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Pyridine nucleotides in teleost islets
Comp. Biochem. Physiol., 1967, 17ol. 22, pp. 297 to 301. Pergamon Press. Printed in Great Britain
SHORT COMMUNICATION PYRIDINE
NUCLEOTIDES
IN TELEOST
ISLETS*
ARNOLD W. L I N D A L L Department of Anatomy, T h e Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A. (Received 23 J a n u a r y 1967)
A b s t r a c t - - 1 . Pyridine nucleotide concentrations were surveyed in a variety of tissues of the toadfish and goosefish including the islets of Langerhans. 2. Compared to the rat the teleosts have lower concentrations of the pyridine nucleotides. T h e islets have much lower concentrations of these cofactors than the corresponding livers. 3. In the goosefish islet, there appears to be more N A D P + N A D P H t relative to the total pyridine nucleotides than is found in the toadfish. A similar feature is observed in the activities of glutathione reductase. 4. Goosefish islet has almost twice the activity of toadfish islet and also has almost twice as much N A D P H . INTRODUCTION
THE ISLETSof Langerhans in certain teleost fish, notably the toadfish (Opsanus tau) and the goosefish (Lophius araericanus), have been studied in detail. These fish are convenient experimental animals because the islets are separated from the acinar pancreas into a discrete organ (Rennie, 1904; McCormick, 1924) which is ideal for physiological and chemical studies. Studies on the enzyme content (Lazarow et al., 1964), the isolated insulin secretion granule fraction (Lindall et al., 1963) and the biosynthesis of insulin (Bauer et al., 1966) of islets have been reported. In the present work, the pyridine nucleotide concentrations of a variety of tissues from goosefish and toadfish were compared to the islet. The enzyme, glutathione reductase (IUB number 1.6.4.2), was measured in the islets of the two fish. MATERIAL AND METHODS
The pyridine nucleotides were determined by a fluorometric method previously modified in our laboratory (Lindall & Lazarow, 1964). In order to accommodate the small tissue samples in the islet, optic lobes and pituitary of the toadfish, the extraction procedure outlined in Table 1 was used. For goosefish islet, a total * This work was supported by Diabetes training grant AM-(TI)-5127 and special fellowship 2 F I I NB 1068-02. t T h e abbreviations used are N A D and N A D H for the oxidized and reduced forms of nicotinamide adenine dinucleotide respectively and N A D P and N A D P H for the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate respectively. 297
298
ARNOLD W . LINDALL
volume of 1 ml of extracting medium was used (step IV of Table 1) with all additions scaled down tenfold from the previously reported procedure. TABLE 1 - - P Y R I D I N E NUCLEOTIDE EXTRACTION PROCEDURE FOR SMALL TISSUE SAMPLES
NAD and NADP TrisoHCl
II III 1V V
VI VII
NADH and NADPH Tris-NaOH
100/zl 0'1 N Tris, pH 8-2 100/~1 0-1 N Tris, pH 8'2 60/~1 H 20 60/zl H 20 10/d 2-0 N HCI 10/~1 2"0 N NaOH Preheat briefly in boiling water bath. 2-10 mg tissue to each microhomogenizer, crush with pestle, heat 30 min in bath. Remove homogenizer and grind tissue for 1 rain while hot. Cool quickly in ice water. 10 pl 2"0 N NaOH 10/~1 2"0 N HCI Add directly to homogenizer 5 pl glucose-6-PO4 dehydrogenase (0"2 units, Sigma Chemical Co.) 5/zl 0"1 M glucose-6-POa 5/xl 0'035 M EDTA 5/zl ADH (0"01 mg Sigma Chemical Co.) 5/zl 0"4% acetaldehyde Incubate 20 rain at room temperature; spin 10 rain 2000 rev/min at room temperature. Analysis by routine procedure (Lindall & Lazarow, 1964).
In this study, live fish kept in running sea water at the Marine Biological Laboratory, Woods Hole, were stunned by a blow on the head and tissue samples were removed, weighed and quickly placed in the appropriate extracting media preheated in a boiling water-bath. T h e tissue was 1-2 min post mortem at the time of extraction. T h e method for glutathione reductase was adapted from Ciotti & Kaplan (1957). The procedure is as follows: to a microcuvette for the Beckman model DU were added 0.1/~moles oxidized glutathione, 0.033 t~moles N A D P H , 10-20 ~1 of a onetenth tissue homogenate in 0.1 M K2HPO4 buffer, pH 7.5. The total volume was adjusted to 300 tA with the same buffer. T h e buffer, oxidized glutathione, and N A D P H were added first; a baseline reading at 340 mt~ was taken for 3 min. T h e n the tissue was added and readings were continued for 4 min. Tissue activity was expressed as ~moles N A D P H oxidized.min -1 (g of tissue) -1 under the conditions described. T h e reaction was carried out at room temperature. All enzymes and cofactors were purchased from Sigma Chemical Company. RESULTS In comparison to rat liver, analysed under similar conditions, the fish livers have lower concentrations of pyridine nucleotides (Table 2). Toadfish and goosefish livers had 41 and 20 per cent as much total pyridine nucleotide respectively
6
Rat Liver
33 39 10 9 9 9
561 _+ 52
157+24 55+13
359 + 288 + 214 + 189+ 114+ 83 + --* --*
NAD
228 _+20
58+15 24+4
99 + 11 91 + 13 72 + 10 33+ 4 22+ 2 --* 31 +_ 5 17 + 3
NADH
* Below t h e sensitivity of t h e m e t h o d . U n i t s : m/zmoles/g f r e s h tissue _+s t a n d a r d error.
5 6
9 10 10 9 14 6 6 6
No. o f animals
29
27 30
22 24 25 15 16
% reduced
106 _+9
9+3 4+1
24 + 3 6+2 14 + 2 17+2 13+4 14 + 8 --* ~*
NADP
R A T LIVER INCLUDED FOR COMPARISON
487 _+38
49+8 42+3
76 + 7 17 + 3 31 + 7 39+3 29+2 m* 35 __ 5 29 _+2
NADPH
82
85 91
76 74 69 70 69
% reduced
1382
278 125
570 401 331 272 178 m ---
Total
100 45
--
100 70 58 48 31 --
liver (total)
% of
NUCLEOTIDE CONCENTRATION OF TOADFISH AND GOOSEFISH I " I S S U ~ I N THE ORDER OF TOTAL CONCENTRATION.
Goosefish Liver Islet
Toadfish Liver Heart Kidney Pituitary Islet Abdominal muscle O p t i c lobes Gill
TABLE 2--PYRIDINE
tO ~O
t"
t~
Z
300
ARNOLDW. LINDALL
as rat liver. There would also appear to be proportionately less of the phosphorylated forms ( N A D P + N A D P H ) in the fish tissues, 18 and 21 per cent for the toadfish and the goosefish livers respectively compared to 43 per cent in the rat liver. Liver contained the highest total concentration of pyridine nucleotides in the toadfish. The other tissues studied ranged (as a percentage of the liver value) from 9 per cent in the gill to 70 per cent in the heart ventricle. The two endocrine organs, the pituitary and islet, had 48 and 31 per cent of the liver value respectively. Generally, the total NAD (NAD + NADH) had about the same distribution as the total pyridine nucleotide in toadfish. The total NADP ( N A D P + NADPH) was highest in liver followed by pituitary, kidney and islet in decreasing order. The abdominal muscle, optic lobes and gill are peculiar in some respects. Abdominal muscle had almost no detectable reduced forms while both the optic lobes and gill had almost no oxidized forms. These three tissues had the lowest total pyridine nucleotide concentration. Only two tissues were studied in the goosefish, liver and islet. Total islet pyridine nucleotide concentration was only 45 per cent of the value for goosefish liver. However, there appeared to be a large quantity of total NADP which amounted to 80 per cent of the goosefish liver value (compared to only 42 per cent in the toadfish). The glutathione reductase activity in goosefish and toadfish islet respectively was 1.55+0.1 (standard error) and 0.92_+0.13/~moles NADPH oxidized . (g of tissue) -1 min -1 (six animals each). It is significant that the NADPH, one of the substrates for this enzyme, had a similar distribution (42 _+3 and 29 + 2 m/~moles/g tissue for goosefish and toadfish respectively). The differences between the islets for both the enzyme and coenzyme are highly significant (P< 0-005). In contrast to the higher glutathione reductase and NADPH in goosefish islet, the total pyridine nucleotide is higher in the toadfish islet. DISCUSSION The teleost tissues studied have lower pyridine nucleotide concentrations than measured in rat liver by the same procedure. This would be expected since the fish tissues generally have lower enzyme activities (Lazarow et al., 1964). The islet of the toadfish does not seem unique, except for a decreased percentage reduced NAD. On the other hand, goosefish islet had a relatively large proportion of total NADP, mainly in the reduced form. No direct evidence exists for involvement of pyridine nucleotides in insulin synthesis; however, an enzyme has been found in rat liver which will reduce insulin with reduced glutathione as a donor (Katzen & Stetten, 1962). This enzyme has been called a glutathione-insulin transhydrogenase. The source of reduced glutathione in tissue is probably glutathione reductase and NADPH. Thus, the presence of a glutathione reductase could be of importance in insulin metabolism. Goosefish islet contains about 160 m/zmoles/g of reduced glutathione (Lazarow, 1948). Falkmer (1961) estimated oxidized glutathione to be about one-fourth of
PYRIDINE NUCLEOTIDES IN TELEOST ISLETS
301
the reduced glutathione in Cottus scorpius. I f this is true in goosefish islet, one would expect about 40 m # m o l e s of oxidized glutathione. T h e total glutathione content would then be about fourfold higher than the total N A D P content. T h e glutathione reductase activity is well above the substrate level in islet if one does not take into account compartmentalization. SUMMARY Pyridine nucleotides were measured in a n u m b e r of tissues f r o m two teleost fish. Glutathione reductase was measured in the islets of Langerhans. Acknowledgement--The author wishes to acknowledge the kind support of Arnold Lazarow, head of the Anatomy Department, University of Minnesota.
REFERENCES BAUER G . E., LINDALL A. W . , DIXIT P. K., LmTER G. & LAZAROW A. (1966) Studies o f
insulin biosynthesis. J. Cell Biol. 28, 413-421. CIOTTI M. M. & KAPLAN N. O. (1957) Methods in Enzymology. p. 894. Academic Press, New York. FALKMERS. (1961) Experimental diabetes in fish. Acta Endocrinol. Supplement 59. KATZENH. M. & STETTEND. (1962) Hepatic glutathione-insulin transhydrogenase. Diabetes 11, 271-280. LAZAROWA. (1948) Further studies on the mechanism of alloxan action; the reaction of alloxan with sulfhydryl groups; the glutathione content of islet tissue. Biol. Bull., Woods Hole 95, 276. LAZAROWA., DIXIT P. K., LINDALLA. W., MORANJ., HOSTETLERF. & COOPEaSaXlN S. J. (1964) Symposium on the Structure and Metabolism of Pancreatic Islets (Edited by BROLIN S. E., HELLMANB. & KNUTSONH.) pp. 203-221. Pergamon Press, Oxford. LINDALL A. W., BAUER G. E., DIXIT P. K. & LAZAROWA. (1963) Isolation of an insulin secretion granule fraction. J. Cell Biol. 19, 317-324. LINDALLA. W. & LAZAROWA. (1964) A critical study of pyridine nucleotide concentrations in normal fed, normal fasted, and diabetic rat liver. Metabolism 13, 259-271. McCORMICK N. A. (1924) Distribution and structure of islands of Langerhans in certain fresh water and marine fish. Trans. R. Can. Inst. 15, 57-58. RENNIE J. (1904) The epithelial islets of the pancreas in teleostei. Quart. J. microsc. Sci. 48, 379-405.
SHORT COMMUNICATION PYRIDINE
NUCLEOTIDES
IN TELEOST
ISLETS*
ARNOLD W. L I N D A L L Department of Anatomy, T h e Medical School, University of Minnesota, Minneapolis, Minnesota, U.S.A. (Received 23 J a n u a r y 1967)
A b s t r a c t - - 1 . Pyridine nucleotide concentrations were surveyed in a variety of tissues of the toadfish and goosefish including the islets of Langerhans. 2. Compared to the rat the teleosts have lower concentrations of the pyridine nucleotides. T h e islets have much lower concentrations of these cofactors than the corresponding livers. 3. In the goosefish islet, there appears to be more N A D P + N A D P H t relative to the total pyridine nucleotides than is found in the toadfish. A similar feature is observed in the activities of glutathione reductase. 4. Goosefish islet has almost twice the activity of toadfish islet and also has almost twice as much N A D P H . INTRODUCTION
THE ISLETSof Langerhans in certain teleost fish, notably the toadfish (Opsanus tau) and the goosefish (Lophius araericanus), have been studied in detail. These fish are convenient experimental animals because the islets are separated from the acinar pancreas into a discrete organ (Rennie, 1904; McCormick, 1924) which is ideal for physiological and chemical studies. Studies on the enzyme content (Lazarow et al., 1964), the isolated insulin secretion granule fraction (Lindall et al., 1963) and the biosynthesis of insulin (Bauer et al., 1966) of islets have been reported. In the present work, the pyridine nucleotide concentrations of a variety of tissues from goosefish and toadfish were compared to the islet. The enzyme, glutathione reductase (IUB number 1.6.4.2), was measured in the islets of the two fish. MATERIAL AND METHODS
The pyridine nucleotides were determined by a fluorometric method previously modified in our laboratory (Lindall & Lazarow, 1964). In order to accommodate the small tissue samples in the islet, optic lobes and pituitary of the toadfish, the extraction procedure outlined in Table 1 was used. For goosefish islet, a total * This work was supported by Diabetes training grant AM-(TI)-5127 and special fellowship 2 F I I NB 1068-02. t T h e abbreviations used are N A D and N A D H for the oxidized and reduced forms of nicotinamide adenine dinucleotide respectively and N A D P and N A D P H for the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate respectively. 297
298
ARNOLD W . LINDALL
volume of 1 ml of extracting medium was used (step IV of Table 1) with all additions scaled down tenfold from the previously reported procedure. TABLE 1 - - P Y R I D I N E NUCLEOTIDE EXTRACTION PROCEDURE FOR SMALL TISSUE SAMPLES
NAD and NADP TrisoHCl
II III 1V V
VI VII
NADH and NADPH Tris-NaOH
100/zl 0'1 N Tris, pH 8-2 100/~1 0-1 N Tris, pH 8'2 60/~1 H 20 60/zl H 20 10/d 2-0 N HCI 10/~1 2"0 N NaOH Preheat briefly in boiling water bath. 2-10 mg tissue to each microhomogenizer, crush with pestle, heat 30 min in bath. Remove homogenizer and grind tissue for 1 rain while hot. Cool quickly in ice water. 10 pl 2"0 N NaOH 10/~1 2"0 N HCI Add directly to homogenizer 5 pl glucose-6-PO4 dehydrogenase (0"2 units, Sigma Chemical Co.) 5/zl 0"1 M glucose-6-POa 5/xl 0'035 M EDTA 5/zl ADH (0"01 mg Sigma Chemical Co.) 5/zl 0"4% acetaldehyde Incubate 20 rain at room temperature; spin 10 rain 2000 rev/min at room temperature. Analysis by routine procedure (Lindall & Lazarow, 1964).
In this study, live fish kept in running sea water at the Marine Biological Laboratory, Woods Hole, were stunned by a blow on the head and tissue samples were removed, weighed and quickly placed in the appropriate extracting media preheated in a boiling water-bath. T h e tissue was 1-2 min post mortem at the time of extraction. T h e method for glutathione reductase was adapted from Ciotti & Kaplan (1957). The procedure is as follows: to a microcuvette for the Beckman model DU were added 0.1/~moles oxidized glutathione, 0.033 t~moles N A D P H , 10-20 ~1 of a onetenth tissue homogenate in 0.1 M K2HPO4 buffer, pH 7.5. The total volume was adjusted to 300 tA with the same buffer. T h e buffer, oxidized glutathione, and N A D P H were added first; a baseline reading at 340 mt~ was taken for 3 min. T h e n the tissue was added and readings were continued for 4 min. Tissue activity was expressed as ~moles N A D P H oxidized.min -1 (g of tissue) -1 under the conditions described. T h e reaction was carried out at room temperature. All enzymes and cofactors were purchased from Sigma Chemical Company. RESULTS In comparison to rat liver, analysed under similar conditions, the fish livers have lower concentrations of pyridine nucleotides (Table 2). Toadfish and goosefish livers had 41 and 20 per cent as much total pyridine nucleotide respectively
6
Rat Liver
33 39 10 9 9 9
561 _+ 52
157+24 55+13
359 + 288 + 214 + 189+ 114+ 83 + --* --*
NAD
228 _+20
58+15 24+4
99 + 11 91 + 13 72 + 10 33+ 4 22+ 2 --* 31 +_ 5 17 + 3
NADH
* Below t h e sensitivity of t h e m e t h o d . U n i t s : m/zmoles/g f r e s h tissue _+s t a n d a r d error.
5 6
9 10 10 9 14 6 6 6
No. o f animals
29
27 30
22 24 25 15 16
% reduced
106 _+9
9+3 4+1
24 + 3 6+2 14 + 2 17+2 13+4 14 + 8 --* ~*
NADP
R A T LIVER INCLUDED FOR COMPARISON
487 _+38
49+8 42+3
76 + 7 17 + 3 31 + 7 39+3 29+2 m* 35 __ 5 29 _+2
NADPH
82
85 91
76 74 69 70 69
% reduced
1382
278 125
570 401 331 272 178 m ---
Total
100 45
--
100 70 58 48 31 --
liver (total)
% of
NUCLEOTIDE CONCENTRATION OF TOADFISH AND GOOSEFISH I " I S S U ~ I N THE ORDER OF TOTAL CONCENTRATION.
Goosefish Liver Islet
Toadfish Liver Heart Kidney Pituitary Islet Abdominal muscle O p t i c lobes Gill
TABLE 2--PYRIDINE
tO ~O
t"
t~
Z
300
ARNOLDW. LINDALL
as rat liver. There would also appear to be proportionately less of the phosphorylated forms ( N A D P + N A D P H ) in the fish tissues, 18 and 21 per cent for the toadfish and the goosefish livers respectively compared to 43 per cent in the rat liver. Liver contained the highest total concentration of pyridine nucleotides in the toadfish. The other tissues studied ranged (as a percentage of the liver value) from 9 per cent in the gill to 70 per cent in the heart ventricle. The two endocrine organs, the pituitary and islet, had 48 and 31 per cent of the liver value respectively. Generally, the total NAD (NAD + NADH) had about the same distribution as the total pyridine nucleotide in toadfish. The total NADP ( N A D P + NADPH) was highest in liver followed by pituitary, kidney and islet in decreasing order. The abdominal muscle, optic lobes and gill are peculiar in some respects. Abdominal muscle had almost no detectable reduced forms while both the optic lobes and gill had almost no oxidized forms. These three tissues had the lowest total pyridine nucleotide concentration. Only two tissues were studied in the goosefish, liver and islet. Total islet pyridine nucleotide concentration was only 45 per cent of the value for goosefish liver. However, there appeared to be a large quantity of total NADP which amounted to 80 per cent of the goosefish liver value (compared to only 42 per cent in the toadfish). The glutathione reductase activity in goosefish and toadfish islet respectively was 1.55+0.1 (standard error) and 0.92_+0.13/~moles NADPH oxidized . (g of tissue) -1 min -1 (six animals each). It is significant that the NADPH, one of the substrates for this enzyme, had a similar distribution (42 _+3 and 29 + 2 m/~moles/g tissue for goosefish and toadfish respectively). The differences between the islets for both the enzyme and coenzyme are highly significant (P< 0-005). In contrast to the higher glutathione reductase and NADPH in goosefish islet, the total pyridine nucleotide is higher in the toadfish islet. DISCUSSION The teleost tissues studied have lower pyridine nucleotide concentrations than measured in rat liver by the same procedure. This would be expected since the fish tissues generally have lower enzyme activities (Lazarow et al., 1964). The islet of the toadfish does not seem unique, except for a decreased percentage reduced NAD. On the other hand, goosefish islet had a relatively large proportion of total NADP, mainly in the reduced form. No direct evidence exists for involvement of pyridine nucleotides in insulin synthesis; however, an enzyme has been found in rat liver which will reduce insulin with reduced glutathione as a donor (Katzen & Stetten, 1962). This enzyme has been called a glutathione-insulin transhydrogenase. The source of reduced glutathione in tissue is probably glutathione reductase and NADPH. Thus, the presence of a glutathione reductase could be of importance in insulin metabolism. Goosefish islet contains about 160 m/zmoles/g of reduced glutathione (Lazarow, 1948). Falkmer (1961) estimated oxidized glutathione to be about one-fourth of
PYRIDINE NUCLEOTIDES IN TELEOST ISLETS
301
the reduced glutathione in Cottus scorpius. I f this is true in goosefish islet, one would expect about 40 m # m o l e s of oxidized glutathione. T h e total glutathione content would then be about fourfold higher than the total N A D P content. T h e glutathione reductase activity is well above the substrate level in islet if one does not take into account compartmentalization. SUMMARY Pyridine nucleotides were measured in a n u m b e r of tissues f r o m two teleost fish. Glutathione reductase was measured in the islets of Langerhans. Acknowledgement--The author wishes to acknowledge the kind support of Arnold Lazarow, head of the Anatomy Department, University of Minnesota.
REFERENCES BAUER G . E., LINDALL A. W . , DIXIT P. K., LmTER G. & LAZAROW A. (1966) Studies o f
insulin biosynthesis. J. Cell Biol. 28, 413-421. CIOTTI M. M. & KAPLAN N. O. (1957) Methods in Enzymology. p. 894. Academic Press, New York. FALKMERS. (1961) Experimental diabetes in fish. Acta Endocrinol. Supplement 59. KATZENH. M. & STETTEND. (1962) Hepatic glutathione-insulin transhydrogenase. Diabetes 11, 271-280. LAZAROWA. (1948) Further studies on the mechanism of alloxan action; the reaction of alloxan with sulfhydryl groups; the glutathione content of islet tissue. Biol. Bull., Woods Hole 95, 276. LAZAROWA., DIXIT P. K., LINDALLA. W., MORANJ., HOSTETLERF. & COOPEaSaXlN S. J. (1964) Symposium on the Structure and Metabolism of Pancreatic Islets (Edited by BROLIN S. E., HELLMANB. & KNUTSONH.) pp. 203-221. Pergamon Press, Oxford. LINDALL A. W., BAUER G. E., DIXIT P. K. & LAZAROWA. (1963) Isolation of an insulin secretion granule fraction. J. Cell Biol. 19, 317-324. LINDALLA. W. & LAZAROWA. (1964) A critical study of pyridine nucleotide concentrations in normal fed, normal fasted, and diabetic rat liver. Metabolism 13, 259-271. McCORMICK N. A. (1924) Distribution and structure of islands of Langerhans in certain fresh water and marine fish. Trans. R. Can. Inst. 15, 57-58. RENNIE J. (1904) The epithelial islets of the pancreas in teleostei. Quart. J. microsc. Sci. 48, 379-405.