Faculty Bibliography

We have established a rat model to investigate the relationship among serum thyroid hormones, nuclear iodothyronine receptors, and biological responses in thyrotoxic and euthyroid rats after surgical stress. Euthyroid or hyperthyroid rats (1.0 microgram T3/ml drinking water for 14 days) were subjected to surgical stress (ether anesthesia, laparotomy plus 50 mg talc, ip). Groups of control or stressed rats were killed 1, 2, and 3 days after surgical stress for measurement of thyroid hormone-responsive hepatic enzymes, alpha-glycerophosphate dehydrogenase (alpha GPD) and cytosol malic enzyme, serum T3, T3 nuclear receptors, and GH mRNA. Thyrotoxic rats had a 3.8-fold increase in alpha GPD compared to euthyroid rats before surgical stress; alpha GPD increased further to 5.9-fold the euthyroid value 1 day after surgery (P less than 0.001) to 5.1-fold after 2 days (P less than 0.05) and was similar to control after 3 days. Malic enzyme activity increased 10.5-fold before surgical stress and decreased slightly after surgical stress perhaps due to multifactorial regulation of that enzyme. No increases in T3 nuclear receptor or GH mRNA occurred after surgery in hyperthyroid rats or in any of the above parameters after surgical stress in euthyroid rats. Our findings suggest that increased alpha GPD after surgical stress in thyrotoxic rats was not due to either increased serum total T3 or free T3 or to increased T3-nuclear receptor complexes. Increased alpha GPD, therefore, appeared to be a consequence of postreceptor amplification of this thyroidal response.

Cultured rat somatotrophic cells have been useful models for the study of thyroid hormone action. A consensus of previous reports has indicated that approximately 0.2 nM T3 results in 50% occupancy of T3 nuclear receptors as well as half-maximal stimulation of several T3 responses. To characterize the nature of thyroid hormone responses in GC cells, we studied in detail the T3 dose relationships between nuclear receptor occupancy and three thyroid hormone responses (cell growth, GH production, and T3 nuclear receptor regulation). The dose response to T3 for each parameter was unique, and none was identical to the dose response for receptor occupancy. Respective T3 concentrations and percentage of T3 nuclear receptor occupancy resulting in 50% of the maximal response for GC cell growth were 0.05 +/- 0.02 nM and 15 +/- 3% (four experiments), 0.15 +/- 0.04 nM and 27 +/- 3% for GH production (three experiments), and 2.1 nM and 69% for down-regulation of T3 nuclear receptors (two experiments). We conclude that the dose response for occupancy of the T3 nuclear receptor covers a wide range of T3 concentrations. Within the wide dose-response range for nuclear occupancy a spectrum of biological responses are regulated by distinct thyroid hormone dose ranges. These data suggest that the impact of T3 nuclear receptor occupancy on T3 responses might be variable and that the mechanisms involved may be clarified through studies in GC cells.

To continue our studies on the influence of T3 on TSH regulation in the Walker 256 carcinoma-bearing rat model of nonthyroidal disease, we measured the effect of T3 on pituitary content of beta TSH mRNA and rat (r) TSH in hypothyroid control (C) and tumor-bearing (T) rats. The effect of T3 on TSH regulation was compared to effects on GH mRNA and rGH in the same animals. mRNA content was normalized to a pool of pituitaries from euthyroid rats (= 1.0). beta TSH mRNA increased 18-fold in both hypothyroid C and T rats and then decreased similarly with increasing T3 infusion to a value of 0.1. GH mRNA content decreased to 0.11 +/- 0.01 in hypothyroid C rats, but to only 0.38 +/- 0.02 in T rats (P less than 0.001). The pituitary contents of GH mRNA and rGH in hypothyroid T rats was significantly greater than those in C rats at all T3 infusion rates. These data together with our previous report of decreased nuclear T3 in T rats suggest that regulation of beta TSH mRNA by T3 is intact in T rats, but occurs at a lower concentration of nuclear T3. In contrast, the GH mRNA response is enhanced, displaying differential regulation of these two T3-responsive gene products in this model of nonthyroidal illness.

Rats bearing the Walker 256 carcinoma have decreased pituitary nuclear T3 but normal pituitary TSH content and response to experimental hypothyroidism. To elucidate further the role of T3 receptor occupancy and biological response in the tumor-bearing rat model of nonthyroidal disease, we measured the concentration of T3 nuclear receptors, rTSH and rGH and beta-TSH mRNA and GH mRNA in the anterior pituitary of euthyroid rats bearing the Walker 256 carcinoma. The abundance of T3 nuclear receptors was decreased in tumor-bearing rats and was associated with a decrease in mRNA content for beta-TSH and GH. alpha-tubulin mRNA was decreased to a comparable degree. The pituitary content of rTSH and rGH was, however, the same as in control animals. Since tumor rats have normal regulation of TSH secretion by thyroid hormone, the present findings suggest that TSH secretion in T rats is maintained by a lower T3 nuclear receptor occupancy than in controls. The decrease in beta-TSH mRNA may precede a decrease in TSH synthesis and changes in pituitary TSH stores. Since the decrease in GH mRNA was comparable to the decrease in alpha-tubulin mRNA, it does not appear to be specifically related to decreased T3 nuclear receptor occupancy. We conclude that, in the tumor-bearing rat model of nonthyroidal disease, decreases in beta-TSH mRNA occur despite a decreased T3 receptor occupancy. Both thyroid-dependent and thyroid-independent factors may be involved in regulating beta-TSH mRNA.

To facilitate studies of cell growth regulation by T3, we developed a variant GC cell line (V-GC) characterized by normal growth in T3-depleted (-T3) medium. The doubling time (dt) of V-GC cells was 28.8 h (-T3) and 28.0 h (+0.2 nM T3), respectively, whereas the dt of the parent GC cells, 24.0 h (+0.2 nM T3), increased to more than 100 h (-T3). The dt of V-GC cell was unaffected even by maximal T3 (5 nM). Cell protein (micrograms) per microgram DNA increased in GC cells in a T3 concentration-dependent manner, whereas V-GC cell protein was unaffected by T3. GH production appeared partially independent of T3 in V-GC cells. GH production (nanograms per 10(6)/h) in V-GC cells maintained for 3 months in -T3 medium was 3.3- to 4.6-fold greater than that in GC cells after 4 days in -T3 medium (P less than 0.001). Addition of T3 resulted in similar maximal GH production in both cell lines. The binding capacity and Ka of nuclear T3 receptors were similar in GC and V-GC cultures, and receptor down-regulation in response to added T3 occurred similarly in both cultures. Lastly, studies employing conditioned medium indicated that T3-independent growth of V-GC cells did not result from production of an autocrine growth factor. Our findings raise the possibility that overexpression of a transacting cell-specific gene-regulating protein that variably affects thyroid hormone-dependent genes may account for the phenotype of the V-GC cultures.

We have previously proposed that the effects of heat shock on thyroid hormone-responsive rat pituitary tumor (GC) cells may be a model relevant to the in vivo effects of nonthyroidal disease on thyroid hormone action. To determine the effects of heat shock on thyroid hormone responses, GC cells (normally cultured at 37 C) were studied after incubation at 41 C. After 18 h at 41 C there was enhanced synthesis of proteins (mol wt, 70,000 and 90,000) considered to be universal markers of the cellular response to heat shock. Incubation at 41 C also resulted in a significant decrease in GC cell viability and (after 24 h) arrest of GC cell growth. However, the induction of GH synthesis by T3 was significantly enhanced in GC cells stressed by incubation at 41 C. The addition of 5 nM T3 to thyroid hormone-depeleted GC cells resulted in a significantly greater (P less than 0.001) accumulation of GH (2642 +/- 280 ng/18 h) during 41 C incubation than during 37 C incubation (1223 +/- 175 ng/18 h). The enhanced T3-induced production of GH was coincident with a proportional increase (P less than 0.05) in cellular GH mRNA determined by dot hybridization analysis. Thus, the stress of 41 C incubation elicits a heat shock response in GC cells characterized by decreased viability and growth arrest, but enhanced accumulation of GH mRNA in response to T3. Our recent report on the identical effects due to the stress of implantation of the Walker 256 carcinoma on T3-induced rat pituitary GH mRNA in vivo suggests that heat shock of cultured GC cells is a valid in vitro model of nonthyroidal disease.

The effect of Zn(II) in 3,5,3'-triiodo-L-thyronine (T3) binding to nuclear receptors was studied in dialyzed 0.4 M NaCl extracts of nuclei from cultured GC cells. Addition of ZnCl2 to nuclear extracts resulted in a time- and concentration-dependent dissociation of T3 from nuclear receptors. Half-maximal dissociation occurred at 6 microM ZnCl2. Addition of ZnCl2 also resulted in a concentration-dependent inhibition of binding of T3 to nuclear receptors. Half-maximal inhibition of binding occurred at 1-3 microM ZnCl2. Scatchard analysis indicated that Zn(II) addition decreased kA and did not alter receptor concentration. These effects of Zn(II) were prevented when ZnCl2 was added to nuclear extracts in the presence of 5 mM EDTA or 5 mM dithiothreitol. Moreover, Zn(II)-induced inhibition of T3 binding was reversed by the addition of 5 mM EDTA. The inhibitory effect of Zn(II) on T3 binding seemed specific for nuclear receptors; no effect of Zn(II) on the binding of T3 to proteins in rat serum or GC cell cytosol or to rabbit anti-T3 serum was observed. Cd(II) had a similar concentration-dependent inhibition of T3 binding to nuclear receptors which was reversible. Our findings suggest that Zn(II) may play a role in T3 binding to nuclear receptors as well as its putative role in the binding of receptor to DNA.

The heat shock (HS) response is a characteristic disruption of protein synthesis which occurs in cells exposed to a variety of noxious stimuli. The effects of HS on thyroid hormone-responsive GC cells were studied in an attempt to devise an in vitro model for the adaptive changes in thyroid hormone action caused by nonthyroidal disease. HS enhanced GC cell synthesis of 70 K and 90 K proteins in a manner previously described as characteristic of the HS response in many tissues. A step-wise decrease in GC cell viability occurred when cells were exposed to 45 C for 10 to 35 min. HS (45 C, 20 min) resulted in a rapid decrease in binding of T3 to nuclear receptors. Two hours after HS, analysis of T3 binding to isolated nuclei showed a 50% fall in binding capacity (240 fmol/100 micrograms DNA) compared to non-HS control cells (540 fmol/100 micrograms DNA); no difference in dissociation constant (Kd) was observed. The effect of thyroid hormone on cell viability after HS was then determined. Thyroid hormone depletion (less than or equal to 0.02 nM T3) resulted in significantly (P less than 0.05) enhanced cell viability compared to cells cultured with physiological T3 (0.2 nM) after incubation at 45 C for intervals of 10-35 min. This inverse relationship between medium T3 content and cell tolerance of HS occurred over a wide range of T3 concentrations. Mean cell viability after exposure to 45 C for 20 min was 44 +/- 3% in T3-depleted cultures (less than or equal to 0.02 nM), 27 +/- 1% to 32 +/- 5% in cultures containing 0.07-0.5 nM T3, and 13 +/- 3% in cultures containing 5 nM T3. Our results thus characterize the response to HS in GC cells and the relationship of this response to medium T3. Similar to the effect of various nonthyroidal diseases on rat hepatocytes in vivo, HS resulted in a decrease in T3 nuclear receptors. Similar to the adverse effect of thyroid hormone on morbidity in animals with experimental diseases or injury, GC cell viability after HS was inversely related to medium T3 content. Thus the HS response in GC cells may be a valuable in vitro model relevant to the effect on thyroid hormone action caused by nonthyroidal disease.