Acta Veterinaria Hungarica 57 (2), pp. 319–330 (2009) DOI: 10.1556/AVet.57.2009.2.12

0236-6290/$ 20.00 © 2009 Akadémiai Kiadó, Budapest

EFFECTS OF ENERGY RESTRICTION ON THYROID HORMONE METABOLISM IN CHICKENS

Andrea GYŐRFFY1, Ahmed SAYED-AHMED2, Attila ZSARNOVSZKY1, Vilmos László FRENYÓ1, Eddy DECUYPERE3 and Tibor BARTHA1*

1Department of Physiology and Biochemistry, Faculty of Veterinary Science, Szent István University, István u. 2, H-1078 Budapest, Hungary;

2Department of Anatomy, Faculty of Veterinary Medicine, Alexandria University, Damnhur Branch, Egypt; 3Laboratory for Farm Animals, Catholic University of Leuven,

Heverlee, Belgium

(Received 22 May 2008; accepted 1 October 2008)

Energy restriction induces changes in thyroid hormone economy in the form of a complex adaptation mechanism, in order to conserve energy storage and protein reserves. In the present work, thyroid hormone serum concentrations, he-patic deiodinase enzyme activities and hepatic deiodinase mRNA expression were examined after feed restriction and fasting. We demonstrate that during energy re-striction, T3 concentration is lowered due to a decreased T4 activation and in-creased T3 inactivation. We show that hepatic type-I deiodinase (D1) is not af-fected by energy restriction, however, hepatic D2 is decreased on both transcrip-tional and enzyme activity levels. Furthermore, hepatic D3 is increased after feed restriction in the liver. We also show that the hypothalamic feedback is not in-volved in the changes in serum T3 and T4 concentrations. Our data indicate that D2 enzyme contributes to the special hormone-exporting role of the chicken liver and this enzyme can be modulated by feed restriction.

Key words: Chicken, liver, deiodination, energy restriction

Thyroid hormones are essential for many biological processes including normal development, growth and metabolism. The thyroid gland is the sole source of thyroid hormones. Its main product is thyroxine (T4), while most of the relevant biological effects can be attributed to triiodothyronine (T3). T3 is also produced by the thyroid gland; however, it can be formed in extrathyroidal tis-sues as well, by an enzymatic conversion of T4 into T3, accounting for more than 99% of the total T3 production in the chicken (Darras et al., 2006). There is an-

*Corresponding author; E-mail: bartha.tibor@aotk.szie.hu; Fax: 0036 (1) 478-4165

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other thyroid hormone, reverse-T3 (r-T3), believed to have hypometabolic effects and to reduce the hypermetabolic properties of T3 (Bobek et al., 1993).

The peripheral conversion of thyroid hormones is catalysed by deiodinase enzymes. Three different deiodinases are known: type-I, type-II, and type-III (D1, D2 and D3) enzymes. All these enzymes have been cloned and character-ised: all contain selenium in the active site and remove single iodine atoms from the hormone molecules. D1 and D2 remove iodine from the outer ring (5' deiodi-nation, 5'D) producing T3 from T4, therefore 5'D is called the activating pathway (Bianco et al., 2002; Darras et al., 2006). D3 and in certain cases D1 remove io-dine from the inner ring (5 deiodination, 5D) degrading T3 or converting T4 into r-T3; therefore, 5D is called the inactivating pathway. The two activating en-zymes show different characteristics. D1 deiodinase monomer is an integral membrane protein, while D2 resides in the endoplasmic reticulum. This differen-tial subcellular localisation explains why there is such a minimal contribution of the T3 generated by D1 to the intranuclear T3 in contrast to the large fraction of D2-generated T3 to this compartment (Bianco and Larsen, 2005).

In terms of thyroid control, tissues containing D2 are relatively independ-ent of the circulating hormone concentrations and the T3 that is produced has mostly local, intracellular effects. The high D2 level of the brain helps to maintain nearly normal T3 levels in neurons even in severe hypothyroidism (Rudas et al., 1994). Organs with high D1 deiodinase activity, however, are active contributors in maintaining the active thyroid hormone concentration of the circulation. These organs are the so-called ‘hormone-exporting’ organs (Hulbert, 2000). Producing a considerable amount of circulating T3, the liver is one of the most important hor-mone-exporting organs. Liver deiodinases have an important role in adjusting the metabolic rate of the body to the energy content of the ingested food. The liver is directly informed about the energy uptake as energy equivalents absorbed from the gut reach the liver through the portal circulation. Gastrointestinal hormones also inform the liver about amino acids, carbohydrates and fatty acids present in the stomach and the small intestines to regulate the digestion (Bartha et al., 1989).

The role of hepatic deiodinases in adapting to different energy conditions has been widely examined. Data were collected mainly from mammals, espe-cially from rats. It was found that hepatic D1 and D3 enzymes are important in responding to different feeding conditions. D2 enzyme was not examined as D2 is not present in mammalian hepatocytes. Birds, however, show certain differ-ences as compared to mammals. It was shown that D1 enzyme does not react to energy restriction, while D3 activity increases significantly. It was concluded that due to energy restriction the available T3 concentration decreases because of the increased enzymatic inactivation (Van der Geyten et al., 1999; Buyse et al., 2000; Reyns et al., 2002).

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After cloning the chicken D2, its tissue distribution was examined by Northern blot technique. D2 deiodinase was found in the chicken liver as well (Gereben et al., 1999).

The aim of this study was to describe the effects of energy restriction on thyroid hormone economy in the chicken, including peripheral hormone activation, with the main focus on liver deiodination, especially on D2 as well as the central feedback regulation.

Materials and methods

Experimental animals

In the first set of experiments 180 male, one-day-old Hunniahybrid chicks were sorted into groups of 10 individuals. The chicks were provided with feed and water ad libitum. The commercial feed and the animals were purchased from Bábolna Co., Bábolna, Hungary. The chicks were exposed to normal lighting re-gime (16L:8D). On day 28, birds were allotted to three major groups: (1) ad libi-tum fed control (100%), (2) feed intake restricted to 70% of control (70%), and (3) feed intake restricted to 85% of control (85%). Six, 12, 24, 36, 48 and 96 h after the beginning of feed restriction, liver and blood samples were collected. Before sacrificing the chicks 5 out of 10 birds were used for TRH provocation test in each group. In the second set of experiment 30 animals with the same conditions as the control group above were used for measuring D1, D2 and D3 activities and to perform real-time PCR studies. Ten birds formed each group, the ad libitum fed control (100%), the 6-hour fasted and the 12-hour fasted ones. The birds were kept according to the welfare regulations of Szent István Univer-sity, Budapest (registry number: 475/001/2003).

TRH provocation test

Blood samples were taken just before and 30 min after an iv. injection of thyrotropin-releasing hormone (2 µg TRH/100 g of body weight) and assayed for thyroid hormone concentrations. Changes in T4 and T3 concentrations after TRH (Sigma-Aldrich Inc. St Louis, MO, USA) induction were determined.

Thyroid hormone serum concentrations

T4 and T3 concentrations in the blood were determined by a radioimmunoas-say (RIA) procedure adapted for chicken as described by Rudas and Pethes (1986).

Deiodinase activities

The hormone activation (5'D) was assessed according to a procedure de-scribed earlier (Bartha et al., 1989). In short, liver homogenates were incubated at 37 °C with T4 in the presence or absence of 1 mM 6-propyl-2-thiouracil (PTU,

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Sigma-Aldrich Inc., St. Louis, MO, USA), a specific blocker of D1 deiodinase. After 60 min the incubation was terminated by ethanol, and after centrifugation the supernatant was used for the determination of T3.

For determination of D1, D2 and D3 activities 125I-labelled r-T3, T4 and T3 (specific activity: 5 MBq/sample; Isotope Institute Ltd., Budapest, Hungary) were used, respectively. The final hormone concentrations were set to 2 µM, 2 nM and 10 nM for r-T3, T4 and T3, respectively. Two hundred µg of microsomal protein was incubated with radiolabelled hormones in the presence of 50 μM di-thiothreitol. To determine D2 activity 200 nM T3 was added in order to block D3. To determine D2 and D3 activities 1 mM PTU was added in order to block D1. For a review see St Germain and Galton (1997).

Q-PCR

Total RNA was isolated from the tissue samples using SV Total RNA Iso-lation Kit (Promega Co., Madison, WI, USA) according to the manufacturer’s instructions. Reverse transcription polymerase chain reaction was performed as described by Sayed-Ahmed et al. (2004). Chicken gene sequences available in GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) were used for designing oligonucleotide primers (Light Cycler Probe Design Software; F. Hoffmann-La Roche Ltd, Basel, Switzerland). Primer sequences are available upon request. Hepatic gene expression concentrations were quantified in quantitative PCR reactions (QPCR; LightCycler 2.0, F. Hoffmann-La Roche Ltd, Basel, Switzerland) using SYBRGreen I fluorescence dye (Hoffmann-La Roche Ltd, Basel, Switzerland). Aliquots of cDNAs were dispensed according to the manufacturer’s instructions. QPCR cycles were constructed based on the manufacturer’s instructions and were optimised for each primer pair. Amplified products were identified by agarose gel electrophoresis, melting point and se-quence analysis (Applied Biosystems ABI 3100 Genetic Analyzer, Agricultural Biotechnology Center, Gödöllő, Hungary)

Statistical analysis

One- and two-way analysis of variance and t-tests were carried out by the freeware R program (http://www.r-project.org/). Differences are considered to be significant if the probability levels were below 5%.

Results

According to the analysis of variance, feed restriction had an overall sig-nificant (P < 0.0001) effect on serum T3 concentration. Serum T3 concentrations were decreased in the feed-restricted groups (Fig. 1).

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Fig. 1. Serum concentrations of T3. Bars show the T3 concentrations (mean ± SEM) in 70%, 85%,

and ad libitum fed control (100%) groups in the first 96 hours after feed restriction. Feed restriction has a significant (P < 0.0001, ANOVA) effect on serum T3 level. The decrease in T3 level is

already significant at hour 6 (*P < 0.05, **P < 0.01, ***P < 0.001, t-test)

The liver deiodination activity was examined by T4-to-T3 conversion ca-

pacity (Fig. 2). 5'D activity was affected significantly by feed restriction (P < 0.0001, ANOVA) and time (P < 0.0001). Enzyme activities were lower in the feed-restricted groups as compared to the controls. There was an interaction between time and feed restriction (P < 0.001). The time dependence could be traced by t-test at each sampling time. Significant changes appeared at hour 6. The most striking difference could be observed at hour 12. The differences in conversion capacity gradually disappeared, and no significant changes existed at hour 96.

Serum T4 concentrations are shown in Fig. 3. Analysis of variance showed that feed restriction had a significant (P < 0.0001) effect on the concentration of serum T4. The T4 concentrations were higher in the feed-restricted groups than in the controls. After 6 h of feed restriction there was already a 2-fold elevation in serum T4 concentration in the 70% group, and this difference increased to 3-fold at hour 96. Comparing individual groups with t-test, the difference was signifi-cant as early as 6 h after the onset of feed restriction between the 70% and the 100% group. Both experimental groups were significantly different from the control group from 24 h on.

The response in serum T4 concentrations 30 min after TRH injection was also investigated. Data in Fig. 4 show the percent increase of serum T4 concen-tration. Feed restriction significantly (P < 0.0002, ANOVA) changed T4 concen-trations induced by TRH. There was a blunted TRH response in the feed-restricted groups. No significant time effect could be detected; however, signifi-cant (P < 0.05, ANOVA) time × feed restriction interaction was found in T4 re-sponse to TRH. The differences between the experimental groups became sig-nificant (according to the t-test) from 48 h on.

100% 70% 85%

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Fig. 2. Net T4 to T3 conversion by liver deiodinases. Bars show the 5'D enzyme activity (mean ± SEM) in the three different groups (ad libitum fed control [100%], 70%, and 80% groups) in the first 96 h after the start of the experiment. Feed restriction decreases the net liver deiodination

activity and hence the net T3 production significantly (P < 0.0001, ANOVA). The differences are significant up to hour 48 (*P < 0.05, **P < 0.01, ***P < 0.001, t-test)

Fig. 3. Serum concentrations of T4. Bars show the serum T4 concentrations (mean ± SEM) in the

70%, 85% and ad libitum fed control (100%) groups in the first 96 h of feed restriction. Feed restriction has a significant (P < 0.0001, ANOVA) effect on serum T4 concentrations. Serum T4 concentrations increase significantly even after 6-h exposure to relative fasting

(*P < 0.05, **P < 0.01, ***P < 0.001, t-test)

Changes in serum T3 concentrations were also measured after TRH injec-tion. While TRH treatment increased serum concentrations of T3, no significant group or time effect was found by analysis of variance (Fig. 5).

Hepatic D1-3 measurement data after 6- or 12-h fasting are illustrated in Fig. 6. Panel A shows that there were no significant differences in D1 deiodina-tion activity between the fasted and ad libitum fed groups. Panel B shows that

100% 70% 85%

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D2 activity was significantly (P < 0.001, t-test) lower in the fasted groups than in the control one. Panel C shows that D3 activity was also affected significantly (P < 0.001, t-test) by fasting.

Fig. 4. Changes of T4 concentrations after TRH induction. Bars show the increase in serum T4 concentrations (mean ± SEM) 30 min after exogenous TRH (100%: no change) in the different experimental groups (ad libitum fed control [100%], 70%, and 80% groups]). Feed-restricted

groups show a decreased response to TRH (P < 0.0002, ANOVA). This is the trend at each time point; however, the differences are significant after two days (***P < 0.001, t-test)

Real-time PCR data are shown in Fig. 7. The amount of D2 mRNA in

hepatocytes of chickens fasted for 6 h was 18 times, while after 12-h fasting it was 28 times less than in the control. These differences were significant (P < 0.001) according to the t-test.

Fig. 5. Changes of serum T3 concentrations after TRH induction. Bars show the increase in serum T3

concentrations (mean ± SEM) 30 min after exogenous TRH (100%: no change) in the different experimental groups (ad libitum fed control [100%], 70%, and 80% groups). Serum T3 concentrations increase after injecting TRH. No significant group or time effect was confirmed by statistical analysis

100% 70% 85%

100% 70% 85%

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Fig. 6. Hepatic D1, D2 and D3 activities as measured individually after 6 and 12 h of fasting. Bars show the hepatic deiodinase activity (mean ± SEM) in the different experimental groups (ad libitum fed control [100%], 6-h fasting, 12-h fasting) as expressed by the percentage of specific substrate

converted. D1 enzyme shows no change after short-term fasting (panel A), while D2 enzyme activity (panel B) significantly decreases and D3 activity (panel C) significantly increases in

response to starvation (***P < 0.001, t-test)

Fig. 7. Changes in hepatic D2 mRNA expression levels after 6- and 12-h fasting. Bars show D2

mRNA levels (mean ± SEM) measured by quantitative PCR. Experimental groups are compared to the ad libitum fed control (100%). Six and 12 h of fasting significantly decreases D2 mRNA by 15

and 28 times, respectively (***P < 0.001, t-test)

Discussion

Decreased energy uptake and/or fasting turns on energy-saving mecha-nisms. Thyroid hormones, which regulate oxygen consumption, are involved in the adaptation mechanism to prepare the body for energy shortage. In the present study

Panel A Panel B Panel C

100% 6 h 12 h

100% 6 h 12 h 100% 6 h 12 h 100% 6 h 12 h

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we examined how thyroid hormone economy reacts to energy restriction. Both the central regulation (hypothalamo-hypophysis-thyroid axis) and the peripheral hor-mone conversion (liver deiodinating activities, especially D2) were examined.

Our results show that thyroid hormone economy in the chicken reacts to feed restriction within hours. The observed changes tend to decrease the avail-ability of the only biologically active thyroid hormone, T3. T3 that binds to nu-clear receptors might come from two different sources. In the brain tissue the lo-cal production covers the physiological needs of the nervous tissue; however, most other tissues rely on blood-borne T3; therefore, the latter group is referred to as ‘hormone-importing’ tissues (Hulbert, 2000). Energy restriction decreases the serum T3 concentrations (Fig. 1) providing less available T3 for ‘hormone-importing’ cells. Even 24-h 15 to 30% feed restriction results in a significant de-crease in the amounts of circulating T3.

As mentioned in the introduction, only a small portion of the circulating T3 is produced by the thyroid gland; the rest originates from the enzymatic conver-sion of T4. Blocking deiodination activity by iopanic acid decreases the T3/T4 ra-tio to less than 30% (Decuypere et al., 1982). Buyse et al. (2002) and Reyns et al. (2002) stated that the net result of fasting is a reduction of T4-to-T3 conversion and a decrease in the calorigenic effect of thyroid hormones presumably due to altered T4 to T3 ratios. Decuypere and Kühn (1984) could halt circadian rhythmic changes of thyroid hormone blood concentrations with two days of fasting and could restore them by re-feeding.

Decreased net hormone activation in the liver was found in the feed-restricted groups, and the deiodinase system is sensitive enough to react to even slight energy restriction. Deiodinase enzymes react quickly, within hours, but the differences between experimental and control groups tend to disappear after 4 days (Bartha et al., 1989).

As a consequence of the decreased 5'D activity, less T4 is converted to T3, which leads to an increase in the circulating concentration of T4 (Darras et al., 2006). We also found increased T4 concentrations in the restricted groups as compared to controls. Increased thyroid T4 output might be the other explanation for the increased T4 serum concentration. The TRH provocation test was used to test this hypothesis. TRH is one of the most important stimulating factors in the hypothalamic-pituitary control of TSH secretion. This is so in mammals and in young birds, whereas in adult birds TRH plays a different role (Decuypere et al., 2005; Kühn et al., 2005). The major inhibitory influence on TSH secretion is an increase in the concentration of circulating thyroid hormones, especially T4, which desensitises the pituitary to the stimulatory effects of TRH (Hulbert, 2000). In feed-restricted groups, the ability to respond to TRH has indeed been reduced and may concomitantly be invoked for a reduced response to endoge-nous TRH; hence a relatively low TSH production and consequently a lower se-cretion rate in the thyroid gland exist. Results obtained in the TRH groups sug-

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gest that in addition to lower enzymatic consumption of T4, lower T4 production occurs in the thyroid gland.

In the present experiment feed restriction did not alter T3 response upon TRH injection. The 4- to 5-week-old chickens used in the experiments show the pattern to react to TRH, which is characteristic of young birds. Decuypere and Scanes (1983) have shown that the magnitude of T3 responses to TRH increase with age, while that of T4 decreases. Decuypere et al. (2005) and Kühn et al. (2005) found that TRH is not thyrotropic (does not increase plasma concentra-tion of T4), but somatotropic [increases growth hormone (GH) plasma concentra-tion] in adult chickens. TRH administration is followed by increased circulating concentration of GH, and GH decreases 5D (Darras et al., 2006), while it proba-bly increases 5'D (Geris et al., 1998; Kühn et al., 2005). In this way, serum T3 concentrations are not only decreased due to the reduced T4-to-T3 conversion but also by the increased T3-to-T2 degradation. Furthermore, it was also shown that energy restriction affects D3 only, while D1 activity remains unaffected (Van der Geyten et al., 1999; Buyse et al., 2000; Reyns et al., 2002).

Fig. 8. Regulation of thyroid hormone economy during energy restriction in the chicken. Decreased

energy absorption decreases the hormone activation and increases hormone inactivation in the liver. As a result, serum T3 concentration decreases, while T4 concentration increases. Increased

serum T4 concentration renders the central regulation relatively insensitive to TRH

Our observations, however, strongly suggest that thyroid hormone activa-

tion is also affected by the energy concentration of the feed. Increased T4 con-centration in the feed-restricted groups cannot be explained by decreased TRH sensitivity and unaffected T4-to-T3 conversion. In such a case T4 concentration should drop rather than increase. Although D1 is not affected, the PTU-insensitive 5'D decreases during energy restriction (data not shown). Finding the PTU-insensitive D2 in the chicken liver gave a new prospect in understanding the role of liver hormone activation.

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In a new set of experiments the short-term effects of energy restriction were examined after 6 or 12 h of fasting. Pilot experiments showed that this ex-perimental setup is suitable for examining all the above observed changes result-ing from energy restriction. We measured D1, D2 and D3 activity in all three groups. Figure 6 shows that D1 activity does not react to short-term energy re-striction (panel A), while D2 activity decreases significantly (panel B). We con-clude that the change in total 5'D activity can be attributed to the changes in D2 and not to D1. Our results are in accordance with the findings of other research-ers (Van der Geyten et al., 1999; Buyse et al., 2000; Reyns et al., 2002), who found that only D3 activity (panel C) changes during feed restriction and there is no change in D1 deiodinase activity. In these studies liver D2 activity, however, was not measured.

We also investigated whether D2 activity is reflected in D2 mRNA ex-pression level. D2 mRNA expression levels were determined using quantitative PCR. Our data show that the regulation of D2 enzyme is already present at the transcriptional level (Fig. 7).

Figure 8 summarises the regulation of thyroid hormones during fasting in the chicken. The thyroid gland produces less T4 for the circulation, meanwhile less T4 is consumed by 5'D activity. As a net result, more T4 remains in the circu-lation, thereby increasing the circulating amount of T4. The slowed activating pathway produces less T3, while more T3 is consumed by inactivation. Finally, less T3 remains in the circulation providing less available T3, thus decreasing the metabolism. We demonstrated that D2 has a special role in chickens. D2 is pre-sent in hepatocytes and produces T3 for the circulation. Furthermore, in the adap-tation to energy restriction, thyroid hormone activation is decreased in the liver and the entire regulatory role in hormone activation (5'D) can be attributed to the activity of the D2 enzyme.

Acknowledgements

This work was supported by the Hungarian Scientific Research Fund grants OTKA 46914, OTKA 72186, GVOP-3.2.1.-2004-0225/3.0, and by the PhD programme funds of the Catholic University of Leuven, Belgium. Special thanks to Zsuzsanna Szikora and Péter Rákoss for their excellent technical assistance.

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