Guido G. Guidotti

Amino acid signaling through the mammalian target of rapamycin (mTOR) pathway: Role of glutamine and of cell shrinkage.

Mammalian target of rapamycin (mTOR) mediates a signaling pathway that couples amino acid availability to S6 kinase (S6K) activation, translational initiation and cell growth rate, participating to a versatile checkpoint that inspects the energy status of the cell. The pathway is activated by branched‐chain amino acids (BCAA), leucine being the most effective, whereas amino acid dearth and ATP shortage lead to its deactivation. Glutamine‐ or amino acid‐deprivation and hyperosmotic stress induce a fast cell shrinkage (with marked decrease of the intracellular water volume) associated to mTOR‐dependent S6K1 dephosphorylation. Using cultured Jurkat cells, we have measured the changes of cell content and intracellular concentration of ATP, of relevant amino acids (BCAA) and of ninhydrin‐positive substances (NPS, as measure of NH2‐bearing organic osmolytes) under conditions that deactivate (leucine‐deprivation, glutamine‐deprivation, amino acid withdrawal, sorbitol‐induced hyperosmotic stress) or reactivate a previously deactivated, mTOR‐S6K1 pathway. We have also assessed the mitochondrial function by measurements of mitochondrial transmembrane potential in cells subjected to hypertonic stress. Our results indicate that diverse control signals converge on the mTOR‐S6K1 signaling pathway. In the presence of adequate energy resources, the pathway senses the amino acid availability as inward transport of effective amino acids (as BCAA and especially leucine), but its activation occurs only in the presence of an extracellular amino acid complement, with glutamine as obligatory component, and does not tolerate decrements of cell water volume incapable of maintaining adequate intracellular physicochemical conditions.

Cell size reduction induced by inhibition of the mTOR/S6K-signaling pathway protects Jurkat cells from apoptosis

In Jurkat cells, the decreased cell growth rate associated with a long-lasting deactivation of the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K)-signaling pathway generates a cell population of progressively reduced cellular mass and size. When promoted by rapamycin as prototype inhibitor, the mTOR deactivation-dependent cell size reduction was associated with slowed, but not suppressed, proliferation. Small-size cells were significantly protected from apoptosis induced by Fas/Apo-1 death-receptor activation (as shown by reduced procaspase cleavage and decreased catalytic activity of relevant caspases) or by stress signals-dependent mitochondrial perturbation (as shown by reduced cleavage of caspase-2, lower dissipation of mitochondrial membrane potential and decreased release of cytochorome c and apoptosis-inducing factor from mitochondria). Protection faded when reactivation of the mTOR/S6K pathway promoted the cell recovery to normal size. These results suggest that cells induced to reduce their mass by the mTOR deactivation-dependent inhibition of cell growth become more resilient to lethal assaults by curbing the cells suicidal response.

Cell susceptibility to apoptosis by glutamine deprivation and rescue: survival and apoptotic death in cultured lymphoma-leukemia cell lines.

Human leukemia/lymphoma cells maintained in culture medium without provision of fresh nutrients lose viability and die by a process resembling apoptosis within a few days. Upon incubation in an FCS‐supplemented RPMI 1640 medium containing 2 mM L‐glutamine CEM, Namalwa, HL‐60 and U937 cells, seeded at initial densities of 0.2 to 1 × 106 cells/ml, ceased growing within 3–5 days and progressively entered an apoptotic pathway, as assessed by nucleosomal DNA fragmentation and morphology. Both the major energy‐source nutrients in the medium, glucose and glutamine, became rapidly exhausted during the incubation. Further studies were performed using CEM cells. Incubation in glutamine‐free or glucose‐free medium renewed every 24 h showed that glutamine deprivation is associated with cell death by apoptosis independent of energetic failure, whereas glucose deprivation is followed by rapid loss of mitochondrial function with sharp drop of intracellular ATP and cell death by necrosis. A 12–24 h incubation in glutamine‐depleted medium was required to direct the cells toward the apoptotic pathway. Growth arrest followed by apoptotic death was detected in CEM cells when medium glutamine concentration remained below 0.3–0.4 mM for at least 24 h, but a reinstatement of medium glutamine to 2 mM within this period rescued the cells from growth arrest and death.

Amino Acid Transporters: Systematic Approach and Principles of Control

All living cells, from autonomous protists to constrained components of the complex multicellular mammalian organisms, require amino acids for replacing their structure, synthesizing essential proteins and supplying sources of metabolic energy. Transport of these molecules across the membranes that fix the boundaries of the cell and generate intracellular discontinuities is one of the determinants of cell survival.

Regulation of amino acid transport in chick embryo heart cells. IV. Site and mechanism of insulin action

Abstract The regulation of amino transport by insulin in chick embryo heart cells has been studied. Experiments were designed to identify the system(s) of mediation involved in this regulation and to investigate the mechanisms of the hormone action. Results and conclusions based on the adopted experimental approach include the following: 1. 1. Among the four systems (A, ASC, L, Ly+) found to be operative in cardiac cells for the transport of neutral and basic amino acids (Gazzola, G. C., Franchi-Gazzola, R., Ronchi, P. and Guidotti, G. G. (1973) Biochim. Biophys. Acta 311, 292–301), only the A mediation is responsive to insulin. 2. 2. The hormone enhances the activity of this mediation by increasing the maximal velocity ( V ) of transport (without substantial changes in K m ) even under conditions of inhibited protein synthesis. 3. 3. The rate of degradation of protein components of the A mediation (as estimated by measurements of transport activity under conditions of inhibited protein synthesis) is decreased when cells are incubated in the presence of insulin. The hormone does not affect this rate for systems ASC, L and Ly+. 4. 4. Cells previously incubated in a medium containing cycloheximide (phase of inhibited translation) subsequently exhibit a net increase of transport activity (A mediation) when transferred into a medium containing actinomycin D (phase of inhibited transcription). The presence of insulin during pre-incubation in cycloheximide has no effect on the subsequent rate of change in transport activity; when added to the second phase, the hormone definitely enchances transport activity. 5. 5. These observations have been interpreted to indicate that insulin modulates the activity of the A transport system by acting at two different sites; at the cell membrane by protecting specific transport proteins against degradation and at a post-transcriptional level by increasing the rate of synthesis of these transport proteins.

Role of Amino Acid Transport System A in the Control of Cell Volume in Cultured Human Fibroblasts

Human fibroblasts shrink and are unable to recover their initial volume when incubated in hypertonic saline solutions, whereas an efficient volume restoration takes place in hypertonic media containin

The transport of l-glutamine into cultured human fibroblasts

The transport of L-glutamine has been studied in diploid human fibroblasts in culture. Mathematical discrimination by nonlinear regression, competition analysis, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of this amino acid. The adopted criteria showed that L-glutamine enters the fibroblast by the Na(+)-dependent systems ASC and A and by a Na(+)-independent route identified as system L. The relative contribution of these agencies to the total saturable uptake of glutamine varied with the concentration of the amino acid and with the nutritional state of the cell. At amino acid concentrations approaching those encountered in human plasma: (1) system ASC represented the primary mediation for entry of L-glutamine in human fibroblasts; (2) the contribution of system A was lower, though significant, in unstarved repressed cells and became predominant in starved derepressed cells; (3) the Na(+)-dependent system L accounted for less than one-fifth of glutamine uptake in either nutritional condition. The changes in the relative contribution of the various systems to the uptake of glutamine as a function of its concentration may have implications in pathophysiology under conditions associated with enhanced glutamine concentrations in the extracellular fluids.

Effect of extracellular potassium on amino acid transport and membrane potential in fetal human fibroblasts

The distribution ratio of the lipophilic cation tetraphenylphosphonium (TPP+) has been used to estimate the electrical potential difference across the plasma membrane in cultured human fibroblasts. These cells exhibit a membrane potential markedly influenced by the diffusion potential of K+. High extracellular potassium concentrations depolarize human fibroblasts and depress the activity of transport systems A, ASC (both serving for zwitterionic amino acids), X-AG (for anionic amino acids), and y+ (for cationic amino acids). High doses (100 microM) of the K+-ionophore valinomycin hyperpolarize the cells. This condition enhances the activity of systems A, ASC and y+. Transport systems L (for neutral amino acids) and x-C (for anionic amino acids) are insensitive to changes in extracellular K+ or to valinomycin. System X-AG is inhibited by the addition of 100 microM valinomycin, but the effect of the ionophore appears to be potential-independent. These results indicate that: (a) the activity of systems L and x-C is potential-independent and (b) the activity of systems A, ASC, X-AG and y+ is sensitive to alterations of external [K+] associated to changes in membrane potential.

Effect of insulin on the activity of amino acid transport systems in cultured human fibroblasts

The regulation of amino acid transport by insulin has been studied in cultured human fibroblasts. Among the six amino acid transport systems operating in cultured human fibroblasts, two systems (A and X-C) are strongly stimulated by insulin and four (ASC, X-AG, y+ and L) are essentially not sensitive to the presence of the hormone in the incubation medium. The hormonal stimulation of system A and system X-C became significant after 3 h of incubation and increased up to 12 h. The stimulatory effect was related to insulin concentration, with a half-maximal stimulation at 10(-9) M hormone concentration. Insulin enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in Km values.

Phorbol esters stimulate the transport of anionic amino acids in cultured human fibroblasts.

The effect of phorbol esters on the transport of amino acids has been evaluated in cultured human fibroblasts. The activity of the Na(+)-dependent system XAG- for anionic amino acids is selectively and markedly stimulated by phorbol esters. The effect is maximal within 15 min; it is attributable to an increase in transport maximum (Vmax) and not prevented by inhibitors of protein synthesis. The half-maximal stimulation is observed at concentrations of phorbol 12,13-dibutyrate lower than 100 nM. Prolonged incubations in the presence of 1 microM phorbol 12,13-dibutyrate lower the binding of the ligand to its receptor with a loss of the stimulatory effect on transport. The results presented indicate that the stimulation of amino acid transport through system XAG- by phorbol esters requires the activation of protein kinase C.