0013-7227/91/1293-1370$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 3 Printed in U.S.A.
Quinone Reductase Enzyme Activity in Pancreatic Islets* MICHAEL J. M A C D O N A L D Childrens Diabetes Center, University of Wisconsin Medical School, Madison, Wisconsin 53706
ABSTRACT. A water-soluble quinone, coenzyme Qo (CoQ0), was shown to stimulate insulin release, and dicumarol, an inhibitor of quinone reductase, inhibited glucose-induced insulin release in pancreatic islets. Since this suggested that quinone reductase might play some role in physiological insulin release, this enzyme was characterized in islets. More than 90% of the total activity was located in the cytosol, but the specific enzyme activity was highest in the microsomal fraction. The relative rates of activity with various substrates (CoQ0 =* durohydroquinone — hydroquinone — dichloroindophenol > menadione >
P
REVIOUS studies of pancreatic islets have implicated site II of the mitochondrial respiratory chain as being important for insulin release. Pancreatic islets (1) and insulinomas (2) are rich in the mitochondrial glycerol phosphate dehydrogenase. Succinate (when added to islets as its methyl ester to promote cellular penetration) has been shown to be an insulin secretagogue (3-5). Both succinate dehydrogenase, which is the first enzyme of succinate's metabolism, and glycerol phosphate dehydrogenase transfer electrons to ubiquinone [coenzyme Qi0 (CoQio)] at site II of the electron transport chain. It was, therefore, reasonable to ask whether CoQio, being a lipid-soluble mobile electron carrier, might exit the mitochondria and in some way interact in the cytosol to participate in insulin secretion. An obvious participant in this process might be quinone reductase (EC 1.6.99.2), previously called diaphorase, a flavin-containing cytosolic enzyme. The current study provides some suggestive evidence to implicate this enzyme in insulin release. It shows that water-soluble quinones are potent stimulators of insulin release and that dicumarol, an inhibitor of quinone reductase, inhibits glucose-induced insulin release. To implicate quinone reductase as a participant in insulin release, its properties in islets needed to be described. Since pancreatic islets exhibit a high amount of flavin-containing protein in their cytosol (6), and only one flavin enzyme in islet
duroquinone > CoQ6 = CoQio > ferricyanide) were similar to those described previously for quinone reductase from liver. Dicumarol, chlorpromazine, and T3 were much more potent inhibitors of the enzyme when NADPH was the coenzyme than when NADH was the coenzyme. Dicumarol was the most potent inhibitor. The enzyme was not inhibited by rotenone. Islets ranked second to liver in quinone reductase activity, but the activity in islets was much closer to that found in all other tissues examined. Quinone reductase may play a role in insulin secretion. (Endocrinology 129: 1370-1374,1991)
cytosol has been identified (glutathione reductase) (7), other flavin enzymes must be present in islet cytosol. In the current study quinone reductase was characterized as to intracellular location, substrate specificity, susceptibility to inhibitors, and activity relative to that in other tissues.
Materials and Methods Isolation of islets Pancreatic islets were isolated from well-fed 200- to 250-g Sprague-Dawley rats by digesting minced pancreas with collagenase (8) and separated from the bulk of the pancreas by centrifuging the digested pancreas in a discontinuous gradient ofFicoll(9). Insulin release Insulin release was studied according to a standard procedure, previously described (1, 5). Cytosol Cytosol was the supernatant fraction obtained from centrifuging a homogenate of tissue at 105,000 X g for 1 h. Tissues were homogenized in 230 mM mannitol, 70 mM sucrose, and 5 mM potassium 4-(2-hydroxyethyl-l)piperazine ethanesulfonic acid, pH 7.5, as previously described (10). Mitochondria and microsomes were isolated by differential centrifugation, as previously described (10,11). Quinone reductase activity
Received March 11,1991. Address all correspondence and requests for reprints to: Michael J. MacDonald, University of Wisconsin Medical School, 1300 University Avenue, Room 3459, Madison, Wisconsin 53706. * This work was supported by NIH Grant DK-28348.
Quinone reductase enzyme activity was measured spectrophotometrically at 37 C in 1 ml reaction mixture containing tissue cytosol or islet subcellular fraction, 50 mM Tris buffer (pH 7.6), 200 MM NADH or NADPH, and the concentrations 1370
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 17:29 For personal use only. No other uses without permission. . All rights reserved.
QUINONE REDUCTASE IN ISLETS of electron acceptor and/or inhibitor described. Oxidation of NAD(P)H was monitored at 340 nm (12).
TABLE 2. Effect of dicumarol on CoQ0-induced and glucose-induced insulin release
DEAE chromatography
Protein Protein was determined by the method of Lowry et al. (13). Materials NAD and NADH were obtained from P-L Laboratories (Milwaukee, WI). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI) in the highest purity available.
Results Insulin release CoQo stimulated significant insulin release at a concentration of 50 nM. Maximal insulin release was observed at 0.5 mM CoQo (Table 1). Benzoquinone and hydroquinone also stimulated insulin release. Dicumarol inhibited glucose-induced insulin release, but not CoQ0induced insulin release (Table 2). Quinone reductase activity The major quinone reductase activity in islets has many of the characteristics of DT diaphorase (12) More than 90% of the total activity is located in the cytosol (Table 1), and activities with NADH and NADPH are approximately equal (Tables 3 and 4 and Fig. 1). The specific activity was highest in the microsomal fraction. TABLE 1. Stimulation by QuinonesQ of insulin release from pancreatic islets Insulin release insulin/5 islets • h) 29 ± 12 (10)
None CoQo 50 MM 0.5 mM 1 mM Benzoquinone 50 MM 1 mM Hydroquinone (1
Insulin release insulin/5 islets-h)
Conditions
Cytosol (200 n\) representing about 1500 islets was mixed with 1.5 ml 10 mM potassium phosphate buffer, pH 6.4, and applied to a DEAE-Sephacyl column (0.125 cm2 X 6 cm) previously equilibrated with the same buffer. The column was washed with 1.5 ml 10 mM phosphate buffer, and then the quinone reductase was eluted from the column with a continuous gradient of 1.5 ml potassium phosphate, pH 6.4, (10-500 mM). Fifty-microliter samples were collected.
Quinone cone.
1371
169 ± 29 (4) 385 ± 33 (10) 293 ± 64 (15) 124 ± 60 (4) 260 ± 59 (5) 295 ± 31 (5)
Results are the mean ± SD. The numbers of observations is given in parentheses.
No addition
28 ± 2 0 221 ± 41 271 ± 31
CoQo (100 fiM)
CoQo (100 MM) + dicumarol (10 (iM)
Glucose (20 mM) Glucose (20 mM) and dicumarol
131 ± 44 50 ± 16
(10/iM)
Results are the mean ± SD. There were five replicate incubations for each condition. TABLE 3. Intracellular distribution of enzyme activities in pancreatic islets that reduce CoQ0 or ferricyanide in the presence of either NADH or NADPH Total activity (nmol/min) fraction
Homogenate Nuclei/cell debris Mitochondria Microsomes Cytosol
SA (nmol/min • mg protein)
NADPH NADH NADH NADPH NADH NADH CoQo
CoQo Fe(CN)6
109 4.6
117 17.5
6.8 2.5 97
13.8 12.5 78
CoQo
CoQo Fe(CN)6
399 87.2
157 21
168 81
596 402
106 54 0
58 143 217
118 345 173
926 1483 0
There was no appreciable enzyme rate in the presence of NADPH and ferricyanide in any fraction, and thus, these data are not shown. TABLE 4. Quinone reductase-like enzyme activity in pancreatic islets assayed in the presence of various substrates
Substrate Homogenate of whole islets CoQo (50 MM) Duroquinone (100 MM) Durohydroquinone (100 MM) Hydroquinone (100 MM) Menadione (100 MM) Dichloroindophenol (50 mM) CoQ6 (100 MM) CoQ10 (50 MM) Islet cytosol CoQo Potassium ferricyanide (100 MM)
Relative enzyme activity NADH
NADPH
100 38 60
90 52 120
74
106
52 101 NT NT
58 104 0 0
100 0-72°
90 0
Rates are expressed relative to the rate with CoQo and NADH as equal to 100. Results are the mean of two to four observations. Measurements were repeated with two to seven different batches of islets with similar results. 0 Rate apparently depends on the amount of contamination with mitochondrial or microsomal enzymes. The relative rates of activities of the cytosolic enzyme with various substrates (CoQ0 - durohydroquinone ^= hydroquinone ^ dichloroindophenol > menadione >
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 17:29 For personal use only. No other uses without permission. . All rights reserved.
QUINONE REDUCTASE IN ISLETS
1372
Endo•1991 Voll29«No3
tors of the enzyme when NADPH was the coenzyme than when NADH was the coenzyme (Table 5). Other than this difference, the pattern with inhibitors resembled that of the classical DT diaphorase. The islet diaphorase was inhibited the most by dicumarol and the least by T 3 and chlorpromazine; it was not inhibited at all by rotenone. Similarly to early reports (12), liver cytosol contained by far the highest quinone reductase activity. Islets ranked second in quinone reductase activity, but the activity in islets (one seventh of that in liver) was closer to the range of activities found in all other tissues examined (Table 6). 1
10
20
30
35
Discussion
FRACTION NUMBER FIG. 1. Profile of quinone reductase activity eluted from a DEAESephacyl column. Enzyme activity in each fraction was measured with CoQo (50 MM) and either NADH (—; 100 M M) or NADPH (- - -; 100 MM) as substrates. Activity in each fraction with NADH or NADPH plus ferricyanide as substrates was zero and is, therefore, not shown in the figure. The apparent small peak of activity with NADH as substrate in fractions 23-26 was not observed in four repeat experiments.
CoQ6 = CoQio > ferricyanide) are similar to those described previously for DT diaphorase (12). Upon chromatography on DEAE-Sephacyl, only one peak of enzyme activity was obtained (Fig. 1), which suggests that a single enzyme is responsible for the islet quinone reductase activity. The activity with NADH was about 5-10% higher than that with NADPH, but the profiles of activity with CoQ0 and in respect to either NADH or NADPH as a substrate were identical. Depending on the individual preparation, cytosol contained a variable amount of enzyme activity when ferricyanide was the electron acceptor and NADH was the coenzyme (Tables 3 and 4). This activity varied between completely absent to 70% of the rate attributable to quinone reductase. This rate in the presence of ferricyanide was probably due to variable contamination of the cytosol fraction by the very active mitochondrial NADH dehydrogenase (14-18) or to a NADH ferricyanide dehydrogenase found in membrane fractions in tissues, such as liver (19). These activities with ferricyanide were not due to quinone reductase, because they could be separated from quinone reductase by chromatography on DEAE-Sephacyl (Fig. 1) and because an enzyme rate was never observed when NADPH was the substrate with ferricyanide. Even though this rate was occasionally as high as 70% of quinone reductase, it represented only a small fraction (
Vol. 129, No. 3 Printed in U.S.A.
Quinone Reductase Enzyme Activity in Pancreatic Islets* MICHAEL J. M A C D O N A L D Childrens Diabetes Center, University of Wisconsin Medical School, Madison, Wisconsin 53706
ABSTRACT. A water-soluble quinone, coenzyme Qo (CoQ0), was shown to stimulate insulin release, and dicumarol, an inhibitor of quinone reductase, inhibited glucose-induced insulin release in pancreatic islets. Since this suggested that quinone reductase might play some role in physiological insulin release, this enzyme was characterized in islets. More than 90% of the total activity was located in the cytosol, but the specific enzyme activity was highest in the microsomal fraction. The relative rates of activity with various substrates (CoQ0 =* durohydroquinone — hydroquinone — dichloroindophenol > menadione >
P
REVIOUS studies of pancreatic islets have implicated site II of the mitochondrial respiratory chain as being important for insulin release. Pancreatic islets (1) and insulinomas (2) are rich in the mitochondrial glycerol phosphate dehydrogenase. Succinate (when added to islets as its methyl ester to promote cellular penetration) has been shown to be an insulin secretagogue (3-5). Both succinate dehydrogenase, which is the first enzyme of succinate's metabolism, and glycerol phosphate dehydrogenase transfer electrons to ubiquinone [coenzyme Qi0 (CoQio)] at site II of the electron transport chain. It was, therefore, reasonable to ask whether CoQio, being a lipid-soluble mobile electron carrier, might exit the mitochondria and in some way interact in the cytosol to participate in insulin secretion. An obvious participant in this process might be quinone reductase (EC 1.6.99.2), previously called diaphorase, a flavin-containing cytosolic enzyme. The current study provides some suggestive evidence to implicate this enzyme in insulin release. It shows that water-soluble quinones are potent stimulators of insulin release and that dicumarol, an inhibitor of quinone reductase, inhibits glucose-induced insulin release. To implicate quinone reductase as a participant in insulin release, its properties in islets needed to be described. Since pancreatic islets exhibit a high amount of flavin-containing protein in their cytosol (6), and only one flavin enzyme in islet
duroquinone > CoQ6 = CoQio > ferricyanide) were similar to those described previously for quinone reductase from liver. Dicumarol, chlorpromazine, and T3 were much more potent inhibitors of the enzyme when NADPH was the coenzyme than when NADH was the coenzyme. Dicumarol was the most potent inhibitor. The enzyme was not inhibited by rotenone. Islets ranked second to liver in quinone reductase activity, but the activity in islets was much closer to that found in all other tissues examined. Quinone reductase may play a role in insulin secretion. (Endocrinology 129: 1370-1374,1991)
cytosol has been identified (glutathione reductase) (7), other flavin enzymes must be present in islet cytosol. In the current study quinone reductase was characterized as to intracellular location, substrate specificity, susceptibility to inhibitors, and activity relative to that in other tissues.
Materials and Methods Isolation of islets Pancreatic islets were isolated from well-fed 200- to 250-g Sprague-Dawley rats by digesting minced pancreas with collagenase (8) and separated from the bulk of the pancreas by centrifuging the digested pancreas in a discontinuous gradient ofFicoll(9). Insulin release Insulin release was studied according to a standard procedure, previously described (1, 5). Cytosol Cytosol was the supernatant fraction obtained from centrifuging a homogenate of tissue at 105,000 X g for 1 h. Tissues were homogenized in 230 mM mannitol, 70 mM sucrose, and 5 mM potassium 4-(2-hydroxyethyl-l)piperazine ethanesulfonic acid, pH 7.5, as previously described (10). Mitochondria and microsomes were isolated by differential centrifugation, as previously described (10,11). Quinone reductase activity
Received March 11,1991. Address all correspondence and requests for reprints to: Michael J. MacDonald, University of Wisconsin Medical School, 1300 University Avenue, Room 3459, Madison, Wisconsin 53706. * This work was supported by NIH Grant DK-28348.
Quinone reductase enzyme activity was measured spectrophotometrically at 37 C in 1 ml reaction mixture containing tissue cytosol or islet subcellular fraction, 50 mM Tris buffer (pH 7.6), 200 MM NADH or NADPH, and the concentrations 1370
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 17:29 For personal use only. No other uses without permission. . All rights reserved.
QUINONE REDUCTASE IN ISLETS of electron acceptor and/or inhibitor described. Oxidation of NAD(P)H was monitored at 340 nm (12).
TABLE 2. Effect of dicumarol on CoQ0-induced and glucose-induced insulin release
DEAE chromatography
Protein Protein was determined by the method of Lowry et al. (13). Materials NAD and NADH were obtained from P-L Laboratories (Milwaukee, WI). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) or Aldrich Chemical Co. (Milwaukee, WI) in the highest purity available.
Results Insulin release CoQo stimulated significant insulin release at a concentration of 50 nM. Maximal insulin release was observed at 0.5 mM CoQo (Table 1). Benzoquinone and hydroquinone also stimulated insulin release. Dicumarol inhibited glucose-induced insulin release, but not CoQ0induced insulin release (Table 2). Quinone reductase activity The major quinone reductase activity in islets has many of the characteristics of DT diaphorase (12) More than 90% of the total activity is located in the cytosol (Table 1), and activities with NADH and NADPH are approximately equal (Tables 3 and 4 and Fig. 1). The specific activity was highest in the microsomal fraction. TABLE 1. Stimulation by QuinonesQ of insulin release from pancreatic islets Insulin release insulin/5 islets • h) 29 ± 12 (10)
None CoQo 50 MM 0.5 mM 1 mM Benzoquinone 50 MM 1 mM Hydroquinone (1
Insulin release insulin/5 islets-h)
Conditions
Cytosol (200 n\) representing about 1500 islets was mixed with 1.5 ml 10 mM potassium phosphate buffer, pH 6.4, and applied to a DEAE-Sephacyl column (0.125 cm2 X 6 cm) previously equilibrated with the same buffer. The column was washed with 1.5 ml 10 mM phosphate buffer, and then the quinone reductase was eluted from the column with a continuous gradient of 1.5 ml potassium phosphate, pH 6.4, (10-500 mM). Fifty-microliter samples were collected.
Quinone cone.
1371
169 ± 29 (4) 385 ± 33 (10) 293 ± 64 (15) 124 ± 60 (4) 260 ± 59 (5) 295 ± 31 (5)
Results are the mean ± SD. The numbers of observations is given in parentheses.
No addition
28 ± 2 0 221 ± 41 271 ± 31
CoQo (100 fiM)
CoQo (100 MM) + dicumarol (10 (iM)
Glucose (20 mM) Glucose (20 mM) and dicumarol
131 ± 44 50 ± 16
(10/iM)
Results are the mean ± SD. There were five replicate incubations for each condition. TABLE 3. Intracellular distribution of enzyme activities in pancreatic islets that reduce CoQ0 or ferricyanide in the presence of either NADH or NADPH Total activity (nmol/min) fraction
Homogenate Nuclei/cell debris Mitochondria Microsomes Cytosol
SA (nmol/min • mg protein)
NADPH NADH NADH NADPH NADH NADH CoQo
CoQo Fe(CN)6
109 4.6
117 17.5
6.8 2.5 97
13.8 12.5 78
CoQo
CoQo Fe(CN)6
399 87.2
157 21
168 81
596 402
106 54 0
58 143 217
118 345 173
926 1483 0
There was no appreciable enzyme rate in the presence of NADPH and ferricyanide in any fraction, and thus, these data are not shown. TABLE 4. Quinone reductase-like enzyme activity in pancreatic islets assayed in the presence of various substrates
Substrate Homogenate of whole islets CoQo (50 MM) Duroquinone (100 MM) Durohydroquinone (100 MM) Hydroquinone (100 MM) Menadione (100 MM) Dichloroindophenol (50 mM) CoQ6 (100 MM) CoQ10 (50 MM) Islet cytosol CoQo Potassium ferricyanide (100 MM)
Relative enzyme activity NADH
NADPH
100 38 60
90 52 120
74
106
52 101 NT NT
58 104 0 0
100 0-72°
90 0
Rates are expressed relative to the rate with CoQo and NADH as equal to 100. Results are the mean of two to four observations. Measurements were repeated with two to seven different batches of islets with similar results. 0 Rate apparently depends on the amount of contamination with mitochondrial or microsomal enzymes. The relative rates of activities of the cytosolic enzyme with various substrates (CoQ0 - durohydroquinone ^= hydroquinone ^ dichloroindophenol > menadione >
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 16 November 2015. at 17:29 For personal use only. No other uses without permission. . All rights reserved.
QUINONE REDUCTASE IN ISLETS
1372
Endo•1991 Voll29«No3
tors of the enzyme when NADPH was the coenzyme than when NADH was the coenzyme (Table 5). Other than this difference, the pattern with inhibitors resembled that of the classical DT diaphorase. The islet diaphorase was inhibited the most by dicumarol and the least by T 3 and chlorpromazine; it was not inhibited at all by rotenone. Similarly to early reports (12), liver cytosol contained by far the highest quinone reductase activity. Islets ranked second in quinone reductase activity, but the activity in islets (one seventh of that in liver) was closer to the range of activities found in all other tissues examined (Table 6). 1
10
20
30
35
Discussion
FRACTION NUMBER FIG. 1. Profile of quinone reductase activity eluted from a DEAESephacyl column. Enzyme activity in each fraction was measured with CoQo (50 MM) and either NADH (—; 100 M M) or NADPH (- - -; 100 MM) as substrates. Activity in each fraction with NADH or NADPH plus ferricyanide as substrates was zero and is, therefore, not shown in the figure. The apparent small peak of activity with NADH as substrate in fractions 23-26 was not observed in four repeat experiments.
CoQ6 = CoQio > ferricyanide) are similar to those described previously for DT diaphorase (12). Upon chromatography on DEAE-Sephacyl, only one peak of enzyme activity was obtained (Fig. 1), which suggests that a single enzyme is responsible for the islet quinone reductase activity. The activity with NADH was about 5-10% higher than that with NADPH, but the profiles of activity with CoQ0 and in respect to either NADH or NADPH as a substrate were identical. Depending on the individual preparation, cytosol contained a variable amount of enzyme activity when ferricyanide was the electron acceptor and NADH was the coenzyme (Tables 3 and 4). This activity varied between completely absent to 70% of the rate attributable to quinone reductase. This rate in the presence of ferricyanide was probably due to variable contamination of the cytosol fraction by the very active mitochondrial NADH dehydrogenase (14-18) or to a NADH ferricyanide dehydrogenase found in membrane fractions in tissues, such as liver (19). These activities with ferricyanide were not due to quinone reductase, because they could be separated from quinone reductase by chromatography on DEAE-Sephacyl (Fig. 1) and because an enzyme rate was never observed when NADPH was the substrate with ferricyanide. Even though this rate was occasionally as high as 70% of quinone reductase, it represented only a small fraction (
