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4. Lernmark, A. (1974) Diabetologia 10,431-438 5 . Hamill, 0. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F. J. ( 1 98 1 ) Pfliigers Arch. 3 9 1,85 - I 00 6. Rink, T. J., Montecucco, C., Hesketh, T. R. & Tsien, K. Y. ( 1980) Biochim. Biophys. Acta 595, 15-30 7. Arkhammar, P., Nilsson, T., Rorsman, P. & Berggren, P.-0. (1987)J. Biol. Chem. 262,5448-5454 8. Kanatsuna, T., Lernmark, A., Rubenstein, A. H. & Steiner, D. F. (1981) Diabetes30.231-234 9. Katada, T. & Ui, M. ( 1 979) J. Biol. C’hem. 254,469-479 10. Ahren, B., Arkhammar, P., Berggren, P.-0. & Nilsson, T. (1986) Biochem. Biophys. Res. Commun. 140, 1059- 1063 I 1. Sussman, K. E., Leitner, J. W. & Draznin, B. (1987) Diabetes 36,571-577 12. Nilsson, T., Arkhammar. P., Rorsman. P. & Berggren, P.-0. ( 1988)J. B i d . Chem. 263, 1855- I860 13. Nilsson, T., Arkhammar, P., Rorsman, P. & Berggren, P.-0. ( 1989)J. Biof. C’hem. 264,973-980 14. Jones, P. M., Stutchfield, J. & Howell, S. L. (1985) FEBS Lert. 191, 102-106 15. Tamagawa, T., Niki. H. & Niki, A. (1985) FEBS Leu. 183, 430-432 16. Hughes. S. J. & Ashcroft. S. J. H. ( I 988) Biochem. J. 249, 825-830 17. Metz, S. A. ( 1988) Diabetes 37. 3-7 18. Cook, D. L. & Perara, E. ( 1 982) IXabetes 31.985-990 19. Santana de Sa, S., Ferrer, R., Rojas, E. & Atwater, I. ( 1983) Q. J. I’hysiol. 68, 247-258 20. Luini, A,, Lewis, D., Guild, S., Schofield. G. & Weight, F. ( 1986) J . Neurosci. 6, 3 128-3 I32 2 1. Marchetti, C., Carbone, E. & Lux, H. D. (19x6) I’J’iigersArch. 406, 104- 1 I I 22. Tsunoo, A,, Yoshii, M. & Narahashi. T. ( 1986) /’roc. Natl. Acad. Sci. U.S.A.83,9832-9836

23. Rorsman, P., Arkhammar, P., Berggren, P.-0. & Nilsson, T. (1987)Diabetologia 30,575A 24. Ahren, B., Rorsman, P. & Berggren, P.-0. (1988) FEBS Lert. 229,233-237 25. Fosset. M., Schmid-Antomarchi, H., de Weille. J. R. & Lazdunski, M. ( 1 988) FEBS Left. 242.94-96 26. Ronner, P., Smith-Maxwell, C. & Kimmich, G. A. (1988) Diabetes 37 (Suppl. I), 7A 27. de Weille, J., Schmid-Antomarchi, H., Fosset, M. & Lazdunski, M. ( 1988) Proc. Narl. Acad. Sci. U.S.A.85. 1 3 12- I 3 16 28. Ahren, B., Berggren, P.-O., Bokvist, K. & Korsman, P. ( 1 9 8 9 ) I’eptides 10,453-457 29. Dunne, M. J., Bullett, M. J.. Li, G., Wollheim, C. €3. & Petersen, 0. H. ( 1989) EMBO J . 8.4 13-420 30. Trube, G., Rorsman, P. & Ohno-Shosaku, T. ( I 986) Iy7iigers Arch. 407,493-499 31. Tamagawa, T. & Henquin. J.-C. (1983) Am. J. l’hyiol. 244. E245-E252 32. Henquin, J.-C. & Meissner, H. P. ( 1982) Biochem. /’hurmucol. 31,1407-1415 33. Ui, M. (1984) Trends Biochem. Sci. 7,277-279 34. Brown, A. M. & Birnbaumer, L. ( 1 988) Am. J . I’hysiol. 254, H40 1 -H4 10 35. Barrowman, M. M., Cockcroft, S. & Gomperts, B. D. (1986) Narure (London)319,504-507 36. Vallar, L., Biden, 7’.J. & Wollheim, C. B. ( 1987) J. Hiol. (%em. 262,5049-5056 37. Fernandez, J. M., Neher, E. & Gomperts, B. 11. ( 1984) Nhtitre (London) 312,453-455

Received 24 July 1989

Biosynthesis and storage of insulin J O H N C . HUTTON,* E L A I N E M. BAILYES, CHRISTOPHER J. RHODES, NICHOLAS G. RUTHERFORD, SUSAN D. A R D E N and PAUL C. G U E S T Department of Clinical Biochemistry, Acldenbrooke h Hospital, Hills Road, Cumbridge c‘B2 2QR, V.K. T h e insulin storage granule of the pancreatic B-cell is comprised of at least 100 different polypeptides. These play essential roles in the morphogenesis of the organelle, posttranslation modification of granule proteins and in the exocytotic release of the hormone. Investigation of the process of insulin storage at the molecular level entails a description of the structure and function of individual constituent proteins of the secretory granule, studies of their biosynthesis, post-translational modification and segregation to the secretory granule and, in the case of granule membrane proteins, examination o f their fate after exocytosis. A n important question arises as to how proportionality of the various granule constituents is maintained through regulation of gene expression and how co-ordination is achieved in the, recruitment of newly synthesized and recycled proteins to the nascent granule structure. T h e fact that rates of insulin granule biogenesis can fluctuate more than 20-fold over time intervals of less than an hour suggest that complex regulatory processes are involved. We consider here the regulation of the biosynthesis of four granule proteins which differ in their function o r location in the insulin granule, namely the hormone insulin, carboxypeptidase H, an enzyme involved in prohormone conversion, chromogranin A , precursor of an autocrine regulator, and SGM 1 10, a granule membrane protein. *To whom correspondence should be addressed.

Carboxypeptidase tl

Carboxypeptidase H is a relativcly abundant component protein comprising approx. 5% of the total granule protein. A procedure has been developed for the purification of the enzyme from a transplantable insulinoma [ 1) based upon affinity chromatography using p-aminobenzoylarginine coupled to Sepharose 4 B [2] which gives a 700-fold purification and 42% yield. T h e purified protein is a monomer of 53-54 kDa under either reducing or non-reducing conditions. It binds strongly to concanavalin A , indicating the presence o f mannosccontaining carbohydrate side-chains, and somewhat less strongly to wheatgcrm agglutinin, indicating that somc o f the Carbohydrate component is of the complex o r hybrid type. T h e total carbohydrate determined by deglycosylation with trifluoromethane sulphonic acid is 4-6%,(w/w). Immunofluorescence microscopy shows that carboxypeptidase H in the pancreas is localized in islet tissue and present in all islet cells. Immunoreactivity was also found in the pituitary and was particularly abundant in the median eminence. Western blot analysis showed that the immunoreactive forms in all these tissues were of a similar molecular size to the purified insulinoma protein (P. C. Guest, L. Orci & J. C. Hutton, unpublished work). Post-translational modifcation. Biosynthetic labelling experiments conducted with insulinoma cells demonstrated that carboxypeptidase H is initially synthesized as a 56 kDa protein which is rapidly transformed into a 53 kDa protein at about the time that newly synthesized insulin is traversing the Golgi apparatus. Treatment of the 56 kDa form with endoglycosidase H o r F produces a protein of 5 2 kDa, that is a molecular form which is similar in size to the native deglycosylated protein. Endoglycosidase H removes the high1990

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NUTRIENT REGULATION OF INSULIN SECRETION mannose glycans that are initially added in the endoplasmic reticulum. Endoglycosidase F can remove these and, in addition, hybrid and biantennary complex glycans which are formed by further modification of the glycosyl chains in the Golgi apparatus. Endoglycosidase F treatment of the molecular form which appears after 30 min produces a similarly sized protein, but endoglycosidase H treatment only caused a 52% conversion to the 52 kDa form. It would seem from these results that a factor contributing to the changes in size on maturation of the carboxypeptidase H is the trimming of carbohydrate residues. The fact that these changes occurred at a time when it is known that insulin is passing through the Golgi, and, that carboxypeptidase-H is co-secreted with insulin, suggests that proinsulin and carboxypeptidase-H traverse the route from the endoplasmic reticulum to the secretory granule at the same rate. Cloning and primary structure. Amino acid sequence information was obtained for a number of peptides purified by h.p.1.c. after digestion of insulinoma carboxypeptidase H with trypsin or staphyloccocal V8 protease. Probes consisting of a mixed oligonucleotide of 17 bases corresponding to one of these peptides, and also bovine carborypeptidase H cDNAs were used to screen rat hippocampal, hypothalamic, pancreatic islet cell and insulinoma libraries constructed in Igt 10 and I 1. The cDNAs clones obtained appeared to be derived from a single mRNA and covered a 1989 bp stretch of DNA which encoded a 476 amino acid protein of approx. 54 kDa 131. The encoded protein had a typical hydrophobic leader sequence and a sequence of five arginine residues commencing at amino acid position 38. The latter may constitute the site of endoproteolytic processing of the protein from a proform to the mature enzyme. Two possible sites of N glycosylation are evident, consistent with the observed presence of carbohydrate residues on the molecule [2]. The rat sequence showed an overall sequence similarity of 9 1.6% with bovine pituitary carboxypeptidase H, suggesting that the protein is very strongly conserved between species and between tissues engaged in regulated release of polypeptides. The sequence could also be aligned with the exocrine pancreas metalloexopeptidases, carboxypeptidase A and carboxypeptidase B. Individual amino acids thought to be important for zinc binding and the catalytic function of carboxypeptidase B were in similar relative positions in the carboxypeptidase H molecule. The sequence similarity between carboxypeptidase A and B (48%),however, is much greater than between A and H (20%) or B and H ( 17"/0).

Cloning arid primary structure. To obtain further sequence information, an insulinoma expression vector cDNA library constructed in Agtl 1 was screened with antisera to the native 21 kDa protein [7]. This produced a number of overlapping clones covering 1696 bp of DNA and encoded the complete sequence of the precursor protein. The authenticity of the clones was confirmed by a series of antibody controls and gas-phase sequencing of tryptic fragments of the original protein. The sequence encoded a protein o f 448 amino aicds ( S 1 kDa). A total of 10 different dibasic sites were present in the molecule, one of which, a lysine-arginine sequence, appeared just beyond the most C-terminal peptide fragment that had been sequenced from betagranin [ 41. Two consensus sequences for N-linked glycosylation were observed, consistent with apparent glycosylation of the betagranin molecule. Granule membrane SGMllO Much of the diversity among insulin granule proteins resides within the granule membrane. Most of the components are of low abundance and their purification is thus extremely difficult by conventional chromatographic techniques. An alternative approach has been to generate monoclonal antibodies to the granule membrane at random, to use these to characterize the antigen further, and then undertake the purification based o n immunoaffinity chromatography 18, 91. One such monoclonal antibody recognizes protein bands on Western blot analyses of I 1 0 kDa and SO kDa (collectively termed SGM 1 1 0 ) . Immunofluorescence microscopy showed that SGM 1 10 has a punctate intracellular distribution in islet cells consistent with its localization to granules. SGM 110 appeared to be an integral membrane protein and its epitopes appeared to be exposed on the inner face of the granule membrane. lmmunoreactivity was detected in secretory granule preparations from rat adrenal medulla and pituitary and also with a liver vesicle preparation, though in the latter case the antigen was a different size (a single protein of 97 kDa). Biosynthesis arid post-translations/ modificution. SGM I 10 is synthesized initially as a 97 kDa glycoprotein and transformed within 60 min to a more diffusely migrating 1 10 kDa protein 191. The change in size was attributed to further glycosylation. The SO kDa immunoreactive form evident on Western blots appeared much later ( > 12 h) and possibly results from crinophagy of senescent granules 1101 or degradation of the 1 10 kDa molecule after exocytosis.

Chromogranin A Among the minor insulin secretory granule matrix constituents is a 21 kDa protein termed betagranin [4]. Immunofluorescence microscopy using antisera raised to the purified protein showed that the antigen is restricted to cells of the neuroendocrine system, and hence appears in the islet, pituitary, adrenal and cells scattered throughout the intestinal tract IS]. In the islet it was present in most pancreatic endocrine cells, but concentrated in subpopulations A- and B-cells. N-terminal amino acid sequence analysis showed that betagranin exhibited sequence similarity with the much larger co-secreted protein of the adrenal chromaffin cell, chromogranin A 141. Riosynthesis and post-translational modification. Pulsechase labelling experiments showed that betagranin is synthesized as a larger precursor of the same size as rat adrenal chromogranin A and that this is converted in time to peptides identical in size to the native 21 kDa protein 161. The conversion paralleled that of proinsulin to insulin, suggesting that it was subject to similar post-translational proteolytic events. VOl. 18

.Short-term regulation of granule protein biosynthesis A considerable amount of information has been obtained concerning the short-term regulation of insulin biosynthesis in secretagogue-stimulated islets. Glucose induces a rapid 20-fold increase in insulin labelling arising principally from regulation of the translation of preformed insulin mRNA [11]. Such regulation involves changes in the rate of initiation, release of signal-recognition particle arrest and stimulation of peptide chain elongation rate [ 121. The availability of antibodies to carboxypeptidase H, chromogranin A and SGMl 10 now permits us to address the question of whether the synthesis of different proteins is co-ordinated under different physiological states. Glucose stimulation of rat pancreatic islets increased the synthesis of both chromogranin A and of SGMIlO to an extent similar to that of insulin [9, 131 (Fig. 1). In contrast, little or no effect on carboxypeptidase H synthesis was observed. Inhibition of glucose metabolism by the addition of mannoheptulose to the media blocked the biosynthesis of SGMllO, chromogranin A and insulin, suggesting that a

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Fig. 1. Biosynthetic response of different insulin secretory granule proteins to stimulation of isolated rat islets with glucose

granule constituents, likewise is not correlated to the biosynthetic response to glucose. Since control of the biosynthesis of chromogranin A and SGMl 10 so closely parallels that of insulin, it is tempting to conclude that regulation also occurs at the level of mRNA translation. It is possible that there are common response elements within the mRNAs encoding these proteins which are recognized by the synthetic machinery in a manner different to that of mRNAs encoding non-responsive granule proteins and other cellular proteins. Such elements, if they eixst, are not readily discernible from comparison of their cDNA sequences. Gene fusion studies may, in the future, provide an experimental means of testing for the existence of such response elements. These studies were supported by the British Diabetic Associa-

Groups of 100-200 islets were incubated for 20 min at 37°C tion, the Medical Research Council of Great Britain, the Wellcome and Nordisk Insulinlaboratorium. C.J.R. is a research fellow of in 100 pl of Krebs buffer containing 150 pCi of [?SS]meth- Trust the Juvenile Diabetes Foundation. ionine, then lysed and the indicated proteins immunoprecipitated, electrophoresed and subjected to fluorography 1. Chick, W. L., Warren, S., Chute, R. N., Like, A. A., Lauris, V. & and densitometry [6, 8, 131. Results are expressed relative to Kitchen, K. C. (1977) Proc. Natl. Acad. Sci. U.S.A. 14, the incorporation determined under basal conditions (2.8 628-632 mwglucose). m, 2.8 mM-glucose; e, 16.7 mM-glucose; M, 16.7 2. Davidson, H. W. & Hutton, J. C. (1987) Hiochem. J . 245, mM-glucose plus 1 mM-EGTA; 0, 16.7 mM-glucose plus 20 575-582 3. Fricker, L. D., Adelman, J., Douglas, J.. Thompson, R., von mM-mannoheptulose. Strandmann, R. P. & Hutton, J. C. (1989) J. Mol. Endocrinol. 3, 666-673

common signal arising from glucose metabolism is involved. Removal of extracellular Ca2+, which inhibits glucoseinduced insulin release, did not alter the biosynthetic response of insulin, chromogranin A or SGM 1 10. Conversely, secretion could be stimulated by tolbutamide without induction of a biosynthetic response. Thus the activation of the synthesis of these proteins is not obligatorily coupled to exocytosis, but occurs as a primary response to a signal generated by the secretagogue. The biosynthesis of insulin, chromogranin A and SGMl10 show the same glucose concentration dependency and the same delay in activation of the response (20 min) after introduction of the stimulus, further suggesting that similar regulatory processes are involved. A common structural theme among these proteins is the presence of pre- and pro-sequences which are removed by limited proteolysis [ 141. However, pre- and pro-sequences are found in other proteins which follow the same secretory pathway as far as the trans-Golgi network, but are not segregated to secretory granules or regulated in the same manner. Glycosylation, which occurs on some, but not all, of these

4. Hutton, J. C., Hansen, F. & Peshavaria, M. (1985) FEHS Lett. 188,336-340 5. Hutton, J. C., Peshavaria, M., Johnston, C. F., Ravazzola, M. & Orci, L. (1988) Endocrinology 122, 1014-1020 6. Hutton, J. C., Davidson, H. W., Grimaldi, K. A. & Peshavana, M. (1987) Biochem. J. 244,449-456 7. Hutton, J. C., Nielsen, E. & Kastern, W. (1988) FEBS Lett. 236, 269-274 8. Grimaldi, K. A., Hutton, J. C. & Siddle, K. (1987) Biochem. J. 245,567-573 9. Grimaldi, K. A,, Siddle, K. & Hutton, J. C. (1987) Biochem. J . 245,557-566 10. Halban, P. A. & Wollheim, C. B. ( 1 980) J. Biol. Chem. 255, 6003-6006 1 I . Morris,G. E.& Korner,A.(1970) FEBSLerr. 10, 165-168 12. Welsh, M., Scherberg, G., Gilmore. R. & Steiner, D. F. (1986) Biochem. J. 235,459-467 13. Guest, P. C., Modes, C. J. & Hutton, J. C. (1Y89) Biochem. J . 257,43 1-437 14. Docherty, K. & Steiner, D. F. ( 1 982) Annu. Rev. Physiol. 44, 625-638

Received 24 July 1989

Defective regulation of insulin secretion in diabetes and insulinoma PETER R. FLATT Department of Biological and Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry BT52 ISA, Northern Ireland, U.K.

In view of the general interest in disorders of insulin secretion and glucose homoeostasis, and the cost of such diseases in terms of treatment and quality of life, much research has been devoted to understanding the physiology and pathophysiology of insulin secretion. Studies of insulin secretion in diabetes and insulinoma, which in their various forms affect up to 4% and 0.02%, respectively, of the population, have almost exclusively used islets or tumour B-cells from animal models. In the present paper, the results of such studies will be considered in the framework of our current understanding of the insulin secretory mechanism.

Insulin secretory mechanism

The mechanism by which glucose and other nutrient fuels provoke insulin secretion from normal pancreatic B-cells has been considered in detail elsewhere in this Colloquium. In brief, it is generally believed that glucose metabolism leading to the generation of cellular ATP provides the stimulus to insulin secretion through a sequence of ionic events triggered by the closure of ATP-sensitive K + channels in the B-cell plasma membrane. This results in a decrease of K + permeability which in turn leads to membrane depolarization, opening of voltage-dependent C a 2 + channels and Ca2+ influx. The resulting increase in cytoplasmic Ca2 triggers exocytosis by affecting enzyme activities, electrostatic membrane charges, microtubules and microfilaments. Nonmetabolizable secretagogues such as hormones and +

1990