Thyroid status, but not insulin status, affects expression of
avian uncoupling protein mRNA in chicken
Anne
Collin1,2,
Mohammed
Taouis1,
Johan
Buyse2,
Ndey
B.
Ifuta4,
Veerle M.
Darras3,
Pieter
Van
As2,
Ramon D.
Malheiros2,
Vera M. B.
Moraes2,5, and
Eddy
Decuypere2
1 Station de Recherches Avicoles, Institut National
de la Recherche Agronomique, F-37380 Nouzilly, France;
2 Laboratory for Physiology of Domestic Animals,
Department of Animal Production, Katholieke Universiteit Leuven,
B-3001 Leuven and 3 Laboratory of Comparative
Endocrinology, Katholieke Universiteit Leuven, B-3000 Leuven,
Belgium; 4 Département de Biologie,
Institute Supérieur de la Gombe, Kinshasa/Gombe, République
Démocratique du Congo; 5 Faculdade de Ciencas
Agrárias e Veterinárias, Universidade Estadual
Paulista-Jaboticabal, Departamento de Zootecnia, Jaboticabal-Sao Paulo,
Brazil 14870 - 000
 |
ABSTRACT |
The aim of this
study was to investigate the hormonal regulation of the avian homolog
of mammalian uncoupling protein (avUCP) by studying the impact of
thyroid hormones and insulin on avUCP mRNA expression in chickens
(Gallus gallus). For 3 wk, chicks received either a standard
diet (control group), or a standard diet supplemented with
triiodothyronine (T3; T3 group) or with the thyroid gland
inhibitor methimazole (MMI group). A fourth group received injections
of the deiodinase inhibitor iopanoic acid (IOP group). During the 4th
wk of age, all animals received two daily injections of either human
insulin or saline solution. The results indicate a twofold
overexpression of avUCP mRNA in gastrocnemius muscle of T3 birds and a
clear downregulation (
74%) in MMI chickens compared with control
chickens. Insulin injections had no significant effect on avUCP mRNA
expression in chickens. This study describes for the first time
induction of avUCP mRNA expression by the thermogenic hormone
T3 in chickens and supports a possible involvement of avUCP
in avian thermogenesis.
thyroid hormones; thermogenesis; muscle
 |
INTRODUCTION |
IN MAMMALS,
mitochondrial uncoupling proteins (UCPs) are known to uncouple
phosphorylation from oxidation and, hence, to be involved in energy
metabolism. Brown fat UCP1 has also been reported to be involved in
heat production (for review see Ref. 32). The impact of
thyroid hormones on UCP expression is well documented (16, 23,
25), and UCP3 could be one mediator of the thermogenic effect of
triiodothyronine (T3) in mammalian skeletal muscle
(10). However, the implication of UCP3 in thermogenesis in
mammals is controversial (17, 33).
As in mammals, T3 has been reported to have a role in
thermoregulatory mechanisms in birds by stimulating heat production (9, 34). However, the involvement of uncoupling mechanisms in such regulation is still unclear. A recent study (31)
showed that a UCP homolog called avian (av)UCP is expressed in chicken and duckling muscle. The authors suggest that avUCP is structurally close to mammalian UCP2 and UCP3 but that its function could be nearer
to that of UCP1 (expressed exclusively in brown adipose tissue) in
mammals. Indeed, the avUCP messenger is overexpressed in cases of cold
acclimatization in ducklings and in cockerels from the R+ line
presenting a high diet-induced thermogenesis (31). Recent
results obtained in our laboratory (6) also suggest that
the induction of avUCP mRNA expression in cold-exposed chicks is
associated with increased plasma T3 concentrations and heat
production. Moreover, avUCP mRNA expression has been shown to be
inhibited in chickens early conditioned to heat (37), characterized by low plasma T3 concentrations
(39). However, the influence of thyroid hormones on avUCP
expression in chickens has not previously been clearly demonstrated.
Insulin is also known to increase mRNA expression of UCP1 in brown
adipocytes (38) and UCP2 and UCP3 in rat skeletal muscle in vitro (30). It may thus also be suggested that insulin
could regulate avUCP expression in chickens.
The aim of the present study was to investigate hormonal regulation of
mRNA expression of avUCP, a gene potentially involved in thermogenesis,
by insulin, thyroid hormones, and thyroid hormone metabolism inhibitors
in chicken muscle.
 |
MATERIALS AND METHODS |
Experimental design.
Forty-eight 1-day-old male broiler chicks (Gallus gallus)
from a commercial meat-type line (Ross) were purchased from a local hatchery (Avibel, Zoersel, Belgium) and reared in a
temperature-controlled pen. The temperature was set at 30°C for the
1st wk and was gradually lowered by 2°C/wk. The lighting schedule
provided 23 h of light each day, and wood shavings were used as
litter. Commercial starter feed (see Ref. 4 for diet
composition) was provided ad libitum until 7 days of age.
At 7 days of age, animals were randomly allocated to four floor pens
and given one of the following treatments (Table
1). Two groups received a commercial
grower diet in small pellets. A hypothyroid group (MMI group) received
the same commercial diet mixed with 1 g/kg methimazole (Sigma Chemical,
St. Louis, MO). This product inhibits the production of thyroid
hormones in the thyroid gland (7). A hyperthyroid group
(T3 group) received the commercial diet mixed with 1 mg of
T3/kg feed (Sigma Chemical). Treatments remained unchanged
during the 3rd wk of age, except for one group with the control diet
that received two daily subcutaneous injections of 20 mg/kg body wt of
iopanoic acid (IOP group; Sigma Chemical). This product is an inhibitor
of deiodinase and thyroid hormone metabolism, especially of the
conversion of thyroxine (T4) to T3 (9,
29). For the last 5 days of the experiment (week 4),
chickens were subjected to their initial treatment, with addition of
twice daily intramuscular injections of 0.9% saline solution or 4 U of
human insulin/kg body wt (Novo Nordisk, Brussels, Belgium). All groups
were fed ad libitum from week 1 to week 4. On
day 5 of 4 wk of age (day 26), injections
(insulin, saline solution, or iopanoic acid) were planned for birds to
be slaughtered 2 h later.
Measurements and blood and tissue sampling.
Individual body weights and group feed intakes were measured every
week. In week 3 for the IOP group only and for all groups in
week 4, body weights were measured every 2 days to calculate the amounts of insulin or IOP to be injected. Amounts were calculated from estimated body weights on the nonweighing days.
On the sampling day (day 26), blood was drawn from a
brachial vein with a heparinized syringe and collected in ice-cold
tubes to check the effects of the thyroid and insulin treatments on plasma T4 and T3 concentrations and glycemia.
After the animals were killed by cervical dislocation, part of the
liver and gastrocnemius muscles were excised, frozen in liquid
nitrogen, and stored at
80°C for further analysis.
Expression of avUCP.
avUCP mRNA expression was determined in gastrocnemius muscles by
reverse transcription-polymerase chain reaction (RT-PCR). Total RNAs
were isolated using an InstaPure kit (Eurogentec, Angers, France)
according to the manufacturer's recommendations. RNA concentrations were estimated by measuring the absorbance at 260 nm, and purity was
assessed by 260/280-nm absorbance by means of a spectrophotometer (Eppendorf, Hamburg, Germany). For RT-PCR analysis, 1 µg of total RNA
was reverse transcribed with 200 U of Superscript II RT (Invitrogen, Cergy-Pontoise, France) in the presence of random hexamer primers (0.5 µg/µl, Promega). RT was carried out in the presence of 0.5 mM dNTP
mix (Sigma, St-Quentin Fallavier, France) and human placenta RNAguard
RNAse inhibitor (40 U, Amersham Pharmacia Biotech, Les Ulis, France).
The reaction was assessed at 25°C for 15 min and at 42°C for 45 min. PCR was carried out on one-tenth of total RT product in the
presence of two sets of primers flanking a 321-bp fragment of avUCP
(forward: CTCTACGACTCTGTGAAGCA, reverse: TGTGTCCTTGATGAGGTCGTA), and a 148-bp fragment of 18S (forward: CGCGTGCATTTATCAGACCA, reverse: ACCCGTGGTCACCATGGTA). Annealing and extension were carried out at 58 and 72°C, respectively, over 35 cycles followed by 7 min at 72°C.
Negative-control RT-PCR with DNA-free water was included in all
experiments. PCR products were electrophoresed on a 1% agarose gel
containing 0.075 µl/ml Vistra green (Amersham Pharmacia Biotech). The
intensity of RT-PCR bands was determined using a STORM apparatus
(Molecular Dynamics).
Plasma analysis.
Plasma 3,3',5-triiodothyronine (T3) and T4
concentrations were measured by radioimmunoassay as described by Darras
et al. (8). Intra-assay coefficients of variation were 4.5 and 5.4% for T3 and T4, respectively. Antisera
and T3 and T4 standards were purchased from
Byk-Belga (Brussels, Belgium).
Plasma glucose and triglyceride concentrations were determined by use
of commercially available kits from Instrumentation Laboratory
(Lexington, KY). Free fatty acid concentrations in plasma were measured
using kits from Wako Chemicals (Neuss, Germany) modified for use with
the Monarch Chemistry System (Instrumentation Laboratories,
Zaventem, Belgium).
Because glycemia was not depressed in the MMI birds receiving the
insulin treatment, insulin concentrations were measured by
radioimmunoassay on plasma samples from this group treated with insulin
and from all groups treated with saline solution, as described by Simon
et al. (35) by use of a guinea pig anti-porcine insulin
serum (Ab 27-6, gift of G. Rosselin, Hôpital Saint-Antoine, Paris, France) and chicken insulin as standard. The intra-assay coefficient of variation was 1.7%.
All measurements were run in the same assay to avoid interassay variation.
Thyroid hormone hepatic concentrations.
In normal chickens, liver is the main contributor to plasma
T3 through intracellular conversion of T4 to
T3 by type I deiodinase. Therefore, the efficiency of the
treatment with the deiodinase inhibitor IOP can be evaluated from the
effect on intrahepatic T3 and T4
concentrations. Thyroid hormones were extracted from livers by
homogenization in methanol, extraction in chloroform-methanol (2:1) and
two back-extractions in chloroform-methanol-CaCl2 0.05% (3:49:48) as described by Gordon et al. (18). The extracts
were purified on Bio-Rad AG 1 × 2 resin columns and eluted in
70% acetic acid (24, 27). The addition of labeled
[125I]T4 and
[131I]T3 immediately after homogenization and
eluate counting after purification allowed the calculation of an
extraction yield, ranging from 50 to 80%, that was used for final
calculations. Extracted T3 and T4
concentrations were measured by radioimmunoassay (8).
Statistics.
Values for individual body weights and body weight gains for the first
period (thyroid treatment weeks 2 and 3) and the
second period (thyroid and insulin treatment week
4) were analyzed by one-way ANOVA with the thyroid treatment
as main effect (Statview, version 5.0; SAS Institute, Cary, NC),
followed by a Student-Newman-Keuls test.
Due to high heterogeneity of variance between groups, the effects of
insulin treatment and thyroid treatment on thyroid hormone concentrations, plasma glucose, triglyceride, and free fatty acid concentrations, and avUCP mRNA expression were analyzed by
nonparametric tests, including Kruskal-Wallis tests followed by
Mann-Whitney tests. Pearson correlation coefficients were calculated
between plasma T3 concentrations and avUCP mRNA
expressions, between plasma and liver thyroid hormone concentrations,
and between avUCP mRNA expressions and plasma free fatty acid concentrations.
 |
RESULTS |
Plasma and hepatic thyroid hormone concentrations.
Plasma thyroid hormone concentrations on day 26, after both
thyroid and insulin treatments, are presented in Fig. 1, A
and B. As expected from the
pharmacological treatments, plasma T3 concentrations in
saline-treated chickens were lower in IOP and MMI groups compared with
control birds (1.87 and 0.24 vs. 3.17 pmol/ml; P < 0.01). As expected, the T3 treatment induced a threefold increase in plasma T3 concentrations (P < 0.05) compared with saline-treated birds. Plasma T3
concentrations were significantly affected by insulin treatment in the
control group (P < 0.01) and the MMI group
(P < 0.05): T3 concentrations were
depressed in the insulin-treated control group (2.3 vs. 3.2 pmol/ml),
whereas they were enhanced in the insulin-treated MMI group (0.3 vs.
0.2 pmol/ml). Plasma T3 levels tended to be reduced in the
insulin-treated T3 group compared with the saline-treated T3 group
(
50%, P = 0.10).

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Fig. 1.
A: plasma triiodothyronine (T3)
concentrations (±SE) at 26 days of age in chickens treated with either
saline (open bars) or insulin (filled bars) injections. B:
plasma thyroxine (T4) concentrations (± SE) at 26 days of
age in chickens treated with either saline (open bars) or insulin
(filled bars) injections. Animals received a control diet (control
treatment), or a diet containing 0.1% methimazole (MMI treatment) or
containing 1 ppm/kg T3 (T3 treatment), or a control diet
and 2 daily injections of 20 mg/kg body wt of iopanoic acid (IOP
treatment). Different letters represent statistically different values
between thyroid treatments in saline-treated chickens.
*P < 0.05 and **P < 0.01 between
saline- and insulin-treated birds within a thyroid treatment.
|
|
In saline-treated chickens, methimazole and T3 treatments
clearly depressed T4 plasma concentrations compared with
control chickens (1.95 and 2.70 vs. 8.17 pmol/ml, respectively; Fig.
1B). Iopanoic acid-treated birds exhibited slightly but
nonsignificantly higher T4 plasma concentrations than
control chickens. Plasma T4 concentrations were not
affected by insulin treatment.
Patterns of thyroid hormone concentrations were very similar in the
liver and plasma, correlation coefficients being 0.92 for both
T3 and T4. In saline-treated chickens, hepatic
T3 concentrations were not different in IOP and control
birds, whereas concentrations were 91% lower in MMI and 100% higher
in T3 chickens than in control chickens (P < 0.01;
Fig. 2A). Insulin treatment
depressed hepatic T3 concentrations only in control and T3
chickens compared with saline-treated chickens of these groups. The
pattern for hepatic T4 concentrations (Fig. 2B)
was the same as the one observed for plasma T4
concentrations (Fig. 1B).

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Fig. 2.
A: liver T3 concentrations (±SE)
at 26 days of age in chickens treated with either saline (open bars) or
insulin (filled bars) injections. B: liver T4
concentrations (±SE) at 26 days of age in chickens treated with either
saline (open bars) or insulin (filled bars) injections. Animals
received control, MMI, T3, or IOP treatment. Different letters
represent statistically different values between thyroid treatments in
saline-treated chickens. *P < 0.05 between saline- and
insulin-treated birds within a thyroid treatment.
|
|
Plasma glucose, free fatty acid, and triglyceride concentrations.
Thyroid treatments did not significantly affect plasma glucose
concentrations in saline-treated chickens, except for IOP birds, which
exhibited slightly lower glycemia than control birds (2.05 vs. 2.63 g/l; Fig. 3). As expected, insulin
treatment decreased glycemia in the control (1.38 vs. 2.63 g/l;
P < 0.0001), IOP (1.01 vs. 2.05 g/l, P < 0.05), and especially in the T3 (0.62 vs. 2.44 g/l,
P < 0.0001) groups. However, glycemia was not affected
by insulin treatment in MMI chickens.

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Fig. 3.
Plasma glucose concentrations (±SE) at 26 days of age in
chickens treated with either saline (open bars) or insulin (filled
bars) injections. Animals received control, MMI, T3, or IOP treatment.
Different letters represent statistically different values between
thyroid treatments in saline-treated chickens. *P < 0.05, ****P < 0.0001 between saline- and
insulin-treated birds within a thyroid treatment.
|
|
In saline-treated chickens, thyroid treatment significantly affected
plasma triglyceride and free fatty acid concentrations (Table
2): T3 chickens had lower triglyceride
concentrations than IOP and control chickens (P < 0.05) and lower free fatty acid concentrations than control and MMI
birds (P < 0.01). In insulin-treated chickens, thyroid
treatment did not affect plasma triglyceride concentrations, whereas it
affected plasma free fatty acid concentrations (P < 0.05): MMI chickens had lower free fatty acid concentrations than
control and T3 chickens.
Expression of avUCP.
avUCP mRNA expression was positively correlated with plasma
T3 concentrations (y = 0.0127x + 0.0421, R2 = 0.4071, P < 0.001). In saline-treated birds, avUCP
mRNA expression was significantly lower in MMI chickens (
74%) and
twice as high in T3 chickens as in control birds (Fig.
4). Treatment with iopanoic acid did not
affect avUCP expression (P = 0.37). avUCP mRNA
expression was eight times higher in T3 than in MMI chickens. Insulin
treatment did not significantly affect avUCP mRNA expression,
irrespective of the thyroid treatment.

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Fig. 4.
A: avian uncoupling protein (avUCP) mRNA expression
relative to 18S mRNA expression (±SE) in gastrocnemius muscle of
chickens at 26 days of age injected with either saline (open bars) or
insulin (filled bars) solutions. B: RT-PCR products obtained
from avUCP and 18S RNA. Products were stained using Vistra green
(Amersham Pharmacia Biotech) and quantified with a Storm apparatus
(Molecular Dynamics). Animals received control, MMI, T3, or IOP
treatment. Different letters represent statistically different values
between thyroid treatments in saline-treated chickens.
|
|
avUCP mRNA expressions were not correlated with fatty acid
concentrations (coefficient of
0.03, P = 0.86) in the
present experiment.
Feed intake and growth.
Overall feed intakes during weeks 2 and 3 of the
experiment were the highest in the control group and the lowest in the
MMI group, these chickens eating 50% less than the control group in week 3. During the 4th wk of the experiment, feed intakes in
birds receiving saline solution injections were 30, 81, 112, and 80 g · day
1 · animal
1
for the MMI, IOP, control, and T3 groups, respectively. Insulin injections reduced feed intakes in MMI, IOP, control, and T3 groups by
32, 9, 22, and 20%, respectively (Table
3).
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Table 3.
Feed intake and body weight gain during weeks 2 and 3 of experiment
(1st period) and week 4 (2nd period)
|
|
Growth was affected by thyroid status. Especially during the 3rd wk of
the experiment, body weight gains of MMI, IOP, and T3 chickens were 68, 25, and 29% lower than that of control chicks, respectively, and the
same tendency was observed during the 4th wk of the experiment in
saline- or insulin-treated chickens. However, body weights did not
differ significantly between animals receiving saline solution or
insulin on day 26 (data not shown).
 |
DISCUSSION |
Treatment with T3 or thyroid inhibitors induced clear
changes in thyroid status and dramatic changes in avUCP expression in chickens. As expected, the methimazole treatment strongly depressed both plasma and liver T4 and T3 levels as a
consequence of the thyroid gland dysfunction (7). The
effect of iopanoic acid on T3 concentrations was less
pronounced than that of methimazole, probably because of residual
production of T3 by the thyroid gland (21,
22). In addition, the iopanoic treatment might not have been
strong enough to inhibit deiodinases completely. This is suggested by
the similar liver T3 and T4 concentrations in
IOP and control chickens. It is likely that total suppression of the peripheral degradation of T4 in IOP birds would have
significantly increased its plasma levels compared with control birds
(9). Nevertheless, T3 addition in the diet had
a clear hyperthyroid effect in T3 chickens, causing a dramatic increase
in plasma and liver T3 concentrations and a decline in
plasma and liver T4 levels. This is probably a consequence
of the negative feedback of plasma T3 on the pituitary
release of thyroid-stimulating hormone and, hence, on thyroidal release
of T4 (20).
Thyroid treatment clearly affected avUCP mRNA expression in the same
way as plasma T3 concentrations, in view of the high correlation coefficient between both parameters. avUCP mRNA expression was markedly enhanced by T3 treatment, whereas it was
slightly (but nonsignificantly) depressed by IOP and dramatically
depressed by MMI treatment. The poor effect of iopanoic acid on avUCP
mRNA expression might be the result of the moderate decline in plasma T3 concentrations. The results of avUCP gene expression
would have been strengthened by measurements of protein expression by Western blot, but assays made with a heterologous antibody were not
good, and there is still a need for a specific anti-avUCP antibody. The
increase in avUCP mRNA expression in gastrocnemius muscles of
hyperthyroid chickens is consistent with previous results showing the
enhancement of UCP3 mRNA expression in skeletal muscle of
T3-stimulated rats (10, 16, 25). However, the
implication of UCP3 in thermogenesis in mammals is still being debated
(10, 17, 33). Our results are consistent with an
involvement of avUCP in thermogenesis in poultry, as suggested by
Raimbault et al. (31), where glucagon (a thermogenic
hormone) strongly stimulates the expression of this gene. The mRNA
expression of avUCP is also markedly increased in cold-acclimatized
ducklings and in chickens from the R+ energy-inefficient laying strain
(31), both of which present high heat production (1,
11, 14). In contrast, avUCP mRNA expression is reduced in early
heat-conditioned chicks, which exhibit lower internal temperatures than
nonconditioned chicks (37). It is likely that, as in
mammals with UCP1, the enhancement of thermogenesis observed in
T3-treated chickens (9) is partly mediated by
increased expression of avUCP. The enhancement of the avUCP gene
expression, together with the increases in T3
-receptor
mRNA expression,
-oxidation, and cytochrome oxidase activities,
mitochondrial respiration, and ATP synthesis observed by Mouillet
(28) in muscle of ducklings treated with thyroid hormones,
could contribute to the thermogenic action of thyroid hormones in birds.
During the last few years, many studies have focused on the roles of
mammalian uncoupling proteins UCP2 and UCP3 (12, 17, 33).
In particular, Dulloo et al. (12) suggested that UCP3 was
more involved in the regulation of lipids as fuel substrate than in
thermogenesis, and several authors have suggested that UCPs could
facilitate electrophoretic translation of fatty acid RCOO
anions through the mitochondrial internal membrane (15,
36). Indeed, UCP3 mRNA is reported to be upregulated during
fasting, which contradicts a role in maintaining thermogenesis through uncoupling mechanisms. Furthermore, nutritional factors [high-fat diet, lipid infusions, fasting (2, 19, 26)] that
upregulate the expression of this gene in skeletal muscle also
simultaneously enhance plasma free fatty acid levels (17,
40). Evock-Clover et al. (13) recently showed that
fasting and the subsequently increased plasma free fatty acid levels
were correlated with high mRNA expression of avUCP in chicken. However,
in the present study, avUCP mRNA expressions were not correlated with
plasma free fatty acid concentrations, which contradicts a role for
these plasma metabolites in the mediation of the effects of
T3 or of inhibitor of thyroid metabolism on avUCP mRNA
expression in chicken.
In the present experiment, insulin treatment did not significantly
affect avUCP mRNA expression either in control or in
thyroid-manipulated chickens. This result contrasts with the results
observed in mammals, where clear increases in UCP1 mRNA expression were
described in brown adipocytes (38) and in UCP2 and UCP3
mRNA expression in rat skeletal muscle in vitro (30). It
is possible that the insulin treatment in the present experiment was
not strong enough to have clear metabolic effects. However, the results
shown in Fig. 3 demonstrate the dramatic effects of insulin treatment
on glycemia in control, IOP, and T3 chickens, consistent with the
strong effect of insulin in T3-treated birds already
observed by Buyse et al. (3). The question of the effect
of insulin injections remained for insulin-treated MMI birds that
presented glycemia similar to that of saline-treated MMI birds. Yet
plasma insulin concentrations measured by radioimmunoassay were more
than ten times higher in insulin-treated MMI chickens than in
saline-treated MMI birds (665 vs. 43 µU/ml). Thus the absence of
effect of insulin in MMI chickens probably results from a resistance to
exogenous insulin. Moreover, the present results show that plasma
T3 concentrations tended to be reduced after insulin
treatment and that glycemia was the most affected by insulin treatment
in the T3 group. This is consistent with recent findings suggesting
that glucose is a major driving force for the regulation of peripheral
T3 formation (5). The interactions between
thyroid status and insulin signaling must therefore be further
investigated in chicken muscle. Our results suggest that, in chickens
in vivo, insulin is not a strong regulator of avUCP mRNA expression.
However, it is difficult to know whether a single insulin injection or
a physiological insulin infusion would also have resulted in unchanged
avUCP mRNA expression.
In conclusion, avUCP mRNA expression was strongly regulated by
triiodothyronine and the thyroid gland inhibitor methimazole but not by
insulin treatment in our conditions. However, further studies are
necessary to demonstrate the role of avUCP in uncoupling mitochondria
and the relationship with thyroid hormone status.
 |
ACKNOWLEDGEMENTS |
We thank G. Nackaerts, C. Borgers, A. Respen, L. Noterdaeme, W. van
Ham, F. Voets, G. Reyns, and B. Six in Leuven, and S. Crochet and M. Derouet in Tours for their skilled technical assistance. R. D. Malheiros received a postdoctoral fellowship from Conselho Nacional de
Desenvolvimento Científico e Tecnológico, Brazil.
 |
FOOTNOTES |
Address for reprint requests and other correspondence:
A. Collin, Station de Recherches Avicoles, Institut
National de la Recherche Agronomique, F-37380 Nouzilly, France
(E-mail: collin{at}tours.inra.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 10, 2002;10.1152/ajpendo.00478.2002
Received 31 October 2002; accepted in final form 2 December 2002.
 |
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