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Review

BBALIP 53714

Metabolic aspccts of peroxisomal/3-oxidntion Harald Osmundsen ~, Jon Bremer -"and Jan

I. P e d e r s e n

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i DcptJrlt~lclll ¢!f Ph.~i,,h~,ey a n d Bitnhcmi~t~3", Dr'Ill,It ,~'1hocg, L m r ~'r~ll~ ot ()~lo, (~lt~ (,~'onvat). 2 ]tl~tilttlt' ~ff,~lt'di~ttl i~i~l(ht'ltli3H~ ('till t'ldtY t~] t)~hl ()~1¢~ (.\:ot~ ~e~I ,tiP/ t l~,ltllll~' ~lr \~Hlli(m Rt'~t'~lrtI1, /~'~tH~'r~tt) o I ()do. (1~[¢,

{Received 6 Dccumbcr lt)t~(l) (Revised nlZlnu~cripl received 23 ,\pril I'~l[)

Key ~nrll~: Fatty :~cid ilxitl~lion: Pe!o'ti~ml~ll ~-~id,~litm:

Hik'

acid hlrmutinn

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141

II

Fatty acid oxid~lt ion .............. A. Induction tit per¢)xi~m:ll /3-oxid:ttilm . . . . . . . . . . . . B. Chaln-shortening . . . . . . C. Met abo[ic producls tff l~croxi~om al /J-oxidl titln . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pcroxi~tlmal ~-oxidalion of un~aluratcd fatty u~id~ . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Transport itcrt~s the i~.'roxisom~ll mcmbr~ule . . . . . . . . . . . . . . . . . . . . . . F. Whlch fatty ~¢[ds are rcaUy oxldized by pero d~nl~d ~ Llxid;itK,n ? . . . . . . . . . . . . . . . G. Are peroxisumes involved in oxidatintl of ph~.hluic ilcid7 . . . . . . . . . . . . . . . . . . . . . . . . . .

142 142 ~1.4 14h 14~. 147 147 IJS

IlL

/~-Oxidntiun i~tdiczlrboxvlic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Formalion of dic:lrboxy;i¢ ;icids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. /J-Oxidatitm (chain shc~rlening) ot dicarboxylic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . C , End prtlducls t ff dic~lrhox~ lic ~lcid tlxid~llitm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Gluctmcogcacsis [trim tally aeid~.' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Rate of felly acid ~o-oxitl;Ition . . . . . . . . . . . . . . . . . . . . . . . F, ~-Oxidalion of xenobi0tic fatty acid5 . . . . . . . . . . . . . . . . . . . .

14S [4.~ 14~) 14~1 1511

Clxid~llit~nof chol¢~lcr~d side chitin und bile ~lCid tt,rmalion . . . . . . . . . . . . . . . . . . . . . A. Cleawlgc uf the ~temid side chain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Pcrt~xisonud disorders ~lnd bile ~lcid f~rnlzltion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Rcl~ltionship bet~ce[i pcroxi~mal I'alI~. acld t~xid~lgon :ttld bile acid lolmtlllOll . . . . . . . . . D. Ctmjugat k~n ~1' bd¢ ;~cld~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

152 152 1~3 153 154

IV.

150

151

Summary and (~ndusion~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154

References

155

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction M a m m a l i a n p e m x i s o m a l . B - o x i d a t i o n w a s c l e a r l y est a b l i s h e d b y L a z a r o w a n d d e D u v c in 1976 [1]. S u b s e quently, .B-oxidative clcawtge of the cholesterol sidechain during bile-acid Iormation was also lound to

Correspondence: J, Brcmer. Institute of ~vtcdical Bil3chcmistl~. University tff Oslo. Box I 112 81indern, 11316 Oslo 3. Ntll~'ay

t~ccur in liver p c r o x i s o m e s [2]. E a r l i e r l i t e n ~ t u r c d e scribes phenomena w h i c h , in h i n d - s i g h t , m o s t likely wcrc duc to pcroxisnmal /3-oxidation. The work of F i c c c h i c t al. [3]. s u g g e s t e d t h e p r e s e n c e o f a low level o f c y t o s o l i c .B-oxidative a c t i v i l y a n d t h a t o f O s h i n o cl ill. [4] d e m o n s t r a t e d f a t t y a c i d - d e p e n d e n t H _ , O : - g e n c r a t i o n in p c r f u s c d livers. I n t h i s r e v i e w w e will d i s c u s s t h e p h y s i o l o g i c a l s i g m l i c a n c e a n d s p e c i f i c i t y , o f p e r o x i s o m a l . B - o x i d a t l o n in t h e i n l a c t ceil. T h e r o l e o f p e m x i s n m c s in e t h e r - l i p i d

142 synthesis will not be included. As extensive reviews on peroxisomal metabolism recently have been published [5,6], we do not attempt a complete coverage of the literature. Although the chemical modifications made to the fatty acyI-CoA substrates daring peroxisomal and mitoehondrial ,8-oxidation are identical, the enzyme-proteins involved arc. all non-idcmical. Another difference is that peroxisomal B-oxidation can operate in relative independence of celhdar energy status, because the reduced acyl-CoA oxidase is directly re-oxidised by molecular 0 2 (see Fig. l). This arrangement is ideally suited for a process the main function of which is elimination of poorly metabolizable compounds (i.e., xenometabolism, detoxificution), rather than genera-

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Fig. 2. ESsential features of maochondrial ,g*oxidaaon. A schematic

p~senlatinn of the main metabolic features of mltochondrial .8oxidation. 3"h¢ enzymes of /3-oxidation are contained within the H20~

iH~CICH3 ~CHa C ~ C H

Bt(TttlfcnctlonalezL~ym¢

~O'S'C0~

inner maochondrial membrane tsnlid line). ATP is formed by reoxidation of N A D H and F A D H z by the respiratot~ chain. Chain-

shortened intermediates are immediately re-zycled through the ~oxidation sequence to be completelydegradedto acetyI-CoA, tion of A T P (see Figs. 1 and 2). Mitochondria1,8-oxidation, in contrast, is directly coupled to synthesis of ATP. 11. Fatty acid oxidation

II-A. Induction of peroxisomal E-oxidation

Fig, I, E~ential fealmus of pcroxi~omal fi-oxidabon. A schematic premmalion of the main metabolic fcalurcs of pcroxisoma] /$-oxidabon The enzymesof pcroxi~m~l /3-~xidation arc contained within the perogisoma] membrane (solid lincl. Possible end-products of /3 oxidation are indicated at Ihe bollom of Ihe Figure. The IHOOCI io the left of the sub~trate acyI-CoA ester, indicates Ihat monaCo:A-esters of di¢~trht,xylic acids arc also suhstrates for pcroxi~ma~ /Loxidation An incorrect stoichiomelry of catalase-dependenl dccl~mpc~silion of HzO ~ is used tar the sake of brevity. The arrows pointing down zo aeytcarniline/FFA/aeybCoA, or to acetylcarnltme/acetatc/'accty6CoA, are meant to indicate possible alternative forms of export ~ff chain-shmlcned fany acids out of Ihe peroxisome (FFA = free fult~ acidg The oxidant (R) ot NADH active in vivo remains m be established With isolated peroxisomes pyrm'ate (and lactate dehydrogenase)can function in this capacity Ih3,7.tl.

In normal rat liver peroxisomal 0-oxidation is oresent at a very low level of activity, a feature which probably allowed th~ process to remain undetected until 1976. An outstanding feature of pesoxisomal ,8oxidation is the powerful induction which occurs upon treatment with many hypolipidaemle agenls (more than 10-fold is commonly observed). This phenomenon was initially noted following treatment of rats with doffbrute [1] and several related compounds [7]. The list of compounds has subsequently grown steadily and it now includes plasticizcrs, pcrfluorinated fatty acids, alkylsu[phonic acids and ethyl 2[5(4-chlorophenyl)pentyl] oxirane-2-carboxylate (an inhibitor of carnitine palmitoyltransferase) (see Table I). Induction was initially found in liver, but was later shown to occur also ht heart and kidney [8,9].

143

TABLE I ¢;vh~tanccs, o r trea~,ct, t~. ~*hxcJt cause md~,t'tuln oJ h¢l~t:tl, pt,r,,usoreal ,~-oxi~htlioB

Substance/treatment Clonbrate Tibric acid Cipronbrate Nafenopin Tiadenol/Niadenate

Di(2~thylhexyl)derlvalives Ethyl 2tSl4~hlorophenynpenlyU oKirane-2-carbo~hae (PCA) Hyperthyroidism Vitamin E deficiency Perfluorlnated fatty acids Peffluorinated alkane sulphonic acid Methyl-subsitutedcarbox~lic acids Methyl-substituted alkanes 3-Thia-fauy acids Trichloroacetic acid Diehloroaeeti¢acid Partially hydrogenated fish oil Adrentmortocotropin Dexamethasone & ACTH Dehyd~piandrosteronc

Species Ral Rat Monkey. cat, chicken, pigeon Mouse Rat Rut

Reference [ 7

Ral Ral Rat Rat

49 51) 5I 52

47 32 26 48

Rat

It)

Rat Ral Rat Mouse Mouse

53 54, I,~ 2a

Rat Guinea pig Guinea pig Rat

I t. 13 56 5"1 58.59

55

In livers of rats fed on high fat diets peroxisomal B-oxidation is also induced, in particular when the diet contains a high proportion of very-tong-chain fatty acids known to be poorly B-oxidized by mitochondrial Boxidation [10-12]. In liver the more powerful induction occurs with a diet containing partially hydrogenated fish oil [12], while in the heart rape-seed oil and partially hydrogenated fish oil are equally effective [8]. Although the myocardial peroxisoma[ B-oxidative activity is low, relative to that of the liver, it can b e . significant for B-oxidation of very-long.chain fatty acids

[8]. It is now clear that partial hydrogenation of many dietary oils, e.g., soybean-oil, rape-seed oil and fish-oil, generates dietary fats with enhanced ability to induce peroxisoma[ B-oxidation [13]. These partially hydrogenated oils have been shown to contain a high proportion of very-long-chain mono-unsaturated fatty acids [14] which are poorly oxidized hy mitochondria[ Boxidation [15,16]. Most of the compounds demonstrated to cause induction of peroxisomal B-oxidation are earboxylic acids (or will be converted into carboxylie acids in vivo), pos essing substituents which completely, or partially, block B-oxidation. The powerful induction observed with methyl-substituted Mug-chain dicarboxylic acids is an excellent example in this respect [17]. This suggests that induction of peroxisomal B-oxidation is associated

with presence of non-B-oxidizable carboxylie acids in th~ body. It has also beret reported that heptamethylnonane causes indoctior, of peroxisomal B-oxidation [18]. The major metabolite isolated was heptamethylnonanedioic acid, ~'hieh again cannot be /3-oxidized. Induction of peroxisomal B-oxidation by pernuorihated octane sulphonic acid lit)] deviates from this general picture. This suggests that non-B-oxidizable acids, other than carboxylic acids, also can bring about induction of peroxisomal B-oxidation. Treatment of rats with thia-substituted fatty acids has supplied further information regarding this conundrum. 3-Thia-,atty acids, which are blocked for ,8oxidation, arc powerful inducers of peroxlsomal Boxidation [20]. In contrast, 4-thia-fatty acids, which are partially B-oxidized and cause inhibition of mitochondrial B-oxidation [21]. are weak inducers of peroxisomal B-oxidation [20]. Accordingly, inhibition of mitochondrial B-oxidation is not a pre-requisim for induction of pcroxisomal B-oxidation. Valproi¢ acid (dipropylaeetie acid) is a short-chain fatty acid. which also is blocked for B-oxidation. In one study no valproate-dependem peroxisomal proliferation was found [22]. However, with a high dosage (1%, w/wk added to the fodder/ about 5-fold increase in peroxisomal B-oxidation was measured in rat liver [23]. Valproic acid is, in contrast to most other inducers of peroxisomal B-oxidatlon. a relatively hydrophilic compound. This implies that relatively hydrophoblc carboxylic (or sulphonic) acids cause the more powerful induction. Alternatively, sobstances which in vivo are converted into such compounds, will also give powerful induction of ,B-oxidation. This is in line with the findings of Vecrkamp and Zevenbergen [24], that effective induction by high fat diets is primarily a function of the content of C2u- and C2z-fmty acids, the amounts of t r a n s C t~-fatty acids being of no particular significance. h has been suggested that a high level of long-chain acyI-CoA esters (or xenobiotic acyI-CoA esters) may ~,;~s~, ;,,d~do,, [25 27]. , ~ pctfluorodecylsulphonie acid, which evidently cannot be activated to an acylCoA ester, has been shown to induce peroxisomal /J-oxidation [ 19] this hypothesis is rendered less attractive. Tl;e presence of an amphipathic, hydrophobie and poorb' metabolizable acid, in the cell therefore appears sufficient to trigger induction. Inducti(m caused by hypolipidaemic agents involvcs selectively increased transcriptional activity, as judged by increased levels of m R N A from genes coding for enzymes of pcroxisomal /3-oxidation, e.g., hifunctional enzyme [28,29]. The amount of m R N A coding for catalase was, however, not significantly increased. The mechanism of induction remains unclear. I1 is possible that the mechanism behind induction caused by pardally hydrogenated fish oil is somewhat differcnL The life-time for acyI-CoA oxidase is increased in li~ ers of

144 rats fed on diets containing partially hydrogenated fis;i oil [30]. Recently, Horie and Suga [31] have shown thai s[muitaneous treatment of rats with clofibrate and partially hydrogenated fish oil have additive effects a~ regards hepatic levels of acyl-CoA oxidase activity. even though the fish-oil-dependent increase in acyiCoA oxidase mRNA levels was a fraction of that observed with c/ofibratc alone. It was suggested th,"t two different inductive mechanisms were operating. one at the level of mRNA, the other affecting the stability of the acyI-CoA oxidasc activity. A recent finding thaL e.g., clofibrate and nafenopin activate a nuclear receptor protein related to the nuclear steroid hormone-receptor superfamily [32], sup-; ports the suggestion that a receptor-mediated mocha-. nism for induction is involvcd [33]. An extensive discussion of possible inductive mechanisms has been given by Lock e t a l . [34]. It is pcrtill~ut to point out that also non-pcroxisomal enzymes ot lipid metabolism are induced by hypolipidaemie agents, e.g., aeyI-CoA hydrolascs, glycerophosphate acyhransfcrase, acyI-CoA synthetase, and carnitine palmitoyltransfcrase [20]. Cloflbrate-dependent stimulation of palmitate oxidation in man was observed in 1970 [35]. Using liver homogenates and hepatocytes, isolated from rats treated with clofibrate enhanced rates cff ketogcncsis [36] and of mitochondrial .6-oxidation were demonstrated [37,38]. Mitoehondrial .6oxidation of palmitate is stimulated by hypolipidaemic agents and by high fat diets, to a much smaller extent than is pcroxisomal /~-oxidation (about 50% increase in activity in animals fed on a dict containing 20% by wt. of partially hydrogenated fish oil) [12,39]. In general, treatment with clofibrate approximately doubles the mitochondrial .6-oxidative activity [36,411]. An auxiliary enzyme of .6-oxidation of unsaturated fatty acids, 2,4-dienoyI-CoA reductase is also induced by clofibrate [41], by high fat diets [42] and by growthhormone [43]. This is the reason why rates of mitochoitdria] /J-oxidation of many polyunsaturated fatty acids arc stimulated up to 5-fold following treatment with clofibratc [39,44]. In endoplasmic rcticulunt laurie acid co-hydroxylase is powerfully induced by elofibrate [45] and by high fat diets [46]. II-B. CTlain-shorzening While mitochondrial ,6-oxidation is known to /3oxidize fatty acids completely to acctyl-CoA, available data unequivocally show that pemxison'tal ,f:l-oxidation is incomplete. This phenomenon wa~ initially observed by Lazarow and de Duve [I], who found that palmitoyI-CoA was subjected to five cycles of B-oxidation using isulutcd, solubilized, pcroxisomal fractions. It was subsequently suggested [6(I] Ihat partial .6-oxidation, or chain-shortening, might b c a physiologically significant

pcro~:isomal function, particularly with respect to verylong-chain fatty acids which arc poorly oxidized by rnitoehondria] .6-oxidation. This process can be physiologically relevant because the chain-shortened products would be excellent substrales for mitoehondrial ,6oxidation. Treatments known to cause induction of peroxisomal .6-oxidation stimulate chain-shortening of veryic;~-chain fatty acids by perfused liver [61], or by isolated hep;,~t~ cvtes [10]. Erucic acid was chain-shortened by at lea'~t three cycles cf B-oxldation, the product from the t~¢o first cycles (i.e., oleatel constituting the major product [10,61]. A similar pattern of chainshortening of (14-14C]eruco:~l-CoA was found using isolated rat liver peroxisoma', fractions [62,63]. Indeed, the ability of isolated hepatocytcs to chain-shorten [14J4C]erueie acid was shown to increase in parallel with increasing induction of peroxisomal E-oxidation [121. The literature presents diver~e opinions regarding the quantitative contributions of mitochondrial and peroxlsomal /~-oxidation to cellular fatty acid oxidation. Peroxisomal .6-oxidation (measured as rate of acetyI-CoA production) has been estimated to contribute from 10% [64] to 30% [65] of total cellular .6-oxidation of palmitate in normal rat hepatocytes. These estimates will also include acetyI-CoA formed from .O-oxidized fatty acids (see Section Ill-E). The higher contribution of peroxisomal /]-oxidation is achieved only when the concentration of fatty acid in the incubation-medium is I mM or higher [66] (see section II-F). It is now reasonable to state that the physiologically significant aspect of peroxisomal iS-oxidation is chainshortening, while the major function of mitochondrial .6-oxldation is to generate aeetyl-groups. The peroxisomal hepatic capacity to chain-shorten, e.g., erueie acid can be estimated to be several.fold higher than the mitochondrial capacity to ~6-oxidize this fatty acid [25,67,68}. Nevertheless, when mitochondrial and peroxisomal ~6-oxidation is compared in terms of classical .6-oxidative capacity, i.e., ability to generate acctylgroups, the mitoehondrial capacity is massive compared to that of the peroxisomes [25,67,68]. The importance of chain-shortening, however, is illustraled by the transient nature of the cardiac lipidosis found in rats fed on diets containing high amounts of erucic acid [69}. The disappearance of lipidosis coincides with induction of peroxisomal ff-c.xidation [8]. The precise extent of chain-shortening of a given fatty acid remains ambiguous. Palmitoyl-CoA has been reporte'.l to he chain-shortt.ued by three to seven cycles of /3-t:,~idation using ist~lated peroxisomal fractions [1,62,67]. With other l~atty acids, i.e., erueic, oleic, !inoleic, linolenic, docosatctrucnoic ( 2 2 : 4 l n - 6)] and docosahexaenoic t 2 2 : 6 ( n - 3)), two to three cycles of

145

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ann

4oo

Conc. of [U-r'C] hexadecanoate (pM) Fig. 3. The effect of cuncentralilm nf [U-14C]palmdalcnn exlcnt at chain-shortening of [U-taC]palmitate by isolated rat fiver peroxihomes. Isolated peroxi~mes w~re incubated in the presence of the various concentratiol~s of [U-14CJpalmilate(about 20(11)11dora per nmol) shown in the Fig. After 15 min of incubation at 37°Cexlent of chain-shortening was mea~u~d by analysis of the radioaclive acylCoA-esters found in the incubations, as described [731 The fractkm of acyI-CoA esler; of chain-lengths four In ten carhon-aloms has been expressed as per cent of the tolal amount of chain-shtmen¢o acyl-CoA esters (i.e.. 4-14 carbon-atoms} found in the incubaliu~ The data presented here has been compiled tram results presen'ed in [7S]. "rbe peroxlsomal fraction was isolated by density-gradient eentrifugation using a selfgenerated Percoll gradient [12]. q'be peroxisomes 11 m g of protein/ml incubation} were incubated in a medium ¢onlaining KCI, 13n raM; l-lepes{pl-I 7.21, 10 mM; EGTA. 0.1 raM; HAD+, 0.5 raM; NADP'. U.I mM: dilhiolhreUol, I raM; CoA, 0.2 raM. MgCI,.5 raM; ATP. 5 raM; pyru~ate, 2 raM: lactate dehydrogenase, l tl/mk defatted b~wine serum albumin, 2 mg/ml: antimycin a. 10ug/ml.

/~-oxidation have been observed using i:*olated rat liver peroxisomes [2,70]. This is in line with data obtained using perfused rat liver [61]. rat heart [71,72], or isolated rat hepatocytes [10]. Similarly Veerkamp and Van Moerkerk [9] suggested that palmitate is subiected to about two cycles of/3-oxidation. Henceforth, two to three cycles of ~-oxidation appears to be the rule, Using isolated rat liver peroxisomes the observed extent of chain-shortening of palmitate is a function of the concentration of palmitate [73] Chain-shortening down to butyryI-CoA was observed (i.e., six cycles of g-oxidation). At low concentrations (20-50 /.tM) of palmitate intermediates of chain-lengths shorter than 12 carbon atoms constituted up to 60% of the total amount of chain-shortened products. At a high concentration of palmitate (400 ~M), however, the contributions from these short-chain intermediates drooped to about 16% of the total (F;g. 3). At the higher concentration of palmitate, therefore, the extent of chainshortening approaches two cycles of/3-oxidation. This phenomenon can be interpreted to demonstrate that oxidation of medium chain-length intermediates is prevented as long as a high concentration of long-chain

acyI-CoA is present. Also earlier results, based o n measurements of paimitoyI-CoA-depend.:n: generation of H e 0 : by light mitochondrial-fractions, suggested that chaln-shortening of palmitate decreased from ahout two to one cycle of/3-oxidation with increasing concentration of palmitoyI-CoA [74]. With isolated rat liver peroxh,omcs the rate of pcroxisomal /3-oxidation was stimulated by the presence of an NAD*-regenerating system and the composition of chain-shortened intermediates from [U-taC]pahnitatc was aim influenced [63.73.75]. in the presence of pyrerate and lactate dehydrogenase no hydmxylated or monounsaturated intermediates were observed while such inlcrmcdiates were readily apparent in the absence or pyruvate and lactate dehydrogenase [63,73,75]. These findi.ga suggest that re-oxidation of N A D H is rat~-limiting for /3-oxidation, In the presence of pyrevale and lactate dehydrogenase, hox~,ever, 2-oxohexadecanoyl-('oA can be seen as an intermediate suggesting that thiolase can become rate-limiting for B-oxidation [73]. Using isolated rat hepatocytes [-lagve and Christopherscn [7b] showed that adrenic acid ( 2 2 : 4 ( n - 6)) and docosahexaenoic acid are chain-shortened by one cycle of ~-oxi,tation only, causing their retroconversion to arachidonic and eicosapentaenoic acid (20: 51n - 3}), respectively. Retroconversion may therefore be another physioh*gically significant function of pcroxisoreal/3-oxidation. Arachidonit: and eicosapentaenoic acid are themselves substrates for pcruxisomal /3-oxidation [70,77]. They are IxJth /3-oxidized at relatively low rates in view of their extensive degree of unsaturation. It appears that an initial double bond in the iS-position renders the fatty acids more resistant to pero!dsomal /3-oxidation [77]. The net efii:ct may be to shield such physiologically essential fatty acids from peroxisomal /3oxidatitm and may be the reason why no significant chain-shortened products were found from arachidonic acid added to isolated bepatocytes [76]. However, using isohncd pemxisomes it has been shown that prostaglandins [metabolites derived from, e.g., arachidtmlc acid} arc chain-shortened by one to two cycles of 0-oxidation [78]. Peroxisomal /3-oxidation has been implicatcu in the catabolism of the very long-chain fatty acids which arc abundant in neural tissues (e.g., nervonic acid {24: Iln - 15)} and lignoceric acid (24:0) [79]. Studies of Ooxidation of thcse fatty acids are inherently difficult because of their very low solubility in the aqueons phase. Rates of /3-oxidation measured with liguoccric acid are often less than I% of that observed with, e g., palmitic acid. To what extent these low rates of /3oxidation arc due to physico-cbemical phenomena, or tt~ a genuine property of the tfiological system, remains to he answered.

146 Chain-shortening of these poorly metabolized fatty acids would convert them into fatty acids which will be rcadily B-oxidized by mitochondria. Current data, from studies of inborn crror~ of metabolism, suggest that lignoceric acid is mainly B-oxidized by peroxisomal fl-oxidafion [80,81] and that a separate, pemxisomal, lignoceroyI-CoA synthetasc is responsible for its activation [82]. The physiological significance of this enzyme and of peroxisomal chain-shortening of very-long-chain fatty acids, is illustrated by rite severity of the symptoms of X-linkod adrenoleukodystrophy, an inherited disease in which the peroxisomal lignoceroyl-CoA synlhclasc appears missing [82]. The metabolic dependence on rite peroxisomal lignoceroyI-CoA synthetase for activation of very-long-chain fatty acids is, however, probably not absolute as cultured fibroblasts isolated IYom patients with X-linked adrcnoleukodystrophy Boxidize [1-1"~C]Iignoceric acid [83],Their rate of oxidation is only diminished when the cells ate exposed to high concentrations of lignoeetic acid [83]. Most likely, the pahnitoyI-CoA synthetase present in mitochondria, microsomes and peroxisomes shows some activity towards llgnoccrlc acid and other very-long-ehaln fatty acids. In line with the above findings chain-shortening of erucic- and adrcnic acid has been shown to occur in fibroblasts from patients with X-linked adrenoleukodystrophy, while it was almost completely absent in fibroblasts from patients with Zellweger syndrome [8486i. In conclusion, it therefore appears that the mctabolic dcpendcnce on peroxisomes for chain-shortening of very-long-chain fatty acids is absolute, while the dependence on lignoceroyI-CoA synthetase for their activation is not.

II C. Metabolic prodtlcts of peroxisomal l~-oxidation The crucial metabohte derived from peroxisomal B-oxidation is the chain-shortened carboxylic acid. We kmlw that chain-shortened fatty acids can be further oxidized in Ihc mitochondria and that they can be incor0oratcd into lipids [lll,15l. The details of transfer of peroxisomal products to other sub-cellular compartmcms are, however, not known. Is the chain-shortenod acyI-CoA ester transferred as such~ or is it hydrolysed by an acyl-CoA hydrolase prior to transfer out of the pcro×isomal matrix, or is it released into the cytosol as fatty aeylcarnitine osier? These alternatives are all possible as pcroxisomes are known to possess acyI-CoA hydrolascs [87,88], as well as acylcarniline transferases. Both acetyl- and octanoyl-carnitine transferase have been found [89,90] and thc prcsencc of a palmitoylcarnitine transferase has also been reported [911. Chaln-shortcncd products which cannot be further /3-oxidized must be excreted, following hydrolysis of the acyI-CoA ester.

Acetyl-CoA is also a mandatory product of peroxisoreal B-oxidation, the ultimate fate of which has been assumed to be transfer to mitochondria. We have recently found, however, that peroxisomal E-oxldation occurring in isolated hepatocytes results in production of free acetate [92]. It is, therefore, likely that pcroxisoreal B-oxidation contributes to acetate present in the general circulation, even though the gut flora is a major contributor [93]. As liver peroxisomes contain earnitine aeetyltransferase, as well as acetyl-CoA hydrolase, acetyl-groups may leave the organelle as acetylcarnitinc (for transfer to mitcchondria), free acetate or as aeetyI-CoA. Free acetate may also be formed in the cytosol [94] by a cytoplasmic acetyI-CoA hydrolase [95]. It remains to be decided what is the major mechanism of export of peroxisomal acetyl-groups. The absence of ketogenesis from dicarboxylie acids (see Section lit-B) indicates that the carnitine-dependem transfer to the mitoehondria is of minor significance in the liver.

II-D. Peroxisomal B-oxidation of unsaturated fatty acids During the last few years it has been demonstrated using isolated peroxisomes that some unsaturated fatty acids are particularly good substrates for peroxisomal /3-oxidation [70,77,96,97]. Studies with isolated hepatocytes have likewise suggested that, e.g., adranie acid is rapidly chain-shortened by peroxisomal/3.oxidation [76] (see also previous section II-B). The scheme for Boxidation of polyunsaturated fatty acids has also been revised in view of findings accumulated during the last decade, It is now well established that a reductive step is obligatory if mitochondrial B-oxidation is to proceed beyond a double bond positioned at an even numbered carbon-atom (for review see Ref. 98). In -'aitochondria the reaction is eatalysed by an NADPH-dependent 2,4-dienoyI-CoA reduetase (EC 1.3.1.34) [99]. Induction of this enzyme is probably important for the increased rate of ketogenesis from polyunsaturated fatly acids found in hepatocytcs from rats treated with clofibrate, or fed on high fat diets [39]. The presence of a peroxisomal 2,4-dienoyI-CoA reduetase activity has also been documented [100] and demonstrated to be involved during B-oxidation of polyunsaturated fatty acids [70]. By analogy with the mitochondrial reductase the peroxisomal enzyme is also presumed to be inducible. B-Oxidation of docosahexaenoate was stimulated by several-fold on inclusion of NADPH to isolated peroxisomes [70]. Results from a recent investigation [lOt] suggest that, as yet, only the mitoehondrial reductase has been purified. Although 3-hydroxyacyI-CoA epimerase (EC 5.1.2.3) classically has been implicated in B-oxidation of unsaturated fatty acids, the involvement of 2,4-dienoyI-CoA

147 reductase during their ,0-oxidation eliminates the requirement for epimerase [98]. In line with this argument it now appears that epimerase is not present in mitochondria [81[ an¢~ that the peroxisomal 'epirnerase' activity [102] is comp~,.~ed ,if two separate 2-enoyI-CoA hydratases [103,1(14]. Arachidonate has also been demonstrated tu be chain-shortened by at least two cycles of ~-oxidation when incubated with isolated peroxisomal fractions [70]. This suggests that also pcroxisomal 8-oxidation can proceed beyond double bonds at odd-numbered positions (arachidonate has an initial double bond in .a 5-position) and that peroxisomes must have a .I a, .a 2enoyI-CoA isomerasc (EC 4.2.1.171 activity. Patosaari and Hiltunen [105] have presented evidence which strongly suggests that there is no separate peroxisomal isomerase. Their results indicated that the peroxisomal bifunefional enzyme, known to possess enoyI-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activity [106], also has a~,~2-enoyI-CoA isomerasc activity ] 195] The bifunctlonal enzyme may therefore he trlfunctional. A similar trifunctional enzyme has also been found in Candida tropicalis [1117]. Does the use of a multifunctional enzyme endow peroxisomal .fl-oxidation with capabilities not available to mitoehondrial /3-oxidation? Typical of peroxisomal g-oxidation is the relatively high rates of t'3-oxidation of trans-fatty acids. With elaidoyI-CoA rates of /3-oxidation similar to that of oleoyI-CoA is observed [62,10gL This is strikingly different from mitoehondria where rates of /]-oxidation of trans-isomers often are about 50% of rates observed with corresponding c/s-isomers [44,109], although the difference depends somewhat on the position of the tram-double bond in the carbonchain [I 10]. It has been suggested that this feature of peroxisomal /]-oxidation is due to intermediate-channelling on the bi(tri)functional enzyme [111].

II-E. Transport across the peroxisomal t~tembrtme Isolated peroxisomes appear freely permeable to substrates and cofactors, e.g., acyl-CoA, CoA, N A D ' , ATP and carnitine. This has been explained by the presence of a non-specific pore-formlng protein in the peroxisomal membrane [112]. However. pcroxisomes may also possess a membrane-bouod ATP-ase. suggested to be involved in active transport of metabolites across the peroxisomal membrane [113]. Active transport may explain the observed ATP-depcndent stimulation of fl-oxidation of acyI-CoA by light mitochondrial fractions [741. In spite of the permeability of the membrane to CoA the peroxisomal matrix contains CoA which is not lost on isolation of peroxisomes [114]. This phenomenon has been demonstrated to be caused by firm binding of CoA to matrix proteins [115]. It is presum-

ably this CoA which enables isolated peroxisomes to /J-oxidize added acyI-CoA in the absence of exogenous CoA [116]. On the contrary, when acyI-CoA esters arc used as sub~trates, added CoA can be shown tc. cause inhibition of /C-oxidation [63,67]. Fatty acids destined for peroxisomal /3-o'ddation can be activated by a long-chain acyI-CoA syathctase located on the eytostrlic side of the peroxisomal membrane [117]. This location therefore explain," i_he absolute requirement for added CoA when free fatty acids are substrates for /~-oxidation [I 18]. The lignoceroylCoA synthetase is claimed to be located on the matrix-side of the membrane [119]. With isolated hepatocytes inhibition of mitochondrial /~-oxidation by addition of ( + }dceanoylcarnitinc led to increased recovery of chain-shortened products from [14-t*C]crucic acid fill[. This was presumed to demonstrate the carnitine-indepeodent nature of peroxisomul chain shortening of very long-chain fatty acids. Recently, I + klecanoylcarnitine, ho~vever, was sho~,~n to protect peroxisomal thiolase activity against inactivation by 2-bromo-substituted fatty acids [1211]. The same protection was furnished hy telradecylglycidic acid which is an inhibitor of carnitine palmitoyltransferase 1 [120]. These results were interpreted to demonstrate carnitinc-dcpendent transport of fatty acyl-substrates into the peroxisomes [120]. It has also been proptx-;ed that ratty acid-binding protein (Z-protein) is involved during transport of fatty acyI-CoA esters across the pcroxisomal membrane. The suggestion was based on the ability of flavispidic acid to inhibit /3-oxidation of addod aeyI-CoA by non-solubilized peroxisomes, hut not by solubilized peroxisomes [121]. More recent data, however, suggest that the fatty acid-binding protein may shuttle fatty acids to the peroxisome, although not necessarily across the peroxisomal membrane [122]. The current state of knowledge regarding possible peroxisomal transport-phenomena and their regulalion, is thcrcforc at present best described as unsettled.

II-l~ Which fatty acids art" really oxidized by treroxi.~u~al [~.,z~:idation ? As with many other metabolic phenomena, the ultimate remaining question is: Does it really occur in viva? More often than m)t the question is extremely difficult to answer directly. As regards peroxisomal /:l-oxidation such attempts have been made using perfused rat liver. Using the technique of organ spectrophutomctry Fucr~ter et aL [123] was able to directly monitor fatty acid-dependent generation of H,O_, in the perfused rat liver. The pattern of fatty acid oxidation which emerged appeared somewhat surprising. Erucic acid, in line with in vitro findings, was lound to bc ,~-oxidizcd at an appreciable rate. Palmitate, in contrast to all in vitro findings, appeared not to bc

148 subjected to peroxisomal ,B-oxidation in viva. However. fatty acids of shorter ehain-lcngtl~s generated HeO,. These experiments were carried out using relatively low concentrati~e'~ of fatty acids in the perfusate. Handler and Thurman [66] have clearly demonstrated that the pcroxisomal eonlribution to Lcpatic fatty acid oxidation is dependent on the concentration of fatty acid in the pcrfusate. They showed that mitochondrial `6oxidation of oIeate is practically saturated with I).5 mM tncatc in the pcrfusate. At this concentration only mirror oleate-dependent stimulation of H202-generations was observed. On increasing the concentration of oIcatc to I mM marked stimulation of H202-generalions was found [66]. From these results it follows that the conventional mitl~chondrial substrates, e.g., palmitale or oleate, arc preferentially utilized by mitochondrial B-oxidation (in addition to lipid biosynthesis). Fatty acids for which the mitochondrial B-oxidative capacity is low, e.g., erucic acid, are more likely to be subjected to pcroxisomal chain-shortening because their acyI-CoA esters will more readily accumulate in the cytosol. As medium-chain-length fatty acids are preferentially oxidized in mitochondria, exogenous mediumchain-h. :gth fatty acids may funnel endogenous longchain fatty acids into pet,lxisomal B-oxidation. Alternatively, medium-chain fatty acid~ may be oJ-oxidized, as a laurie acid ~o-hydroxylase activity is also induced on treatment with clofibratc [124]. As the resulting dicarboxylic acids are eminent substrates for peroxisomal B-oxidation, medium-chain fatty acid-dependent generation of H ~O 2 may have been due to oxidation of dicarboxylic acids (see section Ill-B). These cogitations serve to illustrate the complexity involved in the assessnlcnt of an ill vivo experimental situation. In vitro studies have revealed that medium chain acyI-CoA esters are substrates for pcroxisomal .8oxidation, although the apparent Kin-values were much higher than that of, e.g, palmitoyl-CoA [125]. Also, using isolated, intact peroxisomcs it has been demonstrated tlmt peroxisomal B-oxidation showed no evidenec of substrate inhibition by concentrations of oleate or linolcnatc up to I).8 mM [70]. This reinforces the impression of peroxisomal B-oxidatlon as a process designed to operate under u high metabolic fatty acid load. `6-Oxidation of polyunsaturated fatty acids has also been investigated using organ s0cctrophotometry [70]. These studies showed that all lkltty acids investigated (e.g., (fleatc, linoleate, Imolenate and docosahex~lenoale) traused reduction of mitoehondrial flavoproteins and increased die level of catalas¢ compound 1. suggesting that both peroxisomal ,6-oxidation and mitochondrial B-oxidation wcrc involved in their catabolism. In these experintenls the absence of albumin in the pcrfusate-fluid may have led to enhanced

rates of peroxisomal B-oxidation, in spite of the use of low concentrations of fatty acids in the perfusate. As peroxisomal ,6-oxidation also leads to formation of free acetate [92]. it is likely that measurement of formation of acetate may supply additional inlbrmation regarding extents of hepatic pcroxisomal ,6-oxidation of various fatty acids.

II-G. Are peroxisomes hll oh'ed in oxidation of phytanic acid? The discovery of accumulation of phytanic acid in patients with peroxisomal disorders, e.g., infantile Refsum disease [126A27] and Zellweger syndrome [128] raised the question about the role of peroxisomes in phytanic acid degradation. Because of the presence of the ,6-methyl group `6-oxidation of phytanie acid is blocked. Since the work of Steinberg the classical view is that phytanic acid oxidation starts with a-hydroxylarian followed by decarboxylation and formation of pristanic acid, which can then be `6-oxidized (for review see Ref. 129). a-Oxidation was found to be localized in mitochondria in rat [129], guinea-pig [130] and human liver [131]. Refsum disease was considered to be due to defective o~-hydroxylase activity [129]. Measurement of a-oxidation activities of 1-14C-labelled phytanic acid by collection of 14CO2 has shown a lack of activity in fibroblasts from classical Refsum disease, infantile Refsum syndrome and other peroxisomal disorders [132]. These new findings deafly indicate that peroxisomes are involved in the degradation of phytanic acid. At present we can only speculate on how these findings can be reconciled with the old view. One explanation may be that the a-hydroxylation is mitochondrial, while the decarboxylation reaction depends on peroxisomes. The finding that a-ketophytanie acid is oxidized with formation of H202 by rat kidney peroxisomes [133] may point in this direction, Another possibility is that both a-oxidation and decarboxylation occur in mitochondria and that the resulting pristanie acid is /Soxidized in peroxisomes. In the absence of active peroxisomal B-oxidation, accumulation of pristanic acid could bring about feed-back inhibition of a-oxidation [134]. IlL fl-Oxldation of dicarboxylle acids

IliA. Formation of dicarboxylic acids Excretion of diearboxylic acids in urine after ingestion of medium-chain fatty acids was detected by Vcrkade, who suggested the term co-oxidation for this conversion [135]. Dicarboxylie acids, mainly adipic (C6) and suberic acid (C8), are normally found in urine and their cxeletion is increased during lusting, in diabetes [136], in

149 carnitinc deficiency [137] and in some inborn errors of metabolism with impaired fatty acid oxidation, e.g., Zcllwegcr syndrome [13g,1391. Their excretion is also elevated when medium-chain fatty acids are fed in the diet [ 1411].Small amounts of 3-hydroxydicarboxylic acids are also found [141,142]. Experiments with [I-laC]- and [16-14C]palmitic acid have shown that short and medium-chain dicarboxylic acids in urine arc normally formed from long-chain falty acids [143], evidently by ~o-oxidation fi~llowed by chain-shortening by .6-oxidation. (o-Oxldation is initiated with an ~0- 1or ~,~ I Ihydroxylation of the fatty acid catalysed by a group of closely related cytochrome P-450 enzymes located in the endoplasmic rcticulum of liver and kidney [1441. Thcsc cytochrome P-4511s also hydroxylate fatty acid related compounds, e.g., prnstaglandins. The conversion of the (o-hydroxy-acids to dicarboxylic acids is catalysed by an alcohol dehydrogenase, fi.~llowed by an aldehyde dehydrogenase. In the liver the cytosolic alcohol (ethanol) dehydrogenase and the acetaldehyde dehydrogcnase appear most important in ~a-oxidation of ordinary long-chain fatty acids, but ram-characterized alcohol- and aldehyde dehydrogenases in the endoplasmie retie((lure may also participate [145]. ~o-Hydro×yfatty acyI-CoA esters are rapidly oxidized in isolated mitochondria in the presence of carnitine [146]. However, it is unknown to what extent the eJ-hydroxy fatty acids are converted to dicarboxylic acids in the intact cell. The high activity of the alcohol dehydrogenase(s) makes it likely that ~o-hydroxy-acids arc rapidly converted to dicarboxylic acids, at least in liver.

III-B. #.Oxidation (chahl-shortening) of dicarhoxylic acids Isolated mitochondria and peroxisomes can ,6oxidize diearhoxylic acids [146-148]. It hits been assumed therefore that both organellcs arc important in their metabolism [5]. However, several observations suggest that dicarboxylic acids are ,8-oxidized almt~st exclusively in the peroxisomes in the intact cell: ( 1) The apparent Kin-values for ,8-oxidation of dicarhoxylic acids by isolated mitochondria are 15-40-fold higher than K~ values found with isolated peroxisomes. The specific rates of oxidation in mitochondria are only 1/5 to 1/111 of those in pcroxisomes [147]; (21 earn(tint-dependent /3-oxidation of dicarboxylic acids has been demonstrated in isolated mitochondria [147]. However, the formation oL e.g., hexadecanedioylcarnitine monoester from the corresponding CoA-cster and its ,8oxidation by isolated mitochondria, is very slow compared to the rates obtained with, e.g., palmitate [148]. In isolated hcpatocytes the oxidation of hmg dicarboxylie acids, e,g., dodecanedioic acid. is relatively rapid compared to the ,8-oxidation of palm((ate. { 4 )De-

canoylcarnitinc, an inhibitor of earn(((no-dependent fatty acid oxidation, does not inhibit the '8-oxidation of dodccancdioic acid. while '8-oxidation of palm(tare is strongly inhibited [ 149]. Thus, the '8-oxidatitm of dicarboxybc acids seems to be rapid and carnitinc-independent, in the intact liver cell; (3) in intact rat hepatocytcs medium- and long-chain diearboxylic acids stimulate t~rmation of H202 (a prcxtuct of the peroxisomal acyI-CoA oxidasc), while no ketone bodies are formed [1511.92]. The latter are formed when fatty acids are #-oxidized in mitochondrim (4) N M R studies on the metabolism of I~C-labelled diearbo~lic acids in the liver of intact rats havc shown that no intra-mitochondrial [~~C]accty[-CoA is formed, cxcluding utittx:hondrial '8-oxidation [151]; and (5) recently wc hax,e found that '8-oxidation of dicarboxylic acids in isolated hepatocytes gives rise to free acetate, while no ketone bodies are formed. Als~l, the lormation of I¥ce ~lcetate corrchttcs closely with the formation of hydrogen peroxide [92]. The generation of flee acetate has previously been shov, n to be mainly an extra-mitochondrial process [94]. Recent thlx-studies have demonstrated that an extra-mitochondrial source of acetyI-CoA mum function in isolated hepatocytes lo explain rates of acetate production observed in the presence of, e.g., ( l-hydroxycitratc [152]. Presumably acctyI-CoA formed in the peroxisomes is hydrolysed by the cytosolic ATPregulated acctyI-CoA hydrolasc [95], or by acetyI-CoA hydrolasc present in the peroxisomes (Htwik and Osmundsen, unpublished data). This explains the lack of ketogcncsis, in spite o[ ample hepatic capacity for '8-oxidation of hmg-ehain dicarlx~xylic acids [151),92]. Altogether these observations indicate that diearboxylic acids are a-oxidized mainly, if not exclusively, in pcroxisomes in the intact eelh Kinetic and immunological studies have shown that the diearhoxylic acids are oxidized by the same acyI-CoA oxidasc as long chain fatty acids [153]. These results raise the question aboul the origin of the odd- and even-numbered mcdum-chain dicarboxylic acids excreted by patients with Zcllwcger syndrome [154.155]. One possibility is that the lack of pcroxisomes cause such extensive intra-ccllular accumulalion of hmg-chain diearboxylylCoA esters that their mitochondrial '8-oxidation mmethcless is facilitated, in spite of Ihcir slow conversion into acylcarnitincs [148]. The dicarboxylic acids with an odd number of carbons may be formed by ~-oxidation [ 156].

II1-('. End pr~Muct.~ of dic'arbc~rylic ac'id tzridatiott In man, ingested short-chain dicarboxylic acids arc rapidly excreted in urine and only it smalI fraction ~s oxidized to CO 2 [I 57]. When subcrie acid is injected to rats, it is rapidly excreted in the urine and again only a

15(1 small fractiou is oxidized to CO~ [158], T h e s e results arc explained by a poor activation of short-chain dicarboxylic acids to their CoA esters [159] and by a rapid. active, excretion of short-chain dicarboxyIic acids by the kidneys [158]. Suberic acid is also poorly metabolized by isolaled hepatoeytes [149]. With long-chain dicarboxylic acids a different picture is obtained. W e have already mcmioned that free acetate seems to he the main product of .8-oxidation in liver [92]. W h e n d,~decanedioic acid is given to rats only sholter dicarboxylic acids and no unchanged dodccanedioic acid, are found in urine [158,160]. In normal rats the sum of shortened dicarboxylic acids in urine amounted to about 311 molar % of the injected dose. In rats treated with clofibrate only 5 % of ingested dodccancdioic acid was recovered in urine as shortened products. This implies that most of the injected dicarboxylic acid was chain-shortened to succihate. Since elofibrate has a strong inducing effect on pcroxisomal B-oxldatlon [161], the abated excretion of dicarboxylic ackls in urine indicates that the peroxisprees are responsible Ior B-oxidation of both longchain- and short-chain dicurboxylic acids.

2 ~ ?

£

In man a greater fraction of ingested dicarboxylic acids is recovered as shorter dicarboxyllc acids in urine than in the rat [162], suggesting species-differences as regards metabolism of dicarboxylic acids.

III-D. Ghlconeogenesis from fauy acids? The complete breakdown of long-chain fatty acids by