glucose Uptake and Metabolism in Splenocytes from Diabetic and ...

Vol. 266, No. 6, Issue of ‘February 25, pp. 3675-3681, 1991 Printed in U.S.A.

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Enhanced 2-Deoxy-~-glucoseUptake and Metabolismin Splenocytes from Diabetic and Diabetes-prone BB rats FURTHER EVIDENCE TO SUPPORT PRIOR

IN VIVO ACTIVATION* (Received for publication, June 29, 1990)

Catherine J. Field$, Reuben Chayoth,Marie Montambault, andErrol B. Marlisss From the McGill Nutrition and Food Science Centre, The Royal Victoria Hospital, Montreal, Quebec H3A IAI, Canada

Glucose metabolism in splenocytes from the BB rat diabetic syndrome with many homologies with the human was studied for the presenceof abnormalities in [‘“C] disease, and has become a widely studied animal model. It is 2-deoxy-~-glucose (2-dGlc) uptake, [U-’4C]glucose characterized by profound immunological defects, including conversion to“COZ, and the productionof lactate and T-cell lymphopenia affecting both the CD4 (helper) andCD8 pyruvate. Cells were studied freshly isolated (“rest- (suppressor) phenotype (13, 14). It is functionally immunoing”), and following culture both unstimulated (con- deficient with delayedrejection of skin allografts and inA (ConA) or creased susceptibility toinfection, reduced mixed lymphocyte trol) and stimulated with concanavalin phorbol myristate acetate (PMA) + ionomycin. Both reactions, and responses to T-cellmitogens (15-17). The BB resting and controlcells from diabetic (BBd) and dia- rat also has abnormalities characteristic of autoimmunity: 1) betes-prone (BBdp) rats transported more ( p < 0.05) the presence of insulitis (mononuclear cell infiltration into 2-dGlc than did cells from nondiabetes-prone (BBn) the islets with selective (3 cell destruction); 2) an association rats. Consistent with priorin vivoactivation, sustained in vitro, lactate production was higher ( p < 0.05) with the major histocompatibility complex type RT1“; 3) the under control conditions BBd in and BBdp than inBBn modification or prevention of the syndromeby treatment with cells. Lactate production increased less with ConA and antilymphocyte serum and monoclonal antibodies, immunosilica treatment todeplete PMA + ionomycin in both BBd and BBdp than in BBn suppression, neonatal thymectomy, cells. PMA + ionomycin increased 2-dGlc uptake as macrophages, or bone marrow transplants from normal rats; much in BBd and BBdp cells as in BBn cells. Elevated 4) an altered humoral immune function with autoantibodies rates of pyruvate production were observed in BBd directed against islet cells, spleen cell surface determinants, cells under resting, control, and (especially) ConA con- and other tissues; and 5 ) young previously normoglycemic ditions, suggestingan abnormality in pyruvate converdiabetes-prone rats (BBdp)’become diabetic following injecsion to lactate. Few changes were observed in 14C02 tion of mitogen-stimulated immune cells from diabetic rats production. The presence of similar abnormalities in (BBd) (reviewed in Refs. 16-22). These observationstogether BBdp cells to those of the BBd cells suggests that the suggest that BBdp and BBd rats possess an “activated” imdiabetic state is not causal, and the absence of an in mune system, at least insofar as the targeted islet cells are vitro effect of 15 mmol/liter glucose in BBn cells fur- concerned. ther tends to exclude hyperglycemiaas a cause of these We have recently reported enhanced glucose metabolism in alterations. splenocytes of BBd animals compared to those of nondiabetes-prone (BBn) rats, thatis consistent with the concept of prior in vivo activation (22). This abnormality in nondiabeticBBdphasnot been sought previously. The present An early event of mitogen stimulation in lymphocytes is a study was conducted to: 1) further examine glucose metabodramatic enhancement in the rates of glucose transport (1-6) lism in both resting and mitogen-stimulated splenocytes from and metabolism (6-10). Increased anaerobic glycolysis, the BBd and BBdp as compared to that of BBnanimals; 2) conversion of glucose to lactate, may be important for both identify a possible defectin glucose metabolism to explain the the initiation and continuation of the highly active metabolic subnormal proliferative response to mitogen stimulation prestate induced by immune stimuli (4, 8) as blocking glucose viously observed in cells from BBd animals; and3) study the transport completely obliterates the mitogenic response (4, effect on glucose metabolism of a mitogen stimulus, thecom11, 12). Despite these observations, little isknown about the bination of phorbol myristate acetate (PMA)with ionomycin role of glucose transport and metabolism by lymphocytes in that was recently found to increase but notcompletely restore disease states, including those with immunological abnormal- the DNA synthetic responses in BBd splenocytes.’ ities. The BB rat spontaneously develops an insulin-dependent EXPERIMENTALPROCEDURES * These studies were supported by Grant MT-6540from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $Recipient of a Postdoctoral Fellowship from the Medical Research Council of Canada. 5 To whom reprint requests should be addressed: Royal Victoria Hospital, 687 Pine Ave. W., Montreal, Quebec H3A 1A1, Canada. Tel.: 514-843-1665.

Animals and Cell Preparation-Male and female BBdp, BBd and BBnrats were obtained from Dr. P. Thibert(Animal Resources Division, Health Protection Branch, Ottawa, Ontario).Animals were The abbreviations used are: BBdp, diabetes-prone BB rats;BBd, diabetic BB rats; BBn, nondiabetes-prone BB rats; PMA, phorbol 12-myristate13-acetate;HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; ConA, concanavalin A. M.-D. M6troz-Dayer, E. B. Marliss, C . J. Field, and P. Poussier, unpublished data.

3675

3676

Glucose Metabolism in Splenocytes from BB Rats

housed in a light(12-hlight-dark cycles) and climate-controlled figures were used for resting and culturestudies; cells from the same facility (humidity 70%, temperature 20 "C) in laminarflow hoods and spleen were used for as many different assays as permitted by the fed ad libitum Purina Rat Chow (Ralston Purina, Woodstock, On- requirements of the different endpoints measured. Results are pretario, Canada). To maintain normal weight gain and minimize gluS.E. from experiments performedover 1-5 sented as the mean cosuria, diabetic animals (studied 4-14 days postonset) were main- different days. Within the conditions, cells from each animal were tained on single daily subcutaneous injections (2-5 units) of insulin studied a t 4 and 15 mmol/literglucose. One-way analysis of variance (Iletin, ultralente insulin, Eli Lilly, Indianapolis, IN). Immediately was performed using a Primer Biostatistics (McGraw Hill Co., Monafter exsanguinationby cardiac puncture under light ether anesthesia, treal) package on a Hewlett-Packard Vectramicrocomputer (Sunnysplenectomies were performed and splenocytes prepared aspreviously vale, CA), and significant differences between groups was identified described (21) in a pH 7.4, Krebs-Ringer HEPES (KRH)buffer with by the Newman-Keuls multiple-range testat the ( p < 0.05) level of 0.5% (w/v) bovine serum albumin (22). For cell culture experiments, significance. splenocytes were prepared under sterile conditions using RPMI 1640 (10 mmol/liter glucose) supplemented with4% (v/v) heat-inactivated RESULTS pooled BBn rat serum, 2.5 pmol/liter2-mercaptoethanol(Sigma), 2-Deoxy-D-glucose Uptake-Time course analysis of 2-dGlc 4000 pmol/literglutamine,penicillin(100 units/ml), streptomycin (100 rg/ml), and25 mmol/liter HEPES. RPMI and all supplemented uptake indicated a linearuptake for the resting conditionand ingredients were obtained from Flow Laboratories (Mississauga, On- curvilinear uptake for the control, ConA, and PMA ionotario, Canada). Cell viability was assessed by trypan blue exclusion. mycin conditions. As the 5-min time pointused in the assay All assays were performed in duplicate or triplicate as indicated and falls on the curvilinear portion of the curve, the increased data expressed per lo6 viable cells. uptakes of 2-dGlc in control cells and those stimulated with Glucose Conversion t o Lactate, Pyruuate, and C02-Glucose oxidaionomycin are likely to be underestimates tion was determined by a modification of the methodof Rodbell (23). ConA or PMA Splenocytes (2-8 X 106/ml) freshly isolated (resting) or from culture of actual uptake rates; this must be taken into account in (fmitogens) were incubated in1 ml of KRH buffer (described above) interpreting estimates of uptake kinetics. In the resting and in siliconized testtubes (10 ml)containing 0.3pCi/ml D-[u-"c] control states, cells from BBd and BBdp transported more 2glucose (Du Pont-New England Nuclear) and unlabeled glucose to dGlc than did cells from BBn (Fig. 1). The difference in achieve final glucose concentrations of 4 and 15 mmol/liter. Before 500-pl uptake ratesbetween BBn and BBd in thiscondition appears capping vials were gassedbriefly with Oz, andanempty primarily due to a higher VmaXin BBd cells (Fig. 2). It is microcentrifuge tube was suspended in each tube. Tubes, shaking interesting to note that the K , for BBd in the resting condicontinuously, were incubated 1-5 h at 37 "C, and,justpriorto incubation being terminated by the injection of 250 p1 of 2 N sulfuric tion appearsgreater than thatof BBn. However, the apparent acid through the stopper into themedium, 200 pl of methylbenzeth- nonlinear response by BBd cells both resting (Fig. 2 A ) and onium hydroxide (Sigma) was injected through the stopper into the ConA (Fig. 2 0 ) does not allow for an accurate estimate of microcentrifuge tube. Shaking was continued for a further 60 min to trap all 'TO,evolved. The microcentrifuge tubes were then placed in transport kinetics. With PMA ionomycin no difference was vialscontaining acidified (1% v/v aceticacid)scintillation fluid noted in the rate of 2-dGlc uptake among groups (Fig. 1). However, when stimulated with ConA, uptake rates were (Universol CocktailTM,ICN) and counted in a 1217 LKB Rackbeta counter (Pharmacia LKBBiotechnology Inc.). The scintillation fluid significantly higher in BBncells than inBBd and BBdp (Fig. was acidified to result ina neutral pH once the methylbenzethonium 1).The differences in uptake between BBn and BBd appear hydroxide was added, in order to minimize chemiluminescence. Sam- primarily due to lower K, for BBn cells (Fig. 2). ples towhich sulfuric acid was added beforethe additionof cells were Culture per sehad no significant effect in 2-dGlc uptake in used as blanks. All assays were performed in duplicate. The incubation media were stored at -20 "C for later assay of lactate and pyruvate by automated enzymaticfluorimetric procedures (24) using a a two-channel TechniconAutoAnalyzer (Technicon Inc., Tarrytown, NY) and two Turner 430 spectrofluorophotometers (Sequoia-Turner Corporation, Mountain View, CA). 2-Deoxy-D-glucoseUptake-To aliquots of cell suspensions (250 111 each) containing 5-15 X lo6 cells, 5 p1of a solution containing 0.25 NS pCi of ["C]2-dGlc (Du Pont Canada, Montreal) with enough unlabeled 2-dGlc (Sigma) to achieve a final concentration of 3.4 mmol/ liter was added. Tubes were incubated in triplicate in a shaking water bath at 37 "C and transport was stopped after 5 min with 0.5 ml of cold 0.5 mmol/liter phloretin (Sigma). Uptake a t time zero was assessed by adding the described 2-dGlc to cells to which phloretin had already been added. From each tube200-111 aliquots were rapidly added to three500-pl microcentrifuge tubes containing 200 p1 of 10% w/v bovine albumin in the KRH buffer described above (pH 7.4). Tubes were spun for 15 s in a microcentrifuge (model 235B, Fisher), the supernatant aspirated, and the three microcentrifuge tubes for each assay cut tocollect the pelletfor counting as described above in 5-ml scintillation vials containing 3 ml of scintillation fluid. Uptake was assessedby subtracting the amount of 2-dGlc transported at time zero from that at 5 min and expressed asnmol/min/106 cells. Culture Conditions-Splenocytes from BBn, BBd, and older BBdp " n d p n d P n d p n d p (120-180 days) ratswere cultured (2.0 X lo6cells/ml in 100-ml flasks) ( 2 8 7 1 7 7 1 0 4 6 7 1 0 8 7 a t 37 "C in the RPMI mediumdescribedabove withorwithout PMA+lono Con A Resting Control concanavalin A (ConA, 5 pg/ml, Sigma) for 96 h, or with or without FIG. 1. The mean k S.E. rate of 2-dGlc uptake (3.4 mmol/ phorbol myristate acetate (PMA, 20 ng/ml, Sigma) plus ionomycin (0.5 rmol/liter, Calbiochem) for 48 h. The flasks were placed in an liter 2-dGlc) by splenocytes from BBn (m, n ) , BBd (R, d ) , and incubator (Forma Scientific, Marietta, OH), with ahumidified at- BBdp (0,p ) when freshly isolated (Resting),cultured unstimmosphere, a t 37 "C in the presence of 5% CO,. Cells were collected ulated 2-4 days (Control),or cultured 2 days with PMA + and washed two times in KRH buffer before assaying for glucose ionomycin or 4 days withConA. Bars thatdo not sharea common metabolism or 2-dGlc uptake as described above. As no significant letter within each condition are significantly different ( p < 0.05). differences were observed betweenthe production ratesof any metab- Bars that do not share a common symbol for a given animal group olites measured at 2 and 4 days in cells cultured without mitogens indicate significantdifferences ( p < 0.05) between culture conditions (Control, PMA + lono, C o d ) . The number of animals is indicated (control), the datawere combined. below the bars. lono, ionomycin. Statistical Analysis-The numbers of animals indicated on the

*

+

+

+

1

I

3677

Glucose Metabolism in Splenocytes from BB Rats Resting

B. Control

A.

is1

IS1

C. PMA*iono

D. Con A

FIG. 2. Hanes-Woolf plots of 2-dGlc uptake forBBn (*) and BBd (0)splenocytes. Values represent the mean of four replicates performed on a pool of splenocytes from five BBn and five BBd animals. A, resting; B , control; C, +PMA ionomycin; D, +ConA. [SI, 2-dGlc concentrations measured at 0.1,0.5, 1.0,5.0,10.0,and 20.0 mmol/liter; u, uptake of 2-dGlc at 5 min measured in nanomoles/min/106 cells.

+

Control

Resting BBd BBn

K , (millimoles/liter)

vmax (millimo1es/min/lO6)

4.2 0.18

BBd BBn

7.0 0.41

BBn and BBdp cells; however, a significantly ( p < 0.05) higher transport rate was observed for control BBd cells as compared to resting, possibly the result of both a decrease in K , and an increase in V,,, (Figs. 1 and 2). The rates of 2dGlc uptake with mitogen stimulation (PMA ionomycin or ConA) were greater than control (unstimulated) for BBn and BBd, but not BBdp cells (Fig. 1).For BBd cells, compared to the control condition, ConA appeared to increase both the VmaXand the K, of 2-dGlc uptake. For BBn cells, ConA stimulation increased the Vmarand decreased the K, (Fig. 2). Stimulation with PMA ionomycin appeared to increase transport for both BBn and BBd cells by increasing the V,,,,,. Uptake rates with PMA ionomycin for BBn cells were less than with ConA. Glucose Conversion toC02-Except for a higher production rate by BBdp cells at 15 mmol/liter glucose in the resting condition, the rateof 14C02production from D-[U-'4C]glucose did not differ among groups or change with mitogen stimulation (Fig. 3). Lactate Production-No differences in lactate production

+

+ +

2.7 0.21

PMA + Ion0 BBd BBn

2.5

2.8

0.68

0.92

Con A BBd BBn

2.5 0.94

0.9

5.5

0.90

0.82

were observed in the resting condition; however, in the control condition BBd and BBdp cells produced significantly ( p < 0.05) more lactate than did BBn cells (Fig. 4). With PMA ionomycin, BBn cells produced more lactate than did BBd cells. BBdp cells incubated with PMA + ionomycin produced lactate at rates not different from BBn cells. With ConA the rates of lactate production for both BBd and BBdp were less than for BBn cells. Lactate production for all three groups was significantly ( p < 0.05) higher in control as compared to resting cells. For all three groups, incubation with both mitogens increased lactate production over that of control cells, to a significantly greater extent with PMA + ionomycin (Fig. 4). Pyruvate Production-In the resting and control conditions, BBd cells produced more pyruvate than BBn cells (Fig. 5). In the resting condition the rate of pyruvate production by BBdp was significantly greater than by BBn but less than BBd cells. In the control condition pyruvate production by BBdp cells was less than BBd but not different from that of BBn cells. No differences in pyruvate production were ob-

+

3678

Glucose Metabolism in Splenocytes from BB Rats A

\

.

n d pn d pn d pn d

I

B

n d pn d pn d pn d p

ndp ndp ndp nd 1 8 1 2 1 11 47 4

Resting

6 6 4 10 6

Control PM* +lonoCon A

FIG. 3. Glucose conversion to COS (mean f S.E.). Conversion at (4 mmol/liter glucose ( A ) and 15 mmol/liter glucose ( B ) by n ) , BBd (@, d ) , and BBdp (0,p ) for the splenocytes from BBn (a, conditions described in Fig. 1. Bars that do not sharea common letter withineachconditionare significantly ( p < 0.05) different. No significant differences were found between culture conditions within each animal group. The number of animals is indicated below the bars.

1 0 1 1 1 21 4 7 7

0 5 4 107 5

Resting Control

,:Yl”

Con A

FIG. 4. Lactate production (mean f S.E.). Production a t 4 mmol/liter glucose ( A ) and 15 mmol/liter glucose ( B )by splenocytes from BBn (a, n ) ,BBd (B,d ) , BBdp ( Q p ) for the conditionsdescribed in Fig. 1. Bars that do not share a commonletter withineach condition are significantly ( p < 0.05) different. N S indicates no significant differences. Bars that do not share a common symbol for a given animal group indicatesignificant differences ( p < 0.05) between culture conditions (Control, PMA + ionomycin, ConA ). The number of animals is indicated below the bars. DISCUSSION

+

served among groups with PMA ionomycin; however, with ConA the rate of production was %fold greater for BBd than BBn or BBdp cells. Culture conditions (resting versus control) significantly increased pyruvate production for BBn and BBd cells. As compared to control, no significant differences were observed in the rateof pyruvate production for BBn or BBdp cells with either mitogen (Fig. 5). For BBd cells, compared to control, PMA ionomycin reduced (2.5-fold) and ConA increased (1.5-fold) pyruvate production. LactatelPyruvate Ratio-In the resting condition, the lactate/pyruvate ratio was lower for BBd than BBn cells (Fig. 6). A t 4 mmol/liter glucose the lactate/pyruvate ratio for resting BBdp cells was similar to thatof BBd cells and, at 15 mmol/liter glucose, similar to thatof BBn cells. In thecontrol condition, the lactate/pyruvate ratio for BBdp cells was greater than for BBn and BBd cells. Incubation with either mitogen resulted in lactate/pyruvate ratiosfor BBd that were less than for BBn cells. For BBdp cells, incubation with PMA + ionomycin resulted in alactate/pyruvate ratio midway between that of BBn and BBd cells; however, with ConA the lactate/pyruvate ratio of BBdp was less than thatof BBn and not different from that of BBd cells.

+

Enhanced glucose transport activity and metabolism have been associated with mitogen-induced anabolic activity such as the synthesis of protein, RNA, and DNA in cells of the immune system (2, 8). Based on our findings of enhanced spontaneous proliferation of certain T (W3/13’)-cells in culture’ that have the ability to passively transfer diabetes (25) and the identification of “activated” cells by demonstration of certain cell surface markers (26),the concept of prior in viuo activation of BBd cells has been proposed. The enhanced rate of 2-dGlc uptake observed in cells from both BBd and BBdp animals, compared to BBn, in the resting state supports the concept of prior in vivo activation of at least some of the multiple different cell types known to be present (17, 26, 27).2 The enhanced 2-dGlc uptake rate by unstimulated BBd cells appears to be due to an increase in the V,,, (Fig. 2, A and B ) . The enhanced transport rate by cells from both BBd and BBdp animals was sustained in these cells when cultured withoutmitogen stimulation by 2-4 days (control). Similarly, lactate, the major product of glucose metabolism in lymphocytes (6-8), was also observed to be higher in BBd and BBdp than in BBn cells in the controlculture condition. (Although no significant differences in lactateproduction in the resting state were found between groups, we have previously found that when the incubation time is in-

3679


I

b

A

A

b

“1

b

NS

n d b

B

n d p n d p n d p n d p 1 4 9 1 2

9

Resting Control

n d pn d pn d pn d p

4

7

4

6

4

PMA+

lono

9

6

4

Con A

FIG. 6. Lactate/pyruvateratio(mean Z SE). Ratios a t 4 mmol/liter glucose ( A )and 15 mmol/liter glucose ( B ) in splenocytes from BBn (M, n ) , BBd (N, d ) , and BBdp (0,p ) for the conditions described in Fig. 1. Bars that do not share a common letter within each condition are significantly ( p < 0.05) different. The number of animals studied is indicated below the bars.

to those from BBn and BBd (Fig. 1).The BBdp animalsused in this experiment,aged 120-180 days, were beyond the usual age of development of overt diabetes. Such animals may have achieved an equilibrium between the susceptibility to autoimmunity and responses mounted to “control” the autoaggressive process. Mitogen-inducedlymphocyte transformations provide a useful model for the studyof the metabolic events associated with antigen stimulation of lymphocytes. Consistent with previous reports in lymphocytes and thymocytes using phycreased beyond 2 h (3-5 h) significantly greater amounts of tohemagglutinin (8) and ConA (2, 5, 9), mitogen stimulation lactate are produced by BBd compared to BBn resting cells significantly ( p < 0.05) enhanced the rates of 8-dGlc uptake (21).) Lactate production was increased by cells of all three (Fig. 1) and lactate production(Fig. 4) by cells from BBn and BBdcells boththe mitogenic groups during culture, and pyruvate production was increased BBd animals. InBBnand stimuli used appeared to increase the VmaX (Fig. 2, C and D). by BBn and BBd cells (control uers‘sus resting conditions). One possible reason for these changes during culture could be ConA, but not PMA + ionomycin, may also have reduced the an alteration in the proportions of cell types present, infavor K , of transport by BBn cells. For BBdcells PMA ionomycin of those more metabolically active. The role of in uiuo hyper- did not change theK,,, of transport, whereas ConA increased glycemia in the diabetic state can beexcluded as an explana- it. These results suggest the mechanismof increased transport tion, because: 1)increasing thein uitro glucose concentration may be different for BBn and RBd cells. A poor mitogenic response t o T-cell mitogens such as ConA is a well defined t o 15 from 4 mmol/liter did not consistently increase the glucose metabolites measured; 2)maintaining culture medium characteristic of lymphocytes from BBd and BBdp animals glucose concentration a t 20 mmol/liter did not significantly (16, 17, 27, 29, 30). The failure to respond to ConA is illusincrease subsequent lactate or pyruvate production by BBn trated further in the present study by the magnitude of the cells (data not shown); and 3) glucose metabolism of cells increase from the control condition in 2-dGlc uptake (2- us. from BBn rats rendered diabetic by streptozotocin was pre- 7.5-fold) and lactate production (2- us. 5-fold) by BBd cells as compared to BBn.Although glucose metabolism by BBdp viously reported tobe unaltered (28). a role cells was not measured after ConA stimulation at 4 mmol/ Although it is well established that autoimmunity has in thepathophysiology of the BB rat diabetes(16, 17,19, 20), liter glucose, both the rate of 2-dGlc uptake and the amount the mechanism(s) underlying the autoimmunity and the idenof lactate produced (at 15 mmol/liter glucose) were found to tity of the specificcells involved remains unproven. It is be similarto thoseof BBd cells (Fig.4). The observation, that noteworthy that in the resting stateeven an greater rateof 2- mitogen stimulation had as a major metabolic effect an indGlc uptakewas found in cells fromBBdp animals compared crease in lactateproduction, suggests that the augmented FIG. 5. Pyruvate production (mean -C S.E.). Production a t 4 mmol/liter glucose ( A ) and 15 mmol/liter glucose ( B )by splenocytes d ) , and BBdp (13,p ) for the conditions from BBn (m,n ) , BBd (E, described in Fig. 1. Bars that do not share a common letter within each condition are significantly ( p < 0.05) different. N S indicates no significant differences. Bars that do not sharea common symbol for a given animalgroupindicatesignificant differences ( p < 0.05) between culture conditions (Control, PMA + ionomycin, ConA). The number of animals is indicated below the bars.

+


3680

glucose metabolism that occurs flows largely toward lactate. It is well established that one of the earliest effects of mitogen action on lymphocytes is an enhancementof glucose transport (1, 9, 30-32). However, the mechanisms by which the primarily T-cell mitogen ConA and the nonspecific mitogens PMA ionomycin increase glucose transport have not been defined. As noted above, in normal BBn cells, 96 h of culture with ConA (Fig. 2C) appeared to increase both the Vmax(4-fold) and decrease the K, @-fold) of 2-dGlc uptake compared to control conditions (Fig. 2B). Short term incubations (30-60 min) of bovine lymphocytes (33) or human peripheral blood monocytes (4) with another T-cell mitogen, the lectin phytohemagglutinin, have been reported to increase the VmaXwithout a significant change in K,. Whether this difference isdue to the use of different cell populations, duration of exposure to culture conditions, or specific differences in mechanism due to phytohemagglutinin versus ConA remains to be established. It has been suggested that ConA enhances 3-0-methylglucose transport by activating the glucose transporter (1).Compared to the control condition for BBn cells, PMA ionomycin appeared to increase transport primarily by an increase in the VmaXwith no change in K,,,. Recently it was reported that when thymocytes were incuof 3-0-methylglucose bated (60 min) with PMA the initial rate transport was increased by an increased V,,, with no change in K,,, (4) and 2-dGlc uptake increased by a decrease in K,,, but no change in V,,, (32). With an elaborate experimental design these authors concluded that short term PMA incubation may increase the coupling coefficient between the membrane glucose transporter and the hexokinase enzyme. However, the effect of long term exposure sufficient to produce proliferation of the responsive cells, as in the present study, has not been analyzed in this manner. Thus, we are unable to distinguish if increased 2-dGlc uptake is due to increased glucose transport, increased hexokinase activity,or both. Cell activation represents a complicated series of events that involves changes initially in the plasma membrane followedby the cytosol, mitochondria, and nucleus. One approach to identifying the cell surface structures that initiate T-cell activation isto “bypass” the cell structures involved in the activation by stimulating the cell with pharmacological agents known to activateparticular signal pathways. The phorbol ester PMA acts synergistically with the Ca2+ ionophore ionomycin, to activate protein kinaseC (via phosphorylation of serine and threonineresidues) and toincrease intracellular calcium concentrations. However, additional effects of these compoundson otherpathways suchas phosphatidylinositol turnover have been reported (34). In the present study in BBn and BBd cells, stimulation with PMA + ionomycin increased 2-dGlc uptake over control, and significantly greater amounts of lactate were produced than by stimulation with ConA for all three groups of cells. Although also increased, the amount of lactate produced by BBd cells with PMA ionomycin remained less than thatproduced by BBn cells. This observation is consistent with our finding that PMA ionomycin greatly improves the reduced [3H]thymidine incorporation by BBd splenocytes and T-lymphocytes.’ However, PMA is known to alter both accessory cell (Blymphocyte and macrophage) function as well as that of Tcells (35). Therefore, the stimulation of the accessory cells present in the splenocyte preparation may have contributed to the increase in the overall responses. The rate of lactate production by BBdp cells fell midway between that of BBn and BBd, suggesting that theabnormal mitogen response also is present but perhaps not to the same extent.

+

+

+ +

The higher levels of pyruvate observed for BBd cells compared to BBn and BBdp cells (Fig. 5) is a surprising observation, especially the 4-fold increase despite a smaller increase in lactateproduction with ConA stimulation. For pyruvate to be reduced to lactate via lactate dehydrogenase, sufficient NADH must be available, a situation obtained in the stimulated BBn cells whose lactate/pyruvate ratioincreased 6-10fold. The increased pyruvate levels in BBd cells (resting, control, and with ConA) resulted in eithera markedly smaller or noincrease in lactate/pyruvate ratio, suggesting a relatively more oxidized cytoplasmic redox state (lower NADH/NAD) was maintained. The BBdp cells tended to show results between those of BBn and BBd(with the exception of the control condition). In contrast, with PMA ionomycin, the amount of pyruvate produced by BBd cells was equal to that of BBn and BBdp cells, perhaps because this stimulus bypasses a membrane-receptor-mediated step. PMA ionomycin likewise increased lactate production more than with ConA in BBd cells, and the lactate/pyruvate ratio increased toward that of BBn and BBdp cells. This combination of results would be consistent with a BBd defect in generation of reducing equivalents in their i n vivo “preactivated” state, as well as in response to stimulation by a lectin that acts via a membrane-receptor. The heterogeneity within the splenocyte population and differences both at the onset and during culture among BBn, BBd, and BBdp animals (14, 16, 17) render attribution of the differences observed to specific subsets of cells impossible a t present. Reportedrates of substrate utilization by other mixed mononuclear cell populations (36,37) aredifficult to compare with the present results as study conditions were not comparable (reviewed in Ref. 21). It is nonetheless noteworthy that an increased proportion of natural killer cells has been identified in BBd and BBdp spleens (30).3 The contribution of this subset to the overall functional abnormalities reported remains to be assessed. Glutamine is both an oxidative substrate (38) and an important source of nitrogen for de novo synthesis of pyrimidine and purine nucleotides, and amino sugars in lymphocytes (39). We have recently observed enhanced metabolism of glutamine via “glutaminolysis” and oxidation to COz in splenocytes from BBd animals (40). Glutamine was not present in the media of the 2-h incubations of the presentstudy, but was supplemented to 4 mmol/liter in theculture media. However, we have found similardifferences between BBd and BBn cells in terms of lactate and pyruvate concentrations in culture media, as found in the shortincubations of the presentstudy (21). Therefore the absence of other substrates in our incubations is unlikely to have created an artefact. Substrate utilization by immune cells, although critically important to their function, has received little attention in immunodeficient states, such as the BB model of insulindependentdiabetes. The observation of enhanced glucose metabolism by splenocytes from diabetic animals in thepresent study is consistent with the hypothesis of enhanced in vivo immune activation.Partial correction of abnormal lactate production and normalization of 2-dGlc uptake and pyruvate production with PMA ionomycin provides additional information on thepossible site responsible for the abnormal ConA response. The abnormal pyruvate production rates and corresponding lactate/pyruvate ratios in BBd cells suggest a further possibility, that of an innateredox anomaly in certain cells.

+

+

+

C. J. Field, R. Chayoth, M. Montambault, and E. B. Marliss, unpublished data.

Glucose Metabolism in Splenocytes from BB Ruts Acknowledgments-We wish to acknowledgeDr. P. Poussier for his valuable input in formulating the studyprotocol and thank Shirley Tam for secretarial assistance. Lernmark, REFERENCES 1. Whitesell, R. R., Hoffman, L. H., and Regen, D. M. (1977) J . Biol. Chem. 252,3533-3537 2. Wang, T., Marquardt, C., and Foker, J. (1976) Nature 261,702705 3. Reeves, J. P. (1975) J. Biol. Chem. 250,9413-9420 4. Jacobs, D. B., Lee, T.-P., Jung, C. Y., and Mookerjee, B. K. (1989) J . Clin. Invest. 83, 437-443 5. Whitesell, R. R., Johnson, R. A., Tarpley, H. L., and Regen, D. M. (1977) J. Cell Biol. 72,456-469 6. Helderman, J . H. (1981) J. Clin. Invest. 67, 1636-1642 7. Brand, K., Williams, J. F., and Weidemann,M. J. (1984) Biochem. J . 221,471-475 8. Roos, D., and Loos, J. A. (1973) Exp. Cell. Res. 77, 127-135 9. Hume, D. A., Radik, J. L., Ferber, E., and Weidemann, M. J. (1978) Biochem. J. 174, 703-709 10. Brand, K., Leibold, W., Luppa, P., Schoerner, C., and Schulz, A. (1986) Immunobiology 173.23-34 11. Resch, K., Prester, M., Ferber, E., and Gelfand, E. W. (1976) J. Immunol. 117, 1705-1710 12. Mookerjee, B. K., and Jung,C. Y. (1982) J. Immunol. 128,21532159 13. Yale, J.-F., Grose, M., and Marliss, E. B. (1985) Diabetes 34, 955-959 14. Poussier, P., Nakhooda, A. F., Falk, J. A., Lee, C., and Marliss, E. B. (1982) Endocrinology 110, 1825-1827 15. Yale, J.-F., and Marliss,E. B. (1984) Clin. Exp. Immunol. 57, 111 16. Prud'homme, G. J., Colle, E., Fuks, A., Goldner-Sauve, A., and Guttman, R. D. (1985) Immunol. Today 6, 160-162 17. Mordes, J. P., Desemone, J., and Rossini, A. A. (1987) Diabetes Metab. Rev. 3, 725-750 18. Poussier, P., Nakhooda, A. F., Sima, A. A. F., and Marliss, E. B. (1983) Metabolism 32, Suppl. 1, 73-79 19. Dean, B. M., Bone, A. J., Varey, A.-M., Walker, R., Baird, J. D.,

3681

and Cooke, A. (1987) Clin. Exp. Immunol. 69, 308-313 20. Dyrberg, T.,Poussier, P., Nakhooda, A. F., Marliss, E. B., and A. (1984) Diabetologia 26, 159-165 21. Field, C. J.. Wu, G., Mktroz-Dayer, M.-D., Montambault, M., and Marliss, E . B: (1990) Biochem. J. 272,445-452 22. Whitessell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899 23. Rodbell, M. (1964) J . Biol. Chem. 239, 375-380 24. Lloyd, B., Burrin, J., Symthe, P., and Alberti, K. G. M. M. (1978) Clin. Chem. 24, 1724-1729 25. Mktroz-Dayer, M.-D., Mouland, A., Brideau, C., Duhamel, D., and Poussier, P. (1990) Diabetes 39, 928-932 26. Francfort, J. W., Naji, A,, Markmann, D. P., Silver, W. K., and Barker, C. F.(1985) Surgery ( S t . Louis) 98, 251-258 27. Jackson, R., Kadison, P., Buse, J., Rassi, N., Jegasothy, B., and Eisenbarth, G. S. (1983) Metabol. Clin. Exp. 32, Suppl. 1, 8386 28. Wu, G., Field, C. J., and Marliss, E. B. (1990) in Lessons from Animal Diabetes I I I (Shafrir, E., ed) John Libbey, London, in press 29. Prud'homme, G. J., Fuks, A., Colle, E., Seemayer, T. A., and Guttman, R. D. (1984) J. Exp. Med. 159,463-478 30. Woda, B. A., and Biron, C. A. (1986) J. Immunol. 137, 18601866 31. Naftalin, R. J., and Rist, R. J. (1990) Biochem. J. 265,251-259 32. Naftalin, R. J., and Rist, R. J. (1989) Biochem. J. 260, 143-152 33. Peters, J. H.,and Hausen, P. (1971) Eur. J . Biochem. 19, 509513 34. Weiss, A,, and Imboden, J. B.(1987) Ado. Immunol. 41, 1-38 35. Davis, L., and Lipsky, P. E. (1985) J. Immunol. 135,2946-2952 36. Ardawi, M. S. M., and Newsholme, E. A. (1985) Essays Biochem. 21, 1-44 37. Newsholme, P., Curi, R., Gorden, S., and Newsholme, E. A. (1986) Biochem. J. 239,121-125 38. Ardawi, M. S. M., and Newsholme, E. A. (1983) Biochem. J . 212, 835-842 39. Szondy, Z., and Newsholme, E. A. (1989) Biochem. J . 261, 979983 40. Wu, G., Field, C. J., and Marliss, E. B. (1991) Biochem. J., in press