Human CD4(+)CD25(hi)Foxp3(+)CD127(-) Treg and CD4(+)CD25(-)Foxp3(-) Tconv cell functions are governed by their metabolic requirements. Here we report a comprehensive comparative analysis between ex vivo human Treg and Tconv cells that comprises analyses of the proteomic networks in subcellular compartments. We identified a dominant proteomic signature at the metabolic level that primarily impacted the highly-tuned balance between glucose and fatty-acid oxidation in the two cell types. Ex vivo Treg cells were highly glycolytic while Tconv cells used predominantly fatty-acid oxidation (FAO). When cultured in vitro, Treg cells engaged both glycolysis and FAO to proliferate, while Tconv cell proliferation mainly relied on glucose metabolism. Our unbiased proteomic analysis provides a molecular picture of the impact of metabolism on ex vivo human Treg versus Tconv cell functions that might be relevant for therapeutic manipulations of these cells.

The present studies were undertaken to examine the factors that influence the growth of cells of endocrine gland origin, particularly the possible interactions between "nonspecific" growth factors and the trophic hormone for a target endocrine cell. As a model system, we explored the individual and conjoint effects of insulin-like growth factor-I (IGF-I) and TSH on the growth of FRTL5 cells, a nontransformed line of cloned rat thyroid follicular epithelium. In these cells, IGF-I and TSH each produced a dose-dependent enhancement of DNA synthesis and cell proliferation. When added together, IGF-I and TSH were markedly synergistic in stimulating DNA synthesis, producing increases in 3H-thymidine incorporation that were far greater than the sum of the effects of each alone. A similar effect of IGF-I was evident in the case of the stimulation of DNA synthesis produced by immunoglobulin G (IgG) preparations from the blood of patients with Graves' disease. Such IgG bind to the TSH receptor and mimic the actions of TSH therein. It is suggested, therefore, that there exist in the FRTL5 cell line at least two mechanisms for the regulation of growth, one activated at the level of the IGF-I receptor and the other at the level of the TSH receptor. When the two pathways are activated concurrently, a synergistic enhancement of DNA synthesis takes place. The findings indicate that the FRTL5 cell line is an excellent model in which to study these complex interactions and that IGF-I may be a determinant of thyroid cell growth, both normally and in certain thyroid diseases.

Using a clonal line of TSH-dependent functional rat thyroid cells, the effects of TSH on cAMP production and cell growth, as measured by cell number and [¹⁴C]thymidine incorporation, were compared. The withdrawal of TSH from the medium stopped the growth of cells, as measured by cell number. After 24 h in TSH-free medium, an acute challenge with 1 × 10^-11 M pure TSH caused a 40-fold increase in the cAMP level; this response increased to greater than 80-fold as TSH withdrawal from the culture system was prolonged over 10 days. In contrast to these acute effects on cAMP levels, an increase in [¹⁴C]thymidine incorporation required 20-fold higher TSH concentrations and waned to an unresponsive level after 10 days of TSH withdrawal. After withdrawal of TSH from the medium for 10 days, readdition of TSH could return cell growth to normal rates only after a 2-day lag period. In terms of the lag, the TSH effect on cell number was paralleled by its effect on thymidine incorporation, not cAMP levels; (Bu)₂-cAMP at concentrations as high as 1 × 10^-3 M could not duplicate the effect of TSH on either cell number or thymidine incorporation. The effect of TSH on thymidine incorporation was blocked by anti-TSH, but not by an antibody to contaminant proteins in crude TSH preparations. Crude TSH could increase thymidine incorporation, but was nearly 1/100th less effective based on its bioactivity measurements, i.e. contained an inhibitor in addition to an activator of thymidine incorporation. The inhibitor activity affected the cAMP response in a parallel manner. These observations are consistent with separate bioeffects of TSH mediated through different domains of the TSH receptor or different transmission pathways. The data suggest that discriminant separation of these bioeffects in primary cultures of human thyroid cells, wherein TSH challenge augments cAMP production but fails to elevate [¹⁴C]thymidine incorporation, does not reflect the absence of a TSH mitogenic activity but, rather, demonstrates a condition wherein the TSH receptor is uncoupled from the message transmission pathway important for cell growth. The data also suggest that the TSH effect on cell growth requires an action beyond that of activating the adenylate cyclase system

In previous studies we have demonstrated that bovine TSH (bTSH) and insulin-like growth factor I (IGF-I) independently stimulate both the incorporation of [3H]thymidine into DNA and replication in quiescent FRTL5 cells. In the case of TSH, evidence was presented that these responses are cAMP mediated. In addition, responses of thymidine incorporation are greatly amplified when particular concentrations of the two agents are added together, but this effect diminishes as the concentration of either bTSH or IGF-I is increased. The present experiments were undertaken to obtain further information concerning the mechanism of the independent mitogenic effects of bTSH and IGF-I and to explore the nature of the biphasic synergistic interaction with respect to thymidine incorporation that occurs when bTSH and IGF-I are added together. Verification that the increases in [3H] thymidine incorporation induced by bTSH and IGF-I, alone and together, are truly reflective of increases in DNA synthesis was obtained in experiments in which labeled nuclei were counted in cultures of cells grown in the presence of one or both mitogenic agents to which [3H]thymidine had been added. In these studies the number of cells with labeled nuclei was increased markedly by each of the two agents, and the response when the two mitogens were added together was far greater than the sum of their individual effects. Over a range of concentrations which included those that elicit a mitogenic response in FRTL5 cells, IGF-I, unlike bTSH, failed to increase cAMP generation when added alone. Moreover, IGF-I did not significantly enhance the cAMP response to varying concentrations of bTSH. A concentration-dependent increase in the incorporation of [3H]thymidine into DNA was induced by culturing cells in the presence of the cAMP analog (Bu)2cAMP (Bt2cAMP), the phosphodiesterase inhibitor isobutylmethylxanthine, and the stimulator of adenylate cyclase forskolin. When increasing concentrations of these agents were added together with IGF-I, a biphasic pattern of response of DNA synthesis, mimicking that produced by the combination of IGF-I and increasing concentrations of bTSH, was observed. Further evidence that cAMP mediates the mitogenic response to bTSH was the observation that adenosine inhibited the stimulation of both cAMP generation and DNA synthesis that bTSH produced. Although preincubation of quiescent FRTL5 cells for 24 h in the presence of bTSH resulted in only a small increase in DNA synthesis, measured during the last 3 h of a subsequent 24-h incubation carried out in the absence of bTSH, it greatly amplified the response to IGF-I added alone during the second incubation.(ABSTRACT TRUNCATED AT 400 WORDS)