The inhibition of bovine heart hexokinase by 2-deoxy-D-glucose-6-phosphate: characterization by 31P NMR and metabolic implications.

The glucose analog, 2-deoxy-D-glucose (2DG), has been used widely for studying the initial steps in the metabolism of glucose by radio-isotope tracer methods and by 31P NMR. In the rat heart perfused with acetate/2DG (both 5 mM) plus insulin, trapping of phosphorus by 2-deoxy-D-glucose-6-phosphate (2DG6P) results in a steady state exhibiting high 2DG6P (55 mM) and low ATP concentrations but near-normal function, as observed in an earlier 31P NMR study. In order to understand how the 2DG6P concentration is stabilized, we studied the inhibition of a mammalian hexokinase by 2DG6P in vitro by a 31P NMR technique. Inhibition, previously unobserved, was found. It is similar to inhibition by G6P in that it is competitive with ATP and not competitive with 2DG, but the inhibition constant (1.4 mM) is much larger. The experimental protocol includes provisions for enzymatic destruction of stray inhibitors such as G6P. The results show that the high 2DG6P and low ATP concentrations found in the steady state of the perfused heart should strongly reduce the rate of phosphorylation of sugars by hexokinase.     

Accumulation of 2-deoxy-D-glucose-6-phosphate as a measure of glucose uptake in the isolated perfused heart: a 31P NMR study

The accumulation of 2-deoxy-D-glucose-6-phosphate (2DG6P), detected using 31P NMR spectroscopy, has been used as a measure of the rate of glucose uptake, yet the accuracy of this measurement has not been verified. In this study, isolated rat hearts were perfused with different substrates or isoproterenol for 30 min before measurement of either 2DG6P accumulation or [2-3H]glucose uptake, without and with insulin. Basal contractile function and metabolite concentrations were the same for all hearts. The basal rates of 2DG6P accumulation differed significantly, depending on the preceding perfusion protocol, and were 38-60% of the [2-3H]glucose uptake rates, whereas insulin-stimulated 2DG6P accumulation was the same or 71% higher than the [2-3H]glucose uptake rates. Therefore the ratio of 2DG6P accumulation/[2-3H]glucose uptake rates varied from 0.38 to 1.71, depending on the prior perfusion conditions or the presence of insulin. The rates of 2DG6P hydrolysis were found to be proportional to the intracellular 2DG6P concentrations, with a K(m) of 17.5mM and V(max) of 1.4 micromol/g dry weight/min. We conclude that the rates of 2DG6P accumulation do not accurately reflect glucose uptake rates under all physiological conditions in the isolated heart and should be used with caution.     

Effects of 2-deoxy-D-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods.

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30 degrees C in a standard pyrophosphate medium containing 4.5 10(7) cells/ml. 31P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by 13C- and 1H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-D-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-13C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 +/- 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of 2-deoxy-d-glucose on the glucose metabolism in Saccharomyces cerevisiae studied by multinuclear-NMR spectroscopy and biochemical methods

The effects of various concentrations of deoxyglucose (DG) on the aerobic metabolism of glucose in glucose-grown repressed Saccharomyces cerevisiae cells were studied at 30°C in a standard pyrophosphate medium containing 4.5 10⁷ cells/ml. ³¹P-nuclear magnetic resonance (NMR) spectroscopy was used to monitor DG phosphorylation and the formation of polyphosphates. The production of soluble metabolites of glucose was evaluated by ¹³C- and ¹H-NMR and biochemical techniques. The cells were aerobically incubated with 25 mM of glucose and various concentrations of DG (0, 5 and 10 mM) in order to determine the DG concentration leading to optimum of 2-deoxy-d-glucose 6-phosphate (DG6P) formation without over-inhibiting the synthesis of other metabolites. The production of DG6P increased by about 25% when the external DG concentration was doubled (from 5 to 10 mM). The formation of polyphosphates (polyP), on the other hand, was found to be mainly conditioned by the DG concentration. The amount of polyP decreased by a factor of four upon addition of 5 mM DG and became undetectable in the presence of 10 mM DG. The glucose consumption and the production of soluble metabolites of [1-¹³C]glucose were then evaluated as a function of time in both the absence and presence of 5 mM DG. The effect of DG is to decrease the glucose consumption and the formation of polyphosphates, ethanol, glycerol, trehalose, glutamate, aspartate and succinate while stimulating the formation of arginine and citrate. Upon co-addition of 25 mM glucose and 5 mM DG, the ratio between the initial rates of glucose consumption (0.16 mM/min) and DG6P production (0.027 mM/min) is about (5.9 ± 1.2), not very different from the ratio of the initial concentration of glucose and DG (= 5.0). Therefore, hexokinase can phosphorylate deoxyglucose as well as glucose. However, after 100 min of incubation, the glucose concentration in the external medium decreased by about 64% while only 10% of DG was phosphorylated. DG6P was formed and quickly reached the limiting value about 30 min after co-addition of glucose and DG. Nevertheless, when the maximum quantity of DG6P was obtained, the DG consumption became negligible. By contrast, the glucose consumption and the production of ethanol and glycerol, although substantially reduced by about 42%, varied linearly with time up to 80 min of incubation. Thus even in the presence of an excess of DG, glycolysis is only slowed but not gradually or completely inhibited by DG. The reasons why DG6P cannot accumulate indefinitely in cells are discussed, together with the reasons why the consumption of DG, but not glucose, becomes negligible after 30 min of incubation. In the absence of DG, the amount of polyphosphates (polyP) increased regularly with time as long as glucose was sufficiently present (≥ 5 mM) in the suspension. When glucose was exhausted, long chain polyphosphates disappeared to give rise, at first, to polyP with shorter chains and finally to inorganic phosphate. In the presence of 5 mM DG, the reduction in quantity of polyP can be explained by the fact that ATP, normally used for the polyP synthesis, is now diverted to phosphorylation of DG to DG6P. The presence of 5 mM DG also had significant effects on the glutamate C2, C3 and C4 signal intensity and the production of all aminoacids. The results seem to indicate that the enzymes involved in the Krebs cycle are also affected by the presence of DG.     

Nonradioisotope assay of glucose uptake activity in rat skeletal muscle using enzymatic measurement of 2-deoxyglucose 6-phosphate in vitro and in vivo

We investigated a nonradioisotope method for the evaluation of glucose uptake activity using enzymatic measurement of 2-deoxyglucose 6-phosphate (2DG6P) content in isolated rat soleus muscle in vitro and in vivo. The 2DG6P content in isolated rat soleus muscle after incubation with 2-deoxyglucose (2DG) was increased in a dose-dependent manner by insulin (ED(50) = 0.6 mU/ml), the maximum response being about 5 times that of the basal content in vitro. This increment was completely abolished by wortmannin (100 nM), with no effect on basal 2DG6P content. An insulin-mimetic compound, vanadium, also increased 2DG6P content in a dose-dependent manner. In isolated soleus muscle of Zucker fa/fa rats, well known as an insulin-resistant model, insulin did not increase 2DG6P content. The 2DG6P content in rat soleus muscle increased after 2DG (3 mmol/kg) injection in vivo, and conversely, the 2DG concentration in plasma was decreased in a dose-dependent manner by insulin (ED(50) = 0.11 U/kg). The maximum response of the accumulation of 2DG6P in soleus muscle was about 4 times that of the basal content. This method could be useful for evaluating glucose uptake (transport plus phosphorylation) activity in soleus muscle in vitro and in vivo without using radioactive materials.     

Preconditioning enhanced glucose uptake is mediated by p38 MAP kinase not by phosphatidylinositol 3-kinase

Ischemia is reported to stimulate glucose uptake, but the signaling pathways involved are poorly understood. Modulation of glucose transport could be important for the cardioprotective effects of brief intermittent periods of ischemia and reperfusion, termed ischemic preconditioning. Previous work indicates that preconditioning reduces production of acid and lactate during subsequent sustained ischemia, consistent with decreased glucose utilization. However, there are also data that preconditioning enhances glucose uptake. The present study examines whether preconditioning alters glucose transport and whether this is mediated by either phosphatidylinositol 3-kinase (PI3K) or p38 MAP kinase. Langendorff-perfused rat hearts were preconditioned with 4 cycles of 5 min of ischemia and 5 min of reperfusion, with glucose as substrate. During the last reflow, glucose was replaced with 5 mM acetate and 5 mM 2-deoxyglucose (2DG), and hexose transport was measured from the rate of production of 2-deoxyglucose 6-phosphate (2DG6P), using (31)P nuclear magnetic resonance. Preconditioning stimulated 2DG uptake; after 15 min of perfusion with 2DG, 2DG6P levels were 165% of initial ATP in preconditioned hearts compared with 96% in control hearts (p < 0.05). Wortmannin, an inhibitor of PI3K, did not block the preconditioning induced stimulation of 2DG6P production, but perfusion with SB202190, an inhibitor of p38 MAP kinase, did attenuate 2DG6P accumulation (111% of initial ATP, p < 0. 05 compared with preconditioned hearts). SB202190 had no effect on 2DG6P accumulation in nonpreconditioned hearts. Preconditioning stimulation of translocation of GLUT4 to the plasma membrane was not inhibited by wortmannin. The data demonstrate that ischemic preconditioning increases hexose transport and that this is mediated by p38 MAP kinase and is PI3K-independent.     

A nonradioisotope, enzymatic assay for 2-deoxyglucose uptake in L6 skeletal muscle cells cultured in a 96-well microplate

A nonradioisotope, 96-well-microplate assay to evaluate glucose uptake activity in cultured cells has been developed. 2-Deoxyglucose (2DG) was detected by measuring a potent fluorophore, resorufin, generated after incubation with a single assay solution containing hexokinase, adenosine 5'-triphosphate, glucose 6-phosphate dehydrogenase, beta-nicotineamide adenine dinucleotide phosphate, diaphorase, and resazurin. This amplifying detection system could detect the fluorescence intensity induced by uptake of 2DG into L6 skeletal muscle cells, even at the level of cells cultivated in individual wells in a 96-well microplate. Using this assay system, the effects of insulin, cytochalasin B (hexose uptake inhibitor), LY294002 (inhibitor of glucose transporter translocation), and pioglitazone hydrochloride (insulin-sensitizing agent) on 2DG uptake into L6 myotubes could be assessed clearly. Therefore, our simple method may be useful for in vitro high-throughput screening and for evaluating regulators of glucose uptake.     

Glucose Metabolism Assessed with 2-Deoxyglucose and the Effect of Glutamate in Subdivisions of Rat Hippocampal Slices

A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2-deoxyglucose (DG), after which the tissue levels of DG and 2-deoxyglucose-6-phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of approximately 0.5 microgram. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4-fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.     

Alternative pathways of glucose transport in Prevotella bryantii B(1)4(1)

Prevotella bryantii B(1)4 grew faster on glucose than mannose (0.70 versus 0.45 h(-1)), but these sugars were used simultaneously rather than diauxically. 2-deoxy-glucose (2DG) decreased the growth rate of cells that were provided with either glucose or mannose, but 2DG did not completely prevent growth. Cells grown on glucose or mannose transported both (14)C-glucose and (14)C-mannose, but cells grown on glucose had over three-fold higher rates of (14)C-glucose transport than cells grown on mannose. The (14)C-mannose transport rates of glucose- and mannose-grown cells were similar. Woolf-Augustinsson-Hofstee plots were not linear, and it appeared that the glucose/mannose/2DG carrier acted as a facilitated diffusion system at high substrate concentrations. When cultures were grown on nitrogen-deficient (excess sugar) medium, isolates had three-fold lower (14)C-glucose transport, but the (14)C-mannose transport did not change significantly. (14)C-glucose and (14)C-mannose transport rates could be inhibited by 2DG and either mannose or glucose, respectively. The (14)C-glucose transport of mannose-grown cells was inhibited more strongly by mannose and 2DG than those grown on glucose. Cells grown on glucose or mannose had similar ATP-dependent glucokinase activity, and 2DG was a competitive inhibitor (K(i)=0.75 mM). Thin layer chromatography indicated that cell extracts also had ATP-dependent mannose phosphorylation, but only a small amount of phosphorylated 2DG was detected. Glucose, mannose or 2DG were not phosphorylated in the presence of PEP. Based on these results, it appeared that P. bryantii B(1)4 had: (1) two mechanisms of glucose transport, a constitutive glucose/mannose/2DG carrier and an alternative glucose carrier that was regulated by glucose availability, (2) an ATP-dependent glucokinase that was competitively inhibited by 2DG but was unable to phosphorylate 2DG at a rapid rate, and (3) virtually no PEP-dependent glucose, mannose or 2DG phosphorylation activities.     

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

Methods for 2-deoxyglucose (2-DG) and 2-deoxyglucose 6-phosphate (DG6P) are described which are based on the fact that DG6P is oxidized by glucose-6-phosphate dehydrogenase (G6PDH), but at a rate 1000-fold slower than for glucose 6-phosphate, whereas hexokinase phosphorylates 2DG and glucose at comparable rates. Therefore, by adding the two enzymes in a suitable order, and in appropriate concentrations, 2DG, glucose, DG6P, and glucose 6-P can all be separately measured. To avoid a side reaction from the use of a high level of G6PDH, when measuring DG6P, glucose is first removed with glucose oxidase plus aldose reductase.     

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

Microdetermination of 2-Deoxyglucose and 2-Deoxyglucose 6-Phosphate to Determine Glucose Utilization Rates in Single Neurons and Small CNS Regions After Injecting Nontracer Amounts of 2-Deoxyglucose

A nontracer amount (0.25 mmol/kg of body weight) of 2-deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. Freeze-dried samples of CNS regions and cell bodies of spinal motor neurons were prepared, and the concentrations of glucose, glucose 6-phosphate, DG, and DG 6-phosphate (DG6P) in them were microassayed after 3,000-1,500,000-fold amplification using an enzymatic amplification reaction, NADP cycling. Based on the time course of glucose, DG, and DG6P concentrations in arterial plasma and the anterior horn of the spinal cord, the Sokoloff-type rate equations for DG and DG6P concentrations were mathematically solved, and the resultant DG and DG6P concentration functions were fitted to the data points using the nonlinear least-squares fitting SALS package program. This fitting provided four rate constants for the functions and supported the theoretical basis for our calculations of glucose utilization rate (GUR) when DG was administered in nontracer amounts. The GUR was highest in the spinal motor neurons and lowest in the white matter of the cerebellum. Neuron-rich structures, such as the cerebellar molecular and granular layers and the anterior horn of the spinal cord, had higher GUR values than the white matter of the cerebellum and spinal cord.     

A nonradioisotope, enzymatic microplate assay for in vivo evaluation of 2-deoxyglucose uptake in muscle tissue

To determine 2-deoxy-D-glucose (2DG) and 2-deoxy-D-glucose 6-phosphate (DG6P) in mouse tissue after injection of 2DG, we have developed a novel assay. This assay is a simple procedure involving incubation of samples with four independent, single reaction mixtures followed by measurement of fluorescence. From differences between the values obtained with the four reactions, each of glucose, glucose 6-phosphate, 2DG and DG6P were able to be quantified in a sensitive manner. Using this assay system, glucose and 2DG in blood and DG6P-accumulation in muscle were easily determined. Therefore, this assay may be useful for measuring in vivo glucose uptake without the use of radioisotopes.     

Glucose uptake in vivo in skeletal muscles of insulin-injected chicks

Glucose uptake across the plasma membrane in animal cells plays a crucial role in whole-body glucose homeostasis. Insulin-stimulated glucose transport activity in vivo in several tissues was estimated using the 2-deoxy-D-[1-(3)H]glucose ([(3)H]2DG) uptake determination method. A tracer dose of [(3)H]2DG was injected intravenously into 8-day-old chicks (Gallus gallus) administered simultaneously or previously with porcine insulin (40 microg/kg BW). After 10 or 20 min, several major tissues, including skeletal and cardiac muscle, were sampled and their 2-deoxy-D-[1-(3)H]glucose 6-phosphate content analyzed. Plasma glucose concentration and [(3)H]2DG radioactivity were lowered by insulin within 20 min of [(3)H]2DG administration, while the plasma [(3)H]2DG/glucose ratio was not significantly different between chicks injected with insulin and their control counterparts. A marked uptake of 2DG was observed in cardiac tissue and brain, followed by kidney and skeletal muscles. In skeletal muscles, insulin increased the 2DG uptake in soleus, extensor digitorum longus and pectoralis superficialis muscles. On the other hand, no significant increases in insulin-induced 2DG uptake were detected in cardiac muscle or adipose tissue compared to controls. The results show that glucose transport across the plasma membrane in vivo in most skeletal muscles tested, but not cardiac muscle, was increased by insulin administration to chicks. These findings suggest that an insulin-responsive glucose transport mechanism is present in chickens, even though they intrinsically lack GLUT4 homologous gene, the insulin-responsive glucose transporter in mammals.     

The Action of the Hexokinase Inhibitor 2‐deoxy‐d‐glucose on Cryptosporidium parvum and the Discovery of Activities against the Parasite Hexokinase from Marketed Drugs

Cryptosporidium parvum is one of the major species causing mild to severe cryptosporidiosis in humans and animals. We have previously observed that 2‐deoxy‐d ‐glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG‐6‐phosphate (2DG6P), the anti‐cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme glucose‐6‐phosphate isomerase (CpGPI) nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. We also screened 1,200 small molecules consisting of marketed drugs against CpHK, from which four drugs were identified as CpHK inhibitors with micromolar level of anti‐cryptospordial activities at concentrations nontoxic to the host cells (i.e. hexachlorphene, thimerosal, alexidine dihydrochloride, and ebselen with EC₅₀ = 0.53, 1.77, 8.1 and 165 μM, respectively). The anti‐CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase.     

The effect of 2-deoxy-d-glucose on Werner syndrome RecQ helicase gene

Caloric restriction (CR) is known to effectively elongate mammalian life-spans. The compound 2-deoxy-d-glucose (2DG), which is often used as an inhibitor of glucose utilization, is a mimetic agent of CR. In this study, we examined the changes of telomerase and Werner’s syndrome RecQ (WRN) helicase after treatment with 2DG, because of the involvement of recQ helicase in the regulation of telomeres. Interestingly, 2DG treatment increased the expression of WRN protein in accordance with induction of its promoter activity and gene expression. Furthermore, the activation of telomerase was observed after 2DG treatment, whereas it resulted in the reduction of cell proliferation. These results suggest that 2DG could up-regulate telomere maintenance factors accompanied with suppression of proliferation.     

A measurement of cerebral glucose uptake rate by 31P MRS

A method for the measurement of cerebral glucose uptake rate by in vivo 31P Magnetic Resonance Spectroscopy (MRS) is proposed. The initial rate of 2-deoxy-glucose (DG) uptake after DG administration is measured by the increase of 2-deoxy-glucose-6-phosphate (DG6P) signal at a chemical shift of 7.2 ppm with respect to phosphocreatine (PCr). The values for four different metabolic states of rat brain (two levels of epileptic seizures induced by bicuculline, nitrous oxide analgesia and pentobarbital anesthesia) were in good agreement with the previously reported ones by radioisotope methods. This method appears to be useful for measuring cerebral glucose uptake rate.     

Desmodium gangeticum: a potent anti-ulcer agent

The present study was designed to investigate anti-ulcerogenic property of ethanolic extract of Desmodium gangeticum (DG) against cold restraint (CRU, 2 hr cold restraint stress), aspirin (ASP, 150 mg/kg orally), alcohol (AL, absolute alcohol 1 ml/200gm) and pyloric ligation (PL, 4 hr pylorus ligation) induced gastric ulcer models in Sprague Dawley rats, and histamine (HST, 0.25 mg/kg) induced duodenal ulcer in guinea pigs. We found that DG at a dose of 200mg/kg, (orally), markedly decreased the incidence of ulcers in all the above models. DG showed significant protection against CRU (68.37%), AL (88.87%), ASP (38.2%), PL (40.63%) and HST (63.15%) induced ulcer models, whereas standard drug omeprazole (OMZ) showed protection index of 83.86, 56.35, 70.31 and 84.21%, respectively in CRU, ASP, PL and HST models. Sucralfate as standard drug showed 92.64% protection in AL model. DG significantly reduced acid secretion 41.61%, whereas OMZ produced 43.13% reduction. Treatment with DG showed increase in mucin secretion by 56.17%, whereas OMZ showed 12.45% increase. Anti-ulcer effect of DG may be due to its cytoprotective effect along with antisecretory activity and could act as a potent therapeutic agent against peptic ulcer disease.     

An enzymatic photometric assay for 2-deoxyglucose uptake in insulin-responsive tissues and 3T3-L1 adipocytes

An enzymatic assay adapted to photometric analysis with 96-well microplates was evaluated for the measurement of 2-deoxyglucose (2DG) uptake in insulin-responsive tissues and differentiated 3T3-L1 adipocytes. For in vivo measurements, a small amount of nonradiolabeled 2DG was injected into mice without affecting glucose metabolism. For photometric quantification of the small amount of 2-deoxyglucose 6-phosphate (2DG6P) that accumulates in cells, we introduced glucose-6-phosphate dehydrogenase, glutathione reductase, and 5,5′-dithiobis(2-nitrobenzoic acid) to the recycling amplification reaction of NADPH. We optimized the enzyme reaction for complete oxidation of endogenous glucose 6-phosphate (G6P) and glucose in mouse tissues in vivo and serum as well as in 3T3-L1 adipocytes in vitro. All reactions are performed in one 96-well microplate by consecutive addition of reagents, and the assay is able to quantify 2DG and 2DG6P in the range of 5–80 pmol. The results obtained with the assay for 2DG uptake in vitro and in vivo in the absence or presence of insulin stimulation was similar to those obtained with the standard radioisotopic method. Thus, the enzymatic assay should prove to be useful for measurement of 2DG uptake in insulin-responsive tissues in vivo as well as in cultured cells.     

Effect of sphingoid bases on basal and insulin-stimulated 2-deoxyglucose transport in skeletal muscle.

Incubation of rat soleus muscles with 50 microM sphingosine or 50 microM sphinganine augmented basal 2-deoxy-D-glucose (2DG) transport 32%, but reduced the response to 0.1 and 1.0 mU insulin/ml by 17 and 27%, respectively. When the muscles were incubated with 50 microM phytosphingosine, a 63-93% increase in basal 2DG transport was observed. However, this treatment had no effect on insulin-stimulated 2DG transport. The phytosphingosine-induced increase in basal 2-DG transport was inhibited 93 and 98% with 35 and 70 microM cytochalasin B, respectively, suggesting that it is mediated by glucose transporters. Cellular accumulation of L-glucose, which is not mediated by glucose transporters, was not affected by phytosphingosine. It is concluded that (a) both sphingosine and sphinganine increase basal 2DG transport in muscle but diminish insulin-stimulated transport, and (b) phytosphingosine stimulates basal 2DG transport in muscle by a mechanism involving glucose transporters.