Overnight food deprivation in normal and diabetic mice markedly enhances thromboxane production by arachidonate stimulated platelet rich plasma and markedly increases platelet phospholipase A2 activity

THROMBOSIS RESEARCH 31; 557-564, 1983 0049-3848/83 $3.00 + .OO Printed in the USA. Copyright (c) 1983 Pergamon Press Ltd. All rights reserved.

OVERNIGHT FOOD DEPRIVATION IN NORMAL AND DIABETIC MICE MARKEDLY ENHANCES THROMBOKANE PRODUCTION BY ARACHIDONATE STIMULATED PLATELET RICH PLASMA AND MARKEDLY INCREASES PLATELET PHOSPHOLIPASE A2 ACTIVITY

William I. Rosenblum, M.D., Paul D. Hirsh, M.D. and Richard C. Franson, Ph.D. Departments of Pathology (Neuropathology), Medicine (Cardiology) and Biochemistry, Medical College of Virginia and School of Basic Sciences, Virginia Commonwealth University Richmond, VA 23298 Received 5.4.83; Accepted in revised form 20.5.83 by Editor K.M. Brinkhous ABSTRACT These studies report the effect of overnight food deprivation on thromboxane production by mouse platelets stimulated with arachidonic acid. Phospholipase activity was also measured. We used radioimmunoassay to measure thromboxane B2 (TxB ) in platelet rich plasma (PRP) stimulated with 0.5 mM sodium aract idonate. TxB levels increased from 30 to 180 seconds after stimulation, an?Iat 90 and 180 seconds were approximately twice as high in PRP from food deprived mice as compared with levels in PRP from fed animals (PC.01). The same period of food deprivation (17-21 hours) doubled the phospholipase A2 (PLA2) activity of the mouse platelets. No significant differences were observed between the TxB levels in PRP from normal mice and the PRP from mice with streptozotocin diabetes, although the marked elevation of TxB levels produced by food deprivation was somewhat greater in the Pi? P from diabetics. No differences were observed in PLA2 activity in diabetic as compared with control platelets. The marked enhancement of PLA2 activity produced by food deprivation, was seen in both control and diabetic platelets.

INTRODUCTION --Approximately 20 hours of food deprivation have been reported to enhance platelet aggregation in injured cerebral microvessels of mice with streptozotocin diabetes (1). These in vivo results prompted the in vitro studies reported here. The latter include investigations of thromboxane A2 (TxA2) Key Words: --

Prostaglandin synthesis, platelet phospholipids, Diabetes, Food deprivation. 557

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production and phospholipase A (PLA ) activity in platelets of fed and fasted mice, diabetic and non 2iabetzc. The production of TxA may be related to aggregation either because TxA itself is a stimula$or of aggregation or because it is an indicator of c&looxygenase activity, and this activity is associated with the production of proaggregatory precursors of TxA2 (2). Phospholipase A2 and phospholipase C are enzymes responsible for liberating arachidonate from phospholipids in the platelet cell membrane (3,4). Both have been implicated in platelet aggregation (3,4). Arachidonic acid is the cyclooxygenase substrate. The present studies measure the activity of a phospholipase with the properties of PLA2. The present work was performed with platelet rich plasma (PRP). A previous study utilizing washed mouse platelets (5) found that thromboxane production was significantly reduced by food deprivation, a result that was opposite that predicted from the in vivo effects of a fast. The present studies were initiated because of the possibility that PRP may be more representative than washed platelets, of platelets in vivo. While these experiments were in progress, a report appeared showing a significant increase in thromboxane production by clotting rat blood, following only 12 hours of food deprivation (6). METHODS Blood for PRP was obtained from the carotid artery of male mice, ICR strain (Flow Laboratories, Virginia) as previously described (5,7). The blood was centrifuged at 100 X g for 20 minutes. Platelet poor plasma (PPP) was prepared by further centrifugatlon of the PRP at 1400 X g for 30 minutes. The pooled blood from 5-10 mice provided the PRP required for the studies of phospholipase. In the thromboxane studies the blood from a single animal provided sufficient PRP for TxB2 determination following stimulation by arachidonate. 3In these studies the platelet count in PRP was adjusted to 400,000 per mm by adding PPP. Counts were made with a Coulter counter. The PPP was taken from normal mice or from diabetic mice, for addition to normal or diabetic PRP, respectively. Thromboxane synthesis was induced by causing aggregation of platelets in PRP. Sodium arachidonate (Nuchek) 0.5 mM final concentration was the aggregating stimulus, added 2 minutes after PRP was placed in the cuvette of an aggregometer (Chronolog) at 37. This dose of arachidonate was chosen in order to insure a good aggregation in each sample. Moderate reductions in concentration (e.g. 0.1 mM) do not always produce aggregation in mouse PRP. Radioimmunoassay was employed for determination of TxA2 synthesis, via measurement of its stable metabolite, TxB . PRP was placed in the aggregometer and fifty microliters of PRP were w z thdrawn just before (0 times and at 30, 90 and 180 seconds after addition of arachidonate. Aggregation was monitored continuously. Each 50 ~1 aliquot was placed into 450 ul of phosphate buffered saline (pH 7.4) containing 0.1% PVP-40 and 0.1% sodium azide, and quickly frozen by immersion of the tube into a dry ice - methanol mixture. The frozen sample was kept at -70°C until assayed with antibodies produced in rabbits and generously provided by Dr. William Campbell. The rabbits were immunized against a prostaglandin-thyroglobulin conjugate produced with the mixed anhydride method of Jaffe and Behrman (8). Levels of sensitivity and antibody cross reactivity have been previously reported (9). Results are exprPssed in nanograms per 50 pl which is equivalent to nanograms per 2 x 10 platelets.

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Phospholipase A2 activity was measured as previously reported (10,ll) following extraction of a platelet pellet using a modification of an earlier (11) procedure. The pellet from mouse PRP was resuspended in 1 ml of ice was homogenized with a Potter-Elvehjem homogenizer, and cold 0.18 N H SO after 1 hour it 4°C was rehomogenized and centrifuged at 10000 X g for 5 minutes in a Beckman Macrofuge. The supernatant was assayed for PLA activity using either f4radiolabelled autoclaved E. coli whose phosp8olipids were labelled with [l- C] oleate glmost exclusively (10) in the -2- position (5 nmols/6000 CMP) or I-acyl 2 - [ H] - aracidonyl - sn - glycero - 3 ghosphoethanolamine (51 nmoles and 30,000 cpm). More than 95% of the H-arachidonate was in the 2-acyl position as determined by snake venom hydrolysis (Crotalus adamanteus). Reaction mixtures containing substrate and platelet extract were incubated for 2 hours at 37°C when E. coli substrate was used and for 4 hours when tritiated substrate was used. The 0.5 ml incubate contained 100 mM tris-maleate buffer pH 7.5, 5 mM CaCl and 30-120 pg of protein. Reactions were stopped with three volumes of me 8hanol-chloroform (2:1, V/V). Hydrolysis products were extracted by the method of Bligh and Dyer (12). Radioactive lipids were separated by thin layer chromotography as previously described (10). Results are expressed as nmols/hr/mg protein. Protein was determined using the method of Bradford (13). Mice were rendered diabetic by injecting streptozotocin (Sigma) 200 mg/kg via tail vein 4-5 weeks prior to use. The streptozotocin was dissolved in citrate buffer pH 4.5. Control mice received buffer alone. Diabetes was confirmed by demonstration of glucose positive urine (Testape, Lilly Co.) and blood sugar values in excess of 350 mg/dl. The latter were obtained with a spectrophotometric method employing glucose oxidase (Glucinet, Sclavo Labs). Fed mice, both diabetic and normal, received laboratory chow and water ad libitum. Fasted mice were deprived of food at qprnand used between garn and 3Pm on the following day (17-21 hours of food deprivation).

RESULTS The effect of fasting on TxB2 production from arachidonate stimulated PRP is shown in Table 1 for both normal and diabetic mice. Since our primary purpose was to study the effect of fasting rather than to compare normals with diabetics we analyzed the data from normals separately from the diabetic data using an analysis of variance (ANOVA) for each. Both ANOVA showed a highly significant increase of TxB2 from 30 to 180 seconds (pc.01) and both showed a marked increase in TxB production by PRP from fasted mice (p<.Ol). By 180 seconds following onset20f aggregation, TxB values were more than double the values seen in the fed group , whether dzabetic or non diabetic. Because the values from fasted diabetics were somewhat greater than those from fasted controls, at each point in time, it was of interest to subject this observation to statistical analysis. This was done by testing all data from the four treatment groups with a single ANOVA (pc.01) for treatments) and then using a Bonferroni test for multiple comparisons (24) to analyze the differences between fasted controls and fasted diabetics at 30, 90 and 180 seconds. No significant differences (pp.05) were found. Phospholipase activity was markedly increased in platelets from fasted mice, whether diabetic or non diabetic and no effect of diabetes could be detected. The results are summarized in Table 2 which shows the results of

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two separate studies each comparing pooled platelets of fed normal mice with pooled platelets of food deprived, but otherwise normal mice. The Table also shows data from one study of diabetic mice, comparing pooled platelets from food deprived animals with those from fed animals. The results are the same in all three studies. Fasting markedly increased PLA , the levels of activity in samples from fasted mice being 195 to 272% of $he levels in control samples. All of the results in the Table were obtained using the phospholipid substrate made from E. coli. A repetition of the first experiment utilizing aliquots of the same platelet extracts but the tritiated arachidonyl substrate showed a 213% enhancement of PLA2 activity in the platelets from fasted mice. TABLE 1 Thromboxane B in Mouse Platelet Rich Plasma Stimulatgd with 0.5 mMArachidonate Fed Control 0.3+0.1(12) 6.6+3.7(11)* 11.0+5.3(11) 11.9+5.7(11)

Seconds 0 30 90 180

Nanograms per 50 ~1 Food Deprived Fed Control Diabetic 0.2+0.1(11) 0.2+0.1(11) 8.8+6.4(11) 6.4+2.7(11)X 20.3+9.2(11) 9.1?3.2(11) 24.9’9.6(11) 11.2+3.5(10)

Food Deprived Diabetic 0.2+0.2(12) 12.2+6.7(12) 22.2?5.9(12) 28.5?8.0(12)

) - Number of animals ( *The TxB in the medium increased with time following stimulation of the p z atelets. In both control and diabetic groups, fasting significantly increased TxB production (p<.OOl) in separate ANOVAfor control and diabe $ its, from 30 to 180 sec.) TABLE 2 Phospholipase Experiment 1 2 3

#

A2 Activity

in Platelets

PLA2 Activity nmoleslhrlmg

Source of Platelets Normal Normal Normal Normal Diabetic Diabetic

Fed Mice Fasted Mice Fed Mice Fasted Mice Fed Mice Fasted Mice

from Fed and Fasted Mice

(10) (10) ( 5) ( 5) t ‘5;

Ratio Fasted/Fed

2.84 7.73 1.41 2.99 1.74 3.38

2.72 2.12 1.94

In each of 3 studies, food deprivation increased PLA activity by approximately two-fold or better. Num 3 ers in parenthesis are numbers of mice contributing to the platelet pool. DISCUSSION These data show a profound effect of a relatively brief period of food deprivation on TxB2 production by arachidonate stimulated mouse PRP. The fast doubled TxB2 production with virtually no overlap of values between fed

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and fasted mice. Since exogenous arachidonate was the stimulus for platelet aggregation it is probable that this exogenous arachidonate provided most of the substrate for platelet cyclooxygenase, rather than the platelets own endogenous arachldonic acid (14). Nevertheless, the data suggest that food deprivation either markedly enhanced platelet cyclooxygenase activity or markedly stimulated other enzymatic pathways leading to thromboxane production (e.g. thromboxane synthetase). The marked increase of TxB, production in PRP of fasted animals contrasts with our earlier flndfng of a significant decrease jn TxA production bv washed mouse nlatelets from fasted animals. It may be tiat washed platelets-are abnormal, perhaps as a result of excessive handling or suspension in an inappropriate medium (15). It may also be that platelets function differently in the presence of plasma than in its absence (14,16). Phosphollpase A2 (PLA ) is another enzyme whose activity may control thromboxane production. PEA liberates endogenous arachidonate from platelet phosphollpids, therefore a c%ange in PLA, activity would not explain the profound effect of fasting on TxB2 producti& from exogenous arachidonate. Nevertheless we looked at PLA..activity in fed and fasted mice because an increase in activity would p&de a &ond path in addition to enhanced cyclooxygenase activity, by which TxA production from endogenous substrate could be increased. The precise rela8ionship between PLA2 activity and release of endogenous arachidonate appears unsettled and may be quite complex (394). Since phospholipase A2 activity has been reported Increased in human diabetic platelets (17), we measured its activity. We used two substrates to test PLA2 activity. The use of substrate from autoclaved E. coli which is rich in phosphatldylethanolamine (PE) has been described in detail elsewhere (10,18). This substrate has been used extensively to quantitate PLA activity in human platelets (ll), rabbit leukocytes (19) and alveolar macropiages (20). Use of the substrate has several advantages (18): it is stable and inexpensive; and the physical form of the substrate appears fixed and more closely resembles a naturally occurring biological membrane than do other substrates such as phosphatidyl choline (PC). PC is the endogenous substrate for PLA2. However when PC is used in vitro a detergent or organic solvent is required to change its surface properties before it is readily cleaved by PLA2. In addition the hydrolysis of phospholipids by PLA2 depends on net charge of the substrate. Since PE has a net negative charge at physiologic pH, PLA will cleave PE in preference to PC (21). Therefore under our assay conditzons little or no hydrolysis of PC occurs. Others have shown that platelet PLA2 has no specificity for a particular fatty acid in the 2 position and will preferentially hydrolyze PE as compared to PC (22,23). Our measurements of PLA activity depend upon cleavage of the E. coli substrate at the 2-acyl position and the capacity of mouse platelet PLA to act at this position was confirmed by our use of PE labelled with arac& idonate in that position. Since, as noted above, platelet PLA has no specificity for a particular fatty acid in the 2 position nothing wou1d be gained from using PC with the addition of nonphysiologic detergents or solvents. Our studies of PLA activity were not directed at the effect of fasting on phospholipid compositzon or mobilization from the cell membrane. Rather, we examined PLA activity with exogenous substrate for which PE by itsel
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mice. However in all 3 studies, fasting increased PLA2 activity by 200 to 300 percent. The failure of diabetic platelets to display a different PLA2 activity from that of normal platelets need not be considered contradictory to the work of Takeda et al (17) for several reasons. First they studied human diabetics while we studied mice. Second they did not monitor cleavage of exogenous phospholipid for their measurements of PLA2 activity but monitored changes in the composition of endogenous platelet phospholipid instead, As they recognized in their discussion they may have measured phospholipase activity other than PLA2. Third their data show only a slight (25%) albeit significant increase in diabetic phospholipase activity and this increased activity was found only in washed platelets, and disappeared when platelets were resuspended in plasma. The data reported here shows that fasting enhanced TxB2 production to a greater degree in diabetic than on non diabetic mice. However, this difference was not statistically significant. Thus the data does not exactly parallel the in vivo data which stimulated the present study. That in vivo data showed that food deprivation significantly enhanced aggregation only in diabetic mice and not in non diabetic controls. Moreover, one of us (WIR) has recently been unable to show (unpublished) the proaggregatory effect of fasting reported by him in the earlier in vivo study (1). Instead streptozotocin diabetic mice now show more rapid aggregation in vivo than controls, even in the absence of a fast and fasting has no additional stimulatory effect on in vivo aggregation. Our current failure to demonstrate a proaggregatory effect of fasting in vivo in either diabetes or controls is contrary to the prediction one would make from the concurrent chemical data presented here , which demonstrates in both types of mice markedly enhanced thromboxane production and phospholipase A2 activity produced by the fast. This discordance indicates the dangers inherent in interpreting the biological significance of in vitro investigations. Nevertheless the profound effect of modest food deprivation we demonstrate in both diabetic and non diabetic mice is of great interest, and apparently has been reported on only one prior occasion (6). In the latter instance a marked increase of TxB production was seen in the clotting blood of fasted rats. This data, togeti: er with the present results concerning TxB2 production and PLA2 activity should help alert others to the variability which brief periods of food deprivation can produce in data involving platelet phospholipid metabolism. The data indicate that starvation is not required to cause profound biochemical alterations, and should stimulate investigations of the consequences of overnight food deprivation on other organs and on other parameters.

Acknowledgements The authors received excellent technical assistance from Ms. Deborah Weir, Ms. LeAnne Haskin and Mr. Alfred Allen. The work was supported by grants HL-23560, and HL-24907 and AI-18644 from the National Institutes of Health, and a grant from the A.D. Williams Fund of Virginia Commonwealth University. Dr. Franson is recipient of NIH Research Career Development Award, PII.-00561. Reprint requests to Dr. Rosenblum, Division of Neuropathology, Medical College of Virginia, Box 17, MCV Station, Richmond, Virginia 23298.

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REFERENCES 1.

ROSENBLUM, W.I. and EL-SABBAN, F. Proaggregatory effects of fasting on platelet aggregation in the microcirculation of mice with streptozotocin diabetes. Arteriosclerosis L, 127-133, 1981.

2.

SMITH, J.B. The prostanoids in hemostasis and thrombosis. A review. Am. J. Path. 2, 743-804, 1980.

3.

BELL, R.L. and MAJERUS, P.W. Thrombin induced hydrolysis of phosphatidylinositol in human platelets. J. Biol. Chem. -- 225, 1790-1792, 1980.

4.

BILLAH, M.M., LAPETINA, E.G., and CUATRECASAS, P. Phospholipase A2 activity specific for phosphatidic acid. J. Biol. Chem. 256, 5399-5403, 1981.

5.

ROSENBLUM, W.I., ELLIS, E.F. and COCKRELJ,,C. Effect of fasting on production from exogenous arachidonic acid of thromboxane and 12- hydroxy 5, 8, 10, 14 eicosatetraenoic acid (12-HETE) by washed platelets of normal and streptozotocin diabetic mice. Thromb. Res. g, 713-719, 1982.

6.

SULLIVAN, L.M. and MATHIAS, M.M. Eicosanoid production in rat blood as affected by fasting and dietary fat. Prostaglandins, Leukotrienes and Medicine% 223-233, 1982.

7.

ROSENBLUM, W.I., EL-SABBAN, F. and ELLIS, E.F. Aspirin and indomethacin enhance platelet aggregation in mouse mesenteric arterioles. Am. J. Physiol. 239, H220-H226, 1980.

8.

JAFFE, B.M. and BEHRMAN, H.R. ed. Methods of Hormone Radioimmunoassay. Acad. Press, N. Y. p 19-34, 1974.

9.

HIRSH, P.D., HILLIS, L.D., CAMPBELL, W.B., FIRTH, B.D. and WILLERSON, J.T. Release of prostaglandins and thromboxane into the coronary circulation in patients with ischemic heart disease. N. Engl. J. Med. 304, 685-691, 1981.

10.

FRANSON, R., DOBROW, R., WEISS, J., ELSBACH, P. and WEGLICKI, W.B. Isolation and characterization of a phospholipase A2 from an inflammatory exudate. J. Lipid. Res. 19, 18-23, 1978.

11.

JESSE, R.L. and FRANSON, R.C. Modulation of purified phospholipase A2 activity from human platelets by calcium and indomethacin. Biochem. Biophysic. Acta 575, 467-470, 1979.

12.

BLIGH, E.G. and DYER, W.J. A rapid method of total lipid extraction. Am. J. Physiol. 37, 911-917, 1959.

13.

BRADFORD, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254, 1976.

14.

GIANNESSI, D., DE CATERINA, R., GAZZETTI, P., BERNINI, W., MASINI, S. and ZUCCHELLI, G.C. Methodological assessment and applications of a radioimmunoassay for thromboxane (TxB ) to measurement of TxB2 release by platelets. J. Nucl. Med. Allied. 5ci. 26, 25-33, 1982.

564

EFFECTS OF FOOD DEPRIVATION

Vo1.31, No.4

15. DAY, J.J., HOLMSEN, H. and ZUCKER, M.B. Report of the working party on platelets. Thrombos. Haemostas. 36, 263-267, 1976. 16.

ELDOR, A., MERIN, S. and BAR-ON, H. The effect of streptozotocin diabetes on platelet function in rats. Thromb. Res. u, 703-714, 1978.

17.

TAKEDA, H., MAEDA, H., FUKUSHIMA, H., KANAMURA, N. and UZAWA, H. Increased platelet phospholipase activity in diabetic subjects. Thromb. Res. 2, 131-141, 1981.

18.

FRANSON, R.C. Intracellular metabolism of ingested phospholipids Ch 12 in Liposomes: from Physical Structure to Therapeutic Applications. ed. E. Knight, Elsevier Press, N.Y., 1981.

19.

ELSBACH, P., WEISS, J., FRANSON, R.C., BECKERDITE-QUAGLIATA, S., SCHNEIDER, A. and HARRIS, L. Separation and purification of a potent bactericidal/permeability increasing protein and a closely associated phospholipase A from rabbit polymorphonuclear leukocytes. J. Biol. Chem. 254, 1100&11009, 1979.

20.

LANNI, C. and FRANSON, R.C. Localization and partial purification of a neutral-active phospholipase A2 from BCG-induced rabbit alveolar machrophages. Bioch. Bilophys. Acta 658, 54-63, 1981.

21.

FRANSON, R. and WAITE, M. Relation between calcium requirement, substrate charge and rabbit polymorphonuclear leukocyte phospholipase A2 activity. Biochemistry ,l7, 4029-4033, 1978.

22.

BILLAH, M.M., LAPETINA, E.G. and CUATRECASAS, P. Phospholipase A and phospholipase C activities of platelets. J. Biol. Chem. 255, 102%710231, 1980.

23.

KANNAGI, R. and KOIZUMI, K. Phospholipid deacylating enzymes of rabbit platelets. Arch. Bioch. Biophys. 12, 534-542, 1979.

24.

WALLENSTEIN, S., ZUCKER, C.L. and FLEISS, J.L. Some statistical methods useful in circulation research. Circ. Res. 47, l-9, 1980.