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Effect of X-irradiation on the hepatic synthesis of fatty acids and cholesterol
516
SHORT COMMUNICATIONS
sc 2159
Effect of X-irradiation on the hepatic synthesis of fatty acids and cholesterol I t is well-established that whole-body X-irradiation leads to increased hepatic synthesis of f a t t y acids and cholesterol 1,2. The observed relationship between liver glycogen and lipogenesis 3,4 and the possible shifts in the glucose metabolism from the glycolytic to the phosphogluconate oxidative pathway are unable to explain thisS, 6. As the previous dietary intake markedly influences lipogenesis 7-9 and irradiation induces severe loss of appetite 1°, 11 fasted or pair-fed rats were used for lipogenic studies1, 2. However, unlike fasted non-irradiated controls, the irradiated fasted animals have a higher liver glycogen lz, 13, show hyperglycemia 14 and utilise glucose more efficiently 15 with the make-up of carbohydrate-metabolising enzymes similar to that of a fed animaP ~. Thus, as f a t t y acid synthesis is known to be influenced by the concurrent carbohydrate metabolism in m a n y ways 17,18, it appeared conceivable that the irradiation-induced increases in hepatic synthesis of fatty acids and cholesterol could have been due to an increased carbohydrate metabolism of liver prevailing in an irradiated fasted animal, similar to a fed animal. A fall in the hepatic synthesis of fatty acids and cholesterol which accompany stalvation 19,~° was thus prevented by irradiation. This possibility was tested by studying the hepatic synthesis of these lipids under conditions of similar availability of carbohydrate. Male albino rats, 5 in each group, weighing 15o-2oo g and maintained on standard laboratory diet and water ad libitum, were used. The rats were placed in a perforated wooden box and received total-body X-irradiation of iooo R including back scatter in a single dose at a rate of 6o R/min. The X-rays were delivered from an Escorts Westinghouse deep-therapy unit operated at 2oo kV and 15 mA with o.5-mm copper and i - m m aluminium filters from a target distance of 4 ° cm from the upper skin in air. Each irradiated rat was paired with a sham-treated animal of similar weight. All animals unless otherwise mentioned were force fed with glucose (8o % solution in water). Following irradiation 5 ml of glucose solution was given to irradiated and nonirradiated animals twice at 3-h intervals and then 4 ml of glucose solution every 4 1l until the animals became glycosuric. Irradiated animals developed glycosuria 14-16 h after irradiation. The force feeding with glucose to non-irradiated rats was continued, the last feed being given 4 h before they were killed. All animals force fed with glucose were glycosuric at the time of death. The animals had free access to water at all times and no other food was allowed. In addition to the animals force fed with glucose, normal rats fed ad libitum with stock diet were also included in this study. 22 h after irradiation, all rats were injected with sodium [I-14C]acetate (IO /~C per IOO g body wt.) intraperitoneally. 2 h later, the rats were killed, livers were removed and analysed for ~4C incorporation into fatty acids and cholesterol as described by BHATTATItIRYet al. 21. F a t t y acids were estimated by a modification 22 of BRAGDON'S method 2a and non-saponiflable lipids as cholesterol b y ZLATKIS procedure 24. Force feeding of glucose to animals was undertaken with a view to eliminating the possible effects on lipid synthesis of the known differing availability of carbohydrate between fasted irradiated animals and non-irradiated fasted controls. As irradiated and non-irradiated rats force fed with glucose were glycosuric, it is assumed Biochim. Bioph3's. Acta, ~)5 (I962) 5I~,-5~8
517
SHORT COMMUNICATIONS
that they had received about the same amounts of available carbohydrate for metabolism prior to killing. Under these conditions the [I-14Clacetate incorporation into fatty acids and cholesterol was almost the same in both irradiated and non-irradiated controls (Table I). The reported increase of 300 and 400 % in the synthesis of fatty acids ~5 and cholesterol 2e, respectively, in irradiated fasted animals could thus be due to the greater availability of carbohydrate resulting from an increased breakdown of body proteins 27,~8. There was a tendency for the animals force fed with glucose to incorporate [I-l*Clacetate into fatty acids and cholesterol to a greater extent than the animals fed the stock diet ad libitum. Similar observation was made by MASORO4. TABLE I INCORPORATION OF SODIUM [I-14CTACETATE INTO FATTY ACIDS AND CHOLESTEROL OF LIVER OF IRRADIATED RATS FORCE FED WITH GLUCOSE (Ig), NON-IRRADIATED RATS FORCE FED WITH GLUCOSE (Cg) A N D
Description of the animal
Total fatty acids in liver (rag)
RATS
FED
THE
STOCK
Counts/rain incorporated into fatty acids Total
I o*
Specific activity
DIET
(C)
Total cholesterol in liver (rag)
Counts/rain incorporated into cholesterol Total
z os
(per rag)
I
1I
Specific activity (per rag)
Ig Cg C
147 159 155
41.o4 49.88 15.48
2800 3118 IOlO
16.9 16.3 17
88.26 92.76 32.61
5589 5897 1929
Ig Cg
283 242 261
36.43 24.74 18.98
1292 lO23 728
16. 5 15. 3 9.7
29.84 6.48 5 .08
1854 424 539
168 181 176
35.42 42.54 12.56
1958 2249 714
14. 5 14.75 17.2
IOO.OO lO5.5o 56.17
6986 777 ° 3264
C
Ig III
Cg C
IV
Cg C
168 228 220
22.90 23.96 19.14
1368 lO53 868
18.9 12 9.1
16.4o 12.47 6.66
862 lO33 731
[g Cg C
157 202 124
38.65 27.07 13.35
2448 1345 1335
13. 3 13.8 14.6
60.60 68.25 69.30
6986 5000 4706
Ig
V
It is possible that the decreases in the activities of the enzymes synthesising fatty acids2°, 3° and cholesterol m and alterations in the regulatory factors 32-34 which accompany starvation are prevented as a result of irradiation. These aspects of the problem are under investigation. Our thanks are due to Dr. R. VISWANATHAN for his interest; to the International Atomic Energy Agency, Vienna, for financial assistance and the Irwin Hospital, New Delhi, for irradiation facilities.
Vallabhbhai Patel Chest Institute, University of Delhi, Delhi (India)
S. V. P A N D E A . RAMAIAH
T. A. 1 S. 2 K. 25 3 E.
VENKITASUBRAMANIAN
R. LERNER, W'. L. WARNER AND C. ENTENMAN, Federation Proc., 12 (1953) 85. A. TRETYEKOVA AND D. E. GRODZENSKII, Biokhimia, 25 (196o) 399 ; t r a n s l a t e d in B i o c h e m i s t r y , (1961) 307 . S. HAUGAARD AND \¥. C. STADIE, J. Biol. Chem., 199 (1952) 741.
Bioehim. Biophys. Acta, 65 (1962) 516 518
518
SHORT COMMUNICATIONS
E . J. MASORO, A. i . COHN AND S. S. PANAGOS, ,4m. J. Physiol., 179 (1954) 45 I. 5 j. G. CONIGLIO, J. C. K1RSCHMAN AND G. Vvr. HUDSON, ./Jm. J. Physiol., 19o (1957) 385 . 6 j . G. CONIGLIO, D. L. GATE AND G. \V. HUDSON, Federation Proe., 18 (1959) 206. 7 G. E . BOXER AND D. STETTEN, J. Biol. Chem., 153 (1'944) 607. s I. LYON, M. S. MASRI AND I. L. CHAIKOFF, J. Biol. Chem., 196 (1952) 25. 9 j. T. VANBRUGGEN, T. T. HUTCHENS, C. K. CLAYCOMB, W . J. CATHEY AND E. S. WEST, J . Biol. Chem., 196 (1952 ) 389. a0 C. L. PROSSER, E . F. PAINTER, H. LISCO, A . M . BRUES, L. O. JACOBSON AND ~'~. N. SWIFT, Radiology, 49 (1947) 299It D. E . SMITH AND E. B. TYREE, Am. J. Physiol., 177 (1954) 2.57. 12 M. G. ORB AND L. A. STOCKEN, Physiol. Revs., 33 (1953) 356. 13 M. K. NERURKAR AND M. B. SAHASRABUDHE, Brit. J. Expll. Pathol., 4 ° (1959) 318. 14 R . E . KAY AND C. ENTENMAN, Proc. Soc. Exptl. Biol. Med., N . Y . , 91 (19.56) 143. ,5 p. V. VITTORIO, \V. P. SPENCE AND J. JOHNSTON, Can. J. Biochem. Physiol., 37 (I959) 7~7 . 16 M. F. WALTER, I. L. CHAIKOFF AND l~. HILL, Arch. Biochem. Biophys., 94 ( I 9 6 I ) 387 . 17 I. B. FRITZ, Physiol. ]~evs., 4 ~ ( I 9 6 I ) 52. 18 j . TEPPERMAN AND H. M. TEPPERMAN, Am. J. Physiol., 200 (196I) IO69. 19 E . J. MASORO, I. L. CHAIKOFF, S. S. CHERNICK AND J. M. FELTS, J. Biol. Chem., 185 (195o) 845. 20 I{. J, EMERSON, W. C. BERARDS AND J. T. VAN BRUGGEN, dr. Biol. Chem., 234 (I959) 435. 21 E . ]). M. BHATTATHIRY, T. A. \rENTIKASUBRAMANIAN AND 17~. VISWANATHAN, Indian .[. CheJ Diseases, 3 (19611 20. 22 S. V. PANDE, R. P. KHAN AND T. A. VENKITASUBRAMANIAN, submitted for publication. 23 j . H . BRAGDON, J. Biol. Chem., I9O (I95 I) 513 . 24 A. ZLATKIS, B. ZAK AND A. J. BOYLE, J. Lab. Clin. Med., 41 (1953) 486. 25 j . G. CONIGLIO, D. B. MCCORMICK AND (~. W . HUDSON, Am. J. Physiol., 185 (1956) 577. 26 S. GARATTINI, P. PAOLETTI AND ]:~. PAOLETTI, i n G. POP JACK, Biochemistry of Lipids, Pergamon Press, Inc., Oxford, 196o, p. I84. 27 G. GERBER, G. GERBER, I(. ]. ALTOMAN AND L. H . HEMPLEMANN, Intern. J. Radiation and Biol., I (1959) 277. 18 L. F. NIMS AND R. E. THURBER, Federation Proc., 18 (I959) 114. 2s D. M. GIBSON AND D. D. HUBBARD, Biochem. Biophys. Research Communs., 3 (196o) 531. 3o S. NUMA, M. MATHUSASHI AND F. LYNEN, Bioehem. Z., 334 (I961) 203. 31 N. L. ]~. BUCHER, P. OVERATH AND F. LYNEN, Biochim. Biophys..4cta, 4 ° (196o) 4 9 I . 32 G. N. CATRAVAS AND H. S. ANKER, Proc. Natl. Acad. Sci. U.S., 44 (1957) lO97. 13 E. J. MASORO, E . PORTER AND H. M. KORCttAK, Federation Proe., 20 (196I) 2 7 2 F . ~4 B. B. MIGICOVSKY, Can. J. Biochem. Physiol., 39 (196I) 747.
Received July 9th, 1962 Biochim. Bioph.vs. Acta, 65 (1962) 516 5 I S
s¢
2171
Deacetylation of a-keto-~-acetamidocaproic acid by ,-lysine acylase In spite of the fact that some ophioxidases can oxidize L-lysine at a considerable rate x, it has been known for some time that mammalian L-amino acid oxidase (EC 1.4.3.2 ) catalyzes the oxidation at a very low rate 2, 3. It has been demonstrated, however, that enzymic oxidative deamination of D- and L-lysine and transamination of L-lysine involving the a-amino group take place readily when the e-amino group is substituted. Thus, it has been postulated that substitution of the e-amino group may be a prerequisite for the degradation of this amino acid. Indeed, the formation of a-hydroxy-e-acetamidocaproic acid has been demonstrated in Neurospora a. In accordance with the above hypothesis, an enzyme which is capable of hydrolyzing e-N-acyl-L-lysine was identified ~. This enzyme, e-lysine acylase, has been shown to be highly specific and has been purified to a high degree from rat and hog kidney 5,6. However, the biochemical significance of this enzyme has not yet been elucidated. We present here evidence that e-lysine acylase deacetylates ~-keto-e-acetamidocaproic Hiochim. tDiophvs..4eta, ~5 (1962) 5 1 8 - 5 z °
SHORT COMMUNICATIONS
sc 2159
Effect of X-irradiation on the hepatic synthesis of fatty acids and cholesterol I t is well-established that whole-body X-irradiation leads to increased hepatic synthesis of f a t t y acids and cholesterol 1,2. The observed relationship between liver glycogen and lipogenesis 3,4 and the possible shifts in the glucose metabolism from the glycolytic to the phosphogluconate oxidative pathway are unable to explain thisS, 6. As the previous dietary intake markedly influences lipogenesis 7-9 and irradiation induces severe loss of appetite 1°, 11 fasted or pair-fed rats were used for lipogenic studies1, 2. However, unlike fasted non-irradiated controls, the irradiated fasted animals have a higher liver glycogen lz, 13, show hyperglycemia 14 and utilise glucose more efficiently 15 with the make-up of carbohydrate-metabolising enzymes similar to that of a fed animaP ~. Thus, as f a t t y acid synthesis is known to be influenced by the concurrent carbohydrate metabolism in m a n y ways 17,18, it appeared conceivable that the irradiation-induced increases in hepatic synthesis of fatty acids and cholesterol could have been due to an increased carbohydrate metabolism of liver prevailing in an irradiated fasted animal, similar to a fed animal. A fall in the hepatic synthesis of fatty acids and cholesterol which accompany stalvation 19,~° was thus prevented by irradiation. This possibility was tested by studying the hepatic synthesis of these lipids under conditions of similar availability of carbohydrate. Male albino rats, 5 in each group, weighing 15o-2oo g and maintained on standard laboratory diet and water ad libitum, were used. The rats were placed in a perforated wooden box and received total-body X-irradiation of iooo R including back scatter in a single dose at a rate of 6o R/min. The X-rays were delivered from an Escorts Westinghouse deep-therapy unit operated at 2oo kV and 15 mA with o.5-mm copper and i - m m aluminium filters from a target distance of 4 ° cm from the upper skin in air. Each irradiated rat was paired with a sham-treated animal of similar weight. All animals unless otherwise mentioned were force fed with glucose (8o % solution in water). Following irradiation 5 ml of glucose solution was given to irradiated and nonirradiated animals twice at 3-h intervals and then 4 ml of glucose solution every 4 1l until the animals became glycosuric. Irradiated animals developed glycosuria 14-16 h after irradiation. The force feeding with glucose to non-irradiated rats was continued, the last feed being given 4 h before they were killed. All animals force fed with glucose were glycosuric at the time of death. The animals had free access to water at all times and no other food was allowed. In addition to the animals force fed with glucose, normal rats fed ad libitum with stock diet were also included in this study. 22 h after irradiation, all rats were injected with sodium [I-14C]acetate (IO /~C per IOO g body wt.) intraperitoneally. 2 h later, the rats were killed, livers were removed and analysed for ~4C incorporation into fatty acids and cholesterol as described by BHATTATItIRYet al. 21. F a t t y acids were estimated by a modification 22 of BRAGDON'S method 2a and non-saponiflable lipids as cholesterol b y ZLATKIS procedure 24. Force feeding of glucose to animals was undertaken with a view to eliminating the possible effects on lipid synthesis of the known differing availability of carbohydrate between fasted irradiated animals and non-irradiated fasted controls. As irradiated and non-irradiated rats force fed with glucose were glycosuric, it is assumed Biochim. Bioph3's. Acta, ~)5 (I962) 5I~,-5~8
517
SHORT COMMUNICATIONS
that they had received about the same amounts of available carbohydrate for metabolism prior to killing. Under these conditions the [I-14Clacetate incorporation into fatty acids and cholesterol was almost the same in both irradiated and non-irradiated controls (Table I). The reported increase of 300 and 400 % in the synthesis of fatty acids ~5 and cholesterol 2e, respectively, in irradiated fasted animals could thus be due to the greater availability of carbohydrate resulting from an increased breakdown of body proteins 27,~8. There was a tendency for the animals force fed with glucose to incorporate [I-l*Clacetate into fatty acids and cholesterol to a greater extent than the animals fed the stock diet ad libitum. Similar observation was made by MASORO4. TABLE I INCORPORATION OF SODIUM [I-14CTACETATE INTO FATTY ACIDS AND CHOLESTEROL OF LIVER OF IRRADIATED RATS FORCE FED WITH GLUCOSE (Ig), NON-IRRADIATED RATS FORCE FED WITH GLUCOSE (Cg) A N D
Description of the animal
Total fatty acids in liver (rag)
RATS
FED
THE
STOCK
Counts/rain incorporated into fatty acids Total
I o*
Specific activity
DIET
(C)
Total cholesterol in liver (rag)
Counts/rain incorporated into cholesterol Total
z os
(per rag)
I
1I
Specific activity (per rag)
Ig Cg C
147 159 155
41.o4 49.88 15.48
2800 3118 IOlO
16.9 16.3 17
88.26 92.76 32.61
5589 5897 1929
Ig Cg
283 242 261
36.43 24.74 18.98
1292 lO23 728
16. 5 15. 3 9.7
29.84 6.48 5 .08
1854 424 539
168 181 176
35.42 42.54 12.56
1958 2249 714
14. 5 14.75 17.2
IOO.OO lO5.5o 56.17
6986 777 ° 3264
C
Ig III
Cg C
IV
Cg C
168 228 220
22.90 23.96 19.14
1368 lO53 868
18.9 12 9.1
16.4o 12.47 6.66
862 lO33 731
[g Cg C
157 202 124
38.65 27.07 13.35
2448 1345 1335
13. 3 13.8 14.6
60.60 68.25 69.30
6986 5000 4706
Ig
V
It is possible that the decreases in the activities of the enzymes synthesising fatty acids2°, 3° and cholesterol m and alterations in the regulatory factors 32-34 which accompany starvation are prevented as a result of irradiation. These aspects of the problem are under investigation. Our thanks are due to Dr. R. VISWANATHAN for his interest; to the International Atomic Energy Agency, Vienna, for financial assistance and the Irwin Hospital, New Delhi, for irradiation facilities.
Vallabhbhai Patel Chest Institute, University of Delhi, Delhi (India)
S. V. P A N D E A . RAMAIAH
T. A. 1 S. 2 K. 25 3 E.
VENKITASUBRAMANIAN
R. LERNER, W'. L. WARNER AND C. ENTENMAN, Federation Proc., 12 (1953) 85. A. TRETYEKOVA AND D. E. GRODZENSKII, Biokhimia, 25 (196o) 399 ; t r a n s l a t e d in B i o c h e m i s t r y , (1961) 307 . S. HAUGAARD AND \¥. C. STADIE, J. Biol. Chem., 199 (1952) 741.
Bioehim. Biophys. Acta, 65 (1962) 516 518
518
SHORT COMMUNICATIONS
E . J. MASORO, A. i . COHN AND S. S. PANAGOS, ,4m. J. Physiol., 179 (1954) 45 I. 5 j. G. CONIGLIO, J. C. K1RSCHMAN AND G. Vvr. HUDSON, ./Jm. J. Physiol., 19o (1957) 385 . 6 j . G. CONIGLIO, D. L. GATE AND G. \V. HUDSON, Federation Proe., 18 (1959) 206. 7 G. E . BOXER AND D. STETTEN, J. Biol. Chem., 153 (1'944) 607. s I. LYON, M. S. MASRI AND I. L. CHAIKOFF, J. Biol. Chem., 196 (1952) 25. 9 j. T. VANBRUGGEN, T. T. HUTCHENS, C. K. CLAYCOMB, W . J. CATHEY AND E. S. WEST, J . Biol. Chem., 196 (1952 ) 389. a0 C. L. PROSSER, E . F. PAINTER, H. LISCO, A . M . BRUES, L. O. JACOBSON AND ~'~. N. SWIFT, Radiology, 49 (1947) 299It D. E . SMITH AND E. B. TYREE, Am. J. Physiol., 177 (1954) 2.57. 12 M. G. ORB AND L. A. STOCKEN, Physiol. Revs., 33 (1953) 356. 13 M. K. NERURKAR AND M. B. SAHASRABUDHE, Brit. J. Expll. Pathol., 4 ° (1959) 318. 14 R . E . KAY AND C. ENTENMAN, Proc. Soc. Exptl. Biol. Med., N . Y . , 91 (19.56) 143. ,5 p. V. VITTORIO, \V. P. SPENCE AND J. JOHNSTON, Can. J. Biochem. Physiol., 37 (I959) 7~7 . 16 M. F. WALTER, I. L. CHAIKOFF AND l~. HILL, Arch. Biochem. Biophys., 94 ( I 9 6 I ) 387 . 17 I. B. FRITZ, Physiol. ]~evs., 4 ~ ( I 9 6 I ) 52. 18 j . TEPPERMAN AND H. M. TEPPERMAN, Am. J. Physiol., 200 (196I) IO69. 19 E . J. MASORO, I. L. CHAIKOFF, S. S. CHERNICK AND J. M. FELTS, J. Biol. Chem., 185 (195o) 845. 20 I{. J, EMERSON, W. C. BERARDS AND J. T. VAN BRUGGEN, dr. Biol. Chem., 234 (I959) 435. 21 E . ]). M. BHATTATHIRY, T. A. \rENTIKASUBRAMANIAN AND 17~. VISWANATHAN, Indian .[. CheJ Diseases, 3 (19611 20. 22 S. V. PANDE, R. P. KHAN AND T. A. VENKITASUBRAMANIAN, submitted for publication. 23 j . H . BRAGDON, J. Biol. Chem., I9O (I95 I) 513 . 24 A. ZLATKIS, B. ZAK AND A. J. BOYLE, J. Lab. Clin. Med., 41 (1953) 486. 25 j . G. CONIGLIO, D. B. MCCORMICK AND (~. W . HUDSON, Am. J. Physiol., 185 (1956) 577. 26 S. GARATTINI, P. PAOLETTI AND ]:~. PAOLETTI, i n G. POP JACK, Biochemistry of Lipids, Pergamon Press, Inc., Oxford, 196o, p. I84. 27 G. GERBER, G. GERBER, I(. ]. ALTOMAN AND L. H . HEMPLEMANN, Intern. J. Radiation and Biol., I (1959) 277. 18 L. F. NIMS AND R. E. THURBER, Federation Proc., 18 (I959) 114. 2s D. M. GIBSON AND D. D. HUBBARD, Biochem. Biophys. Research Communs., 3 (196o) 531. 3o S. NUMA, M. MATHUSASHI AND F. LYNEN, Bioehem. Z., 334 (I961) 203. 31 N. L. ]~. BUCHER, P. OVERATH AND F. LYNEN, Biochim. Biophys..4cta, 4 ° (196o) 4 9 I . 32 G. N. CATRAVAS AND H. S. ANKER, Proc. Natl. Acad. Sci. U.S., 44 (1957) lO97. 13 E. J. MASORO, E . PORTER AND H. M. KORCttAK, Federation Proe., 20 (196I) 2 7 2 F . ~4 B. B. MIGICOVSKY, Can. J. Biochem. Physiol., 39 (196I) 747.
Received July 9th, 1962 Biochim. Bioph.vs. Acta, 65 (1962) 516 5 I S
s¢
2171
Deacetylation of a-keto-~-acetamidocaproic acid by ,-lysine acylase In spite of the fact that some ophioxidases can oxidize L-lysine at a considerable rate x, it has been known for some time that mammalian L-amino acid oxidase (EC 1.4.3.2 ) catalyzes the oxidation at a very low rate 2, 3. It has been demonstrated, however, that enzymic oxidative deamination of D- and L-lysine and transamination of L-lysine involving the a-amino group take place readily when the e-amino group is substituted. Thus, it has been postulated that substitution of the e-amino group may be a prerequisite for the degradation of this amino acid. Indeed, the formation of a-hydroxy-e-acetamidocaproic acid has been demonstrated in Neurospora a. In accordance with the above hypothesis, an enzyme which is capable of hydrolyzing e-N-acyl-L-lysine was identified ~. This enzyme, e-lysine acylase, has been shown to be highly specific and has been purified to a high degree from rat and hog kidney 5,6. However, the biochemical significance of this enzyme has not yet been elucidated. We present here evidence that e-lysine acylase deacetylates ~-keto-e-acetamidocaproic Hiochim. tDiophvs..4eta, ~5 (1962) 5 1 8 - 5 z °