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isolated pancreatic islets to mannoheptulose: evidence for insulin deg- radation in the p-cell. Biochem Pharmacol 39:2625-33. 1980. 10. Peterson PA, Rask L, ...
Rapid Publications High-Glucose Stimulation of 64,000-/Wr Islet Cell Autoantigen Expression OLLE KAMPE, ARNE ANDERSSON, ELISABETH BJORK, ANDERS HALLBERG, AND F. ANDERS KARLSSON

This study examined the effect of various extracellular glucose concentrations on the expression of a previously described 64,000-/Wr islet cell autoantigen associated with insulin-dependent diabetes mellitus. The protein was precipitated from patient serums incubated with Triton X-100 lysates of [35S]methioninelabeled rat pancreatic islets that had been cultured in 5, 11, or 28 mM glucose for 6 h or 3 days. In both types of experiment, 28 mM glucose was the most efficient stimulator of 64,000-Mr autoantigen production. In contrast, the class I antigens of the major histocompatibility complex, precipitated by a rabbit polyclonal antiserum, were not influenced by differences in glucose concentrations. Our data indicate that expression of islet cell antigens may be increased during the course of hyperglycemia and suggest that the functional activity of islet cells influences their antigenicity. Diabetes 38:1326-28, 1989.

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nsulin-dependent diabetes mellitus (IDDM) is regarded as an autoimmune endocrine disease, characterized by lymphocytic infiltration of the islets and the formation of autoantibodies (1). Serums of patients with new-onset IDDM usually, but not always, contain antibodies that recognize a 64,000-/Wr protein of unknown function in detergent lysates of [35S]methionine-labeled islets of human or rat origin (2,3). Knowledge about the nature and production of the 64,000-/Wr antigen is limited. In this study, we examined the effects of various glucose concentrations on the expression in vitro of islet cell antigens identified from serums of patients

Glucose 1 mM = 18 mg/dl From the Department of Internal Medicine, University Hospital, and the Department of Medical Cell Biology, Biomedical Center, Uppsala University, Uppsala, Sweden. Address correspondence and reprint requests to F. Anders Karlsson, Department of Internal Medicine, University Hospital, S-751 85 Uppsala, Sweden. Received for publication 8 June 1989 and accepted in revised form 24 July 1989.

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with new-onset IDDM. A stimulatory effect of high glucose on the 64,000-Mr antigen was found.

RESEARCH DESIGN AND METHODS Islets from 250-g male Wistar-Furth rats were isolated by handpicking from collagenase digests of pancreases (4) and were cultured free floating in RPMI-1640 medium containing 10% (vol/vol) calf serum (SBL, Stockholm), 100 U/ml penicillin, and 100 |xg/ml streptomycin (5). Glucose concentration was adjusted to 5, 11, or 28 mM. For short-term culture, freshly isolated islets were transferred to the corresponding methionine-free medium and incubated with [35S]methionine (>500 Ci/mmol, New England Nuclear, Boston, MA) for 6 h. In long-term cultures, the islets were kept in the different glucose concentrations for 72 h. Mediums were then changed, and radiolabeling was performed during a 6-h incubation (500 islets/incubation). All incubations were performed at 37°C in humidified air containing 5% CO2. After labeling, islets were washed twice with ice-cold medium without calf serum. Islets were lysed at 4°C in 20 mM Tris-HCI buffer (pH 7.4) containing 0.15 mM NaCI, 1% (vol/ vol) Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 1% (vol/vol) Trasylol (Bayer, Leverkusen, FRG). Insoluble material was removed by centrifugation at 100,000 x g for 60 min at 4°C. The lysates were precleared overnight at 4°C with human control serum and subsequently incubated in equal aliquots for 24 h with the different antiserums. Immunoprecipitates were collected as described by Sege et al. (6). In brief, a 20% suspension of formalinized, heatkilled Staphylococcus aureus (Cowan I strain) was added in fivefold excess over the volume of serum used. After 30 min, the bacteria were collected by centrifugation at 10,000 x g for 2 min. The pellets were subsequently washed five times in ice-cold 20 mM Tris-HCI buffer (pH 8.0) containing 0.15 M NaCI, 0.2% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, and 1 % Trasylol. Finally, the bacteria were washed once in distilled water. The bound material was eluted by heating the bacteria at 95°C for 3 min in gel sample buffer consisting of 0.1 mM Tris-HCI (pH 8.8), 3% sodium dodecyl sulfate (BDH, Poole, DIABETES, VOL. 38, OCTOBER 1989

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KAMPE AND ASSOCIATES

lysates from 6-h incubations. Furthermore, a 38,000-/Wr component that was stimulated by increasing glucose concentrations was detected in lysates of islets cultured for 3 days. However, this precipitate was not disease specific, because it was found not only with the two patient serums chosen for the detailed studies but also with a rabbit anti-rat major histocompatibility complex (MHC) class I antiserum. In contrast, we detected no influence of variations in glucose concentration on the amount of the cell membrane-bound MHC class I proteins (a heavy 47,000-/Wr chain and a light 11,000-Mr chain). Likewise, a 45,000-M, protein that was recognized by the serum precipitating the 18,000Mr component was not influenced by differences in glucose RESULTS In initial experiments, 10 serums from patients with new- concentration. In an experiment with islets cultured for 3 days, we exonset IDDM were examined by immunoprecipitation of radiolabeled rat pancreatic islets cultured for 3 days in 11 mM amined the biosynthesis of (pro)insulin in samples of islets glucose. Six of 10 serums reacted with a double band of in parallel to studies with radiolabeling and immunoprecip-64,000 M,. Two serums precipitated an 18,000-/Wr protein itation. Islets cultured with 5, 11, or 28 mM glucose synthebut not the 64,000-M, doublet. In 2 serums, no specific sized (pro)insulin at mean rates of 2.0, 4.2, and 5.3 kdpm • 1 1 bands, i.e., different from those precipitated with serums of 20 islets" • 2 h" , respectively. Corresponding figures for healthy individuals, were found (data not shown). In exper- the mean contribution of (pro)insulin to the amount of newly iments aimed at a detailed study of glucose effects on synthesized protein were 20, 32, and 29%. A stimulatory islet cell autoantigen expression, 1 serum recognizing the effect of glucose on the 64,000- and 18,000-Mr proteins was 64,000-/Wr protein and 1 serum recognizing the 18,000-Mr also found by separate analysis of immunoprecipitates (not shown) in this experiment. band were used. Short-term incubation of islets in 5, 11, or 28 mM glucose for 6 h caused a glucose-dependent increase of the 64,000- DISCUSSION Mr doublet. A more pronounced influence of glucose on the We report a stimulatory effect of high glucose concentrations labeling of this antigen was observed after culturing the islets on two different antigens in isolated rat pancreatic islets—an for 3 days (Fig. 1). The expression of the 18,000-Mr antigen -64,000 M, double band and an 18,000-/Wr antigen. The was also glucose dependent. However, it was only precip- 64,000-/V/r double band corresponds to the previously deitated from lysates of islets cultured for 3 days but not from scribed 64,000-M, protein identified in serums of patients

UK), 10 mM dithiothreitol (Sigma, St. Louis, MO), 10% sucrose, and trace amounts of bromophenol blue. After removing the bacteria by centrifugation, iodoacetamide (Sigma) was added to a final concentration of 50 mM then incubated for at least 15 min before applying the samples to the gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was carried out on 9%- to 14%-gradient slab gels as described (7). Fluorography was accomplished on dried gels (8) with Fuij RX film. The biosynthesis of (pro)insulin was determined with an immunoprecipitation technique described by Halban et al. (9).

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FIG. 1. A: fluorograph of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of [35S]methionine-labeled lysates of rat islets. Islets were incubated for 6 h in 5 (lane a), 11 (lane b), or 28 (lane c) mM glucose and precipitated with polyclonal rabbit anti-MHC class I antiserum (left), serum from patient with insulin-dependent diabetes mellitus (IDDM) containing antibodies against 64,000-Mr antigen (middle), and serum from patient with IDDM containing antibodies against 18,000-Mr antigen (right). B: immunoprecipitates of rat islet lysates cultured for 3 days in 5,11, and 28 mM glucose and analyzed with serums as in A.

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GLUCOSE REGULATION OF ISLET CELL ANTIGENS

with IDDM (2,3). In our experiments, this antigen consistently appeared as a double band of similar mobility, presumably due to the high resolution of the gel system employed. The nature of this antigen has remained elusive. It is tempting to believe that by inducing an increased expression of the 64,000-M, protein, as was done in this study by culturing the islets at a high glucose concentration, the means have been attained by which the amount of protein can be increased to facilitate its chemical characterization. Furthermore, this demonstration of a correlation between glucose stimulation of (pro)insulin biosynthesis and 64,000-Mr antigen expression and results of ongoing studies with other p-cell stimulants suggest that the expression of this protein is dependent on the function of the islet (3-cells. In contrast to the 64,000M, antigen, cell membrane-bound MHC class I proteins, which consist of a heavy 47,000-/Wr chain and a light 11,000M, chain (p2-microglobulin), were unaffected by variations of the glucose concentration (10). To our knowledge, the 18,000-Mr band has not been described previously. However, antibodies against this protein do not seem to be confined to IDDM, because we have observed antibodies against the 18,000-/Wr antigen in 1 of 5 serums from patients with systemic lupus erthematosus, 1 of 5 serums from patients with primary biliary cirrhosis, but in none of 5 serums from patients with rheumatoid arthritis and none of 10 serums from healthy individuals (unpublished observations). The prevalence of serums from patients with IDDM recognizing the 18,000-Mr band and a possible relationship to the 64,000-Mr antigen are subjects of further study. In autoimmune thyroid diseases, antibodies against thyroglobulin, the thyroid-stimulating hormone (TSH) receptor, and thyroperoxidase are present (11,12). These antigens are all proteins important in thyroid cell function. In autoimmune gastritis, the parietal cell antigen has been found to correspond to H+-K+-ATPase (13), the acid-producing enzyme of the stomach. Thus, it appears as if antigens in autoimmune endocrine diseases are associated with key functions of the target cells, and it might be speculated that antigens of insulin-producing cells are proteins of importance in their differentiated function. Indeed, the concomitant and stimulatory effects of glucose on the 64,000-M, antigen and (pro)insulin biosynthesis found in this study support such a view. Lowering TSH levels seems to reduce antibody levels against the thyroid microsomal antigen in autoimmune thyroiditis (14), and in vitro stimulation by TSH has been shown to increase the production of the microsomal antigen in rat thyroid cells (15). A lowering of the functional activity of the p-cells in vivo might make them less antigenic. It is possible

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that such a reduction contributes to the induction of a "honeymoon" period, characterized by improved insulin secretion from the islets, often induced after correction of hyperglycemia at the start of insulin therapy in IDDM.

ACKNOWLEDGMENTS

We thank Margareta Engkvist for technical assistance, Dr. Per Ivar Gaarder for supplying an islet cell antibody reference serum, and Prof. Lars Rask for the gift of a rabbit antirat MHC class I antiserum and Staphylococcus aureus bacteria. This study was supported by Swedish Medical Research Council Grants 4996, 109, and 8982; the Pharmacia Foundation; and Torsten och Ragnar Soderbergs Foundation.

REFERENCES 1. Rossini AA, Mordes, JP, Like AA: Immunology of insulin-dependent diabetes mellitus. Annu Rev Immunol 3:289-320, 1985 2. Baekkeskov S, Nielsen JH, Marner B, Bilde T, Ludvigsson J, Lernmark A: Autoantibodies in newly diagnosed diabetic children immunoprecipitate human pancreatic islet cell proteins. Nature (Lond) 298:167-69,1982 3. Kanatsuna T, Baskkeskov S, Lernmark A, Ludvigsson J: Immunoglobulin from insulin-dependent diabetic children inhibits glucose-induced insulin release. Diabetes 32:520-24, 1983 4. Howell SL, Taylor KW: Potassium ions and the secretion of insulin by islets of Langerhans incubated in vitro. Biochem J 108:17-24, 1968 5. Andersson A: Isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. Diabetologia 14:397-404, 1978 6. Sege K, Rask L, Peterson PA: Role of beta2-microglobulin in the intracellular processing of HLA antigens. Biochemistry 20:4523-30, 1981 7. Blobel G, Dobberstein B: Transfer of proteins across membranes. II. Reconstitution of functional rough microsomes from heterologous components. J Cell Biol 67:852-62, 1975 8. Bonner WM, Laskey RA: A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46:83-88, 1974 9. Halban PA, Wollheim CB, Blondel B, Renold AE: Long-term exposure of isolated pancreatic islets to mannoheptulose: evidence for insulin degradation in the p-cell. Biochem Pharmacol 39:2625-33. 1980 10. Peterson PA, Rask L, Lindblom JB: Highly purified papain-solubilized HL-A antigens contain beta2-microglobulin. Proc Natl Acad Sci USA 71:35-39, 1974 11. Volpe R (Ed.): Autoimmune thyroid disease. In Autoimmunity and Endocrine Diseases. New York, Dekker, 1985, p. 109-286 12. Czarnocka BJ, Ruf M, Ferrand P, Carayon P, Lissitzky S: Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid disease. FEBS Lett 190:147-52, 1985 13. Karlsson FA, Burman P, Lb'o'f L, Mardh S: The major parietal cell antigen in autoimmune gastritis with pernicious anemia is the acid-producing H,K-ATPase of the stomach. J Clin Invest 81:47.5-79. 1988 14. Jansson R, Karlsson FA, Dahlberg PA: Thyroxine, methimazole and thyroid microsomal autoantibody titres in hypothyroid Hashimoto's thyroiditis. Br MedJ 290:11-12, 1985 15. Chiovato L, Vitti P, Lombardi A, Kohn LD, Pinchera A: Expression of the microsomal antigen on the surface of continuously cultured rat thyroid cells is modulated by thyrotropin. J Clin Endocrinol Metab 61:12-16,1985

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