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F Ishihara, T Aizawa, N Taguchi, Y Sato and K Hashizume

Abstract

Insulin release, glucose utilization (3H2O formation from [5-3H]glucose), and glucose oxidation (14CO2 formation from [4C(U)] glucose) were determined in pancreatic islets from 96-h fasted rats at 37 ° C and those from fed rats at 22 ° C, using the islets from fed rats incubated at 37 ° C as controls. In the islets from 96-h fasted rats and those from fed rats incubated at 22 ° C, we could not demonstrate significant insulin release in response to high glucose concentrations of up to 16·7 mmol/l. However, 16·7 mmol/l glucose clearly augmented insulin release caused by a depolarizing concentration (50 mmol/l) of K+ in these islets: i.e. 16·7 mmol/l glucose plus 50 mmol/l K+ produced significantly greater insulin release than 50 mmol/l K alone. Glucose utilization and oxidation by the islet cells were suppressed by 96-h fasting of the rats or by lowering the incubation temperature to 22 ° C, and depolarization with K at 50 mmol/l did not at all augment glucose utilization and oxidation by the islets. Thus we conclude that reduction of glucose metabohsm in islets from fasted rats and in those incubated at low temperature eliminated initiation, but not augmentation, of insulin release by 16·7 mmol/l glucose. The data indicate that the metabolic threshold for the initiation of insulin release is significantly higher than it is for the augmentation of release by glucose.

Journal of Endocrinology (1994) 143, 497–503

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A. Sakurai, K. Ichikawa, K. Hashizume, T. Miyamoto, K. Yamauchi, H. Ohtsuka, Y. Nishii and T. Yamada

ABSTRACT

The effects of histone subfractions on rat liver thyroid hormone receptor–DNA interaction were examined using an in-vitro DNA-cellulose binding assay. H1 histones bound to DNA showed reversible and potent inhibition of receptor–DNA binding without affecting receptor–hormone binding. Poly-lysine, bovine serum albumin, ovalbumin and cytochrome c did not alter receptor–DNA binding. H1 histone subfractions (calf thymus lysine-rich histone (CTL)-1, CTL-2 and CTL-3) showed potent inhibition of receptor–DNA binding indistinguishable from each other. The quantity of H1 histone subfractions bound to DNA was the same. Although each subfraction has different functional properties, inhibition of receptor–DNA binding was a common feature of all the H1 histone subfractions, which is important for the non-random distribution of the receptor in chromatin.

Binding of the receptor to core histones was investigated; it was found to bind to core histones more potently than to other proteins (H1 histone, ovalbumin and cytochrome c). Among core histone subfractions, H4 histone bound to the receptor most potently and is the candidate to be one of the acceptor sites of the receptor in chromatin.

Journal of Endocrinology (1989) 121, 337–341

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K. Ichikawa, K. Hashizume, T. Miyamoto, Y. Nishii, K. Yamauchi, H. Ohtsuka and T. Yamada

ABSTRACT

An aqueous two-phase partitioning study of partially purified nuclear thyroid hormone receptor from rat liver was performed. Stability of 3,5,3′-tri-iodo-l-thyronine (T3)–receptor complex and T3-binding activity in the presence of dextran or polyethylene glycol were assessed in order to determine the amount of occupied or unoccupied receptors in each phase. Partition coefficients were calculated as the ratio of receptor concentration in the upper polyethylene glycol-rich phase H2O and that in the lower dextranrich phase H2O. The partition coefficient was a sensitive function of the salt at pH above 6·1 and below 5·1. The salt had no effect on the partition coefficient at pH around 5·6. These results suggest that the isoelectric point of the thyroid hormone receptor is about 5·6, confirming previous determinations using isoelectric focusing. The partition coefficient of the receptor decreased upon T3 binding, regardless of the salt composition. In contrast, the partition coefficient of thyroxine-binding globulin increased upon T3 binding. Free T3 preferentially partitioned into the upper polyethylene glycol-rich phase and gave a partition coefficient higher than 1·0. These results strongly suggest that the decrease in the partition coefficient of the receptor upon hormone binding reflects conformational changes or changes in electrostatic properties of the receptor upon hormone binding. Such an alteration may be involved in biological activation of the receptor upon hormone binding.

J. Endocr. (1988) 119, 431–437

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K. Ichikawa, J. Brtko, L. J. DeGroot, K. Hashizume and T. Yamada

ABSTRACT

Rat liver nuclear thyroid hormone receptor lost 3,5,3′-tri-iodo-l-thyronine (T3)-binding activity with a half-life of 14 days, 4 h, 139 min, 62 min, 16 min or 6 min at 0, 36, 38, 40, 43 or 45 °C respectively, when present in crude nuclear extracts. Glycerol increased the half-life of the receptor during heat inactivation. Protection was reversible by removing the glycerol. The receptor was unstable at a pH below 6·0 or above 10·0. We also found a loss of the receptor activity during the separation of bound and free hormone using the resin test. Of several conditions tested for the separation of bound and free hormone, the addition of heated nuclear extract gave the most accurate estimation of bound hormone when using the resin test. Using these characteristics of the receptor, we purified the receptor to 1220 pmol T3-binding capacity/mg protein with a final yield of 14·6 μg/4 kg rat liver.

Journal of Endocrinology (1989) 120, 237–243

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T Nagasawa, K Ichikawa, K Minemura, M Hara, H Yajima, A Sakurai, H Kobayashi, K Hiramatsu, S Shigematsu and K Hashizume

Abstract

Cellular and nuclear uptake of tri-iodothyronine (T3) and thyroxine (T4) was examined using the cultured cell line derived from rat liver, clone 9, and rat hepatoma, dRLH-84. The saturable cellular uptake of T3 and T4 was demonstrated in these cells. First we examined the cell cycle-dependent alteration of thyroid hormone uptake. Cellular T3 uptake was minimal in the early G1 phase and increased in the late G1 phase, reaching a maximal level in the S phase. Alterations in nuclear T3 uptake were in accordance with the changes in cellular T3 uptake. On the other hand, cellular and nuclear T4 uptake was unchanged throughout the cell cycle, suggesting the T3 specificity of the cell cycle-dependent alteration of cellular hormone transport. Next we examined the effect of sodium butyrate on the cellular transport of thyroid hormones. After treatment with 5 mm sodium butyrate, cellular and nuclear uptake of T3 was increased, reaching a maximal level (four- to sevenfold increase) after 48 h. When cells were incubated for 48 h with various concentrations of sodium butyrate, T3 uptake was enhanced by 1 mm sodium butyrate, reaching a maximal level with 5 mm. Although cellular T4 uptake was also increased after treatment with sodium butyrate, the degree and time-course of the increase were different from those of T3. The maximal increase in cellular T4 uptake (two- to threefold increase) was attained 20 h after treatment. Despite the increase in cellular T4 uptake, nuclear T4 uptake was decreased after treatment with sodium butyrate. For both T3 and T4, the enhanced cellular uptake was due to the increased Vmax without changes in the Michaelis–Menten constant. These data indicate that cellular transport of T4 is different from that of T3 in rat hepatic cells.

Journal of Endocrinology (1995) 147, 479–485

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Y. Nishii, K. Hashizume, K. Ichikawa, T. Miyamoto, S. Suzuki, T. Takeda, K. Yamauchi, M. Kobayashi and T. Yamada

ABSTRACT

Changes in the amount of cytosolic 3,5,3′-tri-iodo-l-thyronine (T3)-binding protein (CTBP) and its activator during administration of l-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor.

These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor.

Journal of Endocrinology (1989) 123, 99–104

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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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S Yamada, M Komatsu, T Aizawa, Y Sato, H Yajima, T Yada, S Hashiguchi, K Yamauchi and K Hashizume

When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.

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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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T Takeda, K Ichikawa, M Kobayashi, T Miyamoto, S Suzuki, Y Nishii, A Sakurai, T Nagasawa, M Katai, K Nakajima and K Hashizume

Abstract

In order to study whether peripheral action of thyroid hormones is altered in insulin deficiency and to elucidate the biological consequences of alteration of the cytosolic 3,5,3′-tri-iodo-l-thyronine (T3) binding protein (CTBP), we measured malic enzyme, T3-responsive nuclear n protein, CTBP and nuclear thyroid hormone receptor in the liver and kidney of streptozotocin (STZ)-induced diabetic rats that were treated with or without insulin and/or a receptor-saturating dose of T3. The following results were obtained. 1. Induction of malic enzyme by T3 was apparently diminished in diabetic rats. However, supplementary injection of insulin enabled previously given T3 to take effect in diabetic rats. 2. T3-responsiveness of other hepatic proteins (n protein and CTBP) was not altered by insulin in diabetic rats. 3. The level of n protein was increased by insulin in diabetic rats in vivo and in perfused rat liver, indicating that the hepatic n protein is a novel insulin-responsive protein. T3 and insulin increased the level of n protein non-synergistically in diabetic rat liver. 4. Hepatic nuclear receptor levels were not altered in diabetic rats. 5. Hepatic CTBP levels were decreased in diabetic rats. This was not due to the toxic effect of STZ. Low CTBP level was only partially increased by insulin after 30 days of diabetic period. Renal CTBP levels were not altered in diabetic rats with or without insulin treatment. These results indicate that reduction of CTBP did not influence the hepatic response to a receptor-saturating dose of T3, although CTBP may regulate the nuclear T3 transport, and that fundamental action of a receptor-saturating dose of T3 was not attenuated in diabetic rat liver.

Journal of Endocrinology (1994) 143, 55–63