- Email: [email protected]
Effect of insulin on the levels of oxidized and reduced nicotinamide-adenine dinucleotides in the perfused rat heart
347 sc 23089 Effect of insulin on the levels of oxidized and reduced nicotinamide-adenine dinucleotides in the perfused rat heart SHORT COMMUNICATIONS
Since the original observation of GEMMILLI that insulin increased the glucose uptake and glycogen synthesis of the isolated rat diaphragm, it has been established that this effect is caused by an increased permeability of the cell membrane to glucose. Studies with uniformly labeled [14C]glucose in the perfused rat heart have shown that insulin stimulates each of the pathways of glucose metabolism to glycogen, lactate and 14C0~, and also increases the incorporation of 14C isotope into intermediates other than glycogen which remain in the heart 2. Extensive investigations with the isolated rat diaphragm have shown that insulin also enhances the incorporation of labelled amino acids and pyruvate into protein 3-5, nucleotide incorporation into nucleic acid 6, and [~4C]acetate into trigiyceride and phospholipidL These effects are independent of the action of insulin in facilitating the transport of glucose, although in general greater effects are obtained in the presence of glucose. Many biosynthetic pathways require a supply of NADPH 2 and high-energy phosphate compounds, hence insulin may promote certain anabolic reactions by increasing the supply of one or both of these essential cofactors, possibly by stimulation of the pentose-phosphate pathway of glucose metabolism. The purpose of the present study was to investigate this hypothesis, and to examine a possible effect of insulin on the tissue levels of nicotinamide-adenine dinucleotides and ATP in the perfused rat heart. Hearts from male albino rats (220-28o g) fed ad libitum prior to sacrifice, were perfused as previously described~, 8. Analytical techniques and the techniques used for the isotope perfusion experiments have also been described ~. Nicotinamide-adenine dinucleotides in the oxidized and reduced form were determined by modifications of the methods used by ]~STABROOKAND MAITRA9. Attempts to detect qualitatively the presence of the pentose-phosphate pathway in the rat heart by determination of the ratio of the specific yields (14C02 output divided by the glucose uptake) z° from [I-I4C]- and [6-i4C]glucose were unsuccessful. Results are shown in Table I. An experimental time interval from 3 ° to 60 min of perfusion was chosen in the present experiment in order to avoid the initial lag in TABLE I OXIDATION OF [I-laG]GLUCOSE AND [6-14C]GLUCOSE BY THE PERFUSED RAT HEART H e a r t s were p e r f u s e d for 60 m i n w i t h 15 or 2o m l of K r e b s b i c a r b o n a t e buffer c o n t a i n i n g 5 mM Ex4C]glucose (o.I/*C/ml). M e t a b o l i c c h a n g e s were m e a s u r e d o v e r t h e t i m e i n t e r v a l 30-60 ra i n a n d the r e s u l t s are m e a n ± S.E. of the m e a n of s i x h e a r t s e x p r e s s e d i n / , m o l e s p e r g d r y wt. glucose equivalents.
Label in glucose
Insulin (munits/ml)
I-1tC 6-14C
o o
1-14C 6-1~C
2 2
Glucose uptake
35.6 i 5.o 32.2 ± 4.7 z 2 7 ± 5.7 II9:hi2
Medium count decrease
laCOz output
32.3 -¢- 2.6 28.3 J: 2.0
25.2 + 3.8 23.5 ± 3.2
87.5 2_ 5. 2 94.5!5.1
57.3 4- 5.4 51.3±4.7
iaC r e c o v e r e d
[z-14C_]/[6-i4C] ratio (specific yield)
in glycogen
inheart intermediates
1.9 ± o.z 2.4 ± 0.5
25.8 ± 1.8 23. 7 4- 2. 5
0.97
34 4-2 33:L2
0.95
2oj_2 I8±1
Biochim. Biophys. Acta, 97 (I965) 347-349
348
SHORT COMMUNICATIONS
TABLE II EFFECTS
OF INSULIN,
DINUCLEOTIDES,
ATP
GLUCOSE, ANn
ANOXIA
GLUCOSE
AND
AMYTAL
6-PHOSPHATE
ON THE LEVELS IN
PERFUSED
OF NICOTINAMIDE--ADENINE
RAT
HEART
H e a r t s were p re-perfused for 15 rain w i t h s u b s t r a t e - f r e e K r e b s b i c a r b o n a t e buffer a n d t h e n perfused for a f u r t h e r 15 m i n w i t h 15 ml buffer c o n t a i n i n g s u b s t r a t e s a d d e d as shown. The pe rfus i on w a s t e r m i n a t e d b y r a p i d l y freezing t h e h e a r t s w i t h a m e t a l c l a m p cooled in l i q u i d N 2 (ref. 12). R e s u l t s e x c e p t for A T P are e x p r e s s e d in m/~moles p e r g d r y wt. J~ S.E. of t h e me a n, w i t h four h e a r t s in each group. A T P c o n t e n t is e x p r e s s e d i n / * m o l e s p e r g d r y wt.
Substrate
Insulin (munils/ml)
NA D
NA DH 2
5 mM glucose o 3972==46 I46ili 5 mM glucose 2 4oI6~ II2 296±23 None o 4o47 ~ 28 175±14 20 mM glucose o 414 ° ~ 114 21o ± 7 i o mM a c e t a t e o 3902 ==44 370±36 i o mM a c e t a t e 2 3767 ~ 97 352 ± 9 IO mM glucose o 388o ~c I 6 I 84±15 iomMglucose o 29oo==14o 1 5 2 8 ± 1 2 6 (anaerobic) i o m M glucose o 2836 ~ 5 8 1358--27 (i rain a f t e r 3 mM a m y t a l )
NA DP
,,N\4DPH,~
267i2 215~9 192±3 2oi ± 7 I98i6 206 ± 3 299i48 156±23
271ii 7 337±19 308±20 264 ~ 19 341 ± 2 5 346 ~ 24 214i3I 338-c27
I 5 5 ~ 17
347±31
Glucose 6-phosphate 35o~17 I9OO ~ 5 I 313±23 432 ~ 19 334±3 ° I99 ± 21
-
,4 T P
21.7±o. 4 22.4 ~ o. 3 --21.1 ± i . o 20. 4 ~- 0.6 21.6 i 1.2 I7.3 ~ I. 5 2 t . 4 ~ 1.9
the release of 14C02 (ref. 2). The production of 14C0 2 in the absence of insulin accounted for 75 % of the counts removed from the medium, which in turn represented 9 ° % of the glucose uptake. In the presence of insulin, glucose uptake was stimulated 4-fold, and 14C02 production accounted for 61-71% of the counts removed from the medium. The total recovery of I~C isotope ranged from 80 to 92 %. However, the ratio of the specific yields of 14C0~ from [I-14C] - and E6-14Clglucose were not significantly different from unity both in the presence and absence of insulin. Similar results with rat hearts perfused in the absence of insulin have recently been reported by SHIPP et al. n, although in their experiments 14C0 2 production accounted for only 21-24 % of the glucose uptake. The content of nicotinamide-adenine dinucleotides, glucose 6-phosphate and ATP in rat hearts are shown in Table II. These results represent a series of experiments performed over a period of several months, hence each experimental variation should be compared with the appropriate control. Insulin in the presence of glucose significantly decreased the content of NADP (P < 0.005) and increased that of N A D P H 2 by an equivalent amount. NADH~ was increased 2-fold. These effects were not obtained when the concentration of glucose in the medium was increased to 20 mM in the absence of insulin, or when glucose was replaced by acetate. The increased content of N A D P H 2 in hearts perfused with glucose and insulin was associated with a 4-fold increase of glucose 6-phosphate, while ATP remained unchanged. Glucose alone (20 mM) increased glucose 6-phosphate slightly, while insulin in the presence of acetate produced a decrease in the content of glucose 6-phosphate, and no change of ATP. The effects of anaerobiosis and amytal in the presence of IO mM glucose are included in Table I I for comparison. These treatments increased the levels of NADH2 16-18 times, but caused only a small increase in the levels of NADPHz. In general the sum of the oxidized and reduced nicotinamide-adenine dinucleotides showed good Biochim. Biophys. Acla, 97 (1965) 347-349
349
SHORT COMMUNICATIONS
agreement between different experiments. The level of ATP in hearts perfused anaerobically for 15 min was decreased by 20 %, and the hearts continued to beat, although with a decreased force of contraction. ATP levels remained constant I min after the addition of amytal, but the hearts stopped beating after 20-3o sec, presumably due to effects other than inhibition of the respiratory chain. In the present experiments the ratio of NADPH~ to NADP varied over the range 0.7-2.2, the highest ratio being obtained with anoxia and amytal. These results are in agreement with those reported by BURCI~ et al. 13, but contrast strongly with earlier values of GLOCK AND McLEAN 14 who found ratios of 8. This discrepancy can be explained by recent improvements of the analytical techniques and particularly rapid freezing of the tissue TM. The content of NADP in rat heart is, however, only about 5 % that of NAD and hence may limit the activity of the pentose-phosphate pathway. Tissues such as liver and kidney in which the pentose-phosphate pathway is more pronounced have a much greater relative proportion of NADP 13. Results reported in the present paper suggest that the pentose-phosphate pathway may be indirectly stimulated by insulin as a result of the elevated glucose 6-phosphate concentration, even though the presence of this pathway could not be demonstrated from the ratio of 14C02 yields from LI-14CI- and I6-14C~glucose. Activity of the first two NADP-dependent enzymes of the pentose-phosphate pathway has been demonstrated in homogenates of rat heart by GLOCK AND MCLEAN15, and recently confirmed in this laboratory (E. A. JONES, unpublished observations). CREVASSE et al. TM have also demonstrated that homogenates of dog heart, suitably fortified with NADP, possess the enzymatic capacity for the oxidation of glucose by the pentose-phosphate pathway. Undoubtedly the activity of this pathway in cardiac muscle is very low compared with the Embden-Meyerhof and non-triose-phosphate pathways, but nevertheless may still represent a significant cytoplasmic NADPH 2 generating system in normal cardiac metabolism. This study was supported by grants from the U.S. Public Health Service (TI AM-5o77-o7 and PHS 122o2-ol).
Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pa. (U.S.A.) Baker Clinic Research Laboratory, Harvard Medical School, Boston, Mass. (U.S.A.) I 2 3 4 5 6 7 8 9 Io ii 12 13 14 15 16
J O H N R. WlLLIAMSON ROBERT A. KREISBERG
C. L. GEMMILL, Bull. Johns Hopkins Hosp., 66 (194o) 232. J. R. WILLIAMSON, J. Biol. Chem., 239 (1964) 2721. F. G. YOUNG, Proc. Roy. Acad. Sci., London, I 5 7 B (I962) i. K. L. MANCHESTER AND F. G. YOUNG, Vitamins Hormones, 19 (1961) 95. I. G. WOOL AND M. E. IfRAHL, Biochim. Biophys. Acta, 82 (1964) 606. I. G. WOOL, Am. J. Physiol., I99 (196o) 719 . I{. L. MANCHESTER, Biochim. Biophys. Acta, 7 ° (1963) 208. J. R. WlLLIAMSON AND H. A. KREBS, Biochem. J., 80 (1961) 54 o. R. W. ESTABROOK AND P. If. MAITRA, Anal. Biochem., 3 (1962) 369 . J. KATZ AND N. G. \•OOD, J. Biol. Chem., 238 (1963) 517 . J. c. SHIPP, I-[. I~.. DELCHER AND L. E. CREVASSE, Biochim. Biophys. Acta, 86 (1964) 399. A. WOLLENBERGER, O. RISTAU AND G. SCHOFFA, Arch. Ges. Physiol., 270 (196o) 399. H. B. BURCH, O. H. LOWRY AND P. VON DIPPE, J. Biol. Chem., 238 (1963) 2828. G. E. GLOCK AND P. MCLEAN, Biochem. J., 61 (1955) 388. G. E. GLOCK AND P. MCLEAN, Biochem. J., 56 (1954) 171. L. E. CREVASSE, J. C. SHIPP AND H. If. DELCHER, Biochim. Biophys. Acta, 86 (1964) 402.
Received October 5th, 1964 Biochim. Biophys. Acta, 97 (1965) 347-349
Since the original observation of GEMMILLI that insulin increased the glucose uptake and glycogen synthesis of the isolated rat diaphragm, it has been established that this effect is caused by an increased permeability of the cell membrane to glucose. Studies with uniformly labeled [14C]glucose in the perfused rat heart have shown that insulin stimulates each of the pathways of glucose metabolism to glycogen, lactate and 14C0~, and also increases the incorporation of 14C isotope into intermediates other than glycogen which remain in the heart 2. Extensive investigations with the isolated rat diaphragm have shown that insulin also enhances the incorporation of labelled amino acids and pyruvate into protein 3-5, nucleotide incorporation into nucleic acid 6, and [~4C]acetate into trigiyceride and phospholipidL These effects are independent of the action of insulin in facilitating the transport of glucose, although in general greater effects are obtained in the presence of glucose. Many biosynthetic pathways require a supply of NADPH 2 and high-energy phosphate compounds, hence insulin may promote certain anabolic reactions by increasing the supply of one or both of these essential cofactors, possibly by stimulation of the pentose-phosphate pathway of glucose metabolism. The purpose of the present study was to investigate this hypothesis, and to examine a possible effect of insulin on the tissue levels of nicotinamide-adenine dinucleotides and ATP in the perfused rat heart. Hearts from male albino rats (220-28o g) fed ad libitum prior to sacrifice, were perfused as previously described~, 8. Analytical techniques and the techniques used for the isotope perfusion experiments have also been described ~. Nicotinamide-adenine dinucleotides in the oxidized and reduced form were determined by modifications of the methods used by ]~STABROOKAND MAITRA9. Attempts to detect qualitatively the presence of the pentose-phosphate pathway in the rat heart by determination of the ratio of the specific yields (14C02 output divided by the glucose uptake) z° from [I-I4C]- and [6-i4C]glucose were unsuccessful. Results are shown in Table I. An experimental time interval from 3 ° to 60 min of perfusion was chosen in the present experiment in order to avoid the initial lag in TABLE I OXIDATION OF [I-laG]GLUCOSE AND [6-14C]GLUCOSE BY THE PERFUSED RAT HEART H e a r t s were p e r f u s e d for 60 m i n w i t h 15 or 2o m l of K r e b s b i c a r b o n a t e buffer c o n t a i n i n g 5 mM Ex4C]glucose (o.I/*C/ml). M e t a b o l i c c h a n g e s were m e a s u r e d o v e r t h e t i m e i n t e r v a l 30-60 ra i n a n d the r e s u l t s are m e a n ± S.E. of the m e a n of s i x h e a r t s e x p r e s s e d i n / , m o l e s p e r g d r y wt. glucose equivalents.
Label in glucose
Insulin (munits/ml)
I-1tC 6-14C
o o
1-14C 6-1~C
2 2
Glucose uptake
35.6 i 5.o 32.2 ± 4.7 z 2 7 ± 5.7 II9:hi2
Medium count decrease
laCOz output
32.3 -¢- 2.6 28.3 J: 2.0
25.2 + 3.8 23.5 ± 3.2
87.5 2_ 5. 2 94.5!5.1
57.3 4- 5.4 51.3±4.7
iaC r e c o v e r e d
[z-14C_]/[6-i4C] ratio (specific yield)
in glycogen
inheart intermediates
1.9 ± o.z 2.4 ± 0.5
25.8 ± 1.8 23. 7 4- 2. 5
0.97
34 4-2 33:L2
0.95
2oj_2 I8±1
Biochim. Biophys. Acta, 97 (I965) 347-349
348
SHORT COMMUNICATIONS
TABLE II EFFECTS
OF INSULIN,
DINUCLEOTIDES,
ATP
GLUCOSE, ANn
ANOXIA
GLUCOSE
AND
AMYTAL
6-PHOSPHATE
ON THE LEVELS IN
PERFUSED
OF NICOTINAMIDE--ADENINE
RAT
HEART
H e a r t s were p re-perfused for 15 rain w i t h s u b s t r a t e - f r e e K r e b s b i c a r b o n a t e buffer a n d t h e n perfused for a f u r t h e r 15 m i n w i t h 15 ml buffer c o n t a i n i n g s u b s t r a t e s a d d e d as shown. The pe rfus i on w a s t e r m i n a t e d b y r a p i d l y freezing t h e h e a r t s w i t h a m e t a l c l a m p cooled in l i q u i d N 2 (ref. 12). R e s u l t s e x c e p t for A T P are e x p r e s s e d in m/~moles p e r g d r y wt. J~ S.E. of t h e me a n, w i t h four h e a r t s in each group. A T P c o n t e n t is e x p r e s s e d i n / * m o l e s p e r g d r y wt.
Substrate
Insulin (munils/ml)
NA D
NA DH 2
5 mM glucose o 3972==46 I46ili 5 mM glucose 2 4oI6~ II2 296±23 None o 4o47 ~ 28 175±14 20 mM glucose o 414 ° ~ 114 21o ± 7 i o mM a c e t a t e o 3902 ==44 370±36 i o mM a c e t a t e 2 3767 ~ 97 352 ± 9 IO mM glucose o 388o ~c I 6 I 84±15 iomMglucose o 29oo==14o 1 5 2 8 ± 1 2 6 (anaerobic) i o m M glucose o 2836 ~ 5 8 1358--27 (i rain a f t e r 3 mM a m y t a l )
NA DP
,,N\4DPH,~
267i2 215~9 192±3 2oi ± 7 I98i6 206 ± 3 299i48 156±23
271ii 7 337±19 308±20 264 ~ 19 341 ± 2 5 346 ~ 24 214i3I 338-c27
I 5 5 ~ 17
347±31
Glucose 6-phosphate 35o~17 I9OO ~ 5 I 313±23 432 ~ 19 334±3 ° I99 ± 21
-
,4 T P
21.7±o. 4 22.4 ~ o. 3 --21.1 ± i . o 20. 4 ~- 0.6 21.6 i 1.2 I7.3 ~ I. 5 2 t . 4 ~ 1.9
the release of 14C02 (ref. 2). The production of 14C0 2 in the absence of insulin accounted for 75 % of the counts removed from the medium, which in turn represented 9 ° % of the glucose uptake. In the presence of insulin, glucose uptake was stimulated 4-fold, and 14C02 production accounted for 61-71% of the counts removed from the medium. The total recovery of I~C isotope ranged from 80 to 92 %. However, the ratio of the specific yields of 14C0~ from [I-14C] - and E6-14Clglucose were not significantly different from unity both in the presence and absence of insulin. Similar results with rat hearts perfused in the absence of insulin have recently been reported by SHIPP et al. n, although in their experiments 14C0 2 production accounted for only 21-24 % of the glucose uptake. The content of nicotinamide-adenine dinucleotides, glucose 6-phosphate and ATP in rat hearts are shown in Table II. These results represent a series of experiments performed over a period of several months, hence each experimental variation should be compared with the appropriate control. Insulin in the presence of glucose significantly decreased the content of NADP (P < 0.005) and increased that of N A D P H 2 by an equivalent amount. NADH~ was increased 2-fold. These effects were not obtained when the concentration of glucose in the medium was increased to 20 mM in the absence of insulin, or when glucose was replaced by acetate. The increased content of N A D P H 2 in hearts perfused with glucose and insulin was associated with a 4-fold increase of glucose 6-phosphate, while ATP remained unchanged. Glucose alone (20 mM) increased glucose 6-phosphate slightly, while insulin in the presence of acetate produced a decrease in the content of glucose 6-phosphate, and no change of ATP. The effects of anaerobiosis and amytal in the presence of IO mM glucose are included in Table I I for comparison. These treatments increased the levels of NADH2 16-18 times, but caused only a small increase in the levels of NADPHz. In general the sum of the oxidized and reduced nicotinamide-adenine dinucleotides showed good Biochim. Biophys. Acla, 97 (1965) 347-349
349
SHORT COMMUNICATIONS
agreement between different experiments. The level of ATP in hearts perfused anaerobically for 15 min was decreased by 20 %, and the hearts continued to beat, although with a decreased force of contraction. ATP levels remained constant I min after the addition of amytal, but the hearts stopped beating after 20-3o sec, presumably due to effects other than inhibition of the respiratory chain. In the present experiments the ratio of NADPH~ to NADP varied over the range 0.7-2.2, the highest ratio being obtained with anoxia and amytal. These results are in agreement with those reported by BURCI~ et al. 13, but contrast strongly with earlier values of GLOCK AND McLEAN 14 who found ratios of 8. This discrepancy can be explained by recent improvements of the analytical techniques and particularly rapid freezing of the tissue TM. The content of NADP in rat heart is, however, only about 5 % that of NAD and hence may limit the activity of the pentose-phosphate pathway. Tissues such as liver and kidney in which the pentose-phosphate pathway is more pronounced have a much greater relative proportion of NADP 13. Results reported in the present paper suggest that the pentose-phosphate pathway may be indirectly stimulated by insulin as a result of the elevated glucose 6-phosphate concentration, even though the presence of this pathway could not be demonstrated from the ratio of 14C02 yields from LI-14CI- and I6-14C~glucose. Activity of the first two NADP-dependent enzymes of the pentose-phosphate pathway has been demonstrated in homogenates of rat heart by GLOCK AND MCLEAN15, and recently confirmed in this laboratory (E. A. JONES, unpublished observations). CREVASSE et al. TM have also demonstrated that homogenates of dog heart, suitably fortified with NADP, possess the enzymatic capacity for the oxidation of glucose by the pentose-phosphate pathway. Undoubtedly the activity of this pathway in cardiac muscle is very low compared with the Embden-Meyerhof and non-triose-phosphate pathways, but nevertheless may still represent a significant cytoplasmic NADPH 2 generating system in normal cardiac metabolism. This study was supported by grants from the U.S. Public Health Service (TI AM-5o77-o7 and PHS 122o2-ol).
Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pa. (U.S.A.) Baker Clinic Research Laboratory, Harvard Medical School, Boston, Mass. (U.S.A.) I 2 3 4 5 6 7 8 9 Io ii 12 13 14 15 16
J O H N R. WlLLIAMSON ROBERT A. KREISBERG
C. L. GEMMILL, Bull. Johns Hopkins Hosp., 66 (194o) 232. J. R. WILLIAMSON, J. Biol. Chem., 239 (1964) 2721. F. G. YOUNG, Proc. Roy. Acad. Sci., London, I 5 7 B (I962) i. K. L. MANCHESTER AND F. G. YOUNG, Vitamins Hormones, 19 (1961) 95. I. G. WOOL AND M. E. IfRAHL, Biochim. Biophys. Acta, 82 (1964) 606. I. G. WOOL, Am. J. Physiol., I99 (196o) 719 . I{. L. MANCHESTER, Biochim. Biophys. Acta, 7 ° (1963) 208. J. R. WlLLIAMSON AND H. A. KREBS, Biochem. J., 80 (1961) 54 o. R. W. ESTABROOK AND P. If. MAITRA, Anal. Biochem., 3 (1962) 369 . J. KATZ AND N. G. \•OOD, J. Biol. Chem., 238 (1963) 517 . J. c. SHIPP, I-[. I~.. DELCHER AND L. E. CREVASSE, Biochim. Biophys. Acta, 86 (1964) 399. A. WOLLENBERGER, O. RISTAU AND G. SCHOFFA, Arch. Ges. Physiol., 270 (196o) 399. H. B. BURCH, O. H. LOWRY AND P. VON DIPPE, J. Biol. Chem., 238 (1963) 2828. G. E. GLOCK AND P. MCLEAN, Biochem. J., 61 (1955) 388. G. E. GLOCK AND P. MCLEAN, Biochem. J., 56 (1954) 171. L. E. CREVASSE, J. C. SHIPP AND H. If. DELCHER, Biochim. Biophys. Acta, 86 (1964) 402.
Received October 5th, 1964 Biochim. Biophys. Acta, 97 (1965) 347-349