Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate

M.‐Y., Maggie; Pusateri, Mary Ellen; Carter, Joyce G.; McDougal, David B.; Lowry, Oliver H.

doi:10.1016/0003-2697(87)90481-7

Cited by 35 publications

(19 citation statements)

References 4 publications

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“…The effect of glucose was mimicked by 2-deoxyglucose (Fig. 2a), a glucose analog which is phosphorylated by cytoplasmic hexokinase but cannot be metabolized further in the glycolytic pathway (15). The same effect was observed when glucose was combined with 2-deoxyglucose.…”

Section: Resultsmentioning

confidence: 74%

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H⁺-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Sautin

Gaugler

et al. 2005

Molecular and Cellular Biology

207

213

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Vacuolar Hϩ -ATPases (V-ATPases) are a family of electrogenic ATP-driven proton pumps which function in almost every eukaryotic cell. V-ATPases are required to maintain proton gradients between intracellular compartments and for proton secretion from the plasma membrane of certain specialized cells (reviewed in references 32, 42, and 43). In the intracellular compartments, V-ATPases acidify early and late endosomes, lysosomes, and Golgi-derived secretory vesicles, providing the motive force and optimal pH for internalization and dissociation of ligand-receptor complexes, pH-driven secretion, and activation of lysosomal enzymes for protein processing and degradation (43, 48). Plasma membrane VATPases are essential for acid secretion and bicarbonate transport in the proximal tubule and collecting duct of the kidney, pH homeostasis in macrophages and neutrophils, acidification of the extracellular environment by certain tumor cells, and K ϩ secretion in insect midgut cells, (24, 43). V-ATPase-dependent acidification of the extracellular compartment between the osteoclast ruffled membrane and the bone surface is crucial for bone remodeling (23).V-ATPase is a large, multisubunit protein. It is composed of at least 13 subunits with a total molecular mass of about 900 kDa and has two domains, the V 1 domain (catalytic, membrane-associated, 640 kDa) and V o domain (membrane spanning, 260 kDa). The V 1 domain is responsible primarily for ATP hydrolysis. It consists of eight different subunits (A to H) in a stoichiometry of A 3 B 3 CDEFG 2 H 1-2 . V 1 attaches to the proton-translocating V o domain, which is composed of five subunits (a, b, c, cЈ, and cЉ) in a stoichiometry of abcЈcЉc 4 (20, 32, 43). Regulation of V-ATPase function in response to physiological stimuli is thought to be a multilevel process. It includes control of the expression of V-ATPase subunit genes (34, 62), intracellular targeting and translocation from vesicles to the plasma membrane (2, 9, 10), and reversible dissociation of the V o and V 1 domains, entailing inactivation of the pump (30,31,55).Several extrinsic regulatory factors have been reported to control V-ATPase-mediated proton transport. They include bicarbonate concentration and the closely related parameters pH and pCO 2 , mineralocorticoid hormones, endothelin, angiotensin II (reviewed in references 23 and 24), and cytokines, such as interleukin-1 (IL-1) (7) and IL-4 and -13 (59). The underlying intracellular signaling mechanisms remain largely unknown. Involvement of G proteins and protein kinases A and C has been implicated in several studies (23,24). The E subunit of V-ATPase is able to interact directly with the Dbl homology domain of the guanine nucleotide exchange factor

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Section: Resultsmentioning

confidence: 74%

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H⁺-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Sautin

Gaugler

et al. 2005

Molecular and Cellular Biology

207

213

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“…5-thio-glucose is a competitive inhibitor of hexokinase, it cannot be phosphorylated and therefore cannot be effectively used as a hexokinase substrate (Wilson and Chung 1989). In contrast, 2-deoxy-glucose can be phosphorylated but not further metabolized (Chi et al 1987).…”

Section: The First Committed Step Of Glycolysis Is Sufficient For Aktmentioning

confidence: 96%

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Gottlob¹,

Majewski²,

Kennedy³

et al. 2001

Genes Dev.

866

704

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The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival. Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release. However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism. Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism. We provide evidence that such coupling serves as a downstream effector function for Akt. First, Akt increases mitochondria-associated hexokinase activity. Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase. Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis. We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane. Extrinsic signals emanating from cell-surface growth factor and cytokine receptors are major determinants of mammalian cell survival. The transduction of these signals oppose both basal intrinsic proapoptotic activity, as well as external proapoptotic stimuli (Raff 1992;Raff et al. 1993). Analysis of downstream signaling pathways has shown that activation of the PI-3 kinase/Akt(PKB) pathway plays a major role in cell survival induced by cell surface receptors. Following the initial demonstration that activation of the serine/threonine kinase Akt promotes cell survival (Dudek et al. 1997; KauffmannZeh et al. 1997;Kennedy et al. 1997), mounting reports established Akt as a major determinant of cell survival. Akt has been reported to mediate cell survival by various growth factors and cytokines in a variety of cell types and blocks apoptosis induced by multiple apoptotic stimuli (for review, see Datta et al. 1999;Kandel and Hay 1999). Various specific targets of Akt have been proposed to mediate the antiapoptotic activity of Akt (for review, see Datta et al. 1999;Kandel and Hay 1999). However, because growth factors promote cell survival via maintenance of the metabolic function of mitochondria , it is likely that Akt exerts its effect through similar mechanisms, which may be more fundamental and generally conserved.In mammalian cells, apoptosis has been described as a multistep process that can be initiated by a variety of stimuli. Mitochondria play a major role in this process through the release of cytochrome c and other proapoptotic proteins that normally reside in the intermembrane space between the inner and outer mitochondrial membranes (for review, see Gross et al. 1999;Desagher and Martinou 2000). Cytochrome c release is considered an ea...

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“…G6P does not accumulate to high levels because it rapidly enters the glycolytic and pentose phosphate pathways. By contrast, 2-DG6P accumulates because it cannot be processed further by glycolysis and only enters the pentose phosphate pathway at a very reduced rate (8). As such, we suggest that under physiological signaling conditions MondoA senses G6P levels, accounting for the partial affect of glucose on MondoA nuclear localization.…”

mentioning

confidence: 88%

Glucose sensing by MondoA:Mlx complexes: A role for hexokinases and direct regulation of thioredoxin-interacting protein expression

Stoltzman

Peterson

Breen

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

247

387

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Glucose is a fundamental metabolite, yet how cells sense and respond to changes in extracellular glucose concentration is not completely understood. We recently reported that the MondoA: Mlx dimeric transcription factor directly regulates glycolysis. In this article, we consider whether MondoA:Mlx complexes have a broader role in sensing and responding to glucose status. In their latent state, MondoA:Mlx complexes localize to the outer mitochondrial membrane, yet shuttle between the mitochondria and the nucleus. We show that MondoA:Mlx complexes accumulate in the nucleus in response to glucose and 2-deoxyglucose (2-DG). Furthermore, nuclear localization of MondoA:Mlx depends on the enzymatic activity of hexokinases. These enzymes catalyze conversion of glucose to glucose-6-phosphate (G6P), which is the first step in the glycolytic pathway. Together, these findings suggest that MondoA:Mlx monitors intracellular G6P concentration and translocates to the nucleus when levels of this key metabolite increase. Transcriptional profiling experiments demonstrate that MondoA is required for >75% of the 2-DG-induced transcription signature. We identify thioredoxin-interacting protein (TXNIP) as a direct and glucose-regulated MondoA:Mlx transcriptional target. Furthermore, MondoA:Mlx complexes, via their regulation of TXNIP, are potent negative regulators of glucose uptake. These studies suggest a key role for MondoA:Mlx complexes in the adaptive transcriptional response to changes in extracellular glucose concentration and peripheral glucose uptake. metabolism ͉ mitochondria ͉ transcription

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Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate

Cited by 35 publications

References 4 publications

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H⁺-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H⁺-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Glucose sensing by MondoA:Mlx complexes: A role for hexokinases and direct regulation of thioredoxin-interacting protein expression

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Enzymatic assays for 2-deoxyglucose and 2-deoxyglucose 6-phosphate

Cited by 35 publications

References 4 publications

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase

Glucose sensing by MondoA:Mlx complexes: A role for hexokinases and direct regulation of thioredoxin-interacting protein expression

Contact Info

Product

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Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H⁺-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells

Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H⁺-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells