Glucose metabolism as a target of histone deacetylase inhibitors.

The therapeutic efficacy of histone deacetylase inhibitors (HDACI) is generally attributed to their ability to alter gene expression secondary to their effects on the acetylation status of transcription factors and histones. However, because HDACIs exhibit similar transcriptional effects in most cells, the molecular basis for their therapeutic selectivity toward malignant cells is largely unknown. In this study, we report that HDACI, of distinct chemotypes, quantitatively inhibit glucose transporter 1 (GLUT1)-mediated glucose transport into multiple myeloma cells through both down-regulation of GLUT1 and inhibition of hexokinase 1 (HXK1) enzymatic activity. Unexpectedly, however, this inhibition of glucose utilization is accompanied by an increase in amino acid catabolism with no increase in fatty acid oxidation. Our findings suggest that an HDACI-induced change in carbon source preference could contribute to the therapeutic efficacy of these drugs by creating a pattern of fuel utilization that is incompatible with rapid tumor growth and survival. Furthermore, these results, which implicate glucose metabolism as a target of HDACI, suggest that caution should be exercised in attributing effects of this class of drug to primary alterations in gene transcription.

Autophagy provides nutrients but can lead to Chop-dependent induction of Bim to sensitize growth factor-deprived cells to apoptosis.

Tissue homeostasis is controlled by the availability of growth factors, which sustain exogenous nutrient uptake and prevent apoptosis. Although autophagy can provide an alternate intracellular nutrient source to support essential basal metabolism of apoptosis-resistant growth factor-withdrawn cells, antiapoptotic Bcl-2 family proteins can suppress autophagy in some settings. Thus, the role of autophagy and interactions between autophagy and apoptosis in growth factor-withdrawn cells expressing Bcl-2 or Bcl-xL were unclear. Here we show autophagy was rapidly induced in hematopoietic cells upon growth factor withdrawal regardless of Bcl-2 or Bcl-xL expression and led to increased mitochondrial lipid oxidation. Deficiency in autophagy-essential gene expression, however, did not lead to metabolic catastrophe and rapid death of growth factor-deprived cells. Rather, inhibition of autophagy enhanced survival of cells with moderate Bcl-2 expression for greater than 1 wk, indicating that autophagy promoted cell death in this time frame. Cell death was not autophagic, but apoptotic, and relied on Chop-dependent induction of the proapoptotic Bcl-2 family protein Bim. Therefore, although ultimately important, autophagy-derived nutrients appear initially nonessential after growth factor withdrawal. Instead, autophagy promotes tissue homeostasis by sensitizing cells to apoptosis to ensure only the most apoptosis-resistant cells survive long-term using autophagy-derived nutrients when growth factor deprived.

An essential role for the Glut1 PDZ-binding motif in growth factor regulation of Glut1 degradation and trafficking.

Cell surface localization of the Glut (glucose transporter), Glut1, is a cytokine-controlled process essential to support the metabolism and survival of haemopoietic cells. Molecular mechanisms that regulate Glut1 trafficking, however, are not certain. In the present study, we show that a C-terminal PDZ-binding motif in Glut1 is critical to promote maximal cytokine-stimulated Glut1 cell surface localization and prevent Glut1 lysosomal degradation in the absence of growth factor. Disruption of this PDZ-binding sequence through deletion or point mutation sharply decreased surface Glut1 levels and led to rapid targeting of internalized Glut1 to lysosomes for proteolysis, particularly in growth factor-deprived cells. The PDZ-domain protein, GIPC (G(alpha)-interacting protein-interacting protein, C-terminus), bound to Glut1 in part via the Glut1 C-terminal PDZ-binding motif, and we found that GIPC deficiency decreased Glut1 surface levels and glucose uptake. Unlike the Glut1 degradation observed on mutation of the Glut1 PDZ-binding domain, however, GIPC deficiency resulted in accumulation of intracellular Glut1 in a pool distinct from the recycling pathway of the TfR (transferrin receptor). Blockade of Glut1 lysosomal targeting after growth factor withdrawal also led to intracellular accumulation of Glut1, a portion of which could be rapidly restored to the cell surface after growth factor stimulation. These results indicate that the C-terminal PDZ-binding motif of Glut1 plays a key role in growth factor regulation of glucose uptake by both allowing GIPC to promote Glut1 trafficking to the cell surface and protecting intracellular Glut1 from lysosomal degradation after growth factor withdrawal, thus allowing the potential for a rapid return of intracellular Glut1 to the cell surface on restimulation.

Mechanisms and methods in glucose metabolism and cell death.

Glucose metabolism represents a critical physiological program that not only provides energy to support cell proliferation, but also directly modulates signaling pathways of cell death. With the growing recognition of regulation of cell death by glucose metabolism, many techniques that can be applied in the study have been developed. This chapter discusses several protocols that aid in the analysis of glucose metabolism and cell death and the principles in practicing them under different conditions.

Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival.

Lymphocytes require glucose uptake and metabolism for normal survival and function. The signals that regulate the expression and localization of glucose transporter 1 (Glut1) to allow glucose uptake in T cells are now beginning to be understood. Resting T cells require extracellular signals, such as cytokines, hormones, and growth factors, or low-level TCR stimulation to take up adequate glucose to maintain housekeeping functions. In the absence of extrinsic signals, resting T cells internalize and degrade Glut1 and cannot maintain viability. Activated T cells have dramatically increased metabolic requirements to support the energy and biosynthetic needs necessary for growth, proliferation, and effector function. In particular, glucose metabolism and aerobic glycolysis fuel this demand. Therefore, activation of T cells causes a large increase in Glut1 expression and surface localization. If glucose uptake is limited, glycolytic flux decreases to a level that no longer sustains viability, and proapoptotic Bcl-2 family members become activated, promoting cell death. However, excessive glucose uptake can promote hyperactive immune responses and possible immune pathology. Tight regulation of glucose uptake is required to maintain immune homeostasis, and understanding of these metabolic pathways may lead to therapeutic strategies to target some forms of cancer or autoimmunity.

Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways.

T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active myristoylated Akt increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent up-regulation of Glut1 and Akt-dependent Glut1 cell surface trafficking. In support of this model, coexpression of Glut1 and myristoylated Akt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism for T cell activation and proliferation.

IL-7 promotes Glut1 trafficking and glucose uptake via STAT5-mediated activation of Akt to support T-cell survival.

Lymphocyte homeostasis requires coordination of metabolic processes with cellular energetic and biosynthetic demands but mechanisms that regulate T-cell metabolism are uncertain. We show that interleukin-7 (IL-7) is a key regulator of glucose uptake in T lymphocytes. To determine how IL-7 affects glucose uptake, we analyzed IL-7 signaling mechanisms and regulation of the glucose transporter, Glut1. The IL-7 receptor (IL-7R) stimulated glucose uptake and cell-surface localization of Glut1 in a manner that required IL-7R Y449, which promoted rapid signal transducer and activator of transcription 5 (STAT5) activation and a delayed yet sustained activation of Akt. Each pathway was necessary for IL-7 to promote glucose uptake, as Akt1(-/-) T cells or PI3-kinase inhibition and RNAi of STAT5 led to defective glucose uptake in response to IL-7. STAT5 and Akt acted in a linear pathway, with STAT5-mediated transcription leading to Akt activation, which was necessary for STAT5 and IL-7 to promote glucose uptake and prevent cell death. Importantly, IL-7 required glucose uptake to promote cell survival. These data demonstrate that IL-7 promotes glucose uptake via a novel signaling mechanism in which STAT5 transcriptional activity promotes Akt activation to regulate Glut1 trafficking and glucose uptake that is critical for IL-7 to prevent T-cell death and maintain homeostasis.

Glycogen synthase kinase 3alpha and 3beta mediate a glucose-sensitive antiapoptotic signaling pathway to stabilize Mcl-1.

Glucose uptake and utilization are growth factor-stimulated processes that are frequently upregulated in cancer cells and that correlate with enhanced cell survival. The mechanism of metabolic protection from apoptosis, however, has been unclear. Here we identify a novel signaling pathway initiated by glucose catabolism that inhibited apoptotic death of growth factor-deprived cells. We show that increased glucose metabolism protected cells against the proapoptotic Bcl-2 family protein Bim and attenuated degradation of the antiapoptotic Bcl-2 family protein Mcl-1. Maintenance of Mcl-1 was critical for this protection, as glucose metabolism failed to protect Mcl-1-deficient cells from apoptosis. Increased glucose metabolism stabilized Mcl-1 in both cell lines and primary lymphocytes via inhibitory phosphorylation of glycogen synthase kinase 3alpha and 3beta (GSK-3alpha/beta), which otherwise promoted Mcl-1 degradation. While a number of kinases can phosphorylate and inhibit GSK-3alpha/beta, we provide evidence that protein kinase C may be stimulated by glucose-induced alterations in diacylglycerol levels or distribution to phosphorylate GSK-3alpha/beta, maintain Mcl-1 levels, and inhibit cell death. These data provide a novel nutrient-sensitive mechanism linking glucose metabolism and Bcl-2 family proteins via GSK-3 that may promote survival of cells with high rates of glucose utilization, such as growth factor-stimulated or cancerous cells.

Cytokine stimulation promotes glucose uptake via phosphatidylinositol-3 kinase/Akt regulation of Glut1 activity and trafficking.

Cells require growth factors to support glucose metabolism for survival and growth. It is unclear, however, how noninsulin growth factors may regulate glucose uptake and glucose transporters. We show that the hematopoietic growth factor interleukin (IL)3, maintained the glucose transporter Glut1 on the cell surface and promoted Rab11a-dependent recycling of intracellular Glut1. IL3 required phosphatidylinositol-3 kinase activity to regulate Glut1 trafficking, and activated Akt was sufficient to maintain glucose uptake and surface Glut1 in the absence of IL3. To determine how Akt may regulate Glut1, we analyzed the role of Akt activation of mammalian target of rapamycin (mTOR)/regulatory associated protein of mTOR (RAPTOR) and inhibition of glycogen synthase kinase (GSK)3. Although Akt did not require mTOR/RAPTOR to maintain surface Glut1 levels, inhibition of mTOR/RAPTOR by rapamycin greatly diminished glucose uptake, suggesting Akt-stimulated mTOR/RAPTOR may promote Glut1 transporter activity. In contrast, inhibition of GSK3 did not affect Glut1 internalization but nevertheless maintained surface Glut1 levels in IL3-deprived cells, possibly via enhanced recycling of internalized Glut1. In addition, Akt attenuated Glut1 internalization through a GSK3-independent mechanism. These data demonstrate that intracellular trafficking of Glut1 is a regulated component of growth factor-stimulated glucose uptake and that Akt can promote Glut1 activity and recycling as well as prevent Glut1 internalization.

T cell homeostasis requires G protein-coupled receptor-mediated access to trophic signals that promote growth and inhibit chemotaxis.

Signals that regulate T cell homeostasis are not fully understood. G protein-coupled receptors (GPCR), such as the chemokine receptors, may affect homeostasis by direct signaling or by guiding T cell migration to distinct location-restricted signals. Here, we show that blockade of Galphai-associated GPCR signaling by treatment with pertussis toxin led to T cell atrophy and shortened life-span in T cell-replete hosts and prevented T cell homeostatic growth and proliferation in T cell-deficient hosts. In vitro, however, neither GPCR inhibition nor chemokine stimulation affected T cell atrophy, survival, or proliferation. These findings suggest that GPCR signals are not trophic stimuli, but instead may be required for migration to distinct trophic signals, such as IL-7 or self-peptide/MHC. Surprisingly, while chemokines did not affect atrophy, atrophic T cells displayed increased chemokine-induced chemotaxis that was prevented by IL-7 and submitogenic anti-CD3 antibody treatment. This increase in migration was associated with increased levels of GTP-bound Rac and the ability to remodel actin. These data suggest a novel mechanism of T cell homeostasis wherein GPCR may promote T cell migration to distinct location-restricted homeostatic trophic cues for T cell survival and growth. Homeostatic trophic signals, in turn, may suppress chemokine sensitivity and cytoskeletal remodeling, to inhibit further migration.