Proceedings of the National Academy of Sciences Vol. 68, No. 1, pp. 20-24, January 1971 Hypoglycin A: A Specific Inhibitor of Isovaleryl CoA Dehydrogenase KAY TANAKA, EDITH M. MILLER, AND KURT J. ISSELBACHER Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Mass. 02114 Communicated by Herman M. Kalckar, August 27, 1970 ABSTRACT Evidence is presented for the specific in vivo and in vitro inhibition of isovaleryl CoA dehydro- genation by hypoglycin A and its derivative, a-keto- methylenecyclopropylpropionic acid. a-Methylbutyryl CoA dehydrogenation was also impaired, but the degree of inhibition was much lower. Isobutyryl CoA dehydro- genation was not inhibited. 4-Pentenoic acid inhibited none of these reactions. It is concluded that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase, contrary to previous assumptions that it is dehydrogenated by green acyl CoA dehydrogenase. The present concept agrees with our previous findings in isovaleric acidemia, a genetic disorder in which a specific defect of isovaleryl CoA dehydrogenase was observed. It was also demonstrated that isovaleric acidemia can be induced in experimental animals by the administration of hypoglycin A. Furthermore, some symptoms of "the vomiting sickness of Jamaica" appear to be due to iso- valeric acid accumulation secondary to the ingestion of hypoglycin A. Isovaleric acidemia is a genetic defect of leucine metabolism (1-4) in which isovaleric acid accumulates in blood in large amounts. Based on the biochemical findings in this hereditary disorder, we proposed that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase (1, 3) and that this enzyme is deficient in patients with isovaleric aci- demia (Fig. 1). It was previously believed that isovaleryl CoA, butyryl CoA, and hexanoyl CoA were dehydrogenated by a single enzyme, acyl CoA dehydrogenase (5, 6). It has subsequently been shown that oxidation of leucine is inhibited by hypoglycin A (7, 8). In contrast, oxidation of valine and isoleucine are not significantly inhibited by this compound (8). However, the specific step in the pathway of leucine metabolism that is inhibited by hypoglycin A has not been precisely identified. Hypoglycin A is an unusual amino acid having the structure of a-aminomethylenecyclopropylpropionic acid (9). It has been extracted from unripe fruits of ackee grown in Jamaica and has been identified as the cause of the "vomiting sickness of Jamaica" (10). Extreme hypoglycemia (11) and depletion of liver glycogen (12) have been observed in the cases of this disease. These same in vivo effects have been observed in ex- perimental animals after injection of hypoglycin A (13, 14). Several groups of investigators subsequently concluded that the hypoglycemia and depletion of glycogen were due to the decreased gluconeogenesis resulting from impairment of long chain fatty acid metabolism (15-17). In these studies, hypoglycin A or its metabolite, methylenecyclopropylacetic acid, and also 4-pentenoic acid (4-PE) were shown to inhibit long-chain fatty acid oxidation; whereas oxidation of straight short chain fatty acids including butyrate, hexanoate, and octanoate was not inhibited (15-18). Based on the data cited above plus the similarity in chemical structure between isovaleric acid and methylenecyclopropyl- acetic acid we postulated that hypoglycin A or its metabolite is a specific inhibitor of isovaleryl CoA dehydrogenase. The present studies have shown this to be the case. MATERIALS AND METHODS Hypoglycin A and a-ketomethylenecyclopropylpropionic acid (KMCPP) were generous gifts from Dr. P. H. Bell, Lederle Laboratories. Fasted male Sprague-Dawley rats weighing about 150 g were used throughout. In vivo experiments Amino acids were given by stomach tube 30 min after intra- muscular injection of the inhibitor. The blood samples were drawn by heart puncture 150 min after injection of the inhibi- tor. Short chain fatty acid analyses were performed by gas- liquid chromatography (GLC) (1). In vitro experiments Incubations were for 3hr at 37°C. The tissues and the incuba- tion medium were homogenized after acidification with 0.5 ml of 2 N H2SO4 and extracted with 20 vol of chloroform-meth- anol 2:1. The filtered extract was separated into two phases by mixing with 0.2 N NaOH (0.2 vol of the extract). Un- labeled carrier was added to the homogenate before extraction. Only the upper alkaline layer, which contains most of the water soluble salts of carboxylic acids and acidic conjugates, was analyzed. The steam-distillable fractions after acid hy- drolysis were designated as total fractions; the fractions steam- distilled without acid hydrolysis were referred to as free acid fractions. Conjugated fractions were calculated from the differ- ence between the total and the free acid fractions. For the total fraction, the residue after evaporation of the upper layer was redissolved in 2 ml of 6 N HCl and hydrolyzed for 17 hr at 1100C. The hydrolysate was alkalinized with 2.5 ml of 5 N NaOH, reacidified with 5 M o-phosphoric acid to pH 2.8-3.0, and then steam-distilled (1).N-isovalerylglycine in the incuba- tion medium was analyzed by GLC (3). Teflon tubing (2 mm X 1 ft) cooled with dry ice-acetone, was used to trap the effluent. 20 Abbreviations: 4-PE, 4-pentenoic acid; KMCPP, a-keto- methylenecyclopropylpropionic acid; iVGly, N-isovalerylglycine. Downloaded from https://www.pnas.org by 27.79.75.39 on February 15, 2023 from IP address 27.79.75.39.
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Hypoglycin A: A Specific Inhibitor of Isovaleryl CoA DehydrogenaseProceedings of the National Academy of Sciences Vol. 68, No. 1, pp. 20-24, January 1971
Hypoglycin A: A Specific Inhibitor of Isovaleryl CoA Dehydrogenase
KAY TANAKA, EDITH M. MILLER, AND KURT J. ISSELBACHER Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Mass. 02114
Communicated by Herman M. Kalckar, August 27, 1970
ABSTRACT Evidence is presented for the specific in vivo and in vitro inhibition of isovaleryl CoA dehydro- genation by hypoglycin A and its derivative, a-keto- methylenecyclopropylpropionic acid. a-Methylbutyryl CoA dehydrogenation was also impaired, but the degree of inhibition was much lower. Isobutyryl CoA dehydro- genation was not inhibited. 4-Pentenoic acid inhibited none of these reactions. It is concluded that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase, contrary to previous assumptions that it is dehydrogenated by green acyl CoA dehydrogenase. The present concept agrees with our previous findings in isovaleric acidemia, a genetic disorder in which a specific defect of isovaleryl CoA dehydrogenase was observed. It was also demonstrated that isovaleric acidemia can be induced in experimental animals by the administration of hypoglycin A. Furthermore, some symptoms of "the vomiting sickness of Jamaica" appear to be due to iso- valeric acid accumulation secondary to the ingestion of hypoglycin A.
Isovaleric acidemia is a genetic defect of leucine metabolism (1-4) in which isovaleric acid accumulates in blood in large amounts. Based on the biochemical findings in this hereditary disorder, we proposed that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase (1, 3) and that this enzyme is deficient in patients with isovaleric aci- demia (Fig. 1). It was previously believed that isovaleryl CoA, butyryl CoA, and hexanoyl CoA were dehydrogenated by a single enzyme, acyl CoA dehydrogenase (5, 6).
It has subsequently been shown that oxidation of leucine is inhibited by hypoglycin A (7, 8). In contrast, oxidation of valine and isoleucine are not significantly inhibited by this compound (8). However, the specific step in the pathway of leucine metabolism that is inhibited by hypoglycin A has not been precisely identified. Hypoglycin A is an unusual amino acid having the structure
of a-aminomethylenecyclopropylpropionic acid (9). It has been extracted from unripe fruits of ackee grown in Jamaica and has been identified as the cause of the "vomiting sickness of Jamaica" (10). Extreme hypoglycemia (11) and depletion of liver glycogen (12) have been observed in the cases of this disease. These same in vivo effects have been observed in ex- perimental animals after injection of hypoglycin A (13, 14). Several groups of investigators subsequently concluded that the hypoglycemia and depletion of glycogen were due to the decreased gluconeogenesis resulting from impairment of long chain fatty acid metabolism (15-17). In these studies,
hypoglycin A or its metabolite, methylenecyclopropylacetic acid, and also 4-pentenoic acid (4-PE) were shown to inhibit long-chain fatty acid oxidation; whereas oxidation of straight short chain fatty acids including butyrate, hexanoate, and octanoate was not inhibited (15-18). Based on the data cited above plus the similarity in chemical
structure between isovaleric acid and methylenecyclopropyl- acetic acid we postulated that hypoglycin A or its metabolite is a specific inhibitor of isovaleryl CoA dehydrogenase. The present studies have shown this to be the case.
MATERIALS AND METHODS
Hypoglycin A and a-ketomethylenecyclopropylpropionic acid (KMCPP) were generous gifts from Dr. P. H. Bell, Lederle Laboratories.
Fasted male Sprague-Dawley rats weighing about 150 g were used throughout. In vivo experiments Amino acids were given by stomach tube 30 min after intra- muscular injection of the inhibitor. The blood samples were drawn by heart puncture 150 min after injection of the inhibi- tor. Short chain fatty acid analyses were performed by gas- liquid chromatography (GLC) (1). In vitro experiments Incubations were for 3 hr at 37°C. The tissues and the incuba- tion medium were homogenized after acidification with 0.5 ml of 2 N H2SO4 and extracted with 20 vol of chloroform-meth- anol 2:1. The filtered extract was separated into two phases by mixing with 0.2 N NaOH (0.2 vol of the extract). Un- labeled carrier was added to the homogenate before extraction. Only the upper alkaline layer, which contains most of the water soluble salts of carboxylic acids and acidic conjugates, was analyzed. The steam-distillable fractions after acid hy- drolysis were designated as total fractions; the fractions steam- distilled without acid hydrolysis were referred to as free acid fractions. Conjugated fractions were calculated from the differ- ence between the total and the free acid fractions. For the total fraction, the residue after evaporation of the upper layer was redissolved in 2 ml of 6 N HCl and hydrolyzed for 17 hr at 1100C. The hydrolysate was alkalinized with 2.5 ml of 5 N NaOH, reacidified with 5 M o-phosphoric acid to pH 2.8-3.0, and then steam-distilled (1).N-isovalerylglycine in the incuba- tion medium was analyzed by GLC (3). Teflon tubing (2 mm X 1 ft) cooled with dry ice-acetone, was used to trap the effluent.
20
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W g(KMCPP Action Site)
,G-METHYLCROTONYL CoA
FIG. 1. Scheme of the site of action of a-ketomethylenecyclopropylpropionic acid in the pathway of leucine oxidation.
RESULTS
In vitro studies
1. Inhibition of Leucine Oxidation and Fractionation of the Products by Steam Distillation. We assumed that if leucine oxidation is inhibited at the step of isovaleryl CoA dehydro- genation, isovaleryl CoA might alternatively be converted to N-isovalerylglycine (iVGly), as has been observed in pa-
tients with isovaleric acidemia (Fig. 1) (3). Therefore, liver slices were incubated in 2 ml of Krebs-Ringer bicarbonate buffer containing 2.8 mM glucose and 0.76 mM D,L-[2-14C]- leucine, with and without glycine. As shown in Table 1, ["4C]- CO2 production was inhibited almost 90% by 0.70 mM KMCPP, a transamination product of hypoglycin A. At the same concentration of 4-PE ["4C]C02 production was
inhibited only 16% (Table 1). In the presence of KMCPP, the increased radioactivity in the conjugated fraction was 60 times the control when the incubation was performed in the presence of 1.5 mM glycine. Radioactivity in the total steam- distillable fraction was also increased in the presence of 1.5
mM glycine. Therefore, further analyses were made only on
the group where glycine was added.
2. Identification of Inhibition Product as N-isovalerylgly- cine. To identify the product of the inhibited reaction as iVGly an aliquot of one incubation medium with inhibitor was analyzed by silicic-acid chromatography (3) (Fig. 2). The radioactivity peak of the product corresponded with that of unlabeled iVGly (assessed by titration). Much less radioactiv- ity was observed around the region of iVGly in the same chro- matographic system from an incubation medium without in- hibitor. Therefore, the fractions of the main radioactive peak for each sample were combined and the product was recrystal- lized several times (Table 2). The iVGly crystals from the in- cubation medium with the inhibitor showed a constant, high specific activity through four recrystallizations. In contrast, almost all of the radioactivity of the control was lost in the mother liquor from the first recrystallization, and the specific activity in the iVGly crystals decreased with each subse- quent recrystallization. In a subsequent experiment, it was
TABLE 1. Incorporation of 14C into carbon dioxide and steam-distiUable fractions from D,1,[2-14C]leucine
[14C]C02 14C Incorporation into steam-distillable fractions Glycine Inhibitor Produced Inhibition Total Free Conjugated [amol] [Mmol] [nmol] % [nmoll
O 0 67.1 4- 1.3 14.8 -- 0.4 11.3 - 0.5 3.5 -- 0.9 0 KMCPP 1.40 8.7* it 0.8 87 43.6t±- 5.0 38.0*-- 0.6 5.1 a 4.4
3.0 0 88.8 ± 0.7 9.8 ±- 0.7 9.0 ±- 0.5 0.8 ±- 0.4 3.0 KMCPP 1.40 10.4* It 0.5 88 66.2*-±7.0 22.2* It 2.0 44.0* 4- 7.8 0 0 63.0 ±- 3.1 0 4-PE 1.40 52.9 ±- 1.1 16
Each incubation mixture contained liver slices (about 100 mg) in 2 ml of Krebs-Ringer bicarbonate buffer containing 5.6 jsmol glucose. The amount of the substrate was 1.5 lsmol (1 1sCi). Results are expressed as the mean ± standard error of four experiments. Numbers marked are significantly different from corresponding controls: * P < 0.001; t P < 0.005, as appraised by Student's t test.
Vol. 68, 1971
100- CONrROL
80- l
MINI] TES
(Left) FIG. 2. Silicic acid chromatography of incubation product after addition of carrier N-isovalerylglycine. Each incubation me-
dium contained liver slices (about 100 mg) in 2 ml of Krebs-Ringer bicarbonate buffer containing 5.6 ,mol of glucose, 3.0 /mol of glycine, and 1.5 /Amol (1 ,.Ci) of D,L-[2-'4Clleucine. Four-tenths of each homogenate was used for the analyses. 20 mg of unlabeled N-isovaleryl-
glycine was added as a carrier to the aliquots of the extracts. Elution solvents were chloroform-methanol mixtures as follows: 100:0, 40
ml; 99: 1, 60 ml; 98: 2, 80 ml; 96.5: 3.5, 60 ml; 95: 5, 40 ml. 10-ml fractions were collected. N-isovalerylglycine was eluted in the 9X: 2 fraction. 1.5 ml of each fraction was used for titration and 4 ml was counted for radioactivity. The small peak of radioactivity in the con-
trol was not N-isovalerylglycine (see recrystallization studies, Table 2).
(Right) FIG. 3. Radio gas chromatography of the incubation products. Aliquots of the same samples as in Table 1 were analyzed. Only the samples that contained 3.0 /Amol of glycine were analyzed. 500 jug of unlabeled N-isovalerylglycine was added as carrier; the peak seen in
the figure is N-isovalery'glycine. Results are expressed as mean ±t standard error of four experiments. The difference in the N-isovaleryl-
glycine fraction was significant (P < 0.005) by Student's t test.
confirmed by GLC, that the iVGly fraction was highly labeled in the four incubations containing KMCPP, whereas radio- activity of this fraction was very low in the control group (Fig. 3).
3. Demonstration of Radioactivity in the Isovaleryl Moiety of iVGly. Incorporation of 14C into the isovaleric acid moiety was confirmed by GLC of short-chain fatty acids after acid hydrolysis. As shown in Table 3, most of the radioactivity was found in the isovaleric-acid fraction in the samples from in- cubations containing KMCPP. Very little label was found in this fraction in the control group. Essentially no activity was found in the j3-methylcrotonic acid fraction in either group.
4. Effect of KMCPP on Oxidation of Isoleucine and Valine. To test the specificity of the KMICPP inhibition, experiments were performed using valine and isoleucine as substrates. At the concentration of 0.7 mM KMCPP, [14CICO2 produc- tion from D,[-14-4C]valine was not significantly inhibited.
TABLE 2. Specific activity of N-isovalerylglycine upon repeated recrystallization
KMCPP (1.4 umol) Control Mother Mother liquor, liquor,
Crystals, total Crystals, total specific radio- specific radio-
Recrystal- activity activity activity activity lization (cpm/mg) (cpm) (cpm/mg) (cpm)
1st 650 2216 18 1477 2nd 636 844 14 64 3rd 631 584 9 31 4th 623 694
One-half of each sample from tubes 7-11 of Fig. 2 were com- bined. 10 mg of iVGly was added before the first recrystallization.
[14CIC02 productionwas inhibited 60% with 2.1 mrNI KTMCPP, but radioactivity did not increase significantly in the steam- distillable fractions. By GLC it was found that '4C-incorpora- tiol) into isobutyric acid was not significantly different from control incubations (Table 3). KMCPP showed inhibitory effects (20%) on isoleucine oxidation at a concentration of 2.1 mMA. Analysis of the incubation by GLC showed accumulation of labeled a-methylbutyrate (Table 3).
TABLE 3. GLC analyses of the incubation products after acid hydrolysis
KMCPP Acetate Isovalerate i3-Methyl- Substrates added crotonate
(.amol) (JAmol) (nmol/100 g tissue)
D,I, [2-14C]- Leu,1.5 0 1.0 ± 0.1 0.1 ± 1.6 0
DALE [2-14C] -
a-Methyl- butyrate Tiglate
Ile,1.5 4.2 2.3 ± 0.2 3.8* ± 0.5 0.3 ± 0
Meth- Isobutyrate acrylate
Val,1.5 0 2.5 ± 0.2 3.3 ± 0.8 3.4 ± 0.5 DIL [4-14C] -
Val,1.5 4.2 1.5 ± 0.2 5.1 ± 0.5 S.6 ± 1.1
Each incubation mixture contained liver slices (about 100 mg) in 2 ml of Krebs-Ringer bicarbonate buffer containing 5.6 Mumol glucose and 3.0 Mmol glycine. Results are mean ± SE of four experiments. Numbers marked are significantly different from corresponding controls: *P < 0.001 (see Table 1).
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TuBE NUMBER
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Isovaleryl Dehydrogenase Inhibition 23
TABLE 4. Effect of amino acids, administered orally with and without inhibitors, on plasma short-chain fatty acids (mg per 100 ml)*
Amino acid Inhibitor Number of (60 mg/100 g) (mg/100 g) experiments Isobutyrate n-Butyrate Isovalerate n-Hexanoate
None None 4 0.06 ±t 0 0.05 i 0 0.05 ±4 0.01 0.15 i 0.03 None Hypoglycin (10 mg) 4 -0.06 ± 0 0.71 ±- 0.18 2.32t i 0.36 0.33 ± 0.11 Leu None 5 0.01 ±0 0.03 ±0 0.10 ± 0.02 0.12 ±00 Leu Hypoglycin (10 mg) 5 0.06 ±t 0.01 1.16 ±t 0.08 19.2t i 1.1 0.44± 0.01 Leu 4-PE(30mg) 4 0.02±0 0.05±0 0.06±0.01 0.14±0 Ile None 4 0.06 ± 0.02 0.06 ±E 0.02 0.23t 4 0.04 0.12 ±00 Ile Hypoglycin (10 mg) 4 0.06 ± 0.01 0.45 ± 0.04 6.59tt ±L 0.58 0.28 ± 0.01 Val None 4 0.26 ± 0.04 0.14 ± 0.03 0.18 ± 0.03 0.26 ± 0.05 Val Hypoglycin 4 0.33 ±00.04 0.47 ±00.05 0.94 ±00.12 0.49 ±00.03
Data on the direct metabolite of each amino acid and n-butyrate are described. No significant change was observed in the amounts of propionate and n-valerate. No f3-methylerotonate, tiglate, or methacrylate was found in significant amounts after amino acid administra- tion.
* Values expressed as mean ±J SE for four experiments (except n = 5 for Leu, none and Leu, hypoglycin experiments). t P < 0.001; t a-methylbutyrate.
Comparison of inhibitory effect of hypoglycin A and 4-PE in vitro was made using uniformly labeled ileucine, L-iso- leucine, and L-valine. Hypoglycin A (1.4 mM) inhibited [14C1C02 production from L-leucine most strongly. i-Isoleu- cine oxidation was inhibited less, while L-valine oxidation was unaffected. In contrast, 4-PE, up to 4.2 mMA, did not inhibit leucine oxidation significantly. In vivo effects of hypoglycin A on metabolism of L-leucine, L-isoleucine, and L-valine
The administration of each amino acid to rats at a dose of 60 mg per 100 g of body weight resulted in no significant increase of the corresponding short-chain fatty acid in the plasma. However, when hypoglycin A was given (10 mg per 100 g of body weight) 30 min before ileucine, the plasma content of a branched-chain pentanoic acid increased up to 200 times that of the control group (Table 4). In a second control group that was given only hypoglycin A, the same branched-chain pentanoic acid increased 45-fold. The product was identified as isovaleric acid on a DOP-PA column (1). When isoleucine was given after hypoglycin A administra-
tion, a branched-chain pentanoic acid increased 30-fold. By the use of a DOP-PA column, this peak was identified as a- methylbutyric acid (1). In both the leucine and isoleucine experiments, there was also some increase in plasma n-butyric acid. Note that in the L-valine-hypoglycin group, in which isobutyric acid should have increased if the same kind of inhibition had occurred, no significant increase in isobutyric acid was found. When these experiments were carried out using 4-PE as an
inhibitor (30 mg per 100 g of body weight), followed by ileu- cine administration, no significant increase in plasma iso- valeric acid was observed (Table 4).
DISCUSSION The acctumulation of N-isovalerylglycine in the medium when liver slices were incubated with [2-'4C]leucine and KMCPP indicated that leucine oxidation was inhibited by KM\CPP at the isovaleryl CoA dehydrogenation step. This inhibition was further confirmed by in vivo experiments showing the accumu- lation of isovaleric acid after the administration of hypoglycin A and leuciine. N-Isovalerylglycine and fl-hydroxyisovaleric
acid were found in urine samples of rats after these treatments (unpublished results); this finding is similar to that in patients with isovaleric acidemia (3, 19). The amount of leucine added as substrate in the in vitro experiments was small. Therefore, most of the isovaleryl CoA that could not be dehydrogenated because of the inhibition was readily conjugated with the ex- cess of glycine (3). In the in vivo experiments, this conjugation system appears to be overloaded with the amount of leucine given. As a result, isovaleryl CoA is hydrolyzed resulting in an accumulation of free isovaleric acid in the blood, again similar to the findings in patients with isovaleric acidemia (1, 3, 19). Although isoleucine oxidation was inhibited at the a-methyl- butyryl CoA dehydrogenation step, the degree of the inhibi- tion was significantly lower. No inhibition of isobutyryl CoA dehydrogenation occurred either in vivo or in vitro. It may be reasonable, therefore, to assume that CoA esters of these branched-chain fatty acids are dehydrogenated by three differ- ent dehydrogenases, and that isovaleryl CoA dehydrogenase is specifically inhibited by hypoglycin A or its metabolites. This conclusion agrees with the findings in isovaleric acidemia where a more specific decrease in isovaleryl CoA dehydro- genase has been observed (1, 3).
Bressler and co-workers have suggested that the inhibition of long chain fatty acid oxidation by methylenecyclopro- pylacetic acid and 4-PE is due to the ability of these inhibitors to form nonmetabolizable esters with CoA and (-)-carnitine (17, 20). As a result, free CoA and (-)-carnitine become less available. Since (-)-carnitine is necessary for the oxidation of long-chain fatty acids in mitochondria, but not for short- chain fatty acid oxidation, this has been proposed as the basis for the specific inhibition of long-chain fatty acid oxidation while short chain fatty acid oxidation remains unaltered (21).
Methvlenecyclopropylacetic acid and 4-PE share two chemical structures in common, namely a five-carbon chain and a vinyl group separated by two carbons from the car- boxyl group. These structures are believed to be necessary for their hypoglycemic action (9) and, also, for their inhibition of long-chain fatty acid oxidation (17, 20). However, there is a striking difference in the structure of these two compounds, namely the presence of the cyclopropane ring. The inhibitory effect of hvpoglycin A on isovaleryl CoA dehydrogenase ap-
Vol. 68, 1971
24 Medical Sciences: Tanaka et al.
pears to be, in part, due to the presence of a cyclopropane ring since 4-PE failed to show this action. In our preliminary ex-
periments, cyclopropane carboxylic acid, which is analogous to isobutyric acid, only slightly inhibited dehydrogenation of isobutyric acid in vivo. It appears, therefore, that the methy- lenecyclopropane group is responsible for the inhibition of isovaleryl CoA dehydrogenation. The inhibition of isovaleryl CoA dehydrogenase by hypo-
glycin A has important clinical implications. One of these is the experimental production of isovaleric acidemia. In addi- tion, the present findings provide a pathophysiological ex-
planation for some of the symptoms of the "vomiting sickness of Jamaica." In this disease, profound hypoglycemia occurs
following the ingestion of unripe ackee fruit; the hypoglycemia has been attributed to ingested hypoglycin A. Some of the patients with this disorder die in spite of glucose infusions. This lack of response has been attributed to irreversible brain damage occurring during the duration of hypoglycemia prior to the glucose infusions (11, 12). However, it would appear
that increased concentrations of isovaleric acid induced by the ingested hypoglycin A probably account for some of these ob- servations. Congenital isovaleric acidemia is sometimes fatal (4). It is also noteworthy that when cats and dogs are given hypoglycin A, they manifest symptoms similar to those…
Hypoglycin A: A Specific Inhibitor of Isovaleryl CoA Dehydrogenase
KAY TANAKA, EDITH M. MILLER, AND KURT J. ISSELBACHER Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Mass. 02114
Communicated by Herman M. Kalckar, August 27, 1970
ABSTRACT Evidence is presented for the specific in vivo and in vitro inhibition of isovaleryl CoA dehydro- genation by hypoglycin A and its derivative, a-keto- methylenecyclopropylpropionic acid. a-Methylbutyryl CoA dehydrogenation was also impaired, but the degree of inhibition was much lower. Isobutyryl CoA dehydro- genation was not inhibited. 4-Pentenoic acid inhibited none of these reactions. It is concluded that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase, contrary to previous assumptions that it is dehydrogenated by green acyl CoA dehydrogenase. The present concept agrees with our previous findings in isovaleric acidemia, a genetic disorder in which a specific defect of isovaleryl CoA dehydrogenase was observed. It was also demonstrated that isovaleric acidemia can be induced in experimental animals by the administration of hypoglycin A. Furthermore, some symptoms of "the vomiting sickness of Jamaica" appear to be due to iso- valeric acid accumulation secondary to the ingestion of hypoglycin A.
Isovaleric acidemia is a genetic defect of leucine metabolism (1-4) in which isovaleric acid accumulates in blood in large amounts. Based on the biochemical findings in this hereditary disorder, we proposed that isovaleryl CoA is dehydrogenated by a specific enzyme, isovaleryl CoA dehydrogenase (1, 3) and that this enzyme is deficient in patients with isovaleric aci- demia (Fig. 1). It was previously believed that isovaleryl CoA, butyryl CoA, and hexanoyl CoA were dehydrogenated by a single enzyme, acyl CoA dehydrogenase (5, 6).
It has subsequently been shown that oxidation of leucine is inhibited by hypoglycin A (7, 8). In contrast, oxidation of valine and isoleucine are not significantly inhibited by this compound (8). However, the specific step in the pathway of leucine metabolism that is inhibited by hypoglycin A has not been precisely identified. Hypoglycin A is an unusual amino acid having the structure
of a-aminomethylenecyclopropylpropionic acid (9). It has been extracted from unripe fruits of ackee grown in Jamaica and has been identified as the cause of the "vomiting sickness of Jamaica" (10). Extreme hypoglycemia (11) and depletion of liver glycogen (12) have been observed in the cases of this disease. These same in vivo effects have been observed in ex- perimental animals after injection of hypoglycin A (13, 14). Several groups of investigators subsequently concluded that the hypoglycemia and depletion of glycogen were due to the decreased gluconeogenesis resulting from impairment of long chain fatty acid metabolism (15-17). In these studies,
hypoglycin A or its metabolite, methylenecyclopropylacetic acid, and also 4-pentenoic acid (4-PE) were shown to inhibit long-chain fatty acid oxidation; whereas oxidation of straight short chain fatty acids including butyrate, hexanoate, and octanoate was not inhibited (15-18). Based on the data cited above plus the similarity in chemical
structure between isovaleric acid and methylenecyclopropyl- acetic acid we postulated that hypoglycin A or its metabolite is a specific inhibitor of isovaleryl CoA dehydrogenase. The present studies have shown this to be the case.
MATERIALS AND METHODS
Hypoglycin A and a-ketomethylenecyclopropylpropionic acid (KMCPP) were generous gifts from Dr. P. H. Bell, Lederle Laboratories.
Fasted male Sprague-Dawley rats weighing about 150 g were used throughout. In vivo experiments Amino acids were given by stomach tube 30 min after intra- muscular injection of the inhibitor. The blood samples were drawn by heart puncture 150 min after injection of the inhibi- tor. Short chain fatty acid analyses were performed by gas- liquid chromatography (GLC) (1). In vitro experiments Incubations were for 3 hr at 37°C. The tissues and the incuba- tion medium were homogenized after acidification with 0.5 ml of 2 N H2SO4 and extracted with 20 vol of chloroform-meth- anol 2:1. The filtered extract was separated into two phases by mixing with 0.2 N NaOH (0.2 vol of the extract). Un- labeled carrier was added to the homogenate before extraction. Only the upper alkaline layer, which contains most of the water soluble salts of carboxylic acids and acidic conjugates, was analyzed. The steam-distillable fractions after acid hy- drolysis were designated as total fractions; the fractions steam- distilled without acid hydrolysis were referred to as free acid fractions. Conjugated fractions were calculated from the differ- ence between the total and the free acid fractions. For the total fraction, the residue after evaporation of the upper layer was redissolved in 2 ml of 6 N HCl and hydrolyzed for 17 hr at 1100C. The hydrolysate was alkalinized with 2.5 ml of 5 N NaOH, reacidified with 5 M o-phosphoric acid to pH 2.8-3.0, and then steam-distilled (1).N-isovalerylglycine in the incuba- tion medium was analyzed by GLC (3). Teflon tubing (2 mm X 1 ft) cooled with dry ice-acetone, was used to trap the effluent.
20
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W g(KMCPP Action Site)
,G-METHYLCROTONYL CoA
FIG. 1. Scheme of the site of action of a-ketomethylenecyclopropylpropionic acid in the pathway of leucine oxidation.
RESULTS
In vitro studies
1. Inhibition of Leucine Oxidation and Fractionation of the Products by Steam Distillation. We assumed that if leucine oxidation is inhibited at the step of isovaleryl CoA dehydro- genation, isovaleryl CoA might alternatively be converted to N-isovalerylglycine (iVGly), as has been observed in pa-
tients with isovaleric acidemia (Fig. 1) (3). Therefore, liver slices were incubated in 2 ml of Krebs-Ringer bicarbonate buffer containing 2.8 mM glucose and 0.76 mM D,L-[2-14C]- leucine, with and without glycine. As shown in Table 1, ["4C]- CO2 production was inhibited almost 90% by 0.70 mM KMCPP, a transamination product of hypoglycin A. At the same concentration of 4-PE ["4C]C02 production was
inhibited only 16% (Table 1). In the presence of KMCPP, the increased radioactivity in the conjugated fraction was 60 times the control when the incubation was performed in the presence of 1.5 mM glycine. Radioactivity in the total steam- distillable fraction was also increased in the presence of 1.5
mM glycine. Therefore, further analyses were made only on
the group where glycine was added.
2. Identification of Inhibition Product as N-isovalerylgly- cine. To identify the product of the inhibited reaction as iVGly an aliquot of one incubation medium with inhibitor was analyzed by silicic-acid chromatography (3) (Fig. 2). The radioactivity peak of the product corresponded with that of unlabeled iVGly (assessed by titration). Much less radioactiv- ity was observed around the region of iVGly in the same chro- matographic system from an incubation medium without in- hibitor. Therefore, the fractions of the main radioactive peak for each sample were combined and the product was recrystal- lized several times (Table 2). The iVGly crystals from the in- cubation medium with the inhibitor showed a constant, high specific activity through four recrystallizations. In contrast, almost all of the radioactivity of the control was lost in the mother liquor from the first recrystallization, and the specific activity in the iVGly crystals decreased with each subse- quent recrystallization. In a subsequent experiment, it was
TABLE 1. Incorporation of 14C into carbon dioxide and steam-distiUable fractions from D,1,[2-14C]leucine
[14C]C02 14C Incorporation into steam-distillable fractions Glycine Inhibitor Produced Inhibition Total Free Conjugated [amol] [Mmol] [nmol] % [nmoll
O 0 67.1 4- 1.3 14.8 -- 0.4 11.3 - 0.5 3.5 -- 0.9 0 KMCPP 1.40 8.7* it 0.8 87 43.6t±- 5.0 38.0*-- 0.6 5.1 a 4.4
3.0 0 88.8 ± 0.7 9.8 ±- 0.7 9.0 ±- 0.5 0.8 ±- 0.4 3.0 KMCPP 1.40 10.4* It 0.5 88 66.2*-±7.0 22.2* It 2.0 44.0* 4- 7.8 0 0 63.0 ±- 3.1 0 4-PE 1.40 52.9 ±- 1.1 16
Each incubation mixture contained liver slices (about 100 mg) in 2 ml of Krebs-Ringer bicarbonate buffer containing 5.6 jsmol glucose. The amount of the substrate was 1.5 lsmol (1 1sCi). Results are expressed as the mean ± standard error of four experiments. Numbers marked are significantly different from corresponding controls: * P < 0.001; t P < 0.005, as appraised by Student's t test.
Vol. 68, 1971
100- CONrROL
80- l
MINI] TES
(Left) FIG. 2. Silicic acid chromatography of incubation product after addition of carrier N-isovalerylglycine. Each incubation me-
dium contained liver slices (about 100 mg) in 2 ml of Krebs-Ringer bicarbonate buffer containing 5.6 ,mol of glucose, 3.0 /mol of glycine, and 1.5 /Amol (1 ,.Ci) of D,L-[2-'4Clleucine. Four-tenths of each homogenate was used for the analyses. 20 mg of unlabeled N-isovaleryl-
glycine was added as a carrier to the aliquots of the extracts. Elution solvents were chloroform-methanol mixtures as follows: 100:0, 40
ml; 99: 1, 60 ml; 98: 2, 80 ml; 96.5: 3.5, 60 ml; 95: 5, 40 ml. 10-ml fractions were collected. N-isovalerylglycine was eluted in the 9X: 2 fraction. 1.5 ml of each fraction was used for titration and 4 ml was counted for radioactivity. The small peak of radioactivity in the con-
trol was not N-isovalerylglycine (see recrystallization studies, Table 2).
(Right) FIG. 3. Radio gas chromatography of the incubation products. Aliquots of the same samples as in Table 1 were analyzed. Only the samples that contained 3.0 /Amol of glycine were analyzed. 500 jug of unlabeled N-isovalerylglycine was added as carrier; the peak seen in
the figure is N-isovalery'glycine. Results are expressed as mean ±t standard error of four experiments. The difference in the N-isovaleryl-
glycine fraction was significant (P < 0.005) by Student's t test.
confirmed by GLC, that the iVGly fraction was highly labeled in the four incubations containing KMCPP, whereas radio- activity of this fraction was very low in the control group (Fig. 3).
3. Demonstration of Radioactivity in the Isovaleryl Moiety of iVGly. Incorporation of 14C into the isovaleric acid moiety was confirmed by GLC of short-chain fatty acids after acid hydrolysis. As shown in Table 3, most of the radioactivity was found in the isovaleric-acid fraction in the samples from in- cubations containing KMCPP. Very little label was found in this fraction in the control group. Essentially no activity was found in the j3-methylcrotonic acid fraction in either group.
4. Effect of KMCPP on Oxidation of Isoleucine and Valine. To test the specificity of the KMICPP inhibition, experiments were performed using valine and isoleucine as substrates. At the concentration of 0.7 mM KMCPP, [14CICO2 produc- tion from D,[-14-4C]valine was not significantly inhibited.
TABLE 2. Specific activity of N-isovalerylglycine upon repeated recrystallization
KMCPP (1.4 umol) Control Mother Mother liquor, liquor,
Crystals, total Crystals, total specific radio- specific radio-
Recrystal- activity activity activity activity lization (cpm/mg) (cpm) (cpm/mg) (cpm)
1st 650 2216 18 1477 2nd 636 844 14 64 3rd 631 584 9 31 4th 623 694
One-half of each sample from tubes 7-11 of Fig. 2 were com- bined. 10 mg of iVGly was added before the first recrystallization.
[14CIC02 productionwas inhibited 60% with 2.1 mrNI KTMCPP, but radioactivity did not increase significantly in the steam- distillable fractions. By GLC it was found that '4C-incorpora- tiol) into isobutyric acid was not significantly different from control incubations (Table 3). KMCPP showed inhibitory effects (20%) on isoleucine oxidation at a concentration of 2.1 mMA. Analysis of the incubation by GLC showed accumulation of labeled a-methylbutyrate (Table 3).
TABLE 3. GLC analyses of the incubation products after acid hydrolysis
KMCPP Acetate Isovalerate i3-Methyl- Substrates added crotonate
(.amol) (JAmol) (nmol/100 g tissue)
D,I, [2-14C]- Leu,1.5 0 1.0 ± 0.1 0.1 ± 1.6 0
DALE [2-14C] -
a-Methyl- butyrate Tiglate
Ile,1.5 4.2 2.3 ± 0.2 3.8* ± 0.5 0.3 ± 0
Meth- Isobutyrate acrylate
Val,1.5 0 2.5 ± 0.2 3.3 ± 0.8 3.4 ± 0.5 DIL [4-14C] -
Val,1.5 4.2 1.5 ± 0.2 5.1 ± 0.5 S.6 ± 1.1
Each incubation mixture contained liver slices (about 100 mg) in 2 ml of Krebs-Ringer bicarbonate buffer containing 5.6 Mumol glucose and 3.0 Mmol glycine. Results are mean ± SE of four experiments. Numbers marked are significantly different from corresponding controls: *P < 0.001 (see Table 1).
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TuBE NUMBER
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Isovaleryl Dehydrogenase Inhibition 23
TABLE 4. Effect of amino acids, administered orally with and without inhibitors, on plasma short-chain fatty acids (mg per 100 ml)*
Amino acid Inhibitor Number of (60 mg/100 g) (mg/100 g) experiments Isobutyrate n-Butyrate Isovalerate n-Hexanoate
None None 4 0.06 ±t 0 0.05 i 0 0.05 ±4 0.01 0.15 i 0.03 None Hypoglycin (10 mg) 4 -0.06 ± 0 0.71 ±- 0.18 2.32t i 0.36 0.33 ± 0.11 Leu None 5 0.01 ±0 0.03 ±0 0.10 ± 0.02 0.12 ±00 Leu Hypoglycin (10 mg) 5 0.06 ±t 0.01 1.16 ±t 0.08 19.2t i 1.1 0.44± 0.01 Leu 4-PE(30mg) 4 0.02±0 0.05±0 0.06±0.01 0.14±0 Ile None 4 0.06 ± 0.02 0.06 ±E 0.02 0.23t 4 0.04 0.12 ±00 Ile Hypoglycin (10 mg) 4 0.06 ± 0.01 0.45 ± 0.04 6.59tt ±L 0.58 0.28 ± 0.01 Val None 4 0.26 ± 0.04 0.14 ± 0.03 0.18 ± 0.03 0.26 ± 0.05 Val Hypoglycin 4 0.33 ±00.04 0.47 ±00.05 0.94 ±00.12 0.49 ±00.03
Data on the direct metabolite of each amino acid and n-butyrate are described. No significant change was observed in the amounts of propionate and n-valerate. No f3-methylerotonate, tiglate, or methacrylate was found in significant amounts after amino acid administra- tion.
* Values expressed as mean ±J SE for four experiments (except n = 5 for Leu, none and Leu, hypoglycin experiments). t P < 0.001; t a-methylbutyrate.
Comparison of inhibitory effect of hypoglycin A and 4-PE in vitro was made using uniformly labeled ileucine, L-iso- leucine, and L-valine. Hypoglycin A (1.4 mM) inhibited [14C1C02 production from L-leucine most strongly. i-Isoleu- cine oxidation was inhibited less, while L-valine oxidation was unaffected. In contrast, 4-PE, up to 4.2 mMA, did not inhibit leucine oxidation significantly. In vivo effects of hypoglycin A on metabolism of L-leucine, L-isoleucine, and L-valine
The administration of each amino acid to rats at a dose of 60 mg per 100 g of body weight resulted in no significant increase of the corresponding short-chain fatty acid in the plasma. However, when hypoglycin A was given (10 mg per 100 g of body weight) 30 min before ileucine, the plasma content of a branched-chain pentanoic acid increased up to 200 times that of the control group (Table 4). In a second control group that was given only hypoglycin A, the same branched-chain pentanoic acid increased 45-fold. The product was identified as isovaleric acid on a DOP-PA column (1). When isoleucine was given after hypoglycin A administra-
tion, a branched-chain pentanoic acid increased 30-fold. By the use of a DOP-PA column, this peak was identified as a- methylbutyric acid (1). In both the leucine and isoleucine experiments, there was also some increase in plasma n-butyric acid. Note that in the L-valine-hypoglycin group, in which isobutyric acid should have increased if the same kind of inhibition had occurred, no significant increase in isobutyric acid was found. When these experiments were carried out using 4-PE as an
inhibitor (30 mg per 100 g of body weight), followed by ileu- cine administration, no significant increase in plasma iso- valeric acid was observed (Table 4).
DISCUSSION The acctumulation of N-isovalerylglycine in the medium when liver slices were incubated with [2-'4C]leucine and KMCPP indicated that leucine oxidation was inhibited by KM\CPP at the isovaleryl CoA dehydrogenation step. This inhibition was further confirmed by in vivo experiments showing the accumu- lation of isovaleric acid after the administration of hypoglycin A and leuciine. N-Isovalerylglycine and fl-hydroxyisovaleric
acid were found in urine samples of rats after these treatments (unpublished results); this finding is similar to that in patients with isovaleric acidemia (3, 19). The amount of leucine added as substrate in the in vitro experiments was small. Therefore, most of the isovaleryl CoA that could not be dehydrogenated because of the inhibition was readily conjugated with the ex- cess of glycine (3). In the in vivo experiments, this conjugation system appears to be overloaded with the amount of leucine given. As a result, isovaleryl CoA is hydrolyzed resulting in an accumulation of free isovaleric acid in the blood, again similar to the findings in patients with isovaleric acidemia (1, 3, 19). Although isoleucine oxidation was inhibited at the a-methyl- butyryl CoA dehydrogenation step, the degree of the inhibi- tion was significantly lower. No inhibition of isobutyryl CoA dehydrogenation occurred either in vivo or in vitro. It may be reasonable, therefore, to assume that CoA esters of these branched-chain fatty acids are dehydrogenated by three differ- ent dehydrogenases, and that isovaleryl CoA dehydrogenase is specifically inhibited by hypoglycin A or its metabolites. This conclusion agrees with the findings in isovaleric acidemia where a more specific decrease in isovaleryl CoA dehydro- genase has been observed (1, 3).
Bressler and co-workers have suggested that the inhibition of long chain fatty acid oxidation by methylenecyclopro- pylacetic acid and 4-PE is due to the ability of these inhibitors to form nonmetabolizable esters with CoA and (-)-carnitine (17, 20). As a result, free CoA and (-)-carnitine become less available. Since (-)-carnitine is necessary for the oxidation of long-chain fatty acids in mitochondria, but not for short- chain fatty acid oxidation, this has been proposed as the basis for the specific inhibition of long-chain fatty acid oxidation while short chain fatty acid oxidation remains unaltered (21).
Methvlenecyclopropylacetic acid and 4-PE share two chemical structures in common, namely a five-carbon chain and a vinyl group separated by two carbons from the car- boxyl group. These structures are believed to be necessary for their hypoglycemic action (9) and, also, for their inhibition of long-chain fatty acid oxidation (17, 20). However, there is a striking difference in the structure of these two compounds, namely the presence of the cyclopropane ring. The inhibitory effect of hvpoglycin A on isovaleryl CoA dehydrogenase ap-
Vol. 68, 1971
24 Medical Sciences: Tanaka et al.
pears to be, in part, due to the presence of a cyclopropane ring since 4-PE failed to show this action. In our preliminary ex-
periments, cyclopropane carboxylic acid, which is analogous to isobutyric acid, only slightly inhibited dehydrogenation of isobutyric acid in vivo. It appears, therefore, that the methy- lenecyclopropane group is responsible for the inhibition of isovaleryl CoA dehydrogenation. The inhibition of isovaleryl CoA dehydrogenase by hypo-
glycin A has important clinical implications. One of these is the experimental production of isovaleric acidemia. In addi- tion, the present findings provide a pathophysiological ex-
planation for some of the symptoms of the "vomiting sickness of Jamaica." In this disease, profound hypoglycemia occurs
following the ingestion of unripe ackee fruit; the hypoglycemia has been attributed to ingested hypoglycin A. Some of the patients with this disorder die in spite of glucose infusions. This lack of response has been attributed to irreversible brain damage occurring during the duration of hypoglycemia prior to the glucose infusions (11, 12). However, it would appear
that increased concentrations of isovaleric acid induced by the ingested hypoglycin A probably account for some of these ob- servations. Congenital isovaleric acidemia is sometimes fatal (4). It is also noteworthy that when cats and dogs are given hypoglycin A, they manifest symptoms similar to those…
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