Harold L. Schwartz

Replacement dose, metabolism, and bioavailability of levothyroxine in the treatment of hypothyroidism. Role of triiodothyronine in pituitary feedback in humans.

A change in the formulation of the levothyroxine preparation Synthroid (Flint) in 1982 prompted us to reevaluate the replacement dose of this drug in 19 patients with hypothyroidism. The dose was titrated monthly until thyrotropin levels became normal. The mean replacement dose (+/- SD) was 112 +/- 19 micrograms per day, significantly less (P less than 0.001) than the dose of an earlier formulation--169 +/- 66 micrograms per day--used in a similar study (Stock JM, et al. N Engl J Med 1974; 290:529-33). The fractional gastrointestinal absorption of a tablet of the current formulation is 81 percent, considerably higher than the earlier estimate of 48 percent. Using high-performance liquid chromatographic analysis, we found that the current tablet contains the amount of thyroxine stated by the manufacturer. By measuring the bioavailability of the earlier type of tablet in five patients, we inferred that the strength of the previous tablet had been overestimated. In the present study, the thyrotropin levels of patients on replacement therapy returned to normal when serum triiodothyronine concentrations were not significantly different from those of controls (122 vs. 115 ng per deciliter [1.87 vs. 1.77 nmol per liter]), but when serum thyroxine levels were significantly above those of controls (11.3 vs. 8.7 micrograms per deciliter [145 vs. 112 nmol per liter], P less than 0.001). These findings suggest the possibility that in humans, serum triiodothyronine may play a more important part than serum thyroxine in regulating the serum thyrotropin concentration.

Selective alterations in hepatic enzyme response after reduction of nuclear triiodothyronine receptor sites by partial hepatectomy and starvation.

Abstract Partial hepatectomy results in a decrease in the nuclear triiodothyronine binding capacity of rat liver. Although we confirmed reports that starvation causes a reduction in binding capacity, starvation alone did not fully account for the decrease after hepatectomy. Reduction in nuclear binding sites both by hepatectomy and starvation failed to impair the response to triiodothyronine administration as measured by an increase in the enzyme activity of mitochondrial α-glycerophosphate dehydrogenase, but significantly blocked the triiodothyronine response as measured by cytosolic malic enzyme. These findings indicate that triiodothyronine response is influenced by currently undefined metabolic factors and raises the possibility that such factors cause a selective decrease in receptors associated with the gene for malic enzyme.

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.

Stereospecific transport of triidothyronine to cytoplasm and nucleus in GH1 cells

We have recently demonstrated substantial stereospecific nuclear/cytosolic free triiodothyronine (T3) gradients within T3 responsive rat tissues in situ. These studies have now been extended to examine T3 transport in a rat pituitary tumor cell line, GH1. L-T3 had a 7.6-fold higher affinity for the nuclear receptor when assayed in whole cell incubations in comparison to isolated nuclei, though D-T3 affinity was not altered under these conditions. An apparently higher number of receptors for D-T3 was explained by racemic contamination of the isotopes used. Measurement of free hormone concentration ratios for both enantiomers revealed a small step up from medium to cytosol for L-T3 (1.65) but a reverse ratio for D-T3 (0.46). The nuclei were able to concentrate both enantiomers, though stereospecificity was maintained (nucleus/cytosol, L-T3, 4.5, D-T3 1.7). Transport of L-T3 at both boundaries could be inhibited by monodansylcadaverine. Thus, stereospecific transport functions are found within GH1 cells, though the magnitude of the free nucleus/cytosol gradient is reduced from those seen in rat tissues in situ.

TSH response to TRH in euthyroid, hypercholesterolemic patients treated with graded doses of dextrothyroxine

In an attempt to determine the minimum dose of D-Thyroxine (D-T4) which will suppress pituitary TSH response to TRH, we have treated 6 euthyroid, hypercholesterolemic patients with graded doses of D-T4. TSH response was suppressed in 3 patients with 3 mg and in the remaining 3 patients with 4 mg D-T4 administered once daily. The mean TSH suppressive dose of 3.5 mg, as determined in this study, is considerably less than the 6 mg daily dose given to patients treated with D-T4 in the Coronary Drug Project. This suggests that the adverse effects observed with D-T4 treatment in the Coronary Drug Project may have been due to mild, undetected hyperthryroidism. D-T4 treatment in our patients was not associated with an increase in heart rate or ventricular ectopic beats as determined by Holter monitoring. However, bile samples obtained at the time of TSH suppression showed a significant increase in lithogenic index. In four patients, TSH suppressive doses of D-T4 were associated with a 12% decrease in mean cholesterol and a 17% decrease in mean LDL cholesterol concentrations.

Inhibition of malic enzyme induction by triiodothyronine in the diabetic rat: reversal by fructose feeding.

Abstract In order to determine whether an increase in insulin is a prerequisite for induction of malic enzyme (ME) by triiodothyronine (T 3 ), we examined the ability of various diets to affect the T 3 induction of ME in diabetic rats. Forty-eight hours after streptozotocin, rats were placed on either a regular diet, a 60% glucose diet, or a 60% fructose diet. The next day selected groups received 200 μg T 3 /100 g body weight. Twenty-four hours later serum was collected and the liver 100,000 × g supernatant fraction was assayed for ME. Compared to intact controls, the level of ME in the basal state as well as in the T 3 stimulated state was significantly less in diabetic rats (in U/mg, basal: intact, 19.3; diabetic, 7.1; T 3 -treated:intact, 56.6; diabetic, 15.6). Fructose feeding in the diabetic rats led to no significant increment in ME (1.1 U/mg) whereas the intact rat fed fructose showed a significant increment in ME (39.4 U/mg). The combined administration of fructose and T 3 in the diabetic rat however, resulted in a substantial increment of ME (30 U/mg), which was significantly greater than the increment induced by T 3 and glucose (2 U/mg). Moreover, the increment in ME induced by T 3 and fructose in diabetic animals was not significantly different from that achieved in intact animals (54.9 U/mg). All groups of diabetic rats showed a decrease in insulin from the start of the dietary manipulations to the end of the experiment (28.7 μU/ml fasting to 10.9 μU/ml when killed in the fed state). Thus ME induction by T 3 can occur in the absence of an increase in insulin secretion. Fructose feeding can supply, at least in part, the necessary factors that permit T 3 to induce ME in diabetic rats.

Comparison of age-related decreases in the basal and carbohydrate inducible levels of lipogenic enzymes in adipose tissue and liver

Abstract An age-related decrease in the level of four lipogenic enzymes—malic enzyme (ME), fatty acid synthetase (FAS), glucose-6-phosphate dehydrogenase (G-6-PD), and 6-phosphogluconate dehydrogenase (6-PGD)—was compared in the adipose tissue and liver of rats. In adipose tissue the activity of ME per milligram of protein at one month exceeded that observed at one year by a factor of 27 or more. The bulk of the fall occurred between one and three months. Adipose FAS, G-6-PD, and 6-PGD showed a similar but less precipitous age-related decline. The extent of age-related changes in the adipose tissues consistently exceeded those observed in liver preparations from the same animal. For both fat and liver, administration of a high carbohydrate fat-free diet for four days also elicited a substantially greater incremental ME response in young rats than in old animals. The age-related changes in basal ME activity were proportional to the increments produced by dietary stimulation. Thus, as previously proposed by us for liver, the decrease in basal ME in adipose tissue may be the result of a reduced generation from dietary carbohydrate of a stimulus responsible for the induction of lipogenic enzymes. Additional experiments were performed in animals provided with 10% glucose in their drinking water and infused with high doses of insulin for 4 to 12 days. In the fat of older animals, such treatment resulted in the induction of adipose ME activity to levels almost equivalent to those achieved with similar treatment in younger animals. Whereas maximal levels of ME in the younger animals could be attained simply with the glucose in the drinking water, insulin administratin was required in the older animals. Presumably, the insulin response evoked by glucose in the younger animals elicited a maximal ME increment. In the older animals, however, the endogenous insulin response to glucose failed to achieve a comparable degree of ME induction in the fat. Since the concentrations of insulin attained in the older animals were equal to or greater than those attained in the younger rats, both in the basal and the stimulated state, the diminished ME response in the older animals can be considered to be a form of tissue insulin resistance. Diminished glucose metabolism in the adipocytes of older rats is well established even in the presence of normal plasma glucose concentrations. Therefore, the results suggest that the level of hepatic and adipose ME activity may be useful as an index of such changes.

Effect of dichloroacetic acid on rat hepatic messenger RNA activity profiles

We have previously suggested that the ability of glucose to induce rat hepatic lipogenic enzymes is mediated by the mitochondrial oxidation of pyruvate. In part, this hypothesis is supported by the finding that an activator of pyruvate dehydrogenase, dichloroacetic acid (DCA), is capable of inducing malic enzyme in hepatocyte cultures. In order to further test this hypothesis, we compared the mRNA responses induced by carbohydrate feeding in vivo and by glucose administration to hepatocytes in culture with those mRNA responses induced in DCA both in vivo and in culture. DCA administration to rats resulted in a significant increase in liver:body weight ratio. It was, in addition, a potent inducer of malic enzyme. Hepatic mRNA activity profiles were examined by two-dimensional gel electrophoretic analysis of in vitro translation products. Six of the seven mRNAs altered by carbohydrate feeding were similarly altered by DCA feeding in vivo. In cultured hepatocytes 10 mmol/L DCA significantly increased four of six glucose-induced mRNAs. The mRNA for malic enzyme was among those mRNA sequences induced both in vivo and in culture. Increasing glucose concentrations in the culture medium resulted in an expected rise in pyruvate levels, whereas DCA caused a significant decrease in the concentration of this intermediate. It is likely, therefore, that augmentation of the flux of pyruvate through pyruvate dehydrogenase rather than alterations in pyruvate levels per se, is a proximal event leading to the induction of multiple mRNAs. The marked overlap in mRNA response to both carbohydrate and DCA indicates that the signal regulating the content of the carbohydrate responsive mRNAs is derived from mitochondrial pyruvate oxidation.

Interaction of T3 and carbohydrate in the induction of lipogenic enzymes

Abstract The response of hepatic lipogenic enzymes to T3 in animals on a regular diet is amplified. On a high carbohydrate diet the response becomes linear with a leftward shift in the T3 dose response curve. This interaction between thyroid hormone and carbohydrate feeding is not a primary result of an increase in insulin secretion. Additional studies in the aging animal have shown that the known decline in basal lipogenic enzymes is paralleled by a decline in response to thyroid hormone as well as a decline in response to carbohydrate feeding. Nevertheless, the amplification phenomenon persists suggesting that T3 interacts with a carbohydrate generated signal in a multiplicative fashion. The decline in response to T3 and carbohydrates is probably due to an age-related decrease in the generation of this signal. Preliminary studies in isolated hepatocytes have shown that increases in glucose concentrations perse can induce malic enzyme. These cells also exhibit a multiplicative interaction between T3 and glucose.