Role of 2-deoxy-D-glucose in the inhibition of phagocytosis by mouse peritoneal macrophage

2-Deoxy-D-glucose inhibits Fc and complement receptor-mediated phagocytosis of mouse peritoneal macrophages. To understand the mechanism of this inhibition, we analyzed the 2-deoxy-D-glucose metabolites in macrophages under phagocytosis inhibition conditions and conditions of phagocytosis reversal caused by glucose, mannose and 5-thio-D-glucose, and compared their accumulations under these conditions. Macrophages metabolized 2-deoxy-D-glucose to form 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucose 1-phosphate, UDP-2-deoxy-D-glucose, 2-deoxy-D-glucose 1, 6-diphosphate, 2-deoxy-D-gluconic acid and 2-deoxy-6-phospho-D-gluconic acid. The level of bulk accumulation as well as the accumulation of any of these 2-deoxy-D-glucose metabolites did not correlate with changes in macrophage phagocytosis capacities caused by the reversing sugars. 2-Deoxy-D-glucose inhibited glycosylation of thioglycolate-elicited macrophage by 70-80%. This inhibition did not cause phagocytosis inhibition, since (1) the reversal of phagocytosis by 5-thio-D-glucose was not followed by increases in the incorporation of radiolabelled galactose, glucosamine, N-acetylgalactosamine or fucose; (2) cycloheximide at a concentration that inhibited glycosylation by 70-80% did not affect macrophage phagocytosis. The inhibition of protein synthesis by 2-deoxy-D-glucose similarly could not account for phagocytosis inhibition, since cycloheximide, when used at a concentration that inhibited protein synthesis by 95%, did not affect phagocytosis. 2-Deoxy-D-glucose lowered cellular nucleoside triphosphates by 70-99%, but their intracellular levels in the presence of different reversing sugars did not correlate with the magnitude of phagocytosis reversal caused by these sugars. The results show that 2-deoxy-D-glucose inhibits phagocytosis by a mechanism distinct from its usual action of inhibiting glycosylation, protein synthesis and depleting energy supplies, mechanisms by which 2-deoxy-D-glucose inhibits other cellular processes.     

Effects of the probiotic strain Lactobacillus johnsonii strain La1 on autonomic nerves and blood glucose in rats

Oral administration of Lactobacillus casei reportedly reduces blood glucose concentrations in a non-insulin-dependent diabetic KK-Ay mouse model. In order to determine if other lactobacillus strains affect glucose metabolism, we evaluated the effect of the probiotic strain Lactobacillus johnsonii La1 (LJLa1) strain on glucose metabolism in rats. Oral administration of LJLa1 via drinking water for 2 weeks inhibited the hyperglycemia induced by intracranial injection of 2-deoxy-D-glucose (2DG). We found that the hyperglucagonemic response induced by 2DG was also suppressed by LJLa1. Oral administration of LJLa1 for 2 weeks also reduced the elevation of blood glucose and glucagon levels after an oral glucose load in streptozotocin-diabetic rats. In addition, we recently observed that intraduodenal injection of LJLa1 reduced renal sympathetic nerve activity and enhanced gastric vagal nerve activity, suggesting that LJLa1 might affect glucose metabolism by changing autonomic nerve activity. Therefore, we evaluated the effect of intraduodenal administration of LJLa1 on adrenal sympathetic nerve activity (ASNA) in urethane-anesthetized rats, since the autonomic nervous system, including the adrenal sympathetic nerve, may be implicated in the control of the blood glucose levels. Indeed, we found that ASNA was suppressed by intraduodenal administration of LJLa1, suggesting that LJLa1 might improve glucose tolerance by reducing glucagon secretion via alteration of autonomic nerve activities.     

Replacement of intracellular C-terminal domain of GLUT1 glucose transporter with that of GLUT2 increases Vmax and Km of transport activity.

The intracellular C-terminal domain is diverse in size and amino acid sequence among facilitative glucose transporter isoforms. The characteristics of glucose transport are also divergent, and GLUT2 has far higher Km and Vmax values compared with GLUT1. To investigate the role of the intracellular C-terminal domain in glucose transport, we expressed in Chinese hamster ovary cells the mutated GLUT1 protein whose intracellular C-terminal domain was replaced with that of GLUT2 by means of engineering the chimeric cDNA. Cytochalasin B, for which GLUT2 protein has much lower affinity, bound to this chimeric protein in a fashion similar to GLUT1. In contrast, greater transport activity was observed in this chimeric glucose transporter compared with the wild-type GLUT1 at 10 mM 2-deoxy-D-glucose concentration. The kinetic studies on 2-deoxy-D-glucose uptake revealed a 3.8-fold increase in Km and a 4.3-fold increase in Vmax in this chimeric glucose transporter compared with the wild-type GLUT1. Thus, replacement of the intracellular C-terminal domain confers the GLUT2-like property on the glucose transporter. These results strongly suggest that the diversity of intracellular C-terminal domain contributes to the diversity of glucose transport characteristics among isoforms.     

Elevation of plasma triglyceride levels due to 2-deoxy-D-glucose in conscious rats

Hypertriglyceridemia due to 2-deoxy-D-glucose administration was observed in conscious rats. Plasma triglyceride levels were elevated dose-dependently 2 or 3 hrs after administration of 2-deoxy-D-glucose (5-40 mg/100 g body weight). Prior to the rises in triglyceride, plasma epinephrine levels were elevated rapidly, whereas plasma insulin was not increased depspite continuous hyperglycemia. Elevation of plasma triglyceride was suppressed by addition of phentolamine, whereby insulin release was remarkably enhanced. Plasma lipoprotein lipase release by heparin infusion was significantly suppressed 2 hr after 2-deoxy-D-glucose administration. In conclusion, it is suggested that the hypertriglyceridemic effect of 2-deoxy-D-glucose may be mediated by decreased clearance of endogeneous lipoprotein particles (mostly chylomicrons) attributable to a lowered lipoprotein lipase activity.     

Metabolic responses to glucoprivation induced by 2-deoxy-D-glucose in<Emphasis Type="Italic"> Brycon cephalus</Emphasis> (Teleostei,Characidae)

The effects of functional cytoglucopenia provoked by 2-deoxy-D-glucose (2-DG) were studied in adult Brycon cephalus, an omnivorous fish from the Amazon Basin in Brazil. Glycogen content in liver and muscle as well as plasmatic glucose, free fatty acids (FFA), insulin, and glucagon were measured. After 48 h fasting, an intraperitoneal saline injection (NaCl 0.6 g/100 ml) was administered to control fish, whereas the experimental group received 2-DG, dissolved in saline, in the dosage of 80 mg/kg (0.487 mmol/kg) or 150 mg/kg (0.914 mmol/kg) body weight; injection volume was 5 ml in all treatments. Blood and tissue samples were taken immediately before, and 2, 8, 10, and 24 h after administration of the drug or saline. Fish injected with both doses of 2-DG showed a marked increase in glycemia levels. Liver and muscle glycogen decreased after 2-DG administration and reached their lowest values 10–24 h after injection, while in control animals no significant changes were observed. Elevation in plasma glucagon was observed only in response to the maximum dosage of 2-DG administered, especially 10 h and 24 h post-injection. Plasma insulin levels were lower in animals treated with the glucose analogue but only statistically significant 24 h after drug administration. In conclusion, the administration of the non-metabolizable glucose analogue 2-DG in B. cephalus is a stimulus to generate responses towards an increase in the glucose available to tissues, which is a characteristic of a fasting situation. All the above data support the interest of 2-DG administration as a model to study carbohydrate metabolism adjustment mechanisms in fish.Abbreviations 2-DG 2-deoxy-D-glucose - FFA free fatty acids Communicated by G. Heldmaier     

Effect of 2-deoxy-D-glucose on lipolytic processes in blood and adipose tissue of rat

The effect of 2-deoxy-D-glucose on lipolytic processes in the blood and adipose tissue was studied. Rats treated with this antimetabolite showed a significant increase in serum glucose, FFA and glycerol level, as well as in the lipid mobilizing activity. On the other hand, the lipolytic activity of rat serum decreased when compared to control group. From these results it may be concluded that during hypothermia induced by administration of 2-deoxy-D-glucose intracellular, but not intravascular, lipolysis is enhanced.     

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.     

The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae

The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy-D-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy-D-glucose (6-dGlc). Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose. These uptake velocities can be increased again by raising the ATP level. These results establish a role of ATP in sugar transport. Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells. Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged. It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes.     

Nitric oxide (NO) is involved in modulation of non-insulin mediated glucose transport in chicken skeletal muscles

The biosynthesis and release of nitric oxide (NO) from skeletal muscle plays a crucial role in transport and utilization of glucose. There are, however, no reports concerning the effects of NO on the transport of glucose in skeletal muscles of chickens characterized by hyperglycemia and insulin resistance. The present study was undertaken to investigate whether a NO donor or a nitric oxide synthase (NOS) inhibitor influences basal or insulin-mediated glucose uptake in vivo in skeletal muscles of chickens. Single administration of NOC12, a NO donor at 1125 microg/kg body mass (BW) to 14 days old chicks caused an increase in plasma NO concentration, while it did not affect plasma glucose concentration. In contrast, a single injection of NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) at 300 mg/kg BW reduced plasma NO concentration, while it did not effect plasma glucose concentration. Chicks were also treated with or without NO modifier and/or insulin to estimate glucose transport activity, which was estimated by the 2-deoxy-D-glucose (2DG) uptake method. NOC12 treatment significantly increased basal glucose uptake, with no insulin stimulation, in extensor digitrorum longus (EDL) muscle (P<0.01), while it caused no significant changes in insulin-stimulated glucose uptake in the skeletal muscles assayed. Injection of L-NAME at 300 mg/kg BW resulted in a significant decrease in the basal glucose uptake in gastrocnemius muscles (P<0.01). No significant changes in the insulin-stimulated glucose uptake by L-NAME were observed in any skeletal muscles studied. The results suggest that NO plays a lesser role in the modulation of glucose transport in chicken skeletal muscle compared to mammals and may be involved in non-insulin mediated glucose transport.     

Influence of energy metabolism on the repair of X-ray damage in living cells

Summary Effects of the glucose antimetabolite, 2-deoxy-D-glucose (2DG) on liquid-holding reactivation (LHR) in X-irradiated yeast cells were studied. It has been observed that LHR in respiratory-deficient mutants is completely inhibited at a molar concentration ratio 2DG/glucose equal to 1, whereas for the wild-type this ratio is 10. Significance of these results for a radiochemotherapy of tumors is discussed.Dedicated to Prof. Dr. Dr. h. c. mult. B. Rajewsky on the occasion of his 80th birthday.     

Effect of glucose uptake on growth rate of mouse 3T3 cells

The objective of this investigation was to determine whether the rate of glucose uptake by mouse 3T3 cells was a primary determinant of growth rate. The experimental approach was to control the rate of glucose uptake into intracellular pools by supplying this sugar at varying concentration in minimal Eagle's medium with dialyzed serum in the absence and presence of 6-deoxy-D-glucose, a metabolically inert homomorphic analog of D-glucose that competitively inhibits the uptake of D-glucose. Total hexose (D-glucose and 6-deoxy-D-glucose) concentration was maintained at the physiological concentration of 5.5 mM, in order to maintain saturation and maximum activity of the D-glucose transport system; thus the flux of D-glucose into the cell was controlled by adjusting its concentration relative to its competing nonmetabolizable analog. It was found that even when the concentration of D-glucose was reduced to 0.7 mM, one eighth of the “normal” level of 5.5 mM. and 6-deoxy-D-glucose was present in sevenfold excess (4.8 mM), conditions under which glucose uptake was reduced to 20% of that shown by cells in the presence of 5.5 mM D-glucose, and intracellular pools of glucose and phosphorylated sugars derived from glucose were reduced to approximately 14% of normal, there was not a significant decrease in growth rate. These data support the view that the rate of glucose uptake is not a primary determinant of growth rate under the usual conditions of cell culture.     

Differential regulation of the GLUT-1 and GLUT-4 glucose transport systems by glucose and insulin in L6 muscle cells in culture

The regulation by glucose and insulin of the muscle-specific facilitative glucose transport system GLUT-4 was investigated in L6 muscle cells in culture. Hexose transport activity, mRNA expression, and the subcellular localization of the GLUT-4 protein were analyzed. As observed previously (Walker, P. S., Ramlal, T., Sarabia, V., Koivisto, U.-M., Bilan, P. J., Pessin, J. E., and Klip, A. (1990) J. Biol. Chem. 265, 1516-1523), 24 h of glucose starvation and 24 h of insulin treatment each increase glucose transport activity severalfold. Here we report a differential regulation of the GLUT-4 and GLUT-1 transport systems under these conditions. (a) The level of GLUT-4 mRNA was not affected by glucose starvation and was diminished by prolonged (24 h) administration of insulin; in contrast, the level of GLUT-1 mRNA was elevated under both conditions. (b) Glucose starvation and prolonged insulin administration increased the amount of both GLUT-4 and GLUT-1 proteins in the plasma membrane. (c) In intracellular membranes, glucose starvation elevated, and prolonged insulin administration reduced, the GLUT-4 protein content. In contrast, the GLUT-1 protein content in these membranes decreased with glucose starvation and increased with insulin treatment. Glucose transport was rapidly curbed upon refeeding glucose to glucose-starved cells, with half-maximal reversal after 30 min and maximal reversal after 4 h. This was followed by a marked decrease in the levels of GLUT-1 mRNA without major changes in GLUT-4 mRNA. Neither 2-deoxy-D-glucose nor 3-O-methyl-D-glucose could substitute for D-glucose in these effects. It is proposed that glucose and insulin differentially regulate the two glucose transport systems in L6 muscle cells and that the rapid down-regulation of hexose transport activity by glucose is regulated by post-translational mechanisms.     

Induction of sugar transport in chick embryo fibroblasts by hexose starvation. Evidence for transcriptional regulation of transport.

Incubation of chick embryo fibroblasts in glucose-free medium resulted in a dramatic increase in the rate of 2-deoxy-D-glucose transport. The greatest increase in rate occurred during the first 20 hours of incubation in glucose-free medium and was blocked by actinomycin D, dordycepin, or cycloheximide. The conditions of 2-deoxy-D-glucose concentration and time of incubation with the sugar were determined where transport rather than phosphorylation was rate-limiting in sugar uptake. These studies demonstrated that the transport of 2-deoxy-D-glucose was rate-limiting for only 1 or 2 min when the concentration of sugar in the medium was near the Km for transport, i.e. 2mM. No difference was found in the level of hexokinase activity in homogenates prepared from cells incubated glucose-free medium or standard medium when either 2-deoxy-D-[14C]glucose or D-glucose was used as substrate. A kinetic analysis of the initial rates of 2-deoxy-D-glucose transport by Lineweaver-Burk plots showed that the Vmax for sugar transport increased from 18 to 95 nmol per mg of protein per min when fibroblasts were incubated in glucose-free medium for 40 hours. The Km remained constant at 2 mM. Analysis of the initial rates of 3-omicron-methyl-D-glucose transport by Lineweaver-Burk plots further substantiated that the increase in sugar transport was due to an increase in the Vmax for transport with the Km remaining constant. The activation energy for the transport reaction calculated from an Arrhenius plot was 17.4 Cal per mol for cells cultured in the standard medium and 17.2 Cal per mol for cells cultured in the glucose-free medium. These results are consistent with the interpretation that the Vmax increase observed in hexose-starved cells is due to an increase in the number of transport sites.     

Regulation of glycolysis and sugar phosphotransferase activities in Streptococcus lactis: growth in the presence of 2-deoxy-D-glucose.

Streptococcus lactis K1 has the capacity to grow on many sugars, including sucrose and lactose, in the presence of high levels (greater than 500 mM) of 2-deoxy-D-glucose. Initially, growth of the organism was transiently halted by the addition of comparatively low concentrations (less than 0.5 mM) of the glucose analog to the culture. Inhibition was coincident with (i) rapid accumulation of 2-deoxy-D-glucose 6-phosphate (ca. 120 mM) and preferential utilization of phosphoenolpyruvate via the mannose:phosphotransferase system, (ii) depletion of phosphorylated glycolytic intermediates, and (iii) a 60% reduction in intracellular ATP concentration. During the 5- to 10-min period of bacteriostasis the intracellular concentration of 2-deoxy-D-glucose 6-phosphate rapidly declined, and the concentrations of glycolytic intermediates were restored to near-normal levels. When growth resumed, the cell doubling time (Td) and the steady-state levels of 2-deoxy-D-glucose 6-phosphate maintained by the cells were dependent upon the medium concentration of 2-deoxy-D-glucose. Resistance of S. lactis K1 to the potentially toxic analog was a consequence of negative regulation of the mannose:phosphotransferase system by two independent mechanisms. The first, short-term response occurred immediately after the initial "overshoot" accumulation of 2-deoxy-D-glucose 6-phosphate, and this mechanism reduced the activity (fine control) of the mannose:phosphotransferase system. The second, long-term mechanism resulted in repression of synthesis (coarse control) of enzyme IImannose. The two regulatory mechanisms reduced the rate of 2-deoxy-D-glucose translocation via the mannose:phosphotransferase system and minimized the activity of the phosphoenolpyruvate-dependent futile cycle of the glucose analog (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982). Phosphoenolpyruvate was thus conserved for transport of the growth sugar and for generation of ATP required for biosynthetic and work functions of the growing cell.     

Chloramphenicol decreases brain glucose utilization and modifies the sleep-wake cycle architecture in rats

We studied the effects of chloramphenicol on brain glucose utilization and sleep-wake cycles in rat. After slightly anaesthetized animals were injected with [18F]fluoro-2-deoxy-D-glucose, we acquired time-concentration curves from three radiosensitive beta microprobes inserted into the right and left frontal cortices and the cerebellum, and applied a three-compartment model to calculate the cerebral metabolic rates for glucose. The sleep-wake cycle architecture was analysed in anaesthetic-free rats by recording electroencephalographic and electromyographic signals. Although chloramphenicol is a well-established inhibitor of oxidative phosphorylation, no compensatory increase in glucose utilization was detected in frontal cortex. Instead, chloramphenicol induced a significant 23% decrease in the regional cerebral metabolic rate for glucose. Such a metabolic response indicates a potential mismatch between energy supply and neuronal activity induced by chloramphenicol administration. Regarding sleep-wake states, chloramphenicol treatment was followed by a 64% increase in waking, a 20% decrease in slow-wave sleep, and a marked 59% loss in paradoxical sleep. Spectral analysis of the electroencephalogram indicates that chloramphenicol induces long-lasting modifications of delta-band power during slow-wave sleep.     

The hemodynamic and metabolic effects of 2-deoxy-D-glucose in waking Wistar rats]

The effect of 2-deoxy-D-glucose (2-DG) in a dose of 250 mg/kg as a stressogenic factor on changes in hemodynamic parameters and metabolism in awake rats was quantified during 6 hours. It was found that a single 2-DG injection causes lowering of blood pressure on min 40 and 120. Heart rate tended to slow down. Diminished glucose concentration in the brain observed on experiment minute 15 induced a number of adaptive reactions in the body. Epinephrine plasma levels increased sharply on min 15 and 40. Norepinephrine concentrations elevated slightly only at the beginning of the experiment. The maximal glucose level in blood plasma was observed on min 40 and 120 and that of lactate 40 min following 2-DG injection. The level of immunoreactive insulin rose. Glucose content in the heart came up sharply on min 15 and 40. Lactate concentration in the heart increased continuously.     

Control of sugar transport in human fibroblasts independent of glucose metabolism or carrier-substrate interaction

Transport regulation by different metabolizable and nonmetabolizable sugars was studied in human fibroblasts. Sugars were classed as glucose-like (D-mannose, 3-0-methyl-D-glucose, thio-D-glucose, and D-allose) and starvation-like (D-galactose, D-fructose, L-glucose, D-xylose, 6-deoxy-D-glucose and 2-deoxy-D-glucose) based on their competence in curbing glucose starvation enhanced transport. No significant correlation existed between the ability of a sugar to curb hexose transport and the KI of that sugar in inhibiting hexose transport. Independence of the transport curb from glucose metabolism was observed since nonmetabolizable analogs of D-glucose when substituted for D-glucose in the culture medium effected glucose [i.e. 3-0-methyl-D-glucose (3-OMG)] and starvation-like (i.e. 6- and 2-deoxy-D-glucose) effects. The KI of inhibition pf 2-deoxy-D-glucose transport for 3-OMG was 8.5 mM, similar to those obtained for 6-deoxyglucose and 2-deoxyglucose on 2-deoxyglycose transport (7.5 and 3.5 mM, respectively) and on 3-0-methylglucose transport (3.5 and 2.5 mM, respectively). An equimolar mixture of D-glucose and 3-OMG (5.55 mM each) was more effective than 11.1 mM D-glucose or 3-OMG alone in curbing hexose transport or reversing hexose starvation induced increases in transport. The effect of 3-OMG may be independent of glucose metabolism but it is possible that 3-OMG structurally mimics a metabolite of glucose that may interact with intracellular regulators of carrier degradation and or expression.     

Novel phosphoenolpyruvate-dependent futile cycle in Streptococcus lactis: 2-deoxy-D-glucose uncouples energy production from growth.

The addition of 2-deoxy-D-glucose to cultures of Streptococcus lactis 133 that were growing exponentially on sucrose or lactose reduced the growth rate by ca. 95%. Inhibition did not occur with glucose or mannose as the growth sugar. The reduction in growth rate was concomitant with rapid accumulation of the analog in phosphorylated form (2-deoxy-D-glucose 6-phosphate) via the phosphoenolpyruvate-dependent mannose:phosphotransferase system. Within 5 min the intracellular 2-deoxy-D-glucose 6-phosphate concentration reached a steady-state level of greater than 100 mM. After maximum accumulation of the sugar phosphate, the rate of sucrose metabolism (glycolysis) decreased by only 30%, but the cells were depleted of fructose-1,6-diphosphate. The addition of glucose to 2-deoxy-D-glucose 6-phosphate preloaded cells caused expulsion of 2-deoxy-D-glucose and a resumption of normal growth. S. lactis 133 contained an intracellular Mg2+-dependent, fluoride-sensitive phosphatase which hydrolyzed 2-deoxy-D-glucose 6-phosphate (and glucose 6-phosphate) to free sugar and inorganic phosphate. Because of continued dephosphorylation and efflux of the non-metabolizable analog, the maintenance of the intracellular 2-deoxy-D-glucose 6-phosphate pool during growth stasis was dependent upon continued glycolysis. This steady-state condition represented a dynamic equilibrium of: (i) phosphoenolpyruvate-dependent accumulation of 2-deoxy-D-glucose 6-phosphate, (ii) intracellular dephosphorylation, and (iii) efflux of free 2-deoxy-D-glucose. This sequence of events constitutes a futile cycle which promotes the dissipation of phosphoenolpyruvate. We conclude that 2-deoxy-D-glucose functions as an uncoupler by dissociating energy production from growth in S. lactis 133.     

Novel method to differentiate 3T3 L1 cells in vitro to produce highly sensitive adipocytes for a GLUT4 mediated glucose uptake using fluorescent glucose analog

Adipocytes play a vital role in glucose metabolism. 3T3 L1 pre adipocytes after differentiation to adipocytes serve as excellent in vitro models and are useful tools in understanding the glucose metabolism. The traditional approaches adopted in pre adipocyte differentiation are lengthy exercises involving the usage of IBMX and Dexamethasone. Any effort to shorten the time of differentiation and quality expression of functional differentiation in 3T3 L1 cells in terms of enhanced Insulin sensitivity has an advantage in the drug discovery process. Thus, there is a need to develop a new effective method of differentiating the pre adipocytes to adipocytes and to use such methods for developing efficacious therapeutic molecules. We observed that a combination of Dexamethasone and Troglitazone generated differentiated adipocytes over fewer days as compared to the combination of IBMX and Dexamethasone which constitutes the standard protocol followed in our laboratory. The experiments conducted to compare the quality of differentiation yielded by various differentiating agents indicated that the lipid droplet accumulation increased by 112 % and the GLUT4 mediated glucose uptake by 137 % in cells differentiated with Troglitazone and Dexamethasone than in cells differentiated traditionally. The comparative studies conducted for evaluating efficient measurable glucose uptake by GOPOD assay, radioactive ³H-2-deoxy-D-glucose assay and by non-radioactive 6-NBDG (fluorescent analog of glucose) indicated that the non-radioactive method using 6-NBDG showed a higher signal to noise ratio than the conventional indirect glucose uptake method (GOPOD assay) and the radioactive ³H-2-deoxy-D-glucose uptake method. Differentiated 3T3 L1 cells when triggered with 2.5 ng/mL of Insulin showed 3.3 fold more glucose uptake in non-radioactive method over the radioactive ³H-2-deoxy-D-glucose uptake method. The results of this study have suggested that a combination of Dexamethasone and Troglitazone for 3T3 L1 cell differentiation helps in better quality differentiation over a short period of time with increased sensitivity to Insulin. The application of these findings for developing new methods of screening novel Insulin mimetics and for evaluating the immunological responses has been discussed.     

In vivo effect of 2-deoxy-D-glucose on glucose-6-phosphate dehydrogenase activity in the cytosol of liver, heart and skeletal muscle of rats

2-deoxy-D-glucose (2-DG), the unmetabolizable analogue of glucose induces a series of metabolic, hormonal and behavioral responses, causing cellular glucoprivation. According to in vitro studies, 2-DG inhibits phosphofructokinase in cultured human cells. The present investigations deal with changes in the cytosolic glucose-6-phosphate dehydrogenase activity following in vivo 2-DG administration. A single dose of 2-DG (600 mg/kg) has no influence on the activity of glucose-6-phosphate dehydrogenase in the cytosol of liver, heart and skeletal muscle of the rat. The concomitant increase in serum glucose, lactate and FFA concentrations observed in the study indicates indirectly a stimulation of adrenergic system. After three days of successive administration of 2-DG to rats, dehydrogenase activity decreased in the liver by approx 57% and in the skeletal muscle by approx 82% in comparison with control animals. Moreover the in vivo effect of 2-DG was found to be fully reversible, probably when the total amount of the inhibitor was excreted.