Long-term in vitro exposure to high glucose increases proinsulin-like-molecules release by isolated human islets F Bertuzzi 1 , K Saccomanno 5 , C Socci 1 , A M Davalli 2 , M V Taglietti 3 , C Berra 3 , E Dalcin 4 , L D Monti 3 , G Pozza 3 and A E Pontiroli 2 1 Istituto Scientifico San Raffaele, Chirurgia 2 a , 2 Unita` Malattie Metaboliche, 3 Medicina Interna, 4 Anatomia Patologica, Universita` degli Studi di Milano, Milan, Italy and 5 Istituto di Scienze Endocrine, Ospedale Maggiore, Milan, Italy (Requests for offprints should be addressed to F Bertuzzi, Istituto Scientifico San Raffaele, Via Olgettina 60, I-20132 Milan, Italy) Abstract The aim of this study was to determine the effect of long-term in vitro exposure to high glucose on the release and content of proinsulin and insulin in human islets. After 48 h culture in CMRL medium at 5·5 mM (control islets) and 16·7 mM glucose (experimental islets), islets were perifused and acutely stimulated with 16·7 mM glucose, followed by 3·3mM glucose. Compared with control islets, experimental islets showed a higher basal release of true insulin and proinsulin-like-molecules (PLM), with no increase of true insulin and PLM release in response to 16·7 mM glucose, and a paradoxical true insulin release in response to 3·3 mM glucose; the PLM/total insulin ratio increased significantly after 16·7 mM glucose. Moreover these islets showed a decreased true insulin content and an increased PLM/total insulin ratio. Quantitative ultra- structural analysis of granules, supported by double gold immunostaining with monoclonal antibodies against proinsulin and insulin, showed an increased proinsulin to insulin ratio in -cells from experimental islets. These data support in vitro what was recently shown in vivo, and further confirm that culture in high glucose is a useful tool to mimic the effect of in vivo chronic hyperglycemia on human -cell function. Journal of Endocrinology (1998) 158, 205–211 Introduction Proinsulin-like-molecules (PLM) consist of several molecules on the pathway of production of insulin from proinsulin (C-peptide and intact and partially processed proinsulin) with a low insulin-like biological activity (Temple et al. 1989). PLM have been found elevated in type II diabetic patients (NIDDM) (Polonsky & Rubenstein 1989, Temple et al. 1989), in newly diagnosed type I diabetic patients (IDDM) (Snorgaard et al. 1990), in healthy siblings (Lindgren et al. 1993) or twins of IDDM patients (Heaton et al. 1988), as well as in elderly subjects (Shimizu et al. 1996). Different hypotheses have been proposed to understand the etiology of high PLM levels: (1) a primary dysfunction in insulin secretion (Porte & Kahn 1989); (2) a consequence of chronic hyperglycemia; and (3) a decreased proinsulin clearance (Polonsky & Rubenstein 1989, Porte 1991, Rhodes & Alarcon 1994). Primary islet dysfunction as the cause of high PLM is supported by the finding of hyperproinsulinemia without hyperglycemia in identical relatives of IDDM patients (Heaton et al. 1988), or by clinical situations of increased metabolic demand without hyperproinsulinemia such as in patients with pharmacologically induced insulin resistance (Kahn et al. 1989a), in partial pancreatectomized dogs (Ward et al. 1988) or in obese Pima Indians (Kahn et al. 1989b). High serum levels of PLM could be related to an increased secretory demand placed on -cells, as it has recently been shown to occur after hemipancreatectomy (Seaquist et al. 1996), in the presence of glucose intolerance (Shiraishi et al. 1991), in cystic fibrosis (Hartling et al. 1988), or after prolonged intravenous glucose administration (Davis et al. 1993). Cross-sectional data in NIDDM have shown that increased PLM occurs after the onset of hyperglycemia (Birkeland et al. 1994). Finally, many but not all studies have found partial to full normalization of the PLM in patients with hypoglycemic therapies (Yoshioka et al. 1989). This hypothesis was also confirmed in animal models of NIDDM in which hyper- proinsulinemia has been described as the result of the premature release of proinsulin before it could be fully processed (Alarcon et al. 1995, Gadot et al. 1995). An immunocytochemical study showed an impaired process- ing of proinsulin due to a chronic secretory state (Bendayan et al. 1995). We have previously shown that in vitro culture of isolated human islets in the presence of high glucose is a suitable model to mimic the effects of in vivo chronic hyperglycemia (Davalli et al. 1991, 1992). The aim of the 205 Journal of Endocrinology (1998) 158, 205–211  1998 Society for Endocrinology Printed in Great Britain 0022–0795/98/0158–0205 $08.00/0