Effects of glucose on the biochemical changes in rat liver in acute ethionine intoxication

EXPEIUMENTAL

ASD

MOLEC'ULAR

Effects of Glucose Liver ALBERTO

on the

in Acute N.

14, 178-183

P.~THOLOGY

RAICK,

A.

Biochemical

Ethionine s.

(1071)

Changes

in Rat

Intoxication’

XARAYAKAN,

A. C.

AND

RITCHIE

WXen glucose is giren to female rats 6 hours after a dose of ethionine. there is rapid reversal of the inhibition of RiYA qnthesis induced in lhc liver by the ethionine. Three hours after the administration of glucose, thr lr~wl of RNA1 synthesis is 72% of normal, as compared n-it.11 a lewl of 15% of normal in rats given ethionine but. no glucose. The inhibition of protein synthesis is reT-crsed by glucose more slowly, reaching 27%’ of nornnd in 3 hours, and 54% in 18 llours. In rats given rthionine. but not glucose. tile le,-el is 18% at 3 hours and 27s’ at 18 hours. Glucose failed to alter sipnificxntly the level of r\TP in the liar. or to incrrasc tlrc r:itc of ATP turnover. The significnnw of these results is discussed.

Ethionine, the ethyl analog of methionine, causes a w:cLll-established and reproducible sequence of biochemical and morphological changes in female rat liver (Baglio and Farber, 1965; Bartels and Hohorst, 1963; Farber, 1967; Farber et al., 1959; Farber et nl., 1964; Miyai and Steiner, 1967; Olivercrona, 1962; Schlenk and Lombardi, 1969; Shinozuka et al., 1968; Shull, 1962; Shull et nl., 1966; Stekol, 1963; Villa-Trevino and Farber, 1962; Villa-Trevino et OZ.: 1963; Villa-Trevino et nl., 1964; Villa-Trevino et al., 1966). The effects of acute ethioninc intoxirat’ion are readily rcverscd by the administration of mcthionine, ATP, or the precursors of ATP (Farber et al., 1965; Shull et nl., 1966; VillaTrevino and Farber, 1962; Villa-Trerino et rrl., 1963). Glucose and sucrose can also prevent or rcversc the fatt,y liver inducctl by ethioninc ( Campannri-Visconti et al., 1962; Farber et al., 1959), but unlike met,hionine, ,4TP and its precursors, do not affect the inhibition of protein synthesis induced by ethionine (Simpson et al., 1950). The ultrastructural changes which occur in rat’ liver when the fatty change induced by cthioninc is prevented or reversed by glucox have been previously reported from this laboratory (Miyai and Raick, 1969; 1,Iiyai et al.. 1970). This paper presents the biochcmicnl changes vssocinted with the revttrsal of the ethioninc effects induced by glucose. MATERIALS

BND

METHODS

Female, white Wistar rats (Woocllyn Farms, Guelph, Ontario), weighing 140 to 160 gm were fasted overnight prior to treatment and kept without food throughout t,he experiment. Water was allowed ad liDI:t~~l. The experimental ’ This

work

was supported

by grant-in-aid

of the Nat,ional

176

Cancer

Institute

of &nada

EFFECT

OF

GLUCOSE

IN

ACUTE

ETHIONINE

INTOXICATION

177

groups each contained six rats. nn-Ethione (25 mg/ml in physiologic saline) was injected intraperitoneally in a dose of 1 mg per gram body weight between 6 and 7 AM. Six hours after the injection of ethionine, 16 mmole of D-&lCOSe dissolved in 4 ml of normal saline was given by stomach tube under ether anaesthesia. Control rats received saline in corresponding volume by a similar route in place of the ethionine, glucose, or both. The rats were killed 3, 6, or 12 hours after the administration of glucose. Fragments of the left lateral lobe of the liver were immediately removed, homogenized with a Teflon pestle in 10 ml of ice-cold 10% trichloroacetic acid (TCA) for isolation of RNA or protein, or in 2% perchloric acid for ATP extraction. The RNA and protein were isolated according to the method of Schneider (1945). The RNA was determined by the Orcinol reaction (Majbaum, 1939)) the protein by the method of Lowry et nZ. (1951), and the ATP by the luciferin-luciferase reaction (Strehler and McElroy, 1957). Labelled

precursor

incorporation

studies

Orotic-B 14C acid hydrate (39.6 mCi/mmole) (New England Nuclear Corporation, Boston, Massachusetts), in a dose of 4 &$‘rat was injected intravenously through the jugular vein 1 hour before sacrifice, and L-leucine-l-14C (25.6 mCi/ mmole) in a dose of 3 &i/rat was injected 30 minutes prior to sacrifice. For adenosine triphosphate (ATP) turnover studies, 3”P01 (Atomic Energy Commission of Canada Ltd.) was administ’ered intraperitoneally in a dose of 20 #&‘rat, 30 minutes prior to sacrifice. The radioactivity of aliquots in 10 ml of Bray’s solution (Bray, 1960) were measured in a Packard Tricarb liquid scintillation counter. Isolation

and determination.

of ATP

For turnover studies, the ATP in t’he perchloric acid extract was adsorbed on activat,ed charcoal, and then eluted by four 5-ml volumes of 50% ethanol containing 1% ammonia. The washings were evaporated to dryness under vacuum in a rot,ary evapo-mix and loaded on a Dowex 1-Xg column (0.5 x 6 cm) in a formate form. The column was successively washed with 25-ml volumes of water, 2.5 N formic acid, and 0.1 M ammonium formate (pH 5.0), then finally with 15 ml of 1.1 M ammonium formate (pH 5.0) to yield ATP. The eluate was concentrated under vacuum in a rotary evapo-mix. The ATP thus obtained was further purified by paper chromatography along with standard ,4TP, using the solvent system isobutyric acid, 1 M ammonia, 0.01 M ethylene diamine tetra acetate (sodium salt) (100:60: 1.6 v/v). The ATP spot was located by UV light, and eluted with water. The concentration of ATP was determined by absorbance at 260 mp, and suitable aliquots were taken for measurement of radioactivity. RESULTS The effects of ethionine and glucose on the total liver RNA are given in Table I. When ethionine was followed by saline, the rate of RNA synthesis dropped to 15% of the control group (saline + saline) in 9 hours, and then increased to 43% of normal after 18 hours. When glucose was given 6 hours after the ethionine, RNA was synthesized at 72% of the rate in the control group 9 hours after

1%

RAICK.

NARAYANAN,

AND

RITCHIE

RXA synthesis count/min/mg

9

Percentage synthesis

of

SaliJle + salillr l~:thionitie + saline Ethioninc + glur~)sr SaliJle + saline I’:thiouitrr + saliJJe I~;thiorlitJr + gliIcose Salille + saliJle I:thioJJirJe + saline EthioJJiJJe + glucose

!J !J 12 12

12 1x IS IX

a ltat,s were injected ip with ethioilinc il rngjgm body weight) or s:tliJJe at, zero time. ;ii.\ later they were give11 u~g1J1cosc (l(i mmole ;rat) or saline by st c,Jnach tube. Orot ic6-W acid lJydrat,e (4 pCi,‘rat,) was ilrjrcted illto the jllglllar veil1 1 hol~r bcforr sacrifice. l’:ach gr,j,JlJ was of six rats.

huurs

cthionine (3 hours after glucose), and gradually increased to 80% of the control value 18 hours after ethionine (12 hours after glucose). Ethionine followed by saline reduced the rate of protein synthesis to 18:; of the control (saline + saline) value after 9 hours, and to 27% of the control value after 18 hours (Table II). %%en ethionine was followed by glucose, the rate was 27% of t’he cont’rol value 9 hours after ethionine (3 hours after glucose) and 54:; of the control value 18 hours after et,hionine (12 hours after glucose). That is to say, the reversal of the inhibition of protein synthesis induced by glucose islower than is the reversal of the inhibit’ion of RNA synthesis, and much 1~;:: complete.

Treatment

9 !J !I

12 11’ 12 18 18 1s a Rats were injected later they were given pC!i/rat) was iJJjected rats.

Protein synthesis count/&/mg

Percentage synthesis

of

----

Saline + saliJJe Ethionine + saliJJe E;thionine + glucose Saline + saline Ethionine + saline Ethionine + glucose Saline + saline l.:thioJline + saline I?thionine + glucose

155G *

119

275 415 1x93 403 tiso 907 344

f zk f xk f III

57 33 143 !J2 93 40

f f

14 10

4%

100 17.i Y2fi.i 100 21,:s 364

100 2G !I 53.0

-

ip with ethioJJine (mg/gm body weight) or saline at zero time. Sk holes n-glucose (lBmmole/rat) or saline by stomach tube. L-leucine-l-“C’ (3 into the jugular vein 30 minutes before sacrifice. Each group was of six

EFFECT

OF

GLUCOSE

IN

ACUTE TABLE

Hours after glucose or saline 3 3 3 ti 0 G 12 12 12

ETHIONINE

9 9 9 12 1% 12 1X 18 1X

1SU

III

Hours after

ethionine or saline

INTOXICATION

Treatment

Saline + saline Ethionine + saline Ethionine + glllcose Saline + saline Ethionine + saline Ethionine + glucose Saline + saline Ethionine + saline Ethionine + glucose

ATP content mg/gm liver

O.G-19 f 0.209 f 0.272 f 1.364 +I 0.123 A 0.5% f 1.389 f O.-l35 f 0.G33 f

0.041 O.Ml O.OOi 0.222 0.050 0.05fi 0.078 0.030 0.030

Percentage of content

100 38.2 41.9 100 31.0 40.0 100 31.3 -15.6

(I Experimental conditions were similar to Tables I anti 11. Each grollp was of sis rats. A fragrtlent of the left lateral lobe of the liver was rapidly excised and homogenized iu 2qb perchloric acid. The ATP concentration was determined by the Irlciferin-lllc,iferase reaction.

The possibility that the reversal of the effect of ethionine induced by glucose could be due t.o increased availability of ATP after glucose feeding, prompted us to compare the levels of ATP in t,he liver in the experimental groups. Table III shows that when ethionine was followed by saline the ATP content was reduced to 33% of the control value after 9 hours and remained so at 12 and 18 hours. After glucose treatment when ethionine was followed by glucose, the ATP level was 42% of the control value in 9 hours after ethionine (3 hours after glucose), 40% 12 hours after ethionine, and 45% 18 hours after. As the recovery of RNA synthesis is marked (Table I) while the level of ATP is almost unchanged, (a recovery of 10% when compared with ethionine treated groups) it was though that the marked restoration of RNA synthesis by glucose might be achieved by a higher turnover rate of ATP. To test this hypothesis, the turnover of ATP was &died hy the determination of incorporation of 32P into ATP. Table IV indicatc>s that, contrary to what might be expected, the rate of ATP turnover is reduced to about 65% of the control value in both the ethionine + saline and the ethionine + glucose groups. DISCUSSION This study demonstrates that glucose fed to female rats 6 hours after a dose of &hionine can reverse the inhibition of RNA and protein synthesis induced in the liver by the ethionine. This reversal induced by glucose differs from the reversal of the ethionine effect which is induced by methionine, adenine, ATP, and the precursors of ATP. These agents all reverse rapidly the inhibition of both RNA and protein synthesis, and with all there is a close parallelism between the degree of recovery of RNA and protein synthesis, the ATP level in the liver and liver triglyceride levels (Farber et al., 1964). In contrast, with glucose there is a rapid reversal of

RAICK,

180

NARAYANAN, TABLE

Hours after glucose Or

saline

HOLXS after ethionine or saline

AND

RITCHIE

IV

Incorporation into ATP count/min/mg

Percentage incorporation

the inhibition of RNA synthesis, a slo\l-er and less complete reversal of protein bynthesis with but little effect on the iZTP level. There is no correlation bctTyeen the degree of reversal of RNA synthchis, the degree of reversal of protein synthesis and the levels of ATP in the liver. Nor dots glucose affect the rate of a4TP synthesis in the liver. The incorporation of 32P in ATP was reduced to 65% of the control value in both the groups given ethionine + saline and those given ct’hionine + glucose. As reported in our previous study (Miyai and Raick, 1969; Miyni et al., 1970), glucose, like adenine and mcthionine, revcrscs many of the fine 5tructural changes induced by cthionine in the hcpntocytes, its most striking effect being the restitution of the nucleolar ultrns.tructure. The rcvc>rsal of the cytoplnsmic changes induced by ethionine is slower and is incomplete in a great number of the hepatocytes 12 hours after the administration of glucose (Miyai et al., 1970). The observations now reported agree with the electron microscopic data and suggest that the ultrastructural changes induced in the nucleolus by ethionine are associated with the inhibition of RNA synthesis it produces. Furthermore, the observation that nucleolar restitution occurs despite the absence of significant recovery in the ATP level or turnover, shows that the nucleolnr disaggregation induced by ethionine is not directly related to the degree of hTP deficiency in the hcpatocytcs, as was suggested by Shinozuka et al. (1968). The observation by Shinozuka and Farber (1968) that act,inomycin D given together with adenine 8 hours after ethionine, inhibits completely the nucleolar reformation, corroborates our suggest,ionthat the recovery of RNA synthesis and the restoration of the fine structure of the nucleoli are closely relat’ecl. A similar conclusion is suggestcclby the findings of Reddy and Svoboda (1968). The dissociation in the degree and time of recovery of protein and RNA synthesis which occurs after glucose feeding indicates that t’he recovery in protein synthesis probably is not essential for the restoration of nucleolar structure. The observat,ion that the morphology of the nucleolus is restored by adenine in the presence of a marked

EFFECT

OF GLUCOSE

IN ACUTE

ETHIONINE

INTOXICATION

181

inhibition of protein synthesis induced by cyclohexamide (Shinozuka and Farber, 1969) corroborates this suggestion. The observation that glucose induces a recovery of RNA and protein synthesis in ethionine-treated rats without a corresponding recovery in the ATP level in the liver seemsto conflict with the hypothesis that ATP deficiency is the basis of the changes induced by acute intoxication with ethionine (Farber et al., 1964). It is, however, possible that the small increase in ATP concentration observed after glucose feeding is sufficient to surpass a critical level, or that the small increase in the synthesis of ATP induced by glucose is preferentially utilized for RNA synthesis. The mechanism by which glucose reverses some of the acute effects of ethionine thus remains to be elucidated. It is, perhaps, pertinent however to draw attention to the relationship between glucose and insulin. Glucose restores the polysomal aggregation in fasting rats but not in alloxan diabetic rats (Floyd et al., 1966), while insulin restores the polysomal profile in alloxan diabetic rats, but not in fasted rats (Wittmann et al., 1969). Glucose and insulin together, but not separately, stimulate the incorporation of 14C-Ieucine into liver protein in alloxan diabetic rats (Penhos and Krahl, 1962, 1963). Insulin stimulates RNA synthesis and RNA polymerase activity in the liver in alloxan diabetic rats (Steiner and King, 1964, 1966)) and increases the activity of the enzymes of the pentose phosphate pathway in both diabetic rats (Steiner and King, 1964; Weber and Covery, 1966) and nondiabetic rats (Weber and Covery, 1966). Ethionine induces marked hypoglycemia in rats (Coombes and Schrenker, 1966) and glucose is known to cause insulin release (Fajans et al., 1964; Floyd et al., 1966; Mayhan et al., 1969). It is possible that the increase in RNA and protein synthesis caused by glucose is related to its stimulation of insulin release. REFERENCES C. M.. and FARBER, E. (1965). Correspondence between ribosome aggregation patterns in rat liver homogenates and electron micrographs following administration of ethionine. J. Mol. Biol. 12,466-467. BARTELS, H., and HOHORST, H. J. (1963). Zur Wirkung des Aethionins auf den Metabolitstatus der Rattenleber. Biochem. Biophys. Acta 71,214-216. BRAY, G. A. (1960). A simple efficient liquid scintillator, for counting aqueous solutions in a liquid scint.illation counter. Anal. Rio&em. 1,279-285. CAMPONARI-VISCONTI, L., CAMPONARI, F.. and KOCH-WESER, D. (1962). Inhibition by glucose of the ethionine induced fatty liver. Proc. Xoc. Ezp. Biol. Med. 111, 479-482. COMBES, B., and SCHRENKER. S. (1966). Eth’oi nine-induced hypoglycemia. Nature (London) 209,911-912. FAJANS, S. S.. FLOYD, J. C., JR., KNOPF, R. F., and CONN, J. W. (1964). A comparison of leucine and aceto acetate-induced hypoglycemia in man. J. Cl&. Invest. 43, 2003-2008. FARBER, E. (1967). Ethionine fatty liver. Advan. Lipid Res. 5, 119-183. FARBER, E., SHULL, K. H., MCCONO~Y, J. M., and CASTILM, A. E. (1965). The effects of inosine and aminoimidazole carboxamide upon the ethionine fatty liver. Biochem. Phnrmacol.14,761-767. FARBER, E., SHULL, K. H., VILLA-TREVINO. S., LOMBARDI, B., and THOMAS, M. (1964). Biochemical pathology of acute hepatic adenosine triphosphate deficiency. Nature (London) 203,34-40. FARBER, E., SIMPSON, M. V., and TARVER, H. (1959). Studies on ethionine. II. The interference with lipid metabolism. J. Biol. Chem. 182, 91-99. BAGLIO,

182

RAICK,

NARAYANAN,

AND

RITCHIE

J. C.. JR., FAJUS, S. S., COSN. J. W.. KNOPF, R. Ii‘., and RTLL, J. (1966). Stimulaof insulin secretion by amino acids. J. C/in. Iwest. 45, 1487-1502. LOTTRY, 0. H., ROSEBROUGH, N. J., F.u~R, A. L., and RANDALL, R. J. (1951). Protein measurement with folin phenol reagent. J. 13iol. Chem. 193,265-275. MAJBAUM, W. (1939). nber die Bestimmung Kleiner Pentosc-mengen, insbesonderc in Derivaten der Adenylsaiire. Hoppe-Seyler’s 2. Physiol. Chem. 258, 117-120. Regulation of insulin secretion. MAYHEW, D. $., UPRIGHT, P. H.. and ASHMORE, J. (1969). Phammacol. Re,u. 21, 183-212. Mayan, R., BARRT, J. M., and RIVER.~, E. M. (1966). Stimulation by insulin of the formation of ribonucleic acid and protein by mammary tissues i/L ,vil~o. Biochem. J. 99, 68% 693. MIYAI. K., and Raws, A. N. (1969). Reversal by glucose of the nucleolar changes induce~l by ethionine. Fed. PTOC. Fed. Amer. SOC. Exp. Biol. 28, 940. MIYAI, K., RAICH. A. N., and RITCHIE, A. C. (1970). Effects of glucose on the subcellul:~r structure of rat liver cells in acute ethionine intoxication. Lab. Invest. 23, 268-277. MIYAI, Ii., and STEIKER, J. W. (1967). Fine structure of interphase liver cell nuclei in acute ethionine intoxication. Lab. Invest. 16,677-692. OLIVERCRONA, T. (1962). Plasma and liver lipids of ethionine twatcd rats. Acta I’l~~siol. Stand. 55,291-302. PENHOS, J. C., and KRAHL: M. E. (1962). Insulin stimulus of leucine incorporalion in rat liver protein. Amer. J. Physiol. 202,349-352. PENHOS, J. C., and KRAHL, M. E. (1963). Stimulus of leucinr incorl)oration into per’fused liver protein by insulin. Amer. J. Ph?/siol. 204, 140-142. REDDY, J., and SVOBODA, D. (1968). The relationship of nucleolnr segregation to ribonurlei,* acid synthesis following t.hc administration of selected hepatocarcinogens. Lrtb. Inwst. 19, 132-145. SCHLEA-E(. F. F., and LOMBaRDI, B. (1969). On the cllrioninP-inducrd fatty-liver in mall, :m(l female rats. Lab. Invest. 17,299-307. SCHXEIDER, W. C. (1945). Phosphorous compounds in nnimnl tissws. I. Extraction :Ind estimation of desosy pentose nucleic acid and of pentose nucleic acid. J. Biol. Chrm. 161, 293-303. SHINOZUU, H., and FARBER. E. (1968). R e f ormation of the liver nucleolus in ctlrioninc treated rats in t,hc absence of significant protein synthesis. Fed. Proc. Fed. Amer. SOC. E.r/‘. Biol. 27, 605. ~IKVOZ~K.~. H., and F.4RHlm. E. (1969). Reformation of nucleoli after cthionin~-inclilt,f,(l fragmentation in the absence of significant, protein synthesis. .I. Cell Biol. 41, 380-256. SHINOZUKA, H.. GOLDBLATT. P. J., and FARBER. E. (1968). Thr disorganization of tlepntic cell nucleoli induced by cthionine and its reversal by adeninc. J. Cell Biol. 36, 313-338. SHULL. K. H. (1962). Hepatic phosphorylase and ndcnosinc iriphosphate levels in ethionine treated rats. J. Biol. Chem. 237, PC 1734. SHULL. K. H., McCo~ow-. J.. VOCT. M.. CASTILLO, A., and F.~RBER, R. (1966). 011 ttlp mechanism of induction of hepatic ndenosinc triphosphate deficiency hy ct.lrioninc. .I. Riol.Chen~.241,5060-5070. Sm. H. Cr.. and Frsrrnr.4~. W:. H. (1968). Stimulation of pentose plloq,hate patl,\v:ay. DC.. hydrogenase enzyme activities in ethionine trcnted mice. Riochenr. J. 106, 769776. SIAIP~OS. M. v., FARBER, E., and TARVER, H. (1950). Studies on ethioninc, 1. Inhibitiorr of protein synthesis in intact animals, /. Biol. Chem. 182, 81-89. %WNER, D. F., and Kr~o. J. (1964). Induced synthesis of hrpntic uridine diphosphate glucose-glycogen glucosyltransferasc after ndminiPtration of insulin to allosnn-,Ii:lhc,tic rats. J. Rio!. Chem. 239, 1292-1298. STEIKER. D. F.. and KINO. J. (1966). Insulin-stimulatrd ribonucleic acid synthesis and RN.1 polymerasc activity in allosan diabetic rat liver. Biochim. Biophys. Acta 119, 519-516. SrnKon. J. A. (1963). Biochemical hnsis for ethioninc effects on tissues. Adun)). Enqmol. Relnt. Areas Xol. Biol. 25, 36$393. FLOYD,

tion

EFFECT

OF GLUCOSE

IN ACUTE

ETHIONINE

INTOXICATION

183

S., and FARBER, E. (1962). The reversal by adenosine triphosphate of induced inhibition of protein synthesis. Biochem. Biophys. Acta 61, 649-651. VILLA-TREVINO, S., FARBER, E., STAEHELIN, T., WETTSTEIN, F. O., and NOLL, H. (1964). Breakdown and reassembly of rat liver ergosomes after administration of ethionine or puromycin. J. Biol. Chem. 239, 38263833. VILLA-TREVINO, S., SHULL, K. H., and FARBER, E. (1963). The role of adenosine triphosphate deficiency in ethionine-induced inhibition of protein synthesis. J. Bid. Chem. 238, 1757-1763. VILLA-TREVINO, S., SHULL, K. H., and FARBER, E. (1966). The inhibition of liver ribonucleic acid synthesis by ethionine. J. Biol. Chem. 241,4670-4674. WEBER, G., and COVERY, H. J. H. (1966). Insulin: inducer of glucose+phosphate dehydrogenase. Life Sci. 5, 1139-1146. WITTMANN, J. B., LEE, K. L., and MILLER, 0. N. (1969). Dietary and hormonal influences on rat liver polysome profiles; fat, glucose and insulin. Biochim. Biophys. Acta 174, 536VILLA-TREVINO,

ethionine

543.