Short-term regulation of hydroxymethylglutaryl coenzyme a reductase by reversible phosphorylation: Modulation of reductase phosphatase in rat hepatocytes

SHORT-TERM REGULATION OF HYDROXYMETHYLGLUTARYL COENZYME A REDUCTASE BY REVERSIBLE PHOSPHORYLATION: MODULATION OF REDUCTASE PHOSPHATASE IN R A T HEPATOCYTES DAVID M GIBSON, REXA PARKER, CAROLES STEWART and KAREN J EVENSON Department of Bmchemtstry, Indmna Umverslty School of Medicine, lndmnapohs, Indmna 46223

INTRODUCTION

Multlvalent Control o / H M G CoA Reductase H y d r o x y m e t h y l g l u t a r y l c o e n z y m e A reductase (EC 1 I I 34) ( H M G C o A reductase, o r simply reductase in this review), whtch is localized m the e n d o p l a s m l c reticulum of most e u c a r y o t i c cells, catalyzes the principal ratelimiting step m the synthesis of cholesterol and a host of isoprene derivatives The v a r i o u s kinds of c o n t r o l m e c h a n i s m s that impinge on this sensitive step, which hmits the rate of p r o d u c t i o n of mevalonate, are s u m m a r i z e d in Figure !. (Reviews I-5.) The classic sterol f e e d b a c k circuit that regulates tissue levels of reductase is o r d i n a r i l y viewed in terms of m e c h a n i s m s involving repression of e n z y m e synthesis (6) a n d / o r s t i m u l a t i o n of reductase d e g r a d a t i o n (7). Action of sterols a n d fatty acid derwatlves as direct m o d u l a t o r s of reductase activity, by affecting the s u r r o u n d i n g m l c r o e n v i r o n m e n t in the m e m b r a n e , is also suggested by certain e x p e r i m e n t a l a p p r o a c h e s [8-10]. In a d d i t i o n , several kinds of p r o t e i n a c t i v a t o r s and inhibitors are now k n o w n to exert an influence on the catalytic c a p a c i t y of reductase ( 11, 12). Provision of m e v a l o n a t e to cells in culture (or to intact animals) leads to a p r o f o u n d , rapid mhibttlon of reductase, not by m e v a l o n a t e itselt, but, m part at least, t h r o u g h the g e n e r a t i o n of cholesterol or o x y s t e r o l derivatives (13-15) C o n t r o l of reductase by h o r m o n e s m a m m a l s is well established (3) The wide d i u r n a l sweeps of reductase levels m liver are keyed principally to the Abbrevlattons m legends and Figures HMG CoA reductase (HR), macttvated HR (HRo), glycogen synthase (GS), glucose-6-phosphate (G-6-P); mevalonlc acid (MVA), mevalonolactone (MVA lactone); MVA phosphate (MVAP), morgamc pyrophosphate (PP~), ethylenedlammetetraacetlc acid (EDTA), dlthiothreltol (DTT), purtfied serum albumm (BSA) 263

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feeding schedule of a n i m a l s (3, 4) The rise in reductase a t t e n d i n g feeding is e x p l a i n e d by the c o n c o m i t a n t increase m circulating Insulin (3) lnsuhn s t i m u l a t i o n of reductase, which is t h o u g h t to be m e d i a t e d t h r o u g h enzyme i n d u c t i o n , is o p p o s e d by glucagon. The influence of g l u c o c o r t i c o l d s and t h y r o x i n on this p a t t e r n has also been e x a m i n e d (3) The role of protein d e g r a d a t i o n in c o n t r o l l i n g reductase levels by e n d o c r i n e cycles has not been a d e q u a t e l y studied Nevertheless, the lnsuhn-signaled induction of reductase places it m the h p o g e n l c set of hver enzymes that c o o r d i n a t e l y rise m response to insulin (16) t h e r e b y insuring a flow of both trlglycerlde and cholesterol for p l a s m a hpoprotexn ( V L D L ) synthesis (17) The r e m a i n i n g vectors in Figure 1 pertain to a relatively new m o d e ot c o n t r o l of reductase activity t h r o u g h reversible p h o s p h o r y l a t l o n of the enzyme (I, 2) As outlined in the sections which follow, the state of p h o s p h o r y l a t l o n of reductase is sensitive to e n d o c r i n e signals Rever~tble Phosphorvlatlon o / R e d u c t a ~ e m Subcellular Sv.stem.s F o l l o w i n g the discover 3, of Beg et al in 1973 that li'~er m l c r o s o m a l reductase was inactivated by p r e t r e a t m e n t with A T P ( M g ) , a protein klnase activity was identified in rat hver cytosol fractlon~ ( F i g 2a) (18, 19) M l c r o s o m a l reductase that had been inactivated with A T P ( M g ) in the presence of reductase klnase, and washed free ot reagents, could be frozen indefinitely in the reduced activity state A d d i t i o n of purified protein p h o s p h a t a s e from liver in a ~ubsequent i n c u b a t i o n c o m p l e t e l y restored

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reductase activity (Fig. 2b) (20) F l u o r i d e and other p h o s p h a t a s e mhlb~tors blocked this effect ( 19, 20). An unexpected o u t c o m e of the lmUal research was the o b s e r v a U o n that the protein p h o s p h a t a s e inactivated reductase klnase m a time and c o n c e n t r a t i o n d e p e n d e n t m a n n e r (Fig 2c) (20) The p h o s p h a t a s e treated klnase was reactlxated with a second kind of cytosohc kmase (reductase klnase kinase) m the presence of A T P ( M g ) (Fig 2d) (20) The absolute r e q u i r e m e n t for A T P ( M g ) and for protein p h o sp h at ase m the m o d u l a t i o n of reductase and reductase kinase led to the proposal of the blcychc system presented m Figure 3 (20). 7his scheme has now been a m p l y c o n f i r m e d m terms of purified enzymes and the d~rect d e m o n s t r a U o n of p h o s p h o r y l a u o n of reductase (21-24) and reductase klnase (23) with [-y32P ] A T P In studies f r o m this l a b o r a t o r y reductase kmase and reductase klnase klnase did not require added c A M P nor were they blocked w~th the protein | n h l b i t o r of c A M P - p r o t e i n klnase (25) Protein p ho sp h at ase acUvlty w~th active glycogen p h o s p h o r y l a s e a, Inactive reductase b, and acu v e reductase klnase a as substrates behaved m a parallel m a n n e r during the purification of the 35,000 d a l t o n p h o s p h a t a s e - C f r o m liver (25). Similarly, in the c o m p a r i s o n of the rat liver 260,000 d a l t o n p h o s p h a t a s e - H with the derived 35,000 dalton

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FIG 2a Time and concentration curves for inactivation of reductase by reductase kinase Standard reductase-klnase deficient mlcrosomes are incubated at 37° without (O) or with 4 mM MgCI2 and 2 mM ATP m the presence of zero (rq), 5#g (A), 10~,g (o), or 20~g (A) of soluble mlcrosomal reductase klnase At the indicated times, reductase activity is assayed as described m Methods Acuv,ty Is expressed as percent of control (334 mU per assay) Similar data pubhshed m (20) This experiment is from lngebntsen (49)

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F I G 2b l-tme and c o n c e n t r a u o n cur,~es for reactl~aUon ol M g A q P - m a c U ~ a t e d reductase M t c r o s o m a l reductase(159 g protein) is incubated at 37 ° m the presence of 0/~g(O),0 28 ~ g ( A ) , 0 7 0 # g (r-I), or 0 98~zg (m) of hver phosphor~lase phosphatase purified through the D E A E S e p h a d e x stage as described by Ingebntsen et al (20) The horizontal hne ((3) is reductase act~lt~ prtor to m a c t ~ a U o n by M g A T P Acuvtty ~s expressed as mU reductase as a funcUon of incubation Ume (20)

phosphatase-C (26) the ratio of activities a m o n g the three substrates was the same Thus, it is entirely possible that reductase b and reductase kmase a are regulated by the same phosphatase e n z y m e in vtvo. In th~s circumstance the blcyhc system would be affected by the square of the concentraUon (acUvlty) of the protein phosphatase e n z y m e (26) Consequently, btologlcal control of protein phosphatase a c u w t y would exert a profound influence on the state ot phosphorylatlon of reductase

S h o r t - t e r m Regulatton o! Reducta~e in Rat Hepato~ vtes wtth In~uhn a n d Glucagon In the 1977 studies of N o r d s t r o m et al (19) the amount o f m t c r o s o m a l reductase that could be o b t a m e d from rat hver h o m o g e m z e d in the absence of fluoride was a p p r o x i m a t e l y five-fold higher than that from mtcrosomes separated in the presence of 50 m s fluoride In the latter case e n d o g e n o u s protein phosphatases were apparently blocked thus provtdxng a ghmpse of the

CONTROL OF HMG CoA REDUCTASE PHOSPHATASE

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FIG 2c Inactivation of soluble mlcrosomal reductase kinase by phosphorylase phosphatase Reductase kinase is incubated at 37° (under conditions described by lngebntsen et al (25) with zero, 5, or 50 mU of phosphatase At the md]cated times, ahquots are assayed for reductase klnase actwlty Data are expressed as percent of control reductase kmase activity (238 mU per assay) Slm,lar data pubhshed m (25) This experiment Is from Ingebntsen (49)

state of p h o s p h o r y l a t l o n of reductase m v t v o ( E D T A was also e m p l o y e d to prevent the action of protein klnase d u r i n g the isolation ) S u b s e q u e n t o b s e r v a t i o n s (27) c o n f i r m e d that the ratio of e x p r e s s e d reductase activity ( m l c r o s o m e s isolated with fluoride) to t o t a l reductase activity (no fluoride, t r e a t m e n t with ad d ed phosphatase) was lnvarlant in animals e x a m i n e d after a variety of l o n g - t e r m n u t r it io n a l and h o r m o n a l m a n i p u l a t i o n s . Ingebrltsen et al (28), however, d e m o n s t r a t e d that expressed and total m l c r o s o m a l reductase activity (and the E / T ratio) were quite responsive to p r e t r e a t m e n t of isolated rat h ep at o cy t e s with insulin or glucagon m v u r o . Insul,n (Fig 4a) caused a d r a m a t i c rise in expressed reductase activity and slowed the fall in total reductase activity By contrast, g l u c a g o n (Fig. 4b) caused a d r o p m expressed activity, and also e n g e n d e r e d a d i m i n u t i o n in total reductase O f great interest was the o b s e r v a t i o n that expressed c y t o s o h c reductase kinase activity varied inversely with expressed reductase activity (28) These

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FIG 2d Reactwation of reductase kmase Inactwated soluble reductase kmase is incubated at 37 ° under conditions described by lngebrttsen et al (25) m the presence (closed symbols) or absence (open symbols) of MgATP High lomc strength buffer Lsadded to stop the reaction at the md~cated ttmes (e,O) or at t~me zero (re,n) Reductase kmase actwlty m the incubated preparations ~s assayed as described in (25) Slmdar data publ,shed m (25) This experJment Js from Ingebntsen (49)

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o b s e r v a t i o n s showed that the b L c y c h c s y s t e m ( F l g 3) could o p c r a t e w l t h m intact cells. G l u c a g o n signaled the net p h o s p h o r y l a t , o n of both reductase ( m a c t l ~ a t l o n ) and reductase klnase (activation), whereas lnsuhn brought a b o u t d e p h o s p h o r y l a t t o n of both enzymes Changes m total reductase acttx ltv (28) could be interpreted in terms of net proteolysls ( However, changes in the rate ol e n z y m e synthesis were not e x a m i n e d ) The response to low levels ot a d d e d insulin a n d g l u c a g o n w e r e q u l t e r a p l d m a x i m a were r e a c h e d t n 1 0 m m with 0.1-1.0 nM m s u h n ( 2 6 ) (See Table 1, below, for 10 m m r e s p o n s e t o 10-~ M g l u c a g o n ) These results with isolated hepatocytes were recently confirmed in Rodwell's l a b o r a t o r y (29) A fall in the activity of hver reductase, and an increase in the extent of its p h o s p h o r y l a t l o n following g l u c a g o n inJection of rats, was reported by Beg et al (24) In a n o t h e r interesting study with intact a m m a n expressed reductase activity In liver fell after a d m i n i s t e r i n g m e v a l o n a t e (as the lactone) to rats (by s t o m a c h tube) (30). Thts o b s e r v a t i o n by Erlckson et al was a t t r i b u t e d to an inhlbLtton ot reductase p h o s p h a t a s e activity (see below)

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FIG 4b Total (open symbols) and expressed (closed symbols) reductase actl'~ltym mlcrosomes ~solated from hepatocytes pretreated with 10 nM glucagon ( - - - ) at 0, 60 and 120 mm (28)

A c u t e regulation of reductase (and reductase kmase) by m s u h n and glucagon places these enzymes a m o n g the growing hst of intercon~ertlble enzymes of hver that respond, Ln t a n d e m , through d e p h o s p h o r y l a t t o n (alter m s u h n brads to hepatocytes) and phosphorylatLon (alter glucagon) (26). As with the reducible set of hpogemc enz.,,mes (16) (see before), acute m s u h n slgnahng also promotes the flow of metabohtes to tHgl3cerldcs and cholesterol

ME1HODS Enzyme assals Assays ol H M G ( ' o A rcductdsc, reductasc k m a s c , reductase kmase kmase, and reductasc phospbatasc are described m detad h~, Ingebrltsen et al (25) It should be emphasized that the kmasc and phosphatase assays are carried out wtth tsolatcd mtcrosomes as ,l p r e t r e a t m e n t of m e m b r a n e b o u n d H M G C o A reductase, following which mlcrosomes are separated by centrlfugatlon and resuspended m t r e s h med~a

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such that the concentrations of initial substrates (e g. ATP, Mg) and added enzymes are vanishmgly small Furthermore, 30 mM EDTA and 50 mM NaF are routinely added to the final med,um Immediately before the HMG CoA reductase assay. These precautions are necessary to prevent the intervention of any enzyme react,ons other than reductase in the measured flux of (~4C) H MG CoA to ('4C) mevalonate (EDTA and NaF do not affect the reductase assay.) Umts of enzyme act,vlty are expressed in the figure legends. Hepatocytes (26). Methods ,n this section have been described in detail by Ingebrltsen et al. (28) Hepatocytes, obtained from male Wistar rats at the dxurnal maximum for microsomal reductase activity, were isolated by collagenase treatment and incubated (50 mg wet we.ght/ml) m Krebs bicarbonate buffer supplemented w~th 10 mM glucose and serum albumin (35 mg/ml) under 95% oxygen and 5% CO 2 at 37°C. insuhn or glucagon was added at mtervals as indicated in Results During incubation of hepatocyte suspensions, quadruphcate 5 ml ahquots were removed at intervals for separation and somcatlon of hepatocytes (m the presence of 10 mM ethylenedlammetetraacetic acid (EDTA) and 50 mM NaF) After centrifugal separation of mlcrosomes and cytosol, washed mlcrosomal pellets were resuspended in 250 mM NaC1, 5 mM dithiothreltol (DTT), 1 mM EDTA, and 50 mM ~m~dazole w~th and w.thout added 50 mM NaF In samples without NaF, total reductase activity was obtained by prelncubat~on in the presence of added hver phosphorylase phosphatase C, followed by the addition of fluoride and reagents for the reductase assay. Mtcrosomes handled throughout in the presence of EDTA and fluoride were assayed as"expressed reductase", i e., unchanged by protem klnases and phosphatases .n the course of separation of the m~crosomes Cytosohc expressed reductase ktnase actlwty was assayed w~th separately isolated hver mlcrosomes extracted to remove any endogenous reductase kmase activity. EDTA (30 mM) was present in all reductase assays Tissue extracts (26). In studies with whole ammals, rats were injected with insulin or glucagon (as indicated), and livers were removed for homogemzation 20 min later. Glucagon (200 #g/100 g of body weight) or control sahne was injected subcutaneously into rats 6 hr into the dark cycle (reductase activity diurnal maximum). Insulin (120 #g/100 g of body weight) or control sahne was tnlected subcutaneously into rats 4 hr into the light cycle (several hr before reductase diurnal minimum) Glucagon (bowne) was obtained from Sigma and porcine crystalhne insulin was provided by Ldly. Homogenates (3.5 ml/g) were prepared strictly at 0°C in 0.25 M sucrose, 50 mM T n s - H C L (pH 7.4), 5 mM DTT, and 5 mM EDTA. The 17,000× g(10 mm)extract was preincubated during the time intervals indicated m Figure 5a,b. At these points, NaF was added (50 mM) to stop any further reductase phosphatase actiwty, and the incubation mixture was diluted 4-fold with 0.25 M NaC1, 5

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mM D T T , 50 mM lmldazole, 1 mM E D T A , and 50 mM N a F (pH 7 4) Mmrosomes were removed by c e n t r i f u g a t m n and resuspended m the latter buffer for reductase actw~ty d e t e r m m a t m n

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G l u c a g o n p r e t r e a t m e n t of hepatoc).tes leads to a decrease m expressed reductase and an increase m expressed kmase actlvitms (28). The m e c h a m s m of action o[ glucagon ~s considered to be exclusively through c A M P d e p e n d e n t protein k i n a s e ( 3 1 ) Yet reductase k l n a s e ( a n d reductase klnase klnase) are cAM P insensitive For this reason our a t t e n t i o n has shifted to an e x a m l n a t m n of e n d o c r l n e - h n k e d control of liver protein phosphatase, which in theory would affect the state of p h o s p h o r y l a t i o n of both reductase and reductase klnase In these studies (26) rats were pretreated with insulin or glucagon (see Methods) for 20 rain following which the 17,000 × g hver extract was raptdly prepared. This extract, which c o n t a i n s both cytosol and mmrosomes, was incubated at 37 ° in order to follow the rate of f l u o n d e - s e n s m v e a c t u a t i o n oI e n d o g e n o u s m l c r o s o m a l reductase The slope ot the acre.arran curve ~s an estimate of reductase phosphatase activity (Fig. 5a after glucagon, 5b, m s u h n ) The initial n i n e points on the sohd hnes reflect the expressed actwlty of reductase o r d m a r d y observed m hver mmrosomes after glucagon or insulin t r e a t m e n t of ~solated hepatocytes (28) Thus i n d i w d u a l , intact a n i m a l s do show an acute response to these h o r m o n e s The dotted lines m 5a and 5b were o b t a i n e d w~th extracts prepared m the absence ot E D T A An m~tlal dllferentml influence of m s u h n or glucagon is not observed It is p r o b a b l y that d u r i n g the p r e p a r a t i o n of the extracts a m e t a l - d e p e n d e n t process o~errode the e n d o c r i n e - i n d u c e d changes accomphshed m vtvo Similarly, on e x a m i n a t i o n of the rate of increase in expressed reductase activity d u r i n g m c u b a t m n of the extracts it was found that only those prepared with E D T A displayed a s~gmftcant difference between endocrine pretreatment and control A s,mdar result was obtained by Nuttall and Gllboe (32) m following glycogen synthase a c t ~ a t m n in hver extracts after glucagon p r e t r e a t m e n t of rats Overall these results with whole animals indicate that glucagon signals a fall m reductase phosphatase actlxlty while m s u h n brings a b o u t an activation (see also reference 33 tor Insulin activation of glycogen synthase phosphatase) Since expressed reductase actwlt,; m hepatocytcs rises following m s u h n p r e t r e a t m e n t (see before) m w t r o , and falls after glucagon, attempts were made to d e m o n s t r a t e changes in reductase phosphatase activity atter endocrine pretreatment of freshly isolated hepatocytes The relative mlluence of m s u h n and glucagon on the rate of e n d o g e n o u s reductase activation m

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MINUTES FIG 5a a n d b Effect of g l u c a g o n (5a) a n d m s u h n (5b) injecUon m t o rats on the rate of activation of e n d o g e n o u s m l c r o s o m a l reductase m extracts of h',er (see Methods) Half of each hver was h o m o g e m z e d either without ( b r o k e n lines) or with 5 mM E D T A (sohd hnes) Basal expressed reductase a c t w t t y m 5a exceeds that of 5b smce the rats were obtained at extremes ,n the d m r n a l cycle ( M e t h o d s ) (26)

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extracts is presented in Figure 5c In this experiment hepatocytes were preincubated for 30 min in the absence of added hormone, and then lot 10 min with 5 nM g l u c a g o n or 5 nM insulin A 1 7 , 0 0 0 × g extract was prepared ( c o n t a i n i n g 5 mM E D T A ) and then incubated at 30 °. At zero time, and at the indicated time lnter~ als thereafter, m i c r o s o m e s were separated in the pre~cncc of 50 mM fluoride and 5 mM E D T A (All samples were brought to 50 mM fluoride and 30 mM E D T A in the final reductase assay.) S t a r t i n g at a lower expressed reductase activity the extract from the glucagon-pretreated cells displayed a lesser reductase activation slope, especially after the initial 15 min, than the slope in the insulin series Activation of e n d o g e n o u s glycogen

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P I G 5c Effect o f h e p a t o c y t e m c u b a t l o n w l t h m s u h n a n d g l u c a g o n o n e n d o g e n o u s a c t p ~ a t l o n o l H R a n d G S in e x t r a c t s H e p a t o c y t e s p r e p a r e d b) S e g l e n m e t h o d f r o m r a t , , a t t h e d m r n a l p e a k o l H R are i n c u b a t e d with glucose (10 mM), B S A , a n d b a c l t r a c m f o r 30 mmn p r i o r to a d d ~ n o n of h o r m o n e s G l u c a g o n ( 5 nM) or m s u h n ( 5 nM) l s a d d e d a n d cell', f u r t h e r m c u b a t e d l o r 1 0 m m 17,000× g s u p e r n a t a n t s f r o m s o m c a t e d h e p a t o c y t e s are p r e p a r e d m 20 mM T n ~ - H C I , 5 mM E D T A , 5 mM D T T , 200 mM s u c r o s e , p H 7 4 a n d d e s a l t e d o v e r S e p h a d e x (;-25 rote the ,,ame b u t l e r T h e e x t r a c t s are t h e n m c u b a t e d m a n m e c o u r s e a t 3 0 ° At e a c h p o m t , a s a m p l e ~ s d d u t e d w~th cold N a F - c o n t a m m g buffer, a n d f r o m o n e a h q u o t G S a c n v l t y is d e t e r m i n e d (m the presence a n d ab,,ence of G - 6 - P ) by the m e t h o d el T h o m a s et al (50) P r o m a n o t h e r a h q u o t the m l c r o s o m a l f r a e n o n r, o b t a i n e d b} u l t r a c e n t r f f u g a n o n a n d H R acn'~lty is d e t e r m i n e d m the presence el N a F G S acnvlt~ (as p e r c e n t G - 6 - P i n d e p e n d e n t f o r m ) a n d H R specific acnvlt3 a r e e x p r e s s e d as a f u n c n o n of rime of m c u b a n o n of the e x t r a c t

CONI ROL OF HMG CoA REDUCIASE PHOSPHAIASE

275

synthase was also followed m the two hepatocyte series The msuhn-glucagon differential in glycogen synthase activity was seen at zero t~me, but the rate of activation overall was not remarkably different in this experiment. W,th the evtdence at hand ,t is reasonable to conclude that lnsuhn and glucagon do exert a degree of control over the act,wty of fluoride-sensitive acuvatlon of reductase (which wc interpret to be an expression of a protein phosphatase acting on the inactive, or phosphorylated form of the enTyme) Sim,lar studies with phosphorylase a macttvat,on m hepatocytes (34) support the concept that insulin s~gnals a rise in protein phosphatase activity and glucagon a lall The mechanism by which glucagon and insuhn affect protein phosphatase actw,ty m hver has not been eluc,dated. The existence of protein mh~bltors of phosphatase has been described m several t,ssues (35). and these appear to dampen reductase phosphatase actwlty in partially purified systems (25). Of special interest is protein mhlb~tor-I that ~s fully effective only in its phosphorylated state (36). An increase m the degree of phosphorylatlon (and thus activation) of protem phosphatase mhibitor-I has been demonstrated in rabbit skeletal muscle after epinephrine injection (36-38). Here phosphorylat~on was linked to the action of cAMP-dependent protein kinase Insuhn administration brought about net dephosphorylation (inactivation) of the inhibitor (39). The possible involvement of protein phosphataseinhibitor-I prowdes an attractive explanation for the mode of action of glucagon and insulin on protein phosphatase in liver (1, 2, 26). Nevertheless, a functional role for phosphatase inhlbitor-I has yet to be estabhshed m this tissue. Competition among various phosphorylated enzymes and proteins for ex,stmg protein phosphatases would permit a direct influence of c A M P protein kmase on all reversible phosphorylatlon systems (2) The action of msuhn could be interpreted m the framework of its abdlty to diminish the release ot the catalytic subun,t of protein kinase from the holoenzyme (33)

Raptd hThtbttlon o! Reductase Pho.sphatase m Hepatocvtes Pretreated wtth Mevalonate Erickson et al (30) made the interesting observat,on that admlmstratlon of mevalonate (as mevalonolactone) to rats by stomach tube brought about a rap~d fall m reductase act,vtty in m,crosomes that were prepared ,n the absence of fluoride. Yet reductase activity could be restored with added protein phosphatase The 100,000×g cytosol was tested for its abd,ty to activate a preparation of reductase b (phosphorylated form) Freshly prepared (undialyzed) liver cytosol from the mevalonate-treated rats was s~gnlficantly less potent in reactivating reductase b in comparison to the control cytosol. Since reactivation was fluoride sensitive these authors suggested that mevalonate diminished reductase activity by generating m vivo

276

1) M G I B S O N et al

an mhLbitor(s) ol reductase phosphatase kxpet.tments w l t h l n t a c t rats were also carried out by At.ebalo el al. (40) and b.'v Beg et al. (41) Studms ol Edwards el al (13, 14) showed that m e x a l o n a t e ( a s mc'~alonolactone) p r e t r c a t m e n t ol hcpatoc.vtes quickly led to a lall in rcductasc actt\ it\ Since it is k n o w n that m c \ a l o n a t c i s rapidl.,, con\ct.tcd to cholesterol m hepatocytcs, it \~as probable that cholesterol lccdback ~ a s impinging on t.eductase actixit\ Reductasc phosphatasc actp, ity ~as not examined Regaldless ot the mechanism It was clcal that the hcpatocytc s3stcm was ,t uselu[ a p p r o a c h [or following mexalonate-generated metabohtcs that inhibit t.eductase acti\ ~ty In the pt.cscnt study hep,|tocytcs were pretteatcd with 5 mM (R,Y,)me,~alonolactonc o'~er the ttme span indicated m Figure 6 ( M c v a h m o l a c t o n e is h y d r o l s : e d to mcxalonate after tt.anspot,t into hepatocytes ) Mlcrosomcs were isolated and assayed tot- expressed and total t.eductasc actpctty (scc before) At 15 m m thet.c w,as no change m total rcductase actp~ it}' (mict,osomcs treated with purified protein phosphatase-C) Expressed activity how'e~cr (50

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F I G 6 Elle~.t ol M V & l a c t o n e m h e p a t o c ) t e m c u b a t l o n s o n e x p r e s s c d a n d t o t a I H R a c t l ~ l t l c s H e p a t o c ~ t c s a r e p r e m c u b a t e d l o r 30 m m with g l u c o s e ( 10 m,,a), B%A, a n d bacLtracm lo[lowed by the a d d i t i o n ol M V A l a c t o n e ( 5 raM) At the i n d i c a t e d t~me polnt.~, a h q u o t ~ ol cells are s o m c a t e d m f l u o r i d e - E D T A c o n t a i n i n g b u f f e r a n d the m l c r o s o m e s are ~solated by u l t r a c e n m f u g a t l o n E x p r e s s e d a n d t o t a l H R actl~ltms are d e t e r m i n e d as d e s c r i b e d m M e t h o d s H R s p e c H m a c t t v l t y ~s e x p r e s s e d as a f u n c t i o n ol time ol cell i n c u b a t i o n

C O N I-ROI. O F H M G C o A R E D U C I A S E P H O S P H A I

277

A'-,E

mM fluoride) was severely diminished relatwe to :ero trine, l-hat ts, the p h o s p h o r y l a t l o n state of reductase was increased O1 interest m the sequence of changes during treatment of hepatocytes with mevalonolactone ~s the fall m total reductase actwtty which began after expressed rcductase ~s severely dtmlshed (A sLmdar pattern has been observed with glucagon pretreatment ) Since 5 mM mevalonate m itself does not inhibit reductase (also ',ee zero time values m Fig. 6), a product must bc generated during the hepatocytc mcubation pertod that lmmedmtely brings about net phosphorylatlon o[ reductase, whLch m turn may be a precondttlon lot subsequent net loss ot total enzyme act~wty. (See also Arebalo, et al (40) The rapidity of the m~tml response to mevalonatc was reproduc~ble, and c o m p a r a b l e to that seen with glucagon ('1 able I ) A d~rect assay of reductasc phosphatase (reductase b plus added cytosol from prctreated hepatocytes) showed a striking diminution m this activity, m agreement v~lth the results obtained with liver extracts obtained from pretreated rats (30) (Fig 7) Mevalonate ~tself at 5 mM ~¢as not inhibitory to reductase phosphatase assayed with reductase b and purified protein phosphatase-C (prepared from liver). The product(s) of mevalonate m e t a b o h s m that could acutely atlect reductase phosphatase are not known. Figure 8 outhncs the categories that mtght be constdered (5) In the hst of intermediates a number of orgamc pyrophosphates appear early (42, 43). Furthermore, m o r g a m c pyrophosphate (PP,) is generated as a consequence of the multlcondensatlons of these FABLE 1 PREINCUBATION OF HEPATOCYTES INHIBITS EXPRESSED REDUCTASE (I) Expresaed redu~ tase Control Mevalonate Glucagon Total redu~ tare Control Mevalonate Glucagon E~ T ratto Control Mevalonate Glucagon Time (mm)

748 274 .

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.

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WITH MEVAI.ONAIE AC'I IVI I Y

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M e v a l o n a t e , 5 0 mM, G l u c a g o n , 10-8 M H e p a t o e y t e m c u b a t ~ o n s a n d a s s a y s were c a r n e d o u t as d e s c r t b e d m F t g u r e 6 In the hrst t o u r e , ~ p e n m e n t s , m w h i c h h e p a t o c y t e s were p r e m c u b a t e d for 15 m m with 5 mM m e v a l o n o l a c t o n e , e x p r e s s e d r e d u c t a s e actl~,tty w a s inhibited to 51"'f oI c o n t r o l

278

D M GIBSON et al

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FIG 7 Effect of MVA lactone m hepatocyte mcubattons on actwlty of cytosohc reductase phosphatase Hepatocytes are incubated for 15 mm m the presence or absence of 5 mM MVA lactone Cytosol prepared from somcated hepatocytes (m 40 mM KH2PO4, 30 mM EDTA, 50 mM KCI, 100 mM sucrose) ls tested for its abdlty to activate mactwe HRb (previously prepared from the same control hepatocyte preparation) by incubating ahquots of cytosol (3 mg protein) with mlcrosomal HRb (1 3 mg protein) for 0, 5, and 15 mm at 37 °, followed by relsolatlon of mlcrosomes by ultracentnfugatlon and determ, nahon of HR specff, c actlv~ty Data are expressed as actlvatmn of HRb (percent of zero time control) as a funchon of t~me of lncubat,on with cytosol Zero time HR b specific actwlty ----440 mU ,' mg

ATP HMG C o A

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ISOPENTENYL NUCLEOTIDES

SQUALENE POLYISOPRENES DOLICHOL

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UBIQUINONE CHOLESTEROL

FIG 8 Diagram showing products of mevalonate metabohsm m hver (5. 42, 43)

CONTROL OF HMG CoA REDUCTASE PHOSPHA-IASE

279

intermediates m the synthesis of squalene This fact is emphasized since the broad-speoficlty protein phosphatase-C of liver is inhibited by various pyrophosphates (especially PP,) (44-47) Reductase activation m the presence of 35,000 dalton protein phosphatase-C is very sensitive to added PPI (and less so with ATP) (Fig. 9) The level of PP, that effected a 50c/~ lnhlbRlon was approximately 30/aM A similar result was obtained with phosphorylase a as substrate (44, 45). When corrections are made for the ddutlon of[PP,] in the final reductase phosphatase assay this value approaches 1.5 #M (See legend to Fig. 9.) Levels of PP,, generated by 5 mM mevalonate xn hepatocytes, conceivably could reach this value in vttro. Total [PP,] in normal rat hver is 14/aM, and cytoplasmxc levels are approximately 10% of this value (48).

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FIG 9 Effect of PP, and ATP on reductase phosphatase and phosphorylase phosphatasc acllv]tlcs of protein phosphatase-C m v l t r o Ahquots of rat laver protean phosphatase-C (purafied 1,000><) are premcubated wlth a series of concentratlons of pyrophosphate or ATP followed by ddutaon and m e a s u r e m e n t of phosphatase actavataes Condmons arc adapted from Khande]wahl and Kamam (45). SUMMARY

Hydroxymethylglutaryl CoA reductase catalyzes the limiting step in cholesterol synthesis In liver and other ussues. Beginning in 1973 studies with subcellular systems established that mtcrosomal reductase is inactivated with ATP(Mg) and reductase klnase, and restored to full activity w~th phosphoprotein phosphatase. By contrast reductase kmase ~s macttvated with phosphatase and reacttvated with a second protein klnase (reductase klnase

280

I) M GIBSON et al

kmase) This blcychc system has n o ~ been c o n h r m e d in terms ol h o m o g e n e o u s enzyme c o m p o n e n t s and b\ direct re~erslble p h o s p h o r y l a t l o n with [3,~eP]ATP m several labor,ttones S h o r t - t e r m e n d o c r i n e c o n tr o l ol reductase and reductasc kmasc has been d e m o n s t r a t e d in intact rat hepatocytes P r e m c u b , m o n ol cells ~ t h glucagon b r o u g h t a b o u t a lall in the expressed activity of rcductase and a rise in rcductase kmasc consistent w'lth net p h o s p h o r ) latlon ol both c n : y m e s I otal rcductasc levels were also severel) depressed alter glucagon A d d l n o n o[ msuhn to suspcnsLons of hepatocytcs had the r e \er se effect on expressed a c m l t y ol rcductasc (elevated) and reductasc kmase (depressed) lnsuhn also prevented the decay in total reductase act~x~t\ Since both protein kmases l d e n n h c d m th~s system are c A M P-mscnsm~c, ~t ~ a s possible that h o r m o n a l slgnahng ~s mediated t h r o u g h the protein p h o s p h a t a s c that acts on both reductase kmase and rcductasc In recent studies we have s h o wn that the rate of a c n ~ a n o n of e n d o g e n o u s reductase in h e p a t o c y t c extracts ( m l c r o s o m e s plus cytosol) is responsive to h o r m o n a l modulatLon P r e t r e a t m e n t ol hepatocytes w~th insulin increases apparent reductase p h o s p h a t a s e actl~lt)' m extracts ~ h t le glucagon d~mmtshe~ the rate o1 rcductase a c t l ~ a n o n H M G C o A is co n v e r t e d to m e v a l o n a t e b3 the rcductase e n : v m c In h e p a t o c ) t e s me~alonate ~s rapLdb c o n v e r t e d to cholesterol and to a ~anety ol isoprene derivatives Expressed reductase activity falls preclpltousl 3 ~,hen hc pa to cy t es are incubated with m e ~ a l o n a t e (added m the form ol m e~ al o n o lactone) As m the case with g l u c a g o n p r e t r e a t m e n t reductase p h o s p h a t a s e is rapldl 3 diminished ( M e ~ a l o n a t e ~tself ~s not inhibitory to rcductase or reductase p h o s p h a t a s e actlv~t\ m subcellular systems ) It ~s probable that a product of m e ~ a l o n a t e m e t a b o h s m generated m intact cells may act as a rcductase p h o s p h a t a s e inhibitor A m o n g these added mo~gamc p y r o p h o sphate mh~b~ted reductase p h o s p h a t a s e at lo,s c o n c e n t r a n o n s

A C K N O W L E D G E M ENTS l n v e s t i g a n o n s c~ted f r o m this l a b o r a t o r y were s u p p o r t e d by grants f r o m the N a t t o n a l Institutes of Health ( A M 19299 and A M 21278); A m e r i c a n Heart Association, I n d i an a Affiliate; and the G r a c e M S h o w a l t e r F o u n d a t i o n

REFERENCES I 2

D M GIBSON and T S INGEBRITSEN, Mm~re~lew Reversible modulanonofhver HMG CoA reductase, Lt[e S~t 23, 2649-2664 (1978) T S INGEBRITSEN and D M GIBSON, Re,,erslble phosphor~latlonof HMG CoA reductase, pp 63-93 m Molecular Aspect.s o f Cellular Regulatton (P COHEN, ed ). Elsevier/North Holland Biome&cal Press, Amsterdam, Vol I (1980)

CON'IROI. OF HMG CoA REDUCTASE P H O S P H A I A S E 3

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R F DUGAN and J W PORTER. Hormonal regulation of cholesterol synthesis, pp 197-247 In Bto~hemual A¢ttons o/Hormone6 (G LITWACK. ed ). Academic Press. New York (1977) V W RODWELL. J L N O R D S T R O M and J J M[TSCHELEN. RegulatlonofHMG CoA reductase. Adv Lipid Res 14, 1-74 (1976) M S BROWN and J L GOLDSTEIN. Multwalent feedback r e g u l a t l o n o f H M G C o A reductase, a control mechanism coordinating lsoprenold synthesis and cell growth. J Lipid Re~ 21, 505-517 (1980) M H I G G I N S a n d H RUDNEY. Regulatlonofratll..er H M G C o A r e d u c t a s e a c t l v l t y b y cholesterol, Nat New Biol 246, 60-61 (1973) I - Y CHANG, J S LIMANEK and C C Y CHANG, Evidence indicating that inactwatlon of HMG CoA reductase by low density hpoprotem or by 25-hydroxycholesterol requires mediator protein(s) with rapid turno',er. J Btol Chem 256, 6174-6180 (1981) K A MITROPOULOS, S VENKATESAN, B E A REAVES and S BALASUBRAMANIAM, Modulation of HMG CoA reductase and of acyl CoA-cholesterol acyltransferase by the transfer of non-esterlfled cholesterol to rat hver mlcrosomal vesicles, Btochem J 194, 265-271(1981) G LEHRER, S R PANINI, D H R O G E R S a n d H RUDNEY, M o d u l a t t o n o f r a t h v e r HMG CoA reductase by lipid lnhlbttors, substrates and cytosohc factors, J Btol Chem 256, 5612-5619 (1981) A B SIPAT and J R SABINE, Membrane-mediated control of hepatic HMG CoA reductase, Bio~hem J 194, 889-893 (1981) J T B I L L H E I M E R a n d J L GAYLOR, Cytosohcmodulatorsofactlwtlesofmlcrosomal enzymes of cholesterol biosynthesis, J Btol Chem 255, 8128-8135 (1980) T R A M A S A R M A B PATTON and S GOLDFARB, Inactivation of HMG CoA 2+ ' reductase by Fe and a cytosohc protein, Bto~hem Btophts Res Commun I00, 170-176 (1981) P A EDWARDS. G POPJAK. A M FOGELMAN and JOHN EDMOND. Controlof H MG CoA reductase by endogenously synthesized sterols tn vitro and m vtvo. J Btol Chem 252, 1057-1063 (1977) P A EDWARDS. D L E M O N G E L L O . J KANE. I SCHECHTER and A M FOGELMAN. Properties of purified rat hepatic HMG CoA reductase and regulation of enzyme actwlty. J Btol Chem 255, 3715-3725 (1980) T KITA. M S BROWN and J L GOLDSTEIN. Feedback regulation of H M G C o A reductase in hvers of mice treated with Mevmohn. a eompetmve inhibitor of the reductase. J Chn Investtg 66. 1094-1100 (1980) D M GIBSON. R T LYONS. D F S C O T T a n d Y MUTO. Syntheslsanddegradatlonof the hpogemc enzymes of rat liver. Advances m Enzyme Regulatmn 10, 187-204 (1972) E H GOH and M HEIMBERG. Relationship between activity of hepatic HMG CoA reductase and secretion of very-low-denslty-hpoproteln cholesterol by the isolated perfused liver and in the intact rat. Bto~hem J 184, I-6 (1979) Z H BEG. D W A L L M A N N a n d D W G[BSON. M o d u l a t l o n o f H M G C o A r e d u c t a s e activity w~th cA M P and with protein fractions of rat hver cytosol. Bto~hem Btopht's Res Commun 54, 1362-1369 (1973) J L N O R D S T R O M . V W R O D W E L L a n d J J MITSCHELEN. Interconverslonof active and inactive forms of rat liver HMG CoA reductase. J Btol Chem 252, 8924-8934 (1977) T S INGEBRITSEN. H S LEE. R A PARKER and D M GIBSON. Reversible modulation of the activities of both liver mlcrosomal HMG CoA reductase and ~ts inactivating enzyme Evidence for regulation by phosphorylatmn-dephosphorylatlon. Btochem Btoph|~ Re~ Cornmun 81, 1268-1277(1978) Z H BEG, J A S T O N I K a n d B BREWER, HMGCoAreductase Regulatlonofenzymlc activity by phospborylatmn and dephosphorylatlon, Proc Natl A~ad Stt U S A 75, 3678-3682 (1978) M l KEITH, V W RODWELL. D H ROGERS and H RUDNEY, In vitro

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I) M GIBSON et al phosphorylatlon ol HMG CoA reductase Analys~s ol~2P labeled mactl',atcd en:yme, Bto~hem Btophls Res ( o m m u n 90, 969-975 (1979) Z H BEG, J A S T O N I K a n d B B R f W E R , Character,:atkmandrcgulatlonolreductasc klnase, a protem-kmasc that modulates the enTyme actl~ xt) ol H M G CoA rcductase, Pr¢~¢ Natl 4¢ad Set t A 4 76,4375-4379(1979) i H BEG, I A %-lONIKand H B B R E W E R , I n v t t r o a n d m ~ t v o p h o s p h o r j l a t l o n o l r a t h~cl H M G CoA reductase and its modulation by glucagon, J Btol Chem 255, 854 I-~545 (1980) 1 S [ N G E B R I I S E N , R A PARKER and D M GIBSON Regulahon ol h~c.r hydro~,ymeth~lglutaryl-('oA reductase b~ a btcychc phosphorvlatlon system, ,I Btol Chem 256, 1138-1144 (1981) R A PARKER, 1 S INGEBRITSEN, M J H GEFI EN and I) M GIBSON, Shortterm modulation ol HMG CoA reductase act~',~ty m rat hepatoc~tes m response to msuhn and glucagon, pp 609-624 m Cold Sprmg Harbor Con[eren¢ e ~n ( ell Proh/eratton, Vol 8: Protein Phosphor~latlon(O M ROSEN and E G KREBS, eds)(19811 M S BROWN, J l_ G O I D S T E [ N a n d J M D I E I S C H Y , Actl~candmactv, e l o r m s o l HMG CoA reductaseln the [t~er of the rat, J Bull Chem 254, 5144-5149 (1979) T S INGEBRITSEN, M J H GEELEN, R A PARKER, K J EVENSON and l) M GIBSON, Modulation of HMG CoA reductase act~,,~ty, reductase kmase actl~lt~, and cholesterol s5 nthesls m rat hepatoc~,tes m response to msuhn and glucagon, J Btol ('hem 254, 9986-9989 (1979) R H E N N E B E R G a n d V R O D W E l I , A l t e r e d m o d u [ a t l o n s t a t e o l rathepatoc~teHMG CoA reductase m response to msuhn, glucagon, cAMP, cGMP, and epinephrine, Federatton Pro¢ 40, 1604 ( 1981 ) S K ERICKSON, M A SHREWSBURY, R G G O U I D a n d A D COOPER, Studies on the mechamsms of the rapid modulation ol HMG CoA reductase m intact h~er by mevalonolactone and 25-hydroxycholesterol, Btochtm Btophv~ Acta 620, 70-79 11980) G A ROBISON, R W BUTCHER and E W S U T H E R L A N D , C v t h ¢ A M P , Academlc Press (1971) F Q NUTTAI L and D P GILBOE. Liver glJ,cogen sj, nthase phosphatase and phosphorylase phosphatase actlVlttes tn vitro following glucose and glucagon administration, Arch Bzothem Btophvs 203, 483-486 (1980) I G [ A R N E R , K GALASKO, K CHENG, A A DEPAOIA-ROACH, I HUANG, P [)AGG~ and J KELLOGG, Generation by msuhn ot ac.hem~calmedmtorthatcontrols protein phosphorylatton and dcphosphorylat~on. Stten~e 206, 1408-141 I (1979) A R S H A H E D , P P MEHTA, D CHALKER, D W ALLMANN, D M GIBSONand E I HARPER. Stimulation of rat hvcr phosphorylase phosphatase act~;qt 3 b3 msuhn, Btothem Internatwnal I, 486-492 (1980) R I K H A N D E I W A L a n d S M ZINMAN, Punhcat~onand propert~csolaheat-stable protcm inhibitor nl phosphoprotem phosphatase Irom rabb~t h~er, J Btol ¢he m 253, 560-565 (1978) S - H IAO, F I HUANG, A I Y N C H a n d W H G I I N S M A N N ( o n t r o l o l r a t s k c l c t a l muscle phosphorylase phosphatase actv~ty by adrenahne, Bu~them J 176, 347-350(1978) J G F O U L K E S a n d P C O H E N , The hormonal control of glycogen metabohsm, Europ J B~o~hem 97, 251-256 (1979) B S K H A T R A , J - L CHIASSON, H SHIKAMA, J H EXTON and q R SODERLING, Effect of epinephrine and msuhn on the phosphorylat~on of phosphorylase phosphatase mh~b~tor-I m perfused rat skeletal muscle, F E B S Lett 114, 253-255 (1980) J G FOULKES, L S J E F F E R S O N a n d P COHEN, The hormonal control of glycogen metabohsm Dephosphorylat~on of protein phosphatase mhlbttor-I m response to lnsuhn, F E B S Lett 112, 21-24 (1980) R E AREBALO, J E HARDGRAVE, B J NOI AND and 1 J %('AI I EN, In vtvo rcgulat,on ol rat h~er 3-hydro~cy-3-methylglutaryl-coen:jmc A reductase Enzyme phosphorylatlon as an earl_', regulator_~ response after mtragastrlc administration ol me~alonolactone, Pro~ Nail 4~ad S~t U S A 77, 6429-64"~3 (1980)

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Z H BEG, J A S ' I O N I K a n d H B B R E W E R , J r , l n v t v o m o d u l a t l o n o f 3 - h y d r o x y - 3 met hylglutaryl, coenzyme A reductase ( H M GR) phosphorylatlon by cholesterol (CH L) and mevalonolactone (M V L), Federatton Prot 40, 1604 ( 1981 ) E D BEYTIA and J W P O R T E R , Biochemtstry ol polylsoprenold blos,,nthcsls. 4 n n Rev Blochem 45, 113-142(1976) E D M I T C H E L L , Jr a n d J AVIGAN, Control of phosphorylatlon and dccarboxjlatlon of me~ alomc acid and its metabohtes m cultured h u m a n fibroblasts and ~n rat h,cer tn ~tvo, J Btol Chem 256, 6170-6173 (1981) R L KHANDELWAL, Theregulatlonofllverphosphoprotemphosphatasebymorgamc p y r o p h o s p h a t e a n d cobalt, Arch Btothern Biophls 191, 764-773 (1978) R L K H A N D E L W A L a n d S A S KAMAN1, S t u d l e s o n a c t l v a t t o n a n d r e a c t l v a t l o n o f homogeneous rabbit hver phosphoprotem phosphatases by morgamc pyrophosphate and divalent cations, Btothtm Btophv~ A~ta 613,95-105(1980) S C B YAN and D J GRAVES, lnblbttlon and react~,,atlon ol phosphoprotem phosphatase, Federatton Prot 37, 1425 (1978) T S INGEBRITSEN, J G F O U L K E S a n d P COHEN, The broad spectficlty protein phospbatase from m a m m a h a n hver Separatton of the M r 35,000 catalytic subumt into two distinct enzymes. F E B S Left 119, 9-15 (1980) R L VEECH, G A COOK and M T KING, Relationship of lrce cytoplasmic pyrophosphate to hver glucose content and total pyrophosphate to cytoplasmtc phosphorylatton potentml, F E B S Lett 117, S u p p l , K65-K72 (1980) T S INGEBRITSEN, Regulation of H MG CoA reductase by reversible phosphorylatlon Doctoral Thesis, lndmna U ntverslty (1979) J A T H O M A S , K K S C H L E N D E R and J L A R N E R , A rapid filter paper a s s a y t o r UDP-glucose-glycogen glycosyltransferase including an improved biosynthesis of UDP[14C]glucose, A n a l Btochem 25, 486-499 (1968)