L. Krawiec

Hormonal regulation of brain development. III. Effects of triiodothyronine and growth hormone on the biochemical changes in the cerebral cortex and cerebellum of neonatally thyroidectomized rats

Abstract The effects of l -triiodothyronine and bovine growth hormone, administered to neonatally thyroidectomized rats, have been studied in connection with the changes in nucleic acid content and enzymic activities of the cerebral cortex and cerebellum. When administered from the 10th day of age, both triiodothyronine and bovine growth hormone were effective in correcting the alterations produced by neonatal thyroidectomy. On the contrary, when the beginning of treatments were delayed until the 15th day of age, triiodothyronine and/or bovine growth hormone showed no restoring effect. The finding that bovine growth hormone was as effective as triiodothyronine raises the question of whether the effects of early thyroid dysfunction on cerebral development reflect a direct action of thyroid hormone on this process or whether they are indirectly mediated.

Thyroid autoregulation. Inhibition of goiter growth and of cyclic AMP formation in rat thyroid by iodinated derivatives of arachidonic acid

Thyroid autoregulation has been related to intraglandular content of an unknown putative iodocompund. Data from different laboratories have shown that the thyroid is capable of producing different iodolipids, including iodinated derivatives of arachidonic acid; such as 5-hydroxy-6-iodo-8, 11, 14-eicosatrienoic-δ-lactone (IL-δ). Previous results from our laboratory showed that a semi-purified preparation of iodinated arachidonic acid exerts an inhibitory action in vitro on calf thyroid. In the present studies three purified iodinated derivatives of arachidonic acid were synthetized: IL-δ; 14-io-do-15-hydroxy-5, 8, 11-eicosatrienoic acid (I-OH-A) and its corresponding ω-lactone (IL-ω). Their action on MMI-induced goiter was studied in rats. Administration of MMI to rats during 10 days increased thyroid weight by 124%. This effect was significantly inhibited by the simultaneous injection of 5 μg/day of I-OH-A (57% inhibition of MMI action), IL-W (39%), IL-δ (33%) and T3 (95%), while arachidonic acid was without action. No inhibition was found with 1.25 μg/day KI, a dose equivalent to that which could be originated from total dehalogenation of the iodocompounds. These results support the idea that these iodocompounds have an intrinsic biologic activity and that there is a correlation between action and chemical structure. Serum TSH was increased around 15–20 fold after MMI administration. Chronic or acute injection of I-OH-A failed to alter TSH levels, indicating that this iodocompound exerts its action directly on the gland, without altering TSH concentration. Serum T3 and T4 were significantly decreased by MMI and addition of I-OH-A, did not change these values. Thyroidal cyclic AMP content was measured in the rats. Treatment with MMI increased cAMP, while injection of I-OH-A significantly decreased this action. These data suggest that this iodocompound acts at the step of cyclic AMP formation. Dose-response studies showed parallel decreases in gland weight, DNA and cAMP contents after I-OH-A treatment. The lowest effective dose was 3 μg/day. The present data demonstrate, for the first time, that some iodinated derivatives or arachidonic acid mimic the action of iodine on thyroid growth and cyclic AMP production and support the hypothesis that they may play a role in the autoregulatory mechanism.

Studies on the goiter inhibiting action of iodolactones

The thyroid gland synthesizes 6-delta-iodolactone, a compound shown to inhibit goiter growth in vivo and cell proliferation in culture. The present studies were performed to characterize this effect further with the aim of exploring the possible therapeutic action of iodolactones. Prevention assay: rats were treated simultaneously with a goitrogen, methylmercaptoimidazole, and either 6-delta-iodo-lactone or 14-iodo-omega-lactone, a synthetic derivative, given either i.p. or p. o. Both compounds caused a significant decrease in thyroid weight irrespective of the route of administration, but oral administration was less effective. A dose-response relationship was observed, the minimal effective dose (i.p.) being 3 micrograms/day. Involution assay: goiter was first induced with methylmercaptoimidazole and then the iodolactones were injected. Both compounds caused a significant involution, which was dose-related. Acute (10 days) administration of the iodolactones did not produce significant changes in several serum parameters (total T3 and T4, cholesterol, total protein, urea and acetylcholinesterase). These results give further support to the potential therapeutic application of iodolactones.

Turnover of phosphatidylcholine in cell membranes of adult rat brain.

—Incorporation of radioactive choline into subcellular fractions of adult rat brains, after intracranial injection of the precursor, was studied at different times. Maximum labelling of phosphatidylcholine occurred at 5 h in all fractions, the highest specific radio‐activity being found in mitochondria. The specific radioactivity diminished rapidly for a few hours after the peak of maximum incorporation and continued to decrease thereafter at a much slower rate. The type of decay curve found in all fractions except myelin, indicated the possible presence of two populations of phosphatidylcholine with quite different turnover rates. The significance of these findings is discussed.

In Vitro incorporation of [3H] methyl choline into phosphatidyl choline of rat liver subcellular fractions☆

Abstract Incubation of a rat liver total homogenate with radioactive choline and subsequent isolation of subcellular fractions, at different times, showed similar patterns of labeling. Incubation of microsomes, mitochondria and purified nuclei isolated from rat liver, showed that all fractions were able to incorporate the precursor into phosphatidyl choline. The specific activity was higher in mitochondria and increased in all cases with added supernatant. The addition of microsomes to mitochondria diminished the incorporation of label. Contamination of mitochondria by microsomes, was negligible as shown by undetectable amounts of cytochrome P450, while NADPH2 cytochrome c reductase showed a 10% contamination. A certain amount of radioactivity was incorporated in the absence of ATP and oxidizable substrates due to the presence of substrates and cofactors in the fraction and/or the supernatant. Labeled fractions reincubated with unlabeled choline, showed no loss of radioactivity, proving that incorporation was not due to simple exchange processes. It is concluded that although rat liver mitochondria can acquire part of their own provision of phosphatidyl choline by transference from microsomes, all organelles and specially mitochondria, can independently synthesize this phospholipid.

INFLUENCE OF NEONATAL HYPOTHYROIDISM UPON TRANSCRIPTION IN ISOLATED RAT BRAIN AND LIVER NUCLEI

The optimum conditions for measuring the transcriptional activity of endogenous RNA polymerase I and I1 were established in low ionic strength incubation mixtures for liver and cerebrum cell nuclei. Both activities were estimated in the presence of Mg2+ for RNA polymerase 1 and Mn2+ for RNA polymerase I1 under conditions in which the nuclear morphology is prcscrvcd. The UMPIGMP incorporation ratio was used to determine the type of RNA synthesized and the results were confirmed employing specific inhibitors of RNA synthesis, such as a‐Amanitin and Actinomycin‐D. Neonatal thyroidectomy in the rat diminished the normal growth of body and liver more than the cerebral growth. The transcriptional capacity in the presence of Mg2+ in cerebral cell nuclei was not changed by the absence of thyroid function during postnatal development. Contrariwise, the activity the presence of Mn2+ was transicntly diminished between the 10th and 13th day of life. When the assays were performed at high ionic strength there were no differences between normal and hypothyroid animals. In liver, thyroid deficiency affected in an early and permanent way the transcription of an AU‐rich RNA. The endogenous activity measured at high ionic strength and that tested in the presence of Mg2+ were altered from the 20th day onward.

Effect of the interaction of TSH and insulin on the stimulation of 2-deoxyglucose uptake in FRTL-5 cells1

Since thyroid glycogen stores are low, the uptake of glucose is very important in order to maintain cell function (house-keeping). Previous studies have shown that TSH and insulin, independently, are regulators of this parameter. Since their corresponding mechanisms of action are different, we investigated the possible effect of the interaction between TSH and insulin on the stimulation of 2-deoxyglucose (2-DOG) uptake, a non metabolizable derivative of glucose. Confluent FRTL-5 cells were submitted to different treatments, usually for 72 h. In one series of experiments the concentration of TSH was kept constant, at 1 U/l, and the addition of insulin, from 0.16 to 1.6 μmol/l caused a progressive synergic increase in DOG uptake. When insulin concentration was kept constant, increasing amounts of TSH, from 0.5 to 10 U/l), also caused a synergic stimulation of DOG uptake. The effect of insulin was mimicked by IGF-1 (1–10 nmol/l), while that of TSH was mimicked by forskolin. Timecourse studies showed that TSH had a peak at 3 h of incubation, while insulin caused a progressive increase for up to 72 h. At short incubation times, up to 6 h, an additive effect of TSH and insulin was observed, while at longer times the interaction was synergic. The present results suggest that the interaction between the cAMP and the tyrosine kinase pathways on DOG uptake would involve two different mechanisms. At early times the effects of both hormones are additive, while in longer periods it becomes synergic.

Post-Receptor Events in Growth Control

TSH regulates thyroid function and growth. However, both clinical observations and experimental results have suggested that TSH is necessary for, but not the trigger of, gland growth. Several findings support this hypothesis: 1 Glands with decreased iodine stores are hypersensitive to TSH (1,2). 2 Serum TSH is normal in some endemic goiter areas (3) and in most thyroid cancers (4,5). 3 Experimental goiter (LID, MTU, etc.) develops before an increase in serum TSH is detected (6,7). 4 Goiter size in some endemic areas is inversely related to iodine content (8). 5 Compensatory thyroid growth, after hemithyroidectomy, may occur in the absence of TSH (9,10).

Relationship between nuclear ADP-ribosylation and RNA transcription in calf and human thyroid

ADP-ribosylation is a posttranslational modification of proteins that has been related to many cellular events, such as DNA replication and repair, cell proliferation and differentiation. The present studies were performed in order to explore the possible relationship between nuclear protein ADP-ribosylation and RNA transcription in the thyroid gland. Inhibition of RNA transcription by α-amanitin and actinomycin D caused a decrease in ADP-ribossylation of 27 and 17%, respectively. Nicotinamide caused a dose-related inhibition of ADP-ribosylation, which was highest at 2 mM (around 90%). At this dose nicotinamide inhibited total RNA transcription by 46%, while the activity due to RNA polymerase II decreased by 50% and that related to RNA polymerases I+III dropped by 24%. These results suggest that inhibition of total nuclear protein ADP-ribosylation is accompanied by a parallel decrease in RNA transcription. Since our previous work has shown that TSH stimulates both nuclear ADP-ribosylation and RNA transcription it may be concluded that these activities follow parallel changes within the thyroid. When the same activities were assayed in normal human and in glands bearing follicular adenoma, RNA polymerase II was increased 4 fold in the latter group, without change in nuclear ADP-ribosylation. These results would suggest that a mechanism, distinct from ADP-ribosylation, may also be involved in the regulation of RNA transcription. This latter might be altered under this pathologic condition.

Effects of Synthetic Iodolipids on Thyroid Function “In Vitro”: Possible Role in Thyroid Regulation

The inhibitory effects of iodine on the thyroid are mediated by an iodine organic intermediate (see review, 1), whose chemical nature is not known. Although thyroid hormones have been proposed to be such intermediates, their role is still controversial (see ref. 2 for discussion). It is known that, in addition to thyroid hormones, the gland is capable of synthesizing iodolipids (see review, 3), another type of organic iodocompounds. We have recently shown that iodinated free fatty acids comprise most of the iodolipids (4). The results obtained with different inhibitors of arachidonic acid metabolism suggested that iodinated derivatives of arachidonic acid may be a component of iodinated free fatty acids. Moreover, recent data from our laboratory has shown that in calf thyroid slides incubated with 125I, the incorporation of radioiodine into 15-iodo14-hydroxy- and 15-hydroxy-14-iodo-eicosatrienoic acid (I-OH-A) and the corresponding lactones was observed (to be published). Boeynaems and Hubbard (5) have also shown that rat thyroid produces the acid 5-iodo-6hydroxy-eicosatrienoic 8,11,14 delta lactone (IL-δ). In a previous publication, we have demonstrated that a semi-purified preparation of iodoarachidonate inhibits in vitro different thyroid parameters (6). We have also shown that studies with pure preparations of I-OH-A and another derivative with an iodine atom in position 6 (IL-δ).