186 TIPS - May 1986

Hypoglycin, the famous toxin of the unripe Jamaicanackee fruit H. S. A. Sherratt

Unripe fruit of the Jamaican ackee tree, Blighia sapida, contains hypoglycin, L-(methylenecyclopropyl)alanine), a hypoglycaemic toxin which has caused an estimated 5000 deaths. Methylenecyclopropylacetyl-CoA, a metabolite of hypoglycin, inactivates several, but not all, flavoprotein acyl-CoA dehydro- genases causing widespread disturbances of the oxidation of fatty acids and several amino acids leading to secondary inhibition of gluconeogenesis. Despite intensive study for three decades the mechanism of hypoglycin poisoning is still only partly understood. H. S. A. Sherratt reviews these complex effects which have implications for toxicology and the study of metabolic regulation and of many inborn errors of metabolism.

small animals, hypothermia with body temperatures as low as 25C (when the ambient temperature is 20C), are features of hypoglycin poisoning.

Hypoglycin is not readily avail- able and has not been synthesized in useful quantities. It also occurs in a few other plants of the Acer family, including seeds of the common sycamore Acer pseudo- platinatus. Since its isolation there has been a sustained interest in hypoglycin, and this review will mainly describe work during the last 15 years. Space does not allow mention of all workers who have contributed and further details are given elsewhere (Refs 3-7).

Primary metabolic effects: inhibition of fatty acid oxidation

Hypoglycin causes profound

The ackee tree was imported from West Africa to Jamaica where it was named Blighia sapida, this introduction being wrongly attri- buted to Captain Bligh of Bounty fame. Its fruit are about the size of an orange with a thick rind containing seeds resembling chestnuts (Fig. 1). The seeds are surrounded by a fleshy arillus which is a regular feature of the Jamaican diet. Eating unripe ackee fruit often causes vomiting sick- ness with the onset of symptoms about 2-3 h later. Severe hypo- glycaemia and vomiting, depletion of liver glycogen, accumulation of fat in the liver, increased plasma free fatty acid concentrations and death often occur. More recently, massive dicarboxylic aciduria and some organic acidaemia has been observed 1. Between 1886 and 1950 vomiting sickness in Jamaica caus- ed approximately 5000 deaths 2. Although the toxic nature of unripe arrilli is now recognized in Jamaica there are still occasional cases of poisoning.

Two toxic compounds, hypo- glycins A and B, were isolated from ackee seeds by Hassal and Reyle in 1954. Hypoglycin (hypoglycin A) is an unusual amino acid, L- (methyl- enecyclopropyl)alanine, and hypo- glycin B is 7-glutamylhypoglycin

(Fig. 2). Hypoglycin (either given orally or parenterally) causes hypo- glycaemia in fasted animals in doses ranging from 10-150 mg kg -1 body weight in guinea-pigs, rab- bits, dogs, cats, rats and mice with decreasing sensitivity. The toxic dose in man is unknown but may be much less on a per/kg basis. Behavioural depression, and, in

disturbances of the oxidation of fatty acids, some amino acids, and inhibition of gluconeogenesis. Pioneering work by yon Holt established that hypoglycin is converted in vivo to methylene- cyclopropylpyruvate (MCPP) which is then oxidatively decarboxylated to methylenecyclopropylacetate (MCPA) as its CoA ester (MCPA- CoA). MCPA-CoA may be rever- sibly converted to free MCPA or

H. S. A. Sherratt is a reader in biochemical pharmacology at the Department of Pharma- cological Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, UK.

Fig. 1. Ripe ackee fruit, Blighia sapida. The arillus surrounding the seeds is cooked and eaten, for example as 'ackee and salt fish; When unripe the ari/li contain dangerous amounts of hypoglycin and hypoglycin B. During ripening these compounds are translocated into the seeds.

1986, Elsevier Science Pub l i she~ B.V., Amsterdam 0165 - 6147186/$02.00

TIPS - May 1986

conjugated with glycine to MCPA- glycine (Fig. 3). The metabolic disturbances are consequences of the inactivation by MCPA-CoA in the mitochondrial matrix of the general acyl-CoA and butyryl-CoA dehydrogenases involved in ~- oxidation, and the isovaleryl-CoA, 2-methylbutyryl-CoA and glutaryl- CoA dehydrogenases involved in amino acid catabolism 8-1. MCPA- CoA is a suicide inhibitor of the general-acyl-CoA dehydrogen- ase m (and presumably of the other enzymes). An early step in the 2,3- dehydrogenation of acyl-CoA es- ters catalysed by this enzyme is removal of a proton from the 2- position. MCPA-CoA gives an active species which combines with the flavinadenine dinucleo- tide prosthetic groups of the en- zyme to form modified flavin derivatives 1. In addition, mito- chondrial NADPH-dependent 4- enoyl-CoA reductase which is not a flavoprotein may be inhibited. Hypoglycin and MCPP have other, apparently less important effects.

It was shown in 1958 that several carboxylic acids with the structure

CH2=C.C.C.COOH are hypogly- caemic (Fig. 2). Of these, pent-4- enoate has been extensively in- vestigated because it was once believed to be an analogue of MCPA. However its mechanism of action differs from that of hypo- glycin (see Refs 6, 7).

The complete mitochondrial oxi- dation of long-chain fatty acids requires the successive action of palmitoyl-CoA, general acyl-CoA and butyryl-CoA dehydrogenases as the carbon chain is shortened by successive loss of acetyl units. MCPA-CoA inactivates the latter two enzymes so that long-chain acyl-CoA esters are only oxidized as far as butyryl-CoA. Butyryl- CoA accumulated is then con- verted to butyrate and CoASH by a high Km acyl-CoA hydrolase. This partial oxidation by inhibited liver mitochondria is clearly shown with conditions where the acetyl groups formed by ~-oxidation are quantitatively converted to aceto- acetate 9 (Fig. 4). The maximum possible rate of generation of acetyl units from fatty acids is also decreased by about half. This

187

limited rate is largely set by the rate of recycling of CoASH (neces- sary for other stages of ~-oxi- dation) and by the activity of palmitoyl-CoA dehydrogenase. Following administration of hypo- glycaemic doses of hypoglycin to rats, inhibition of butyryl-CoA dehydrogenase is virtually com- plete in liver and fatty acid oxidation is impaired in other tissues 6. Although acyl-CoA/ CoASH ratios are increased in liver, 40-50% of the total CoA remains in the free form (CoASH) (P. P. Koundakjian, unpublished results). Free butyrate is found in plasma during hypoglycin poison- ing (up to 0.7 mM in rats and 3 mM in micen).

Dicarboxylic aciduria Hypoglycin-poisoned rats ex-

crete large amounts of glutaric acid 1. Glutaryl-CoA is an inter- mediate in the degradation of tryptophan and lysine. Inhibition of glutaryl-CoA dehydrogenase by MCPA-CoA, followed by deacyl- ation of accumulated glutaryl-CoA yields glutarate. Rats also excrete

hypoglycin (L-(methylenecyclopropyl)alanine) CH 2

/ \ * CH2=C CH.CH 2.CH(NH2).COOH

hypoglycin B (%'-glutamylhypoglycin)

methylenecyclopropylpyruvate (MCPP)

CH2 / CHz~C ~H.CH2.CH.COOH

I NH.CO.CH2.CH2.CH(NH2).COOH

CHa

CH2=C / * \

CH.CH 2 .CO.CO 2

methylenecyclopropylacetate (MCPA) CH z

CH2=C / ~ ~H.CH..CO~-

3-methylenecyclobutanecarboxylate H2-~H.CO2-

CHz=CH--CH 2

pent-4-enoate CH2=CH.CH~.CH2.CO ~-

Fig. 2. Hypoglycin and some related compounds. Hypoglycin is an L-amino acid, C~ is asymmetric, and the structure is thought to be (+)- (2S: 4S)-2-amino-4,5-methanohexo5-enoic acid. MCPP and MCPA (as its CoA-estor) are derived from hypoglycin in vivo (see Fig. 3). Most preparations of hypoglycin contain up to 20% of leucine, pure hypoglycin is obtained by hydrolysis of hypoglycin B first separated from leucine by ion-exchange chromatography. 3-Methylenecyciobutanecarboxylate is a synthetic analogue of hypoglycin. All these compounds contain the grouping CH2 = CH C.C.COOH. MCPA-CoA (and possibly 3-methylenecyciobutanecarbonyI-CoA) appears to undergo 2-deprotonization by some acyI-CoA dehydrogenases to form suicide inhibitors. Pent-4-enoyI-CoA formed in vivo is a substrato for butyryl- CoA dehydrogenase without inhibiting it, and is partly metabolized to 3-oxopent-4-enoyI-CoA which inhibits mitochondrial ~-oxidation by inactivating the 3-oxoacyI-CoA thiolases.

188 TIPS - May 1986

hypoglycin _ 2-oxoglutarate

glutamate ~'- -- MCPP . MCPA-CoA MCPA

S ~NADH+H+/ NAD ~ AMP + PPi ~ " \ CoASH + ATP

~ -glycine CoASH

MCPA-glycine

Fig. 3. Metabolism of hypoglycin. MCPA-CoA is formed in mitochond/~a by the enzymes which catalyse the degradation of branched-chain amino acids. MCPA-CoA maybe hydrolysed to free MCPA which is partly reconverted to MCPA-CoA, or partly conjugated with glycine. The enzymes catalysing these reactions are: I. Leucine-2-oxoglutarate aminotransferase in the cell cytosol 2. Branched-chain 2-oxoacid dehydrogenase in the mitochondrial matrix 3. Glycine N-acylase in the matrix 4. Medium-chain acyI-CoA hydrolase in the matrix 5. Butyryl- CoA synthase in the mattYx.

several other medium-chain di- carboxylic acids including some saturated sebacic (C10) and suberic (C8) acids, and larger amounts of unsaturated acids particularly 4- cis-decene-l,lO-dioic and 4-cis- octene-l,8-dioic acids. Suberic and sebacic acids are formed when hepatic mitochondrial B-oxidation is overloaded in diabetic keto- acidosis, or is impaired in some inborn errors of metabolism such

as general acyl-CoA-dehydrogen- ase deficiency, or after admini- stration of drugs which inhibit mitochondrial B-oxidation (see Ref. 12). With such conditions some excess long-chain fatty acids supplied to the liver are converted to long-chain dicarboxylic acids by c0-oxidation by mixed function oxidases in the endoplasmic re- ticulum. Mono-CoA esters of these dicarboxylic acids then formed

The relation between gluconeogenesis and fatty acid oxidation

0

glucose

L I

glycemld.ehyi.3"phosphate

1, 3-diphosphoglycerate

phosph~no p~mvate

NAD +

t J3-oxidation J

cytoso]

rm'toch ondn'a/raatr/x

outside the mitochondrial matrix are substrates for partial B-oxi- dation in the peroxisomes where they are chain-shortened to med- ium-chain mono-CoA esters. These are then deacylated to non- toxic medium-chain dicarboxylic acids.

B-Oxidation in peroxisomes dif- fers from that in mitochondria and the 2,3-dehydrogenation steps are catalysed by the flavoprotein acyl- CoA oxidase with the concomitant formation of H202. Acyl-CoA oxi- dase does not appear to be inhibited, although it is not known whether this enzyme is resistant to inhibit ion, or whether any MCPA- CoA occurs outside the mito- chondrial matrix. Experiments with 14C-labelled fatty acids have shown that 4-cis-decene-l,lO-dioic and 4-cis-octene-l,8-dioic acids are derived from the polyunsatur- ated fatty acid, linolenate, but not from stearate or oleate 13. Their formation may be explained as a consequence of inhibit ion of an auxiliary enzyme of B-oxidation necessary for the degradation of polyunsaturated fatty acids with double bonds in the 9,10 and 12,13 positions, probably the NADPH- dependent 4-enoyl-CoA reduc- tase 13.

Inhibit ion of branched-chain fatty acid oxidat ion

The branched-chain amino acids leucine, isoleucine and va- line are transaminated to the cor- responding 2-oxoacids which are then oxidatively decarboxylated to isovaleryl-CoA, 2-methylbutyryl-

TIPS - May 1986

CoA and isobutyryl-CoA respec- tively. Their further catabolism involves 2,3-dehydrogenation by the specific isovaleryl-CoA or 2- methylbutyryl-CoA dehydrogen- ases. MCPA-CoA inactivates these dehydrogenases and some of the branched-chain CoA-esters which accumulate are deacylated by acyl- CoA hydrolase causing a severe organic acidaemia in rats (isovaler- ate plus 2-methylbutyrate up to 5 mM) I'II.

Inhibition of gluconeogenesis Fatty acid oxidation may stimu-

late gluconeogenesis by providing ATP, NADH where necessary, and acetyl-CoA which is an obligatory aUosteric effector for pyruvate carboxylase (Ref. 14). This enzyme is required for glucose synthesis from pyruvate and lactate, and alanine which must first be con- verted to pyruvate. Hypoglycin (after a lag period) strongly in- hibits the synthesis of glucose from pyruvate, lactate and alanine, but not significantly from glycerol, dihydroxyacetone or fructose, in isolated rat hepatocytes 14,zs, and this inhibition might be explained by inhibition of fatty acid oxi- dation. However, gluconeogenesis is not dependent on fatty acid oxidation when alternative sub- strates are available whose oxi- dation also provides ATP, NADH and acetyl-CoA. These substrates include pyruvate and lactate, and the carbon skeletons of some amino acids, which can be parti- tioned between oxidation and glucose synthesis. MCPA-CoA formed from hypoglycin in the mitochondria causes a secondary accumulation of isovaleryl-CoA, 2- methylbutyryl-CoA, glutaryl-CoA and butyryl-CoA. Elevated con- centrations of these esters occur to levels determined by their rates of formation and of removal by deacylation and by conjugation with glycine and carnitine. These acyl-CoA esters competitively in- hibit the activation of pyruvate carboxylase by acetyl-CoA and this is probably the major mechan- ism of inhibition of gluconeo- genesis 14.

Mechanism of whole-body metabolic disturbances caused by hypoglycin

It was suggested many years ago that when fatty acid oxidation is

impaired by hypoglycin, increased utilization of glucose and decreas- ed gluconeogenesis lead to hypo- glycaemia when hepatic glycogen reserves are exhausted 3"4. Poison- ing is also marked by ketosis with a more oxidzzcd state of the plasma acetoacetate-3-hydroxy- butyrate ratio. Since hepatic keto- genesis is thought to be decreased this may be due to greater inhibi- tion of the peripheral utilization of ketone bodies. The physiological stress of hypoglycaemia mobilizes

189

free fatty acids from adipose tissue. More long-chain acyl-CoA esters are formed in the liver than can be oxidized and some are used for triacylglycerol synthesis with ac- cumulation of neutral hepatic lipid. Whole body energy production may be impaired because of limited fatty acid, ketone body oxidation, and limited availability of glucose. However, normal ATP concentra- tions are maintained in liver during hypoglycin poisoning in rats (Ref. 16 and H. S. A. Sherratt, unpub-

mitochondria (10 mg of protein)

< 430

oxygen uptake T

400 ng-atom of 0 /~

1 2min

palmitoyl-carnitine

(79)

~P~ ;palmitoyl-carniline

Fig. 4. Experiment illustrating partial inhibition of mitochondrial ~-oxidatlon (from Ref. 9). A rat liver mitochondrial fraction, 10 i~M paimitoyl-carnitine and 1.0 mM- MCPA were added where indicated to 3.0 ml of medium at 30C containing 100 mM KCI, 2.5 mM phosphate, 5 mM MgCI2, 1.0 mM EDTA, 2.0 mM ADP (to maintain the maximum rate of electron transport), tO mM malonata (to prevent oxidation of acetyl groups by the citrate cycle), 9 mg of defatted bowne serum albumin and 20 mM 3 (morpholino) propanesulphonate (buffer), pH 7.2. Oxygen uptake was recorded potarographically. Rates of oxygen uptake (ngatom O/mg of protein) are given in parenthesas, and the amount of oxygen consumed by vertical arrows. PalmitoyI-CoA is germratdd in the mitochondrial matrix from added palmitoyl-carnitine by the action of the camitine palmitoyltransferasas in the inner membrane. In control incubations palmitoyI-CoA is quantitatively oxidized to aceto- acetate

PalmitoyI-CoA + 702 -* 4Acetoacetate + CoASH

and endogenous respiration is largely suppressed during paimitoyl-camitine oxidation. After incubation for 3 min with MCPA to allow formation of MCPA-CoA in the math'x, both the rate and extent of oxidation of palmitoyI-CoA are decreased:

PalmitoyI-CoA + 602--~ 3Acetoacetate + Butyrate + CoASH

(see text). Similar results were obtained with even-chain acyl-carnitine asters (Ca to C14). If MCPA was incubated with uncoupled mitochondria (in the presence of 0.2 pM trifluormelhoxycarbonylcyanidephenylhydrazone, lOmM arsenate and 0.6mg of valinomycin and absence of ADP) no inhibition was found of acyl-carnitine oxidation indicating that prior A TP-dependent conversion of MCPA to MCPA-CoA is necessary. Similar inhibitions are found in mitochondria prepared from rats which have been injected with hypoglycin ~ ~.

190 TIPS - May 1986

Processes determining blood glucose concentrations

Net rate of [ inputor [ output of = glucose

1 Rateof absorption of dietary glucose

[ 2 Rate of ] 3 Rateof breakdown of hepatic andrenal glycogen

4 Rateof util ization

- of glucose

5 Rateof 6 Rateof glycogen ] [ excretion

- synthesis in] - ofglucose l iverand I I

other [ tissues ]

Constant concentrations of blood glucose are maintained when the rates of input and output into the circulation are equal. Measurements of changes of glucose concentration alone do not give the flux through the glucose pool (see Refs 14, 17). (1) Zero during fasting; (2) variable rate, occurs mainly in the liver but with some contribution from the kidneys, stimulated by glucagon and adrenaline; (3) free glucose is only derived from glycogen in liver and kidney, stimulated by glucagon and adrenaline; (4) mainly converted to CO2 and lactate; lactate formed in extrahepatic tissues is used as precursor for gluconeogenesis (Cori cycle). Glucose uptake by some tissues (particularly muscle and adipose tissue) is increased by insulin. Free fatty acids and ketone bodies compete for oxidation and decrease glucose oxidation; (5) occurs in both liver and many other tissues, stimulated by insulin. Total extrahepatic glycogen reserves are greater than in liver; (6) usually only occurs in pathological conditions (diabetes) when blood glucose concentrations exceed the renal threshold (about 10 mM). The rates of these processes, most of which are under hormonal control, vary widely and change rapidly after a meal or during and after exercise, and the liver can switch rapidly between glucose uptake or output. The system is normally self-adjusting and in the absence of drugs or disease maintains glycaemia within desirable limits. A normal fasting blood glucose concentration of about 5 mM represents about 15 g of glucose in the extracellular fluid of an adult human.

The Scheme shows the requirement for acetyl-CoA, NADH and ATP when pyruvate is converted to glucose

2Pyruvate + 4ATP + 2GTP + 2(NADH + H +) ---, Glucose + 4ADP + 2GDP + 6Pi + 2NAD +

Several steps and the control of the activities of regulatory enzymes by hormones are not shown for clarity.

[3-Oxidation in the mitochondrial matrix provides acetyl-CoA required to activate pyruvate carboxylase, and reducing equivalents for export to the cytosol to reduce 1,3-diphosphoglycerate. B-Oxidation and oxi- dation of some of the acetyl-CoA formed by the citrate cycle also provide NADH and ATP necessary for gluconeogenesis. The oxidation of pyruvate and the carbon skeletons of some amino acids may also provide acetyl-CoA, NADH and ATP, although fatty acid oxidation usually appears to be most important. Details of the 'shuttles' transferring reducing equivalents, ATP and oxaloacetate across the mitochrondrial inner membrane are omitted. In hypoglycin-poisoning, gluconeogenesis is impaired at the stage of the conversion of pyruvate to oxaloacetate by pyruvate carboxylase. The obligatory activation of pyruvate carboxylase by acetyl-CoA is competitively inhibited by unusual medium-chain acyl-CoA esters which accumu- late in the matrix. A decreased availability of cytosolic NADH may also contribute to the inhibition, although ATP concentrations appear to be maintained.

lished observations). ATP has not been determined in other tissues so it is not known if behavioural depression and hypothermia are caused by impaired supply of metabolic energy or by direct pharmacologial effects of accumu- lated organic acids on the CNS, or by both. Behavioural depression may decrease util ization of ATP so that normal concentrations might be maintained even if the absolute rate of oxidative phosphorylation is impaired.

The above interpretation of the effects of hypoglycin, although plausible, is based on circum- stantial evidence. Early attempts to measure whole body metabolism by administering ~4C-labelled fatty acids or glucose to poisoned and control animals and only measur- ing 14CO2 exhaled gave no useful information. The pool sizes of substrates available to tissues are changed by hypoglycin, and fur- ther uncei'tainties are introduced

by hypothermia in small animals (see Ref. 6). An attempt was made .to get quantitative information about glucose metabolism by a kinetic study of the rate of decline of the specific radioactivity of blood glucose following a bolus i.v. dose of [U-14C,2-3H]glucose 16 h after administration of hypo- glycin (100 mg kg -1 body-wt) to fasted rats (body temperatures were artificially maintained) 17. Re- sults indicated that gluconeo- genesis was strongly inhibited since recycling of glucose through the Cori cycle (resynthesis of glu- cose in the liver from lactate formed from glucose in peripheral tissues by glycolysis) was virtually abol- ished and the whole-body content of free glucose was decreased by 70%. The rate of util ization of glucose was decreased by 70%, rather than increased as expected if fatty acid oxidation is impaired. It was concluded that hypoglycaemia resulted from an even greater

inhibit ion of the rate of gluconeo- genesis than of glucose utiliza- tion ~7.

It was also deduced that hepatic glucose 6-phosphatase activity is strongly inhibited in vivo 14"17. It is a mystery how poisoned rats maintain even low concentrations of blood glucose (2 mM) in the apparent absence of glucose 6- phosphatase activity 14 since this enzyme is thought to release glucose into the blood from glu- cose 6-phosphate formed by gluco- neogenesis or by glycogen break- down.

Hypoglycin poisoning partly resembles glutaric aciduria Type II

Hypoglycin poisoning has many similarities to some inborn errors of metabolism involving defective enzymes whose substrates are acyl- CoA esters 7'12. Rats given hypo- glycin provide the best model we have for human hereditary glutaric

TIPS - May 1986

aciduria Type II where there is a defect in the transfer of reducing equivalents from all the mito- chondrial acyl-CoA dehydrogen- ases, perhaps at the level of the electron transferring flavoprotein (ETP) or ETF dehydrogenase, to the respiratory chain 7. This condition is characterized by excretion of isovaleric, 2-methylbutyric, iso- butyric, butyric, some dicarboxylic acids and isovaleryl-glycine. How- ever, this resemblance is only approximate since by contrast with hypoglycin-poisoning where palmitoyl-CoA dehydrogenase re- mains active, there is no hyper- ketonaemia.

Ant idotes to hypoglycin po ison ing It is remarkable that five different

compounds have each been re- ported to decrease the toxicity of hypoglycin by different mechan- isms.

Glucose Early hopes that hypo- glycin might be useful to treat diabetes were soon abandoned because of its toxicity. Conversely, administration of glucose can re- store normal blood glucose con- centrations but does not always prevent death s .

Ribof lav in Administration of ribo- flavin partly protects against chronic hypoglycin poisoning 3. Extra riboflavin may facilitate replacement of chemically altered flavin prosthetic groups of in- hibited enzymes.

Carn i t ine Carnitine is an essential cofactor for the movement of many acyl-groups across the inner mito- chondrial membrane and is of great current interest for the treatment of some metabolic dis- seases TM. Bressler claimed in 1968 that administration of carnitine decreased hypoglycin toxicity in mice, which was an early thera- peutic use of carnitine. However, we tried unsuccessfully several times to repeat this 6.

Glyc ine Tanaka showed that several branched-chain acyl-CoA esters that are poor substrates for the mitochondrial carnitine acyl- transferases are conjugated with glycine and MCPA-glycine, iso- valeryl-glycine, and some butyryl- glycine are excreted in hypoglycin poisoning. The reaction between glycine and acyl-CoA esters in the

mitochondrial matrix is catalysed by glycine N-acylase which has a low affinity for glycine (Kin = 3.3 mM) and tissue concentrations of glycine are not saturating. Ad- ministration of large amounts of glycine increases the rates of con- jugation with glycine ~9. This limits the toxic effects of hypoglycin in rats and minimizes the metabolic disturbances, both decreasing en- zyme inhibition by lowering tissue concentrations of MCPA-CoA, and by decreasing isovaleric acidaemia by increased conversion of any isovaleryl-CoA and still formed to its inert glycine conjugate.

Clof ibrate Clofibrate (ethyl p- chlorophenoxyisobutyrate) is an hypolipidaemic drug. When given chronically in the diet (about 0.5%) it causes a massive increase in the number of peroxisomes and of peroxisomal [~-oxidation in the livers of rodents such as rats and mice, but not in most other spe- cies 2. Clofibrate also increases mitochondrial B-oxidation in both rodent and non-rodent species. Hypoglycin causes remarkable ultra- structural changes in the livers of rats including large increases in mitochondrial volume 5. There was also a 70% decrease in the number of peroxisomes 2. Hypoglycin was therefore given to rats that had been fed a diet containing 0.5% clofibrate for 30 days. This caused dramatic protection against the toxic, hypoglycaemic and hypo- thermic effects and the animals appeared normal. Coupled liver mitochondria from clofibrate-fed rats given hypoglycin oxidized acyl-carnitine esters completely by contrast with rats given hypoglycin alone 2. Curiously, uncoupled mitochondria from both groups only oxidized acyl-carnitine esters as far as butyrate 2. Further, butyryl-CoA dehydrogenase which was inhibited by more than 90% in hypoglycin-treated animals was only inhibited by about 70% after clofibrate feeding. This suggests that clofibrate may induce an additional butyryl-CoA dehydro- genase which is only active in coupled mitochondria, perhaps as a phosphorylated enzyme, and which is not inactivated by MCPA- CoA.

[] [] []

Hypoglycin poisoning iUus- trates the complex metabolic dis-

191

turbances that can be caused by a few primary enzyme inhibitions. However, these disturbances are still only partly understood after three decades of investigation. There have been several mis- understandings in interpretation 6 and some unexpected turns in the story 6"7"14'20. There is still no quan- titative information on the pertur- bation of fatty acid, ketone body or amino acid oxidation nor is it known whether normal ATP con- centrations can be maintained in extrahepatic tissues during hypo- glycin poisoning. The abnormal accumulation of acyl-CoA esters in the mitochondrial matrix may contribute more to the metabolic disturbances than simple limi- tation of fatty acid oxidation. Many problems remain and their eluci- dation will continue to give valu- able information about both nor- mal and abnormal metabolism.

References 1 Tanaka, K. (1972) J. Biol. Chem. 247, 7465--

7478 2 Mitchell, J. C. (1974) Diabetes 23, 919-920 3 Sherratt, H. S. A. (1969) Br. Med. Bull. 25,

250-255 4 Stewart, G. H. and Hanley, T. (1969) in

Oral Hypoglycaemic Agents (Campbell, G. D., ed.), pp. 347-407, Academic Press

5 Kean, E. A. (ed.) (1974) A Symposium on Hypoglycin, Academic Press

6 Sherratt, H. S. A. and Osmundsen, H. (1976) Biochem. Pharmacol. 25, 743-750

7 Sherratt, H. S. A., Bartlett, K. and Turnbull, D. M. (1985) in The Pharmaco- logical Effect of Lipids II (Kabara, J.J., ed.), pp. 247-262, American Oil Chemists' Society

8 Billington, D., Kean, E. A., Osmundsen, H. and Sherratt, H. S. A. (1974) IRCS Res. Biochem. Pharmacol. 2, 1712

90smundsen, H. and Sherratt, H. S. A. (1975) FEBS Lett. 55, 38-41

10 Weng, A., Thorpe, C. and Ghisla, S. (1981) J. Biol. Chem. 256, 9809-9812

11 Billington, D., Osmundsen, H. and Sherratt, H. S. A. (1978) Biochem. Phar- macol. 27, 2891-2900

12 Sherratt, H. S. A. and Veitch, R. K. (1984) J. Inherit. Metab. Dis. Suppl. 2, 7, 52-56

13 Kunau, W. H. and Lauterbach, F. (1978) FEBS Lett. 94, 120-123

14 Sherratt, H. S. A. (1981) in Short-term Regulation of Liver Metabolism (Hue, L. and Van der Werve, G. eds), pp. 199-227, Elsevier

15 Kean, E. A. and Pogson, C. I, (1979) Biochem. J. 182, 789--796

16 Kean, E. A. and Rainford, I.J. (1973) Biochim. Biophys. Acta 297, 31-36

17 Osmundsen, H., Billington, D., Taylor, J.R. and Sherratt, H. S.A. (1978) Bio- chem. J. 170, 337-342

18 Engel, A. G. and Rebouche, C. J. (1984) J. Inherit. Metab. Dis. Suppl 1, 7, 38--43

19 Al-Bassam, S. S. and Sherratt, H. S. A. (1981) Biochem. Pharraacol. 30, 2817-2824

20 Van Hoof, F., Hue, L., Vamecq, J. and Sherratt, H. S. A. (1985) Biochera. J. 229, 387-397