Effect of Endotoxin on Pyruvate Kinase Activity in Mouse Liver

Transcription

1 INFECTION AND IMMUNITY, Aug. 1971, p Copyright 1971 Americn Society for Microbiology Vol. 4, No. 2 Prinzted in U.S.A. Effect of Endotoxin on Pyruvte Kinse Activity in Mouse Liver IRVIN S. SNYDER, MICHAEL DETERS, AND JAMES INGLE Deprtmenit of Microbiology, College of Medicine, University of Iow, Iow City, Iow Received for publiction 9 April 1971 The pyruvte kinse (PK) ctivity of mouse liver increses fter injection of endotoxin. It decreses in nimls given cortisone lone nd remins essentilly unchnged in those given cortisone nd endotoxin t the sme time. CCl4 cuses n increse in liver PK ctivity, but neither it nor endotoxin chnges the ctivity of muscle PK. Addition of octonote to liver homogentes inhibits the ctivity of liver PK. These results suggest tht the rpid depletion in liver glycogen fter dministrtion of endotoxin or CCl4 my be relted to incresed PK ctivity. Induction of tolernce does not prevent the increse in liver PK ctivity in chllenged nimls. A number of metbolic effects produced by endotoxins hve been studied in ttempts to understnd the host ltertions cused by these lipopolyscchrides. Crbohydrte metbolism hs been known for mny yers to be ltered in nimls fter n injection of endotoxin (6, 10, 22). More recently Berry et l. (4) hve shown tht injection of lethl dose of killed Slmonell typhimurium results in reduction of liver glycogen nd lmost complete loss in totl body crbohydrte. Cortisone not only protects mice ginst the lethlity of endotoxin but lso prevents the totl loss of glycogen. Other studies by these workers suggested tht endotoxin-treted nimls re unble to convert body proteins into crbohydrte (5). Subsequent investigtions of phosphoenolpyruvte crboxykinse (PEPCK), n enzyme importnt in synthesis of glycogen, showed tht induction of this enzyme by cortisone ws inhibited by endotoxin (3). Woods et l. (21) nd Giger (7) hve shown incresed glycolysis in cells exposed to endotoxin. More recently Shnds et l. (13) presented dt indicting tht the mjor defect in glucose metbolism in BCG-vccinted mice given endotoxin my be in synthesis of glucose from noncrbohydrte sources. This interprettion would gree with the dt on PEPCK presented by Berry nd co-workers (3). Gluconeogenesis hs s its mjor energy brrier the synthesis of phosphoenolpyruvte. Once this compound is vilble, it is converted into glucose without much difficulty. The reson pyruvte kinse (PK) is importnt to this process is tht it reverses gluconeogenesis nd converts phosphoenolpyruvte (PEP) to pyruvte. PK, therefore, occupies pivotl position in gluconeogenesis. In 138 our experiments we suggest tht n increse in ctivity of liver PK mong mice given endotoxin plys role in glycogen loss. MATERIALS AND METHODS Mice. Mle Swiss Webster mice weighing g were used (Sutter Frms, Springfield, Mo.). Oxytetrcycline hydrochloride (Terrmycin, Pfizer Inc., Brooklyn, New York) ws dded to the drinking wter for 2 dys fter delivery of the nimls from the deler, nd they were used experimentlly 5 dys lter. All mice were used 16 hr fter experimentl tretment nd were fsted during this time. Endotoxin. Het-killed cells of S. typhiimurium strin SR-1i1, suspended in nonpyrogenic sline, were used in ll experiments (1). Doses of this crude type of endotoxin were dministered intrperitonelly s 1 :6 dilutions of the het-killed cell preprtion. The medin lethl dose (LD)5o) ws clculted by the method of Reed nd Muench (12) nd ws found to be pproximtely 1010 cells. In preliminry experiments, the effect of endotoxin (Difco) prepred from S. typhiimuriium by the Westphl method ws used to determine its effect on the systems employed in the study. The results were essen tilly the sme s those obtined with het-killed cells. Thus, het-killed cells were used s the source of endotoxin throughout this study. Production of tolernce. Mice were mde tolernt to the endotoxin by schedule of dily intrperitonel injections of het-killed cells (2). The nimls received 0.1, 0.1, 0.2, 0.2, 0.4, nd 0.4 LD50 doses on successive dys; ech dose ws dministered in 0.5-ml mounts. These nimls were used experimentlly 48 hr fter the lst injection. Ech group of nimls ws tested for tolernce by injection of n LD5o of het-killed cells. Observtions of these mice were mde for 3 dys. Injections. Sterile nonpyrogenic sline ws used s diluent for ll injectble substnces (Mcbiek Compny, Cmbridge, Mss.). One LD50 of endotoxin ws contined in 0.5 ml of nonpyrogenic sline.

2 VOL. 3, 1971 PYRUVATE KINASE ACTIVITY 139 Cortisone cette (The Upjohn Compny, Klmzoo, Mich.) ws injected subcutneously into the interscpulr region in 0.5-ml mounts contining 5 mg of the hormone. CC14 (Fisher Scientific Co., Fir Lwn, N.J.) ws injected subcutneously into the interscpulr region in 0.2-ml mounts. Control nimls received 0.5 ml of nonpyrogenic sline. Mesurement of PK. The livers from mice scrificed by cervicl disloction were quickly weighed, nd pproximtely 1 g of tissue ws dded to homogenizing tube contining 7 ml of cold 0.15 M KCl. The tissue ws homogenized for 1 to 2 min by using Tri-R stirrer. The homogente ws centrifuged t 100,000 X g for 30 min. The superntnt fluid ws removed, plced t 4 C, nd ssyed for PK ctivity by the method of Weber et l. (20). Adenosine diphosphte, PEP (potssium slt), 3-diphosphopyridine nucleotide, nd lctic dehydrogense were purchsed from Sigm Chemicl Compny. The chnges in opticl density t 340 nm were recorded by using Gilford spectrophotometer. Redings were mde every 30 sec for t lest 3 min. Clcultions were mde from the portion of the enzyme curve which ws liner. The ssy ws done t room temperture. The protein content of the enzyme preprtion ws determined by the Folin-Cioclteu method (8). The enzyme ctivity is expressed s the micromolr PEP per minute per milligrm of tissue protein. In n experiment relting enzyme ctivity of homogentes to dry weight, 3 ml of the liver homogente ws dried t 90 C for 18 hr. The dried homogentes were weighed nd corrected for the KCl content. The enzyme ctivity of these preprtions is expressed s micromolr PEP converted per minute per milligrm (dry weight) of homogente. All experimentl vlues represent the men vlue obtined on t lest eight experimentl nimls. Sttisticl method. The significnce of the differences in the men responses ws determined by the t test Ȧll vlues for the ctivity of PK re given s the men the stndrd error of the men. RESULTS Influence of endotoxin on pyruvte kinse ctivity. Mice were inoculted with endotoxin lone, cortisone lone, nd cortisone nd endotoxin simultneously. The ctivity of PK ws followed with time. The vlues in Fig. 1 re expressed s percentge chnges when compred with uninoculted fsted control nimls. All nimls were strved for 16 hr before scrifice. The ctivity of PK ws incresed in mice given het-killed cells t the first time-period ssyed (2 hr) nd reched mximum t round 16 hr. The PK ctivity in nimls receiving cortisone or cortisone nd endotoxin ws lower in the fsted controls. At 20 hr, the vlues for both these groups pproched norml. Tble 1 shows the ctivity of PK in nimls 4 nd 16 hr fter inocultion of endotoxin. The increses t both time intervls re sttisticlly significnt. PK ctivity is incresed 36% bove the vlues obtined with fsted control mice 4 hr fter injection nd 64% bove norml fsted vlues by 16 hr. The PK ctivity of blood ws not ltered in endotoxin-injected nimls. Both vlues were 0.45 units. Compred with the PK ctivity of liver (Tble 1) nd muscle (Tble 3), the ctivity of the enzyme in blood is low. To estblish whether the incresed PK ctivity in the liver cytosol ws reflection of the enzyme ctivity in liver homogentes, comprison of PK ctivity in homogentes nd cytosol ws done. Livers were removed from mice fter 16 hr of experimentl tretment nd homogenized. One hlf of the homogente ws centrifuged t 100,000 X g, nd the enzyme ctivity in the superntnt fluid ws determined. The corresponding homogente ws lso ssyed for enzyme 60 E40-20 t4i ~0 0 z~~~~~~~~~~ -20 OURS AFTERINJECTION HOURS AFTER INJECTION FIG. 1. Chlnges in liver pyruivte kinse fter tretment. Symbols: 0, endotoxin; A, cortisone cette nd endotoxini; 0, cortisone cette. TABLE 1. Comprisoni of pyruivte kinise ctivity in mice givent enzdotoxin Time fter Pyruvte kinse Per cent S injection ctivity increse Sgnficnce (1) 4 Hr i 0.42b vs. 2 (2)Control P < (3)16 Hr vs. 4 (4)Control P < Expressed s micromolr phosphoenolpyruvte per minute per milligrm of protein. b Men i stndrd error of the men. A

3 140 SNYDER, DETERS, AND INGLE INF EC. IMMUN. TABLE 2. Comprison of pyruvte kinse ctivity in liver homogentes nld cytosol of mice Experimentl tretment Liver homogenteb Per cent chnge CytosolC Per cent chnge (1) Fsted.2.70 i i 0.31d (2) Endotoxin.4.14 i vs vs (3) Cortisone vs ± vs (4) Cortisone nd endotoxin vs ± vs. 4-8 Enzyme ssys were done fter 16 hr of experimentl tretment. b Expressed s micromolr phosphoenolpyruvte per minute per milligrm (dry weight). c Expressed s micromolr phosphoenolpyruvte per minute per milligrm of protein. d Men stndrd error of the men. TABLE 3. Comprison of effect of CC14 on pyruvte kinse ctivity of liver nd muscle Tissue Experimentl tretment Pyruvte kinse ctivity Per cent chnge Significnce Liver (1) Fsted 4.45 ± 0.41b 1 vs. 2 P = <0.001 (2) CC i vs. 3 (3) Endotoxin 7.45 i P = <0.001 Muscle (4) Fsted vs. 5 NSc (5) CC ± 2.51 i <1 (6) Endotoxin ± vs. 6 NS Expressed s micromolr phosphoenolpyruvte per minute per milligrm of protein. Men i stndrd error of the men. Sttisticlly not significnt. ctivity. The results (Tble 2) show tht the chnges in PK ctivity for both groups were essentilly unchnged. No enzyme ctivity ws obtined with wshed pellets remining fter centrifugtion t 100,000 X g. The mesurement of PK ctivity in whole homogentes ws not done in subsequent experiments. The sedimenttion of prticulte components contributes to the decrese in dsorbnce, resulting from the decrese in reduced nicotinmide denine dinucleotide, nd requires periodic resuspension during the enzyme ssy. Strvtion nd PK ctivity. Severl workers (9, 15) hve reported tht strvtion cuses lowering of PK ctivity. This is confirmed by dt which show tht the ctivity is decresed in nimls strved for 16 hr. In fed nimls, the PK ctivity (expressed s micromolr PEP per minute per milligrm of protein) ws 4.7 ± 0.21 (men -4- stndrd error of the men); for the strved nimls, the PK ctivity ws Since mice given het-killed S. typhimurium or endotoxin (17) fil to et, it is importnt to observe tht fsting chnges PK ctivity in direction opposite tht seen in Tble 1. Influence of CC14 on PK ctivity. Previously we hve shown tht CCl4 cuses loss of liver glycogen nd sensitizes mice to the lethl effects of endotoxin (14). In ddition, Berry et l. (4) hve shown tht muscle glycogen is ffected somewht less thn liver glycogen in nimls injected with endotoxin. To determine if one might relte glycogen loss to incresed PK ctivity, 0.2 ml of CCl4 ws inoculted into the scpulr re of mice, nd 16 hr fter inocultion the mice were scrificed. Tble 3 shows tht levels of liver enzyme re incresed fter CCl4 injection but to smller degree thn in endotoxin-poisoned nimls. Muscle PK ctivity is not incresed by tretment with CCL4 or endotoxin. Muscle contins much more PK thn does liver when compred on weight bsis. Tolernt stte nd PK ctivity. Berry nd Smythe (2) hve shown convincingly tht in mice mde tolernt to endotoxin the levels of liver tryptophn oxygense re not decresed by inocultion of endotoxin. The effects of tolernce on the levels of PK re shown in Tble 4. The ctivity of the enzyme in nontolernt mice chllenged with endotoxin incresed 35% over control vlues. Tolernt mice, however, showed n increse of 21 %0 over controls. Induction of tolernce cuses

4 VOL. 3, 1971 PYRUVATE KINASE ACTIVITY 141 TABLE 4. Liver pyruvte kinse ctivity in control nd tolernt mice chllenged with endotoxin Tretment nd Pyruvte kinse Per cent Significnce chllenge ctivity chnge (1) Nontolernt sline vs. 2 P <0.001 (2) Nontolernt endotoxin ± vs. 2 (3) Tolernt +35 sline ± vs. 3 1 vs P < (4) Tolernt endotoxin i vs. 4 1 vs P < vs. 4 P < Expressed s micromolr phosphoenolpyruvte per minute per milligrm of protein. b Men -+ stndrd error of the men. TABLE 5. Percentge of inhibition of pyruvte kinse in vitro Experimentl tretment Octonote (M X 10-3) Cortisone Cortisone & endotoxin Endotoxin None % increse in the ctivity of PK when compred with nontolernt mice (Tble 4, lines 1 nd 3). To determine if the nimls were tolernt, groups of mice were chllenged with n LD50 of het-killed cells nd observed for 3 dys. Tolernce ws indeed estblished s indicted by survivl of ll 10 chllenged mice. With nontolernt mice, three of six survived. Similr enzyme ltertions were obtined in nimls mde tolernt by injection of het-killed S. typhimurium nd chllenged with S. mrcescens endotoxin. Inhibition ofpk by free ftty cid. Weber et l. (18) hve shown tht free ftty cids cn ply role in regultion of crbohydrte metbolism. To determine if octonote could inhibit the enzyme from our experimentl nimls, octonote t severl concentrtions ws dded to liver homogentes nd incubted for 30 min before ssy for PK. Different concentrtions of octonote were dded to liver homogentes from fsted nimls, nd from nimls given endotoxin, cortisone, or endotoxin nd cortisone 16 hr before scrifice. The percentge of reduction in PK ctivity (Tble 5) shows tht the incresed enzyme ctivity observed fter injection of endotoxin cn be decresed by free ftty cid. This suggests tht the incresed ctivity of PK might be relted to decrese in the level of those substnces which ply role in controlling the ctivity of this enzyme. DISCUSSION A striking metbolic chnge tht occurs in mice fter injection of endotoxin is the loss of liver glycogen nd the lmost totl depletion of body crbohydrte. Whether this loss of crbohydrte is due to n increse in glycolysis or impired gluconeogenesis hs been uncler. Berry nd coworkers (3, 4) nd Shnds et l. (13) suggested tht the crbohydrte loss is due to the filure of the endotoxin-poisoned nimls to convert gluconeogenic intermedites into glycogen. Specificlly, Berry et l. (3) hve shown tht endotoxin prevents induction of PEP crboxykinse by cortisone, thus confirming by direct enzyme ssy impired conversion of glyconeogenic intermedites to PEP. Our work suggests tht the drmtic loss in crbohydrte my be due not only to decrese in synthesis of PEP but to n ccelerted loss of this crucil compound due to incresed PK ctivity. Woods et l. (21) found tht endotoxin exerts n insulin-like effect on glycolytic processes, nd Pieroni nd Levine (11) discovered tht injection of endotoxin t sublethl doses with insulin results in deth of mice. These dt tend to confirm our work. The bility of cortisone cette to prevent the increse in PK ctivity elicited by endotoxin is consistent with the demonstrtion of the protection of mice ginst the lethlity of endotoxin (4). Weber et l. (19) showed tht glucocorticoids do not induce PK, nd cortisone is known to mintin glycogen levels in mice given endotoxin (3, 4). Snyder et l. (14) demonstrted tht injection of CCl4 not only increses the susceptibility of mice to endotoxin nd stimultes PK, but tht it lowers the ctivity of liver tryptophn oxygense nd cuses loss of glycogen. A difference in the effect of endotoxin nd CC14 on PK in muscle nd liver hs been demonstrted, the liver enzyme being more esily ffected thn the muscle enzyme. The bsence of significnt chnge in enzyme ctivity in the muscle of nimls injected with endotoxin or CCl1 my be relted to the different properties of this enzyme. Tnk et l. (15) demonstrted two types of PK, n M (muscle) nd L (liver) type. The more drmtic loss of liver glycogen s compred with muscle glycogen shown by Berry et l. (4) my be relted

5 142 SNYDER, DETERS, AND INGLE INFEC. IMMUN. to the differences in the muscle nd liver types of PK Ṫhe inbility to mintin the ctivity of PK in the endotoxin-tolernt mouse is surprising. But if the rte of gluconeogenesis is incresed by induction of tolernce, then one might expect mintennce of glycogen in the presence of higher enzyme ctivity. Induction of tolernce cuses greter increse in enzyme ctivity thn did injection of endotoxin into tolernt nimls. The importnce of PK s key enzyme in glycolysis hs been well demonstrted (18). Weber nd ssocites (18) hve shown tht the rtio of the sum of PEPCK nd pyruvte crboxylse to PK is Thus, the importnce of PK control during gluconeogenesis is obvious. Under conditions of gluconeogenesis, the ctivities of PEPCK nd pyruvte crboxylse increse, wheres the ctivity of PK decreses. If the mechnisms for control of ctivity re impired, net synthesis of glycogen cnnot occur. Our dt nd tht of others (3, 13) indicte tht the loss of glycogen in nimls given endotoxin is due not only to n incresed ctivity of PK but lso to inbility of the host to synthesize glycogen from intermedites. The mechnism by which endotoxin cuses n increse in ctivity of PK is unknown. Preliminry experiments indicte tht incresed ctivity is not the result of direct interction between enzyme nd endotoxin. However, ddition of endotoxin to liver homogentes does cuse more rpid loss in glycogen thn is observed in the bsence of endotoxin. Inhibition of denosine triphosphtse ctivity by endotoxin is the only exmple of direct enzyme-endotoxin inhibition (16). Weber nd ssocites (19) demonstrted severl mechnisms which regulte PK ctivity. Inhibition in vitro of PK by octonote grees with the results of Weber et l. (18). This suggests tht similr pproch to study of the metbolic control mechnisms in endotoxin-treted nimls my be meningful. The effects of endotoxin on these control mechnisms re under study in our lbortory. ACKNOWLEDGMENTS This investigtion ws supported by grnt GB-8363 from the Ntionl Science Foundtion nd by the University of Iow College of Medicine NIH Generl Reserch Fund. LITERATURE CITED 1. Berry, L. J., nd D. S. Smythe Effects of bcteril endotoxins on metbolism. VI. The role of tryptophn pyrrolse in response of mice to endotoxin. J. Exp. Med. 118: Berry, L. J., nd D. S. Smythe Some metbolic spects of tolernce to bcteril endotoxin. J. Bcteriol. 90: Berry, L. J., D. S. Smythe, nd L. J. Colwell Inhibition of heptic enzyme induction s senisitive ssy for endotoxin. J. Bcteriol. 96: Berry, L. J., D. S. Smythe, nd L. G. Young Effects of bcteril endotoxin on metbolism. 1. Crbohydrte depletion nid the protective role of cortisone. J. Exp. Med. 110: Berry, L. J., D. S. Smythe, nd L. G. Young Effects of bcteril endotoxin on metbolism. II. 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