Glycogen regulation in LPS-stimulated murine splenocytes

Experimental

Glycogen

Regulation

Cell Research

151 (1984) 306-313

in LPS-Stimulated

DIMITRI MONOS, and HERBERT

Murine

Splenocytes

r IRVING GRAY*** L. COOPER3

‘, ‘Department of Biology, Georgetown University, Washington, DC 20057, and 3Laboratoty of Pathophysiology, National Cancer Institute, National Institute of Health, Bethesda, MD 20205, USA

Enzymatic glycogen regulation in mouse splenocytes cultured in vitro with and without LPS, was studied from O-72 h. Increased [3H]glucose uptake and hexokinase activity demonstrated the activation of cells treated with LPS. There was a greater time-dependent increase of cellular glycogen content in LPS-stimulated cells as compared to control. Glycogen synthetase I in LPS-stimulated cells increased about 200% above control cells to a plateau at 48 h, while in unstimulated cells there was little increase throughout. Glycogen synthetase D increased continually to 72 h in both groups. In the stimulated cells, phosphorylase increased only 90% above control cells up to 48 h. It was concluded that the increased glycogen content of LPS-stimulated cells seen at 48 h may result from an increase in both glycogen synthetase I and D activity compared to lesser increase in hydrolysis. However, between 48 and 72 h, the period of RNA and DNA increased synthesis, the glycogen content of stimulated cells did not increase further, consistent with the observation that synthetase I activity remained constant and synthetase D decreased. Thus, following mitogenic stimulation, the net effect of the enzymatic regulation is to increase cellular glycogen, as an energy source for subsequent events.

Leukocytes, which can be prepared from peripheral blood, present a valuable model for cellular metabolic studies. Attention has been directed to the presence of glycogen in neutrophils, in leukemic cells, and in lymphocytes after the latter have been stimulated by mitogens. Because of the higher concentrations of glycogen in neutrophils, they have been studied more extensively than lymphocytes [l-4]. In neutrophils, glycogen synthesis and degradation proceed rapidly even when the total amount of glycogen in the cell is constant. When endogenous glucose is needed, the glycogen level falls rapidly and when glucose is replaced, the glycogen stores return to normal [2]. In platelets, under conditions of net glycogen degradation, glycogen synthesis is still taking place [3]. An increase in glycogen content has also been reported to occur in mitogen [5,6, 71 or hormonestimulated [8] T lymphocytes after 24-48 h in culture. Yanossy et al. [9] reported that mouse (CBA) splenocytes showed positive glycogen granules after being incubated for 60 h with pokeweed mitogen (PWM). When partially purified subpopulations of mononuclear leucocytes were studied, major differences were detected in the glycolytic energy metabolism of different cell populations [lo]. In order to confirm glycogen increase after stimulation in another subpopulation of *To whom offprint requests should be sent.

Copyright @ 1984 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827184 $03.00

Glycogen

regulation

in activated

lymphocytes

307

lymphocytes, namely B lymphocytes and to better understand the metabolic regulation of the unusual changes in glycogen content following lymphocyte stimulation, enzymatic control has been studied in LPS-stimulated and control B lymphocytes from BALB/c mice. Our results indicate that the activity of the enzymes responsible for glycogen regulation is such that the utilization of glucose effectively conserves glycogen so that there is always a reserve energy source for the various developmental stages of mitogen stimulation.

MATERIALS

AND

METHODS

Male BALB/c mice, 8-12 weeks old (Jackson Memorial Laboratories) were maintained on water ad libitum and standard mouse chow for at least one week in the Department’s animal quarters prior to use. They were sacrificed by cervical dislocation, the spleens removed and teased apart using 32mesh stainless steel. The cells were suspended in 5 ml of Hank’s balanced salt solution (HBSS) and the lymphocytes separated by the method of B#yum [II] as modified by Stewart et al. [12]. The separated lymphocytes were washed three times with HBSS, counted, tested for viability by the Trypan blue exclusion method and finally suspended in a volume at a final concentration of 1 x 10’ cells/ml. The lymphocytes were cultured in 17x100 mm polypropylene culture tubes (Falcon) for the indicated period of time at 37°C in humidified air containing 5 % CO*. The culture medium was RPMI1640 containing 10% heat-inactivated fetal calf serum (FCS) (Gibco), 100 units/ml of penicillin G and 100 mg/ml of streptomycin sulfate. LPS (Escherichia co/i OS5 : BS, Difco) was made up in HBSS to a concentration of 1 mg/ml. The cultures contained 5 ml of medium with lo6 cells/ml. The stimulated cultures also contained LPS in a final concentration of 10 &ml. Cell viability was determined as described above after Ficoll-Hypaque separation and at the completion of the indicated incubation periods. All data were corrected to 100 % live cells. The correction was based upon data collected for each of the parameters measured at the end of the indicated periods using live cells only. The separation of dead from live cells was carried out by placing the 5 ml culture on 3 ml Ficoll-Hypaque and centrifugation at 270 g for 15 min. Live cells remained at the interface, while dead cells sedimented to the bottom of the tube. The data, corrected for viability, were qualitatively the same as the values obtained using live cells. For the study of glucose uptake, LPS was added at the beginning of the incubation and 0.1 ml HBSS containing 50 pCi of [3H]glucose (sp.act. 4.5 Ci/mmol, ICN) were added 1 h prior to the harvest of the ceils. At the end of the incubation period, cells were centrifuged at 500 g for 10 min at 4°C. The pellets were washed twice with 2 ml of HBSS at 500 g for 10 min at 4°C. After each wash the inside of the tubes was carefully wiped with a cotton swab to remove residual radioactive medium. After the second wash, the pellet was resuspended in 0.2 ml of HBSS and transferred to a scintillation vial with 5 ml Ready-Solv HP scintillation fluid (Beckman). The radioactivity was counted in an LS-IOOC Beckman Scintillation counter to an error of less than 5 %.

Preparation of Cells For the analysis of hexokinase, glycogen phosphorylase, and glycogen synthetase activities, at the end of each time period, the cell pellets from four tubes were pooled, washed twice with 2 ml of icecold buffer, 0.05 M ms-HCl, pH 7.5, 100 mM KF, 5 mM EDTA and 0.5 mM dithiothreitol 1131. The cells were lysed by the addition of 0.5 ml of 0.5 % Nonidet, P40 detergent, in 50% glycerol in normal saline [14] to approx. 2x 10’ cells. The preparation was kept at -70°C until the enzymatic assays were performed.

Enzyme Assays Hexokinase was measured at 25°C according to the fluorimetric method of Passonneau (personal communication). One ml of 0.05 M T&buffer, pH 8.0, 1 mM glucose, 2 mM ATP, 5 mM MgCl,, 0.02 % bovine serum albumin (BSA), 0.1 mM NADP’ and 0.1 pg of glucose-&phosphate dehydrogenExp Cell Res 151 (1984)

308

Monos,

Gray and Cooper

Table 1. Glucose

uptake

by BALBIc

mouse splenic

lymphocytes

(nmoleslhl107

cells)” Incubation time (hour)

Control (C)

1 4 6 8 12 24 48 72

0.90+0.05 1.4420.06 1.50+0.03 1.33+0.06 1.55+0.07 1.50+0.11” 3.88rt0.39 6.2lkO.92

Stimulated (LPS) 0.98kO.02 1.62kO.17 2.00+0.03 1.98kO.04 2.70+0.06 3.03f0.2’ 7.62kO.75 11.78k1.62

FI’ 1.09x 1.13x 1.34x 1.49x 1.74x 2.02x 1.96x 2.01 x

a Mean + SE of three expts, each in duplicate. ’ Mean + SE of four expts, each in duplicate. ’ FI, Fractional Increase = [(LPS-C/C] + 1 stimulation >l >inhibition.

ase (G-6-PdH), Boehringer-Mannheim), were added to 3 ml test tubes (10x75 mm). All the above reagents, with the exception of NADP+ and G-6-PdH, were premixed in IO-fold concentration and stored at -20°C. NADP+ and G-6-PdH were added when the enzyme assay was performed. Blank solution (HrO), standards and samples were added in a volume of 2-15 ~1. Standards contained 2-5 nmoles of glucose-6-phosphate (G6P). The reaction was started by the addition of 10-15 ul of sample. After all the samples were added to the tubes, a first reading of the fluorescence was immediately taken and considered as the zero time measurement. After 10 and 20 min, while still on the linear portion of the curve, mesurements were again taken and the activities of the enzyme was calculated and expressed as umolesih/lO* cells. Glycogen phosphorylase was assayed at 25°C according to Lowry et al. [15]. Glycogen synthetase I (independent of glucose-6-phosphate concentration) and glycogen synthetase D (dependent on glucose-dphosphate concentration) activities were measured in a 2-step procedure in which UDP formation was measured fluorimetrically [16].

Cellular Glycogen Measurements At the end of the incubation period, approx. 10’ cells were pooled and glycogen was extracted according to Marchand, Leroux & Cattier [17] and Ptleiderer [ 181. The glycogen was measured according to Passonneau & Lauderdale [ 191. All fluorescence measurements were made on a Fart-and Filter fluorimeter (primary filter, Coming No. 5860, secondary filters, Coming Nos. 4303 and 3387). Statistical analyses were made using Student’s r-test. Fractional increase (FI) was defined as the change in the experimental response as a multiple of the control response, i.e., times (x) control.

RESULTS In the course of 72 h, while there is no net increase in the number of cells, there is a time-dependent progressive decrease in the fraction of viable cells in both groups, particularly among the LPS-stimulated cells, probably because of the toxic effect of LPS itself. Increased uptake of [3H]leucine into TCA-insoluble protein as routinely carried out in our laboratories indicated that lymphocyte metabolism had been Exp Cell

Res

151 (1964)

Glycogen

Table 2. Glycogen (nmolesll08

concentration

regulation in BALBIc

in activated mouse

lymphocytes

splenic

309

lymphocytes

cells)” Incubation time (hour)

Control Stimulated FI

0

24

48

12

7.6+ 1.09 (5) 7.720.67 (4) 1.01

18.7+ 1.04 (3) 42.9k6.07 (2) 2.29

65.7k5.88 (3) 153.6k25.88 (2) 2.34

128.3k16.34 (3) 167.1k13.93 (3) 1.30

In parentheses: No. of expts in duplicate. a Mean + SE.

stimulated by LPS. The FI for leucine uptake was 1.97, 1.88, and 2.08 control at 24, 48, and 72 h, respectively. To confirm LPS stimulation in terms of glucose uptake, the radioactive tracer [‘HIglucose was used in a pulse experiment, as described in Methods. Since our purpose was not the kinetic study of glucose uptake, but rather to maintain normal functions of the cells throughout the 72 h course at the experiment, the concentration of glucose in the medium was at saturation levels. Measurements

Table 3. Activity

of enzymes involved in glycogen metabolisma Incubation time (hours)

Hexokinase (umoles/h/108 cells) C Stimulated (LPS) FI Phosphorylase-a (nmoles/h/lOs cells) C Stimulated (LPS) FI Glycogen synthetase-I (nmoles/h/108 cells) C Stimulated (LPS) FI Glycogen synthetase-D (nmoles/h/108 cells) C Stimulated (LPS) FI

0

24

48

72

2.35f0.27 2.47kO.22 I .05

3.21kO.96 5.1250.68 1.60

4.54kO.44 lO.lf1.21 2.22

7.39f0.52 13.06f0.98 1.77

390.6k26.06 388.5k52.36

335.4k47.2 464.22 19.4 1.38

432.1k28.55 820.0f86.12 1.90

604.2k22.54 1144.0+91.90 1.89

41.80+10.1 38.75k9.19

29.80+10.1 47.00f8.84 1.58

29.9Ok6.47 92.40f13.10 3.09

29.66k7.6 86.42k10.46 2.91

41.5k9.65 49.5k12.48 1.19

95.34k4.85 96.9Odz8.61 1.02

135.80f13.5 117.10+16.76 0.86

174.64k21.79 172.08k9.13 0.99

0 Mean If: SE of three experiments. Exp

Cell

Res I51 (1984)

310 Monos,

Gray and Cooper

of glucose consumption have been used elsewhere to quantitate lymphocyte transformation and closely correlated with [3H]thymidine incorporation [20]. The uptake of glucose is shown in table 1. The [3H]glucose uptake shows increasing radioactivity in the cells with time. The amount in the stimulated cklls exceeds that in control cells at all points. It is apparent from the FI values that during the first 24 h of exposure, LPS increases the uptake of glucose by the stimulated cells, thereafter the fractional increase above the control value remains constant. Cellular glycogen is shown in table 2. The isolation procedure resulted in a 63% recovery of known samples of glycogen. While control cells show an increase in glycogen content from 0 to 72 h, the stimulated cells display a much greater increase during the first 24 h of exposure to the LPS (somewhat similar to that seen with glucose). After 24 h, glycogen in the LPS-treated cells remains higher than in the control cells. However, after 48 h the amount in stimulated cells remains constant while that in the control cells continues to increase up to 72 h. Thus, the FI remains constant from 24 to 48 h and then drops as a result of the continued increase in the non-stimulated cells. Hexokinase activity as a function of time (&72 h) is presented in table 3. There is increased activity in both control and stimulated cells but the latter increase is greater. At 48 h the FI reaches its maximum value. The changes in phosphorylase-a activity are shown in table 3. There is little but continual increase in the control cells to 72 h while the activity in the LPS-treated cells increases somewhat more rapidly. The FI reaches a maximum plateau at 48-72 h. During the entire incubation period, phosphorylase-b activity was essentially unchanged in both groups of cells. In table 3 the data for glycogen synthetase I and D are presented. In the control cells, the I-form remains constant (possibly decreased) during incubation, while in the LPS-treated cells the activity increases from 24 to 48 h and remains elevated to 72 h. Glycogen synthetase D in the control and stimulated cells increases continually from 0 to 72 h.

DISCUSSION Previous studies have shown that mitogenic stimulation of lymphocytes increases the transport of glucose [21, 221. In this study, not only transport is measured but since [3H]glucose was used, also its metabolism. Thus, what is referred to as ‘glucose uptake’ includes transport and non-diffusable metabolites. It is apparent that up to 24 h, the LPS-treated lymphocytes increase glucose uptake at a faster rate than the control cells (table 1). After 24 h, although glucose uptake continues, the uptake rate for both experimental and control cells is the same and no further stimulation by LPS above that achieved at 24 h occurs (FI, table 1). The increase in glycogen content of the cells (table 2) compares with that for Exp Cell

Rrs

ISI (1984)

Glycogen

regulation

in activated

lymphocytes

311

stimulated T lymphocytes and polymorphonuclear leukocytes [6]. While glycogen synthesis in stimulated cells can begin as early as 4 h after exposure to phytohemagglutinin (PHA) [23], the major increase seen in these studies occurs between 24 and 48 h with little change after that time. Over this period of time the control cells also increase their glycogen content. This may be due to non-specific stimulation by culture conditions, such as the presence of FCS. In the first 24 h of incubation, the non-specific activation of the control cells increases the glycogen content by about 2.5 times, while the presence of the mitogen effects an increase of about 5.5 times. The glycogen content increases in about the same proportion for the two groups over the next 24 h. However, after 48 h of incubation the glycogen continues to increase in the control cells, although at a slower rate than earlier, while there is no further increase in activated cells. It is interesting to note that the cessation of glycogen accumulation in activated cells occurs between 48 and 72 h, a time when RNA and DNA synthesis has been significantly increased [24-261. This may be due to increased breakdown of glycogen stores to meet the needs of these energy-requiring reactions. The possibility exists that the plateau in the glycogen content of the cells might result from a ‘saturation’ effect, i.e., the cell can hold no more glycogen because of its high concentration. If, because of the distribution of large and small lymphocytes in the population of stimulated cells we assume an average diameter of 20 urn, the average volume of the cells can be calculated as 4 pl. At 72 h, taking into account the 63 % recovery, the actual glycogen content would be 265 nmoles/108 cells. From these numbers, the concentration of glycogen in the LPS-treated lymphocytes would be approx. 0.66 mM. Brain contains about 89 mg of glycogen/lOO g of tissue, thymus 280 mg/lOO g [32], and muscle 700 mg/lOO g tissue [28]. From these figures, the concentration of glycogen may be calculated to be 0.33, 1.O, and 2.5 mM respectively. In these calculations we have assumed the density of the tissues to be one and the glycogen to have an average of 15 glucosyl residues or a MW of 2700. It would seem therefore that the concentration of glycogen in the lymphocytes is of the same order of magnitude as that in other tissues. Thus, the ‘saturation’ effect might play a role in limiting the glycogen content in the activated lymphocytes at 72 h. However, it is well known that in tissues, i.e., muscle and liver, the concentration of glycogen can vary over a large range. Thus, in LPS-stimulated cells, net glycogen accumulation, while primarily the result of enzymatic regulation, could be limited by the concentration in the cells. It is generally accepted that glycogen synthetase D, the phosphorylated form of synthetase I, is virtually inactive. However, in the presence of available glucose6-phosphate (G6P), glycogen synthetase D is allosterically activated to bring about glycogen synthesis [27]. As has been reported previously [26], LPS stimulation causes an increase in hexokinase activity also seen here (table 3). Soluble hexokinase is inhibited by its product G6P but membrane-bound hexokinase is less sensitive than the soluble enzyme to this inhibition. This may explain why some cells can continue to have elevated hexokinase activity even though their Exp Cell

Res 151 (1984)

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G6P level may have been increased [29, 303. This, together with the stimulated glucose uptake, would make increased G6P available for the allosteric activation of glycogen synthetase D. We have shown that the content of this enzyme increases with the time of incubation for both the control and LP$-stimulated cells. However, because of the greater hexokinase activity and glucose uptake in the LPS cells, which would result in greater G6P availability, greater activity of glycogen synthetase D would be expected in activated than in control cells. Table 3 shows, further, an increase in glycogen synthetase I activity up to 48 h in activated cells while the control cells show no increase (possibly decrease). Thus, with regard to glycogen synthetase, enzymatic conditions consistent with net glycogen synthesis in activated vs control cells appear to be present during the initial 48 h of the incubation period. During the 24 h poststimulation period the active phosphorylase has not increased, compared with T,,. At the same time, hexokinase, glucose, and glycogen concentration have all been observed to have been increased. This would suggest that up to 24 h any increased needs for glucose are apparently satisfied from incoming glucose and not from glycogen stored in the cell. However, from 24 to 48 h, the FI of phosphorylase had reached its maximum value, indicating that the increasing activity of the stimulated lymphocytes was now at the same rate as that of the control cells. The glycogen synthetase had also reached its maximum FI but at 1.5 times greater than that of the phosphorylase. During the same period, both hexokinase activity and glucose-uptake of the stimulated cells continue to increase (tables 3 and 1, respectively) and the major increase in glycogen occurs (table 2). Thus, the cellular conditions would be favorable for a response to the altered relationship of the two key enzymes, glycogen phosphorylase and glycogen synthetase to produce the net increase in cellular glycogen content. This would provide the necessary reserve energy sources to meet the metabolic requirements of increased RNA and DNA synthesis of the stimulated cells [22, 241. Thus, our results indicate that the glycogen content in LPS-stimulated murine lymphocytes is under the simultaneous control of glycogen phosphorylase and glycogen synthase as modulated by glucose and G6P concentration and hexokinase activity in such a way that increased glycogen synthesis is not necessarily accompanied by inactivation of glycogen lysis. It is suggested that this manner of regulation is necessary so the cell, at the initial stages of activation (before 24 h), can utilize the available glucose, while at the same time, excess intracellular glucose can be stored in the form of glycogen (possibly up to a limited concentration) for later metabolic needs of the activated cells. Watanabe & Passonneau have suggested that glycogen regulation is controlled by the relative concentrations of metabolites and co-factors [311. It would appear that the phosphorylase-glycogen synthetase system is a dynamic one and not a simple ‘on-off type [29], where mediation by hexokinase and G6P is probably involved. It is apparent that glycogen, accumulated early in activation, is conserved for the later metabolic needs of the activated cells. Exp

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This work was supported by NIH grant no. ES-02064. The authors wish to thank Dr J V Passonneau, Laboratory of Neurochemistry, NINCDS, NIH, and Dr W Stylos, Laboratory of Molecular Pharmacology, NCI, NIH, for generous assistance and guidance throughout the course of this work.

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