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W Jiang, T Miyamoto, T Kakizawa, T Sakuma, S Nishio, T Takeda, S Suzuki, and K Hashizume

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRalpha and TRbeta, which act as mediators of thyroid hormone in tissues. However, the relative expression of each TR isoform in target tissues is still elusive. Herein, we have developed an RT-PCR and restriction enzyme digestion method to determine the expression of TRalpha1 and TRbeta1. We analyzed the expression of TR isoforms in 3T3-L1 preadipocytes induced to differentiate by an adipogenic cocktail in the presence or absence of 100 nM triiodothyronine (T(3)). The TRalpha1 isoform was predominantly expressed in 3T3-L1 adipocytes, and its expression was increased at the stage of development concomitant with the emergence of lipid droplets. Little, if any, TRbeta1 mRNA was detected in adipocytes. Administration of T(3) to the differentiating 3T3-L1 cells enhanced the accumulation of triglyceride. The expression profile of TRalpha1 in T(3)-treated adipocytes was similar to that in non-treated cells. The transcripts of adipogenic factors, CCAAT/enhancer binding protein beta (C/EBPbeta) and peroxisome proliferator activated receptor gamma (PPARgamma), were not altered by T(3). Lipid binding protein, aP2, that is downstream of these transcription factors was also unaffected by T(3). In contrast, the lipogenic enzyme, glyceraldehyde-3-phosphate dehydrogenase mRNA was significantly increased in the presence of T(3). Therefore, T(3) appears to be a hormone capable of modulating the expression of lipogenic enzyme and augments the accumulation of lipid droplets. We conclude that the TRalpha isoform might play an important role in the generation and maintenance of the mature adipocyte phenotype, regulating the expression of lipogenic enzymes.

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K Ichikawa, T Miyamoto, T Kakizawa, S Suzuki, A Kaneko, J Mori, M Hara, M Kumagai, T Takeda, and K Hashizume

The thyromimetic compound SK&F L-94901 shows more potent thyromimetic activity in the liver than in the pituitary gland or heart when administered to rats. The mechanisms of liver-selectivity of SK&F L-94901 were examined using cultured rat hepatoma cells (dRLH-84) and rat pituitary tumor cells (GH3), both of which showed saturable cellular uptake of tri-iodothyronine (T(3)). When isolated nuclei with partial disruption of the outer nuclear membrane were used, SK L-94901 competed for [(125)I]T(3) binding to nuclear receptors almost equally in dRLH-84 and GH3 cells. SK L-94901 also did not discriminate thyroid hormone receptors (TR) alpha1 and beta1 in terms of binding affinity and activation of the thyroid hormone responsive element. In intact cells, however, SK L-94901 was a more potent inhibitor of nuclear [(125)I]T(3) binding in dRLH-84 cells than in GH3 cells at an early phase of the nuclear uptake process and after binding equilibrium. These data suggest that SK L-94901 is more effectively transported to nuclear TRs in hepatic cells than in pituitary cells and therefore shows liver-selective thyromimetic activity. In conclusion, SK L-94901 discriminates hepatic cells and pituitary cells at the nuclear transport process. The cellular transporters responsible for this discrimination were not evident.

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R Shinohara, T Mano, A Nagasaka, R Hayashi, K Uchimura, I Nakano, F Watanabe, T Tsugawa, M Makino, H Kakizawa, M Nagata, K Iwase, Y Ishizuki, and M Itoh

Free radicals, hydroxyperoxides and H(2)O(2) are all known to damage cell components. This study was designed to compare the concentrations of hydroxyperoxide and free radical scavengers in the cardiac muscles of old rats in the hyper- or hypothyroid condition, to determine whether rates of peroxidation would differ with age, thyroid status, or both. Rats were rendered hyper- or hypothyroid by administration of l-thyroxine or methimazole for 4 weeks. Among the old rats, the lipid peroxide (LPO) concentrations, measured as thiobarbituric acid (TBA) reactants, were significantly greater in the hyperthyroid than in the euthyroid state and the LPO concentrations measured as TBA+Fe(3+) reactants, which may be precursors of LPO, were significantly greater in the hyperthyroid state, whereas in young rats, the LPO concentrations measured by TBA or TBA+Fe(3+) methods did not differ significantly in the hyperthyroid state. In the euthyroid state, the concentration of LPO measured as TBA+Fe(3+) reactants was significantly reduced with age. Xanthine oxidase (XOD) activity also was markedly increased with age, being more pronounced in the hyperthyroid than in the euthyroid state. The Mn and Cu/Zn superoxide dismutase activities were greater in the hyperthyroid than in the euthyroid state. Glutathione peroxidase activity decreased with age in the euthyroid and, particularly, in the hyperthyroid state. Catalase activity was not affected in the old rats. Concentrations of alpha-tocopherol in the old rats were high in the hyperthyroid state and low in the hypothyroid state, whereas the levels of beta- and gamma-tocopherols in these rats were unchanged in both conditions as compared with the euthyroid state findings. Data suggest that the site of free radical generation differs in older rats, with additional shifts in the location of intracellular lipid peroxidation being noted during hyperthyroidism. Thus, as rats age, the reduction of the free radical scavenger system and the increase in LPO and XOD activities might induce myocardial dysfunction.

Free access

T Mokuno, K Uchimura, R Hayashi, N Hayakawa, M Makino, M Nagata, H Kakizawa, Y Sawai, M Kotake, N Oda, A Nakai, A Nagasaka, and M Itoh

The deterioration of glucose metabolism frequently observed in hyperthyroidism may be due in part to increased gluconeogenesis in the liver and glucose efflux through hepatocyte plasma membranes. Glucose transporter 2 (GLUT 2), a facilitative glucose transporter localized to the liver and pancreas, may play a role in this distorted glucose metabolism. We examined changes in the levels of GLUT 2 in livers from rats with l-thyroxine-induced hyperthyroidism or methimazole-induced hypothyroidism by using Western blotting to detect GLUT 2. An oral glucose tolerance test revealed an oxyhyperglycemic curve (impaired glucose tolerance) in hyperthyroid rats (n=7) and a flattened curve in hypothyroid rats (n=7). GLUT 2 levels in hepatocyte plasma membranes were significantly increased in hyperthyroid rats and were not decreased in hypothyroid rats compared with euthyroid rats. The same results were obtained with a densitometric assay. These findings suggest that changes in the liver GLUT 2 concentration may contribute to abnormal glucose metabolism in thyroid disorders.