Presence Of 2DG Research Articles

Inhibiting Glucose Metabolism Results in Herpes Simplex Encephalitis.

This report evaluates how HSV enters the brain to cause herpes simplex encephalitis following infection at a peripheral site. We demonstrate that encephalitis regularly occurred when BALB/c mice were infected with HSV and treated daily with 2-deoxy-d-glucose (2DG), which inhibits glucose use via the glycolysis pathway. The outcome of infection in the trigeminal ganglion (TG), the site to which the virus spreads, replicates, and establishes latency, showed marked differences in viral and cellular events between treated and untreated animals. In control-untreated mice, the replicating virus was present only during early time points, whereas in 2DG recipients, replicating virus remained for the 9-d observation period. This outcome correlated with significantly reduced numbers of innate inflammatory cells as well as T cells in 2DG-treated animals. Moreover, T cells in the TG of treated animals were less activated and contained a smaller fraction of expressed IFN-γ production compared with untreated controls. The breakdown of latency was accelerated when cultures of TG cells taken from mice with established HSV latency were cultured in the presence of 2DG. Taken together, the results of both in vivo and in vitro investigations demonstrate that the overall effects of 2DG therapy impaired the protective effects of one or more inflammatory cell types in the TG that normally function to control productive infection and prevent spread of virus to the brain.

Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia.

SummaryThere is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells.

Abstract 3801: Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells

Abstract Breast cancer stem cells (BCSCs) have been shown to be associated with tumor recurrence, metastasis and drug resistance. We reported that the ganglioside GD2 selectively identifies BCSCs and its over-expression in Triple-Negative Breast Cancer (TNBC). However, the factors regulating GD2+ BCSCs in a growing tumor are not known. Here we hypothesize that nutrient deprivation-induced oxidative stress upregulates GD2+ BCSCs in TNBC. TNBC cell lines including SUM159 and MDA-MB-231 were cultured under different nutrient-deprived conditions and analyzed for GD2 expression by flow cytometry. We have identified a steady increase of GD2+ cells from 5%±2% on day 3 to 20%±5% on day 6 in cells cultured in nutrient deprived condition. We further performed the in vivo by injecting GFP+ MDA-MB-231 cells into NSG mice and measured GD2+ expression in the growing tumor once a week. Interestingly, we also noticed a positive correlation between the percentage of GD2+ cells and tumor volume in vivo with r=0.9829 and p<0.0004 suggesting nutrient deprived conditions in the growing tumors results in higher GD2 expression. Next, the cells were cultured in medium with or without glucose or media containing 2-deoxy glucose (2DG, glycolysis inhibitor). We found that the percentage of GD2+ cells increased 2-fold in the absence of glucose or in the presence of 2DG (10mM) compared to cells cultured with glucose (6g/L) or without 2DG, suggesting that glucose deprivation enhances GD2 biosynthesis in TNBC cells. Global metabolomics profiling using liquid chromatography-mass spectrometry followed by KEGG pathway analysis identified signatures such as glutathione mediated detoxification and glutathione biosynthesis to be most highly upregulated in GD2+ compared to GD2- cells. As glutamine is a key precursor for glutathione biosynthesis, we cultured SUM159 and MDA-MB-231 cells in media with or without glutamine and found that the percentage of GD2+ cells positively correlates with glutamine concentration in the medium. Next, to target glutamine metabolism, we treated TNBC cells with glutaminase inhibitor, CB-839 or glutamine transporter inhibitor, V9302. This resulted in a 70-80% reduction of GD2 expression in a dose dependent manner. CB-839 and V9302 also inhibited mammosphere formation by 40-50% and increased reactive oxygen species (ROS) in a dose-dependent manner in TNBC cells. In conclusion, oxidative stress results in GD2+ BCSC phenotype in TNBC cells. Inhibition of glutamine uptake or its utilization by V9302 or CB-839 inhibits GD2+ BCSC phenotype and alters redox homeostasis in BCSCs by inducing ROS levels. Targeting glutamine transporter or glutaminase could complement conventional chemotherapy by targeting BCSCs in TNBC. Citation Format: Appalaraju Jaggupilli, Stanely J. Ly, Roshan Borkar, Khoa Nguyen, Bin Yuan, Nagireddy Putluri, Michael Andreeff, V. Lokesh Battula. Oxidative stress induces glutamine-dependent GD2+ triple negative breast cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3801.

Abstract 4367: Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1

Abstract Objective: Pancreatic cancer (PanCa) is the third most common cause of cancer-related deaths in the US. Protein Kinase D1 (PKD1) is a kinase molecule which is involved in various important cellular signaling processes. While PKD1 has been reported to play a role in PanCa progression, the molecular mechanisms involved have not been adequately studied. Herein, we investigate the underlying mechanisms which enable PKD1 in enhancing glucose metabolism and further contribute to PanCa aggressiveness. Methods: PanCa cells with high PKD1 expressing (Panc-1 and AsPC-1) and low PKD1 (HPAF-II and BXPC-3) were used in the study. Immunohistochemistry and confocal immunofluorescence were performed to analyze the expression of PKD1 in pancreatic cancer/normal tissues and cells, respectively. The effect of PKD1 gain/loss-in function was investigated on glucose uptake and lactate production in PanCa cells using commercially available kits. Western blotting and real-time PCR experiments were performed to analyze the expression of protein and mRNA levels. MTT assay was conducted to access the influence of PKD1 on cell proliferation. Boyden chamber migration assay and Matrigel invasion assays were performed to determine the migratory and invasive abilities of PanCa cells. The gene silencing was performed using specific siRNAs in the study. Results: Our results demonstrate that PKD1 upregulates glucose metabolism in PanCa cells as indicated by enhanced glucose consumption and lactate production. The invasive characteristics of PKD1 expressing cells are augmented in presence of L-Lactate and reduced in presence of 2DG. PKD1 overexpression demonstrated enhanced phosphorylation of mTOR (ser-2448), 4EBP1, s6kinase and AKT, suggesting the role of PKD1 in the activation of mTOR pathway. We further observed that the phosphorylation of mTOR (ser-2448) and ps6kinase was attenuated on silencing PKD1 or in presence of Rapamycin, suggesting the role of PKD1 in activation of mTORC1 complex, both being the main effectors of mTORC1. Additionally, the inhibition in the phosphorylation of mTOR (ser-2448) in presence of kinase dead PKD1 suggests that PKD1 phosphorylates/activates mTORC1 in PanCa. Decrease in glucose uptake and lactate production in presence of PKD1 overexpression was observed on silencing raptor but not rictor in the cells, further suggesting the involvement of mTORC1 in PKD1 induced metabolic reprogramming in PanCa. PKD1 induced enhanced phosphorylation of mTOR resulted in an activation of HIF-1α and Glut-1 proteins, involved in abberant glucose metabolism. Additionally, our results also demonstrate that altered glucose metabolism leads to chemoresistance and silencing of PKD1 expression sensitizes PanCa cells to gemcitabine. Conclusion: Our studies indicate that PKD1 acts as a key regulator of the glucose metabolism via mTOR activation and facilitates proliferation and invasion of PanCa cells. Citation Format: Sonam Kumari, Sheema Khan, Murali Yallapu, Subhash Chauhan, Meena Jaggi. Protein kinase D1 induces metabolic switch in pancreatic cancer via modulation of mTORC1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4367.

2-Deoxy-d-glucose increases GFAT1 phosphorylation resulting in endoplasmic reticulum-related apoptosis via disruption of protein N-glycosylation in pancreatic cancer cells

The glycolytic inhibitor 2-deoxy-d-glucose (2DG) causes energy starvation, affecting cell viability in a wide range of cancer cell lines. To determine the action of 2DG in pancreatic cancer, we performed proteomic analysis of pancreatic cancer cell line after 2DG treatment. Eighty proteins showed differential expression and among these, proteins involved in phosphohexose metabolism were upregulated. Up-regulation of glutamine: fructose 6-phosphate aminotransferase 1 (GFAT1), which belongs to the hexosamine biosynthesis pathway (HBP) that produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to maintain glycoprotein, was validated by evaluation of mRNA and protein levels. Therefore, we assessed the amounts of total N-glycoproteins. Unexpectedly, we found a reduction of total N-glycoproteins and phosphorylation of GFAT1 by AMP-activated protein kinase (AMPK). These data may shed light on HBP dysfunction. Furthermore, we found endoplasmic reticulum (ER) stress accompanied by increased expression of ER stress markers, such as glucose response protein 78 (GRP78) and C/EBP-homologous protein (CHOP), in 2DG-treated cells. Moreover, the additive activation of AMPK by metformin (Met) synergistically enhanced the reduction of protein N-glycosylation and cell growth inhibition in the presence of 2DG. These results suggest that 2DG reduces N-glycosylation of proteins following the increase of phosphorylation of GFAT1 and results in the inhibition of cell growth mediated by ER stress in pancreatic cancer cells.

Antiproliferative Effects of Metformin in Triple Negative MDA‐MB 231 Breast Cancer Cells Exposed to Glucose‐Starved And 2‐Deoxyglucose

IntroductionWe have previously reported that 50μM of the anti‐diabetic drug metformin protects endothelial cells against hyperglycemia‐induced endothelial dysfunction and senescence thus providing support for the vascular protective benefits of metformin when used in diabetes 1,2. However, when 2mM (but not 50μM) metformin was used in a cell culture protocol with tumor murine microvascular endothelial in a glucose‐starved (GS) or 2‐deoxyglucose (2DG; 5mM) media pro‐survival autophagy and the mTOR pathway were inhibited and cell viability was reduced 3. These data supported other published evidence from both clinical and ex vivo cell‐based studies suggesting the utility of metformin for the treatment of various cancers. We therefore postulated that triple‐negative breast cancer (TNBC) that does not benefit from hormonal or HER2 targeted therapies may also respond to a metformin and GS/2DG protocol and investigated the effects of metformin in MDA‐MB 231 TNBC cells that were cultured in the presence of 2DG.MethodsMDA‐MB 231 cells were treated with 2DG (10mM) in a glucose free media for 48h in the absence/presence of metformin (2mM). Western blot analysis (n=3–4); cell proliferation (MTT assay; n=5); migration (Radius Cell Migration Assay; n=4) and viability (propidium iodide assay; n=5) were performed. Significance was determined by ANOVA.ResultsCell proliferation rate significantly decreased (95%) in 1) GS + metformin when compared to non‐treated GS (88%) and 2) 2DG exposed cells treated with metformin (46%) versus cells that were treated with either metformin (20%) or 2DG (28%) alone. Cell viability significantly (P<0.05) decreased in GS treated with metformin (34%) when compared to non‐treated GS (8%). In addition cell migration in cells exposed to 2DG and treated with metformin was reduced. Western blot analysis revealed that treatment with metformin significantly decreased (fold change) the levels of LC3A‐II (2.4&1.8) and LC3B‐II (2.3&1.6), pmTOR (S2448; 1.6&1.4) and downstream p4E‐BP1(T36/47; 1.5&1.3), pS6(S235/236; 4.7&2.8) and pS6(S240/244; 3.2&1.8) in GS and 2DG‐exposed cells + 2mM metformin.ConclusionResults show that using mM metformin in TNBC cells in a GS media containing 2DG offers a potential adjunct anti‐cancer protocol for the treatment of TNBC.Support or Funding InformationSupported by a Qatar Foundation National Priorities Research Program grant (NPRP: 04‐910‐3‐244) and a Junior Scientist Research Experience Program grant (JSREP: 03‐016‐3‐009)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sestrin-Mediated Inhibition of Stress-Induced Intervertebral Disc Degradation Through the Enhancement of Autophagy

Background/Aims: Intervertebral disc degeneration (IDD) is a pathological process that is the primary cause of low back pain and is potentially mediated by compromised stress defense. Sestrins (Sesn) promote cell survival under stress conditions and regulate AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling. Here, we investigated the expression of Sesn in normal and degraded nucleus pulposus (NP) cells and its potential roles during IDD pathogenesis. Methods: Sesn expression in normal and degraded NP cells was determined by quantitative polymerase chain reaction and immunoblotting and immunohistochemistry, respectively. Sesn function was investigated by using Sesn knockdown and overexpression techniques with analysis of extracellular matrix (ECM), cell apoptosis, autophagy, AMPK, and mTOR activation. Results: In human cultured NP cells, Sesn expression was significantly decreased in degraded NP cells at both the RNA and protein levels. The expression of Sesn1, 2, and 3 increased after stimulation by 2-deoxyglucose (2-DG), an endoplasmic reticulum stress inducer. 2-DG could also increase cell apoptosis, promote extracellular matrix (ECM) degradation, and positively regulate autophagy in NP cells. Sesn knockdown by small interfering RNA increased NP cell apoptosis and ECM degradation under basal culture conditions and in the presence of 2DG. Conversely, Sesn overexpression mediated by plasmid transfection repressed IDD by enhancing autophagy, which was associated with changes in mTOR but not AMPK activation. Conclusions: Sesn expression is suppressed in degraded NP cells. In addition, Sesn inhibits stress-induced cell apoptosis and ECM degradation by enhancing autophagy, which is modulated though mTOR activity. Suppression of Sesn might therefore represent an important cellular dysfunction mechanism in the process of IDD.

Suppression of Sestrins in aging and osteoarthritic cartilage: dysfunction of an important stress defense mechanism

Aging is an important osteoarthritis (OA) risk factor and compromised stress defense responses may mediate this risk. The Sestrins (Sesn) promote cell survival under stress conditions and regulate AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling. This study examined Sesn expression in normal and OA cartilage and functions of Sesn in chondrocytes. Sesn expression in human and mouse normal and OA cartilage was analyzed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. Sesn function was investigated by using small interfering RNA (siRNA) mediated Sesn knockdown and overexpression with analysis of cell survival, gene expression, autophagy, and AMPK and mTOR activation. Sesn mRNA levels were significantly reduced in human OA cartilage and immunohistochemistry of human and mouse OA cartilage also showed a corresponding reduction in protein levels. In cultured human chondrocytes Sesn1, 2 and 3 were expressed and increased by tunicamycin, an endoplasmic reticulum (ER) stress response inducer and 2-deoxyglucose (2DG), a metabolic stress inducer. Sesn1 and 2 were increased by tBHP, an oxidative stress inducer. Sesn knockdown by siRNA reduced chondrocyte viability under basal culture conditions and in the presence of 2DG. Sesn overexpression enhanced LC3-II formation and autophagic flux, and this was related to changes in mTOR but not AMPK activation. These findings are the first to show that Sesn expression is suppressed in OA affected cartilage. Sesn support chondrocyte survival under stress conditions and promote autophagy activation through modulating mTOR activity. Suppression of Sesn in OA cartilage contributes to deficiency in an important cellular homeostasis mechanism.

Suppression of sestrins in aging and osteoarthritic cartilage: Dysfunction of an important stress defense mechanism

Purpose: Aging is an important osteoarthritis (OA) risk factor and compromised stress defense responses may mediate this risk. The Sestrins (Sesn) promote cell survival under stress conditions and regulate AMPK and mTOR signaling. This study examined Sesn expression in normal and OA cartilage and functions of Sesn in chondrocytes. Methods: Sesn expression in human and mouse normal and OA cartilage was analyzed by quantitative PCR and immunohistochemistry. Sesn function was investigated by using siRNA mediated Sesn knockdown and overexpression with analysis of cell survival, gene expression, autophagy, and AMPK and mTOR activation. Results: Sesn mRNA levels were significantly reduced in human OA cartilage and immunohistochemistry of human and mouse OA cartilage also showed a corresponding reduction in protein levels. In cultured human chondrocytes Sesn1, 2 and 3 were expressed and increased by tunicamycin, an endoplasmic reticulum stress response inducer and 2-deoxyglucose, a metabolic stress inducer. Sesn1 and 2 were increased by tBHP, an oxidative stress inducer. Sesn1,2,3 knockdown by siRNA reduced chondrocyte viability under basal culture conditions and in the presence of 2DG. Sesn knockdown and overexpression reduced or enhanced autophagy activation, respectively, and this was related to changes in mTOR but not AMPK activation. Conclusions: These findings are the first to show that Sesn expression is suppressed in OA affected cartilage. Sesn support chondrocyte survival under stress conditions and promote autophagy activation through modulating mTOR activity. In conclusion, Sesn mRNA and protein levels are reduced in OA-affected and aging human and mouse cartilage. Sesn support chondrocyte survival under stress conditions and promote autophagy activation through modulating mTOR activity. Suppression of Sesn in OA cartilage contributes to deficiency in an important cellular homeostasis mechanism.

Altered Subcellular Distribution of the Alzheimer's Amyloid Precursor Protein Under Stress Conditions

Altered metabolism of the Alzheimer's amyloid precursor protein (APP) appears to be a key event in the pathogenesis of Alzheimer's disease (AD), and both altered phosphorylation and oxidative stress appear to affect the production of the toxic Abeta fragment. Our results show that altered processing of APP was observed under conditions of stress induced by sodium azide in the presence of 2-deoxy-D-glucose (2DG). As previously reported, the production of the secreted fragment of APP (sAPP) was inhibited. Using APP-GFP fusion proteins, we show that 2DG causes the accumulation/delay of APP in the endoplasmic reticulum (ER)/Golgi (G). The 751 isoform accumulated preferentially in the G, whereas the 695 isoform was blocked preferentially at the ER. This effect was augmented in the presence of sodium azide. APP subcellular distribution was also affected at the plasma membrane. The involvement of protein phosphorylation in APP subcellular localization was reinforced by the effect of drugs, such as phorbol 12-myristate 13-acetate (PMA), since APP was completely depleted from the membrane in the presence of 2DG and PMA. Thus, the hypothesis that APP is processed in a phosphorylation-dependent manner and that this may be of clinical relevance appears to hold true even under stress conditions. Our results provide evidence for a role of protein phosphorylation in APP sorting under stress conditions and contribute to the understanding of the molecular basis of AD.

Genetic variance contributes to ingestive processes: A survey of 2-deoxy- d-glucose-induced feeding in eleven inbred mouse strains

The feeding response following administration of the anti-metabolic glucose analogue, 2-deoxy- d-glucose (2DG), is conceptualized as an experimental model of glucoprivation, which may contribute to the understanding of inter-individual differences in glucose and carbohydrate intake and, ultimately, obesity. Although variation in the intake of several nutrients as well as food and water are known to be associated with genetic variation, it is not known whether 2DG-induced feeding is similarly genotype dependent. The present study therefore examined 2DG-induced feeding in mice of 11 inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL6/J, C57BL10/J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) strains across a wide range of previously determined effective 2-DG doses (200, 400, 600, 800 mg/kg) and test times (1–4 h). Orderly dose-dependent increases in 2DG-induced feeding occurred after all four doses in outbred CD-1 and inbred DBA/2J mice, across the three highest doses for BALB/cJ, SJL/J and 129P3/J mice, and across the two highest doses for CBA/J and AKR/J mice. Limited instances of 2DG-induced feeding were noted only at the highest dose in A/J and C3H/HeJ mice, or at a moderate dose in C57BL/6J mice. Further, the full 2DG dose range failed to alter food intake in C57BL/10J mice, and produced significant reductions in food intake in SWR/J mice. Food intake after 2DG doses of 200–600 mg/kg, but not 800 mg/kg, displayed significant cross-correlation, suggesting that large 2DG doses may recruit non-specific effects upon food intake. There was no correlation between food intake in the absence (vehicle baseline) of and presence of 2DG, suggesting that the regulation of glucose intake in non-challenged mice does not predict subsequent responses to glucoprivic challenge. The data demonstrate genotype-dependent variability in this glucoprivic response, and may provide the basis for the subsequent identification of trait-relevant genes.

Physiological characterization of Streptococcus bovis mutants that can resist 2-deoxyglucose-induced lysis.

Streptococcus bovis JB1 does not normally lyse, but stationary phase lysis can be induced by including 2-deoxyglucose (2DG) in the growth medium. Isolates deficient in glucose/2DG phosphotransferase activity (PTS-) also lysed when 2DG was present (Lys+) and this result indicated that 2DG phosphorylation via the PTS was not an obligate requirement for 2DG-induced lysis. Cells and cell walls from 2DG-grown cultures lysed faster when proteinase K was added, but glucose-grown cultures and cell walls were not affected. A lipoteichoic acid (LTA) extract (aqueous phase from hot phenol treatment) from glucose-grown cells inhibited the lysis of 2DG-grown cultures, but a similar extract prepared from 2DG-grown cells was without effect. Thin-layer chromatography and differential staining indicated that wild-type and Lys+ PTS- cells incorporated 2DG into LTA, but lysis-resistant cultures (Lys- PTS+ and Lys- PTS-) did not. LTA from lysis-resistant (Lys- PTS+ and Lys- PTS-) cells grown with glucose and 2DG also prevented 2DG-dependent lysis of the wild-type. LTA could not inhibit degradation of cell walls isolated from 2DG-grown cultures, but LTA inhibited the lysis of Micrococcus lysodeikticus (Micrococcus luteus) cells that were exposed to supernatants from 2DG-grown S. bovis cultures. Group D streptococci (including S. bovis) normally have an alpha-1,2 linked glucose disaccharide (kojibiose) in their LTA, but kojibiose cannot be synthesized from 2DG. This observation suggested that the kojibiose moiety of LTA was involved in autolysin inactivation. Wild-type S. bovis had ATP- as well as PEP-dependent mechanisms of 2DG phosphorylation and one lysis-resistant phenotype (Lys- PTS-) had reduced levels of both activities. However, the Lys- PTS+ phenotype was still able to phosphorylate 2DG via ATP and PEP and this result indicated that some other step of 2DG incorporation into LTA was being inhibited. Based on these results, growth in the presence of 2DG appears to prevent synthesis of normal LTA, which is involved in the regulation of autolytic enzymes.

Glucoprivation Attenuates the Hypophagia Induced by Epinephrine in Mice

It is well known that relatively high doses of epinephrine (E) injected intraperitoneally (IP) produce hypophagia, possibly by an action on liver metabolism. The purpose of the present experiment was to find out if lipoprivation with 2-mercaptoacetate (MA, 800 μmol/kg, IP) or glucoprivation with either 2-deoxy-D-glucose (2DG, 500 mg/kg, IP) or 2,5-anhydro-D-mannitol (2,5-AM, 400 mg/kg, IP) were able to modify the anorectic effect of E (300 μg/kg). At the onset of the dark period, mice received a first injection of saline (S) or one of the metabolic blockers mentioned above and, 30 min later, a second injection of S or E; then 30-min food intake was measured. E alone decreased feeding by 80% (p < 0.05); this effect was nearly the same when MA was previously injected. In contrast, in the presence of 2DG or 2,5-AM, E reduced food intake only by 22% and 24%, respectively (not significant). Attenuation of E-induced hypophagia by these blockers suggests the participation of glucose utilization pathways. Because it has been shown that 2,5-AM acts specifically on the liver, we could additionally suggest that E reduces feeding by an action on glucose hepatic metabolism.

Tension responses of sheep aorta to simultaneous decreases in phosphocreatine, inorganic phosphate and ATP.

1. Tension responses of sheep aortae were investigated when different substrates were included in the superfusing medium. The magnitude of tension development was similar whether or not 5 mM glucose was present in the medium. However, the rate of tension development was greater in the absence of glucose. 2. When 5 mM 2-deoxyglucose (2DG) was present in the medium, the magnitude of tension generation was 1.6 times that in the absence of exogenous substrate. A second sequential contraction with 2DG generated tension 1.25 times that in the absence of exogenous substrate. The rate of tension development during the first contraction in the presence of 2DG was similar to that in the absence of substrate. However, the second contraction in the presence of 2DG had a substantially increased rate of tension development. 3. 31P nuclear magnetic resonance (NMR) spectroscopy revealed that, at resting tone, in the presence of 2DG, inorganic phosphate (P(i)) and phosphocreatine (PCr) simultaneously decrease while 2-deoxyglucose-6-phosphate accumulates. During contraction-relaxation cycles, in the presence of 2DG, P(i) and PCr become undetectable while ATP declines to approximately 50% of control values as determined by NMR. During the second contraction in the presence of 2DG, the area of the ADP resonance was similar to that of the alpha-ATP resonance. 4. The increase in the magnitude of tension generation, during 2DG administration, correlated with a decrease in P(i) levels. The rate of relaxation from a contraction, in the presence of 2DG, was slower than in the presence of glucose or in the absence of exogenous substrate. These results are consistent with the role of P(i) in the release of the proposed 'latch-bridge' state of maintained contraction at low energy demand. 5. The increase in isometric tension generation during contraction in the presence of 2DG appears to be related to the decreased levels of P(i). In the presence of 2DG, the reduction of PCr and of ATP occur to a similar extent to that during hypoxia, yet no inhibition of force takes place. The low levels of ATP and PCr reported with 2DG administration in these studies do not energetically limit the contractile apparatus.

Effect of 2-deoxy D-glucose on growth, chlorophyll synthesis and electron transport activities of the cyanobacterium Synechococcus cedrorum

Summary 2-Deoxy D-glucose (2dG) in the growth medium slowed down the cellular multiplication and brought about transient changes in protein and chlorophyll a (Chl a ) content of the cells of Synechococcus . In the presence of 2dG, both protein and Chl a contents of the culture declined to a minimum level on the 6th day after inoculation and increased thereafter. In contrast to Chl a the contents of phycocyanin (PC) was much less affected by 2dG. Analysis of cellular constituents, on per cell basis, however, indicates that, on the 6th day of growth, the 2dG-cells have about 5 to 6 times less Chl a than normal cells and the total protein content of 2dG-cells is less than the normal cells. The electron transport activity of spheroplasts from 6 d old 2dG-cells, expressed on per cell basis, was lower than that of the normal cells. The photosystem II (PSII) dependent electron transport was reduced in 2dG grown cells. The measurement of light saturation curve for DCIP Hill reaction and the p H optimum for DCIP Hill reaction suggest that the presence of 2 dG in growth medium alters the photoelectron transport and pigment-protein constituents characteristics of PSII. Photosystem I (PSI) catalyzed electron flow was also suppressed by the presence of 2dG in the growth medium. 2dG seems to be an useful agent for modulation of cyanobacterial membrane structure and function.

Relationship between expression of herpes simplex virus glycoproteins and susceptibility of target cells to human natural killer activity.

Cells normally insensitive to human natural killer (NK) activity were rendered susceptible by infection with HSV-1. The cytotoxic effector cell was a nonadherent, non-T, non-B lymphocyte. Antibody plus complement treatment, using a monoclonal antibody that recognizes an antigen present on NK cells, removed much of the cytotoxic activity, and a density gradient fraction enriched for NK cells yielded cells of increased virus-specific cytotoxicity. It was concluded that the effector cell active against infected targets possessed characteristics of an NK cell. Blockage of viral protein synthesis during infection inhibited development of increased susceptibility of infected targets to NK activity. When targets were infected with a mutant virus unable to produce viral glycoprotein C (gC), NK activity against these targets was reduced approximately 30% compared with activity against targets infected with wild-type virus. Similarly, activity against targets infected in the presence of 2-deoxyglucose (2dG), which prevents cell surface expression of viral glycoprotein B (gB), was also reduced approximately 30%. An approximately 60% reduction in activity was seen against targets infected with mutant virus in the presence of 2dG; these targets express gD, but neither gB nor gC. When cells expressing various combinations of HSV-1 glycoproteins were used as both labeled targets and cold target competitors, it was found that the susceptibility of a particular target to NK activity was paralleled by its ability to act as a cold target competitor. This indicates that targets with decreased sensitivity to NK cells were less able to bind NK effectors. Further, the amount of interferon produced in co-cultures of NK effectors and infected target cells did not directly correlate with the amount of NK activity generated, and interferon pretreatment of effectors did not decrease virus-specific cytotoxicity. The present results suggest that HSV-1 glycoproteins expressed at the surface of infected targets may act as recognition structures for NK cells.

Epithelial cell motility: the effect of 2-deoxyglucose on cell migration, ATP production, and the structure of the cytoplasmic ground substance in lamellipodia of epithelial cells in culture.

Using a line of epithelial cells (SCCA5) derived from a spontaneous rat carcinoma, the glucose analogue 2-deoxyglucose (2DG) has been shown by time-lapse cinemicrography to produce a cessation of motility by 1 hour that can be reversed by replacement of the 2DG, and does not occur in equivalent media with or without glucose or in 2DG-containing media with added pyruvate and citrate. The effect on the cells at the edge of an epithelial island is to prevent the formation of new lamellipodia and produce a progressive retraction and condensation of lamellipodia already present. This effect of 2DG on motility corresponds with a significant reduction in the level of ATP that is partially restored after 30 minutes in the recovery incubation. Only a slight reduction in protein synthesis occurs in the presence of 2DG. The external morphology and the cytoplasmic ground substance of the cells were studied by scanning electron microscopy and high voltage electron microscopy respectively. It was found that after incubation in 2DG for 1 hour the outline of the free edges of the cells was distorted resulting in redistribution of microvilli, condensation of cytoplasm into strands, and irregular projections from the edges of residual lamellipodia. The structure of the cytoplasmic ground substance in lamellipodia from cells incubated in 2DG for 3 hours was distinctly different from that in cells incubated for 3 hours in 2DG then recovered for 25 minutes, or in cells incubated in glucose-containing medium for 3 hours. In the 2DG-treated cells the lattice-like structure evident in critical-point-dried cells was condensed into short thick strands that terminated in bulbous ends, whereas in cells recovered for 25 minutes the lattice material was elongated and tapering and the interlattice space relatively expanded. The results obtained support the concept of modulation occurring in the structure of the microtrabecular lattice component of the cytoplasmic ground substance coincident with alterations in cell function and metabolic state.