Studies of the development of dibetic ketosis in the rt Jurgen M. Meier, J. Denis McGrry, Gerld R. Floon, Roger H. Unger, nd Dniel W. Foster* Deprtments of Internl Medicine nd Biochemistry, The University of Texs Southwestern Medicl School nd the Veterns Administrtion Hospitl, Dlls, Texs 75235 Abstrct Plsm glucose, free ftty cid, ketone, nd triglyceride concentrtions were mesured t frequent intervls fter the dministrtion of lloxn to rts. Heptic triglyceride levels were determined in the sme nimls. During the second 24-hr period fter lloxn dministrtion, severe ketocidosis developed nd triglyceride concentrtions in the liver becme mrkedly elevted. This finding ws incomptible with the thesis tht enhnced ketogenesis under circumstnces of incresed free ftty cid delivery to the liver requires diminished triglyceride synthesis. Plsm insulin nd glucgon concentrtions were determined t ech time point. Initil chnges in plsm glucose, ketones, free ftty cids, nd triglycerides were ccompnied by fll in insulin concentrtions, but no chnge occurred in glucgon levels. However, concentrtions of the ltter hormone incresed drmticlly in the second 24 hr fter lloxn tretment nd probbly contributed to the development of the extreme hyperglycemi observed during this time period. Supplementry key words lloxn. dibetes. glucgon. insulin. ketogenesis. triglycerides TE INTERRELATIONSHIP between bnormlities in lipid metbolism nd the production of ketone bodies by the liver hs received extensive study. It is now cler tht simple increse in delivery of free ftty cids from peripherl ft stores to the liver is not sufficient in itself to induce ketone production nd tht primry heptic regultory fctors for the control of ketogenesis must exist (1-5). Considerble evidence hs ccrued to suggest tht such regultion resides, t lest in prt, in the * Reserch Creer Development Awrdee 5-K3-AM 9968, U.S. Public Helth Service. 228 Journl of Lipid Reserch Volume 13,1972 cpcity of heptic tissue to utilize incoming ftty cids for triglyceride synthesis; i.e., insofr s peripherl ftty cids re rpidly utilized for esterifiction they would be unvilble to enter the &oxidtion pthwy for the ultimte formtion of ketone bodies (4-6). In this regrd it cn be shown tht triglyceride synthesis from oleic cid is diminished in strvtion ketosis nd restored towrd norml when ketosis is reversed by glycerol nd other ntiketogenic gents (5, 6). On the other hnd, it hs long been known tht the liver in dibetic nimls hs incresed triglyceride concentrtions (7-9), n observtion which on the fce of it would pper incomptible with the thesis tht diminished triglyceride synthesis is necessry for enhnced ketone body formtion. While heptic nd blood lipid vlues hve been reported in dibetes induced by pncretectomy, ntiinsulin ntibodies, nd P-cell destructive gents, it seemed importnt to investigte the temporl reltionship of chnges occurring in the vrious lipid components nd plsm ketone bodies during the induction of dibetic ketosis. Of criticl interest ws the reltionship between triglyceride ccumultion in the liver nd the onset of ketogenesis. In ddition, we hve mesured plsm insulin nd glucgon concentrtions t ech time point in the sequence. MATERIALS AND METHODS Tretment of nimls All experiments were crried out utilizing mle Sprgue-Dwley rts weighing 120-140 g. The nimls were mintined on ft-free diet contining 58.5% sucrose, 21% csein, nd the necessry vitmins nd minerls (Generl Biochemicls, Chgrin Flls, Ohio). Groups of nimls (40-60) were given lloxn mono-
hydrte intrvenously in dose of 60 mg/kg of body weight. The nimls were subsequently llowed free ccess to food nd wter until killed, except for one set of experiments in which the rts were fsted fter injection of the drug. At ech time point, nimls were nesthetized with pentobrbitl, the bdomen ws opened, nd 3-5 ml of blood ws drwn from the ort. The liver ws then immeditely removed nd frozen in liquid nitrogen. Assys on blood Blood ws collected in tubes contining EDTA- Trsylol to llow for hormone ssys (10). After seprtion of the plsm, cetocette nd B-hydroxybutyrte were ssyed enzymticlly (1 1). Nonesterified ftty cids were determined by titrtion s described by Trout, Estes, nd Friedberg (12). Plsm triglycerides were mesured by the method of Vn Hndel nd Zilversmit (13). After their seprtion from the triglyceride frction, phospholipids were quntitted by mesurement of inorgnic phosphorus relesed upon lkline hydrolysis (14). Glucose concentrtions were mesured by the glucose oxidse method (15). Aliquots of plsm from two to four nimls were pooled for determintion of insulin nd glucgon by rdioimmunossy (10, 16). The ltter test utilized n ntibody specific for pncretic glucgon. Assys on liver Frozen livers were extrcted in blender with chloroform-methnol 2 : 1 (v/v) in rtio of 40 ml/g of tissue. Triglyceride nd phospholipid frctions were seprted on silicic cid columns (13) nd ssyed s described bove. Ftty cid nd cholesterol synthesis Synthesis of ftty cid nd cholesterol ws mesured in liver slices utilizing [ L- C]cette s substrte s previously described (1 7). RESULTS The sequence of events tking plce during the onset of cute dibetic ketocidosis ws studied in nimls tht were llowed free ccess to food nd in those tht were fsted. As indicted in Fig. 1, very significnt weight loss, pproching 20%, occurred during the 48 hr fter dministrtion of lloxn. The decrese ws continuous in the fsted nimls, but in the fed nimls the initil weight loss ws followed by weight gin between 12 nd 24 hr, fter which rpid decline ws noted. At 48 hr, no significnt difference existed between the two groups. t- z W v 15- - Fed e-* Fsted W s z o. * # T - *. t 0 I2 24 36 48 FIG. 1. Weight loss fter dministrtion of lloxn. Ech point (men f SEM) represents percentge chnge from initil weight for 12-18 nimls in the fed group nd 6 nimls in the fsted group. no FIG. 2. Chnges in plsm ketone nd glucose concentrtions fter the dministrtion of lloxn. Results re given s mens f SEM for the number of nimls shown in Fig. 1. Plsm glucose nd ketones In the fed nimls, plsm glucose levels were mrkedly elevted by the 6-hr time point, with men vlue of 690 mg/100 ml, s shown in Fig. 2B. In contrst, the fsted rts hd men glucose concentrtion of only 275 mg/100 ml, vlue definitely elevted bove norml fsting vlues but not distinctly different from the initil postprndil level. The wide difference between the two groups reflects the reltive contributions of heptic glucose relese nd dietry crbohydrte t this erly time point. By 48 hr, similr vlues were found in both fed nd fsted groups, suggesting predominnt role for endogenous glucose production in ech cse. The second point of interest is the pronounced drop in plsm glucose concentrtion occurring t 18 hr in both sets of nimls. A second fll ppers to occur t 36 hr, but it is not sttisticlly significnt. Similr ptterns of glucose response to @-cytotoxic gents hve been previously described (18, 19). The reltive hypoglycemi found t 18 hr cn be very severe. Severl nimls in the fsted group died with hypoglycemic convulsions t this time point. Meier, McGtry, Floon, Unger, nd Foster Metbolic Studies in Dibetic Ketosis 229
Totl ketone concentrtions in the plsm (Fig. 2A) were not ffected by dietry intke. Slight elevtions, pproching vlues seen in fsting ketosis, were observed t 12 hr, but the concentrtions returned to ner norml levels t 18 hr. Brisk ccelertion of ketogenesis reppered by 30 hr, nd t 48 hr concentrtions of 17-18 mm were reched. Plsm free ftty cids, triglycerides, nd phospholipids Plsm free ftty cids exhibited chnges remrkbly similr to those seen with plsm glucose, s shown in Fig. 3. Concentrtions more thn doubled by 12 hr nd returned to ner bseline levels t 18 hr. A secondry rise to pek vlues ws seen in the second 24 hr, t which time bsolute levels were higher in the fed group. Chnges in triglyceride levels prlleled those of free ftty cids, with n erly rise followed by return to norml vlues t 18 hr. At 30 hr, only bout hlf the nimls in the fed group hd lipemic plsm (resulting in very lrge stndrd error of the men), while t 36 hr, ll the vlues were elevted. Lipemi ws lso noted in the fsted group, but it ws not lwys present. The higher vlues seen in the fed nimls were not due to ingested triglyceride, since the nimls were fed ftfree diet. Presumbly, the exogenous crbohydrte, which resulted in higher blood glucose levels, ws responsible for the effect, lthough the mechnism is uncertin. While crbohydrte loding cn stimulte heptic triglyceride synthesis (20), the effect could lso be exerted on peripherl disposl of the neutrl lipid (21). It should be noted, lthough the dt re not shown, tht plsm phospholipids were lso mesured nd showed chnges which were qulittively similr to those of the triglycerides, though the mximum increse in bsolute terms ws considerbly less. Plsm insulin nd glucgon Sequentil chnges in plsm insulin nd glucgon concentrtions fter dministrtion of lloxn re shown in Fig. 4. As hs been noted previously (lo), insulin vlues fell initilly, only to rise shrply t 18 hr. The rise in insulin, which ws not ffected by fsting, ws temporlly ssocited with the decrese in plsm glucose, ketones, free ftty cids, nd triglycerides described bove. Plsm immunorective glucgon vlues, on the other hnd, were unchnged until 24-30 hr fter the injection of lloxn. At the 48 hr time point, both fed nd fsted rts hd glucgon concentrtions some 10-1 5 times greter thn control vlues. Heptic triglycerides nd phospholipids Altertions in heptic triglyceride content during the development of dibetic ketocidosis re shown in Fig. FIG. 3. Chnges in plsm free ftty cid nd triglyceride concentrtions fter dministrtion of lloxn. Results re given s mens f SEM for the number of nimls shown in Fig. 1. -Fed ---+Fsted 8. Glucoqon 2000 0 I FIG. 4. Chnges in plsm insulin nd glucgon concentrtions fter dministrtion of lloxn. As indicted in the text, ech hormone ws mesured in pooled smple of from two to four nimls. Since only two or three vlues were obtined t ech time point, SEM were omitted. - e L:.- z 100-01 -Fed --* Fosied 5 P 75- F - 50- ln w E w 25-3 0 12 24 36 48 FIG. 5. Chnges in heptic triglyceride concentrtions fter dministrtion of lloxn. Results re given s mens f SEY for the number of nimls shown in Fig. 1. 5. Initil increses in men neutrl ft content were observed in the fed nimls by 30 hr, nd t the 36 hr point, concentrtions were pproximtely five times greter thn vlues obtined t time zero. Accumultion 230 Journl of Lipid Reserch Volume 13,1972
continued through 48 hr, when men vlue of 79 mg/g of liver ws obtined. While erly time points were not exmined in the fsted rts, it ppers tht similr pttern developed, since no increse ws found t 24 hr but by 48 hr concentrtion of 69 mg/g ws obtined. No sttisticlly significnt chnges were seen in phospholipid content throughout the 48-hr period (vlues not shown). Similr findings were previously reported in dibetes induced by pncretectomy (7). Heptic ftty cid nd cholesterol synthesis Ftty cid synthesis hs long been known to be decresed in dibetes. While erlier studies in fsted rts suggested tht restortion of lipogenesis to norml did not occur rpidly fter feeding (17), we were interested to see if the secondry rise in insulin concentrtion t 18 hr, which ws sufficient to reverse the mjor plsm bnormlities resulting from lloxn dministrtion, would hve n effect on ftty cid synthesis by the liver. The dt shown in Fig. 6 indicte tht rise in ftty cid synthesis took plce between 12 nd 18 hr. However, individul vrition ws sufficient to render the significnce of the results questionble. In the sme studies no obvious chnge in cholesterol synthesis in liver slices ws noted. DISCUSSION The primry purpose of the present investigtion ws to exmine the reltionship of triglyceride ccumultion in the liver to the onset of severe dibetic ketocidosis. This reltionship ws considered to be of importnce in view of the likelihood tht competition between the,&oxidtive nd triglyceride-synthesizing pthwys for incoming ftty cids plys mjor role in the regultion of heptic ketogenesis (5, 22). In view of the fct tht the ketosis of fsting is ssocited with diminished triglyc- eride synthesis in the liver (5, 6, 20) nd tht the reversl of ketosis with ntiketogenic gents such s glycerol nd lctte is ccompnied by n incresed esterifiction of ftty cids (5, 23), emphsis hs been plced on the possibility tht the heptic cpcity for triglyceride synthesis is the key fctor in determining whether or not ketone body formtion is ccelerted. According to this thesis, n incresed trnsport of free ftty cids to the liver from peripherl ft stores would result in enhnced ketogenesis only if the ftty cids could not be utilized for triglyceride synthesis. Under this formultion the oxidtive pthwy is considered to hve cpcity which is fixed nd lrge, nd the genertion of cetyl CoA would be considered limited only by the vilbility of the substrte ftty cid. The dt of the present pper indicte tht this viewpoint my be oversimplified or incorrect. Such conclusion is bsed primrily on the fct tht plsm ketone concentrtions in the fed nimls incresed from 130 f 42 pmoles/100 ml t 24 hr to 815 f 143 pmoles/100 ml t 30 hr, sixfold chnge. In the sme time period, heptic triglycerides lso incresed, but only from 8.2 f 0.78 to 14.0 f 2.92 mg/g of liver. Subsequently, however, triglyceride concentrtion incresed shrply s ketone concentrtions continued to rise. We hve ssumed tht the triglyceride ccumultion reflects in lrge prt incresed heptic synthesis in the liver. While diminished heptic relese of triglycerides hs been shown in isolted perfused livers from lloxn dibetic rts (24), the mrked rise in plsm triglycerides ccompnying heptic ft storge described here suggests tht impired relese does not ply mjor role in vivo.2 If this interprettion is correct, it is cler tht dibetic ketocidosis develops under circumstnces in which triglyceride synthesis is not impired, but ccelerted, nd tht incresed rtes of ketogenesis precede the onset of ccentuted triglyceride f~rmtion.~ Such sequence of events does not fit the simple concept tht rpid ketogenesis follows only fter depression of the triglyceride synthesizing mechnism. At lest two possibilities exist to explin the dt. The first would be tht the mechnisms of ketosis in fsting nd in dibetes re different. In the former, depression of triglyceride synthesis might be primry, while in the dibetic stte no such impirment exists. FI~. 6. Chnges in heptic ftty cid nd cholesterol synthesis fter dministrtion of lloxn in fed rts. Results re given s the mens f SEM for 8-12 nimls t ech time point. An lterntive to this interprettion could be constructed by ssuming tht the lipemic plsm resulted primrily from diminished peripherl utiliztion of triglycerides (21). Should this be true, diminished relese of triglycerides from the liver s lipoprotein might be present despite elevted neutrl ft levels in the plsm. Under these circumstnces, heptic ccumultion of ft might occur with norml or diminished triglyceride synthesis. We consider this sequence possible, but unlikely. *Indeed, severl nimls t the 30- nd 36-hr time points exhibited extremely high ketone concentrtions without concomitnt elevtion in liver triglycerides. Meier, McGrry, Floon, Unger, nd Foster Metbolic Studies in Dibetic Ketosis 231
Presumbly, in the ltter cse free ftty cid mobiliztion would be sufficiently gret to supply substrte for both pthwys. An lterntive possibility would be tht, in contrst to current concepts, the rte of triglyceride synthesis is dependent upon the rte of ftty cid flux into the oxidtive mchinery nd not vice vers. According to this view, triglyceride formtion would be fvored whenever ftty cid oxidtion is limited, s in the fed stte, or when the oxidtive pthwy is sturted becuse of gretly incresed ftty cid lods, s in severe dibetic ketocidosis. The ide tht rtes of ftty cid oxidtion regulte rtes of triglyceride synthesis is not without precedent, since similr conclusions were reched by Wittels nd Bressler (25) nd Wittels nd Spnn (26) in studies of myocrdil ftty cid metbolism. If the cpcity for ftty cid oxidtion is the limiting fctor in the disposl of ftty cids delivered to the liver in fed nimls, the point of regultion would probbly exist prior to the entry of the ftty cids into the oxidtive rections, since octnoic cid, which bypsses the crnitine trnsfer mechnism, is oxidized t the sme rte in livers from fed nd fsted rts (4). As previously noted by other investigtors (19, 27), cler-cut, three-phse response in blood glucose concentrtion followed the dministrtion of the @-cytotoxic gent. The lowering of the blood glucose level t 18 hr correlted well with the incresed insulin relese seen t this time point. The present studies indicte tht similr phenomenon is observed with plsm ketones, free ftty cids,4 nd triglycerides, ll of which begn to rise s the plsm insulin level fell, reverted towrd norml with insulin relese, nd rose gin to pek levels s insulin concentrtions decresed once more. It is of considerble interest tht the erly events of the lloxn dibetic stte re unccompnied by chnges in glucgon concentrtions s mesured by n immunossy specific for the pncretic hormone, while in the second 24-hr periodvluesrise to exceedingly highlevels. Similrly high vlues hve been observed in humns during severe dibetic ketocidosis (1 0). Considerble ttention hs been pid to the role plyed by glucgon in the pthophysiology of dibetes (10, 28). The present dt suggest tht the initil hyperglycemi, mobiliztion of free ftty cids, nd onset of ketosis occur independently of chnges in glucgon concentrtion nd tht they re the primry result of insulin deficiency. While glucgon hs been postulted to ply role in the genertion of ketosis by ctivting heptic lipses (29, 30), such n ctivity would pper to be of little importnce in vivo, In dt not shown, free ftty cid concentrtions were plotted ginst plsm ketone levels in individul nimls. An excellent correltion between these two prmeters ws observed for the first 18 hr fter lloxn tretment in both fed nd fsted groups. Therefter, however, this correltion completely disppered. 232 Journl of Lipid Reserch Volume 13,1972 since pek triglyceride storge occurs in the liver t the very time tht glucgon concentrtions re highest. In this context it is of interest tht pncretectomized rts disply chrcteristics remrkbly similr to those described in the present studies with the exception tht blood glucose concentrtions fter pncretectomy were less thn hlf those observed here (7). Presumbly, the pncretectomized nimls were deficient in pncretic glucgon. It seems likely, therefore, tht the excessive hyperglycemi of the lloxn-treted rts during the second dy ws the consequence of the grossly elevted glucgon levels. It lso seems likely tht the glucgon effect is due totlly or in prt to n ccelertion of gluconeogenesis, conclusion which follows from the observtion tht the considerble differences in plsm glucose concentrtions which exist between fed nd fsted nimls t n erly time point dispper in the second dy, when very high vlues re obtined even in the fsted group. Supported by U.S. Public Helth Service grnt CA 08269 nd by the Dlls Dibetes Assocition. Mnuscript received 9 August 7971; ccepted 2 November 7977. 1. 6. 7. 8. 9. REFERENCES SeyfTert, W. A., Jr., nd L. L. Mdison. 1967. Physiologic effects of metbolic fuels on crbohydrte metbolism. I. Acute effect of elevtion of plsm free ftty cids on heptic glucose output, peripherl glucose utiliztion, serum insulin nd plsm glucgon levels. Dibetes. 16: 765-776. 2. Willimson, D. H., D. Veloso, E. V. Ellington, nd H. A. Krebs. 1969. Chnges in the concentrtions of heptic metbolites on dministrtion of dihydroxycetone or glycerol to strved rts nd their reltionship to the control of ketogenesis. Biochem. J. 114: 575-584. 3. Bieberdorf, F. A., S. S. Chernick, nd R. 0. Scow. 1970. Effect of insulin nd cute dibetes on plsm FFA nd ketone bodies in the fsting rt. J. Clin. Invest. 49: 1685-1693. 4. McGrry, J. D., nd D. W. Foster. 1971. The regultion of ketogenesis from octnoic cid. The role of the tricrboxylic cid cycle nd ftty cid synthesis. J. Bid. Chem. 246: 1149-1159. 5. McGrry, J. D., nd D. W. Foster. 1971. The regultion of ketogenesis from oleic cid nd the influence of ntiketogenic gents. J. Biol. Chem. 246: 6247-6253. Myes, P. A., nd J. M. Felts. 1967. Regultion of ft metbolism in the liver. Nture (London). 215: 716-718. Chernick, S. S., nd R. 0. Scow. 1959. Erly effects of totl pncretectomy on ft metbolism in the rt. Amer. J. Physiol. 196: 125-131. Trrnt, M. E., R. H. S. Thompson, nd P. H. Wright. 1962. 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10. Unger, R. H., E. Aguilr-Prd, W. A. Muller, nd A. M. Eisentrut. 1970. Studies of pncretic lph cell function in norml nd dibetic subjects. J. Clin. Invest. 49: 837-848. 11. McGrry, J. D., M. J. Guest, nd D. W. Foster. 1970. Ketone body metbolism in the ketosis of strvtion nd lloxn dibetes. J. Biol. Chem. 245: 4382-4390. 12. Trout, D. L., E. H. Estes, Jr., nd S. J. Friedberg. 1960. Titrtion of free ftty cids of plsm: study of current methods nd new modifiction. J. Lipid Res. 1: 199-202. 13. Vn Hndel, E., nd D. B. Zilversmit. 1957. Micromethod for the direct determintion of serum triglycerides. J. Lb. Clin. Med. 50: 152-157. 14. Ames, B. N., nd D. T. Dubin. 1960. The role of polymines in the neutrliztion of bcteriophge deoxyribonucleic cid. J. Biol. Chem. 235: 769-775. 15. Sifer, A., nd S. Gerstenfeld. 1958. The photometric microdetermintion of blood glucose with glucose oxidse. J. Lb. Clin. Med. 51: 448-460. 16. Herbert, V., K. S. Lu, C. W. Gottlieb, nd S. J. Bleicher. 1965. Coted chrcol immunossy of insulin. J. Clin. Endominol. Metb. 25: 1375-1384. 17. Foster, D. W. 1967. Studies in the ketosis of fsting. J. Clin. Invest. 46: 1283-1296. 18. Lukens, F. D. W. 1948. Alloxn dibetes. Physiol. Rev. 28: 304-330. 19. Junod, A., A. E. Lmbert, W. Stuffcher, nd A. E. Renold. 1969. Dibetogenic ction of streptozotocin: reltionship of dose to metbolic response. J. Clin. Invest. 48: 2129-2139. 20. Fllon, H..I., nd E. L. Kemp. 1968. Effects of diet on heptic triglyceride synthesis. J. Clin. Invest. 47: 712-719. 21. Bsso, L. V., nd R. J. Hvel. 1970. Heptic metbolism of free ftty cids in norml nd dibetic dogs. J. Clin. Invest. 49: 537-547. 22. Fritz, I. B. 1961. Fctors influencing the rtes of long-chin ftty cid oxidtion nd synthesis in mmmlin systems. Physiol. Rev. 41: 52-129. 23. Wielnd, O., nd F. Mtschinsky. 1962. Zur Ntur der ntiketogenen Wirkung von Glycerin und Fructose. Lye Sci. 2: 49-54. 24. Heimberg, M., A. Dunkerley, nd T. 0. Brown. 1966. Heptic lipid metbolism in experimentl dibetes. I. Relese nd uptke of triglycerides by perfused livers from norml nd lloxn-dibetic rts. Biochim. Biophys. Act. 125: 252-264. 25. Wittels, B., nd R. Bressler. 1964. Lipid metbolism in the hert during fsting. Lb. Invest. 13: 794-799. 26. Wittels, B., nd J. F. Spnn, Jr. 1968. Defective lipid metbolism in the filing hert. 1. Clin. Invest. 47: 1787-1794. 27. Lzrus, S. S., nd B. W. Volk. 1962. The Pncres in Humn nd Experimentl Dibetes. Grune nd Strtton, New York. 88. 28. Muller, W. A., G. R. Floon, E. Aguilr-Prd, nd R. H. Unger. 1970. Abnorml lph-cell function in dibetes. Response to crbohydrte nd protein ingestion. N. Engl. J. Med. 283: 109-115. 29. Bewsher, P. D., nd J. Ashmore. 1966. Ketogenic nd lipolytic effects of glucgon on liver. Biochem. Biophys. Res. Commun. 24: 431-436. 30. Menhn, L. A., nd 0. Wielnd. 1969. The role of endogenous lipid in gluconeogenesis nd ketogenesis of perfused rt liver. Eur. J. Biochem. 9: 182-188. Meier, McGrry, Floon, Unger, nd Foster Metbolic Studies in Dibetic Ketosis 233