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¹ Departments of Biomedical Sciences,
² Veterinary Medicine and Surgery,
³ Animal 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: pbuff{at}ads.msstate.edu)

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 thyroxine were undetectable post-THX. Plasma concentrations of thyrotropin were greater post-THX versus pre-THX (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 (P = 0.02). 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 pony mares, 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 thyroidectomy 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

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Animals

Quarter Horse-type mares (n=7) were maintained with ad libitum access to brome grass pasture or hay and water. Animals were kept at ambient temperature and photoperiod (latitude 38.9° N longitude 92.2° 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 °C. Plasma was stored at –20 °C until analyzed for hormone concentration.

Radioimmunoassays

Plasma samples were analyzed for leptin, in triplicate 200 µl aliquots, using the double-antibody RIA procedures previously 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 µl 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 Corporation, Los Angeles, CA, USA). Plasma samples were assayed in duplicate 25 µl 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 µl 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 1x2 (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 (t_1/2)

Where k=ln(end concentration/start concentration)/time span of pulse.

Decay constant () was calculated with the following equation using t_1/2 as calculated in the previous equation.

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 significance was determined (P<0.05). Results from ANOVA models are reported as least square mean ± S.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

Top Abstract Introduction Materials and Methods Results Discussion References

 
Body weights did not differ between TH and THX (415 ± 12 vs 422 ± 12 kg; P=0.69) 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), with no 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 with RES 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 following THX (Fig. 1). Leptin pulse frequency, amplitude, and pulse area were not different between RES and FED in THX horses (P>0.1, all). Mean t_1/2 of leptin in THX horses was 130.19 ± 21.78 min and was 187.86 ± 31.43 min. No differences in TSH were observed for mean concentration, frequency, amplitude, pulse area, or AUC between the FED and the RES treatments for either TH or THX (P>0.1) 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 (P>0.1, each). TSH pulse amplitudewas greater in THX compared with TH for both FED (P<0.01) and RES (P<0.05). Pulse area for TSH 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 (0.36 ± 0.01 ng/ml vs undetectable; P<0.0001). Concentrations of T₃ were greater in RES compared with FED during the TH state (0.38 ± 0.01 vs 0.33 ± 0.01 ng/ml; P<0.001). Concentrations of T₄ were greater in TH when compared with THX (21.2 ± 1.2 pg/ml vs undetectable; P<0.0001). Concentrations of T₄ were greater in RES compared with FED during the TH state (24.3 ± 1.7 vs 18.2 ± 1.7 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 and TSH were observed in THX horses for either treatment (FED and RES; Table 3). Cross-correlation analysis of TH horses resulted in the greatest correlation occurring at a lag of leptin concentrations by 15 min (r=–0.22, P<0.01) and analysis of THX horses resulted in the greatest correlation with no lag (r=–0.08, P<0.01).

View larger version (15K): [in this window] [in a new window]   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.

View larger version (15K): [in this window] [in a new window]   Figure 2 Representative profiles of TSH in (A) euthyroid ad libitum fed, (B) euthyroid food deprived, (C) thyroidectomized ad libitum fed, and (D) thyroidectomized food deprived horses. Scales differ between euthyroid and thyroidectomized.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 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 thyroidectomy or methimazole treatment (Leonhardt et al. 1999). 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 deprivation 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 thyroidectomy. 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 2006
Accepted 16 November 2006
Made available online as an Accepted Preprint 12 December 2006

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Buff, P. R Articles by Ganjam, V. K Search for Related Content PubMed Articles by Buff, P. R Articles by Ganjam, V. K