Abnormalities in Hepatic Enzyme Activities during Development of Diabetes in Db Mice

Transcription

1 Dabetolog~a, (1970) Abnormaltes n Hepatc Enzyme Actvtes durng Development of Dabetes n Db Mce A.Y. C~A_w~ and D.I. SCI~EIDEI~ Dabetes Research, The Upjolm Company, Kalamazoo, 1Vfchgan, USA Summary. Seven hepatc enzymes were assayed n vtro n dabetc and control C57BLKs mce at 1, 2, and 412 month. An elevaton n phosphoenolpyruvate carboxyknase and glueose--phosphatase was observed before the onset of hyperglycema n db mce at the age of 1 month. At two months, these mce had hyperglycema, hypernsulnema aud hgh levels of phosphoenolpyruvate carboxyknase, glucose--phosphatase, fruetose-l,- dphosphatase, pyruvate knase and -phosphogluconate dehydrogenase. The 412 month old db mce had normal plasma nsuln, severe hyperglycema, exorbtant amount of hepatc gluconeogene enzymes and reduced quantty of glycolytc and pentose-phosphate shunt enzymes. The dsturbance n enzyme actvtes and blood sugar was partally controlled by placng the anmals on a lmted det begnnng at the age of 1 month. In rve studes ndcated that hyperglycema resulted, at least n part, from an accelerated rate of gluconeogeness. The development of dabetes n these mce s dscussed wth reference to these fndngs. Anomales d'actvt~s enzymatques h~patlques pendant le d~veloppement du dab~te chez [a sours db Rdsum~. 7 enzymes hdpatques ont gtd dtuds n vbro chez les sours dab@tques et contr1es de la souehe C57BLKs, ~ l'~ge de 1, 2, et 4 mos et dem. Avant l'apparton de l'hyperglycme, une augmentaton de la phosphonolpyruvate carboxyknase et de la glucose-- phosphatase a dr observde chez la sours db agde d'un mos.k l'&ge de 2 reels, cos sourls prsentaent une hyperglycme, une hypernsuln@me et une actvt accrue de la phosphonolpyruvate carboxyknase, de la glucose-- phosphatase, de la fruetose-l,-dphosphatase, de la pyruvate knase et de la -phosphogluconate d@lydroggnase. Chez los sours db de 4 mos et dem, on a observ des taux normaux d'nsulndme, une hyperglycdme s@v@re, une actvt des enzymes de la gluconog@n~se hpatque et extr@mement rdute de ceux de la glycolyse et du cycle des pentoses. Les dgsordres de ces enzymes ans que Iceux~ du glucose sangun ont contr1es partelleuent ell soumettaut les anmaux ~ une restrcton almentare d@s l'&ge d'un mos. Des tudes n vvo ont ndqu que l'hyperglycme rsulte, ell parte du mons, d'une gluconog@n~se aec@l@r@e. L'apparton du dab&te chez la sours est dscute ~ la lum@re de ees rsultats. Verd~nderungen yon Enzym-Aktlv~ten der JLeber wdh. rend der Entwclclung des Dabetes der dbdb Maus Zusammenfassung. De Bestlmmung der Aktvt~t yon 7 Enzymen der glykolytschen und gluconeogenetschen Ketten n Leberextrakten yon 1, 2 and 4 ~ ~onate alton dbdb l~usen nnd normalen Kontrollteren ergab folgende Resultate: Be noch normoglyks dbdb ~r m Alter yon 1 l~onat waren Phosphoenolpyruvat. Carboxknase und Glucose--Phosphatase-Aktvtt erhht. Im Alter yon 2 l~onaten (manfesto Hyperglyk~me und Hyperlnsulngme) waren auger den Schlsselenzymen der Gluconeogenese auch de A~vt~ten der Pyruvat Knase und der -Phosphogluconat-Dehydrogenase erhht. Be 4 ~ l~onate alten clbdb l~usen (schwere I-lyperglykgme, zur l~orm abgesunkene Plasmalnsulnkonzentratonen) waren de A~ktvtgten der Schl~sselenzyme der Gluconeogenese stark erhht und dejengen der Enzyme der Glykolyse und des Pentose-Shunts ernedrlgt. Enschrgnkung der Nahrungsaufnahme enen l~onat nach Geburt verhnderte das kuftreten deser Vergndertmgen nut telwese.kuf Grund yon n vlvo Expermenten wrd augenonnnen, dal~ de l:s zumndestens telwese, auf verst~rkte Gluconeogenese zurfckzuffhren st. Key.words: Spontaneous dabetes, dabetes n mce, mutaton: dabetes, lver enzymes, glueoneogeness, det and dabetes. The mutant stran dbdb of C 57 BLKs mouse develops dabete syndromes resemblng those found n man [3]. Its dabetc symptoms appear to manfest n two stages. The early perod s characterzed by excessve plasma nsuln, reducton n fl-eell granules, ncrease n glucose oxdaton and elevated level of gluconeogenle and lpogene enzymes, whereas n the later stage, there s a normal plasma nsuln level, retarded glucose oxdaton, and hgh gluconeogene enzyme actvty [3]. The symptomatc changes durng the development of dabetes sgnfy certan causatve bochemcal derangements n the db mouse. Ths paper descrbes some studes on dsturbances n hepatc enzyme actvty that accompany the manfestaton of dabetes n these mce. Furthermore, data obtaned from studes on the rate of gluconeogeness n rve are also reported. Materals and Methods Dabetc (dbdb) and control (++) C 57 BLKs mce were purchased from the Jackson Laboratory. A majorty of the anmals were allowed food and water ad lbtum. Three pars of mce were placed on a lmted det startng at the age of 1 month, and every day 2.25 g Purna Mouse Chow was gven n the afternoon. The dabetc mce usually consumed ther food wthn a short nterval, whereas the raton lasted 24 h for the control mce. Anmals used n glueoneogeness studes were fasted for 1 h. All studes were ntated early n the mornng. Other materals and methods, e.g. determnaton of plasma nsuln, blood glucose, lver protens, enzyme actvtes, and rate of gluconeogeness, were as descrbed prevously [2]. Results Durng the development of dabetes, the lvers of db mce ncreased n sze from 00 to 8% of total body weght between 1 to 2 months of age (Fg. 1). Lver hypertrophy was somewhat attenuated n mce placed on a lmted det. There was no sgnfcant dfference n

2 A.Y. Chang and D.I. Sotmeder: ttepate Enzymes n Db-l~ee 275 I P NS <.001 <.01 N 4 3 Age rues I~,~ 1 2 4* Lver wt. 00 Lver Supernatant proten mgg lver * lmted det [] C578LKs NS NS NS NS <,01 <,05 <.01 NS r Lver Meresomal proten ragg lver Fg. 1. Varous propertes of lvers of dabetc (db) and control C 57BLKs mce. Numbers ndcate the mean of results obtaned from studes on N anmals 2a [] C578LKs P <.001 NS <.1 <.01 NS NS NS NS NS N PI PEP Glu Fru Pyr Glu PG LDH lu Ins CK Paso 1,8 Kase Hex d'ase gpase Kese moo. lver soluble proten content between these two groups of anmals, but the mcrosomal proten level n the db mce appeared to change from lower (1 and 2 months) to hgher (4 ~ months) than that n the controls. The db mce showed sgnfcantly hgher levels of glucose-.phosphatase, and, probably, phosphoenolpyruvate carhoxyknase, at 1 month when blood sugar concentraton n these anmals was normal (Fg. 2a). Hyperglycema and hypernsulnema developed n the 2 month old db mce, whch also had elevated phosphoenolpyruvate carboxyknase, glueose--phosphatase, fructose-,-dphosphatase, pyruvate khaase and -phosphogluconate dehydrogenase actvty (Fg. 2 b). When these mce reached the age of 4 ~ months, they showed severe hyperglycema, normal plasma nsuln, depleton of nsuln n pancreas [4], exorbtan~ amount of hepatc phosphoenolpyrttvate earboxyknase, glu- 2b == 04 ~I C571]Lgs e P <o01 <.001 <.01 <,001 <.GOI ~.01 NS N" BI Pl PEP Gtu Fru Pyr Glu OP[; Glu Ins CK Paso 1, Kase Hex d'ase DPase Kase 2 moo. r~ J NS LBH = [] C570LKs [] dh [] C578UKs I~l db N 20 E P <.001 N r 01 Glu NS <,001 <.001 < PI PEP Glu Fru Ins "cg Pase 1, DPase 4~ moo. <,01 <.02 <.05 < Pyr Glu PG LDH Kese Hex Kase d'ase F <.05 <,01 N PI 2d GIrl Ins <30 <:.02 NS <.01 <3 <.05 NS PEP Glu Fru Pyr Glu OPG LDH CK Paso 1, Kase ltex d'ase ])Paso Kase 4 rues. On lmted det Fg. 2. Actvtes of hepatc enzymes of db and control mce. Numbers ndcate the mean of results obtaned from studes on N anlraals. Blood glucose s n rag%, plasma nsuln ~tuml. Enzyme actvty s defned as m~m substrate turnover per mnute per mg proten n assayed fracton. Abbrevaton for enzymes s as follows: X~EPCK (phosphoenoipyruvate earboxyknase), GluPase (glucose--phosphatase), ~'ru 1, DYase (fructose-l,-dphosphatase), Yyrtt:ase (pyruvate knase), Glv_KexKase (glucose phosphorylatng e~zymes assayed n 0.1 ~ glucose), PGd'ase (-phosphoglueonate dehydrogenase) and LDI~ (lactate dehydrogenase). GluPase was assayed n mcrosomm fractons at 37~ Other enzymes were measured n the soluble fractons at 37~ (PEPCK, Fru 1,D:Pase) or 25~ (Glu~[exl~ase, :Pyr- Kase, PGd'ase and LDI-I)

3 27 A.Y. Chang and D.I. Schneder: Hepatc Enzymes n Db-Mce Dabetologa cose--phosphatase, fructose-l,-dphosphatase and lactate dehydrogenase, and reduced quantty of pyruvate lduase, glucose-phosphorylatng enzymes, and -phosphogluconate dehydrogenase (Fg. 2c). When young db mce were placed on a lmted det, they mantaned a relatvely low blood glucose level and hgh plasma nsuln for a longer perod than those fed ad lb (Fg. 2 d). In addton, the dsturbance n the hepatc enzyme actvty was less pronounced n dabetc mce place on a lmted det. facltated n db mce because they were bgger and had larger blood reserve. One of the seven control mce succumbed durng bleedng, and all db mce survved. The amount of blood zdc-glucose formed from pyruvate-2-zac was consstently hgher n the db mce (Fg. mu% 8 4oo~... 8O0 AGE (mos.} C57BL K s ] db l l~a ~ 9 2 o o 9 4 {L.D.) o * MICtlOSOMAL GLUCOSE -PHOSPUATASE 9 "800I; mpcml ~ 4o. cs?olks ~oo 400 2O0 9 " 00~'" '" ~ " " "" "" l "9 " oe 200 Ks, I I I I,, I, I I? 100 ZOO I GLOOO GLUCOSE-m~% PLASMA INSULIN-pUml Fg. 3. Actvtes of glucose--phosphatase vs. blood glucose and plasma nsuln concentratons L ~ _l... 5 lo 15 MIN. Fg. 5. Concentraton and specfc actvty of blood z~cglucose n db and control mce durng gluconeogeness studes n vvo. Each pont represents the mean of studes on seven db or sx control mce PEP CAI1BOXYKINASE 4O 'I '" 3o 30I; m 20 * 9 ~a ** 10 1OI~*aA, **% ***T* BLOOD GLUCOSE -rob% PLASMA INSULIN -puml Fg. 4. Actvtes of phosphoenolpyruvate carboxyklnase vs. blood glucose and plasma nsuln concentratons Plottngs of glucose--phosphatase aganst blood glucose and plasma nsuln concentratons ndcate that the elevaton of ths enzyme preceded the onset of hyperglycema, and could be lessened somewhat by hgh plasma nsuln level (Fg. 3). A smlar relatonshp was also evdent n the case of phosphoenolpyruvate carboxyknase (Fg. 4). Data obtaned from studes n glueoneogeness n vvo are summarzed n Fgs. 5 to 8. B1eedngs were mpcml p d5 150 ~, C57 db MIN. () (7) ANIMAL WEIGHTS Fg.. Bate of absorpton after I. P. njecton n db and control mce. Fve ~C of pyruvate-2-z~c was admnstered 5b). However, ts specfc actvty was lower than that of control mce (Fg. 5 e) due to the pre-exstng large pool sze of blood sugar (Fg. 5a). The amount of radoactvty appeared n unt volume of blood after

4 Vol., No. 3, 1970 A.Y. Chang and D.I. Schneder: Hepatc Enzymes n Db-Mce 277 I.P. njecton seem to be smlar n these two groups of anmals (Fg. ). However, because the db mce were much bgger and had more blood, the total amount of radoactvty absorbed was larger n the db mce. The rate of gluconeogeness n rve,based on the per cent of total radoactvty n blood found n glucose, was much hgher n the dabetc mce (Fg. 7). Studes on the.~pm Ih] BLOOD 14C-GLUCOSE x 108 dpra ~J WHOLE BLOOD c~ O := 4.0 ~ 2O ~~ 2 E Ml~. Fg. 7. Rate of z~c-glucose formaton from sodum pyruvate-2.14c [] C57BLKs P <'.001 <~.95 <.O1 NS '~.05 %001 <.001 ~..07 r~s hs NS P~ B %~C n PEP Pyr Glu Fru Glu Pyr Hex PG Gu 14C Glu BI Glu CK CB Pase 1, Kase Kase Kase d'ase DPase 31s mos. Fg. 8. Actvtes of hepatc enzymes n mce used n gluconeogcness studes n vvo. The blood glucose and blood lac-glucose concentratons, and % blood z4c n glucose were measured n deprotenzed blood cellected just before decaptaton of anmals. PyrCB abbrevates for pyruvate carboxylase. See legends n Fg. 2 hepatc enzymes n these anmals showed sgnfcant ncrease n the actvty of glucose-.phosphatase, fruetose-l,-dphosphatase and pyruvate earboxylase, but not n phosphoenolpyruvate carboxyknase (Fg. 8). The db mce also had hgher glucoknase actvty, but smlar levels n pyruvate knase, hexoknase and - phosphogluconate dehydrogenase as the control mce. Dscusson Our studes showed that durng the development of dabetes n the db mouse, changes n the metabolc rate of gluconeogeness and glucose oxdaton are often accompaned by alteratons n actvtes of hepatc enzymes regulatng these metabolc pathways. Thus, n the young db mouse, when the rate of glucose oxde. ton s accelerated [3], the level of glyeolyte and penrose phosphate shunt enzymes s also elevated. Conversely, n old db, retardaton of glucose oxdaton [3] s accompaned by depressed actvty of pyruvate knase and -phosphogluconate dehydrogenase. It appears that nsuln may play a crucal role n regulatng these changes becaues of the followng observatons: these enzymes are 1. undsturbed n young db wth normal plasma and panereatc nsuln level, 2. elevated n mce wth hgh plasma nsuln, and 3. depressed n mce wth normal plasma nsuln and depleted pancreatc nsuln [4]. The regulatory role of nsuln on gnconeogene enzymes s less obvous. A hgher than normal amount of these enzymes s already present n the very young db mce, before they show any sgn of dsturbance n blood glucose, or plasma and pancreatc nsuln [4]. Ths suggests an early nvolvement of hepatc gluconeogene enzymes n causng hyperglycema n the db mce. At 2 months, hgh plasma nsuln partally controls the elevaton of gluconeogenc enzymes, whch may reflect a net result of elevated synthess and nsuln-dependent breakdown of these enzymes n the db mce. When the mce reached the stage of nsuln depleton at 4 ~ months, gluconeogenc enzymes are uncontrolled and present at an exorbtant level. Our studes n glucormogeness n vvo ndcate that hyperglycema n db mce results from an accelerated rate of glueoneogeness, n addton to a retarded state of glucose oxdaton as reported by Coleman and Hummel [3]. In contrast to data observed n smlar studes wth dabetc chnese hamsters [2], the accelerated rate of gluconeogeness s reflected by an ncreased level of gluconeogene enzymes. Both phosphoenolpyruvate carboxyknase and glucoknaso are extremely senstve to the detary condton and plasma nsuln level as observed n studes on nsuln effect n streptozotocndabetc rats [1]. Durng fastng, the state of hyponsulnema brngs on a tremendous ncrease n phosphoenolpyruvate carboxyknase and a decrease n glueoknase actvty n the control mce. In the mature db mce, because of depleton of nsuln reserve, the level of these two enzymes remans relatvely unchanged between fed and fastng states. Therefore, a hgher level of ghcoknase n db mce but smlar actvty of phosphoenolpyruvate carhoxyknase n both db and control mce were observed n fastng anmals used n n rve ghconeogeness studes. The fact that dsturbance n hepatc gluconeogenc enzyme level n the db mouse precedes tm onset of such symptoms as hyperglycema, hypernsulnema

5 278 A.Y. Chang and D.I. Schneder: Hepatc Enzymes n Db-Nce and depleton of nsuln n the pancreas, suggests an early nvolvement of lver n the development of dabetes n these anmals. The manfestaton of these dabetc syndromes appears to results of excessve producton of glucose whch may, n turn, arse from an abnormally hgh quantty of hepatc gluconeogenc enzymes. However, whether lver s the prmary ste where mutaton s manfested or a causatve agent s nvolved n stmulatng synthess or retardng degradaton o~ these hepatc enzymes remans to be an. wered. Acknowledgement. The authors gratefully acknowledge the supply of lvers n some studes by Dr. W.E. Duln and Mrs. B. Wyse, and ther permsson to use ther data on blood glucose and plasma nsuln n these anmals. References 1. Chang, A.Y. : Unpublshed results. 2.- Schneder, D.I.: ~r abnormaltes n the pancreatc slets and lvers of the dabetc Chnese hamsters. Dabetologa. In ths ssue. 3. Coleman, D.L., Hummel, K.P.: Studes wth the mutaton, dabetes, n the mouse. Dabetologa 3, ~8 (197). 4. Wyse, B., Duln, W.E.: Influence of age and detary condtons on dabetes n the db mouse. Dabetologa. In ths ssue. A.Y. Chang, t)h. D. Dabetes l~esearch The Upjohu Company Kalamazoo, Mchgan, IJSA