Proc. Natl. Acad. Sci. USA Vol. 92, pp. 11869-11873, December 1995 Biochemistry

Ascorbic acid is essential for the release of insulin from scorbutic guinea pig pancreatic islets WILLIAM W. WELLS*, CHUN-ZHI Dou, LESLIE N. DYBAS, CHE-HUN JUNG, HARRISON L. KALBACH, AND DIAN PENG XU Department of Biochemistry, Michigan State University, East Lansing, MI 48824 Communicated by N. Edward Tolbert, Michigan State University, East Lansing, MI, September 13, 1995

ABSTRACT Pancreatic islets from young normal and dized by freshly derived dehydroascorbic acid. In the absence scorbutic male guinea pigs were examined for their ability to of ascorbate, therefore, cells may synthesize the protein pre- release insulin when stimulated with elevated D-glucose. Islets cursors lacking disulfide formation. To test this hypothesis, we from normal guinea pigs released insulin in a D-glucose- compared the ability of isolated pancreatic islets from normal dependent manner showing a rapid initial secretion phase and and scorbutic guinea pigs to release insulin as a response to three secondary secretion waves during a 120-min period. perifused glucose, in vitro, in the presence and absence of ascorbic Islets from scorbutic guinea pigs failed to release insulin acid as the 2-phosphate. The 2-phosphate group stabilizes ascor- during the immediate period, and only delayed and decreased bic acid (11). The derivative is susceptible to plasma membrane responses were observed over the 40-60 min after D-glucose alkaline phosphatase activity (12), thus releasing metabolically elevation. Insulin release from scorbutic islets was greatly active ascorbic acid for cellular uptake either as ascorbic acid elevated if 5 mM L-ascorbic acid 2-phosphate was supple- directly or as dehydroascorbic acid after extracellular oxidation mented in the perifusion medium during the last 60 min of (13). In the studies reported herein, we demonstrate the abnor- perifusion. When 5 mM L-ascorbic acid 2-phosphate was added mal release of insulin by pancreatic islets from scorbutic guinea to the perifusion medium concurrently with elevation of medium pigs and show, by direct measurement, in vitro, that ascorbic acid, D-glucose, islets from scorbutic guinea pigs released insulin as administered as the precursor, L-ascorbic acid 2-phosphate, stim- rapidly as control guinea pig islets and to a somewhat greater ulates the release of insulin from scorbutic guinea pig islets in the extent. L-Ascorbic acid 2-phosphate without elevated D-glucose presence of elevated glucose. had no effect on insulin release by islets from normal or scorbutic guinea pigs. The pancreas from scorbutic guinea pigs contained 2.4 times more insulin than that from control guinea pigs, EXPERIMENTAL PROCEDURES suggesting that the decreased insulin release from the scorbutic Materials. Guinea pigs were purchased from the Michigan islets was not due to decreased insulin synthesis but due to Department of Public Health and Charles River Breeding abnormal insulin secretion. Laboratories. Ascorbic acid-free diet for guinea pigs (ascorbic acid test-guinea pig) and bovine serum albumin (BSA; RIA Scorbutic guinea pigs have a depressed ability to release insulin grade) were purchased from United States Biochemical. from pancreatic islets when stimulated with glucose (1-3). This Ascorbic acid, collagenase type V, chicken egg albumin, condition is characterized by lowered glucose tolerance (1, 2), guanidine hydrochloride, anti-rabbit IgG (alkaline phos- degranulation of the f cells (3), and decreased deposition of phatase conjugated), p-nitrophenyl phosphate, 5-bromo-4- glycogen in the liver (2), attributed to decreased pancreatic chloro-3-indolyl phosphate, nitroblue tetrazolium, bisbenzamide, insulin content (2). All symptoms of abnormal insulin hypo- calf deoxyribonucleic acid, and DEAE-Sephadex were purchased function are alleviated by treatment of the deficient guinea from Sigma. Sephadex G-50 was purchased from Pharmacia pigs with ascorbic acid. Banerjee et al. (4) observed a decrease LKB. Bio-Gel P-2, Bio-Gel P-30 acrylamide, N,N'-methylenebis- in glutathione (GSH) and an increase in dehydroascorbic acid acrylamide, ammonium persulfate, SDS, and Coomassie brilliant in the pancreas of scorbutic guinea pigs. Thus, ascorbic acid blue R-250 were purchased from Bio-Rad. Nylon mesh (10 gm, and perhaps GSH are implicated in the regulation of insulin pore size) was purchased from Whatman. Standard guinea pig biosynthesis and/or release; yet, the mechanism is unknown. insulin and anti-guinea pig serum were kindly provided by Cecil In recent studies, it was shown that protein disulfide isomer- C. Yip (University of Toronto). Guinea pig pancreas was pur- ase (PDI) has intrinsic dehydroascorbate reductase activity chased from Rockland (Gilbertsville, PA). Difluorodinitroben- (5). A model was proposed in which dehydroascorbic acid zene was a product of Pierce. would cyclically act as an oxidant in the PDI reactions (6). That Scorbutic Guinea Pigs. Male weanling guinea pigs were is, the oxidation of intracellular ascorbic acid by a hypothetical 150-180 g at the beginning of the feeding periods. Control and oxidase or peroxidase was postulated to occur at the surface scorbutic animals were fed the same commercial ascorbic of the endoplasmic reticulum in cells undergoing secretory acid-free diet, ad libitum, throughout the experiments. The protein synthesis despite the presence of a high cytoplasmic control animals received ascorbic acid in their drinking water GSH/oxidized glutathione (GSSG) ratio (7). The resulting prepared daily in the form of a 0.1% solution neutralized with dehydroascorbic acid, moving across the endoplasmic reticu- sodium hydroxide to pH 7.0. Animals were weighed weekly. lum membrane, would oxidize the active center cysteines of Symptoms of weight loss and difficult movements were seen PDI, as shown by Venetianer and Straub (8, 9) and Givol et al. typically between 21 and 25 days after initiation of the dietary (10), which would in turn oxidize the nascent protein sulfhydryl protocol in the animals not supplemented with ascorbate. All groups for the native disulfide conformation. Ascorbic acid, animals were fasted overnight prior to the preparation of islets. reformed from the reaction with PDI, would diffuse back into Isolation of Islets. Islets were immediately isolated from the cytoplasmic space, and the reduced PDI would be reoxi- guinea pig pancreas by collagenase treatment by the proce-

The publication costs of this article were defrayed in part by page charge Abbreviations: GSH/GSSG, reduced/oxidized glutathione; BSA, bo- payment. This article must therefore be hereby marked "advertisement" in vine serum albumin. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 11869 Downloaded by guest on September 29, 2021 11870 Biochemistry: Wells et al. Proc. Natl. Acad. Sci. USA 92 (1995)

dures of Wollheim et al. (14) and Gardner and Jackson (15). the supernatant equivalent to those from 1 mg of fresh tissue Individual islets were hand-picked from the digest mixture in were separated by SDS/PAGE under the reducing conditions Krebs-Ringer bicarbonate (KRB) buffer with 0.1% BSA by described by Ito et al. (23). The separated proteins were then using a Pipetman with the aid of a Leica dissection microscope. electroblotted onto nitrocellulose membranes (0.2 ,tm, pore Islet Perifusions. A typical preparation of 40-60 islets from size) for 2 hr at 70 V. The insulin bands (A and B chains) were either control or scorbutic guineas pigs was placed in a visualized by rabbit anti-guinea pig insulin antibody, anti- chamber made from a plastic 2-ml syringe and sandwiched rabbit IgG antibody (conjugated with alkaline phosphatase), between 300 ,ul of a Bio-Gel P-2 slurry in the perifusion buffer 5-bromo-4-chloro-3-indolyl phosphate, and nitroblue tetrazo- (KRB). The buffers contained 118 mM NaCl, 5 mM KCl, 1.2 lium and quantitated by densitometry (densitometer package mM KH2PO4, 2.5 mM CaCl2, 1.2 mM MgSO4, 5 mM NaHCO3, SW 2000; Ultraviolet Products, San Gabriel, CA). and 10 mM Hepes (pH 7.4). The perifusion buffer consisted of Ascorbic Acid 2-Phosphate Dephosphorylation by Pancreatic KRB with 0.1% BSA. The solution was equilibrated with a Islets. To determine the extent to which typical islet preparations mixture of 95%02/5% CO2 for 15 min and adjusted to pH 7.4 hydrolyze ascorbic acid 2-phosphate, islets from normal and before perifusion. scorbutic guinea pigs were preincubated in 1.0 ml of incubation Tubing was connected to the sealed chamber immersed in a buffer containing 1.7 mM glucose and equilibrated with 95% waterbath at 37°C. In the first experimental series, buffer 02/5% CO2 at 37°C for 15 min. The incubation continued for 30 containing 1.7 mM D-glucose was perifused at a rate of 0.5 min in the same medium containing 20 mM glucose and 5 mM ml/min. Elevation of glucose concentration to 20 mM and ascorbic acid 2-phosphate. The incubation mixture was rapidly addition of 5 mM ascorbic acid 2-phosphate were done at the centrifuged, and 1.0 ml of 10% metaphosphoric acid/i mM indicated time of perifusion (see Figs. 2-4). Fractions of the thiourea/1 mM EDTA was added to the pellet with homogeni- perifusate were collected at 5-min intervals and were imme- zation. Aliquots of the homogenate were analyzed for DNA by diately frozen and stored at -70°C until analyzed for insulin the method of Labarca and Paigen (16). To the supernatant, an content by an ELISA procedure. equal volume of 20% metaphosphoric acid/2 mM thiourea/2 DNA Analysis. To avoid error due to variation in islet size or mM EDTA was added. This solution was centrifuged to remove protein contamination from the BSA-containing medium, we precipitated serum albumin and any solubilized cellular protein. expressed the released insulin on the basis of islet DNA. The The ascorbic acid in each solution was analyzed by HPLC and contents of the chamber (gel and islets) were suspended in 2 ml electrochemical detection as described (21). Control medium of 50 mM sodium phosphate, pH 7.4/2 M NaCl in a plastic tube without islets was analyzed simultaneously. and homogenized with a Tekmar (Cincinnati) Tissumiser at 70 V Electron Microscopy. Isolated islets from control and scor- for 2 min at 4°C. The gel slurry was removed by centrifugation of butic guinea pig pancreas were quickly fixed in 3% (vol/vol) the sample through a 0.22-,um (pore size) filter-containing cen- glutaraldehyde in 0.05 M cacodylate buffer (pH 6.8). The trifuge tube (Costar Spin-X). The DNA content was determined fluorimetrically by the method of Labarca and Paigen (16). samples were post-fixed in buffered 1% osmium tetroxide and stained in half-saturated uranyl acetate. After dehydration, the Guinea Pig Insulin Isolation. Insulin was isolated from samples were infiltrated with epoxy resin. Ultrathin sections frozen pancreas obtained from Rockland by the methods of Zimmerman and Yip (17) and Treacy et al. (18). The purifi- (85 nm) were cut, mounted on 300-mesh copper grids, and stained with acetate and lead citrate. The grids were cation was monitored by dot blot analysis of fractions using uranyl to which were viewed at rabbit anti-guinea pig serum and a secondary anti-rabbit carbon-coated stabilize the sections, antibody conjugated with alkaline phosphatase. 100 kV with a JEOL 100CX II Temscan electron microscope. Insulin Conjugation and ELISA Analysis. Guinea pig insu- Statistics. Statistical analysis of the mean ± SD was con- lin was conjugated with chicken egg albumin as described by ducted with the aid of computer software, INSTAT, from Tager (19) to assure binding to 96-well plates. The conjugated GraphPad (San Diego). guinea pig insulin was purified and stored at -70°C until used for a competitive ELISA analysis after the general procedure RESULTS of Bank (20). The data were calculated from the standard curve plots and expressed as ng of insulin released per 5-min Pancreatic Insulin, Ascorbic Acid, and Total GSH + GSSH. fraction per jig of islet DNA. The ascorbic acid content of pancreas from scorbutic guinea Pancreatic Ascorbic Acid, GSH + GSSG, and Insulin pigs was -1% that of the control animals (Table 1), verifying Analysis. The ascorbic acid content of control and scorbutic the severe ascorbic acid deficiency observed by other criteria guinea pig pancreas was assayed by homogenization of such as measurement of body weight and difficulty of mobility. weighed samples of tissue in 2 ml of 10% (vol/vol) metaphos- In the pancreas from scorbutic guinea pigs, the total GSH + phoric acid/I mM thiourea/1 mM EDTA. Aliquots of the GSSG was -70% that of the control animals. The insulin whole homogenate were taken for protein analysis by the content of the pancreas from scorbutic guinea pigs showed bicinchoninic acid protein assay protocol according to the some variation, two of four being similar to control values manufacturer's direction (Pierce) with BSA as standard. The + remainder of the homogenate was centrifuged and diluted 1:50 Table 1. Comparison of total GSH GSSH, ascorbic acid, and with homogenizing fluid for samples from control animals. insulin content of pancreas from normal and scorbutic guinea pigs The samples from ascorbic acid-deficient guinea pigs were GSH + GSSG, Ascorbic acid, directly analyzed. Ascorbic acid content for both samples was nmol/mg of ,utmol/g of Insulin, analyzed as described (21). For the analysis of total pancreatic Animals protein protein units GSH + GSSH, weighed samples of pancreas were homoge- Control 11.02 + 3.34 7.34 + 1.40 66 ± 32 nized in 5% (wt/vol) sulfosalicylic acid. Aliquots were taken Scorbutic 7.70 ± 3.04 0.08 ± 0.02* 158 ± 102 for protein analysis as described above. Other aliquots were centrifuged and total GSH + GSSG concentration was deter- Eight male guinea pigs 200-250 g were fed a scorbutigenic diet for mined the method of Roberts and Francetic (22). For the 25-28 days. The control group received daily supplements of 0.1% by neutralized ascorbic acid in the drinking water. The pancreas was analysis of pancreatic insulin, 200 mg of guinea pig pancreas rapidly removed after euthanasia and samples of tissue were removed was homogenized in 2 ml of acidified ethanol by using a and assayed for ascorbic acid, total GSH + GSSG, and insulin. Insulin Tekmar Tissumiser (17). The homogenate was centrifuged at is expressed as relative intensity units measured by an Ultraviolet 4000 x g for 30 min and the resulting supernatant fraction was Products densitometer. (n = 4.) further centrifuged at 10,000 x g for 30 min. The proteins in *Statistically different from control animals; P < 0.0001 (n = 4). Downloaded by guest on September 29, 2021 Biochemistry: Wells et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11871

1 2 3 4 5 6 7 R 25

z o

05 Control Scorbutic ~~~~~~~~~T FIG. 1. Western blot analysis of insulin from equal aliquots of control guinea pig pancreas (lanes 1-4) and scorbutic guinea pig -V- pancreas (lanes 5-8). whereas two others were significantly higher. The mean insulin content was 2.4 times higher than that of the control guinea pigs (Table 1 and Fig. 1). 0 15 30 45 60 75 90 105 120 135 150 Insulin Release from Guinea Pig Islet Cells in Response to Perifusion time, min Glucose Perifusion: Effect of Ascorbic Acid. The insulin release by pancreatic islets from normal guinea pigs in re- FIG. 3. Comparison of the effects of elevated D-glucose and sponse to glucose elevation was immediate with an initial delayed ascorbic acid 2-phosphate on insulin release from pancreatic phase followed by secondary waves of insulin secretion over islets of normal (0) and scorbutic (0) guinea pigs. Perifusions were the succeeding 120 min (Fig. 2). In contrast, pancreatic islets initiated with KRB medium supplemented with 0.1% BSA and 1.7 mM D-glucose at a rate of 0.5 ml/min and 37°C. D-Glucose was increased from scorbutic guinea pigs failed to respond immediately to to 20 mM after 30 min (open arrow), and after 90 min, 5 mM ascorbic glucose elevation. A delayed or secondary response (70-80 acid 2-phosphate was added (cross-hatched arrow). Values are the min) was significantly lower than that of the equivalent phase mean ± SD for normal (n = 5) and scorbutic (n = 9) animals. of release for control islet preparations. In the second series (Fig. 3), a pattern similar to the first series was seen for normal incubation medium was assessed by the analysis of ascorbic guinea pig islets-i.e., 5 mM ascorbic acid 2-phosphate had no acid released during a 30-min period as described above. effect on insulin release. However, scorbutic guinea pig islets Ascorbic acid levels in the islets from control and scorbutic responded to 20 mM D-glucose with increased insulin secretion guinea pigs were 0.33 and 0.04 nmol/p,g of DNA, respectively. only after 5 mM ascorbic acid 2-phosphate was added at The ascorbic acid levels of the medium resulting from islet perifusion time, 90 min. The results in Fig. 4 show that phosphatase activity were 0.78 and 0.26 nmol per ml per ,ug of pancreatic islets from scorbutic guinea pigs responded as DNA, respectively. The concentration of ascorbic acid was esti- rapidly as the controls with elevated insulin release when mated to be 3.0-5.0 ,uM and dependent on the number and size ascorbic "acid 2-phosphate was supplemented simultaneously of islets. Thus, the islets from scorbutic guinea pigs (Figs. 3 and with the elevation of glucose levels to 20 mM at perifusion 4) might require only micromolar levels of ascorbic acid for time, 30 min. Islets from scorbutic guinea pigs released in- expression of the glucose (20 mM)-stimulated insulin release. creased amounts of insulin compared with controls especially Electron Microscopy. Electron microscopic evidence (Fig. during the secondary release waves, suggesting they had 5) supports the concept that insulin is present in equal or accumulated elevated insulin deposits in preparation for a higher amounts in the islets from scorbutic animals compared future glucose-mediated signal event, a signal that somehow with islets from normal guinea pigs, in agreement with the data requires ascorbic acid. When the 1.7 mM D-glucose level of the obtained by the Western blot analysis (Fig. 1). perifusion medium was maintained over the entire incubation period, and 5 mM ascorbic acid 2-phosphate was supple- DISCUSSION mented after the first 30 min, no release of insulin from islets from control or scorbutic animals occurred over the next 120 The present study confirms the original observations of Sigal min (data not shown). and King (1) and Banerjee and coworkers (2-4) that the Ascorbic Acid 2-Phosphate Dephosphorylation by Pancre- release of insulin from pancreatic islets of scorbutic guinea pigs atic Islets. The islet-cell membrane phosphatase activity in the 45 25 40 35 20 -0i 30 -o 25 A . 15 -V 20 V L o 10 0. 15 10 "0 S a 5 0 0 15 30 45 60 75 90 105 120 135 150 0 L 0 15 30 45 60 75 90 105 120 135 150 Perifusion time, min Perifusion time, min FIG. 4. Comparison of the effects of elevated D-glucose (20 mM) FIG. 2. Comparison of the effects of elevated D-glucose (20 mM) and L-ascorbic acid 2-phosphate (5 mM) on insulin release from on insulin release from pancreatic islets of normal (0) and scorbutic pancreatic islets of normal (e) and scorbutic (0) guinea pigs. Perifu- (0) guinea pigs. Perifusions were initiated with KRB medium sup- sions were initiated with KRB medium supplemented with 0.1% BSA plemented with 0.1% BSA and 1.7 mM D-glucose at a rate of 0.5 and 1.7 mM D-glucose at a rate of0.5 ml/min and 37°C. D-Glucose was ml/min at 37°C. D-Glucose was increased to 20 mM after 30 min increased to 20 mM concurrently with 5 mM ascorbic acid 2-phosphate (arrow), followed by collection of fractions for 120 min. Values are the after 30 min (arrow). Values are the mean ± SD for normal (n = 3) mean ± SD (n = 3). and scorbutic (n = 5) animals. Downloaded by guest on September 29, 2021 11872 Biochemistry: Wells et al. Proc. Natl. Acad. Sci. USA 92 (1995)

FIG. 5. Electron micrographs of ,B cells from normal (A) and scorbutic (B) guinea pigs. (Bar = 720 nm.) Insulin-containing secretory granules in normal cells (A) are typically close to the plasma membrane (arrows), whereas those from a vitamin C-deficient cell (B) are mostly recessed from the plasma membrane (arrows). is impaired. However, we conclude, in contrast to Banerjee (2), glucose transport across ,B-cell membranes (25). In a recent that the biosynthesis of insulin is not ascorbic acid-dependent study, the inhibitory effect of ascorbic acid on insulin release but, rather, that ascorbic acid is necessary to mediate the from single rat pancreatic islets was reported (26) in which D-glucose-stimulated release of insulin. The discrepancy be- 50% inhibition of insulin secretion from normal rat islets tween our data and Banerjee's data (2, 3) relative to insulin occurred at 200 ,uM ascorbic acid. In addition, the ascorbic content of scorbutic guinea pig pancreas may be explained by acid content of the rat islets used in the previous study (26) was the method used by Banerjee (3), namely, a hypoglycemia 4.4 mM, whereas our pancreatic ascorbic acid values, ex- bioassay of guinea pig pancreas extracts in rabbits. Hypogly- pressed as nmol/g of protein, were 0.08 ± 0.02. This amount cemia bioassay of guinea pig insulin in rabbits may have been is -1% of that in control guinea pig pancreas. We chose the unsuitable considering the currently known wide differences in long-lasting inert provitamin C, ascorbic acid 2-phosphate, chemical structure and immunological properties of guinea pig which was hydrolyzed to low levels of ascorbate (3-5 ,uM) insulin compared with many other species (17, 24). presumably by islet phosphatase activity. This difference may Megadoses of vitamin C delayed insulin response to a explain why we observed the effect of ascorbic acid deficiency glucose challenge in normoglycemic human adults, suggesting on the D-glucose-dependent release of insulin from pancreatic possible conflicts between elevated plasma ascorbic acid and islets and demonstrated a requirement for ascorbic acid in Downloaded by guest on September 29, 2021 Biochemistry: Wells et al. Proc. Natl. Acad. Sci. USA 92 (1995) 11873 glucose-mediated insulin release. The previous study (26) of We thank Ms. Jacqueline I. Wood, Michigan State University inhibition of insulin secretion by levels of ascorbic acid >200 Center for Electron Optics, for electron microscopic analysis and ,uM, however, may provide an explanation for why plasma Carol McCutcheon for typing the manuscript. This work was sup- ascorbate concentrations are normally tightly regulated. ported by National Institutes of Health Grant DK 44456. The use of ascorbic acid 2-phosphate was crucial in the 1. Sigal, A. & King, C. G. (1936) J. Bio. Chem. 116, 489-492. present study since perifusion of ascorbic acid at low concen- 2. Banerjee, S. (1943) Ann. Biochem. Exp. Med. 3, 157-164. trations in the presence of trace transition metals and a pH of 3. Banerjee, S. (1944) Ann. Biochem. Exp. Med. 4, 33-36. 7.4 would likely have resulted in rapid oxidation of ascorbic 4. Banerjee, S., Deb, C. & Belavady, B. (1952) J. Bio. Chem. 195, acid to dehydroascorbic acid (27) and further degradation 271-276. products (28). In addition, Pence and Mennear (29) have 5. Wells, W. W., Xu, D. P., Yang, Y. & Rocque, P. A. (1990)J. Biol. reported the inhibitory effect of 2 mg % ("114 ,tM) dehy- Chem. 265, 15361-15364. droascorbic acid on insulin secretion from mouse pancreatic 6. Wells, W. W., Yang, Y., Deits, T. L. & Gan, Z.-R. (1993) Adv. islets. Although dehydroascorbic acid levels were not analyzed Enzymol. Relat. Areas Mol. Biol. 66, 149-201. 7. Wells, W. W. & Xu, D. P. (1995) J. Bioenerg. Biomembr. 26, in the present study, we believe that since the potential source, 369-377. ascorbic acid, was present at such low levels, the dehydroascor- 8. Venetianer, P. & Straub, F. B. (1964) Biochim. Biophys. Acta 89, bic acid was likewise very minimal. 189-190. The decreased phosphatase activity of islets from scorbutic 9. Venetianer, P. & Straub, F. B. (1965) Acta Physiol. Acad. Sci. animals, compared with normal animals using ascorbic acid Hung. 27, 303-315. 2-phosphate as substrate, is reminiscent of an old observation 10. Givol, D., Goldberger, R. F. & Anfinssen, C. B. (1964) J. Biol. that the alkaline phosphatase activity of the plasma of infants Chem. 239, PC3114-PC3116. and young children suffering from scurvy was low (30). This 11. Nomura, H., Ishiguro, T. & Morimoto, S. (1969) Chem. Pharm. Bull. 17, 387-393. was also seen by several workers in guinea pigs with attempts 12. Hitomi, K., Torii, Y. & Tsukagoshi, N. (1992) J. Nutr. Sci. made to rule out inanition by use of the paired-feeding Vitaminol. 38, 330-337. technique (31). The protocol used for all the present perifusion 13. Welch, R. W., Wang, Y., Crossman, A., Jr., Park, J. B., Kirk, K. L. experiments included uniformly oxygen-equilibrated medium & Levine, M. (1995) J. Bio. Chem. 270, 12584-12592. ruling out potential adverse effects of variable oxygen envi- 14. Wollheim, C. B., Meda, P. & Halban, P. A. (1990) Methods ronments known to affect insulin secretion from pancreatic Enzymol. 192, 188-204. islets (32). Our present results are consistent with a possible 15. Gardner, J. D. & Jackson, M. J. (1977) J. Physiol. (London) 270, role for modification of the redox state of the NADPH/NADP 439-454. 16. Labarca, C. & Paigen, K. (1980) Anal. Biochem. 102, 344-352. and GSH/GSSG systems modulated by entry of D-glucose into 17. Zimmerman, A. E. & Yip, C. C. (1974) J. Biol. Chem. 249, the f3 cells (33), since a decreased but not statistically signif- 4021-4025. icant total GSH + GSSH level was observed in pancreas from 18. Treacy, G. B., Shaw, D. C., Griffiths, M. E. & Jeffrey, P. D. scorbutic animals compared with those from normal animals. (1989) Biochim. Biophys. Acta 990, 263-268. Our original proposal for a role of an ascorbic acid redox 19. Tager, H. S. (1976) Anal. Biochem. 71, 367-375. cycle in protein disulfide formation using insulin biosynthesis 20. Bank, H. L. (1988) J. Immunoassay 9, 135-158. as a model is not supported by the present observations. We 21. Wells, W. W., Rocque, P. A., Xu, D.-P., Meyer, E. B., Chara- have examined the insulin in the pancreas of scorbutic guinea mella, L. J. & Dimitrov, N. V. (1995) Free Radicals Biol. Med. 18, 699-708. pigs by SDS/PAGE under reducing and nonreducing condi- 22. Roberts, J. C. & Francetic, D. J. (1993) Anal. Biochem. 211, tions. No reduced insulin was detected in scorbutic guinea pigs 183-187. under nonreducing SDS/PAGE, and no proinsulin, which 23. Ito, K., Date, T. & Wickner, W. (1980) J. Biol. Chem. 255, might have accumulated if the proper disulfide bond formation 2123-2130. was defective, was detected. Instead, the present work identi- 24. Smith, L. F. (1972) Diabetes 21 (Suppl. 2), 457-460. fied a function for ascorbic acid in enhancing the competency 25. Johnston, C. S. & Yen, M.-F. (1994) Am. J. Clin. Nutr. 60, of glucose in the activation cascade of insulin release (34). 735-738. The broad elements of glucose-induced insulin secretion 26. Bergsten, P., Moura, A. S., Atwater, I. & Levine, M. (1994) J. have been reviewed Glucose-induced Biol. Chem. 269, 1041-1045. extensively (35-37). 27. Feng, J., Melcher, A. H., Brunette, D. M. & Moe, D. K. (1977) insulin release results from vesicular exocytosis, a process that In Vitro 13, 91-99. is triggered by the entry of Ca2+ across the plasma membrane 28. Kazuko, T. & Ohmura, T. (1966) Nippon Nogei Kagaku Kaishi 40, (38, 39). Ca2+ is known to enter through voltage-sensitive 196-200. calcium channels. Accordingly, glucose must generate a signal 29. Pence, L. A. & Mennear, J. H. (1979) Toxicol. Appl. Pharmacol. that involves the depolarization of the 13-cell membrane. 50, 57-65. Current studies reveal a synergistic interaction in ,3 cells 30. Smith, J. & Maizels, M. (1932) Arch. Dis. Child. 7, 149-158. between the glucose-regulated ATP-dependent signaling sys- 31. Todhunter, E. N. & Brewer, W. (1940) Am. J. Physiol. 130, tem and the hormonally regulated 310-318. cAMP-dependent signaling 32. Dionne, K. E., Colton, C. K. & Yarmush, M. L. (1993) Diabetes system. This interaction gives ,B cells the ability to match the 42, 12-21. ambient concentration of glucose to an appropriate insulin 33. Ammon, H. P. T., Grimm, A., Lutz, S., Wagner-Teschner, D., secretory response, a process referred to by Holz and Habener Handel, M. & Hagenloh, I. (1980) Diabetes 29, 830-834. (39) as the induction of glucose competence. Recently, ascor- 34. Matschinsky, F. M. (1990) Diabetes 39, 647-652. bic acid has been reported to modulate calcium channels in 35. Hedeskov, C. J. (1980) Physiol. Rev. 60, 442-509. pancreatic 1 cells by inactivating (IC50 = 1 mM) the slow 36. Zawalich, W. S. & Rasmussen, H. (1990) Mol. Cell. Endocrinol. deactivating calcium channels (40). The observed effect of 70, 119-137. metal mediation the 37. Newgard, C. B. & McGarry, J. D. (1995)Annu. Rev. Biochem. 64, ascorbic acid requires ions, suggesting by 689-714. oxidation product, dehydroascorbic acid, previously known to 38. Wollheim, C. B. & Sharp, G. W. G. (1981) Physiol. Rev. 61, inhibit insulin release (28). Our results offer an explanation for 914-973. the old observations of Sigal and King (1) and Banerjee and 39. Holz, G. G. & Habener, J. F. (1992) Trends Biol. Sci. 17,388-393. coworkers (2-4) that demonstrated an abnormal response of 40. Parsey, R. V. & Matteson, D. R. (1993) J. Gen. Physiol. 102, scorbutic guinea pigs to D-glucose-stimulated insulin release. 503-523. Downloaded by guest on September 29, 2021