; the additional amino acids K and Y

functioning as coupling groups) bound to 200 Agkeyhole limpets hemocyanin (KLH, Sigma-Aldrich,

Taufkirchen, Germany) via the glutardialdehyde re-

action and emulsified in complete Freund’s adjuvant

(Sigma-Aldrich). Nine booster immunisations were

performed with incomplete Freund’s adjuvant (Sig-

ma-Aldrich) in monthly intervals. For the second to

fifth immunisation, half of the amount of antigen

was used and in the last four immunisations 200 Agrecombinant ovine leptin were used omitting the

peptides. The rabbits were bled from an ear vein

1 week after each booster injection and antisera

were stored at � 20 jC.For tracer generation, 40 Ag roLep were biotiny-

lated according to Hennies et al. (2001) using a 100-

fold molar excess of biotinamidocaproate N-hydroxy-

succinimide ester (Sigma-Aldrich).

Microtiter plates (EIA plate 9018, Corning Costar,

Cambridge, MA, USA) were precoated by incubating

150 ng sheep anti-rabbit-Fc fragment antibodies in

100 Al 50 mM sodium hydrogen carbonate, pH 9.6

per well at 4 jC for 20 h. After blocking free binding

sites with 300 Al 2.5% casein in 0.05 M NaCl, pH 7.4

at room temperature for 1.5 h, the plates were washed

five times with 10% PBS, 0.05% Tween 20, filled

with assay buffer and stored at 4 jC up to several

weeks without appreciable loss of sensitivity. The

assay buffer was 0.1% hydrolysed gelatine, 0.12 M

NaCl, 0.02 M Na2HPO4, 0.01 M EDTA, 0.005%

chlorhexidine digluconate (20%), 0.002% phenol red,

200 Al/l proteinase inhibitor cocktail (completek,

Boehringer Mannheim, Germany) and 0.02% ProClin

150R (Supelco, Bellefonte, PA, USA).

For the assay, plates were decanted and leptin

standard or prediluted plasma or serum samples (50

Al) were pipetted in duplicate into the wells. Antise-

rum (50 Al) diluted 1:30,000 with assay buffer con-

taining 2% goose serum were added. All incubation

steps were performed at room temperature. After

preincubation for 16 h, 50 Al of biotinylated leptin

(9 ng/ml assay buffer) were added and incubated for

another 2 h. Bound tracer was quantified via strepta-

vidin-peroxidase as described earlier (Hennies et al.,

2001).

2.2. Radioimmunoassay for IGF-1

Insulin-like growth factor-1 (IGF-1) was deter-

mined by radioimmunoassay (RIA) as described pre-

viously (Daxenberger et al., 1998) using co-incubation

Table 1

Characterisation of the leptin EIA

Antiserum Rabbit anti-ovine leptin

Final dilution 1:90,000

Standard Recombinant ovine leptin

Tracer Biotinylated recombinant leptin

Maximum sample volume 50 AlMeasuring range 0.3–20 ng/ml

50% Binding 2.1 ng/ml

Recovery

1.7 ng/ml 92.5% (n= 4)

5.1 ng/ml 111.7% (n= 4)

14.9 ng/ml 99.9% (n= 4)

CV

Intra-assay 6.3% (n= 12)

Inter-assay 13.9% (n= 41)

H. Sauerwein et al. / Livestock Production Science 87 (2004) 189–195 191

with IGF-2 to minimise potential interferences of IGF

binding proteins. The cross-reactivity to IGF-2 in this

assay was less than 0.01%. The recovery of five

different concentrations IGF-1 added to a sample with

and high and to one with low IGF-1 were 99.2%

(n = 88). Intra-assay and inter-assay coefficients of

variation were 5.1% (n = 32) and 13.4% (n = 64),

respectively.

2.3. Animals, treatments and sample collection

Nineteen lactating Brown Swiss cows received a

500 mg subcutaneous injection of sustained-release

bovine growth hormone (bGH, PosilacR, Monsanto,

USA). Two weeks before and 4 weeks after the

injections, blood samples were collected every second

day. At the time of the bGH injections, the cows were

either not pregnant (98F 47 days postpartum; range:

57 to 202 days, n = 9) or pregnant (158F 47 days

postpartum, range 106–231 days, mean pregnancy

duration was 66 days post insemination; the range was

21 to 147 days; n = 10) and were in their first to sixth

lactation. Pregnancy status was evaluated from the

insemination time and by milk progesterone determi-

nations performed twice per week after the first

insemination. If progesterone remained above 1 ng/

ml throughout the weeks following insemination, the

cows were characterised as pregnant. Retrospectively,

the classification was also confirmed by the calving.

All animals were kept in a cubicle shed, were milked

twice daily in a milking parlour and were fed accord-

ing to their individual production levels. Average milk

yield before the bGH injections was 27.4F 5.9 kg/

day. Blood samples were collected from the udder

vein and plasma was stored at � 20 jC until assayed

for IGF-1 and leptin.

2.4. Statistical analyses

Data were analysed by using the PROC MIXED

model of the SAS 8.01 package. Four covariance

structures (compound symmetry, first order auto-re-

gressive, ante dependence and Toeplitz) were initially

tested and their suitability was assessed by using the

Akaike’s information criterion (AIC). Toeplitz yielded

the lowest AIC values for both the leptin as well as for

the IGF-1 data sets and was therefore applied for all

the subsequent analyses. The independent variables

(i.e. leptin and IGF-1 plasma concentrations) were

log-transformed before statistical analysis. Specific

contrasts were formulated in order to test the hypoth-

eses that (1) there is no effect of GH treatment on

leptin and IGF-1 plasma concentrations, (2) the effect

of the GH treatment on leptin and IGF-1 plasma

concentrations is similar for pregnant and nonpreg-

nant cows, and (3) there is no difference in leptin and

IGF-1 plasma concentrations determined 1 week be-

fore treatment and 2 weeks after GH treatment.

3. Results

3.1. Characterisation and validation of the leptin EIA

Using a combination of recombinant ovine leptin

and peptides out of the bovine leptin sequence,

specific antisera were successfully raised. The antise-

rum with the highest titer against the recombinant

hormone was used to develop the EIA. The criteria of

assay sensitivity, specificity and precision of the

competitive assay developed herein are summarised

in Table 1. The nonspecific binding was less than 5%

of the maximal binding. Accuracy of the assay was

established by ensuring negligible cross-reactivity

( < 0.01%) with other related proteins (ovine prolactin,

ovine placental lactogen and bovine insulin) and an

excellent parallelism between the standard curve and

dilutions from cattle, goat, mufflon, pig and from

horse serum (Fig. 1).

Fig. 1. Standard curve of recombinant ovine leptin and serial

dilutions of serum samples from different species. There was no

relation between the amount of serum assayed and the concentration

measured (ANOVA).

Fig. 2. Plasma leptin and IGF-1 concentrations (LSMeansF S.E.M.) in pr

injection with depot-formulated bGH.

H. Sauerwein et al. / Livestock Production Science 87 (2004) 189–195192

3.2. Leptin and IGF-1 plasma concentrations in cows

before and after the bGH injections

The time course of the leptin and the IGF-1 plasma

concentration for pregnant and nonpregnant cows is

shown in Fig. 2. Type 3 tests of fixed effects on leptin

concentration revealed a significant interaction

(a = 0.0037) between time and pregnancy. Hypotheses

formulated with the contrasts were tested and demon-

strated that leptin concentrations (log transformed)

before and after GH treatment were different for

pregnant cows ( p = 0.0033), but not for nonpregnant

cows ( p = 0.2540). In addition, leptin concentrations

(log transformed) 1 week before and 2 weeks after

treatment were different for pregnant cows

( p = 0.0027) and again not for nonpregnant cows

( p = 0.5805). In pregnant cows, leptin concentrations

decreased after the bGH treatment with a nadir 15

egnant (n= 10) and nonpregnant (n= 9) dairy cows before and after

H. Sauerwein et al. / Livestock Production Science 87 (2004) 189–195 193

days after the injection and returned to pretreatment

levels thereafter. In average, leptin concentrations in

pregnant cows were decreased by 20% after the bGH

injections.

Type 3 tests of fixed effects on the IGF-1 plasma

concentration revealed significant effect of time

( p < 0.0001). bGH treatment lead to a gradual increase

of IGF-1 plasma concentrations with a maximum at

125% of the pretreatment levels during the second

week after the bGH injections and a subsequent

decline to pretreatment levels on day 23 and thereafter

(Fig. 2). In general, Spearman correlations between

plasma IGF-1 and leptin were insignificant. Only

during the pretreatment phase, a weak positive rela-

tionship between leptin and IGF-1 was observed

(r = 0.16, p < 0.05). Milk yield was increased by

bGH treatment by 11% in average.

4. Discussion

The EIA developed herein provides a sensitive and

reliable method to quantify leptin concentrations in

blood serum or plasma from ruminant species. The

concentrations we measured in dairy cows are well in

accordance with leptin values measured by RIA as

reviewed by Chilliard et al. (2001). For the only

enzyme immunological assay published which is

applicable for ruminants (Kauter et al., 2000), no data

for cattle are available. In addition to the advantage of

avoiding the use of radioactive labels, our assay is

valid for a broad range of species, i.e. besides porcine

samples it is also applicable for equine samples and

was already successfully used to characterise peripar-

tal leptin concentrations of breeding mares (Heidler et

al., 2002).

Our experiment in dairy cows provides evidence

that the effect of growth hormone on leptin plasma

concentrations is dependent of the physiological status

of the animal since significant GH-induced changes

were limited to pregnant animals. In contrast to

nonpregnant cows, in which leptin concentrations

remained at a constantly low level throughout the

study, leptin concentrations were decreased after the

GH injections in the pregnant animals. The lack of

reaction in nonpregnant cows cannot entirely be

attributed to lactational stage and thus to differences

in energy balance. Although most of the nonpregnant

cows were at a relatively early stage of lactation, i.e.

days 57 to 89 postpartum (n = 6), there were also three

animals at days 117, 134 and 202. In these animals,

the leptin plasma concentrations were as low as in the

other six cows of the nonpregnant cows and there was

no tendency towards higher concentrations at all.

Although pregnancy was not significantly affecting

leptin plasma concentrations in our study, increased

leptin blood concentrations and leptin mRNA levels in

white adipose tissue have been reported in pregnant

versus nonpregnant ewes (Ehrhardt et al., 2001).

Moreover, the lack of differences in IGF-1 plasma

concentrations between pregnant and nonpregnant

cows suggests that the nutritional status of the animals

was comparable. Based on these observations we

speculate that the leptin response towards GH treat-

ment in pregnant animals may at least in part result

from the specific endocrine conditions of pregnancy

rather than from exclusively nutritional cues. The GH-

induced decrease of leptin plasma concentrations in

pregnant cows we observed fits well into these con-

cepts; however, it is in contradiction to the reports on

GH acting stimulative on leptin mRNA expression in

white adipose tissue from growing cattle (House-

knecht et al., 2000) or growing sheep (Raymond et

al., 1997). The discrepancy between our leptin blood

data and the mRNA expression might be due to the

different physiological status of the animals used: in

contrast to growing animals, we used lactating, mostly

full-grown animals for our experiment. In addition,

concomitant changes within the leptin system (free

leptin, bound leptin, and soluble leptin receptor) might

possibly explain for the discrepancy in leptin response

to GH treatment: Randeva et al. (2002) reported that

GH treatment of GH-deficient humans lead to a fall in

free leptin, a rise in bound leptin and soluble leptin

receptor (sLR). For the RIAs and the ELISAs avail-

able for ruminant leptin, including the assay system

described in the present paper, no information is

available as to whether free and/or bound leptin are

equally recognised. If GH treatment increases both

adipose leptin mRNA expression and sLR, a net

decrease of free leptin may result depending on the

molar ratio of leptin and sLR secreted into the

circulation. In monogastric species, GH treatment

has been demonstrated to either decrease or to have

no effect on (free) leptin blood concentrations (Ran-

deva et al., 2002; Marzullo et al., 2002). However, the

H. Sauerwein et al. / Livestock Production Science 87 (2004) 189–195194

results of these studies might not be directly transfer-

able to healthy organisms since the subjects investi-

gated were affected with complex endocrine disorders,

in particular with GH deficiency.

5. Conclusion

Our results support an inhibiting effect of GH

treatment on leptin plasma concentrations in dairy

cows depending of their endocrine and/or metabolic

status. In view of the discrepancy with reports from the

literature about stimulating effects of GH on adipose

tissue leptin mRNA expression in growing ruminants,

the underlying mechanisms for the decreased leptin

blood concentrations observed remain to be specifi-

cally addressed in the ruminant considering the entire

leptin system and the physiological status of growing,

pregnant or lactating animals.

Acknowledgements

We thank Prof. A. Gertler, Institute of Biochem-

istry, Food Science and Nutrition, Faculty of Agri-

culture, Food and Environmental Quality Sciences,

The Hebrew University of Jerusalem, Rehovot, Israel

for the kind gift of recombinant ovine leptin. The

animal experiment was supported by the Bavarian

Ministry for Nutrition, Agriculture and Forestry

(Bayerisches Staatsministerium fur Ernahrung, Land-

wirtschaft und Forsten). U. Heintges was recipient of

a grant from the Graduiertenforderung des Landes

Nordrhein Westfalen.

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