Selectivity of the 2-Deoxyglucose Transport System in Human and Guinea Pig Polymorphonuclear Leukocytes'

WILLIAM J. LITCHFIELD2 AND WILLIAM W. WELLS* Department of Biochemistry, Michigan State University; East Lansing, Michigan 48824 Received for publication 20 December 1976

To determine whether the deleterious action of -galactose upon phagocyte function could be related to inhibition of glucose uptake, the properties of glucose transport were investigated by following the incorporation of [G-3H]2- deoxyglucose into human and guinea pig polymorphonuclear leukocytes (PMN). Uptake of [G-3H]2-deoxyglucose by guinea pig PMN proceeded in vitro with a Km of 1.8 mM and Vmax of 0.67 nmol/min per 106 cells. This system was competitively inhibited by glucose and mannose but was not significantly affected by galactose, fructose, or 3-O-methylglucose. Maximal uptake of 2-deoxyglucose occurred at 41°C, and phosphorylation was necessary for its intracellular concentration. Transport of 2-deoxyglucose, although not altered by uncouplers of oxidative phosphorylation, was sensitive to inhibitors of glycolysis. Preincuba- tion ofcells with 2 mM iodoacetate for 30 min significantly reduced the uptake of 2-deoxyglucose and the intracellular levels of adenosine-5'-triphosphate without decreasing cell viability. These results indicated that uptake of 2-deoxyglucose in guinea pig PMN occurred by facilitated diffusion with subsequent phosphorylation. Similar results were obtained with PMN isolated from human peripheral blood. Phagocytosis and intracellular killing of mi- [1-_4C]fructose which was obtained from Amersham croorganisms are two primary functions of Searle Corp. All carbohydrates were of the D config- neutrophilic polymorphonuclear leukocytes uration and were purchased from Sigma Chemical (PMN). These activities are of particular im- Co., Mallinckrodt Chemical Works, and Nutritional Biochemicals Corp. Other reagents were primarily portance to the protection of a host against from Sigma with the following exceptions: iodoacetic infection. However, in certain disorders, such acid, Matheson Coleman and Bell Manufacturing as diabetes mellitus (4, 24) and galactosemia Chemists; potassium cyanide, Baker Chemical Co.; (18), the capacity of PMN to ingest and destroy p-chloromercuribenzoate and cytochalasin B, Calbi- bacteria is impaired. Previous studies indicate ochem; phlorizin, Nutritional Biochemicals Corp.; that this impairment directly results from ele- N-ethylmaleimide, Aldrich Chemical Co.; U-80 reg- vated levels of plasma carbohydrate (4, 18, 24) ular Iletin insulin, Eli Lilly and Co.; dextran 250, and is not an effect of insulin or opsonin de- Pharmacia Fine Chemicals, Inc.; guinea pig serum ficiencies (4). and Hanks balanced salt solution, Grand Island Bio- to inhibitors logical Co.; and polystyrene latex particles (1. 1-,u&m Since phagocytosis is sensitive diameter), Dow Diagnostics. All glassware was sili- of glycolysis (25) and glucose transport (9), the conized with a 1% solution of Siliclad (Clay Adams). deleterious effect of galactose on PMN function Cell preparations. PMN were isolated from adult (18) could largely result from a competitive in- male guinea pigs (Connaught Laboratories, Ltd.) hibition of glucose transport. To test this sug- fed a commercial diet and water with 0.04% L-ascor- gestion, we undertook a detailed study of hex- bic acid ad libitum. Guinea pigs were injected intra- ose transport in human and guinea pig PMN, peritoneally with 5.0 ml of sterile 1% caseinate in using the non-metabolizable glucose analogue, saline, and exudates were removed 15 to 20 h later 2-deoxyglucose. The kinetics and selectivity of by flushing peritoneal cavities with Hanks balanced are herein described. salt solution. Exudate cells composed of greater this transport system than 95% PMN were pooled and centrifuged at 250 x MATERIALS AND METHODS g at room temperature for 5 min. Cells were washed and suspended in Krebs-Ringer phosphate solution Materials. All radioisotopes were purchased from without calcium (pH 7.4) (KRPS). New England Nuclear Corp. with the exception of Human PMN were prepared from 10-ml samples of venous blood taken from 15 healthy adult volun- I Michigan Agricultural Experiment Station Journal Ar- with ticle 7901. teers. Samples were pooled, supplemented 2 Present address: Johnson Research Foundation, Uni- 1.2% dextran 250, and allowed to sediment for 1 h at versity of Pennsylvania, Philadelphia, PA 19174. room temperature. Leukocyte-rich supernatants 189 190 LITCHFIELD AND WELLS INFECT. IMMUN. were removed and centrifuged as described above. mined by counting cells in a hemocytometer. Cell To lyse remaining erythrocytes, pellets of leuko- counts were performed at least in quadruplicate cytes were suspended twice and incubated for 1 h at with a Universal model Zeiss microscope using 3700 in 200 ml of 0.015 M tris(hydroxymethyl)amino- phase optics. methane (pH 7.2), containing 0.75% NH4Cl. After Intracellular levels of phosphorylated 2-deoxyglu- the second incubation, cells were again centrifuged cose were measured after the uptake experiments and suspended in 5.0 ml of fresh autologous plasma. were performed. Scintillation fluid from each vial Cells in plasma were then applied to 20 ml of silico- was dried, and carbohydrate was removed from each nized glass beads (0.3-mm diameter), and PMN napthalene residue by extracting with 20 ml of dis- were isolated as described by Rabinowitz (23). Hu- tilled water. Extracts were concentrated under a man PMN of at least 90% purity as judged by visual stream of nitrogen and spotted on Whatman 3MM count were washed and suspended in KRPS. Cell paper for ascending chromatography. After develop- viability was determined by exclusion of 0.04% ment for 15 h with 7:3 (vol/vol) ethanol-i M ammo- trypan blue (22). nium acetate, chromatograms were cut into seg- Oxidation of carbohydrate. Conversion of 14C-la- ments corresponding to fast- and slow-migrating beled carbohydrate to 14CO2 by guinea pig PMN was bands of radioactivity. Phosphorylated 2-deoxyglu- performed in sealed 25-ml Erlenmeyer flasks as pre- cose associated with the latter segments and free 2- viously described (25). Cell suspensions (7 x 106 deoxyglucose plus sucrose present in the former seg- PMN) were incubated for 1 h at 37°C in 1.0-ml ali- ments were eluted with buffer into separate scintil- quots of KRPS containing 10% guinea pig serum and lation vials, dried, and counted as described above. 1.0 mM labeled carbohydrate (0.25 /iCi). Reactions Levels of ATP. PMN (15.2 x 106 cells per assay) were stopped by adding 0.2 ml of 7.3 N H2SO4 to the were incubated either with or without inhibitors at cells, and evolved CO2 was collected on folded strips 37°C in 1.0-ml aliquots of KRPS. After 30 min of (1 by 3 cm) of Whatman 3MM paper with 0.2 ml of incubation, cells were centrifuged for 1 min at 3,700 3.75 N KOH in plastic centerwells. Labeled CO2 was x g. Pellets were immediately frozen by immersing absorbed from the start of each experiment and for 1 tubes in liquid nitrogen, and cells were stored at h after acidifying the medium. Centerwells were -80°C until homogenizing with 0.21 ml of 3 N per- transferred directly to scintillation vials with 10 ml chloric acid. Homogenates were centrifuged at 5,000 of Bray solution and counted. Oxidation of carbohy- < g for 10 min to remove precipitated protein, and drate by phagocytosing cells was determined in the supernatants were neutralized with 0.18 ml of 2 N same manner except that a 20-fold (particle per KOH-0.4 M imidazole-0.4 M KCl. Levels of adeno- PMN) excess of polystyrene latex particles was in- sine 5'-triphosphate (ATP) were determined in each cluded in each flask. supernatant by monitoring the reduction of nicotin- Transport of 2-deoxyglucose. Uptake of [G-3H]2- amide adenine dinucleotide phosphate at 340 nm in deoxyglucose was determined by monitoring the in- the presence of excess glucose-6-phosphate dehydro- corporation of both tritium counts and [U- genase (EC 1.1.1.49) and hexokinase (EC 2.7.1.1) '4C]sucrose counts into pellets of cells. Sucrose, (19). which does not enter PMN (13), was used in each assay to correct for trapped 3H-labeled carbohydrate present in the extracellular space. Such corrections RESULTS were made by multiplying [U'4C]sucrose counts in the pellets by the ratio of tritium to [U-14C]sucrose Oxidation of carbohydrates to '4CO2. Table counts in the supernatants (13). Counts attributable 1 shows the levels of 14CO2 produced by guinea to intracellular [G-3H]2-deoxyglucose were then cal- pig PMN during a 1-h incubation at 37°C with culated by subtracting extracellular tritium counts various '4C-labeled carbohydrates. Values ob- from total tritium counts in each pellet. tained with [1-14C]glucose were higher than Unless otherwise stated, reactions employed ap- those obtained with other carbohydrates, and proximately 4 x 106 PMN suspended in 0.5 ml of participation of the hexose monophosphate KRPS. Suspensions were incubated at 37°C for 30 shunt in this process is indicated by comparing min prior to adding 0.5-ml volumes of buffer con- values for the oxidation of [1-'4C]glucose to taining 0.5 ,uCi of [G-3H]2-deoxyglucose, 0.05 ,uCi of [U-14C]sucrose, and various amounts of nonisotopic those of [6-'4C]glucose. Addition of latex parti- 2-deoxyglucose or inhibitors. After addition of la- cles, which stimulates hexose monophosphate beled carbohydrate, cells were further incubated for shunt activity (1), enhanced "4CO2 production 5 min at the reaction temperature. Since uptake as by the following extents: glucose, 2.6-fold; man- well as loss of labeled 2-deoxyglucose was found nose, 2.6-fold; galactose, 2.8-fold; fructose, 1.3- negligible at temperatures below 4°C (data not fold. Increases in "4CO2 evolution during phago- shown), reactions were stopped by placing tubes cytosis were not observed when '4C-labeled 2- with cells on ice and centrifuging at 3,000 x g at 0°C deoxyglucose, 3-0-methylglucose, or sucrose for 5 min. Supernatants were immediately aspir- ated, and pellets were transferred directly into scin- was employed. The latter carbohydrates were tillation vials with 10 ml of Bray solution. Pellets of not readily converted to "4CO2 by PMN. cells and supernatant fractions were counted with a That [U-'4C]sucrose was not taken up by Beckman CPM 100 liquid scintillation counter set PMN was further demonstrated by monitoring for double label counting. the radioactivity associated with pellets of cells. Quantities of PMN in each assay were deter- This radioactivity (approximately 900 cpm/106 VOL. 16, 1977 2-DEOXYGLUCOSE TRANSPORT IN LEUKOCYTES 191 TABLE 1. Conversion of '4C-labeled carbohydrate to 14CO2 by guinea pig PMNa 14CO2 produced (nmol/h/106 cells + SD) Carbohydrate P Resting Phagocytosing [1-14C]glucose 7.16 ± 1.57 18.5 ± 0.4 <0.01 [1-14C]mannose 2.06 ± 0.16 5.42 + 0.55 <0.01 [1-14C]galactose 0.60 ± 0.14 1.70 ± 0.10 <0.01 [1-14C]fructose 0.26 ± 0.02 0.34 ± 0.01 <0.02 [6-14C]glucose 0.14 ± 0.02 [U-14C]2-deoxyglucose 0.11 ± 0.02 0.11 ± 0.01 NS [U-14C]3-O-methylglucose 0.01 0.01 NS [U-14C]sucrose 0.03 0.04 NS a Cells (7 x 106 PMN) were incubated for 1 h at 37°C in 1.0-ml aliquots ofbuffer containing 10% guinea pig serum and 1.0 mM labeled carbohydrate (0.25 ,uCi). CO2 was collected from th3 start of each experiment on strips of Whatman 3 MM paper saturated with KOH and for 1 h after acidifying the medium. Each experiment was performed in triplicate. Significance levels, P, are given for comparison of values from resting cells to values from cells incub,ated with polystyrene latex particles. Abbreviations: SD, standard deviation; NS, no significance.

cells) did not change significantly over the similar levels of galactose and fructose was not course of a 2-h incubation. In contrast to 3H observed. Addition of 3-O-methylglucose or in- counts, all 14C counts could be removed from sulin had no significant effects on 2-deoxyglu- pellets of cells by washing with cold KRPS cose uptake, and the presence of insulin did not (data not shown). increase the inhibition by glucose. Time course for 2-deoxyglucose uptake. Experiments were then conducted using var- Data in Fig. 1 indicate the time course for ious 2-deoxyglucose levels at fixed concentra- uptake of 5.0 mM [G-3H]2-deoxyglucose into tions of glucose or mannose (Fig. 3A and B, guinea pig PMN. Uptake was essentially linear respectively). Both glucose and mannose were with time for the first 10 min, after which a competitive inhibitors of 2-deoxyglucose up- maximal value of 4.8 nmol/106 cells was take; the former giving a Ki of 2.67 + 0.32 mM reached. Intracellular levels of 2-deoxyglucose and the latter giving aKi of2.28 + 0.33 mM. All were calculated upon dividing uptake by the lines in Fig. 3 were fit to the data by the method intracellular water space of 0.42 1ul/106 PMN ofleast squares using a weighted computer pro- (7). An approximate twofold concentration of gram as previously described (5). sugar then became apparent (Fig. 1, right ordi- Effects of temperature and metabolic in- nate). Intracellular 2-deoxyglucose was 69.1 to hibitors. The velocity of 0.2 mM [G-3H]2-deoxy- 84.9% phosphorylated. Uptake of 2-deoxyglu- glucose penetration rose significantly with in- cose was linear with cell concentration up to 8 creasing temperature until 41°C (Fig. 4). Be- to 106 PMN per ml (data not shown). yond this point velocity decreased and was par- Kinetics of uptake. Values illustrated in ticularly sensitive to temperatures between 48 Fig. 2 were determined by stopping cell incuba- and 530C. tions after 5 min and measuring the penetra- The dependence of 2-deoxyglucose uptake tion of labeled 2-deoxyglucose under conditions upon temperature suggested that this process of varying substrate concentrations. Initial ve- relied, in part, upon metabolic activity. To test locities for uptake were therefore not directly this suggestion, PMN were preincubated for 30 measured but were calculated by dividing the min at 370C with various metabolic inhibitors, average nanomoles of 2-deoxyglucose per 106 and the rates of [G-3H]2-deoxyglucose uptake as cells by the 5-min time interval. Penetration of well as phosphorylation were measured (Table 2-deoxyglucose clearly followed saturation-type 3). In the presence of 2.0 mM iodoacetate, rates kinetics and yielded a Km and Vmax of 1.8 mM of both uptake and phosporylation were signifi- and 0.67 nmol/min per 106 cells, respectively, cantly reduced to 33 and 11% of the control when analyzed by the method of Lineweaver values, respectively. Preincubation of cells and Burk (17). with either 40 mM sodium fluoride or 0.2 mM Effects of heterologous carbohydrates on iodoacetate resulted in similar degrees of inhi- uptake. When PMN suspensions were incu- bition in both uptake and phosphorylation. bated with either 4.0 mM glucose or mannose However, mitochondrial effectors, such as anti- together with 0.2 mM [G-3H]2-deoxyglucose mycin A, potassium cyanide, and dinitrophe- (Table 2), the uptake of labeled sugar was sig- nol, did not show significant effects. Phlorizin, nificantly impaired. However, inhibition by N-ethylmaleimide, and p-chloromercuribenz- 192 LITCHFIELD AND WELLS INFECT. IMMUN. oate produced slight reductions of 2-deoxyglu- theophylline, 3 mM caffeine, 4 mM myo-inosi- cose uptake, but these were only significant tol, 5 mM colchicine, 1 mM 3',5'-cyclic adeno- when the former two agents were employed. In sine monophosphate (cAMP), 1 mM dibutryl subsequent experiments, cytochalasin B at 0.5 cAMP, 1 mM 3',5'-cyclic guanosine monophos- and 1.0 ug/ml also inhibited uptake by 30.3 phate (cGMP), and 1 mM dibutryl cGMP. Pre- and 35.7%, respectively. These differences were incubation of cells with polystyrene latex parti- significant at the P < 0.01 level. cles (200:1, particles:cell) or 0.5 mU of insulin A number of other agents were preincubated did not significantly affect uptake rates. Effects with cells for 30 min but were without signifi- of insulin were not altered by substituting cant effects on [G-3H]2-deoxyglucose uptake. Krebs-Henseleit bicarbonate solution for These included 10 mM sodium barbital, 3 mM KRPS. However, rates for [G-3H]2-deoxyglucose uptake were 30% lower when the former medium was employed. 15 ATP levels. Intracellular levels of ATP in -, 6.0 E guinea pig PMN were significantly lowered by the presence of 2.0 mM iodoacetate (Table 4). (0o 0 Conditions during the incubation of cells were identical with those employed during the inhib- 4O 0 - ; 10 N itor experiments (Table 3). .0 4.0 EC X Effects of heterologous carbohydrates on -j uptake by human PMN. The uptake of 0.2 mM [G-3H]2-deoxyglucose by human PMN was ap- 0.~~~~~~~~25.0 W proximately twofold greater than the uptake by c guinea pig PMN 0.027 versus 0.100 nmol/min a 1-- z per 106 cells). Despite this difference, inhibitors of uptake for human PMN (Table 5) were very similar to those for guinea pig PMN (Table 2). o 20 40 60 Galactose, fructose, and 3-O-methyglucose did TIME (min.) not significantly impair 2-deoxyglucose uptake FIG. 1. Time-course for uptake at 5.0 mM 2-deox- when present at a 20:1 inhibitor-to-substrate yglucose. Assays were performed in quadruplicate ratio, whereas glucose and mannose resulted in using 7 X 106 PMN per experiment at 370C. Line bars significant inhibition. Mannose in this case was indicate standard deviations from the mean. Intra- a slightly better inhibitor than glucose, and cellular concentrations (right ordinate) are derived some inhibition did occur in the presence of 30 from the uptake values (left ordinate). mM galactose.

w = I. 0

0 I- 0 E -J c w U NN

0 w

[2-Deoxyglucose] mM 1/[2-Dooxyglucos.] mMtf FIG. 2. Dependence of 2-deoxyglucose uptake on levels of external homologous carbohydrate. Velocity is plotted (A) against external substrate concentrations, and these values are replotted (B) according to Lineweaver and Burk (17). Each point is a mean of values from three experiments that were performed in triplicate. Line bars indicate standard deviation from the mean. VOL. 16, 1977 2-DEOXYGLUCOSE TRANSPORT IN LEUKOCYTES 193 TABLE 2. Effect of carbohydrate and insulin on The inability of guinea pig PMN to transport uptake of[G-3H]2-deoxyglucose by guinea pig PMNa or oxidize [14C]sucrose is in agreement with Uptake (% ± results on human PMN described by Englhardt Additions SD)5 and Metz (6) and with observations on rabbit None 100 + 13.1 alveolar macrophages described by Gee et al. Insulin 95.7 ± 4.9 NS (9). Both of these studies similarly employed Glucose 42.5 ± 10.1 <0.001 the exclusion of sucrose to correct for extracel- Glucose + insulin 44.5 ± 3.6 <0.001O lular space during hexose transport experi- Mannose 46.9 ± 4.2 <0.001 ments. The use of this technique also corrects Galactose 95.8 ± 7.5 NS for a nonspecific type of uptake encountered Fructose 95.8 ± 6.8 NS during phagocytosis. Esman refers to this as 3-O-methylglucose 87.0 ± 7.1 NS "piggy-back" phagocytosis or the "concomitant a PMN were incubated at 370C in 1.0 ml of buffer. engulfment of extracellular medium with the All additions were made at zero time. 2-Deoxyglu- phagocytosis of particles" (7). Such nonspecific cose was 0.2 mM, whereas levels of carbohydrate uptake increases the insulin space in pellets of and insulin were 4.0 mM and 0.5 mU/ml, respec- leukocytes by 4 to 6% (21) and, thus, could tively. P indicates levels of significance between result in elevated values for 2-deoxyglucose up- values with additions and values without additions. Abbreviations: SD, standard deviation; NS, no sig- take if [14C]sucrose was not also employed. nificance. When 5 mM [G-3H]2-deoxyglucose is pre- b Uptake rate without additions was 0.142 + 0.019 sented to guinea pig PMN, maximal intracellu- nmol/min per 106 cells; N = 6. lar levels of label approach 11.4 mM after 40 e No significance between values obtained with min of incubation (Fig. 1). Since 69 to 85% of glucose and values obtained with both glucose and this label is phosphorylated, the maximal in- insulin. tracellular levels of free 2-deoxyglucose can be calculated to range from 1.7 to 3.5 mM. These DISCUSSION values suggest that entry of 2-deoxyglucose oc- Although the stimulation of [1-_4C]glucose curs in the free form and that phosphorylation oxidation by PMN during phagocytosis has is necessary for 2-deoxyglucose concentration. been frequently reported (11, 12), studies on the These results are in accord with those ofEsman oxidation of other carbohydrates during phago- (7), who found free intracellular glucose in cytosis have not been previously made. Our PMN at low external glucose concentrations. observations (Table 1) demonstrate that [1- Whereas the data in Fig. 1 eliminate an ac- 14C]mannose, [1-14C]galactose, and [1_14C]_ tive transport system for 2-deoxyglucose up- fructose are also metabolized to 14CO2 and that take, those from Fig. 2 and 3 rule out simple this oxidative metabolism is stimulated by diffusion and indicate a facilitated transport phagocytosis. The degree of stimulation of [1- mechanism. Uptake of 2-deoxyglucose dis- 14C]glucose oxidation is comparable to values played saturation-type kinetics and was com- previously reported (12), and the rate of oxida- petitively inhibited by glucose and mannose, tion by phagocytosing cells agrees with the 20 which is suggestive of a common carrier-me- nmol/h per 106 cells observed by Stjernholm et diated transport system. Similar inhibition of al. (26). That a constant degree of stimulation 2-deoxyglucose uptake by glucose occurs in rab- (2.6- to 2.8-fold) occurred, when either labeled bit alveolar macrophages (9), and inhibition glucose, mannose, or galactose (but not fruc- by glucose and mannose, but not galactose and tose) was employed, strongly suggests that the fructose, is reported for rat diaphragm muscle former three hexoses are converted into a com- (14). Studies on guinea pig lymph node cells by mon intermediate, i. e., glucose-6-phosphate, Helmreich and Eisen (10) also imply a common prior to oxidation by the hexose monophosphate carrier mechanism for hexose transport; how- shunt. Support for this suggestion is threefold: ever, this mechanism appears to be more selec- (i) PMN contain a complete Leloir pathway and tive in PMN, since our results did not show are capable ofconverting galactose to glucose-6- competitive inhibition by fructose or 3-0-meth- phosphate (16, 18); (ii) 14C-labeled mannose is ylglucose. converted to lactate, via mannose-6-phosphate Further evidence for facilitated diffusion of 2- and fructose-6-phosphate, with the same label- deoxyglucose arises from the temperature de- ing pattern as lactate derived from 14C-labeled pendence of [G-3H]2-deoxyglucose uptake (Fig. glucose (8); and (iii) PMN are not gluconeo- 4) and from the effects of metabolic inhibitors genic and are not able to convert fructose-1- upon this process (Table 3). Since hexose phos- phosphate or fructose-1,6-bisphosphate to glu- phorylation is required for sequestering of la- cose-6-phosphate (21). bel, inhibition of phosphorylation by depleting 194 LITCHFIELD AND WELLS INFECT. IMMUN.

1/ [Substrate] mMg- FIG. 3. Inhibition of 2-deoxyglucose transport by glucose and mannose. Lineweaver-Burk plots for inhibition by glucose (A) and mannose (B) were generated from data obtained in four and three e-xperiments, respectively, each of which was performed in triplicate. Levels of inhibitor are indicated as follows: *, none; 0, 0.25 mM; A, 0.49 mM; O, 0.98 mM; and *, 1.96 mM. intracellular ATP would be expected to lower 4). These results, together with the lack of sig- the diffusion gradient for free 2-deoxyglucose nificant inhibition by potassium cyanide, anti- and, thus, impair the rate of 2-deoxyglucose mycin A, and dinitrophenol, further demon- entry. We observed significantly lower rates of strate that active glycolysis, and not oxidative entry when guinea pig PMN were incubated at phosphorylation, is the primary source of ATP temperatures below 370C, when an isotonic bi- in PMN. This agrees with previous observa- carbonate solution was substituted for KRPS, tions that PMN contain few mitochondria and and when cells were preincubated with 40 mM that inhibitors of glycolysis can depress phago- sodium fluoride or 2.0 mM iodoacetate. Prein- cytic function (25). cubation with the latter inhibitor not only im- The lack of an in vitro insulin effect on either paired uptake and phosphorylation to the 2-deoxyglucose uptake or the inhibition of 2- greatest extents but also significantly de- deoxyglucose uptake by glucose (Table 2) is creased the intracellular levels of ATP (Table consistent with our findings that the rate of VOL. 16, 1977 2-DEOXYGLUCOSE TRANSPORT IN LEUKOCYTES 195 carbohydrate entry was greater than the rate of TABLE 4. Effect ofiodoacetate and galactose on ATP phosphorylation. However, this also implies levels in guinea pig PMNa that phosphorylation of 2-deoxyglucose is not ATP affected by the presence of insulin. Similar re- AdditionsAdditions cls+S)cells(nmol/10'1±SD) P sults were obtained by Beck (2), who could not None 1.32 ± 0.46 find insulin sensitivity in leukocyte hexoki- lodoacetate (2.0 mM) 0.41 ± 0.33 <0.05 nase, by Englhardt and Metz (6), who did not Galactose (30.0 mM) 0.78 ± 0.27 NS observe an insulin effect upon glucose transport a PMN (15.2 x 106 cells) were incubated with or without additions for 30 min at 37°C. After incuba- w tion cells were immediately centrifuged at 37°C for 1 x g. Pellets were quickly frozen by '0 min at 3,700 0 0.14 - immersing tubes in liquid nitrogen. Cells were stored at -80°C until assay; see Materials and 0 Methods for assay conditions. P refers to levels of E significance between values with additions and val- 0.10I - ues without additions. Abbreviations: SD, standard n deviation; NS, no significance. 0 E 0.06 TABLE 5. Effect of carbohydrate on uptake of[G- 3H]2-deoxyglucose by human PMNa - Additions Uptake (% ± p 0.02 SD) w None 100 ± 20.1 I .-- I 20 30 40 50 Mannose (4.0 mM) 39.1 ± 7.8 <0.02 Glucose (4.0 mM) 50.8 ± 4.6 <0.02 TEMPERATURE (°C ) Fructose (4.0 mM) 78.2 ± 9.9 NS Galactose (4.0 mM) 78.2 ± 6.6 NS FIG. 4. Temperature dependence of0.2 mM 2-de- Galactose (30.0 mM) 64.3 ± 11.9 <0.05 oxyglucose uptake. Line bars indicate standard de- 3-O-methylglucose (4.0 98.8 ± 8.8 NS viations from the mean at values from five experi- mM) ments. a PMN (0.848 x 106 cells per ml) were incubated in TABLE 3. Effect of metabolic inhibitors on rates of 1.0 ml of buffer at 37°C. All additions were made at [G-3H]2-deoxyglucose uptake andphosphorylation by zero time. [G-3H]2-deoxyglucose was 0.2 mM, and guinea pig PMNa uptake rate without additions was 0.227 + 0.046 nmol/min per 106 cells: N = 5. P refers to levels of Uptake Phospho- significance between values with additions and val- rylation ues without additions. Abbreviations: SD, standard Additions (nmoll (nmol/ minlO1) minIlOr deviation; NS, no significance. PM) PMN) into human PMN, and by Helmreich and Eisen None 0.100 0.064 Iodoacetate (0.2 mM) 0.057 (A) 0.018 (10), who were unable to demonstrate an insu- Iodoacetate (2.0 mM) 0.033 (B) 0.007 lin effect upon glucose uptake into guinea pig Fluoride (40 mM) 0.064 (A) 0.017 lymph node cells. On the other hand, Luzzatto Antimycin A (1.0 ,ug/ml) 0.086 (C) (20) observed an increase in xylose penetration Cyanide (6.0 mM) 0.086 (C) 0.042 into leukocytes at a non-physiological level of Dinitrophenol (1.0 mM) 0.096 (C) 0.058 insulin (1 U/ml), and Kalant and Schucher (15) Phlorizin (10.0 mM) 0.069 (D) found an increased disappearance of glucose N-ethylmaleimide (0.2 0.070 (E) when leukocytes were incubated with 0.3 U of mM) insulin per ml. Levels of insulin used in our Chloromercuribenzoate 0.081 (C) studies (0.5 mU/ml) were only slightly higher (0.2 mM) than physiological. a PMN (2.81 x 106 cells per assay) were preincu- Although decreases in cell metabolism, as bated with or without additions for 30 min at 37°C. well as glucose uptake, have been noted in After this period [G-3H]2-deoxyglucose was added to concentrated PMN suspensions, we did not en- a final concentration of 0.2 mM, and uptake rates counter this during our experiments. This so- were measured. Each assay was performed five called "crowding effect" has been attributed to times, and levels of significance between values changes in extracellular pH as a result of lac- with additions and values without additions are in- inhi- dicated by (A) 0.01, (B) 0.005, (C) no significance, tate accumulation and to the subsequent (D) 0.05 and (E) 0.02. bition of phosphofructokinase activity (7). Al- 196 LITCHFIELD AND WELLS INFECT. IMMUN. though Englhardt and Metz (6) observed this in The phagocytic cell in host resistance. Raven Press, New York. their experiments with 7.6 x 107 PMN/ml dur- 2. Beck, W. S. 1958. The control of leukocyte glycolysis. J. ing a 1-h incubation, our experiments avoided Biol. Chem. 232:251-270. this effect by utilizing fewer cells and shorter (5 3. Berger, R. R., and M. L. Karnovsky. 1966. Biochemical min) incubation times. Therefore, lactic acid basis of phagocytosis. V. Effect of phago6ytosis on cellular uptake of extracellular fluid, and on the in- was not allowed to build up, and linearity be- tracellular pool of L-a-glycerophosphate. Fed. Proc. tween 2-deoxyglucose uptake and cell concen- 25:840-845. tration was established. This method also cir- 4. Drachman, R. H., R. K. Root and W. B. Wood. 1966. cumvented any problems associated with exces- Studies on the effect of experimental nonketotic diabetes mellitus on antibacterial defense. J. Exp. Med. sive 2-deoxyglucose-6-phosphate accumulation 124:227-240. which could non-competitively inhibit the hexo- 5. Dunne, C. P., J. A. Gernt, K. W. Rabinowitz, and W. A. kinase reaction (14). Wood. 1973. The mechanism of action of 5'-adenylic Similarities between the uptake of 2-deoxy- acid-activated threonine dehydrase. J. Biol. Chem. 248:8189-8199. glucose by guinea pig PMN and the uptake by 6. Englhardt, A., and T. Metz. 1971. Investigations on human PMN isolated from peripheral blood are glucose uptake in isolated human leukocytes from apparent from comparing values in Table 2 to normal and diabetic subjects. Diabetologica 7:143- those in Table 5. Although the rate of uptake 151. 7. Esman, V. 1972. The diabetic leukocyte. Enzyme 13:32- with human cells was approximately twofold 55. greater than the rate with guinea pig cells at 8. Esman, V., E. P. Noble, and R. L. Stjernholm. 1965. 37°C, uptake in both cases was substantially Carbohydrate metabolism in leukocytes. Acta Chem. inhibited by the presence of glucose or man- Scand. 19:1672-1676. 9. Gee, J. B. L., A. S. Khandwala, and R. W. Bell. 1974. nose. Galactose, fructose, and 3-O-methylglu- Hexose transport in the alveolar macrophage: kinet- cose at 4 mM did not significantly impair 0.2 ics and pharmacologic features. J. Reticuloendothel. mM 2-deoxyglucose entry into either cell type. Soc. 15:394-405. However, uptake was slightly decreased when 10. Helmreich, E., and H. N. Eisen. 1959. The distribution and utilization of glucose in isolated lymph node human PMN were incubated with 30 mM ga- cells. J. Biol. Chem. 234:1958-1965. lactose fhis latter effect was not observed with 11. Karnovsky, M. L. 1962. Metabolic basis of phagocytic guinesA pig PMN under similar conditions but activity. Physiol. Rev. 42:143-168. was found, together with lower ATP levels (Ta- 12. Karnovsky, M. L. 1974. The biochemical basis of the functions of polymorphonuclear leukocytes and mac- ble 4), after a 30-min preincubation with ele- rophages, p. 83-93. In L. Brent and J. Holborrow vated galactose. It therefore appears that galac- (ed.), Progress in immunology II, vol. 4. North- tose may impair 2-deoxyglucose uptake by Holland Publishing Co., New York. blocking its phosphorylation rather than by 13. Khandwala, A. S., and J. B. L. Gee. 1974. Inhibition of 2-deoxy-D-glucose transport by theophylline, caffeine competing with its uptake. Although competi- and papaverine in alveolar macrophages. Biochem. tion in this case with human PMN cannot be Pharmacol. 23:1781-1786. eliminated per se, it seems unlikely that ele- 14. Kipnis, D. M., and C. F. Cori. 1959. Studies of tissue vated levels ofgalactose, as encountered during permeability. V. The penetration and phosphorylation of 2-deoxyglucose in the rat diaphragm. J. Biol. galactosemia, could impair phagocyte function Chem. 234:171-177. by competing with glucose uptake. Thus, im- 15. Kalant, N., and R. Schucher. 1962. Glucose utilization paired phagocytic activity in the presence of and insulin responsiveness of leucocytes in diabetes. galactose (18) may be primarily attributed to Can. J. Biochem. Physiol. 40:899-903. 16. Kalant, N., and R. Schucher. 1963. Concentration of other factors, such as (i) the presence of a futile galactose by leukocytes. Can. J. Biochem. Physiol. adenosine triphosphatase cycle or (ii) the intra- 41:849-858. cellular accumulation of galactose or one of its 17. Lineweaver, H., and D. Burk. 1934. The determination metabolites with subsequent inhibition of gly- of enzyme dissociation constants. J. Am. Chem. Soc. 56:658-666. colysis (18). 18. Litchfield, W. J., and W. W. Wells. 1976. Inhibitory action of D-galactose on phagocyte metabolism and ACKNOWLEDGMENTS function. Infect. Immun. 13:728-734. This investigation was supported by Public Health Ser- 19. Lowry, 0. H., and J. V. Passonneau. 1972. A collection vice Grant AM 10209 from the National Institute of Arthri- of metabolite assays, p. 151-152. In A flexible system tis and Metabolic Diseases. of enzymatic analysis. Academic Press Inc., New W. J. L. was a Public Health Service predoctoral trainee York. of the National Institute of General Medical Sciences (GM 20. Luzzatto, L. 1960. Effects of insulin on xylose transport 1091). in human leukocytes. Biochem. Biophys. Res. Com- mun. 2:402-406. LITERATURE CITED 21. Noble, E. P., R. L. Stjernholm, and L. Ljungdahl. 1961. Carbohydrate metabolism in leukocytes. III. 1. Bachner, R. L. 1975. The growth and development of Carbon deoxide incorporation in the rabbit polymor- our understanding of chronic granulomatous disease, phonuclear leucocyte. Biochim. Biophys. Acta p. 173-195. In J. A. Bellanti and D. H. Dayton (ed.), 49:593-595. VOL. 16, 1977 2-DEOXYGLUCOSE TRANSPORT IN LEUKOCYTES 197

22. Phillips, H. J. 1973. Dye exclusion tests for cell viabil- and V. D. Register. 1973. Role of sugars in human ity, p. 406-408. In P. F. Kruse and M. K. Patterson, neutrophilic phagocytosis. Am. J. Clin. Nutr. Jr. (ed.), Tissue culture methods and applications. 26:1180-1184. Academic Press, Inc., New York. 25. Sbarra, A. J., and M. L. Karnovsky. 1959. The biochem- 23. Rabinowitz, Y. 1964. Separation of lymphocytes, poly- ical basis of phagocytosis. I. Metabolic changes dur- morphonuclear leukocytes and monocytes on glass ing the ingestion of particles by polymorphonuclear columns, including tissue culture observations. Blood leukocytes. J. Biol. Chem. 234:1355-1362. 23:811-828. 26. Stjernholm, R. L., C. P. Burns and H. J. Hohnadel. 24. Sanchez, A., J. L. Reeser, H. S. Law, P. Y. Yahiku, R. 1972. Carbohydrate metabolism by leukocytes. En- E. Willard, P. J. McMillan, S. Y. Cho, A. R. Magie zyme 13:7-31.