In vivo stimulation of sugar uptake in rat thymocytes. An extranuclear action of 3,5,3'-triiodothyronine.

In previous studies we have demonstrated that 3,5,3'-triiodothyronine (T3) in vitro produces a prompt increase in the uptake of the sugar analogue 2-deoxyglucose (2-DG) by freshly isolated rat thymocytes. This effect is prompt, being evident at 20 min after addition of T3, is independent of new protein synthesis, and can be elicited by physiologic concentrations of the hormone. In the present studies, we have sought to determine whether physiologic doses of T3 are capable of inducing an increase in 2-DG uptake in the thymocytes of the living animal. Therefore, 26-28-d-old female rats were injected with increasing doses of i.v. T3, followed 60 min later by 3H-labeled 2-DG. 30 min later, animals were killed, thymocytes were isolated, and their 3H content determined. Uptake of [3H]2-DG was increased by T3 in a dose-dependent manner. The lowest effective dose was 10 ng/100 g of body weight (30% above control) and the maximally effective dose 1 microgram/100 g of body weight (116% above control). The effect of T3 was independent of new protein synthesis in that it was not blocked by a dose of cycloheximide that inhibited the incorporation of [3H]leucine into thymocyte protein by 92-95%. Comparable studies with various thyronine analogues revealed the following rank order of potency: L-T3 greater than L-3,5,3'5'-tetraiodothyronine (L-T4) greater than D-T3 greater than or equal to D-T4 greater than L-3,3'5'-triiodothyronine greater than 3'-isopropyl-3,5-L-diiodothyronine (T2) = 3,5-L-T2. DL-thyronine was without effect. These studies indicate that T3 in physiologic doses acts in vivo to increase the uptake of sugar by rat thymocytes by a mechanism that is extranuclear in origin, in that it is independent of new protein synthesis. The findings support the conclusion that the previously demonstrated effects of T3 on thymocyte sugar uptake in vitro, which seem clearly to be mediated at the level of the plasma membrane, have physiologic relevance

Properties and regulation of the thyrotropin receptor in the FRTL5 rat thyroid cell line.

Despite extensive use of FRTL5 cells in studies of responses to TSH and anti-TSH receptor antibodies, almost nothing is known of the properties of their TSH receptors, possibly because binding of TSH by these cells is negligible when studied in their usual culture medium. In the present studies, we have demonstrated that specific binding of TSH can readily be demonstrated in confluent monolayers of FRTL5 cells if their culture medium is replaced by Krebs-Ringer bicarbonate (KRB) buffer. In keeping with previous observations concerning the effects of cations on the binding of TSH in other thyroid systems, binding of TSH to FRTL5 was far greater when the medium used was a modified KRB in which an isosmotic substitution of sucrose for NaCl had been made. Kinetic studies of TSH binding in both types of medium suggested the presence of two binding sites, one with a higher affinity and lower maximum binding capacity than the other. The influence of NaCl was to decrease the capacity of both sites, that of the low affinity site to a greater extent than that of the high affinity site, whereas the affinities of the two sites remained unchanged. Correlative studies indicated that physiological responses to TSH were associated mainly with occupancy of the higher affinity sites. Experiments in which TSH binding was studied in cells grown to confluence in the presence of TSH from which TSH was then withdrawn and in cells maintained in the absence of TSH to which TSH was then added demonstrated the occurrence of up-and down-regulation, respectively, of receptor concentrations without a change in their affinities. The reduction in maximum binding capacity induced by TSH was proportionately greater in the case of the high affinity than the low affinity receptor. Down-regulation by TSH was concentration dependent and was demonstrable at a TSH concentration of 10(-11) M, considered to be physiological. Further, maximum down-regulation was induced by 10(-9) M TSH, the approximate concentration at which other responses to TSH in these cells reach their peak. Therefore, down-regulation of TSH receptors can be considered to be one of the physiological responses that TSH elicits

Observations on the factors that control the generation of triiodothyronine from thyroxine in rat liver and the nature of the defect induced by fasting.

Studies were performed to explore the mechanism underlying the impaired generation of 125-I-3,5,3'-triiodothyronine (T3) from 125I-thyroxine (T4) (T3-neogenesis)) in preparations of liver from rats fasted for 48 h and the prevention of this effect by the feeding of glucose. T3-neogenesis in livers from fasted animals and those fed chow or glucose was assessed in various mixtures of crude microsomal fractions with either buffer or cytosols. T3-neogenesis was mediated by an enzyme present in the microsomal fraction whose activity was enhanced by cytosolic cofactor(s). In livers from animals fasted for 48 h, the supporting activity of cytosol was decreased, whereas the activity of the enzyme was unaffected. Administration of glucose as the sole nutritional source prevented the decrease in the supporting activity of hepatic cytosol that was regularly observed in the case of animals totally deprived of food. The diminished supporting activity for T3-neogenesis provided by liver cytosol from fasted animals was restored to normal by enrichment with either NADPH or GSH, but the two cofactors appeared to act at different loci. GSH stimulated T3-neogenesis in microsomes incubated in the absence of cytosol, i.e., in buffer, whereas NADPH did not. The stimulatory effect of both agents was blocked by the sulfhydryl oxidant, diamide, which also inhibited T3-neogenesis in mixtures of microsomes with cytosols. Taken together, these observations suggest that GSH acts directly on the enzyme in the crude microsomal fraction, whereas NADPH acts within the cytosol, possibly by increasing the concentration of GSH through the action of the enzyme glutathione reductase, for which NADPH is a cofactor. In this light, the decreased supporting activity of hepatic cytosol from starved animals appears to reflect, at least partly, a decreased concentration of one or both cofactors. The direct stimulation of enzyme activity by GSH, and the apparent lack of inhibition of unstimulated activity by diamide, suggests that the 5'-monodeiodinase for thyroxine that mediates T3-neogenesis may be a GSH transhydrogenase