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Jul 24, 1989 - mM-mannoheptulose. common signal arising from glucose metabolism is involved. Removal of extracellular Ca2+, which inhibits glucose-.
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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

NUTRlENT REGULATION OF INSULIN SECRETION neurotransmitters may potentiate insulin secretion by activation of adenylate cyclase or phospholipase C, leading to changes in intracellular handling and sensitivity to cytoplasmic Ca2 [ 11. +

Defective insulin secretion in diabetes

Nature has provided a wealth of animal models with spontaneous diabetes which may be classified into three main groups according to the severity of the islet defect and the coexistence of obesity [2]. Models with B-cell degeneration and ketosis include the BioBreeding (BB) rat and non-obese diabetic (NOD) mouse. Those with B-cell degeneration and possible ketosis are more numerous including, the diabetes-obese (C57BL/KsJ dbldb) mouse, Chinese hamster spiny mouse, Egyptian sand rat and djungarian hamster. Finally, there is a large group of models with B-cell hyperplasia, obesity and no ketosis which includes the obese hyperglycaemic (ob/ob) mouse, the New Zealand obese mouse, the yellow obese mouse, the KK mouse, the fatty Zucker @/fa) rat and many others. Although no single animal model is an exact match for human diabetes, which itself is a highly heterologous group of disorders, diligent investigation of the range of available animal diabetes syndromes continues to provide important information on the aetiology, pathogenesis and therapeutic approaches to diabetes in man [2]. In the various animal models, glucose stimulation of insulin secretion is defective in magnitude or kinetics, providing the opportunity for interesting studies to be conducted which are impossible to perform for both ethical and technical reasons using islets isolated from diabetic patients. Studies of insulin secretion in spontaneously diabetic animals have stressed that the nature and severity of defective insulin secretion depends on the mutation itself, the genetic background on which the mutation is carried, the age and sex of the animal in question, its diet and nutritional status, and the general environment where it is maintained [2].Some caution must be applied therefore in the integration of individual pieces of knowledge from different laboratories which may not have defined such variables or characterized the diabetic status of the animals employed. The two best characterized and extensively studied models of defective insulin secretion are the diabetes-obese C57BL/KsJ db/dh mouse and the diabetic Chinese hamster. Diabetes-obese C57BLIKsJ db/db mouse. This syndrome which is inherited as an autosomal recessive trait is characterized by obesity, hyperphagia, hyperglycaemia, islet hypertrophy, B-cell hyperplasia and hyperinsulinaemia [2]. Depending on the energy density and carbohydrate content of the diet, adult dbldb mice exhibit a progressive agedependent deterioration of B-cell function potentially culminating in extensive islet necrosis, severe insulin deficiency, marked hyperglycaemia and ketosis [2]. Studies utilizing the perfused pancreas or isolated islets of young adult C57BL/ K d db/db mice have shown that both the dynamics and magnitude of glucose-induced insulin release are defective [3, 41. C57BUKsJ db/db mice exhibit marked and age-dependent alterations in the hormone content and cellular composition of the islets [5]. This undoubtedly interferes with the subtle paracrine interactions involved in insulin release. However, since glucagon secreted locally within the islet can be expected to be an important determinant of basal cyclic AMP formation, it is notable that the basal islet cyclic AMP content is similar to that of control mice [6]. This observation together with the established flow of blood through islet capillaries from the central B-cell core towards A-cells and D-cells in the islet periphery d o not indicate a key role for alterations of cellular composition in the defective response to glucose. Vol. 18

125 Of greater significance in regard to alterations of islet composition is evaluation of defective insulin secretion in terms of metabolic and ionic events in islets which necessarily involves comparison of measurements with control islets containing a different proportion of B-cells. Thus the demonstration in islets of C57BL/KsJ db/db mice of small differences in any individual link in the chain of events thought to underlie glucose-stimulation of insulin secretion is difficult to interpret. Viewed in this context, it must be concluded from available evidence that the glucose-induced increase in islet glycolytic flux is not greatly altered in the mutant [7], and that islet cyclic AMP turnover is consistent with responsiveness to phosphodiesterase inhibitors [6]. In contrast to the lack of data regarding possible abnormalities of metabolic events in islets, there is compelling evidence that defective insulin secretion in C57BL/KsJ db/ db mice is associated with derranged regulation of membrane potential, ion fluxes and cytoplasmic Ca2+ ions. Thus the islets of C57BL/KsJ db/db mice show depolarization and electrical activity at low glucose concentrations [S], abnormally low K + permeability [ O ] , glucose-insensitive efflux of K + and CI- [9, 101, and a lack of effect of glucose on the inhibitory and stimulatory phases of Ca2' efflux [ 11, 121. Since K + permeability is considered the major determinant of membrane potential which is linked to Ca2+ influx through the opening of voltage-dependent Ca2 channels, these data point to a key role of K + channels and their regulation in the secretion defect. Evaluation of ATP-sensitive K channels in C57BL/KsJ db/db mice has not been performed, but it is of interest that a closely related model, namely the Aston ob/ob mouse, exhibits disturbed electrical activity and unresponsiveness to glibenclamide or quinine [ 13, 141. Both of these agents are known to close ATP-sensitive K' channels in normal pancreatic B-cells [ 151. Diabetic Chinese hamster. The inheritance of this syndrome is polygenic and therefore studies with diabetic Chinese hamsters rely on comparison with non-diabetic sublines of hamsters [2, 161. Despite the involvement of different genes, the manifestation and characteristics of the diabetes syndrome in Chinese hamsters is similar in many respects to C57BL/KsJ db/db mice [2].The main difference between the two types of mutant appears to be the limited capacity in Chinese hamsters to temporarily offset the severity of diabetes by a compensatory increase in islet B-cell number [ 171. Insulin is markedly decreased in the diabetic Chinese hamster pancreas in association with an increase of pancreatic glucagon and a decrease of somatostatin [IS]. Studies of the perfused pancreas of diabetic Chinese hamsters have shown age-related defects in both the first and second phases of glucose-stimulated insulin release [ 191. Observations by Matschinsky (cited in [20])indicating that the activities of glucokinase, hexokinase and phosphofructokinase are not compromised in diabetic Chinese hamster islets do not favour the idea that abnormalities in the sequence of metabolic events leading to secretion are responsible for defective insulin secretion. However, further studies examining parameters of islet metabolism, including glucose-induced changes in redox state and cellular ATP content, are clearly required before such an attractive possibility is discounted. The cyclic AMP system appears relatively normal in diabetic Chinese hamster islets as assessed from the ability of theophylline to enhance insulin release [ 191. However, as with C57BL/KsT db/db mice, the participation of Ca2+ions in the stimulus-secretion coupling process is severely compromised. Thus, diabetic Chinese hamster islets d o not respond to glucose with normal changes in the uptake and efflux of Ca2+ ions [21, 221. Recent studies indicate that these disturbances are associaed with abnormalities in the regulation of membrane K + permeability, possibly attribut+

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126 able to an alteration in Ca‘+-activated K + permeability [22]. However, since no change was apparent in the initial suppression of XhRbefflux from islets by glucose, it was concluded that defective regulation of cytoplasmic Ca2+ ions, rather than defective regulation of K + permeability, may be responsible for the impaired insulin secretion [22]. Clearly, electrophysiological studies will help to resolve this issue. Defective insulin secretion in insulinoma

lnsulinomas arise sporadically in many species in addition to man, including the cat, cow, dog, hamster, hagfish, mouse and rat [23]. Several useful models of heritable or serially

transplantable experimentally induced insulinomas have been produced in laboratory animals [24-281. These provide an excellent opportunity for detailed functional studies of defective insulin secretion which are otherwise difficult to perform because of the unpredictable and low incidence of spontaneous insulinomas in man. The five most popular models include spontaneous and BK virus-induced transplantable Syrian hamster insulinomas [24, 261, the transplantable radiation-induced NEDH rat insulinoma [25], the transplantable streptozotocin-nicotinamide-induced Lewis rat insulinoma [27] and the heritable insulinoma induced in transgenic mice through expression of recombinant insulin/simian virus 40 oncogenes [28]. Each syndrome is characterized by defective glucose regulation of insulin secretion, which leads to the development of marked hyperinsulinaemia and severe life-threatening hypogycaemia. The serially transplantable NEDH rat insulinoma has been extensively characterized in terms of its metabolic effects and the nature of the underlying insulin secretion defect. Seriully transplantable NEDH rat insulinoma. Transplantation of small insulinoma fragments into subscapular, pancreatic or hepatic sites of NEDH rats results in hyperphagia, progressive hyperinsulinaemia, defective regulation of insulin secretion and hypoglycaemia, culminating in neuroglycopenic coma within 1 month [29]. Excised tumours contain large amounts of insulin with almost negligible content of other well-known islet peptides [30].The altered hormonal milieu compared with that in normal islets, loss of neural input, lack of integration with surrounding pancreatic tissue, abnormalities in cellular environment and loss of cell-cell contact may contribute to disturbed insulin secretion in vivo [29]. Studies with isolated rat insulinoma cells indicate fundamental disturbances in the normal relationship beween the regulation of cytoplasmic Ca2+ ions and insulin secretion. Thus, whereas the secretory responsiveness to cyclic AMP modulation is retained [31], glucose and a range of other agents believed to trigger secretion through elevation of cytoplasmic Ca?+ do not affect transmembrane Ca” fluxes or insulin release [31,321. The failure of glucose to elicit changes of cytoplasmit Ca2+ and insulin secretion in rat insulinoma cells is likely to partly reflect a deficiency of glucokinase [33] and the lack of effect of glucose on membrane potential as assessed with bisoxonol [311. However, as witnessed by the independence of insulin release on variations of extracellular Ca2+ leading to a 4-fold increase of cytoplasmic CaZ+ concentration, rat insulinoma cells also display grossly abnormal sensitivity to the stimulatory effects of Ca2+ on exocytosis [31]. These observations indicate that rat insulinoma cells exhibit multiple abnormalities in the normal stimulus-secretion coupling mechanism. Regulation of ATP-sensitive K channels has not been assessed in rat insulinoma cells, but it is notable that established chemical probes for such channels, namely sulphonylureas and diazoxide, failed to exert normal effects on transmembrane Ca2+fluxes or insulin release 1321. +

Concluding remarks

Studies using animal models of diabetes and insulinoma have highlighted the involvement of abnormalities of membrane K + permeability and the regulation of cytoplasmic Ca2 ions in defective insulin secretion. Insufficient information is available to judge whether abnormalities of B-cell glucose metabolism contribute to the impaired sequence of ionic events through inept regulation of ATP-sensitive K + channels. No pertinent data exist concerning diabetic human islets, but the few studies in vitro recently performed with human non-responsive medullary-type insulinomas [ 341 indicate that defects in the stimulus-secretion pathway similar to those considered above occur in man. +

Part of this research was supported by a grant from the Cancer Research Campaign (SP 1630).

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NUTRIENT REGULATION OF INSULIN SECRETION 30. Conlon, J . M., Deacon. C. F., Bailey, C. J . & Flatt. P. R. ( 1 986) Diubrlologiu 29, 334-338 31. Flatt, P. R., Abrahamsson, H., Arkhammar, P., Berggren, P.-O., Rorsman, P. & Swanston-Flatt, S. K. (1988) Hr. J. Cancer 58, 22-29 32. Swanston-Flatt. S. K. & Flatt, P. R. (1988) (;en. I'hurrn. 19, 239-242

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127 33. Lenzen, S., Tiedge, M., Flatt, P. R., Bailey, C. J. & Panten, U. (1987)AcrciEndocn'nol.115,514-520 34. Flatt, P. R., Swanston-Flatt, S. K., Powell, C. J. & Marks, V. ( 1 987) Br. J. Ciincer 5 6 , 4 5 9 - 4 6 4 Received 24 July 1989