Choline deficiency activates phospholipases A2 and C in rat liver without affecting the activity of protein kinase C

Choline deficiency activates phospholipases A2 and C in rat liver without affecting the activity of protein kinase C Ushasi Singh, Kinichi Yokota,* Chanda Gupta, and Hisashi Shinozuka D e p a r t m e n t o f Pathology, University o f Pittsburgh School o f Medicine, Pittsburgh, PA, USA

There is evidence to suggest that liver tumor promoters exert their effect through the interference of signal transduction in hepatic cells. Both phospholipase A2 and phospholipase C play important roles in the generation of second messengers and in the activation of Ca 2+ , phospholipid-dependent protein kinase C. Using male Sprague-Dawley rats, we investigated whether liver tumor-promoting regimens of a choline-deficient diet and phenobarbital alter the activities of phospholipase A2 and phospholipase C in the liver, and extended the study to determine the effect of a choline-deficient diet on protein kinase C activities. Feeding a choline-deficient diet for l week increased the activities of both phospholipase A2 (50%) and phospholipase C (22%), and the activities of both enzymes were more than doubled after 4 weeks. Feeding a phenobarbital diet resulted in a slight decrease in phospholipase A2 activities at 4 weeks but no significant changes in PLC activities. The protein kinase C activities and its distribution between soluble and particulate .[?actions remained unchanged after 1. 2, and 4 weeks ,feeding of a choline-deficient diet. Thus. activation of both phospholipase A2 and C is distinct for a choline-deficient diet, not shared by phenobarbital diet. Increased activities of these enzymes were not associated with the activation of protein kinase C under the present experimental condition.

Keywords:choline deficiency;liver; phospholipase A2 and C; protein kinase C; liver tumor promotion

Introduction Choline has been regarded as one of the important dietary components in maintaining the structural and functional integrity of many types of cells in the body. 1,2 In experimental animals, choline deficiency in diets leads to a variety of pathological conditions, including the development of neoplasms in the liver, pancreas, and lung. 3'4 A choline-deficient (CD) diet also enhances the development of chemically induced tumors in a number of experimental models. 5 In the liver, long-term feeding of a CD diet alone leads to the development of hepatocellular carcinomas. 6-8 It is not

Address reprint requests to Hisashi Shinozuka, Department of Pathology, Universityof Pittsburgh Schoolof Medicine, Pittsburgh, PA 15261, USA. *Present address: Third Department of Internal Medicine, Asahikawa Medical College, Japan. Received February 12, 1990; accepted March 9, 1990. 434

J. Nutr. Biochem., 1990, vol. 1, August

certain, however, whether a diet deficient in choline acts as a carcinogenic regimen or merely exerts strong promoting stimuli for tumor development. 9'1° A CD diet and phenobarbital (PhB) served as two prototype liver tumor promoting regimens in experimental animals and have been used extensively for study on the pathogenesis of experimental liver cancers.11,12 The results from this laboratory and others suggest that both a CD diet and PhB altered the properties of liver cell surface receptors for certain growth factors, such as epidermal growth factor and insulin. 13-16 Although the mechanisms by which these two regimens modify the properties of cell surface growth factor receptors are not known, the data strongly suggest that their actions are mediated through the interference of signal transduction pathways in hepatic cells. 15 A phospholipid- and Ca 2+-dependent protein kinase C (PKC) plays a central role in a variety of membranerelated signal transduction events. 17,~s Both phospholi© 1990 Butterworth-Heinemann

Choline deficiency and hepatic phospholipase A2 and C: Singh et al. pase Az (PLAe) and phospholipase C (PLC) are involved in the activation of PKC. Phospholipase Az hydrolyses membrane-bound phosphatidylcholine and liberates free fatty acids, such as arachidonic acid and lysophospholipids, which regulate the activity of P K C . 19-21 It has been shown that certain lysophospholipids activate PKC at a lower concentration and inhibit at a higher concentration. 2~ In the better characterized system, PLC hydrolyses phosphatidylinositol 4,5-diphosphate to 1,2-sn-diacylglycerol (DAG) and to inositol 1,4,5-triphosphate, both of which activate PKC. 22 A CD diet is a well-known dietary regimen which induces changes in phospholipid compositions of cellular membrane. 3'~6 In the present study, we investigated whether a CD diet modifies the activities of PLA2 and PLC in the liver and the results were compared with those induced by PhB diet. Since only a CD diet selectively enhanced PLA2 and PLC, we extended our study to determine whether the diet modifies the PKC activity in the liver.

Materials and methods

Animals and diets Male Sprague-Dawley rats (Zivic Miller Laboratories, Allison Park, PA, USA) weighing 170-180 g at the beginning of experiments were used. They were housed individually in metal wire cages in a room that maintained 12-h light/dark cycles, constant temperature of 20 _+ 2°C and 50 _+ 5% humidity. They were given Purina chow (Ralston Purina, St. Louis, MO, USA) and water ad libitum. Rats were acclimatized to the facility at least 10 days before the start of experiments. Semisynthetic semipurified CD or cholinesupplemented (CS) diets were prepared by Dyet Inc. (Bethlehem, PA, USA) and their compositions have been described previously. 23 The analyses by the manufacturer indicated that the CD and CS diets contained 6.3 and 6942 mg of pure choline per kilogram of diet, respectively. Phenobarbital (Sigma Chemical Co., St. Louis, MO, USA) was incorporated in the basal diet (Dyer Inc.) at a concentration of 0.06%. Rats on various dietary regimens were killed at the indicated time, blood was drained, and the livers were removed and weighed.

Extraction o f the enzymes and their assays Phospholipase A2 assays were performed with slight modifications of the methods previously described. 24 The liver (250 mg/mL) was suspended in 20 mM TrisHC1 buffer containing I mM EDTA and 0.25 M sucrose, pH 8.5, and homogenized for 5-6 s with a Branson cell disruptor. The homogenates were centrifuged at 1000g for 30 min. The upper lipid layers were discarded and the supernatants were kept frozen at - 7 5 °C until used for the assay. The enzyme was diluted to 1 : 10 with homogenizing buffer before assay. At the optimum conditions of assays, the final concentrations of the assay mixture were 30 mM CaC12,

6.6 txM (5 × 10 4 cpm) of [14C]phosphatidylcholine (L-3-phosplatidylcholine, 1-stearoyl-2-[ 1-14C]arachidonyl, sp act 58.3 mCi/mmol) and 20 IxL of enzyme in a total volume of 300 txL of 20 mM Tris-HCl buffer containing 1 mM EDTA, pH 8.5. The reaction was carried out for 10 min at 37°C. The activity was determined by measuring released 14C-labeled fatty acid from phosphotidylcholine. Free fatty acid was separated from phosphotidylcholine25 and an aliquot of the fatty acid layer was counted in a scintillation counter. Protein was determined by the method of Lowry et al. using bovine serum albumin as a standard. 26 The assay of PLC was based on the method described by Rittenhouse 27 with slight modifications. The liver (250 mg/mL) was homogenized with a Branson cell disruptor in 40 mM Tris-HCl, 1 mM EDTA, 120 mM NaC1, and 100 txM phenylmethylsulfonylfluoride, pH 6.5. The homogenates were centrifuged in Beckman centrifuge at 105,000g for 90 min. The supernatant was removed carefully so that the upper fatty layer was excluded and kept at - 7 5 °C until ready for assay. A simple and rapid assay for the detection of phosphatidylinositol-specific PLC was used. The final concentrations of the assay mixtures were 20 txM [3H]phosphatidylinositol (Amersham, 0.02 txCi), 5 mM CaC12, 2 mg/mL deoxycholate-Na-salt, and 100 ~g supernatant protein in a total volume of 300 ~xL of 60 mM Tris-HC1 buffer, pH 6.5. The chloroform solution of radiolabeled phosphatidylinositol was evaporated under N2 and the residue was dissolved in H20 at - 4 ° C using a sonicator. The reactions were started by adding the substrate and continued up to 30 min. At the end of incubation, the reactions were terminated by the addition of 1.5 mL of ice cold chloroform-nbutanol-concentrated HCI (I0: 10:0.06) and 0.4 mL of 1 M HCI. The mixtures were vortexed and then spun at room temperature at 1000 rpm for 5 min, and 250 ~L of the upper aqueous phase was transferred to scintillation vials for counting. The activity of the PLC was estimated from the radioactivity released in the aqueous layer and the results were expressed as picomoles of radioactive phosphatidylinositol hydrolyzed per microgram protein. The products in the aqueous layer were most likely to be PLC-induced inositolphosphate as they were bound to Dowex - 1 column at neutral pH and they were quantitatively eluted by 0.2 M ammonium formate. Even though the assay was developed for the detection of phosphatidylinositol-specific P L C , 27 the possibility remains that the product in the aqueous layer was produced via PLA2 and lysophospholipase pathways. PKC preparations of the soluble and particulate fractions of the liver were done according to the methods of Okamoto et al. 28 and Vaartjes et al. 29 with slight modifications. The liver (50 mg/mL) was suspended in 20 mM Tris-HCl buffer, pH 7.4, containing 10 mM EDTA, 2 mM EGTA, 0.33 M sucrose, 2 mM PMSF, and 50 mM 13-mercaptoethanol (buffer A) and homogenized for 5-6 s with a Branson cell disruptor.

J. Nutr. Biochem., 1990, vol. 1, August

435

Research Communications The homogenate was centrifuged at 100,000g for 1 h at 4°C. The supernatant was used as a soluble fraction, and kept frozen at - 7 5 ° C until ready for loading on column. The pellet was washed and homogenized (pellet from 50 mg liver/mL) with buffer A containing 1% (v/v) NP40 in a glass homogenizer. The homogenate was stirred gently for 40-45 min at 4°C and centrifuged for 1 h at 100,000g. The supernatant was used as solubilized particulate fraction and kept frozen at - 7 5 °C. PKC was partially purified from both soluble and particulate fractions by ion exchange chromatography on DE-52 column according to the method described by Kikkawa et ai. 3° The DE-52 column (1 x 10 cm) was equilibrated with 20 mM T r i s - H C I buffer, pH 7.4, containing 10 mM E D T A , 2 mM E G T A , and 50 mM [3mercaptoethanol (buffer B). Soluble and solubilized particulate fraction (3 mL) was applied on a column and washed with 20 m L of buffer B and then with 20 m L of 20 mM T r i s - H C I buffer, pH 7.4, containing 1 mM E D T A , 1 mM E G T A , and 50 mM [3mercaptoethanol (buffer C). Finally the column was eluted with a linear gradient (100 mL) of 0-0.4 M NaCI in buffer C. The fractions eluted with starting gradient to 0.! M NaC1 were pooled. Protein kinase C activity was measured according to the method of Kikkawa et al. 3° The final concentrations of the assay mixture were 20 mM T r i s - H C l buffer, pH 7.4, 12 mM magnesium acetate, I mM CaCI> 50 mM [3-mercaptoethanol, 3.7 IxM [y-32p]ATP (sp act 0.5-3.0 Ci/mmol), 22.2 txg/mL histone type lI1 S, 37 ixg/mL L-cx-phosphatidyI-L-serine, 18.5 ixg/mL 1,2-dioctanoyl-sn-glycerol, and 100 IxL of pooled enzymes in a total volume of 270 IxL. The control assay contained 4.6 mM E G T A instead of CaCIz, phosphatidylserine, and DAG. An e n z y m e blank was also run during assays. After incubation for 3 min at 30°C, a 50IxL aliquot from assay mixture was transferred onto a 2.5-cm- piece of phosphocellulose paper (Whatman, PC 81) and washed with water, acetone, and petroleum ether. 31 After drying, the paper was counted in a liquid scintillation counter. Protein kinase C activity was obtained after subtracting the control value from the value obtained in the presence of CaCl2, phosphatidylserine, and diacylglycerol. The e n z y m e activity was expressed as picomoles of 3eP incorporated per minute per milligram of protein.

Statistical analyses The data were statistically evaluated using Student's t test, taking p < .05 as the level of significance.

Results

Phospholipase A2 The results of the effect of feeding a CD diet on PLAe activities in the liver are shown in Figure la. After 1 and 2 weeks, the activities were increased 53 and 83%, respectively, in the livers of rats fed a CD diet over that of rats fed a CS diet. After 4 weeks, the PLA2 436

J. Nutr. Biochem., 1990, vol. 1, August

~'*

12-

E

10.

1

8E v

A

c

6-

"6

4-

.<

c-4

5

2.

(3-

0

1W

2W

4W

Weeks 12 E

10 8

E G

6 >

~

4

~

2. 0

1W

LL-2W

4W

Weeks Figure 1 (a) PLA2 activity in the liver of rats fed a CD or CS diet for 1, 2, and 4 weeks. Four rats in each group were sacrificed at each time point, and PLA2 activities were measured as described under Material and methods Each bar represents the mean _~ SEM *Significant at the level o t p < .05: **significant a t p < .01. El, CS; I~, CD. (b) PLA 2 activity in the liver of rats fed a basal or PhB diet for 1,2, and 4 weeks. Four rats in each group were sacriliced at each time point, and PLA2 activities were measured as described under Material and methods. Each bar represents the mean ÷ SEM. **Significant at the level of p <: .05. II, Basat: [~ PhB

activities in the CD liver were more than tripled over those in the CS liver. There were no significant changes in PLA2 activities in the first 2 weeks in the liver of rats fed a PhB diet as compared to rats fed a basal diet (Figure lb). A significant decrease in PLA2 activities occurred in the liver after 4 weeks feeding of a PhB diet.

Phospholipase C The results of the effects of feeding CD and PhB diets on PLC activities in the liver are shown in Figures 2a and b. There was a significant increase in PLC activities after 1 week of a CD diet. After 4 weeks, the activities were doubled in rats fed a CD diet, as compared to rats fed a CS diet (Figure 2a). Feeding a PhB diet had no significant effects on PLC activities as compared to feeding a basal diet (Figure 2b).

Protein kinase C Ca z+-, PS-, and DAG-dependent partially purified PKC was eluted from a DE-52 column at around 0.1 M

Choline deficiency and hepatic phospholipase A2 and C: Singh et al. 16-

S

14. E .c: E "5 E

12-

10, 8

v

6. 4

,4, n

2 0

i. i., 1W

2W

4W

tion of both PLAz and PLC in the liver of rats fed a CD diet may have effects on several metabolic pathways. We recently reported that a CD diet induced a marked elevation of prostaglandin Ez in the liver and the levels of elevation were correlated to the efficacy of the dietinduced tumor promotion. 23 The generation of excess arachidonic acid by enhanced PLA2 activity may contribute to the diet-induced alterations in prostaglandin metabolism. Phospholipase C acts on membrane inositol phospholipids to generate second messengers, that is, diacylglycerol and inositol 1,4,5-triphosphate, both of which activate PKC. Accumulation of 1,2-sn-DAG in

Weeks 16 150-

"~ E "~._. E ~ "5

A

14. 12 10 100

°~_

6

~ o,

4 2.

.0.4

'O

x

1W

2W

4W

• 0.3 50

- 0.2

Weeks

Figure 2 (a) PLC activity in the liver of rats fed a CD or CS diet for 1, 2, and 4 weeks. Four rats in each group were sacrificed at each time point, and PLC activities were measured as described under Material and methods. Each bar represents the mean _+ SEM *Significant at the level of p < .05. II, CS; [], CD. (b) PLC activity in the liver of rats fed a basal or PhB diet for 1, 2, and 4 weeks. Four rats in each group were sacrificed at each time point, and PLC activities were measured as described under Material and methods. Each bar represents the mean _+ SEM. II, Basal: rq, PhB

NaC1 (Figure 3). Specific activities of the enzyme both from soluble and particulate fractions from the liver of rats fed a CS and CD diet at different time intervals are presented in Table 1. Feeding a CD diet for I, 2, and 4 weeks did not result in any difference in the distribution of PKC activity between soluble and particulate fractions when compared with PKC activity after feeding a CS diet for the same period of time.

~mm

z • 0A

o

• 0.0

12

No. of Fraction 3 PKC activity after DE52 column chromatography of a soluble fraction of the liver of a rat fed a CS diet for 2 weeks. A solubilized particulate fraction gave an identical profile. Enzyme activity was measured in 100-#L aliquots from each fraction under the standard condition described under Materials and methods. Peak A: Ca 2. , phosphatidyl serine (PS), dioctonoyl-sn-g~ycerol (DAG) dependent protein kinase. Peak B: Protein kinase independent of Ca 2+, PS, and DAG. A, - (GaOl2, PG, DAG) + EGTA; O, + (CaCI2, PS, DAG) - EGTA; O, NaCI gradient. Figure

Table 1

Specific activity of protein kinase C (PKC) in the liver of rats fed a choline-deficient (CD) or choline supplemented (CS) diet a

Discussion The results of the present study demonstrated that a CD diet, a well-established liver tumor-promoting regimen, induced a more than twofold increase in the activity of both PLA2 and PLC in the liver. By contrast, PhB, another type of well-known liver tumorpromoting agent, had no effects on these enzymes. Based on the observation that two regimens induced similar alterations in hepatocyte surface receptors to certain liver cell growth controlling factors, it has been suggested that both regimens may lead to common patterns of interference with signal transduction. 15 The present results do not support this hypothesis. Activa-

~

Specific activity b (pmol/min/mg protein)

Duration Diet

(weeks)

CS CD CS CD CS CD

1 1 2 2 4 4

So lu b le 169.5 173.4 134.8 147.2 151.7 158.8

-+ 34.8 -+ 15.5 _-z-12.6 ~ 12.9 + 13.9 ~ 415

Particulate 61.3 49.2 32.7 34.4 44.0 38.2

± ± ± ~ ± ~

15.5 19.1 5.4 6.3 11.8 7.5

a Soluble and particulate fractions of PKC were measured in the liver of rats fed a CD or CS diet for 1,2, and 4 weeks by the methods detailed under Material and methods. b Each figure represents the mean +- SEM of 3 rats.

J. Nutr. Biochem., 1990, vol. 1, August

437

Research Communications the liver of rats fed a CD diet has been reported. 32 The hydrolysis products of membrane phospholipids by PLA2, such as arachidonic acid and certain lysophospholipids are known to regulate the activity of PKC. ~s.2~ When these observations are taken together, it is attractive to suggest that feeding a CD diet may activate PKC in the liver, acting as an endogenous tumor promoter. However, the present experiments showed no significant changes in either soluble or particulate PKC activity in the liver of rats fed a CD diet, and there was no shift in the activities between the two fractions. In the present study, a relatively high concentration of metal chelators were used to protect PKC from proteolysis by Ca 2 +- dependent neutral protease, as suggested by Kikkawa et al. 3° Since the ratio of PKC found in cytosol and particulate fractions may be regulated, in part, by calcium, the use of chelators might have contributed to the higher enzyme activity in the soluble fractions. The reasons for the failure to demonstrate PKC activation in the liver of rats fed a CD diet despite enhanced PLA2 and PLC activities and reported accumulation of 1,2-sn-DAG in the liver are not known. Blusztajn and Zeisel also reported that there was no activation of PKC in the liver of rats fed a lipotrope-deficient diet. 33 Certain lysophospholipids generated by the action of PLAz, such as lysophosphatidylcholine, have been reported to be a stimulator or inhibitor of PKC, depending on the concentration. 21 It is possible that there may be excess lysophospholipids in the liver of CD-fed rats interfering with the activity of PKC. Alternatively, the existence of two or three isozymes of PKC in the liver has been reported. 34"3-sThus, a CD diet may shift the activities of these isozymic forms of PKC without affecting total PKC activity as determined in the present experiments. Studies are in progress to assess these possibilities.

6 7 8 9

10

11 12

13

14

15

1162-1165 16

17 18 19

Acknowledgments The study was supported by grants from the National Institute of Health (CA 26556 and CA 40062), and the Pathology Education and Research Foundation. We gratefully acknowledge the technical assistance of Christine M. Tappe and the typing of the manuscript by Christel Lollo.

20

References

23

1 2 3

4 5

438

Kuksis, A. and Mookerjea, S. (1978). Choline. Nutr. Rev. 36, 201-207 Zeisel, S.H. (1981). Dietary choline: biochemistry, physiology and pharmacology. Annu. Rev. Nutr. 1, 95-121 Copeland, D.H. and Salmon, W.D. (1946). The occurrence of neoplasms in the liver, lungs, and other tissues of rats as a result of prolonged choline deficiency. Am. J. Pathol. 22, 1059-1081 Shinozuka, H. and Katyal, S.L. (1984). Pathology of choline deficiency. In Nutritional Pathology (H. Sidransky, ed.), pp. 279-320, Dekker, New York Rogers, A.E. and Newberne, P.M. (1982). Lipotrope deficiency in experimental carcinogenesis. Nutr. Cancer 2, 104112

J. Nutr. Biochem., 1990, vol. 1, August

Mikol, Y.G., Hoover, K.L., Creasia, D., and Poirier, L.A. (1983). Hepatocarcinogenesis in rats fed methyl-deficient amino acid-defined diets. Carcinogenesis 4, 1619-1629 Ghoshal, A.K. and Farber. E. (1984). Induction of liver cancer by a dietary deficiency of choline and methionine withoul added carcinogens. Carcinogenesis 5, 1367-1370 Yokoyama, S., Sells, M.A., Reddy, T N . , and Lombardi, B. (1985). Hepatocarcinogenic and promoting action of a cholinedevoid diet in the rat. Cancer Res. 45, 2834-2842 Shinozuka, H., Katyal, S.L., and Perera, M.I.R. (1986). Choline deficiency and chemical carcinogenesis. In Essential Nutrients in Carcinogenesis (L.A. Poirier, P.M. Newberne. and M.W. Pariza, eds.), pp. 253-267, Plenum, New York Lombardi, B. (1988). The choline-devoid diet model of hepatocarcinogenesis in the rat. In Chemical Carcinogenexis (F. Feo, P. Pani, A. Columbano, and R. Garcia, eds.), pp. 563-581, Plenum, New York Sarma, D.S.K., Rao, P.M., and Rajalakshmi, S. (1986). Liver tumor promotion by chemicals: models and mechanisms. Cancer Surv. 5, 781-798 Pitot, H. (1988). Hepatic neoplasia. In Chemical Induction in the Liver: Biology and Pathohiology (I.M. Arias, W.B. Jakoby, H. Popper, D. Schachter. and D.A. Shafritz, eds.), pp. 1125, Raven, New York Hwang, D.L., Roitman, A., Lev-Ran, A., and Carr, B. (1986). Chronic treatment with phenobarbital decreases the expression of rat liver EGF and insulin receptors. Biochem. Biophys. Res. Commun. 135, 501-506 Betschart, J.M., Virji, M.A., Gupta, C., and Shinozuka, H. (1988). Alterations induced by phenobarbital, a liver tumor promoter, in hepatocyte receptors for insulin and glucagon and glycogen metabolism, Carcinogenesis 9, 1289-1294 Gupta, C., Hattori, A., Betschart, J.M., Virji, M.A., and Shinozuka, H. (1988). Modulation of epidermal growth factors in rat hepatocytes by two tumor-promoting regimens, a choline-deficient and a phenobarbital diet. Cancer Res. 48,

21 22

24 25 26 27

Eckl, P.M., Meyer, S.A., Rive'Whitcombe, W., and Jirtle, R.L. 11988). Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation. Carcinogenesis 9, 479-483 Nishizuka, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 305-318 Blackshear, P.J,, Nairn, A., and Kuo. J.F. (1988). Protein kinases 1988: a current perspective. FASEB J. 2, 2957-2969 McPhait, L.C., Clayton, C.C., and Snyderman, R. (19841. A potential second messenger role for unsaturated fatty acids: activation of Ca-" ~ dependent protein kinase. Science 224, 622-625 Sekiguchi, K., Tsukada, M., Ogita, K., Kikkawa, U., and Nishizuka, Y. (1987). Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids. Bioehem. Biophys. Res. Commun. 145, 797-802 Oishi, K., Raynor, R. L., Charp, P.A., and Kuo, J.F. (1988). Regulation of protein kinase C by lysophospholipids potential role in signal transduction. J. Biol. Chem. 263, 6865-6871 Nishizuka, Y. (1984). The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308, 693 -698 Gupta, C., Banks, M., and Shinozuka, H. (1989). Elevated levels of prostaglandin E 2 in the liver of rats fed a choline deficient diet: possible involvement in liver tumor promotion. Cancer Lett. 46, 129-135 Gupta, C. (1988). Activation of phospholipases during masculine differentiation of embryonic genitalia. Proc. Soc. Exp. Biol. Med. 188, 489-494 Dole, V.P. and Meinertz, H. (1960). Microdetermination of long chain fatty acids in plasma and tissue. J. Biol. Chem. 235, 2595-2599 Lowry, G.H., Rosenbrough, N.J., Farr, A.L., and Randell, R.J. (1957). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 Rittenhouse, S.E. (1982). Preparation of selectively labeled phosphatidylinositol and assay of phosphatidylinositol specific phospholipase C. Methods Enzymol. 86, 3-11

Choline deficiency and hepatic phospholipase A2 and C. Singh et al 28 29

30

31

Okamoto, Y., Nishimura, K., Nakayama, M., Nakagawa, M., and Nakano, H. (1988). Protein kinase C in the regenerating rat liver. Biochem. Biophys. Res. Commun. 151, 1144-1149 Vaartjes, W.J., DeHaas, C.G.M., and Van den Bergh, S.G. (1986). Phorbol esters, but not epidermal growth factor or insulin, rapidly decrease soluble protein kinase C in rat hepatocytes. Biochem. Biophys. Res. Commun. 138, 1328-1333 Kikkawa, U., Minakuchi, R., Takai, Y., and Nishizuka, Y. (1983). Calcium-activated phospholipid dependent protein kinase (protein kinase C) from rat brain. Methods Enzymol. 99, 288-298 Witt, J.J. and Roskoski, R., Jr. (1975). Rapid protein kinase assay using phosphocellulose paper absorption. Anal. Biochem. 66, 253-258

32

33 34

35

Blusztajn, J.K. and Zeisel, S.H. (1989). 1,2-sn-Diacylglycerol accumulates in choline-deficient liver. A possible mechanism of hepatic carcinogenesis via alteration in protein kinase C activity. FEBS Lett. 243, 267-270 Blusztajn, J.K. and Zeisel, S.H. (1988). Accumulation of 1,2sn-diacylglycerol in choline-deficient liver. J. Cell Biol. 107, 277a Azhar, S., Butte, J., and Reaven, E. (1987). Calciumactivated, phospholipid-dependent protein kinases from rat liver: subcellular distribution, purification, and characterization of multiple forms. Biochemistry. 26, 7047-7057 Houweling, M., Vaartjes, W.J., and van Golde, L.M.C. (1989). lsozymic forms of protein kinase C in regenerating rat liver. FEBS Lett. 247, 487-491

J. Nutr. Biochem., 1990, vol. 1, August

439