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1. This study examined the effects of thyroid status on the lipolytic responses of rat white adipocytes to beta-adrenoceptor (beta-AR) stimulation. The beta 1- and beta 3-AR mRNAs and proteins were measured by Northern and saturation analyses, respectively. Glycerol production and adenyl cyclase (AC) activity induced by various non-selective and selective beta 1/beta 3-AR agonists and drugs which act distal to the receptor in the signalling cascade were measured in cells from untreated, triiodothyronine (T3)-treated and thyroidectomized rats. 2. The beta 3-AR density was enhanced (72%) by T3-treatment and reduced (50%) by introduction of a hypothyroid state while beta 1-AR number remained unaffected. The beta 1- and beta 3-AR density was correlated with the specific mRNA level in all thyroid status. 3. The lipolytic responses to isoprenaline, noradrenaline (beta 1/beta 3/beta 3-AR agonists) and BRL 37344 (beta 3-AR agonist) were potentiated by 48, 58 and 48%, respectively in hyperthyroidism and reduced by about 80% in hypothyroidism. 4. T3-treatment increased the maximal lipolytic response to the partial beta 3-AR (CGP 12177) and beta 1-AR (xamoterol) agonists by 234 and 260%, respectively, increasing their efficacy (intrinsic activity: 0.95 versus 0.43 and 1.02 versus 0.42). The maximal AC response to these agonists was increased by 84 and 58%, respectively, without changing their efficacy. 5. In the hypothyroid state, the maximal lipolytic and AC responses were decreased with CGP (0.17 +/- 0.03 versus 0.41 +/- 0.08 mumol glycerol/10(6) adipocytes; 0.048 +/- 0.005 versus 0.114 +/- 0.006 pmol cyclic AMP min-1 mg-1) but not changed with xamoterol. 6. The changes in lipolytic responses to postreceptor-acting agents (forskolin, enprofylline and dibutenyl cyclic AMP, (Bu)2cAMP) suggest the modifications on receptor coupling and phosphodiesterase levels in both thyroid states. 7. Thyroid status affects lipolysis by modifying beta 3-AR density and postreceptor events without changes in the beta 1-AR functionality.

1. This study was undertaken to evaluate in vivo the influence of amiodarone on the effects of triiodothyronine (T3) in brown adipose tissue (BAT) which are independent of thyroid hormone synthesis and of the conversion of thyroxine (T4) to T3. Thyroidectomized rats were given a replacement dose of T3 (0.5 mg kg(-1) p.o. daily for 3 days) with or without amiodarone (50 mg kg(-1) p.o. daily for 1 week). 2. As assessed by RT-PCR, treatment of thyroidectomized rats with T3 caused a 2 fold decrease in beta3-adrenoceptor (beta3-AR) mRNA levels and a 2 fold increase in beta1-AR mRNA levels. 3. Binding studies using [3H]-CGP 12177 as a ligand showed that treatment of thyoidectomized rats with T3 resulted in a 70% decrease in beta3-AR number and in an 80% increase in beta1-AR in BAT membranes. 4. T3-treatment abolished the increase in BAT adenylyl cyclase (AC) activity induced by CGP12177 in thyroidectomized rats. It also decreased the amount of Gi protein (ADP-ribosylation) by 30%. 5. At variance with the literature on the heart, amiodarone administration did not inhibit the positive effect of T3 on beta1-AR expression in BAT in thyroidectomized rats. However, it antagonized the effect of T3 on beta3-AR number, but not on AC activity or on Gi expression. 6. These results indicate that the effects of thyroid hormones on the responsiveness of BAT to catecholamines involves both receptor and post-receptor mechanisms, they also suggest that interaction between amiodarone and thyroid hormones is highly tissue-specific and depends on the beta-AR subtype.

We studied the time course of cell surface beta-adrenoceptors (BAR) in cardiomyocytes in response to a single triiodothyronine (T3) (10(-8) M) stimulation. An early first increase of BAR density was observed within 2 hr (+ 10% versus control cells, P < 0.05), and a plateau was maintained for 17-20 hr. This effect was followed by a much greater, late increase of BAR density, starting around 22 hr and lasting until 48 hr post T3 addition (+40% versus control cells; p < 0.05). Since reverse T3 studied in the same conditions had no effect in this system, we concluded that this T3 effect was specific. We hypothesized that the early response might be nongenomic because the early effect of T3 was still observed in the presence of cycloheximide (2 x 10(-5) M) whereas the late increase was totally suppressed by the drug. The early response to T3 required intact microtubules, since colchicine (2 x 10(-5) M) was able to block the increase in the cell surface BAR number, but it did not involve a change in BAR distribution between external and internal sites, as the external to total BAR ratio remained stable. The measurement of the rate of BAR disappearance from the cell surface allowed us to hypothesize that T3 induced a modification of the turnover in cell surface BAR.

1. The pharmacological features of rat white adipocyte beta-adrenoceptor subtypes were investigated by saturation and beta-agonist competition studies with [3H]-CGP 12177 and by lipolysis induced by beta-agonists as well as their inhibition by CGP 20712A (selective beta 1-antagonist) and ICI 118551 (selective beta 2-antagonist) in an attempt to establish a relationship between the functionality and binding capacity of beta-adrenoceptor subtypes. 2. Two populations of binding sites were identified on adipocyte membranes, one with high affinity (0.22 +/- 0.07 nM) and the other with low affinity (23 +/- 7 nM). The low affinity binding sites constituted 90% of the total binding sites. 3. The competition curves, with 15 nM [3H]-CGP 12177, for the beta-agonists, isoprenaline (Iso), noradrenaline (NA) and adrenaline (Ad), and the selective beta 3-agonist, BRL 37344 (BRL), were clearly biphasic (P < 0.001). The rank orders of agonist potency (pKi) in competing for [3H]-CGP 12177 high affinity and low affinity binding sites, respectively, were Iso (9.28 +/- 0.24) > NA (8.90 +/- 0.12) > Ad (8.65 +/- 0.12) > > BRL (4.53 +/- 0.17) and BRL (7.38 +/- 0.19) > > Iso (2.96 +/- 0.26) > or = NA (2.80 +/- 0.17) > Ad (2.10 +/- 0.11) indicating the expression of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes, respectively. Inversely, competition studies with the selective beta 1-agonist, xamoterol (Xam), provided evidence for a single homogeneous population of binding sites with low density (81 +/- 9 fmol mg-1) and high pKi value (7.23 +/- 0.26) confirming the presence of beta 1-adrenoceptors. 4. To assess a possible contribution of the beta 2-subtype, procaterol (Proc), a selective beta 2-agonist, was used to compete with 2 nM [3H]-CGP 12177. A single low affinity (4.61 +/- 0.07) population of binding sites was identified. The density of these sites (71 +/- 12 fmol mg-1) was similar to the one obtained with Xam, suggesting that Proc displaced [3H]-CGP 12177 from the beta 1-subtype. 5. The functional potency (pD2) order with BRL (9.07 +/- 0.20) and catecholamines (Iso: 7.26 +/- 0.06, NA: 6.89 +/- 0.02 and Ad: 6.32 +/- 0.07) was the same as that found for the low affinity binding sites in competition studies. Xam induced lipolysis with greater potency than dobutamine (Dob), 6.31 +/- 0.06 and 5.66 +/- 0.10, respectively. Proc stimulated lipolysis with a low potency (5.59 +/- 0.21). 6. The lipolytic response to 0.001 microM BRL was inhibited by both, selective beta 1- and beta 2-antagonist, in a monophasic manner with low potencies (CGP 20712A pKi: < 4.5 and ICI 118551 pKi: 5.57 +/- 0.13). Similar monophasic profiles were obtained for inhibition of Xam- and Dob-induced lipolysis. In this case, CGP 20712A was more potent (> 10 times) than ICI 118551. The monophasic inhibition was also observed with ICI 118551 in the presence of 0.05 microM Iso or 0.13 microM NA. In contrast, two populations of sites were identified with CGP 20712A in the presence of Iso as well as NA. The pKi values for the first sites were 8.41 +/- 0.09 and 8.58 +/- 0.17, respectively, and for the second population of sites 4.73 +/- 0.22 and 4.27 +/- 0.27, respectively. The proportion of the first sites was low: 19 +/- 4 and 22 +/- 5%, respectively. Biphasic curves were obtained with both antagonists using 2.5 microM Proc (CGP 20712A: pKi1: 8.17 +/- 0.08, site1: 23 +/- 6%, pKi2: 4.77 +/- 0.14; ICI 118551: pKi1: 7.78 +/- 0.03, site1: 37 +/- 2%, pKi2: 5.35 +/- 0.25). 7. Our results show that the radioligand [3H]-CGP 12177 allows the characterization of beta 1- and beta 3-adrenoceptor subtypes on rat white adipocytes. Lipolysis is highly dependent on beta 1- and beta 3-adrenoceptors. Finally, binding and functional studies confirm that lipolysis is mainly driven by the beta 3-subtype.

The beta-adrenergic effects of catecholamines are potentiated by thyroid hormones in adipose tissue. Amiodarone (AM) is structurally similar to thyroid hormones and was used to explore the mechanism of the triiodothyronine (T3) effect on beta-adrenergic receptors (beta-ARs) in adipose tissue. AM decreases the expression of some T3 sensitive genes in various tissues and antagonizes the effect of T3 on its nuclear receptors. In this study, the T3, AM and AM + T3 effects on the beta 1- and beta 3-AR density were assessed on rat white adipocytes by radioligand binding using [3H]CGP 12177 after characterization of these subtypes by displacement of [3H]CGP 12177 binding by isoproterenol, BRL 37344 and noradrenaline. BRL 37344 was used to study beta 3-AR lipolysis. White adipocytes from hyperthyroid rats had increased responsiveness (Emax x 2) and sensitivity (+ 38%) to BRL 37344, while those given AM alone had decreased values. Moreover, AM antagonized the T3 effect on lipolysis. The beta 1-binding characteristics (receptor density [Bmax]: 45 +/- 4 fmol/mg of proteins; dissociation factor [Kd]: 0.96 +/- 0.10 nM) were not modified by either compound. Finally, T3 significantly increased beta 3-AR density (587 +/- 69 versus 363 +/- 25 fmol/mg of proteins) and Kd (38 +/- 2 versus 23 +/- 3 nM), while AM alone had no effect and did not antagonize the T3 effect on beta 3-AR number. In conclusion, the hyperthyroid state in the rat potentiated the lipolytic response of white adipocytes to a specific beta 3-agonist and increased the beta 3-AR density without changing in beta 1-AR number and affinity. Furthermore, the lack of antagonism between AM and T3 on beta 3-AR expression suggests that T3 does not work directly on the beta 3-AR gene. Moreover, AM induced a functional tissular hypothyroid-like effect and its antilipolytic effect probably occurred at a postreceptor level.

3,3',5-Triiodothyronine (T3), at 10(-8) M, potentiated by 26.4-30.9% the isoproterenol-mediated inotropic effect in chick embryo cardiac myocytes in culture. Amiodarone (10(-6) M) decreased this response by 44.6% only in cells cultured with serum, where the T3 concentration was 10(-13) M. Amiodarone inhibited the potentiating effect of T3. Amiodarone alone had no influence on the beta-adrenoceptor density in cells cultured in serum-free medium. This confirms that the effects of amiodarone on cardiac beta-adrenoceptors are T3 dependent. T3 increased the density of beta-adrenoceptors through two concentration ranges, with an initial 30% increase between 10(-14) and 10(-11) M, followed by a second increase until 10(-7) M. Amiodarone not only inhibited the first positive effect of T3 but also decreased beta-adrenoceptor density far below the control value. The second positive T3 effect was also inhibited by 50% by amiodarone. This study suggests that T3 might increase the number of cell-surface beta-adrenoceptors and modify their cellular traffic through at least two mechanisms, one assumed to be non-genomic, the other being genomic, and that amiodarone could affect the two mechanisms differently.

To assess if the anti-beta-adrenergic effect and the bradycardia induced by amiodarone were mediated by thyroid hormone, we investigated these effects of amiodarone in euthyroid and hypothyroid rats. We studied control rats, thyroidectomized rats, control rats treated with amiodarone (50 mg/kg for 8 days), and thyroidectomized rats treated with amiodarone. At the end of the treatment, free thyroid hormone levels (FT4 and FT3) were determined, and cardiac beta-receptor density (Bmax) and affinity (Kd) were assayed by using (-)-[125I]iodocyanopindolol as radioligand. Resting heart rate (rHR) was also assessed every day in control and thyroidectomized rats, before and after amiodarone. In hypothyroid rats, in which free thyroxine (FT4) was not detectable and free 3,5,3'-triiodothyronine (FT3) was only 16% that of euthyroid rats, Bmax (14.1 +/- 2.5 fmol/mg, n = 7) and rHR (259 +/- 9.7 beats/min, n = 6) were significantly lowered compared with euthyroid rats (Bmax:18.4 +/- 3.4 fmol/mg, n = 7; rHR:277 +/- 4.1 beats/min, n = 5). Amiodarone treatment decreased Bmax (13.6 +/- 2.9 fmol/mg, n = 8) and rHR (252 +/- 5.5 beats/min, n = 5) only in euthyroid rats and did not produce significant cardiac effects in hypothyroid rats. (Values are given as mean +/- SD.) We conclude that a minimum serum thyroid hormone concentration is a necessary condition for amiodarone to produce some of its cardiac effects. An antagonistic reaction to thyroid hormones at the cellular level can be postulated as a mechanism of the cardiac anti-beta-adrenergic action of amiodarone.