1 Division of Endocrinology and Diabetology, Department of Medicine, Faculty of Medicine, University of Geneva, 1211 Geneva 14, Switzerland; and 2 Internal Medicine III, Erasmus University, 3015 GD, Rotterdam, The Netherlands
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ABSTRACT |
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To
assess whether intracerebroventricular leptin administration affects
monodeiodinase type II (D2) activity in the tissues where it is
expressed [cerebral cortex, hypothalamus, pituitary, and brown adipose
tissue (BAT)], hepatic monodeiodinase type I (D1) activity was
inhibited with propylthiouracil (PTU), and small doses of thyroxine
(T4; 0.6 nmol · 100 g body
wt1 · day
1) were
supplemented to compensate for the PTU-induced hypothyroidism. Two
groups of rats were infused with leptin for 6 days, one of them being
additionally treated with reverse triiodothyronine (rT3), an inhibitor
of D2. Control rats were infused with vehicle and pair-fed the amount
of food consumed by leptin-infused animals. Central leptin
administration produced marked increases in D2 mRNA expression and
activity in BAT, changes that were likely responsible for increased
plasma T3 and decreased plasma T4 levels. Indeed, plasma T3 and T4 concentrations were
unaltered by central leptin administration in the presence of
rT3. The additional observation of a leptin-induced
increased mRNA expression of BAT uncoupling protein-1 suggested that
the effect on BAT D2 may be mediated by the sympathetic nervous system.
intracerebroventricular leptin; triiodothyronine; thyroid hormones
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INTRODUCTION |
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THYROID HORMONES have long been known to play an important role in the regulation of energy balance, in particular by stimulating thermogenesis (37). Leptin, an adipocyte-derived hormone acting on hypothalamic neurons to decrease food intake, has also been shown to stimulate energy-dissipating mechanisms (16, 42, 43). It can, therefore, be speculated that leptin may affect thermogenesis through an effect on thyroid hormones. In this respect, it is well documented that food restriction, a condition associated with low leptin levels, has profound effects on the hypothalamo-pituitary-thyroid axis in rats, including low plasma thyroxine (T4) and triiodothyronine (T3) levels, as well as decreased TRH and TSH synthesis in the hypothalamus and the pituitary, respectively (27). Systemic administration of leptin in rats and mice has been shown to prevent the fasting-induced reduction in proTRH mRNA levels occurring in neurons of the paraventricular nucleus, as well as to restore to normal the decreased circulating T4 and T3 levels due to food restriction (1, 24). Intracerebroventricular administration of leptin in rats has also been shown to reverse the marked suppression of TSH secretion measured in food-restricted animals (36). Such a stimulatory effect of leptin on spontaneous TSH secretion during food restriction was shown to depend on the thyroid status, as it was not observed in hypothyroid rats (36).
We have previously demonstrated (8) that central leptin administration in rats prevented the fall in plasma T3 occurring during food restriction, whereas it was without effect on the decrease in serum TSH measured in these conditions. We also demonstrated (8) that hepatic deiodinase type I (D1) activity was decreased by food restriction, and that central leptin infusion completely prevented such a decrease. Under our experimental conditions of central leptin infusion, the peripheral plasma leptin levels were unchanged, indicating that the effects of leptin on hepatic D1 expression and activity had to be centrally elicited. Because central leptin administration has been shown to stimulate the sympathetic nervous system outflow (23), it could be hypothesized that leptin would influence hepatic D1 through an activation of this branch of the autonomic nervous system. However, D1 expression and activity are not known to be influenced by the sympathetic nervous system (or by catecholamines) (19).
In view of these considerations, we postulated that central leptin administration might stimulate deiodinase type II (D2) activity, thus resulting in an increase in plasma T3 levels, which would then be sufficient to increase D1 activity (19). To test the hypothesis of a possible effect of leptin on D2, we used an in vivo approach allowing us to differentiate between the regulation of D1 and D2. This approach was based on the following considerations. The thyroid hormone metabolite reverse T3 (rT3) has been reported to strongly inhibit D2 activity (34). However, the rapid metabolism of rT3 by hepatic D1 in the adult rat (14, 17) hampers its in vivo use as a D2 inhibitor. By inhibiting D1 activity with propylthiouracil (PTU), we could expect to decrease rT3 degradation and to therefore obtain high plasma levels of infused rT3 that would inhibit D2 activity. The use of PTU would also reduce hepatic D1 activity to constantly low levels, excluding the possibility that leptin might affect plasma T3 levels through an activation of this enzyme. Finally, the hypothyroid state induced by PTU would be partly compensated for by moderate T4 supplementation to provide substrate for D2 activity while avoiding inhibition of D2 by T4 (25, 41).
On the basis of the aforementioned considerations and with the use of PTU-treated, T4-supplemented rats, the aims of the present study were 1) to determine whether the intracerebroventricular administration of leptin has an action on D2 in the tissues in which this enzyme is expressed, i.e., in the cerebral cortex, the hypothalamus, the pituitary gland, and brown adipose tissue (BAT); 2) to investigate what the consequences are on plasma T3 levels of such a potential effect of central leptin administration on D2; and 3) to assess whether central leptin has a concomitant stimulatory effect on the sympathetic nervous system activity, as assessed by the measurement of the mRNA expression of BAT uncoupling protein-1 (UCP1).
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MATERIALS AND METHODS |
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Animals. Eight- to nine-week-old male Wistar rats purchased from BRL (Basel, Switzerland) were housed under conditions of controlled temperature (23°C) and illumination (7:00 AM-7:00 PM). They were allowed ad libitum access to water and standard laboratory chow (Provimi Lacta, Cossonay, Switzerland).
Experimental procedure.
All animals were made hypothyroid with PTU (FLUKA Chemie, Buchs,
Switzerland) given in their drinking water at a dose of 0.025% during
the whole experimental period. After 2 wk, the mean body weight of the
animals was 165 ± 2 g, with a daily rate of weight gain of
2.3 ± 1.1 g, indicating that the PTU treatment did not result in weight loss, although the rate of body weight gain was lower
than in the absence of PTU (data not shown). At that time, they were
anesthetized with intramuscular ketamine-xylazine used at 45 and 9 mg/kg, respectively (Parke-Davis and Bayer, Leverkusen, Switzerland),
and equipped with a cannula positioned in the right lateral cerebral
ventricle. After 1 wk of recovery, subcutaneously implanted osmotic
minipumps delivering vehicle or leptin were connected to the
intracerebroventricular infusion cannula via a polyethylene catheter,
as previously described (33). Minipumps (model 2001; Alza,
Palto Alto, CA) infused 10 µg of leptin (Ala-100, human leptin analog
provided by Eli Lilly, Indianapolis, IN) per day for 6 days or its
vehicle, isotonic saline. At the beginning of the respective central
infusions, all PTU-treated hypothyroid rats were supplemented by daily
intraperitoneal injections of T4 for 3 days (0.6 nmol · 100 g body
wt1 · day
1).
Subsequently and for the remaining 3 days of the experiments, the
intraperitoneal T4 injections were replaced by a continuous T4 infusion, at the same dose of 0.6 nmol · 100 g body
wt
1 · day
1, by means
of the subcutaneous minipumps. In one-half of the leptin-infused rats,
a subcutaneous infusion of rT3 was carried out for 3 days at a rate of 25 nmol · rat
1 · day
1
via the same minipumps as those used for T4
supplementation. Three groups of rats were thus investigated, all
animals receiving PTU and T4 substitution: i.e., controls,
leptin-infused, and leptin plus rT3-infused animals
(5-7 rats per group). The saline-infused control group was
pair-fed the amount of food consumed by leptin-infused animals to
determine the impact of decreased food intake per se. The pair-feeding
regimen was performed as follows. Average daily food intake of the
leptin-infused group was calculated; one-third of this amount of food
was given at 8:00 AM, and the remaining two-thirds were given before
the lights were turned off (6:00 PM), on the basis of a preliminary
study of food consumption during the day and the night.
Quantitative RT-PCR procedure. Total RNA was extracted from frozen tissue samples with TRIzol reagent (Life Technologies GIBCO-BRL, Rockville, MD). RNA integrity was assessed by performing a 1% agarose gel electrophoresis in 1× Tris-borate-EDTA, and its concentration was determined by spectrophotometry. cDNA templates for RT-PCR were obtained using 2.5 µg of total RNA. Reverse transcription reaction was performed with random hexamers (Microsynth, Geneva, Switzerland), dNTPs, the RNAse inhibitor RNAsin (Catalys, Promega, Madison, WI), and the MMLV-RT Enzyme Kit (Life Technologies GIBCO-BRL).
The real-time PCR (Lightcycler; Roche Diagnostics, Basel, Switzerland) reaction is an automated quantitative PCR obtained by continuous monitoring of the fluorescence emitted upon binding of the SYBR Green I dye to the double-stranded DNA. Amplification of cyclophilin A, D1, D2, and UCP1 cDNAs was performed with the SYBR Green I DNA master kit (Roche Diagnostics, Mannheim, Germany) according to the light cycler standard protocol, using ~70 ng of template cDNA. Primers for cyclophilin and D1 were used at a final concentration of 0.5 µM, and primers for D2 and UCP1 were used at 0.125 µM. The annealing temperature was 57°C for the D1 and D2 and 55°C for the UCP1 primer sets. After each run, a relative quantification of amplified PCR product in the different samples was performed. This was based on the relative comparison of the PCR products during the log-linear phase of the amplification process. A standard curve was used to obtain the relative concentration of the target gene, and the results were corrected according to the concentration of cyclophilin, used as the housekeeping gene.Primer sequences. Primers for rat cyclophilin A, D1, D2, and UCP1 were designed on-line with Primer 3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and synthesized by Microsynth (Balgach, Switzerland). Primers were as follows: cyclophilin A: sense primer 5'-AGCACTGGGGAGAAAGGATT-3' starting at 166, antisense primer 5'-CATGCCTTCTTTCACCTTCC-3' starting at 471, product size 306; D1: sense primer 5'-CCTCCACAGCTGACTTCCTC-3' starting at 437, antisense primer 5'-TTCCAGAACAGCTCGGACTT-3' starting at 741, product size 305; D2: sense primer 5'-CAGTGAAGCGGAATGTCAGA-3' starting at 2842, antisense primer 5'-AGAGGCATGTTAGGTGGGTG-3' starting at 3380, product size 539; UCP1: sense primer 5'-ACCCTGGCCAAGACAGAAG-3' starting at 369, antisense primer 5'-CAATCCTGAGGGAAGCAAAG-3' starting at 459, product size 91.
The adequacy of the different PCR products was verified by nucleic acid sequencing and agarose gel electrophoresis.D1 and D2 activities.
Tissues were homogenized in 10 volumes of 0.1 M phosphate (pH 7.2), 2 mM EDTA, and 1 mM DTT (P100E2D1 buffer). D2 activity was assayed in
freshly prepared homogenates. Aliquots of homogenates were snap-frozen
and stored at 80°C until analysis of D1 activities. D1 and D2
activities were assayed by monitoring the release of radioiodide from
outer-ring-labeled rT3 and T4, respectively. For hepatic D1 activity, liver homogenates (~50 µg protein/ml) were
incubated for 30 min at 37°C with 0.1 µM rT3 and
105 cpm of [3',5'-125I]rT3 in 0.1 ml of P100E2D10 buffer. Blank incubations were carried out in the
absence of homogenate. Radioiodide production was analyzed as
previously described (32). Deiodinase activity of
homogenates was corrected for nonenzymatic deiodination observed in the
blanks. D2 activities in brain, pituitary, and BAT were determined by incubation of appropriately diluted homogenates for 60 min at 37°C
with 1 nM (105 cpm)
[3',5'-125I]T4 in the presence of 0.1 µM
T3 to block D3 and 0.1 mM PTU to block D1, if
present. Blank incubations were carried out in the absence of
homogenate. Release of 125I was determined and corrected
for nonenzymatic deiodination as described. In the assay of hepatic D1
activity, the samples were diluted 4,000 times, excluding a possible in
vitro effect of the PTU used in vivo to inhibit D1 activity and making
the animals hypothyroid. The same holds true for any putative effect of
in vivo-infused rT3 on the in vitro measurement of D2
activity. Note that D1 and D2 activities were analyzed using substrate
concentrations of 0.1 µM rT3 and 1 nM T4,
respectively, being approximately equal to their
Km values. Very similar changes in hepatic D1
activities were observed in rats with different thyroid states by using
1 µM instead of 0.1 µM rT3 as the substrate. Similarly,
relative differences in BAT D2 activities between groups of rats
subjected to various treatments were identical if D2 activity was
measured at 10 nM instead of 1 nM T4 as the substrate.
These higher substrate concentrations provide near-maximum deiodination rates.
Type 3 deiodinase activity. Type 3 deiodinase (D3) activity was assayed by monitoring the production of radioactive 3,3'-diiodothyronine (3,3'-T2) from outer-ring-labeled T3. Cerebral cortex homogenates (~1 mg protein/ml) were incubated for 60 min at 37°C with 1 nM (2 × 105 cpm) [3'-125I]T3 in 0.1 ml of 100 mM phosphate (pH 7.2), 2 mM EDTA, and 50 mM DTT. Blank incubations were carried out in the absence of homogenate. The reactions were stopped by addition of 0.1 ml of ice-cold methanol on ice. The mixtures were centrifuged, and 0.1 ml of the supernatants was mixed with 0.1 ml of 0.02 M ammonium acetate (pH 4) and analyzed by HPLC. Samples of 0.1 ml were applied to a 250 × 4.6 mm Symmetry C18 column (Waters, Etten-Leur, The Netherlands) connected to an Alliance HPLC system (Waters) and eluted isocratically with a mixture of acetonitrile and 0.02 M ammonium acetate (33:67, vol/vol) at a flow of 1.2 ml/min. Radioactivity in the eluate was monitored on-line using a Radiomatic A-500 flow scintillation detector (Packard, Meriden, CT). Conversion of labeled T3 to radioactive 3,3'-T2 was corrected for nonenzymatic deiodination as observed in the blanks.
Plasma hormones and metabolites. TSH and T4 levels were measured by RIAs (Immulite 2000; Diagnostic Product, Los Angeles, CA; rat TSH: L2KTS6; T4: L2KT4). Plasma T3 and rT3 levels were measured by in-house methods in Rotterdam (Erasmus University) with the following respective intra- and interassay coefficients of variation: T3, 2-6 and 8%; rT3, 3-4 and 9-14%. More specifically, T3 was measured by RIA using a rabbit anti-T3 antiserum (final dilution 1:250,000) and [125I]T3 (20,000 cpm) (Amersham Pharmacia Biotech, Aylesbury, UK). Sample volume was 25 µl, and incubation mixtures were prepared in 1 ml of RIA buffer [0.06 M barbital, 0.15 M HCl, 0.1% BSA, and 0.6 g/l 8-anilino-1-naphthalenesulfonic acid (Sigma)]. Mixtures were incubated in duplicate overnight at 4°C, and antibody-bound radioactivitry was precipitated using Sac-Cel cellulose-coupled second antibody (IDS, Boldon, UK). The lower limit of detection was 0.15 nmol/l. Plasma rT3 levels were determined using an in-house-produced antiserum. The limit of detection was 0.04 nmol/l rT3.
Statistical analysis. The results were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's procedure for multiple comparisons. The calculations were performed using the SigmaStat software (SPSS, Chicago, IL). A P value <0.05 was considered statistically significant.
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RESULTS |
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Because the aim of the present study was to assess a potential
role of leptin on the regulation of D2 with possible consequences on
plasma thyroid hormone levels, the activity of hepatic D1 was inhibited
by PTU, and the hypothyroid state induced by PTU was partly compensated
for by moderate T4 substitution. The pathways targeted by
our experimental design are schematized in Fig.
1. As shown in Table
1, hepatic D1 activity of the animals
studied was consistently low and was ~90% lower than that of
euthyroid rats (see legend of Table 1). This was true for the three
groups of rats investigated, i.e., control animals infused
intracerebroventricularly (icv) with vehicle and pair-fed the
amount of food consumed by the leptin-infused rats (i.e., ~40%
reduction of food intake compared with ad libitum-fed controls),
leptin-infused rats, and leptin-infused rats receiving a 3-day
subcutaneous rT3 infusion. When rats were infused with
rT3, their plasma levels of this thyroid hormone metabolite
reached values that were much higher than those of controls (101.8 ± 5.6 vs. 0.25 ± 0.02 pmol/ml, P < 0.001).
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Figure 2 shows that, relative to pair-fed
controls, the central leptin infusion resulted in a marked increase in
BAT D2 activity (5-fold) and mRNA expression (2.5-fold). The activity
of this BAT enzyme was decreased by rT3 administration to
reach levels that were similar to those observed in the
vehicle-infused, pair-fed control group. In contrast, the D2 mRNA
expression remained elevated in leptin-treated animals receiving
rT3. In tissues other than BAT (e.g., cortex, hypothalamus,
pituitary), and as depicted in Table 2,
central leptin administration had no effect on D2 activity. rT3 alongside leptin brought about a reduction in the
activity of this enzyme in the pituitary only.
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The effect of leptin on plasma levels of thyroid hormones is depicted
in Fig. 3. Compared with pair-fed
controls, central leptin administration resulted in a 50% rise in
plasma T3 levels, an increase that was completely abolished
when leptin was infused in animals concomitantly treated with
rT3 (Fig. 3A). On the contrary, and as further
illustrated by Fig. 3B, plasma T4 levels of
leptin-infused rats were reduced by 30% compared with those of
pair-fed control animals. Such a reduction of plasma
T4 concentrations was only partly normalized by
rT3 administration.
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As can be noted in Table 1, the changes in plasma T3 and T4 levels were associated with a trend for plasma TSH levels to be decreased by the intracerebroventricular leptin infusion, although this change did not reach statistical significance. Plasma TSH levels of leptin-treated rats receiving rT3 were similar to those of pair-fed controls.
The expression of UCP1 mRNA in BAT was determined as a marker of
sympathetic nervous system activity. As shown in Fig.
4, central leptin induced an increase in
BAT UCP1 mRNA expression, an increase that remained present in the
leptin-treated group that received the D2 inhibitor rT3
(Fig. 4).
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D3 activity, determined in the cortex of the three groups of rats, was found to be unaltered by central leptin infusion, whether in the absence or in the presence of rT3 infusion (data not shown).
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DISCUSSION |
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We have previously demonstrated that, when leptin was infused for 6 days into the cerebral ventricles of normal (euthyroid) rats, there was no leptin leakage in the periphery and thus no increase in plasma leptin levels, indicating that the effects produced by this hormone, among which is a sustained decrease in food intake, were centrally elicited (9). By comparing leptin-infused rats with vehicle-infused ad libitum-fed controls, and with vehicle-infused animals given the same amount of food as that consumed by the leptin-treated group (i.e., pair-fed controls to mimic leptin-induced hypophagia), we also demonstrated that central leptin administration prevented the decrease in muscle UCP3 expression observed in response to pair feeding (8, 9). This central leptin effect was, at least partly, mediated by thyroid hormones, as it was not observed in hypothyroid animals (8). Additionally, in normal animals submitted to food restriction, the well documented decreases in hepatic D1 activity and in plasma T3 levels did not occur when the animals were administered leptin intracerebroventricularly (1, 8, 24). This indicated the existence of a stimulatory influence of leptin on hepatic deiodination of T4 to T3 and on plasma T3 levels in euthyroid animals.
D2, a selenoprotein present in the brain, pituitary, and BAT (19), is the other activating enzyme known to catalyze the 5' deiodination of T4 to the metabolically active product T3. The study of a potential effect of central leptin administration on D2 activity in euthyroid animals is difficult, because this enzyme is powerfully inhibited by T4 (40). However, in view of the postulated key role of D2 in the regulation of tissue-specific thyroid hormone-dependent processes, [e.g., the pituitary feedback mechanism, brain developmental processes, BAT thermogenesis, (3, 7, 22)], it was of interest to determine whether, in addition to its stimulatory effect on D1, leptin would also affect D2 activity. The hypothesis that leptin might stimulate D2 activity would be in keeping with the observations that central leptin administration promotes an increased sympathetic nervous system activity (12, 23, 35) and that D2 activity, at the level of BAT in particular, was shown to be stimulated by the sympathetic nervous system (4, 38), whereas such is not known to be the case for hepatic D1 activity (19).
To test the hypothesis that central leptin administration regulates D2 activity, we used an in vivo approach in which D1 activity was strongly inhibited by the use of PTU, which also made the rats hypothyroid. However, to provide substrate for D2 activity, hypothyroid rats were supplemented with T4, at a low substitution rate, to avoid inhibition of D2 by T4.
We observed that, compared with control rats that were given the same amount of food as that consumed by the leptin-treated animals (pair-fed controls), central leptin administration promoted increases in D2 expression and activity in BAT but not in the brain or in the pituitary. Concomitantly, and compared with those of pair-fed controls, plasma T3 levels of leptin-infused rats were higher, whereas their plasma T4 concentrations were lower. Such changes in thyroid hormone levels could be the consequence of the stimulatory effect of leptin on BAT D2, in keeping with the reported observation that BAT D2 is not only a local but also a systemic source of T3 (39). Indeed, we observed that, when leptin stimulation of BAT D2 activity was prevented by rT3, the leptin-induced increase in plasma T3 and the decrease in plasma T4 concentrations failed to occur (Fig. 3). Note that, as rT3 is a known potent inhibitor of D2 activity at a posttranscriptional level (18), it was not surprising to observe that rT3 was without effect on D2 mRNA expression in BAT, which was elevated in response to leptin both in the absence and in the presence of rT3 (Fig. 2).
An additional explanation for the increased T3 plasma
levels in the leptin-infused group can be that leptin not only
increases T3 production but could decrease its degradation
as well through an effect on D3 activity. Indeed, this enzyme is known
to be of importance in the clearance of T3, and its
activity has been reported to be modulated by the nutritional state
(19, 21). However, we observed that leptin was without any
effect on D3 activity in the cerebral cortex. Finally, it is unlikely
that the increase in plasma T3 levels observed in
leptin-infused rats could be due to some residual hepatic D1 activity,
because under our experimental conditions hepatic D1 activity was
inhibited by ~90% in the presence of PTU with 0.6 nmol · 100 g body
wt1 · day
1
T4 substitution.
Plasma TSH levels exhibited a trend toward a decrease in response to intracerebroventricular leptin, although this did not reach statistical significance. This is in contrast to other studies reporting that intracerebroventricular leptin exerts a stimulatory effect on TSH secretion (24, 36). One major difference between these studies and the present one is the timing of leptin infusion. Here, we observe long-term effects of leptin, and it cannot be ruled out that a potential rise in plasma TSH levels could have been masked by the increased plasma T3 concentrations observed in response to central leptin administration. In addition, the moderate hypothyroid state of our rats may have affected the TSH response to leptin, since it has been reported that the stimulatory effect of intracerebroventricular leptin is not present in hypothyroid conditions (36). Yet, in our earlier studies in euthyroid rats, no increase in serum TSH was observed under central leptin infusion (8).
With regard to the mechanism(s) by which central leptin administration could bring about an increase in BAT D2 activity, an involvement of the sympathetic nervous system might be postulated for the reasons mentioned earlier. This is in keeping with the observation made in the present study that central leptin administration stimulates the expression of UCP1 in BAT of T4-substituted hypothyroid rats, as it does in euthyroid animals (6, 9). Furthermore, such an increase in BAT UCP1 mRNA expression was still observed in leptin-infused rats, in which D2 was inhibited by rT3, suggesting that, under our experimental conditions, the increased mRNA expression of UCP1 was not due to local T3 production (2, 10, 29, 31) but to direct adrenergic stimulation (5, 11, 20, 28, 30).
The present data, together with those of our previous study (8), allow us to propose that central leptin could influence the thyroid axis with the following potential series of events. It would enhance the activity of BAT D2 via a likely activation of the sympathetic nervous system, resulting in significant increases in plasma T3 levels. Such an increase in plasma T3 concentrations would favor the activity of hepatic D1, given the high sensitivity of this enzyme to T3 (19). Leptin action could therefore bear on D2 activity in BAT first and subsequently on D1 activity in the liver, ultimately contributing to increased plasma T3 levels. Because in the present study PTU was used to block hepatic D1 activity, leaving D2 as the main site for peripheral T3 production, the relative influence of these two enzymes under physiological conditions cannot be established.
In summary, our data show that central leptin administration brings about a stimulation of BAT D2 activity in T4-substituted hypothyroid rats, thus providing yet another aspect of the well substantiated stimulatory effect of leptin on the hypothalamo-pituitary-thyroid axis (13, 15, 24, 26, 36).
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ACKNOWLEDGEMENTS |
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We thank Ellen Kaptein (Erasmus University, Rotterdam, The Netherlands), Marcella Klein, and Dr. Jean-Daniel Graf (University of Geneva, Geneva, Switzerland) for their excellent technical assistance.
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FOOTNOTES |
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This work was carried out thanks to Grant Nos. 3100-53841.98 and 3100-065416.01 from the Swiss National Science Foundation (Bern, Switzerland), and grants-in-aid from Eli Lilly and Company (Indianapolis, IN), Hoffmann-La Roche (Basel, Switzerland), the Novartis Foundation (Basel, Switzerland), the Roche Research Foundation (Basel, Switzerland), and the "Fondation Prévot" (Geneva, Switzerland). We gratefully acknowledge Eli Lilly and Company for the generous gift of leptin.
Address for reprint requests and other correspondence: F. Rohner-Jeanrenaud, Hôpitaux Universitaires de Genève, Division of Endocrinology and Diabetology, 24, rue Micheli-du-Crest, 1211 Geneva 14, Switzerland (E-mail: F.Rohner-Jeanrenaud{at}hcuge.ch).
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.
July 30, 2002;10.1152/ajpendo.00196.2002
Received 7 May 2002; accepted in final form 10 July 2002.
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