Total hydroxymethylglutaryl CoA reductase activity in the small intestine and liver of insulin-deficient rats

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1 ~ Totl hydroxymethylglutryl CoA reductse ctivity in the smll intestine nd liver of insulindeficient rts Nncy L. Young,' Christopher D. Sudek, nd Srh A. Crwford Deprtment of Medicine, Cornel1 University Medicl College, 13 York Avenue, New York, NY 121 Abstrct We exmined the effect of streptozotocininduced dibetes on the rtelimiting enzyme in cholesterol synthesis, 3hydroxy3methylglutryl CoA reductse (EC ), in liver nd smll intestine of rts. During the cute phse of insulin deficiency (first dy), food intke, plsm cholesterol, nd reductse specific ctivity in liver ll decresed. By 3 dys, food intke, plsm cholesterol, nd reductse ctivity in smll intestine were ll incresing. After 1 week, totl reductse ctivity in smll intestine ws 2.5 times norml, wheres ctivity in liver remined low. Thus dibetes shifted the mjor site of cholesterol synthesis from the liver to the smll intestine. These dt support the proposl tht hyperphgi by dibetic rts leds to incresed input of both dietry nd newly synthesized cholesterol by the smll intestine into thorcic lymph nd thereby contributes significntly to their hypercholesterolemi. The possibility tht dibetes ffected the Finhibitble ctivtion of reductse in vitro ws lso tested. There ws no evidence of n effect in smll intestine, but ctivtion of reductse in vitro ws decresed by 1/3 in liver. These dt suggest tht, in liver, either the ctivity of the ctivtor ws decresed or the frction of reductse in the ctive stte ws incresed fter more thn 12 hr of insulin deficiency.young, N. L., C. D. Sudek, nd S. A. Crwford. Totl hydroxymethylglutryl CoA reductse ctivity in the smll intestine nd liver of insulindeficient rts. J. Lipid Res : Supplementry key words streptozotocin dibetes hyperphgi hypercholesterolemi hypertricylglycerolemi hyperglycemi phosphtse Hypercholesterolemi (14) nd premture therosclerosis (5, 6) re common fetures of dibetes mellitus in humns. In rts, insulin deficiency leds to n increse in cholesterol content of ll plsm lipoproteins (7, 8). This effect is usully ttributed to decresed clernce of lipoproteins (91 1) since cholesterol synthesis in liver is suppressed (12, 13). However, recent work suggests tht the intestine my contribute to hypercholesterolemi in dibetic rts, since synthesis (14, 15), bsorption (16, 17), nd secretion of cholesterol by intestine into thorcic lymph (16) re incresed. The present study describes chnges in severl spects of cholesterol homeostsis with time fter induction of insulin deficiency in rts with the betcytotoxic drug streptozotocin. The prmeters studied include ctivity of the rtelimiting enzyme in cholesterol synthesis (HMGCoA reductse) in liver nd smll intestine; levels of insulin, glucose, nd lipids in plsm; body nd orgn weights; nd food intke. We use new method for ssying HMGCoA reductse in smll intestine (1 8) tht permits estimtion of totl enzyme ctivity nd of the extent of ctivtion in vitro. From the sequence of events fter streptozotocin, we hve considered possible cusl reltionships mong the responses. MATERIALS AND METHODS Tretment of nimls Wistr rts, 225 g (Chrles River Lbortories, Wilmington, MA) were housed with lights on from 6 PM to 6 AM, nd fed Purin Formulb chow #58 d lib. Dibetes ws induced fter 2 weeks when mles weighed c. 3 g nd femles weighed c. 25 g. Streptozotocin (kindly supplied by Dr. John Dulin, Upjohn Co., Klmzoo, MI) (25, 32.5, or 4 mg) ws dissolved in.5 ml of.1 M citrte buffer, ph 6.68, nd 1 ml/kg ws immeditely injected into til vein of rts nesthetized with ether. Control rts received injections of buffer only. Rts tht were to be killed 1 or more dys lter were injected t noon k.5 hr; those to be killed 4 or 12 hr lter were injected t 8 AM or 12 midnight, respectively. After injection nimls hd free ccess to food nd wter. Alterntively, pncretic tissue ws removed with sterile cotton swbs fter bdominl incision under ether nesthesi. The control rts hd n identicl surgicl incision with mnipultion of bdominl contents. Tissue collection On the dy of killing, rts were weighed between 9 nd 1 AM, nd nesthetized with ether t noon k.5 Abbrevitions: HMGCoA, 3hydroxy3methylglutrylCoA; TG, tricylglycerol, ' To whom reprint requests should be ddressed. 266 Journl of Lipid Reserch Volume 23, 1982

2 hr, the middle of their drk period. Plsm smples (19), homogentes, nd subcellulr frctions of liver nd of smll intestine (18) were prepred s described previously. Assys of plsm constituents Glucose ws mesured enzymticlly with glucose nlyzer (Beckmn Instruments, Fullerton, CA). Tricylglycerol ws mesured enzymticlly with regent kit (Dow Chemicl Co., Indinpolis, IN) using glycerol stndrds. Cholesterol ws mesured enzymticlly with regent kit (Beckmn Microbics, Crlsbd, CA) using cholesterol stndrds (Plomr Chemicls, Crlsbd, CA). Insulin ws mesured by rdioimmunossy (2) using '251lbeled porcine insulin (Cmbridge Nucler Rdiophrmceuticl Corp., Belleric, MA), guine pig ntiinsulin serum (Arne1 Products, Brooklyn, NY) nd rt insulin stndrd (Novo Reserch Institute, Bgsverd, Denmrk). Antibody ws diluted 1/1, nd ntibody nd insulins were incubted t 4 C for 48 hr. Assys of HMGCoA reductse, HMGCoA reductse inhibition, nd HMGCoA clevge ctivities Technicl detils nd methods of clcultion re described in the ccompnying report (18). Enzyme specific ctivity, nmol mevlonte/(min X g tissue), expressed in liver microsomes nd in smll intestine homogente ws multiplied by orgn weight to give totl ctivity in ech orgn. Totl ctivity ws then divided by body weight to correct for weight gin in control rts nd slight weight loss in dibetic rts. Activity expressed per g tissue my be converted to ctivity per mg protein using dt for protein recovery in Tble 1. Note tht protein recovery ws quite reproducible nd ws not ffected by dibetes or sex of the rts. Sttistics Dt re expressed s mens f stndrd error of the men. Rtios of mens re presented f7% confidence limits, which were estimted with Fieller's theorem (21). The Student's t test ws used to determine the significnce of difference between mens (21). Vlues of P in the text nd tbles re for comprison of treted nd control vlues unless stted otherwise. RESULTS Responses to streptozotocin dose We first determined the dose of streptozotocin required for significnt responses in prmeters relted to cholesterol metbolism. Streptozotocin ws injected into TABLE 1. Recovery of protein in liver microsomes nd smll intestine homogente Smll Liver Intestine Sex Tretment n Microsomes Homogente Mle (ll times) Dibetic +F 62 F 62 Control +F 63 F 63 Femle (8 Dys) Dibetic +F 5 F 8 Control +F 5 F 8 mg protein/g wet weight 22.7 f f f k f f f f f f 5 14 f f f f k f 9 Dibetic rts were injected with streptozotocin t 65 mg/kg from 4 hr to 22 dys previously (mles), or 8 dys previously (femles). Controls received injections of crrier solution only t the sme time. F ws dded or omitted t the time of homogeniztion of the orgn. Dt re mens f SEM. Differences between mle nd femle, between dibetic nd control, nd between +F nd F re ll insignificnt. Protein ws ssyed by fluorescent method described previously (1 9). mle rts t doses of 5,65, nd 8 mg/kg nd responses t 8 dys were mesured. Growth, orgn weights, nd plsm constituents. At the lowest dose, growth ws impired, plsm glucose ws elevted, nd liver nd smll intestine weights s percent of body weight were incresed (P <.1 ech cse, Fig. 1). As the dose ws rised, there were increments in the levels of TG (P <.1) nd cholesterol (P <.5) in plsm. Body weight continued to decline with higher doses, but orgn weights s percent of body weight did not continue to increse. HMGCoA reductse ctivity. We estimted totl HMGCoA reductse ctivity in liver nd smll intestine to determine effects of dibetes on cholesterol synthesis by ech orgn. To estimte totl ctivity, we ssyed smll intestine homogente nd liver microsomes, preprtions which give the highest yields of enzyme ctivity (18). Totl ctivity divided by body weight ws decresed in liver (P <.2) nd incresed in smll intestine (P <.1) t the lowest streptozotocin dose; incrementl responses with higher doses were not significnt (Fig. 2). The effects on totl ctivity reltive to body weight were due minly to chnges in reductse specific ctivity (Tble 2). The increses in orgn weight reltive to body weight (Fig. 1) tended to mplify the increse in reductse specific ctivity in smll intestine, but tended to nullify the decrese in specific ctivity in liver slightly. The incresed HMGCoA reductse specific ctivity Young, Sudek, nd Crwjord HMGCoA reductse in intestine nd liver of dibetic rts 267

3 I d nl 8 IO I d? P d u 3 J W 2 I ul J r STREPTOZOTOCIN (mqlkq) W J X 4 65 I 1 B 5( 58 Fig. 1 Growth, orgn weights, nd constituents of plsm 8 dys fter injection of streptozotocin t vrious doses. Femle rts weighed 259 * 5 g nd mles weighed g t the time of injection. The number of rts studied (n) t ech dose is shown in the top left hnd pnel. Plsm TG level is shown on log scle. in smll intestine ws observed in sedimentble frctions, including microsomes, s well s in homogente (Tble 2). Fctors ffecting expression of HMGCoA reductse in vitro. Dt bove re for reductse tht ws ctivted in vitro. To determine if dibetes ffected nonctivted ctivity, F ws dded t the time of homogeniztion to inhibit ctivtion. Nonctivted reductse ctivity ws progressively incresed in smll intestine homogente nd progressively decresed in liver microsomes s streptozotocin dose ws incresed from 5 to 65 mg/kg (Tble 3). Activtion ws clculted s the rtio of reductse ctivity without F to tht with F. Dibetes did not ffect ctivtion of reductse in smll intestine homogente, but did decrese ctivtion in liver microsomes (Tble 3). It is pprent, then, tht this decrese contributed to the decrese in ctivity observed in liver microsomes prepred without F (Tble 2). The expression of reductse ctivity in intestinl homogente is inhibited by unidentified fctors in the homogente, possibly including metbolites of HMGCoA produced during the ssy by clevge enzymes (1 8). While inhibition is reduced by preincubtion, it is not entirely eliminted (18). Thus, it is possible tht the increse in reductse ctivity expressed with dibetes ws due to decrese in inhibitory ctivity. This possibility ws tested nd ruled out by mesuring HMGCoA clev ge nd HMGCoA reductse inhibition in intestinl homogente. After 3min preincubtion (when we ssyed reductse), reductse inhibition ws not ffected by dibetes (Tble 4). However, it is interesting to note tht in smll intestine homogente from dibetic rts inhibition ctivity before preincubtion ws lower, nd HMGCoA clevge ctivity ws lso lower both before nd fter preincubtion. In summry, ll prmeters except plsm lipids were significntly chnged t 8 dys fter streptozotocin dose of 5 mg/kg. A dose of 65 mg/kg ws required to significntly increse plsm lipids. Totl HMGCoA reductse ctivity reltive to body weight 8 dys fter injection of streptozotocin ws decresed in liver nd incresed in smll intestine. The effect in liver ws due minly to decrese in the nonctivted level of reductse specific ctivity which ws mplified by decrese in F inhibitble ctivtion in vitro. The effect in smll intestine ws not due to decrese in reductse inhibition ctivity or to n increse in ctivtion, but ws due to n increse in the nonctivted level which ws mplified by n increse in orgn weight nd smll decrese in body weight. Comprison of mles nd femles The responses in femles t 8 dys fter streptozotocin t dose of 65 mg/kg were like those in mles, nd even 268 Journl of Lipid Reserch Volume 23, 1982

4 FEMALE in femles. Reductse ctivity in liver ws much higher in control femles thn in control mles, nd the reltive decrese with dibetes ws smller in femles (Fig. 2, Tble 2). 7 9 IN T' 4 65 STREPTOZOTOCIN (mg/kg ) Fig. 2 Totl HMGCoA reductse ctivity in liver nd smll intestine reltive to body weight 8 dys fter injection of streptozotocin t vrious doses. Body weight (9) t the time of injection in groups of rts from left to right in the figure ws 246 f 4, 251 f 5, 295 f 1, 324 f 5, 31 f 11, 323 f 4. The number of rts (n) in ech group is shown t the top of the bottom pnel. Reductse ctivity ws ssyed in smll intestine homogente nd in liver microsomes prepred without F. more pronounced in severl prmeters. Thus, plsm insulin level ws decresed more (Tble 5), plsm cholesterol nd TG were incresed more (Fig. 1, P <.5 nd P <.1, respectively, compring mles nd femles), nd the reltive increse in HMGCoA reductse ctivity in smll intestine ws lrger (Fig. 2, Tble 2) Responses with time fter streptozotocin injection Dt for vrious times from 4 hr to 3 weeks fter streptozotocin t dose of 65 mg/kg re shown in Fig. 3 with time on log scle to permit visuliztion of both erly nd lte chnges in the sme grph. Erly responses ssocited with decresed plsm insulin nd decresed food intke. Food intke ws decresed during the first 2 dys (P<.2). At 4 hr, plsm glucose ws elevted (P < O.OOl), nd plsm cholesterol ws decresed (P <.5). At 12 hr, insulin ws decresed (P <.5), cholesterol remined low (P <.5), while TG ws incresed (P <.1). HMG CoA reductse specific ctivity in liver ws first decresed t 12 hr (P<.1) nd miniml t 24 hr. At 24 hr there were lso decreses in body weight (P <.1) nd liver weight (P < O.OOl), but plsm cholesterol hd risen to nerly norml. Lte responses ssocited with incresed food intke. At the end of the third dy, food intke (Fig. 3) ws norml but food intke per g body weight (not shown) ws incresed (P <.5). Totl HMGCoA reductse ctivity in intestine per g body nd plsm cholesterol were lso incresed (P <.1 for ech). With continued hyperphgi, reductse specific ctivity in smll intestine ws 57% bove control t 8 dys (P <.5) nd then declined to 33% bove control (P <.5) by 3 weeks. TABLE 2. HMGCoA reductse specific ctivity 8 dys fter injection of streptozotocin HMGCoA Reductse Specific Activity Smll Intestine Liver Streptozotocin 1, g 1, g 1, g Sex Dose n Homogente Pellet Pellet Pellet mg/k nmol mevlonte/(min X g tissue) Femle f f f.5" 5.9 f.7' ***** ******* ** **** f.3".6 f.2.5 f f 1.9d Mle f f f f.3 ***** ******* ns ****** f.5 ****** 1.7 f.7 ******.7 f.3" * 1.6 f.3 **** f f.8.7 f f.4 ****** *** ** **** f.3".7 f.5.4 f f.7'' Reductse ctivity ws ssyed in preprtions without F. Comprison of mles nd femles: ', P <.5; ', P <.2; ', P <.1; ', P <.1. Comprison of dibetic nd controls: *, P <.1; **, P <.5; ***, P <.25; ****, P <.2; *****, P <.1; ******, P <.5; *******, P <.1. Young, Suudek, nd Crwford HMGCoA reductse in intestine nd liver of dibetic rts 269

5 TABLE 3. Nonctivted HMGCoA reductse nd its ctivtion in vitro 8 dys fter streptozotocin injection in mles HMGCoA Reductse Nonctivted Specific Activity Activtion Smll Smll Streptozotocin Intestine Liver Intestine Liver Dose n Homogente Microsomes Homogente Microsomes mg/k nmol meulonte/(mm X g tissue) fold f.12 ******.35 f.8 ****** 3.8 f f.5 *** f f f f.3 *** ** ns f f f f.5 Liver microsomes nd intestinl homogente from ech rt were prepred with nd without F to obtin unctivted nd ctivted HMGCoA reductse, respectively. The rtio of ctivted to unctivted ctivity is the fold ctivtion in vitro. ns, P >.1; **, P <.5; ***, P <.25; ******, P <.5. However, intestine weight continued to increse fter 1 week, thereby sustining t 2.5fold elevtion of totl reductse ctivity per g body during the lst 2 weeks, (P <.1 for ech). Although reductse ctivity in liver remined much below norml, the pttern of chnge fter 1 dy ws remrkbly similr to tht in smll intestine. Thus, there were increses in reductse specific ctivity (P <.5 t 8 dys, P <.1 t 22 dys) nd in liver weight (P <.1 t 8 nd 22 dys) tht led to sustined 2.3fold elevtion in totl reductse ctivity in liver per g body weight during the lst 2 weeks (P <.2) compred to the minimum t 24 hr. During this period plsm insulin remined low nd plsm glucose remined high. Activtion of reductse in vitro. The rtio of ctivtion in dibetics to tht in controls is shown in Fig. 4. Activtion of reductse in smll intestine homogente ws TABLE 4. not significntly ffected by insulin deficiency from 4 hr to 22 dys, verging 3.85 f.13 in controls nd 3.85 rf:.1 1 in dibetics. In contrst, ctivtion of reductse in heptic microsomes ws lower in dibetics fter 1 dy, verging 5.4 k.5 compred to 7.4 &.4 in controls (P <.5, groups from 1 to 22 dys combined). Reductse ctivity in heptic microsomes mesured fter ctivtion (Fig. 3) decresed by 12 hr, hlf dy before the decrese in ctivtion observed t 24 hr (Fig. 4). Effects of insulin nd pncretectomy The possibility tht some of the responses in rts treted with streptozotocin were due not to insulin deficiency but to other toxic effects of the drug ws ruled out by their reversl with exogenous insulin nd their dupliction with pncretectomy (Tble 6). Dily injections of protminezinc insulin normlized food intke, HMGCoA reductse inhibition nd HMGCoA clevge ctivity in intestinl homogente from mles 8 dw fter stremozotocin HMGCoA Reductse Inhibition" HMGCoA Clevge Activityb min preincubtion 3 min preincubtion min preincubtion 3 min preincubtion Streptozotocin Dose n +F F ff F ff F ff F mg/kg % nmol/(min X g tissue) f 3 81 f f f f f 2 2 f 2 *** ns ******* * f 3 81 f 4 17 f 7 23 f f ? f 4 8 ns ns ns 5 85 f 2 81 f 6 17 f 7 19 f f f f 4 3 f 8 ' Inhibition of reductse ctivity by intestinl homogente ws estimted by ssying reductse ctivity in liver microsomes with nd without intestinl homogente s described previously (19). Intestinl homogente ws prepred with (+F) nd without (F) 5 mm NF nd incubted for or 3 min before ssying for reductse inhibition ctivity. " HMGCoA clevge ctivity in the reductse ssy ws mesured s described in Methods. Dt for +F nd F were combined for tests of significnce of difference from control. ns, P >.1; *, P <.1; ***, P <.25; nd *******, P < Journl of Lipid Reserch Volume 23, 1982

6 TABLE 5. Decrese in plsm insulin level 8 dys fter streutozotocin iniection in mles nd femles Mles F e m I e s Streptozotocin Dose n Plsm Insulin n Plsm Insulin mg/k dj/ml u/ml f 9 6 8f3 (P <.2) (P <.5) 9 85? f 9 weight gin, plsm TG level, reductse specific ctivity in liver nd smll intestine, nd totl reductse ctivity in liver t 22 dys fter streptozotocin. Although intestinl weight, totl reductse ctivity in intestine per g body, nd plsm cholesterol were closer to control thn to dibetic vlues, they remined slightly but significntly elevted. One successively pncretectomized rt showed responses 45 dys lter tht were qulittively the sme s those in rts 22 dys fter streptozotocin. DISCUSSION We hve used newly developed ssy of totl HMG CoA reductse ctivity in smll intestine (1 8) to explore fctors which might contribute to the hypercholesterolemi of rts mde insulindeficient with streptozotocin. The most striking finding is tht plsm cholesterol nd reductse ctivity in smll intestine both increse only fter 3 dys of insulin deficiency when the rts begin eting more chow (Fig. 3). This finding suggests tht hyperphgi by dibetic rts contributes to hypercholesterolemi by incresing reductse ctivity in the intestine nd by incresing dietry cholesterol. This possibility is supported by severl dditionl observtions. First, before hyperphgi strts, reductse ctivity in smll intestine is unchnged, while plsm cholesterol nd reductse ctivity in liver re both significntly lower thn norml (Fig. 3). Second, when chroniclly dibetic rts re permitted to et only norml mounts of chow for 13 weeks, reductse ctivity in intestine nd in liver nd plsm cholesterol re ll norml. Third, others hve reported tht cholesterol synthesis by intestinl slices in vitro (1 5) nd secretion of endogenous cholesterol by intestine into thorcic lymph in vivo (16) re incresed in chroniclly dibetic rts eting d lib. Our dt extend the observtions of Nkym nd Nkgw (1 5), who found incresed reductse specific ctivity in intestine of rts with insulin deficiency for 4 Young, N. L., C. D. Sudek, L. Wlters, nd J. Lpeyrolerie. Unpublished observtion. to 5 weeks. They mesured specific ctivity in microsomes isolted from mucosl cells scrped from portions of the intestine, wheres we used homogente of the whole smll intestine nd thus were ble to estimte totl reductse ctivity. Under our conditions, the yield of reductse ctivity is more thn 2 times tht of Nkym nd Nkgw (1 5). In ddition, by voiding the timeconsuming step of scrping mucos, during which uncontrolled ctivtion of reductse cn occur, our method permits ssy of the nonctivted level of reductse ctivity (18). After prolonged insulin deficiency, totl MHGCoA reductse ctivity in the smll intestine increses 2.5fold. This is due, t first, minly to incresed specific ctivity; t lter times, the increse is due minly to incresed orgn weight. When totl ctivity is divided by body weight, the increse is mplified somewht in comprison with controls which continue to grow. However, the effect of the smller body weight is reltively minor. The increse in reductse specific ctivity in smll intestine mesured fter in vitro ctivtion is not due to n increse in ctivtion (Tble 2, Fig. 4) nor to decrese in reductse inhibition ctivity in the ssy (Tble 4) since these prmeters re unffected by dibetes. The chnge in HMGCoA reductse ctivity in liver with time fter streptozotocin, in comprison with chnges observed for other prmeters (Fig. 3), suggests the following physiologicl sequence. Decresed plsm insulin nd/or decresed food intke led to decresed reductse ctivity in liver. When food intke increses, secretion of cholesterol by the smll intestine increses, nd there is prtil recovery of reductse ctivity in liver. Full recovery is prevented by the low level of insulin nd/or the high level of plsm cholesterol. It is known tht cholesterol in remnnts of chylomicrons originting in the smll intestine suppresses reductse ctivity in liver (22), nd it hs been suggested tht this process is heightened in chroniclly dibetic rts (15) where more cholesterol is secreted by intestine into thorcic lymph (1 6). Clerly, this could not ccount for the initil decrese, but my contribute to the continued suppression. The decrese in totl reductse ctivity in liver of dibetic rts is due entirely to decrese in specific ctivity nd not to decresed liver weight (Fig. 3). The initil decrese in specific ctivity occurs between 4 nd 12 hr. Lter (by 24 hr), the in vitro ctivtion lso decreses (Tble 3, Fig. 4) nd therefter decresed ctivtion contributes in minor wy to the decresed specific ctivity mesured fter ctivtion. The decrese in in vitro ctivtion of reductse in heptic microsomes from dibetic rts could be due to decrese in ctivtor ctivity nd/or to n increse in the frction of reductse in the ctive form t the time of Young, Sudek, nd Crwford HMGCoA reductse in intestine nd liver of dibetic rts 271

7 5 W Y ;; \ 25 I I8 I I I 1' 4 l ' 6 J / r 'i.;.. P 6 T IO 17.5 I IO IO 9 IC I 1 1.I7.5 I 3 8 2; T., I c T 1 I 1 I 1 J.I7.5 I 3 2 t DAYS AFTER INJECTION DIABETIC CONTROL Fig. 3 Responses t vrious times fter injection of streptozotocin. Mle rts weighing 298 k 5 were injected with streptozotocin t 65 mg/kg () or with buffer only (). Dily food intke ws estimted from the decrese in weight of chow in food bskets from noon on one dy to noon on the next dy, nd is plotted t.5, 1.5, 2.5, etc. dys fter injection. The dotted line for glucose up to 12 hr is the level in serum from sequentil blood smples from snipped tils. Dt for plsm glucose nd for ll other prmeters were collected from groups of rts killed t 4 or 12 hr, 1, 3, 8, or 22 dys fter injection, but lwys t noon f.5 hr. The number of control (C) nd of dibetic (D) rts in ech group is shown in the bottom middle pnel. HMGCoA reductse ctivity ws mesured in smll intestine homogente nd liver microsomes prepred without F. Dt for smll intestine re given in the row of pnels t the bottom of the figure nd for liver in the row bove. Time nd plsm TG level re shown on log scles; ll other prmeters re shown in liner scles. homogeniztion of the liver. Previous reports (23, 24) in which this frction ws mesured by ctivtion with excess exogenous phosphtse, noted n increse in the ctive frction within 2 hr fter insulin tretment. This is the reverse of wht might be expected from the longterm effects of insulin deficiency we observe, nd implies tht long nd short term effects re different, tht in vivo nd in vitro effects re different, or tht we re seeing 272 Journl of Lipid Reserch Volume 23, 1982

8 2 w cn t I I I I 1 I I 1 I I _.17.5 I 3 22 DAYS AFTER STREPTOZOTOCIN Fig. 4. In vitro ctivtion of HMGCoA reductse t vrious times fter streptozotocin injection. Activtion ws mesured s described in Tble 3 for ech preprtion from ech rt. The rtio of the men vlue for ctivtion in dibetics to the men vlue in controls is shown for liver microsomes (A) nd for smll intestine homogente (). Time is shown on log scle. Other dt for these rts re given in Fig. 3. C decrese in ctivtor ctivity rther thn n increse. u in the ctive frction. N The question of whether the ctivted or nonctivted. e level of reductse ctivity best represents ctivity in vivo. remins open (18). However, we see the increse in re. c U ductse ctivity in smll intestine nd the decrese in c liver regrdless of whether the ctivted or nonctivted. s levels re mesured.. 2 m Turley, Anderson, nd Dietschy (25) hve recently k shown tht, for femle rts in vivo, the liver synthesizes * c twice s much cholesterol s the smll intestine. We find $ w tht totl reductse ctivity in the smll intestine of femle rts eting d lib doubles with dibetes while tht 4 w in liver is hlved. Assuming tht recovery of reductse cl m ctivity is unffected by dibetes nd tht reductse is the rtelimiting step in cholesterol synthesis, then di 8 betes with hyperphgi would shift the burden of cholesterol synthesis from the liver to the smll intestine. Furthermore, the sum of synthesis in the two orgns would be unchnged. The temporl response of plsm cholesterol suggests tht it is influenced t first by the decrese in reductse ctivity in liver nd lter by the increse in ctivity in smll intestine (Fig. 3). However, other fctors re undoubtedly contributing to the lter hypercholesterolemi. One is incresed secretion of dietry cholesterol in intestinl lymph resulting from the combined effects of incresed consumption of diet contining cholesterol nd of incresed frctionl bsorption of cholesterol by the gut (16,l 7).3 Another fctor could be decresed cler x 4 g e, u e, u 4 U 4 d. 5. VI & P b* m 2 E S I. u :" r m $2' I: +I +I $I m U $ x o * m m Zs.5 x B c *z,%.i.z L EZ?.?. 5 E 1 Z E 3 g,; p : 2 2 :. y, g Q +I: +I* +I : R SX.5 g N:b " bc. b m UB Z*Z 6s 2 B p: % 8 C Z E Y $ b w 2.o c m.?. 3 +I: +I +I 2 :.o 4 x 3: b. $ 2 3 $!2 2 b S $2.$. E L E 3.3 m. 2 $ x Z x 2;; g c +I: +I +I 2 3 g 2 C M $?Z g J 5 " 2 " V 2 b +I: +I: +I s s g&* % z:* m x 24 E xtz"2 g.yo: :.%E: z: m 5 3..I mm % *:w 2! b +I: +I +I E z.5 2 b E vim m d b2$2 o*m z 6.5 c v p % %:9*$: &. +I: +I: +I 2 r. 5 E *m*hl Q 4: m+ m %&ON m o o, 9 v u n $,E, ; 4 N 3* m b o* 6.55:!5 E o*,j2* li $2 N* N &iov : F? rg, R +I: tl +I q. m ZZm4 L "9. b m m ;sm: & cli *. (I): B O Q +I +I +I 2 q E V '? y 2 5g $4 2 2.E. M..i:y '? ; i > 2 2 p * ho; Z* E 3ns !2 E 2 R +I: +I +I 2 2. M ; 9: m c9 N 2 cp(i)n. 'u", " e,. s+ :*, * 2*N '? m B(I) 1 : E L : 9 +I: +I: +I 2 2. $,?.e 2..i: ": % 2 ML L hx. k '5.z? c 5:3u 2 4 x i m m *'s 85 % tl: +I +I I I.gz 3 kz b N* w. b b*n N $32 g c w +It +I +I E 5 $3 E: 8 6. d b y:z #?I M g 2.z ; =* U s m m m 2 c S'Z g.m 1.,; ; tl z g Z$.:e. C 2. 2 zs m h l m e, u 3 x w 4 *z,s 2 C 2:". E*% 9 (I) hl m 2. m d N: m m z I+ v. W ML, : P m m E * e, + E$?E, D. 3 m5gf (I) S3 hos g Young, Suudek, nd Cruwjord HMGCoA reductse in intestine nd liver of dibetic rts 273

9 nce of plsm lipoproteins (91 1). Decresed clernce my be responsible for the rise in plsm cholesterol to norml level t 24 hr fter streptozotocin when reductse ctivity is low in liver nd not yet elevted in intestine, nd for the very lrge increse t 3 dys when totl reductse ctivity in intestine nd food intke re only modertely elevted. However, decresed clernce lone is not sufficient to bring bout hypercholesterolemi since without hyperphgi plsm cholesterol is nerly norml t 1 nd 3 weeks fter streptozotocin injection. Plsm TG level increses t 12 hr fter streptozotocin injection when rts re eting less nd plsm cholesterol is significntly low (Fig. 3). This strongly suggests tht the initil elevtion in plsm TG is not dietinduced, in greement with previous report (26), nd tht different mechnisms re responsible for the onsets of high TG nd of high cholesterol level 2% dys lter. It hs been suggested tht the initil elevtion of plsm TG results from incresed synthesis nd secretion of TG by the liver in response to high level of free ftty cids in plsm resulting from lipolysis in dipose tissue (27 29). Decresed clernce occurs lter (91 1) nd, in conjunction with incresed secretion of intestinl lipoproteins (3) s consequence of incresed intke of dietry ft with hyperphgi (Fig. 3), could be responsible for the very high plsm TG level fter 3 dys. Agin, decresed clernce lone is insufficient to rise plsm TG in chronic dibetics since plsm TG is nerly norml when hyperphgi is prevented. The responses to streptozotocin in femle rts were similr to those in mles, nd in some respects even more pronounced. Dibetes ws more severe, s evidenced by lrger decrese in plsm insulin level; consequently plsm lipid levels were higher. Reductse ctivity in liver ws much higher in femle controls thn in mle controls, in greement with previous reports (3133). In humns, whole body cholesterol synthesis is estimted to be norml (34) or incresed (35) when dibetes is poorly controlled. In study of obese dibetics, cholesterol synthesis, plsm cholesterol level, nd clories needed to mintin constnt body weight ll decresed The chow contined.36 mg cholesterol/g. Assuming femle rts consumed 18 g chow/dy nd bsorbed 6% of dietry cholesterol, dietry cholesterol input would normlly be 4 mg/dy. Dibetic rts et t lest twice s much food nd bsorb greter percent of cholesterol, so dietry cholesterol input would be from 8 to 13 mg/dy. Cholesterol synthesis in liver plus smll intestine t middrk is.79 mg/hr. Assuming the rte t midlight is hlf the middrk vlue, the dily verge rte would be bout 14 mg/dy. These preliminry estimtes indicte tht dietry cholesterol input, even from reltively low cholesterol diet such s chow, is incresed by dibetes from bout 3% to 69% of the mount synthesized in liver plus smll intestine. If the sum of synthesis in liver plus smll intestine is unchnged by dibetes, then the increse in dietry cholesterol would increse the totl input from these sources. when control of dibetes ws improved with higher insulin dose (35). These chnges re consistent with the hypothesis tht when insulin is deficient, food intke is incresed thereby leding to incresed cholesterol input from diet nd from synthesis in intestine nd ultimtely to hypercholestero1emi.i We would like to thnk Stewrt Zuckerbrod, Brdley Berger, Demosthenes Tmbkos, nd Mri Lopez for their expert technicl ssistnce. This work ws supported by USPHS grnts RR47, HL2488, nd RR5396. Mnuscript receiued 8 December 198 nd in reutsed form 17 August REFERENCES 1. Kufmnn, R. L., J. P. H. Assl, J. S. Soeldner, E. G. Wilmshurst, J. R. Lemire, R. E. Gleson, nd P. White Plsm lipid levels in dibetic children. Effect of diet restricted in cholesterol nd sturted fts. Dibetes Boyns, D. R., J. N. Crossley, M. E. Abrms, R. J. Jrrett, nd H. 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