The influence of sleep deprivation on thyroid hormone metabolism in rat frontal cortex

Neuroscience Letters, 162 (1993) 145-148 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$06.00

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The influence of sleep deprivation on thyroid hormone metabolism in rat frontal cortex A. C a m p o s - B a r r o s a, R. K 6 h l e r a, F. Miiller ", M. E r a v c P , H. M e i n h o l d a, W. W e s e m a n n c, A. B a u m g a r t n e r *'b ~'Department of Nuclear Medicine ~Radioehemistry ), Universtit6tsklinikum Steglitz. ~'Psychiatric C\linic, Universtitiitsklinikum Rudo([- Virehow. Free University of Berlin. Eschenallee 3, D-14050 Berlin. FRG. ' Department of Physiological Chemistry, University ~[ Marburg, Marburg. FRG

(Received 12 July 1993; Revised version received 9 August 1993:Accepted 12 August 1993) Aey words: Sleepdeprivation: Rat frontal cortex: Thyroid hormone; Deiodinase

The effects of 24 h sleep deprivation (SD) on central thyroid hormone metabolism were investigated in rat frontal cortex. SD induced a significant rise in the activity of iodothyronine type II 5'-deiodinase(5'D-II), which catalyzes the conversion of thyroxine (T4,to triiodothyronine (T~ in the rat central nervoussystem (CNS). Tissue concentrations ofT4 remained unchanged, whereas levelsof T~ increased to more than 150%of the corresponding levels measured in control rats. Serum concentrations of T~ and T, were also significantlyenhanced by SD an effect that has previously been described in depressed patients having undergonethe same procedure. These results suggest that SD can dramatically increase T~concentrations (and possibly function) in rat CNS. Whether or not these findings are of relevance in regard to the well-knownantidepressant effect o| SD in psychiatric patients with major depressive disorders remains to be established.

Since the early seventies it has been well known that sleep deprivation (SD) has a potent, but short lasting antidepressant effect in patients with major depression [15]. The biochemical mechanisms which mediate these effects are, however, as yet unknown. Clinical studies have indicated that changes occuring in the hypothalamic-pituitary-thyroid axis of depressed patients during SD may be involved in the antidepressant action of this form of treatment [e.g. 3, 4]. On the other hand, animal studies have shown that chronic treatment with the tricyclic antidepressant desipramine (DMI) enhances type II 5'- deiodinase activity (which catalyzes the conversion of thyroxine (T4) to triiodothyronine (T3) in the rat central nervous system (CNS)) and cortical T3 concentrations in the rat CNS [2, 7, 8]. Both clinical and animal studies suggest that changes in central thyroid hormone metabolism may be involved in the antidepressant action of these therapies. In order to investigate whether SD likewise influences CNS thyroid hormone metabolism we determined the activities of type I iodothyronine 5'deiodinase (5"D-I), type II iodothyronine 5'-deiodinase (5'D-II) and type III (or inner-ring) iodothyronine 5deiodinase (5D-III), the three isoenzymes regulating *Corresponding author.

CNS thyroid hormone metabolism [e.g. 14, 17, 23], as well as T4 and T3 tissue concentration in the rat frontal cortex after 24 h sleep deprivation. Adult male Wistar rats weighing 200 250 g were used throughout. They were housed in pairs with a 12-h light/ dark cycle (06:00 18:00 h). After an adjustment period of not less than 1 week, SD procedures were conducted as previously described [5]. In brief, each of the 10 rats was placed in one of 10 drums which were rotated at a speed of one revolution per 45 s. The rats were placed in the drums at 10:00 h and remained there for 24 h. Food and water were available ad libitum throughout the whole procedure. Between 10:00 I1 and noon on the next day they were sacrificed by decapitation without anesthesia, together with 10 controls. Their brains were dissected according to Glowinski and Iversen [1 I] and stored immediately at -70°C. Blood was drawn from the decapitation wound, centrifuged and the serum stored at -20°C. For deiodination measurements t¥ontal cortex was homogenized on ice in 5-6 vols. of 0.25 M sucrose, 10 mM HEPES (pH--7.0) containing 10 mM dithiothreitol (DTT) and immediately frozen in a dry ice/acetone bath and stored at - 8 0 ° C until assay. The measurement of 5'D-I , 5'D-II and 5D-111 was

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based on the release of radioiodide from the ~51-1abeled substrates [16]. 5'D-I and 5'D-II activities were determined simultaneously by measuring the release of radioiodide from 100,000 cpm (ca. 2.5 kBq) [5'-J25I]rT3 at 5 nM rT 3, 20 mM DTT, in the presence (for 5'D-II) and absence (5'DI+5'D-II) of 5'D-I-inhibiting 6-n-propyl-2-thiouracil (PTU) [231. 5'D-II was also determined using [5"-1251]~I'4 as substrate in the presence of 6 nM T 4, 30 mM DTT, 1 mM PTU and 1 ¢tM T 3 in order to inhibit the inner-ring deiodination of T 4 [14, 23]. The measurements were conducted after 60 rain incubation at 37°C with 50-100 ¢tg protein from the crude homogenate in 100 ¢tl 0.1 M potassium phosphate buffer (pH = 7.0), 1 mM EDTA. The reaction was started by the addition of the tissue homogenate and stopped by addition of 50 ¢tl ice-cold 5% BSA, 10 mM PTU, followed by 400/.tl 10% ice-cold trichloroacetic acid. After centrifugation for 20 min at 4000 x g, the supernatant containing the ~25I was further purified by cation exchange chromatography on 1.6-ml Dowex 50 WX 8 columns (mesh 100-200) (Serva G m b H and Co., Heidelberg, Germany). The iodide was then eluted with 2 x l ml 10% acetic acid and counted in a gammacounter. For determination of 5D-III (inner-ring deiodinase) 20-70/lg protein were incubated in a final volume of 100 ¢tl 0.1 M potassium phosphate buffer (pH = 7.4), 1 mM EDTA with approximately 1.2 kBq (approx. 50,000 cpm) inner ring-labeled [5-125I]T3 at 50 nM T 3, 20 mM DTT and 1 mM PTU for 60 rain at 37°C. Radioiodide release was measured as described above. Protein was assayed as described by Bradford [6], using reagents from Bio-Rad Laboratories (Richmond, CA) and bovine-?'-globuline as standard. Tissue concentrations of T 3 and T 4 w e r e determined by highly sensitive radioimmunoassays (RIAs) after extraction of the tissue samples as previously described in detail [19]. Briefly, tissue samples were homogenized in 100% methanol containing 1 mM PTU, extracted in chloroform-methanol and back-extracted into an aqueous-phase, which was then purified through Bio-Rad AG 1 x 2 resin columns (Bio-Rad Laboratories, Richmond, CA). The iodothyronines were eluted with 70% acetic acid, evaporated to dryness and taken up in the RIA buffer. The limit of sensitivity was 2.5 pg for T3 and 3.0 pg for T4, the intraassay and interassay variations were less than 10%. Each sample was determined in triplicate at two different dilutions of the extracts. The results were corrected on the basis of individual recovery data obtained after addition of maximum specific activity [125I]T 3 and [131I]T4 t o every sample during the initial ex-

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Fig. 1. The activities of type I iodothyronine 5'-deiodinase (5'D-I), type II iodothyronine 5'-deiodinase (5'D-II) and type III iodothyronine 5deiodinase (5D-III) in the rat frontal cortex after 24 h sleep deprivation (n = 10) and a normal sleep cycle (n = 10); *P < 0.05 vs. control value. Values are given as means + S.E.M. Sleep dep., sleep deprivation.

traction process. The recovery of extracted T 3 ranged between 70 and 80% and that of T 4 between 60 and 70%. All the samples were processed individually. In each experiment all samples of a particular tissue were extracted and/or assayed together. Serum T 4 and T 3 levels were determined by a slightly modified double-antibody radioimmunoassay as previously described for human serum [18]. For the assays of total T 4 and T 3 in the rat sera, standards were set up in iodothyronine-free rat serum. The data are given as means + standard error of the mean (S.E.M.). P-values of less than 0.05 were considered significant. The results for the two different groups of rats were compared using the Mann-Whitney U-test. SD significantly enhanced 5"D-II activity with both rT 3 and T 4 as substrate (127.6 + 7.1 vs. 187.9 _+ 27.8, P < 0.05 (Fig. 1) and 229.2 + 15.9 vs. 319:8_+ 28.8, P -- 0.04 for rT3- and T4-type II 5'-deiodinase activities, respectively). (All data are given as fmol I-/mg pro, tein per hour.) No significant changes were seen in the activities of 5'D-I and 5D-IlI deiodinases (Fig. 1). Tissue concentrations of T 4 remained unchanged, but the levels of T 3 showed a large increase to not less than 157% of the values measured in control rats (1566 + 36 vs. 2466 + 162 pg/g, P < 0.01) (Fig. 2). Serum levels of T3 and T 4 (Fig. 2) were significantly elevated after SD. The T4 concentrations rose from 70.7 + 3.9 to 91.1 + 3.8 nmol/l (P < 0.01), the T3 concentrations from 1.4 + 0,06 to 2.2 + 0.1 nmol/l (P < 0.001). Many different interactions have been found between

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Fig. 2. Frontal cortex and serum (inset) thryoxine (T4) and triiodothyronine (T3) concentrations after 24 h sleep deprivation (n = 10) and a normal sleep cycle (n = 10): **P < 0.01 vs. control value. Values are given as means _+ S.E.M. Sleep dep., sleep deprivation.

the thyroid hormone function and affective disorders. Severe hypothyroidism may lead to all kinds of depressive symptoms [13], administration of T 3 may enhance the effects of antidepressant drugs [20], etc. Furthermore, serum levels of thyroid hormones increase during SD both in depressed patients [e.g. 3, 4] and in rats. Whether or not our findings are relevant for the interpretation of the antidepressant effect of SD in depressed patients remains to be investigated. In the euthyroid rat cerebral cortex aproximately 80% of the T 3 is produced from T4 in a reaction catalyzed by 5'D-II [9]. It is well known that 5'D-II activity in the brain responds very rapidly to variations of serum concentrations of T 4, i.e. decreasing concentrations of T4 lead to enhanced enzyme activity, and vice versa [e.g. 17]. However, our results show that 5'D-II activity may rise in spite of increases in serum concentrations of T4. This strongly suggests that there should be a further regulatory mechanism for this enzyme operating in the brain, in addition to serum levels ofT4. The mechanisms underlying the increase in 5"D-II activity during SD are unknown at present. However, it has been established that 5'D-II activity is stimulated by norepinephrine in brown adipose tissue and the pineal gland [e.g. 12, 21, 22]. Moreover, as mentioned above, we found that chronic treatment with the selective norepinephrine reuptake inhibitor desipramine selectively enhanced 5'D-II only in those areas which received noradrenergic nerve terminals from the locus coeruleus (LC) [2, 7, Campos-Barros,

submitted]. On the other hand, basic research has shown that the noradrenergic activity of the LC is high during waking and low during sleep in all three species investigated (rats, cats and monkeys) [1, 10]. It is therefore reasonable to hypothesize that the fact that 5'D-It activity was enhanced after SD in spite of an elevation of serum levels of T 4 w a s due to an increase in norepinephrine activity. Pharmacological studies must be conducted to obtain further clarification. Finally, it would seem noteworthy that we failed to find a significant increase in 5D-III activity in spite of the pronounced rises in tissue concentrations of T3. 5D-III activity has been found to be enhanced in hyperthyroidism, serving, in cooperation with 5'D-II, as an autoregulatory mechanism keeping brain levels ofT3 within physiological limits [for a review see 17]. Our results show that this compensatory increase in 5D-III does not occur after 24 h SD, again suggesting that parameters other than brain or serum concentrations of thyroid hormones might be involved in the as yet unknown mechanisms of regulation of 5D-III activity. This study was supported by the Deutsche Forschungsgemeinschaft (Grant Ba 932/5-1 I. 1 Aston-Jones, G. and Bloom, F.E., Activity of norepinephrinc-containing locus coeruleus neurons in behaving rats anticipates fluctuations in the sleep-waking cycle, J. Neurosci., 1 ( 1981 } 876 886, 2 Baumgartner, A., Campos-Barros, A., Stula, M. and Meinhold, H., Chronic desimipramine treatment enhances thyroxine deiodination in rat brain, Psychiat. Res., 34 (1990) 217 219. 3 Baumgarmer, A., Gr~if K.J., Kiirten, 1.. Meinhold, H. and Scholz, R, Neuroendocrinological investigations during sleep deprivation. 1, Concentrations of thyrotropin, thyroid hormones, cortisol, prolactin, luteinizing hormone, follicle stimulating hormone, oestradiol. and testosteron in patients with major depressive disorder at 8 a.m. before and after total sleep deprivation, Biol. Psychiatry, 28 (1990) 556, 568. 4 Baumgartner, A., Riemann, D. and Berger, M.. Neuroendocrinological investigations during sleep deprivation. II. Longitudinal measurement of thyrotropin, thyroid hormones, cortisol, prolactin, growth hormone, and luteinizing hormone during night of sleep and sleep deprivation in patients with major depressive disorder, Biol. Psychiatry, 28 (1990) 569 587. 5 Borb61y, A.A. and Neuhaus, H.U., Sleep-deprivation: effecls on sleep and EEG in the rat, J. Comp. Physiol., 133 (1979t 71 87. 6 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding, Anal, Biochem., 72 (1976} 248 254. 7 Campos-Barros, A., Baumgartner, A., Stula, M. and Meinhold, H., Selective alteration of type II 5'-deiodinase activity in the central nervous system of the rat after chronic desipramine treatment. In Gordon, Gross and Hennemann (Eds.), Progress in Thyroid Research, Balkema, Rotterdam, 1991, pp. 915 917. 8 Campos-Barros, A., K6hler, R.. Miiller, F., Meinhold, H. and Baumgartner, A., Chronic desipramine treatment enhances 3,5,3'triiodthyronine concentration in rat cerebral cortex, Ann. d'Endocrinol., 52 (1991) 34. 9 Crantz, F.R., Silva, J . E and Larsen. RR., An analysis of the

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