Cary N. Mariash

A rapid, inexpensive, quantitative technique for the analysis of two-dimensional electrophoretograms

Abstract We describe a rapid, reproducible, and relatively inexpensive method for quantitative analysis of two-dimensional gel radiofluorograms. The equipment required includes a mediumresolution black and white video camera for data acquisition, a video digitizing circuit for data translation, and a microcomputer for data analysis. The system described can also be used to quantitate protein- or DNA-stained one- or two-dimensional gels or chromatograms.

Synergism of thyroid hormone and high carbohydrate diet in the induction of lipogenic enzymes in the rat. Mechanisms and implications.

We have investigated the relationship between the administration of triiodothyronine (T3) and a high carbohydrate (CHO) fat-free diet in the induction of lipogenic enzymes in two rat tissues, liver, and fat. Male thyroidectomized rats were treated with graded daily doses of T3 and either supplemented with a high CHO diet or left on a regular diet. Enzymes studied included malic enzyme (ME), fatty acid synthetase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase. In the liver, all four lipogenic enzymes showed a synergistic response between T3 administration and high CHO feeding. In fat, ME also responded synergistically. The interaction was reflected in an increased sensitivity to T3. The dose of T3 required to achieve 50% maximal response was reduced three- to seven-fold by the high CHO diet. This phenomenon could not be attributed to a dietary-induced alteration either in T3 metabolism or in number or affinity of the T3-nuclear receptors. Moreover, studies of the relative rate of synthesis of ME suggested a simultaneous time of onset in the induction of ME, within 2 h after the application of either T3 or CHO. Thus, it is unlikely that either stimulus is secondary to the other. Since parallel experiments from this laboratory (Towle, Mariash, and Oppenheimer,1980. Changes in hepatic levels of messenger ribonucleic acid for malic enzyme during induction by thyroid hormone or diet. Biochemistry. 19: 579-585.) show that ME induction both by CHO and T3 is mediated by an increase in specific messenger RNA for ME, the interaction of T3 and the dietary factor occurs at a pretanslational level.

Triiodothyronine is a survival factor for developing oligodendrocytes

Thyroid hormone plays an important role in oligodendrocyte development. The studies presented here suggest that thyroid hormone is required for oligodendrocyte survival during development. Oligodendrocyte precursor cells, astrocytes and microglia were cultured in a defined media. Oligodendrocyte precursor cell differentiation was induced by growth factor removal. Time course studies revealed that oligodendrocytes cultured in the presence or absence of triiodothyronine (T3) develop similarly during the first 3 days of development. Oligodendrocytes cultured in the absence of T3, however, die after developmental day 3. TdT-Mediated dUDP Nick End Labeling assay and Hoechst staining indicate that T3 rescues developing oligodendrocytes from death by apoptosis. Apoptosis is likely induced by the presence of the cytokines TNFalpha and IL-1beta. However, expression of these cytokines is not altered by thyroid hormone administration. Thus, thyroid hormone has been demonstrated to effect proliferation, myelin gene expression and now the survival of developing oligodendrocytes.

Glucose and Triiodothyronine Both Induce Malic Enzyme in the Rat Hepatocyte Culture EVIDENCE THAT TRIIODOTHYRONINE MULTIPLIES A PRIMARY GLUCOSE-GENERATED SIGNAL

We have stimulated in a cultured hepatocyte system the synergistic interaction between triiodothyronine (T3) and dietary carbohydrate in the induction of malic enzyme (ME). Kinetic studies revealed that isolated hepatocytes equilibrate with media T3 within 5 min; nuclei equilibrate with media T3 by 45 min; and the half-time of T3 metabolism was 10 h in 10% serum. We demonstrated nuclear T3 receptors in isolated hepatocytes and the induction of ME by T3 in physiological concentrations. However, in the complete absence of T3 glucose could still induce ME. At all concentrations of glucose (100-1,000 mg/dl), T3 (0.3 nM free T3) resulted in a relatively constant (1.4- to 1.7-fold) increase in ME response. The augmentation in ME activity was paralleled by an enhanced rate of enzyme synthesis as determined by [3H]leucine incorporation into immunoprecipitable ME. Cells cultured in serum free media also demonstrated a glucose-dependent increase in ME. Insulin greatly stimulated the glucose induction of ME, whereas dexamethasone had very little influence on ME induction. These studies demonstrate the usefulness of an adult hepatocyte tissue culture model for the study of the effects of T3 on gene expression in cells that are not derived from tumor. They clearly demonstrate that well established effects of T3 can be simulated in such a system at levels of free hormone that approximate those in extracellular body fluids. Our results indicate that an increased concentration of glucose per se can induce the formation of ME in the absence of alterations in extrahepatic hormones or factors. Moreover, our findings confirm inferences from in vivo studies that T3 acts as a multiplier of a glucose-induced signal.

T3 increases Na-K-ATPase activity via a MAPK/ERK1/2-dependent pathway in rat adult alveolar epithelial cells

Thyroid hormone (T3) increases Na-K-ATPase activity in rat adult alveolar type II cells via a PI3K-dependent pathway. In these cells, dopamine and beta-adrenergic agonists can stimulate Na-K-ATPase activity through either PI3K or MAPK pathways. We assessed the role of the MAPK pathway in the stimulation of Na-K-ATPase by T3. In the adult rat alveolar type II-like cell line MP48, T3 enhanced MAPK/ERK1/2 activity in a dose-dependent manner. Increased ERK1/2 phosphorylation was observed within 5 min, peaked at 20 min, and then decreased. Two MEK1/2 inhibitors, U0126 and PD-98059, each abolished the T3-induced increase in the quantity of Na-K-ATPase alpha(1)-subunit plasma membrane protein and Na-K-ATPase activity. T3 also increased the phosphorylation of MAPK/p38; however, SB-203580, a specific inhibitor of MAPK/p38 activity, did not prevent the T3-induced Na-K-ATPase activity. SP-600125, a specific inhibitor of the MAPK/JNK pathway, also did not block the T3-induced Na-K-ATPase activity. Phorbol 12-myristate 13-acetate (PMA) significantly increased ERK1/2 phosphorylation and Na-K-ATPase activity. The PMA-induced Na-K-ATPase activity was inhibited by U0126. These data indicate that activation of MAPK-ERK1/2 was required for the T3-induced increase in Na-K-ATPase activity in addition to the requirement for the PI3K pathway.

Chicken Ovalbumin Upstream Promoter-Transcription Factor (COUP-TF) Modulates Expression of the Purkinje Cell Protein-2 Gene A POTENTIAL ROLE FOR COUP-TF IN REPRESSING PREMATURE THYROID HORMONE ACTION IN THE DEVELOPING BRAIN

The cerebellar Purkinje cell-specific PCP-2 gene is transcriptionally activated by thyroid hormone during the 2nd and 3rd weeks of postnatal life in the rat. In contrast, thyroid hormone has no detectable effects on PCP-2 expression in the fetal rat. We now present data that suggest that the orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor (COUP-TF) represses triiodothyronine (T3)-dependent transcriptional activation of PCP-2 in the immature Purkinje cell. Gel shift assays show that the PCP-2 A1TRE and adjoining sequences (−295/−199 region) bind to rat and mouse brain nucleoproteins in a developmentally regulated fashion and that one of these nucleoproteins could be the orphan nucleoprotein COUP-TF. In support of this hypothesis, in vitro translated COUP-TF binds to the −295/−199 region and COUP-TF represses T3-dependent activation of the PCP-2 promoter in transient transfection analyses. Finally, immunohistochemical studies reveal that COUP-TF is specifically expressed in the immature fetal and early neonatal Purkinje cell and that this expression diminishes coincident with thyroid hormone induction of PCP-2 expression. Our findings are consistent with the hypothesis that the presence or absence of inhibitory proteins bound to the thyroid hormone response element of T3-responsive genes governs the responsivity of these genes to thyroid hormone during brain development.

Beta receptor isoforms are not essential for thyroid hormone-dependent acceleration of PCP-2 and myelin basic protein gene expression in the developing brains of neonatal mice

In rat pups, thyroid hormone dependent brain development coincides with the appearance of the thyroid hormone receptor (TR)beta1 isoform. This finding led to the suggestion that TRbeta1 plays an essential role in brain development. The recent availability of a mouse TRbeta knockout strain allowed us to test this possibility by determining whether TRbeta is essential for the normal developmental pattern of expression of two thyroid hormone regulated brain genes, myelin basic protein (MBP), and Purkinje cell protein 2 (Pcp-2). Northern analysis of total mRNA from the brains of wild-type mice established that, as in the rat pup, the initial rate of rise of the MBP and Pcp-2 mRNA is slowed in the hypothyroid state. Supporting the effectiveness of TRbeta gene deletion was the finding that the thiiodothyronine (T3) nuclear binding capacity in the livers and brains of knockout animals was consistent with the fractional contribution of TRbeta1 to total binding capacity in the wild-type tissues. Further, no TRbeta1 could be detected by isoform-specific immunoprecipitation of nuclear receptor extracts. However, deletion of the functional TRbeta in the TRbeta knockout mice did not affect the normal ontogeny of expression of the Pcp-2 and MBP genes in the postnatal pup. We conclude that TRbeta is not essential for the normal developmental expression of these T3 dependent brain genes.

Local reactions to radioiodine in the treatment of thyroid cancer

PURPOSE To compare the rate of local complications resulting from radioiodine ablation of thyroid cancer in patients with a residual intact thyroid lobe to that in patients who had more extensive surgical treatment prior to radioiodine administration. PATIENTS AND METHODS We retrospectively studied 59 patients who had received 131I between 1979 and 1989. The patients were divided into two groups, depending on the extent of their previous surgical thyroid excision. Group 1 comprised 10 patients with a lobectomy or hemithyroidectomy before the ablative radioiodine dose, and Group 2 comprised 49 patients with more extensive thyroid excision (near-total or subtotal thyroidectomy) before the radioiodine treatment. RESULTS Sixty percent of the 10 patients in Group 1 experienced some degree of neck pain or tenderness following radioiodine ablation of their residual thyroid. In one case, the local reaction was very severe and accompanied by the development of transient hyperthyroidism. There was only a 6% local complication rate in the patients who had undergone more extensive thyroid excision before ablative therapy (p less than 0.001), and none had a severe reaction. CONCLUSIONS Patients with only unilateral surgical excision before radioiodine therapy have a higher rate of local complications than do patients treated with more extensive surgery prior to radioiodine ablation. If radioiodine is to be employed in such patients, they should be informed of this possible complication. Since evidence supports a dose effect in the pathogenesis of the complications, we recommend using a dose of less than 30 mCi for the initial ablation in these patients even though it may be necessary to repeat this dose to complete thyroid ablation.

Stimulation of malic enzyme formation in hepatocyte culture by metabolites: Evidence favoring a nonglycolytic metabolite as the proximate induction signal☆

Recent studies have shown that the addition of increasing concentrations of glucose to the medium of primary adult rat hepatocyte cultures results in the progressive induction of malic enzyme. We have undertaken experiments to determine (1) whether metabolism of glucose was an essential prerequisite for such induction, and (2) whether a specific glycolytic intermediate could be shown to constitute the proximate carbohydrate signal triggering such induction. In line with these objectives we investigated the ability of various sugars and glycolytic metabolites to induce malic enzyme in this system and assessed the influence of insulin, glucagon, and thyroid hormone (triiodothyronine, T3) on this process. Our results show that only those sugars capable of entering the cell and being metabolized induce malic enzyme (glucose, fructose, and galactose). The nonmetabolizable sugars 3-O-methylglucose and 2-deoxyglucose are ineffective. Incubation with 20 mmol/L lactate, pyruvate, dihydroxyacetone, or glycerol resulted in malic enzyme induction, whereas incubation with acetate, citrate, and alpha-ketoisocaproate was without effect. The induction by all sugars and metabolites required presence of insulin. As previously reported for glucose, addition of T3, under all metabolic conditions, resulted in a constant 3.6-fold increase in the rate of malic enzyme induction and further supports the proposal T3 acts to multiply the effect of a common carbohydrate-generated signal. Glucagon administration led to a dose-dependent inhibition of the carbohydrate effect with a half-maximal effect and maximal effect at 2 and 100 nmol/L, respectively. None of the glycolytic metabolites tested could reverse the glucagon inhibition completely.(ABSTRACT TRUNCATED AT 250 WORDS)

Starvation and hypothyroidism exert an overlapping influence on rat hepatic messenger RNA activity profiles.

To assess the effect of starvation and to explore the potential interrelationship of starvation and thyroid status at the pretranslational level, we have analyzed by two-dimensional gel electrophoresis, the hepatic translational products of starved and fed euthyroid and hypothyroid rats. 5 d of starvation resulted in a statistically significant change in 27 of 240 products visualized, whereas hypothyroidism caused a change in 20, both in comparison with the fed euthyroid state. Of considerable interest was that 68% of all changing messenger (m)RNA sequences were common to the hypothyroid and starved groups and showed the same directional shift. Further, both starvation and hypothyroidism yielded comparable decreases in total hepatic cytoplasmic RNA content. Although it has been well established that the level of circulating triiodothyronine (T3) and the level of hepatic nuclear receptors fall in starvation, this reduction cannot account for the observed decrease of total hepatic RNA nor for all of the alterations in the concentrations of specific mRNA sequences. Thus, administration of T3 to starved animals in a dose designed to occupy all nuclear T3 receptors fails to prevent the fall in total RNA and the majority of starvation-induced changes in the level of mRNA sequences. Moreover, starvation of athyreotic animals results in a further decrease in total RNA and in a further change in the level of individual mRNA species. We conclude, therefore, that although the reduced levels of circulating T3 and the nuclear T3 receptors can contribute to the observed results of starvation, the starvation-induced changes are not exclusively mediated by this factor. The striking overlap in the genomic response between hypothyroid and starved animals raises the possibility that those biochemical mechanisms regulated at a pretranslational level by T3 are either not helpful or injurious to the starving animal. The reduction in circulating T3 and nuclear receptor sites together with T3-independent mechanisms initiated in the starved animal may constitute redundant processes designed to conserve energy and substrate in the nutritionally deprived organism.