We have examined the effect of chronic (4 wk) hyperglycemia on insulin secretion in vivo in an awake, unstressed rat model. Three groups of animals were examined: control, partial (90%) pancreatectomy, and partial pancreatectomy plus phlorizin, in order to normalize plasma glucose levels. Insulin secretion in response to arginine (2 mM), hyperglycemia (+100 mg/dl), and arginine plus hyperglycemia was evaluated. In diabetic compared with control animals three specific alterations were observed: (a) a deficient insulin response, in both first and second phases, to hyperglycemia; (b) an augmented insulin response to the potentiating effect of arginine under basal glycemic conditions; and (c) an inability of hyperglycemia to augment the potentiating effect of arginine above that observed under basal glycemic conditions. Normalization of the plasma glucose profile by phlorizin treatment in diabetic rats completely corrected all three beta cell abnormalities. These results indicate that chronic hyperglycemia can lead to a defect in in vivo insulin secretion which is reversible when normoglycemia is restored.

The factors that regulate pancreatic beta cell proliferation are not well defined. In order to explore the role of murine placental lactogen (PL)-I (mPL-I) in islet mass regulation in vivo, we developed transgenic mice in which mPL-I is targeted to the beta cell using the rat insulin II promoter. Rat insulin II-mPL-I mice displayed both fasting and postprandial hypoglycemia (71 and 105 mg/dl, respectively) as compared with normal mice (92 and 129 mg/dl; p < 0.00005 for both). Plasma insulin concentrations were inappropriately elevated, and insulin content in the pancreas was increased 2-fold. Glucose-stimulated insulin secretion by perifused islets was indistinguishable from controls at 7.5, 15, and 20 mM glucose. Beta cell proliferation rates were twice normal (p ‫؍‬ 0.0005). This hyperplasia, together with a 20% increase in beta cell size, resulted in a 2-fold increase in islet mass (p ‫؍‬ 0.0005) and a 1.45-fold increase in islet number (p ‫؍‬ 0.0012). In mice, murine PL-I is a potent islet mitogen, is capable of increasing islet mass, and is associated with hypoglycemia over the long term. It can be targeted to the beta cell using standard gene targeting techniques. Potential exists for beta cell engineering using this strategy.

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic b-cells. Binding of its cytoplasmic tail to b2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca 2+ -dependent proteolysis of ICA512. In vitro m-calpain cleaves ICA512 between a putative PEST domain and the b2-syntrophin binding site, whereas binding of ICA512 to b2-syntrophin protects the former from cleavage. b2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-b2-syntrophin and stimulation of insulin secretion induces the Ca 2+ -dependent, okadaic acid-sensitive dephosphorylation of b2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from b2-syntrophin±utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by m-calpain. Keywords: actin/diabetes/IA-2/secretory granule/tyrosine phosphatase IntroductionThe exocytosis of neurosecretory granules is associated with a focal and transient reorganization of the cortical actin cytoskeleton. Actin may control secretion in a variety of ways. For instance, evidence that depolymerization of actin micro®laments increases stimulated secretion Trifaro, 1990) led to the suggestion that the actin web acts as a barrier that prevents granules from approaching sites of exocytosis (Sontag et al., 1988;Vitale et al., 1991;Nakata and Hirokawa, 1992). The reorganized actin ®laments can also directly support the movement of granules across the subcortical region (Lang et al., 2000). Actin bundles and ®lamentous proteins, in addition, may tether secretory granules to each other and to the plasma membrane, thereby restraining vesicle mobility (Senda et al., 1988(Senda et al., , 1989Lang et al., 2000). Thus, the dynamic relationship of secretory granules with the actin network is likely to account, at least in part, for the existence of different pools of secretory granules, which differ in respect to their readiness to undergo exocytosis upon stimulation of secretion (Trifaro, 1990;Klenchin and Martin, 2000). The identity of the molecule(s) that mediates the interaction between secretory granules and the actin cortex, however, is still unclear.Recent studies from our laboratory have pointed to islet cell autoantigen (ICA) 512, also known as IA-2, as a potential link between secretory granules and the actin cytoskeleton in insulin-secreting cells (b-cells) of the pancreatic islets. ICA512 is an atypical member of the receptor p...

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the postprandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIPPTHrP mice than in their normal littermates.PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that signals in response to extracellular calcium and regulates parathyroid hormone secretion. The CaR is also expressed on normal mammary epithelial cells (MMECs), where it has been shown to inhibit secretion of parathyroid hormone-related protein (PTHrP) and participate in the regulation of calcium and bone metabolism during lactation. In contrast to normal breast cells, the CaR has been reported to stimulate PTHrP production by breast cancer cells. In this study, we confirmed

Islet isolation is a time-consuming process. Islet yields vary, and previous in vitro studies suggest that Ficoll may be an islet toxin. Here, we describe an alternative, Ficoll-free method to purify murine islets by filtration through a cell strainer. Collagenase digestion of pancreata was carried out using standard procedures. The pancreatic digest was divided into aliquots and purified either by Ficoll or by filtration. Following filtration, islets were intact and separated from nondigested tissue. Purity was similar to that achieved using Ficoll. However, purification by filtration was faster, increased islet yield, and resulted in higher insulin secretion in vitro. Moreover, when syngeneic diabetic hosts were transplanted with a marginal islet mass, islets purified by filtration restored normoglycemia significantly faster than those isolated by Ficoll. This suggests that Ficoll exposure negatively impacts islet function. In conclusion, islet filtration is a simple and rapid procedure for purification of islets that demonstrate improved functional mass.

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Treatment with the atypical antipsychotics olanzapine and clozapine has been associated with an increased risk for deterioration of glucose homeostasis, leading to hyperglycemia, ketoacidosis, and diabetes, in some cases independent of weight gain. Because these events may be a consequence of their ability to directly alter insulin secretion from pancreatic ␤-cells, we determined the effects of several antipsychotics on cholinergic-and glucose-stimulated insulin secretion from isolated rat islets. At concentrations encompassing therapeutically relevant levels, olanzapine and clozapine reduced insulin secretion stimulated by 10 mol/l carbachol plus 7 mmol/l glucose. This inhibition of insulin secretion was paralleled by significant reductions in carbachol-potentiated inositol phosphate accumulation. In contrast, risperidone or ziprasidone had no adverse effect on cholinergic-induced insulin secretion or inositol phosphate accumulation. None of the compounds tested impaired the islet secretory responses to 8 mmol/l glucose alone. Finally, in vitro binding and functional data show that olanzapine and clozapine (unlike risperidone, ziprasidone, and haloperidol) are potent muscarinic M 3 antagonists. These findings demonstrate that low concentrations of olanzapine and clozapine can markedly and selectively impair cholinergicstimulated insulin secretion by blocking muscarinic M 3 receptors, which could be one of the contributing factors to their higher risk for producing hyperglycemia and diabetes in humans. Diabetes 54:1552-1558, 2005 R ecent reviews of clinical databases have revealed that olanzapine and clozapine carry a higher risk for producing hyperglycemia, ketoacidosis, and new-onset type 2 diabetes than other second-generation antipsychotics (SGAs) or haloperidol, a first-generation antipsychotic (1-6). The use of olanzapine and clozapine is often associated with notable weight gain and dyslipidemia, which are known risk factors in the development of diabetes. However, several reports have described cases of hyperglycemia following olanzapine and clozapine treatment that were not associated with weight gain (7,8). Furthermore, cases exist where switching to other SGAs, such as ziprasidone or risperidone, resulted in the reversal of olanzapine-or clozapine-associated hyperglycemia, suggesting that fundamental differences exist among the SGAs (9 -11).The mechanisms responsible for the increased diabetes risk of olanzapine and clozapine are not known, but in contrast to other SGAs, both compounds are potent muscarinic receptor antagonists (12). This led us to consider the possibility that disruption of the cholinergic processes regulating insulin secretion is one of the underlying mechanisms for impaired glucose regulation. Therefore, we investigated the effects of several antipsychotics on cholinergic-stimulated insulin secretion and the activation of phospholipase C using isolated rat pancreatic islets. Since the cholinergic activation of insulin release is mediated through muscarinic M 3 receptors on ␤-cells ...