Thioredoxin and thioredoxin reductase in pancreatic islets may participate in diabetogenic free-radical production

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 107, No. 4, 1982

Pages 1412-1418

August 31, 1982

THIOREDOXIN AND THIOREDOXIN REDUCTASE PARTICIPATE

Kjell Grankvist,

IN DIABETOGENIC

IN PANCREATIC

ISLETS MAY

FREE-RADICAL PRODUCTION

Arne Holmgren, Mikaela Luthman and Inge-Bert Tgljedal

Department of Histology and Cell Biology, University of Ume~, S-901 87 Ume$, and Department Institutet,

of Chemistry,

S-I04 01 Stockholm,

Karolinska

Sweden

Received July 2, 1982

Mouse pancreatic islets rich in beta-cells contain about 42 pmol of thioredoxin and 1.8 pmol of thioredoxin reductase per mg islet protein, corresponding to a nominal concentration in the cell water of about 28 and 1.2 ~M. When mixed with alloxan, FeSO 4 and NADPH in a cell-free system, thioredoxin (3.4 pM) and thioredoxin reductase (0.2 ~M) purified from E. coli induced a production of oxy-radicals as evidenced by the emission of light from luminol. Mammalian thioredoxin reductase from calf thymus (0.2 ~M) was capable of eliciting luminescence even in the absence of thioredoxin. The thioredoxin/ thioredoxin reductase system may play important roles in the physiology and pathology of pancreatic beta-cells.

Pancreatic beta-cells have two interesting properties which are probably related. They secrete insulin in response to glucose, and they are especially sensitive to the cytotoxic action of alloxan, become increasingly

clear that reduction-oxidation

role in the mechanisms toxicity

a diabetogenic

of both secretory control

drug (I). It has

processes play a central (2-5) and alloxan cyto-

(6-12). A secretory signal may be transmitted

from glucose metabolism

to the exocytosis machinery by mediation of NAD(P)H-dependent changes

in the beta-cell plasma membrane

thiol-blocking

alloxan

(2-4); experiments with thiols and

agents led to the idea that this signal chain involves the

reduction of membrane-located Membrane

ion-permeability

disulphides

(2,4,13).

thiol groups have also been envisaged as the primary target of

(13,14). Like other reductants,

thiols can convert alloxan to dialuric

acid. Dialuric acid may in turn autoxidize back along with a production of H202, 02 - and OH.

(6-10). The extreme and indiscriminatory

0006-291X/82/161412-07501.00/0 Copyright © 1982byAcademw Press, ~c. Aflrigh~ofreproductioninanyform reserved.

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reactivity of OH.

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BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

may explain the many varied expressions of damage in alloxan-treated betacells. The evidence for the above hypothesis of alloxan action notwithstanding, the identity of the primary reductant is unclear.

In a cell-free system the

12,000 dalton redox protein, thioredoxin, from E. coli or calf thymus is very effective in catalyzing the oxidation of NADPH by alloxan (15). We report here that mouse pancreatic islets rich in beta-cells contain substantial amounts of thioredoxin and thioredoxin reductase.

It is also demonstrated

that the reduction of alloxan by thioredoxin is accompanied by an intense generation of oxy-radicals. MATERIALS AND METHODS Islet homogenates.Collagenase-isolated islets from non-inbred Ume~-ob/ ob-mice were homogenized by ultrasonication in 100-200 ~i of Tris-HCl buffer, pH 7.4. For each homogenate hundreds of islets from the pancreases of about 4 mice were pooled to yield 4-5 mg of protein/ml; protein was assayed (16) with human serum albumin as standard. Thioredoxin and thioredoxin reductase.- Thioredoxin and thioredoxin reductase from E. coli (17,18) and calf thymus (15,19) were purified as described. To measure the analogues in islet homogenates we used an assay (19) based on the reduction of insulin disulphides by the reduced form of thioredoxin (reactions I and II): + thioredoxin + (I) thioredoxin-S 2 + NADPH + H reductase ~ thi°red°xin-(SH)2 + NADP (II)

thioredoxin-(SH) 2 + insulin-S 2

~

thioredoxin-S 2 + insulin-(SH) 2

+

Net:

NADPH + H

+

+ insulin-S 2 '

~

NADP

+ insulin-(SH) 2

The dependence of reactions I and II on thioredoxin-(SH) 2 makes it possible to determine thioredoxin and thioredoxin reductase in crude tissue extracts. Incubations were run for 20 min at 37oc in a final volume of 120 ~I containing: 340 ~M bovine insulin, 80 mM 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid buffer, pH 7.6, 3 mM EDTA, 0.7 mM NADPH and the specified concentrations of calf thymus thioredoxin, calf thymus thioredoxin reductase, and islet homogenate. The reaction was stopped by adding 500 ~I of I mM 5,5'-dithiobis(2-nitrobenzoic acid) in 6 M guanidine-HCl/0.2 M Tris-HCl, pH 8.0. The absorbance at 412 nm was spectrophotometrically determined. A molar extinction coefficient for thionitrobenzoic acid of 13,600 was used in the calculations. Thioredoxin was determined in the presence of 2.3 ~g/ml (20 riM) thioredoxin reductase, and thioredoxin reductase in the presence of 67 ~g/ml (5.6 ~M) thioredoxin. Islet homogenate was added to give an islet-protein concentration of about 0.2 mg/ml in the assay of thioredoxin and 1.6 mg/ml in the assay of thioredoxin reductase. Calf thymus thioredoxin at final concentrations of 11-110 nM and thioredoxin reductase at final concentrations of 0.050.5 nM were incubated as standards in parallel with the islet extracts. Tissue blanks were run without exogenous thioredoxin and thioredoxin reductase. Luminol luminescence.In the presence of molecular oxygen and iron ions the reduction of alloxan may cause the formation of oxy-radicals (20,21). To study the capacity of thioredoxin and thioredoxin reductase to catalyze this

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

process, radical production was monitored with the aid of luminol. Luminol luminescence is dependent on the relaxation to its ground state of a 3-aminophtalate di-anion excited by free radicals (22,23). Measurements were performed as previously (21) except that the reaction volume was I ml instead of 5 ml. Reductants as specified in the text were added to a mixture of 100 ~M alloxan, 10 ~M FeSO 4 and I ~M luminol in a salt-balanced buffer of the same composition as Krebs-Ringer bicarbonate (24) except that bicarbonate was replaced by 20 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid, pH 7.4. The reaction was carried out in the dark in vials that had been kept in the dark overnight. Measurements were made in a liquid-scintillation counter with the coincidence function switched off. Chemicals.NADPH was from Sigma Chemical Co., St. Louis, MO, USA, and Boehringer-Mannheim GmbH, Mannheim, Germany. 5,5'-Dithiobis(2-nitrobenzoic acid) and luminol were from Sigma, GSH from Boehringer, bovine insulin (26.1 IU/mg) from Vitrum AB, Stockholm, Sweden, and alloxan from United States Biochemical Corp., Cleveland, OH, USA. Statistical analysis.Results are given as mean values ± S.E.M. The twotailed Wilcoxon's rank sum test was used to evaluate experimental effects. RESULTS Islet homogenates produced (3 homogenates)

106 ± 7 pmol of thionitrobenzoic

when incubated with NADPH and insulin in the absence of exo-

genous thioredoxin and thioredoxin reductase. is protein

As 8 1 %

of the islet dry weight

(16), this value is 2-4 times the concentration

groups estimated by 6,6'-dithiodinicotinic dithiodipyridine

acid/~g protein

in intact islets

of endogenous thiol

acid in islet homogenates or 2,2'-

(20-40 pmol/~g dry islet; ref. 25). The

difference probably reflects the activity of native thioredoxin/thioredoxin reductase operating under suboptimal conditions. After adding calf thymus thioredoxin to the islet homogenates

the formation

of thiol groups in 20 min rose about 4-fold to 404 ! 64 pmol/~g protein homogenates). reductase/~g

This increase corresponded islet protein

(Fig.

(3

to 1.8 ± 0.4 fmol of thioredoxin

I). When the insulin-reducing

activity was

only corrected for the thiol background measured by 6,6'-dithiodinicotinic acid or 2,2'-dithiodipyridine

(37 pmol/~g islet protein;

ref. 25), the islet

content of thioredoxin reductase was estimated to be 2.2 ± 0.4 fmol/~g protein (3 homogenates). To measure islet thioredoxin,

insulin reduction was determined

presence of calf thymus thioredoxin reductase.

The rate of reduction above

the background recorded without added thioredoxin reductase 42 ± 3 fmol of thioredoxin/~g protein

(3 homogenates).

corresponded

to

When calculated as the

excess above the background measured by 6,6'-dithiodinicotinic

1414

in the

acid or 2,2'-

Vol. 107, No. 4, 1982

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

J

0.5

-."~27

10

20

30

t~0

50

60

70

FEMTOMOLES OF THIOREDOXIN-REDU£TASE

Fig. I. Relationship between A412 and the amount of calf thymus thioredoxin reductase (open circles) in the assay based on insulin reduction and thionitrobenzoic acid formation. The activities observed in parallel incubations of 3 different islet homogenates (solid circles) containing protein as indicated are also shown. The values are corrected for the background recorded when islet homogenates were incubated with NADPH and insulin in the absence of calf thymus thioredoxin. For details, see text. dithiodipyridine,

the results corresponded to 62 ± 5 fmol of thioredoxin/~g

protein. Table I summarizes the experiments with luminol. The basal mixture of alloxan, FeSO 4 and luminol exhibited a certain luminescence. The addition of 100 ~M GSH to this mixture enhances luminescence considerably

(21), but 100

~M NADPH or 3.4 ~M GSH did not. Neither did 3.4 ~M thioredoxin or 0.2 ~M thioredoxin reductase from E. coli enhance luminescence in comparison with NADPH alone. The combination of E. coli reductase with 3.4 ~M GSH caused a slight stimulation of luminescence

(vs. NADPH alone, P < 0.01). As bacterial

thioredoxin reductase has a marked specificity for thioredoxin (18), the effect of GSH probably reflects a contamination of the enzyme preparation by glutathione reductase. An impressive increase of luminescence occurred when E. coli thioredoxin reductase was combined with 3.4 ~M thioredoxin

(vs. NADPH alone or thiored-

oxin plus GSH, P < 0.01). Unlike bacterial thioredoxin reductase, the mammalian enzyme exerts a certain electron-transporting activity in the absence of thioredoxin, as reflected in an abi'lity to directly oxidize NADPH in the

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table I. Chemiluminescence of luminol in the presence of alloxan, FeSO 4 and various organic reductants Additions to basal reaction mixture

Luminescence (thousands of counts/30 s)

None

7.6±0.7

3,4 ~M GSH

7.2±0.7

100 ~M NADPH

4.0±0.5

100 ~M NADPH + 3.4 ~M GSH

4.9±0.7

100 ~M NADPH + 3.4 ~M E. col± thioredoxin

2.9±0.6

100 DM NADPH + 0.2 DM E. col± thioredoxin reductase

5.5±1.1

100 ~M NADPH + 3.4 ~M GSH + 0.2 ~M E. col± thioredoxin reductase 8.4 ± 1.1 100 ~M NADPH + 3.4 ~M E. col± thioredoxin + 0.2 ~M E. col± thioredoxin reductase

26.4 ± 3.0

100 ~M NADPH + 0.2 ~M calf thymus thioredoxin reductase

13.4 ± 2.7

The background reading from buffer alone was 2°2 ± 0.1 thousands of counts/30 s (19 determinations). All values in the table have been corrected for background. Results are mean values ± S.E.M. for 18 (NADPH alone) or 6 (other groups) observations. The significant (P < 0.01) inhibitory effect of NADPH alone is probably due to direct reactions of the nucleotide with H202, 02--. and OH. (21). For statistical testing of other effects, see text.

presence

of 5 , 5 ' - d i t h i o b i s ( 2 - n i t r o b e n z o i c

ance with this broader catalyzed

substrate

a significant

acid)

specificity,

luminescence

or alloxan

the enzyme

in the absence

(15,26).

In accord-

from calf thymus

of thioredoxin

(vs. NADPH

alone, P < 0.01). DISCUSSION The results ase

demonstrate

in pancreatic

the results Apart redoxin

islets.

are probably

As the islets valid

from the sulfurs and thioredoxin

the presence

reductase

and catalytic

standards

had been activated

similar

activation

difficulties unspecific minimum

activity

estimates

involved contain

activity.

reduct-

than 90 % beta-cells

groups

(26). The purified

by treatment

the reaction The reported

of t h i o r e d o x i n

in electron

thiol

(27)

was not p e r f o r m e d

values

for con-

here employed (19,26). because

reductase

regarded

as

A of the

a high background

are therefore

and thioredoxin

both thio-

of importance

proteins

and avoiding

1416

transfer,

w i t h dithiothreitol

of the islet homogenates

in c o n t r o l l i n g thiol

contain more

and thioredoxin

for this cell type.

directly

formation

of thioredoxin

as

in beta-cells.

of

Vol. I07, No. 4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The distribution of thioredoxin and thioredoxin reductase

in various cells

is poorly known. Purification work on E. coli (28) and rat liver Luthman,

to be published)

indicates that thioredoxin

(Holmgren &

is more abundant than

thioredoxin reductase on a molar basis; the difference may be about 20 times. The present finding of 42-62 fmol of thioredoxin and 1.8-2.2 fmol of thioredoxin reductase per ~g islet protein represents reactive thioredoxin has been measured

a similar proportion.

Immuno-

in various tissues of a one-week-old

calf (30). With liver and kidney being the highest and heart the lowest in immunoreactive

activity,

the range of 0.3-0.7 ~g/mg protein covers the present

value of 0.5-0.7 wg thioredoxin/mg

islet protein.

The presence of thioredoxin and thioredoxin reductase

in the beta-cells

interesting in view of the fact that these cells are highly specialized synthesizing

insulin;

10-15 % of their dry weight is insulin

proinsulin are very effective ductant

substrates

(32). Thioredoxin-catalyzed

is

for

(31). Insulin and

for thioredoxin as a disulphide re-

disulphide

formation

in proinsulin has not

yet been studied but from a chemical point of view is a plausible reaction. Moreover, membrane-located secretion

thiol groups have been suggested to control

(2,4,13) and participate

alloxan attack on beta-cells At concentrations

insulin

in the initial steps of the diabetogenic

(13,14).

much lower than those detected

in beta-cells,

purified

thioredoxin and thioredoxin reductase clearly catalyzed a production

of oxy-

radicals from alloxan. The hydrogen-donor

thiored-

oxin reductases

in catalyzing luminescence

alloxan-dependent

oxidation of NADPH

specificities

conform well with data on the

(15). These results support the ideas

that thioredoxin reductase may be responsible dialuric acid from alloxan in vivo dialuric acid autoxidation

of different

for the rapid generation of

(15) and that oxy-radicals

are mediators of the diabetogenic

arising from cytotoxicity

(7-10).

ACKNOWLEDGEMENTS This work was supported by the Swedish Medical Research Council 2288, 13x-3529) and the Swedish Diabetes Association.

1417

(12x-

Vol. 107, No. 4, 1982

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS REFERENCES

I. Rerup, C.C., Pharmacol. Rev. 22, 485-518 (1970). 2. Hellman, B., Idahl, L.-~., Lernmark, A., Sehlin, J., and T~ljedal, I.-B., Excerpta Medica Int. Congr. Ser. 312, 65-78 (1974). 3. Malaisse, W.J., Hutton, J.C., Kawazu, S., Herchuelz, A., Valverde, I. and Sener, A., Diabetologia 16, 331-341 (1979). 4. Ammon, H.P.T., Grimm, A., Lutz, S., Wagner-Teschner, D., Hgndel, M., and Hagenloh, I., Diabetes 29, 830-834 (1980). 5. Henquin, J.-C., Biochem. J. 186, 541-550 (1980). 6. Deamer, D.W., Heikkila, R.E., Panganamala, R.V., Cohen, G., and Cornwell, D.G., Physiol. Chem. Phys. 3, 426-430 (1971). 7. Heikkila, R.E., Winston, B., Cohen, G., and Barden, H., Biochem. Pharmacol. 25, 1085-1092 (1976). 8. Grankvist, K., Marklund, S., Sehlin, J., and Tgljedal, I.-B., Biochem. J. 182, 17-25 (1979). 9. Grankvist, K., Marklund, S., and Tgljedal, I.-B., Nature 294, 158-160 (1981). 10. Fischer, L.J., and Hamburger, S.A., Diabetes 29, 213-216 (1980). 11. Grankvist, K., Marklund, S., and Tgljedal, I.-B., Biochem. J. 199, 393398 (1981). 12. Malaisse, W.J., Malaisse-Lagae, F., Sener, A., and Pipeleers, D.G., Proc. Natl. Acad. Sci. 79, 927-930 (1982). 13. Cooperstein, S.J., and Watkins, D., Biochem. Biophys. Res. Commun. 79, 756-762 (1977). 14. Watkins, D., Cooperstein, S.J., and Fiel, S., J. Pharmacol. Exp. Ther. 208, 184-189 (1979). 15. Holmgren, A., and Lyckeborg, C., Proc. Natl. Acad. Sci. 77, 5149-5152 (1980). 16. Oagerman, E., Anal. Bioehem. 101, 494-497 (1980). 17. Holmgren, A., and Reichard, P., Eur. J. Biochem. 2, 187-196 (1967). 18. Thelander, L., J. Biol. Chem. 242, 852-859 (1967). 19. Engstr6m, N.-E., Holmgren, A., Larsson, A., and SSderhgll, S.J., J. Biol. Chem. 249, 205-210 (1974). 20. Cohen, G., and Heikkila, R.E., J. Biol. Chem. 249, 2447-2452 (1974). 21. Grankvist, K., Biochem. J. 200, 685-690 (1981). 22. Hodgson, E.K., and Fridovich, I., Photochem. Photobiol. 18, 451-455 (1973). 23. O'Brien, P.J., and Hulett, L.G., Prostaglandins 19, 683-691 (1980). 24. DeLuca, H.F., and Cohen, P.P., in Manometric Techniques (Umbreit, W,W., Burris, R.H., and Stauffer, J.F., eds.), pp. 131-133, Burgess, Minneapolis, 1964. 25. Hellman, B., Lernmark, A., Sehlin, J., SSderberg, M., and T~ljedal, I.-B., Endocrinology 99, 1398-1406 (1976). 26. Holmgren, A., J. Biol. Chem. 252, 4600-4606 (1977). 27. Hellman, B., Ann. N. Y. Acad. Sci. 131, 541-558 (1965). 28. Williams, C.H., Jr., Zanetti, G., Arscott, L.D., and McAllister, J.K., J. Biol. Chem. 242, 5226-5231 (1967). 29. Hellman, B., Sehlin., and Tfiljedal, I.-B., Diabetologia ~, 256-265 (1971). 30. Holmgren, A., and Luthman, M., Biochemistry 17, 4071-4077 (1978). 31. Hellman, B., and T~ljedal, I.-B., in Handbook of Physiology, section 7: Endocrinology, vol. I (Steiner, D.F. and Freinkel, N., eds.), pp. 91-110, The Williams & Wilkins Co., Baltimore, 1972. 32. Holmgren, A., J. Biol. Chem. 254, 9113-9119 (1979).

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