T lymphocytes play a critical role in cell-mediated immune responses. whereas

T lymphocytes play a critical role in cell-mediated immune responses. whereas p38 inhibition experienced a much smaller effect. Activation also induced hexokinase activity and manifestation in T cells, and both were similarly dependent on ERK signaling. Thus, the ERK signaling pathway cooperates with PI3K to induce glucose utilization in activated T cells, with hexokinase providing as a potential point for coordinated rules. Introduction T cells are dependent on external materials of glucose to maintain biosynthesis and energy metabolism during activation. Activated T cells adopt a metabolic state of aerobic glycolysis, in which glucose flux through glycolysis is usually high, but only a small proportion of the glucose is usually oxidized in mitochondria [1]C[5]. A comparable phenomenon was acknowledged in tumor cells more than 80 years ago [6], and was originally thought to symbolize a defect in mitochondrial function, perhaps as a result of mutations Rapgef5 that occurred during oncogenic change. However, more recent interpretations suggest that glycolysis is usually a favored metabolic pathway for highly proliferative cells, and the shift to a ZD4054 glycolytic phenotype is usually part of a larger adaptive metabolic program to support growth and proliferation ZD4054 [7]C[9]. Although there is usually growing appreciation for the importance of metabolic control in both ZD4054 immune responses and tumor development, the pathways that regulate glucose metabolism are still not well defined. Resting lymphocytes depend upon growth signals from cytokines and low-level T cell receptor (TCR) activation in order to maintain metabolic homeostasis [10], [11], whereas CD28 costimulation is usually required for induction of high level glucose uptake and glycolysis, in large part via activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway [12], [13]. The inhibitory receptors cytotoxic T lymphocyte antigen-4 (CTLA-4) and programmed death-1 (PD-1) both block CD28-induced Akt activation, and also prevent the increase in glucose utilization [12], [14], suggesting that rules of cellular metabolism might be a component of the inhibitory function of these receptors. Strikingly, overexpression of glucose transporter 1 (GLUT1), the major glucose transporter in hematopoietic cells [10], can partially replace costimulation in the induction of proliferation and cytokine production, and constitutively active Akt synergizes with GLUT1 overexpression [13]. Together, these findings indicate the importance of enhanced glucose utilization as a downstream effect of CD28 signaling. However, ligation of CD28 alone does not induce glucose metabolism [12]. Thus, TCR-initiated signaling pathways must cooperate with PI3K/Akt signaling to regulate glucose metabolism. Ligation of the TCR causes a variety of signaling cascades, several of which are candidates to regulate metabolism. Three key mitochondrial matrix enzymes, pyruvate dehydrogenase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase, are sensitive to calcium levels [15]. This suggests that the quick influx of calcium that occurs after TCR activation may regulate Krebs cycle activity, particularly given the recent evidence that calcium influx in T cells is usually linked to coordinated mitochondrial calcium uptake [16]. However, since most glucose metabolism in T cells does not utilize the Krebs cycle, it is usually likely that metabolic rules by calcium would be more important for option Krebs cycle substrates, such as glutamine [17]C[22]. The mitogen-activated protein kinase (MAPK) signaling pathways are also activated by TCR activation, and have been implicated in control of glucose metabolism in other cell types, particularly in enhancing glycolysis [23]C[25]. We therefore investigated the role of MAPK signaling in T cell glucose metabolism. We found that the enhanced glucose uptake and glycolysis seen in activated T cells is usually dependent on extracellular signal-regulated kinase (ERK) signaling, and that this may be due to the rules of hexokinase manifestation and activity. Results Activation of murine T cells prospects to enhanced glucose metabolism Studies with human peripheral blood T cells have shown that activation via mitogenic lectins or CD3/CD28 ligation prospects to an “aerobic glycolysis” phenotype, highly inducing glucose uptake and glycolysis [2], [12], [14], [26], [27]. In order to further characterize the rules of glucose metabolism in T lymphocytes, we made the decision to switch to the murine system. This would allow us to take advantage of the many genetic and biochemical tools available in the murine system, as well as the lower sample-to-sample variability offered by inbred mouse stresses. To confirm that glucose metabolism in murine T cells follows an induction pattern comparable to that seen in human T cells, we purified splenic T cells from C57BT/6 mice and stimulated them in vitro with anti-CD3 and anti-CD28 antibodies. After 24 hours of activation, glucose uptake by live T cells was assessed by the accumulation of radiolabeled 2-deoxyglucose, a non-metabolizable glucose analog, and glycolysis was assessed by the generation of 3H-labeled H2O from 3H-labeled glucose, at the step catalyzed by enolase. As shown in Physique 1, activated murine T cells increased.