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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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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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T Aizawa, T Kaneko, H Yajima, S Yamada, Y Sato, Y Kanda, S Kanda, M Noda, T Kadowaki, M Nagai, K Yamauchi, M Komatsu and K Hashizume

Oscillation of insulin release by the pancreatic islets was evaluated under stringent Ca(2+)-free conditions for the first time. Isolated single rat islets were exposed to 16.7 mM glucose in the presence of 1.9 mM Ca(2+), or under the stringent Ca(2+)-free conditions (Ca(2+) omission with 1 mM EGTA, 6 microM forskolin and 100 nM phorbol 12-myristate 13-acetate). Fifteen minutes after the initiation of glucose stimulation, effluent was collected at a 6-s interval, insulin was determined in duplicate by a highly sensitive insulin radioimmunoassay, and oscillation and pulsatility of release statistically analyzed. Significant oscillation of insulin release was observed in all islets irrespective of presence and absence of Ca(2+). Significant pulsatility of release was detected in 7 of 11 islets in the presence of Ca(2+) and three of six isl! ets in the absence of Ca(2+). In conclusion, high glucose elicits oscillatory insulin release both in the presence and absence of extracellular Ca(2+).