Characterization of type-II thyroxine 5'-deiodinase activity in rat Harderian gland

Thyroxine 5'-deiodinase activity was studied in male rat Harderian gland homogenates. The reaction rate was proportional to the tissue content in the homogenate and dependent on pH, with an optimum pH of 7.0, and temperature, between 4-37 degrees C. 5'-deiodinase activity was increased by dithiothreitol (DTT) in a dose-dependent manner, and inhibited moderately by propylthiouracil (PTU) and strongly by iopanoic acid (IA). Thyroidectomy enhanced the enzymatic activity (30-fold above the control value) but this increase is totally prevented by the in vivo iopanoic acid treatment. Thyroxine 5'-deiodinase activity was also dependent on T4 concentration (Km = 3.3 nM; Vmax = 10 fmol 125I-released/mg protein/h) and exhibited a nyctohemeral rhythmicity with a maximal activity at 03.00 h (4-fold above basal values) and minimal activity between 12.00-21.00 h.     

Ontogeny of type II thyroxine 5'-deiodinase, N-acetyltransferase, and hydroxyindole-O-methyltransferase activities in the rat Harderian gland

The ontogeny and regulation by isoproterenol of type II thyroxine 5'-deiodinase, N-acetyltransferase, and hydroxyindole-O-methyltransferase activities were studied in the rat Harderian gland. Both 5'-deiodinase and N-acetyltransferase activities exhibited maximal values at the first week of age. These activities gradually decreased till the fourth week. However, hydroxyindole-O-methyltransferase activity did not change during the period of time studied. Neither the different killing times (1600 or 0200 h) nor the photoperiod regimens (darkness or light exposure at night) modified the enzyme activities. On the other hand, isoproterenol, a beta-adrenergic agonist, could not to stimulate 5'-deiodinase at first week of life. Nevertheless, while basal 5'-deiodinase activity was diminishing during development, the enzyme was becoming sensitive to the action of isoproterenol. Thus, isoproterenol elicited increases in Harderian gland 5'-deiodinase activity in rats older than two weeks. However, both N-acetyltransferase and hydroxyindole-O-methyltransferase activities were not affected by isoproterenol treatment in either week studied.     

Type-II thyroxine 5'-deiodinase is present in the rat pineal gland

Thyroxine-5'-deiodinase has been identified in the rat pineal gland. The characteristics of the enzyme are compatible with a Type-II deiodinase which is tissue-specific and presumably related to generating a local action of thyroid hormone. Our data suggest there may be a previously unrecognized role of thyroid hormone in the regulation of pineal activity.     

In vivo stimulation of rat pineal type II thyroxine 5'-deiodinase activity by either norepinephrine or isoproterenol

Herein we show, for the first time, a very marked increase in thyroxine 5'-deiodinase (5'-D) activity in rats injected with norepinephrine (NE) and desmethylimipramine, a drug which inhibits NE uptake by nerve terminals. The response to NE was greater in pineals collected from hypothyroid animals than in glands from euthyroid animals. NE was more effective in stimulating pineal 5'-D than was isoproterenol, suggesting that, in addition to beta-adrenergic receptors, alpha-adrenergic receptors might be involved in the 5'-D activation. However, phenylephrine, an alpha-adrenergic agonist, did not potentiate the effect of isoproterenol on pineal 5'-D activity. The nocturnal increase in pineal 5'-D activity was completely abolished by propranolol, a beta-adrenergic receptor blocker, while prazosin, an alpha-adrenergic receptor blocker, had minimal effect. These results show that the role of alpha-receptors in promoting the NE-mediated rise in rat pineal 5'-D activity is minor in contrast to the role of beta-adrenergic receptors.     

Nocturnal increase of type II thyroxine 5'-deiodinase activity in the Syrian hamster harderian gland is abolished by light exposure and induced by isoproterenol

The presence of type II 5'-deiodinase activity in the Syrian hamster Harderian gland was investigated. This enzyme exhibited an increase of its activity after animals entered the normal dark phase, with maximal activity occurring at 04.00 hr (8 hr after lights off). The nocturnal increase was prevented by maintaining the animals in light during the night. Isoproterenol subcutaneously injected every 2 hr (1.0 mg/kg body wt) from 20.00 hr through 0.400 hr to animals exposed to light during the normal dark period mimicked the effect of darkness, i.e., with this treatment an increase in 5'-deiodinase activity with maximal peak values at 02.00 hr was observed. The results show that 5'-deiodinase activity in the Syrian hamster Harderian gland exhibits a nyctohemeral profile dependent on beta-adrenergic activation of the gland.     

N-Acetyltransferase activity of the rat Harderian gland.

Harderian gland extracts from male rats catalyze the conversion of serotonin to N-acetylserotonin and of tryptamine to N-acetyltryptamine. The reaction is linear up to 14 mg tissue and departs from linearity after 10 min. The pH otpimum with tryptamine as substrate is between 8 and 9. Enzymic activity of the gland in vivo does not show diurnal variations. Enzymic activity of tissue in organ culture is not stimulated by 10 micrometer isoproterenol or 100 micrometer dibutyryl cyclic AMP. Harderian gland tissue in culture can acetylate tryptamine and serotonin and can O-methylate the N-acetylserotonin to form melatonin.     

Harderian gland porphyrin, lysosomal and type II 5'-deiodinase rhythms in Sprague-Dawley and Fischer-344 rats kept under chronic long- or short-photoperiod conditions.

1. Harderian gland porphyrin concentrations were 1.5-fold higher in Fischer-344 male rats than in Sprague-Dawley male rats and there were no differences due to exposure to LD 14:10 (LP) or LD 10:14 (SP) lighting conditions for 8 weeks in either strain. 2. 24-hr periodic regression analyses of porphyrin concentration detected a significant rhythm in both lighting conditions in both strains, with no differences in acrophase due to lighting conditions. 3. In both strains, porphyrin levels were highest in the late phase-early dark period and fell during the early part of the dark period. 4. Acid phosphatase activity did not vary with time (circadian rhythm), strain or photoperiodic lighting condition. 5. Circadian rhythms in beta-glucuronidase, alpha-mannosidase and hexosaminidase activity were present in some instances, but, probably due to the low amplitude to these rhythms, a consistent effect of strain or housing condition was not found. When 24-hr rhythms were observed in either strain, the acrophase occurred during the afternoon-early evening daylight period. 6. A significant effect of strain on mean values of type II 5'-deiodinase activity was noted in Fischer-344 rats. 7. Significant rhythms in type II 5'-deiodinase activity were found in both strains exposed to LD 10:14.     

Mammary gland carcinoma-related increase of type I iodothyronine 5'-deiodinase activity in Sprague-Dawley rats.

Type I, iodothyronine 5'-deiodinase (5'-DI) catalyses deiodination of the prohormone thyroxine (T4) to the metabolically active 3,5,3'-triiodo-L-thyronine (T3). The present study was undertaken to investigate the activity of 5'-DI in rat mammary gland tumours representing various combinations of histologically defined papillary, cribriform or comedo patterns of ductal carcinomas. Female Sprague-Dawley rats were given two doses 50 mg x kg(-1) 1-methyl-1-nitrosourea (MNU) in abdominal parts on the 52nd day and 113th day of age. We have found that in comparison with non-lactating mammary gland, the activity of 5'-DI in all mammary gland tumours studied was significantly (p < 0.0001) increased and that the 5'-DI activity, expressed as pmol of 125I- released per min and per mg of protein, in malignant mammary gland tumours was found to be at least two order higher than that of intact mammary non-lactating gland. From our data, we suggest that thyroid hormone in mammary gland tumours might play a significant role to support high energetic expenditure of neoplastic tissues.     

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.     

Proteome profiling in the rat Harderian gland

The Harderian gland is an orbital gland located behind the ocular bulb in most terrestrial vertebrates probably functioning for production of lipid secretion to protect the eye. We herein present a protein reference database of the rat Harderian gland that may serve as analytical tool for future proteomic work, report lipid and porphyrin handling cascades, address sequence conflicts and report structures that have not been so far described by proteomics methods.     

Immunohistochemical localization of melatonin in the rat Harderian gland.

Using fluorescence and double antibody techniques, melatonin was localized immunohistologically in the secretory cells of the Harderian gland of mature male rats. The presence and quantity of melatonin in the acinar cells seem to correlate with the amount of porphyrins inside the lumen. The specificity was proven by disappearance of yellow fluorescence after saturation of antibody with melatonin or after use of nonspecific antibody only.     

Comparison of the physicochemical properties of type I and type II iodothyronine 5'-deiodinase

The 5'-deiodination of thyroxine is catalyzed by two enzymes which differ in their tissue distribution, substrate specificities, sensitivity to the inhibitor, propylthiouracil, and response to thyroid status. By using the affinity label, N-bromoacetyl-L-thyroxine, both isoenzymes have been found to have substrate binding subunits of approximately 27 kDa. In this study, we compared the substrate binding subunits and hydrodynamic properties of the type I and the type II isozymes using the affinity label, N-bromoacetyl-L-thyroxine, to identify the enzymes. High resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the substrate binding subunit of the type I enzyme had an Mr of 27,000, while that of the type II enzyme had a slightly higher Mr of 29,000. This difference was not accounted for by glycosylation. Partial staphylococcal V8-protease digests of the substrate binding subunit of the type I enzyme yielded fragments of 14.6, 13.7, and 7.0 kDa, while V8-protease digests of the substrate binding subunit for the type II enzyme produced fragments of 28.0, 25.1, 19.0, 9.5, 7.2, and 5.8 kDa. Unique cyanogen bromide fragmentation patterns were also observed for the two substrate binding subunits. Sedimentation coefficients of the detergent-soluble type I and type II holoenzymes were 3.67 and 5.22 S, respectively, as determined by sucrose density centrifugation. The type I enzyme behaved as a globular protein, whereas the type II enzyme showed sedimentation properties typical of asymmetric integral membrane proteins. The Stokes radii were 3.78 and 4.97 nm, respectively. From these data, the calculated Mr for detergent-solubilized type I 5'-iodothyronine deiodinase was 55,400 and for the type II enzyme was 198,700. These data indicate that the two isozymes of iodothyronine 5'-deiodinase are multimeric, differ in holoenzyme size and subunit composition, and that their substrate binding subunits are distinct.     

Iodothyronine 5'-deiodinase activity in rat brown adipose tissue during development

Iodothyronine 5'-deiodinase activity in rat brown adipose tissue has a characteristic pattern of developmental changes that is completely different from that of the liver. Fetal brown fat exhibits an extremely high iodothyronine 5'-deiodinase activity that is approx. 10-fold that in adult rats. Even though brown fat iodothyronine 5'-deiodinase activity falls suddenly at birth, there is a new peak in the activity around days 5-7 of life, whereas it remains very low afterwards. Just after birth, brown adipose tissue iodothyronine 5'-deiodinase activity is already capable of stimulation by noradrenaline. The postnatal peak in brown fat iodothyronine 5'-deiodinase correlates with the known increase in the thermogenic activity of the tissue in the neonatal rat, thus reinforcing the suggestion that local 3',3,5-triiodothyronine generation could be an important event related to thermogenesis in brown adipose tissue. However, the high fetal activity was only slightly related to the thermogenic activity of brown fat. Moreover, the increased iodothyronine 5'-deiodinase activity of brown adipose tissue during fetal and neonatal life suggests a substantial contribution by brown fat in the overall extrathyroidal 3',3,5-triiodothyronine production in these physiological periods.     

Increased pituitary thyroxine 5'-deiodinase activity in adult rats rendered hyper- or hypo-thyroid during perinatal life

Perinatal thyroid dysfunction in the rat leads to permanent alterations in pituitary TSH secretion in the adult animal. Thus, neonatal hyperthyroidism (NH) and perinatal hypothyroidism (PH) both result in apparent increased pituitary sensitivity to the feedback effects of thyroid hormones in the adult rat. To determine if increased intrapituitary generation of triiodothyronine (T3) might account for these observations, we measured thyroxine (T4) 5'-deiodinase activity in pituitary homogenates of adult NH and PH rats. NH was induced by injecting neonatal rats with 12 daily sc injections of T4 (0.4 microgram/g body weight (BW]. Control rats received vehicle alone. PH was induced by administering 0.05% 6-n-propylthiouracil in the drinking water to pregnant dams from the 16th day of gestation through the 12th day postpartum. Thereafter, a normal water supply was substituted. NH and PH rats were allowed to mature and were sacrificed at 105 days of age. Serum T4, T3, and TSH concentrations were measured by radioimmunoassay. Pituitary T4 5'-deiodinase activity was assessed by the measurement of T3 formation by pituitary homogenates incubated in the presence of 0.65 microM T4 and 100 mM dithiothreitol at 37 degrees C for 90 min. Body weights of adult NH and PH rats were slightly but not significantly decreased compared with control rats. Relative pituitary gland weight (milligrams per 100 g BW) was significantly decreased in adult PH rats (P less than 0.005) but not in adult NH rats. In adult NH rats, serum T4 and T3 concentrations were significantly decreased (P less than 0.01) compared with control rats. Serum TSH concentrations were similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairment of cold-induced increase in thyroxine 5'-deiodinase activity in mouse brown adipose tissue by the intracerebroventricular administration of bombesin.

Effects of bombesin on brown adipose tissue (BAT) thyroxine (T4) 5'-deiodinase (5'D) activity and rectal temperature were examined in male mice. Immediately following an intracerebroventricular (ICV) or intravenous (IV) injection of bombesin (0.1-100 ng/animal) or vehicle (20 mM bacitracin dissolved in 0.9% saline), the mice were placed in a room at 4 degrees C or 22 degrees C for 30, 60, 120 or 240 min. The ICV injection of bombesin dose-dependently lessened cold-induced increase in BAT 5'D activity and increased hypothermia determined at 120 min of cold exposure, whereas the IV injection of bombesin was without effect. Bombesin (ICV)-induced hypothermia preceded the inhibition of BAT 5'D activity by at least 30 min at 4 degrees C. BAT 5'D activity was not affected by ICV injection of bombesin in mice kept at 22 degrees C, although the rectal temperature was significantly decreased. Bombesin thus appears to prevent cold-induced increase in T4 5'D activity in mouse BAT by its central effect. Bombesin-induced excessive hypothermia itself and/or the decrease in sympathetic tone of BAT by bombesin might decrease cold-induced increase in BAT 5'D activity.     

Cloning, expression, and functional characterization of the substrate binding subunit of rat type II iodothyronine 5'-deiodinase

Type II iodothyronine 5'-deiodinase catalyzes the bioactivation of thyroid hormone in the brain. In astrocytes, this approximately 200-kDa, membrane-bound enzyme is composed of at least one p29 subunit, an approximately 60-kDa, cAMP-induced activation protein, and one or more unidentified catalytic subunit(s). Recently, an artificial type II-like selenodeiodinase was engineered by fusing two independent cDNAs together; however, no native type II selenodeiodinase polypeptide is translated in the brain or brown adipose tissue of rats. These data suggest that the native type II 5'-deiodinase in rat brain is unrelated to this artificial selenoprotein. In this report, we describe the cloning of the 29-kDa subunit (p29) of type II 5'-deiodinase from a lambdazapII cDNA library prepared from cAMP-induced astrocytes. The 3.3-kilobase (kb) cDNA encodes an approximately 30-kDa, 277-amino acid long, hydrophobic protein lacking selenocysteine. Northern blot analysis showed that a 3.5-kb p29 mRNA was present in tissues showing type II 5'-deiodinase activity such as brain and cAMP-stimulated astrocytes. Domain-specific, anti-p29 antibodies specifically immunoprecipitated enzyme activity. Overexpression of exogenous p29 or a green fluorescence protein (GFP)-tagged p29 fusion protein led to a >100-fold increase in deiodinating activity in cAMP-stimulated astrocytes, and the increased activity was specifically immunoprecipitated by anti-GFP antibodies. Steady-state reaction kinetics of the enzyme in GFP-tagged p29-expressing astrocytes are identical to those of the native enzyme in brain. Direct injection of replication-deficient Ad5-p29(GFP) virus particles into the cerebral cortex of neonatal rats leads to a approximately 2-fold increase in brain type II 5'-deiodinating activity. These data show 1) that the 3.3-kb p29 cDNA encodes an essential subunit of rat type II iodothyronine 5'-deiodinase and 2) identify the first non-selenocysteine containing subunit of the deiodinase family of enzymes.