induction of pulsatile secretion of leptin in horses following thyroidectomy

353

Induction of pulsatile secretion of
leptin in horses followingthyroidectomy
Preston R Buff1, Nat T Messer IV2, Andria M Cogswell2, David A Wilson2, Philip J Johnson2,

Duane H Keisler3 and Venkataseshu K Ganjam1,2

Departments of 1Biomedical Sciences, 2Veterinary Medicine and Surgery, 3Animal Sciences, University of Missouri-Columbia, Columbia, Missouri 56211, USA

(Requests for offprints should be addressed to P R Buff who is now at Department of Animal and Dairy Sciences, Mississippi State University, Box 9815,

Mississippi State, Mississippi 39762, USA; Email: [email protected])

Abstract

Endocrine characteristics of Quarter Horse-type mares were

determined during a 68 h feed deprivation and again in the

same mares following surgical thyroidectomy (THX). A

crossover experimental design was implemented, in which

mares received brome hay available ad libitum (FED) or were

food deprived (RES) for 68 h. Blood samples were collected

every 20 min for 48 h, beginning 20 h after the onset of food

deprivation. Concentrations of triiodothyronine and thyrox-

ine were undetectable post-THX. Plasma concentrations of

thyrotropin were greater post-THX versus pre-THX

Journal of Endocrinology (2007) 192, 353–3590022–0795/07/0192–353 q 2007 Society for Endocrinology Printed in Great

(P!0.001). Plasma concentrations of leptin were greater in

the THX FED group than in the THX RES group (P!0.01). The existence of leptin pulse secretion was found only

in post-THX compared with the same horses pre-THX (PZ0.02). We theorize that non-pulsatile secretion of leptin may

have contributed to the survival of this species, as it evolved in

the regions of seasonal availability of food. Lack of pulsatile

secretion of leptin may contribute to the accumulation of

energy stores by modulating leptin sensitivity.

Journal of Endocrinology (2007) 192, 353–359

Introduction

Leptin, secreted primarily by adipocytes, acts to regulate

energy homeostasis by providing input to the central nervous

system as a signal of energy stored by adipose tissue

(Casanueva & Dieguez 1999, Schwartz et al. 2000). Leptin

acts on hypothalamic receptors to modulate energy balance as

a signal to control food intake and energy expenditure

(Campfield et al. 1995, Ahima et al. 1996). In regulating

energy homeostasis, leptin is the most effective when

circulating concentrations are decreased due to a lack of

food intake. In the fasted state, plasma leptin is reduced,

which activates neuroendocrine and behavioral responses to

restore the energy balance (Ahima et al. 1996). Furthermore,

leptin modulates additional endocrine axes, including the

gonadotropic (Barash et al. 1996), corticotropic (Schwartz

et al. 1996), somatotropic (Carro et al. 1997), and thyrotropic

(Legradi et al. 1997). Leptin secretion by cultured adipose

tissue has been shown to be directly stimulated by thyrotropin

(TSH; Menendez et al. 2003).

An interesting physiological phenomenon of leptin biology

is the detection of a pulsatile release (Licinio et al. 1997). The

concept of a hormone being secreted in a pulsatile manner

from millions of cells at diverse locations is not consistent with

other hormones secreted in pulses. Leptin pulsatile secretion

may be partially controlled by TSH in humans as Mantzoros

et al. (2001) have shown a similar circadian pattern of leptin and

TSH, with peak values occurring at similar time points. Work

performed in our laboratory, evaluating leptin longitudinal

profiles in obese ponymares, demonstrated circadian secretion

of leptin, but pulses were not detected (Buff et al. 2005).

Work with thyroidectomized rodents showed that thyroid

hormones inhibit leptin secretion (Escobar-Morreale et al.

1997) and that leptin secretion did not increase with increased

energy intake (Curcio et al. 1999). Leonhardt et al. (1999)

demonstrated an increase in plasma concentrations of leptin

following thyroidectomy that was attributed to an increase in

leptin synthesis by adipose tissue. To our knowledge, an

investigation of pulsatile leptin secretion following thyroid-

ectomy has not been conducted in any species. In the present

study, we utilized a large animal model that would readily

facilitate extensive frequent sampling of plasma to enable an

accurate reflection of acute changes in peripheral hormone

concentrations. We hypothesized that thyroidectomy would

induce alterations of leptin concentrations in horses during

food deprivation.

Materials and Methods

Animals

Quarter Horse-type mares (nZ7) were maintained with

ad libitum access to brome grass pasture or hay and water.

DOI: 10.1677/joe.1.06989Britain Online version via http://www.endocrinology-journals.org

P R BUFF and others . Pulse secretion of leptin354

Animals were kept at ambient temperature and photoperiod

(latitude 38.98 N longitude 92.28 W) in a group in pasture

and individually in a 0.83 m2 box stalls within sight of one

another. Experimental procedures were approved by the

University of Missouri Animal Care and Use Committee.

Procedures

Experimentation was conducted on mares with thyroid

glands (TH) and following surgical thyroidectomy (THX).

The same horses were used for both experimental states to

serve as their own controls and to strengthen the statistical

power. Surgeries were performed 6 months prior to

experimentation to allow recovery from the procedure.

Animals received the same treatments during TH and

THX. The two phases of the study were conducted 1 year

apart, to minimize any seasonal or photoperiodic influence

on hormone secretion. Mares were placed in individual stalls

at 0800 h on day 1 and provided with brome grass hay. At

0900 h on day 1, each mare was fitted with an i.v. jugular

catheter for collection of blood samples. Water was provided

ad libitum to all animals throughout experimentation.

Treatments of ad libitum brome grass hay (FED) or food

deprivation (RES) were randomly assigned and implemented

at 1200 h on day 1 and continued for 68 h. Blood samples

were collected every 20 min beginning at 0800 h on day 2, to

ensure that animals receiving RES treatment were in a

negative energy balance, and continued for 48 h. Mares were

then returned to pasture for 10 days to recover from

treatment. Following the recovery period, experimentation

was repeated with animals receiving the opposite treatment.

Body weights were measured with a digital scale prior to each

treatment period.

Blood samples were collected in Vacutainer tubes with K3

EDTA additive (Becton Dickinson, Franklin Lakes, NJ, USA)

and placed on ice for transport to the laboratory. Samples

were centrifuged at 3000 g for 25 min at 4 8C. Plasma was

stored at K20 8C until analyzed for hormone concentration.

Radioimmunoassays

Plasma samples were analyzed for leptin, in triplicate 200 mlaliquots, using the double-antibody RIA procedures pre-

viously validated for equine plasma (Buff et al. 2002).

The intra- and inter-assay coefficients of variation (CV)

were !10% and the sensitivity was 0.04 ng/ml. Analysis of

TSH was conducted, in triplicate 200 ml aliquots, with

double-antibody RIA using equine TSH antiserum (AFP-

C33812) and equine TSH antigen (AFP-5144B) provided by

A F Parlow (Harbor-UCLA Medical Center, Torrance, CA,

USA). The intra- and inter-assay CV were !10% and the

sensitivity was 0.02 ng/ml. Thyroxine (T4) was analyzed

using a commercial RIA kit (Diagnostic Products Corpo-

ration, Los Angeles, CA, USA). Plasma samples were assayed

in duplicate 25 ml aliquots following the manufacturer’s

procedures. The intra- and inter-assay CV were !10% and


the sensitivity was 2.9 pg/ml. Triiodothyronine (T3) was

analyzed using a commercial RIA kit (Diagnostic Products

Corporation). Plasma samples were assayed in duplicate

100 ml aliquots following the manufacturer’s procedures.

The intra- and inter-assay CV were!10% and the sensitivity

was 0.34 ng/ml.

Cluster analysis

Pulse characteristics for leptin and TSH were determined for

each animal within each treatment group using the Cluster

pulse analysis program (Veldhuis & Johnson 1986). The

criteria for determining pulsatile secretion were 1!2 (nadir

1, peak 2) cluster size and inter-assay CV (Veldhuis & Johnson

1988). Evaluation was conducted on the following

parameters; area under the curve (AUC), pulse frequency,

pulse amplitude, and peak area.

Leptin pulse analysis

Half-life and decay constant were calculated for each leptin

pulse from data determined by Cluster analysis. Calculations

were performed using the time span of each pulse, start

concentration, and ending concentration of each pulse. The

following equation was used to determine half-life (t1/2).

t1=2 Zlnð1=2Þ

k

Where kZln(end concentration/start concentration)/time

span of pulse.

Decay constant (l) was calculated with the following

equation using t1/2 as calculated in the previous equation.

lZt1=2

ln 2

Statistical analysis

Analyses were performed to determine whether differences

existed in pulsatile characteristics (frequency, amplitude, and

pulse area), mean concentrations, and AUC of leptin and

TSH using the general linear model ANOVA of SAS (SAS

Inst. Inc., V8, Cary, NC, USA). Effects within the model

included individual, thyroid status (TH versus THX), and

treatment (FED versus RES) with residual error used as the

error term. A similar analysis was performed to determine

differences in body weight using the general linear model

ANOVA of SAS. The tested effect was thyroid status (TH

versus THX) with residual error used as the error term.

Repeated measures analyses were performed for T3 and T4

using the mixed model of SAS (Littell et al. 1998). Test effects

for each model included individual, sample, thyroid status

(TH versus THX), and treatment (FED versus RES) with

sample as the repeated variable and individual within

treatment by thyroid status as the subject. Least square

means and differences were generated in each analysis, where

www.endocrinology-journals.org

Pulse secretion of leptin . P R BUFF and others 355

significance was determined (P!0.05). Results from

ANOVA models are reported as least square meanGS.E.M.

Pearson correlation analyses were performed to test the

relationship between leptin and TSH by thyroid status (TH

and THX) within each treatment (FED and RES) using the

software package SAS. A Pearson correlation was also

performed to test the relationship between leptin and TSH

peaks in the THX group. Cross-correlation analyses was

used to test the relationship between leptin and TSH

at various time lags by thyroid status (TH and THX) within

each treatment group (FED and RES) using the software

package SAS.

Results

Bodyweights did not differ betweenTH andTHX (415G12 vs

422G12 kg; PZ0.69) in horses. Leptin and TSH pulse

characteristics, means and AUCs are outlined in Table 1.

Mean concentrations of leptin were greater in THX when

compared with TH during the FED treatment (P!0.05), withno differences observed in the RES treatment. Within thyroid

status, no differences in mean concentrations of leptin were

observed between FED and RES in TH. However, in THX,

mean concentrations of leptin and AUC were greater in the

FED treatment when compared withRES treatment (P!0.01,each). No evidence of pulsatile secretion was observed in any

horse in the TH state, but pulses were present in all horses

followingTHX (Fig. 1). Leptin pulse frequency, amplitude, and

pulse area were not different between RES and FED in THX

horses (PO0.1, all). Mean t1/2 of leptin in THX horses was

130.19G21.78 min and l was 187.86G31.43 min. No

differences in TSH were observed for mean concentration,

frequency, amplitude, pulse area, or AUC between the FED

and theRES treatments for either THorTHX (PO0.1) horses.Mean TSH was greater in THX when compared with TH for

both FED (P!0.001) and RES (P!0.05) groups. No

differences in TSH pulse frequency were observed between

THX and TH in either the FED or RES treatment (PO0.1,

Table 1 Pulse characteristics and area under the curve (AUC) for leptinduring a positive (FED) and negative (RES) energy balance. Values are l

Mean (ng/ml)Frequency(pulse/48 h)

LeptinTH FED 2.5G1.2 0TH RES 1.7G1.2 0THX FED 6.3G1.1† 1.4G0.6THX RES 0.7G1.1* 2.9G0.6†

TSHTH FED 2.8G1.2 1.8G0.8TH RES 2.8G1.2 1.7G0.8THX FED 9.4G1.2† 2.0G0.7THX RES 6.7G1.2† 3.3G0.7

*P!0.05; FED versus RES, within thyroid status. †P!0.05; TH versus THX, withi


each).TSHpulse amplitudewas greater inTHXcomparedwith

TH for both FED (P!0.01) andRES (P!0.05). Pulse area forTSH was greater in THX when compared with TH for both

FED (P!0.05) and RES (P!0.01). The AUC for TSH was

also greater in THX compared with TH for both FED

(P!0.001) and RES (P!0.05). Hormone profiles for leptin

and TSH of the same four horses are illustrated in Figs 1 and 2

respectively. Concentrations of T3 were greater in TH when

compared with THX (0.36G0.01 ng/ml vs undetectable;

P!0.0001). Concentrations of T3 were greater in RES

compared with FED during the TH state (0.38G0.01 vs

0.33G0.01 ng/ml; P!0.001). Concentrations of T4 were

greater in THwhen comparedwith THX (21.2G1.2 pg/ml vs

undetectable;P!0.0001).Concentrations ofT4were greater in

RES compared with FED during the TH state (24.3G1.7 vs

18.2G1.7 pg/ml; P!0.001). A negative correlation existed

between leptin and TSH for TH and THX during both

treatments (FED and RES; Table 2). No correlation between

peaks of leptin andTSHwereobserved inTHXhorses for either

treatment (FEDandRES;Table 3).Cross-correlation analysis of

TH horses resulted in the greatest correlation occurring at a lag

of leptin concentrations by 15 min (rZK0.22, P!0.01) andanalysis of THX horses resulted in the greatest correlation with

no lag (rZK0.08, P!0.01).

Discussion

The current study is the first, to the best of our knowledge, to

show the induction of pulsatile secretion of leptin following

thyroidectomy. Previously, our group reported that leptin was

not secreted in pulses in obese pony mares when analyzed in

the same manner as the current study (Buff et al. 2005). In that

study, leptin was found to be secreted in a non-pulsatile

variable manner. Pulsatile secretion of leptin has been

reported in humans (Sinha et al. 1996, Licinio et al. 1997,

Saad et al. 1997), rats (Bagnasco et al. 2002, Otukonyong et al.

2005), and sheep (Daniel et al. 2002, Recabarren et al. 2002).

and TSH in thyroid intact (TH) and thyroidectomized (THX) horseseast square meanGS.E.M.

Amplitude(ng/ml)

Pulse area(ng/ml min) AUC (ng/ml)

0 0 7279.9G3528.90 0 4935.5G3528.93.0G0.9† 68.6G19.7† 18 208.9G3267.1†

1.4G0.9 51.1G19.7† 2068.5G3267.1*

2.3G2.2 14.7G28.9 7917.6G3566.52.7G2.2 25.5G28.9 8002.7G3566.59.8G2.0† 96.6G26.8† 26 963.1G3301.9†

9.1G2.0† 135.2G26.8† 19 231.1G3301.9†

n energy balance.


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Figure 1 Representative profiles of leptin in (A) euthyroid ad libitum fed, (B) euthyroid food deprived, (C) thyroidectomized ad libitum fed,and (D) thyroidectomized food deprived horses.


The hypothalamic–pituitary–thyroid axis may be

regulated, in part, by leptin as a survival mechanism during

starvation (reviewed by Flier et al. 2000). In a study

investigating the direct effect of TSH on leptin secretion,

Menendez et al. (2003) have clearly shown that leptin

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Figure 2 Representative profiles of TSH in (A) euthyroid ad libitum fed, (B(D) thyroidectomized food deprived horses. Scales differ between euthy


secretion by adipocytes is increased following treatment

with TSH using an in vitro human adipose model. An increase

in plasma leptin concentration and mRNA expression in

adipose tissue has been reported in rats following thyroid-

ectomy or methimazole treatment (Leonhardt et al. 1999).

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) euthyroid food deprived, (C) thyroidectomized ad libitum fed, android and thyroidectomized.


Table 2 Pearson correlation coefficients for leptin and TSH in thyroid intact (TH) and thyroidectomized (THX) horses during a positive (FED)and negative (RES) energy balance

TH THX

FED RES FED RES

Leptin TSH Leptin TSH Leptin TSH Leptin TSHLeptin 1.0 K0.32* 1.0 K0.12* 1.0 K0.46* 1.0 K0.22*TSH 1.0 1.0 1.0 1.0

*P!0.01.

Table 3 Pearson correlation coefficients for leptin and thyrotropin(TSH) peaks in thyroidectomized (THX) horses during a positive(FED) and negative (RES) energy balance

FED RES

Leptin TSH Leptin TSH

Leptin 1.0 K0.01 1.0 0.06TSH 1.0 1.0


These reports support our finding of increased peripheral

leptin concentration following THX in parallel with

increased TSH concentrations. However, this increase

occurred only in the FED treatment. This finding was not

surprising, as we have previously reported a suppression of the

circadian pattern of leptin secretion following food depri-

vation in horses (Buff et al. 2005). This observation, along

with our previous report, would indicate that the suppression

of leptin secretion following food deprivation overrides other

controls in horses.

Mantzoros et al. (2001) have suggested that leptin may

regulate TSH pulsatility in a report correlating leptin and

TSH pulses in humans. Our findings do not agree with the

aforementioned report, as no leptin pulses were observed in

the presence of TSH pulses in TH horses. Additionally, leptin

pulses observed in THX horses did not coincide with TSH

pulses as determined by the Pearson correlation analysis. In

our model, leptin and TSH appear to pulse independently.

However, the ablation of the thyroid gland and subsequent

increases in TSH indicate that the hypothalamic–pituitary–

thyroid axis modulates the pulsatile secretion of leptin. An

explanation for the mechanism of this control is beyond the

scope of our finding. We speculate that thyroid hormones

may act to suppress an unknown leptin pulse generator to

inhibit leptin pulses.

Significant negative correlations were observed between

leptin and TSH indicating that a relationship exists between

these hormones. A greater correlation was present in the FED

treatment in both TH and THX groups. The lesser degree of

correlation in RES treatment could be due to fed deprivation

eliciting a response by leptin and not TSH. The results of the

cross-correlation analysis suggest that leptin secretion in TH

horses lags behind TSH secretion. This provides further

evidence that TSH or the hypothalamic–pituitary–thyroid

axis may, in part, regulate leptin. Analysis of THX horses

resulted in a low cross correlation. This result may be a

reflection of the lack of correlation between leptin and TSH

pulse frequency. In support of our findings, Ghizzoni et al.

(2001) found leptin and TSH correlations with a lag of leptin

in boys and no lag in girls, as only females were used.

In the present study, we did not observe statistical

differences in mean concentrations of leptin or AUC between

FED and RES treatments during TH as expected. The lack of

difference may be attributed to lower concentrations of basal

leptin during this period. Therefore, when horses were food


deprived, leptin concentrations did not decrease. During

THX, mean concentrations of leptin and AUC were

decreased during food deprivation. We did not observe any

pulse differences in frequency, amplitude, or area as a result of

food deprivation in THX horses. However, differences were

observed in these pulsatile variables between TH and THX.

In support of this finding, Bergendahl et al. (2000) reported

that fasting did not affect leptin pulsatility in normal women.

Concentrations of TSH increased following THX as

expected from the lack of negative feedback of thyroid

hormones, which was confirmed by undetectable levels of

thyroid hormones. The pulse frequency of TSH was not

altered by THX or food deprivation, indicating that neither

thyroid nor nutritional status modulates the frequency of

pulses. Pulse amplitude and area were increased following

THX as a result of increased secretion level of TSH. Food

deprivation did not alter measured TSH parameters in either

TH or THX. This observation is consistent with reports in

humans where a 60-h fast did not change TSH levels

(Merimee & Fineberg 1976) and a 10-day total energy

deprivation evoked a minute reduction in serum TSH levels

(Palmblad et al. 1977).

The family Equidae has evolved over the past 55 million

years and throughout the Miocene (23.8 to 5.3 million years

ago), dramatic global climate changes occurred. During this

period, forests declined and grasslands expanded. The species

in the family Equidae that had adapted into grazers survived

and the browsers became extinct (MacFadden et al. 1999).

These grazers evolved into the extant Equus species, which

are grazers. These species evolved in regions of seasonal food

availability and thus reproduced during the season when food

is most plentiful (Epstein 1971). During periods of winter and

drought, the food supply in these grasslands was diminished

and thus mechanisms of survival must have developed,

otherwise extinction would have occurred. Based on the



current theories of endocrine regulation of energy balance, an

animal will regulate its energy intake to maintain energy stores

to meet the demands of energy expenditure. In an

environment where food supply was seasonally scarce, animals

that did not shift their energy balance to a positive state during

periods of abundance would not have survived periods of

food scarceness. Thus, some mechanism must have evolved

that allowed the survival of species reliant on a food supply

that was only available seasonally. We are proposing that the

suppression of a pulsatile secretion of leptin could be such a

mechanism. When a hormone is secreted in pulses, it allows a

refractory period so that receptors are unoccupied and

upregulated to increase the response of the target tissue. If

the brain were less responsive to leptin, it would allow the

energy balance to shift towards the positive state and thus

more energy stores would be available during periods of

dearth. If a hormone is no longer secreted in pulses, the target

tissue will become desensitized from maintaining a steady-

state level (Baulieu 1990). Seasonal photoperiod changes have

been shown by Rousseau et al. (2002) in Siberian hamsters to

modulate the sensitivity to leptin. Horses exhibit seasonal

variations of leptin, which has been reported to decrease as

they transition from summer into autumn and winter without

subsequent weight loss (Gentry et al. 2002, Buff et al. 2005).

In summary, this is the first report to our knowledge

describing pulsatile secretion of leptin following thyroid-

ectomy. Moreover, this species appears to be unique in that

euthyroid individuals do not secrete leptin in pulses. We

believe that this may be a mechanism to regulate metabolism

as a result of evolution of this species, which has allowed

survival during periods of seasonal food shortages. Further

elucidation of this mechanism may further our understanding

of homeostatic regulation of energy balance.

Acknowledgements

This work was supported by a grant from the American

Quarter Horse Association (NTM) and grant from the

National Aeronautics and Space Administration (VKG;

NASA NAG5-12300). The authors express gratitude to D

Lenger and the Middlebush farm crew for their care of the

horses and support of this project. The authors declare that

there is no conflict of interest that would prejudice the

impartiality of this scientific work.

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Received in final form 14 November 2006Accepted 16 November 2006Made available online as an Accepted Preprint12 December 2006


induction of pulsatile secretion of leptin in horses following thyroidectomy

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