Hyperglycemia impairs EAAT2 glutamate transporter trafficking and glutamate clearance in islets of Langerhans: implications for type 2 diabetes pathogenesis and treatment.

Pancreatic endocrine cells employ a sophisticated system of paracrine and autocrine signals to synchronize their activities, including glutamate, which controls hormone release and ß-cell viability by acting on glutamate receptors expressed by endocrine cells. We here investigate whether alteration of the excitatory amino acid transporter 2 (EAAT2), the major glutamate clearance system in the islet, may occur in type 2 diabetes mellitus and contribute to ß-cell dysfunction. Increased EAAT2 intracellular localization was evident in islets of Langerhans from T2DM subjects as compared with healthy control subjects, despite similar expression levels. Chronic treatment of islets from healthy donors with high-glucose concentrations led to the transporter internalization in vesicular compartments and reduced [H3]-d-glutamate uptake (65 ± 5% inhibition), phenocopying the findings in T2DM pancreatic sections. The transporter relocalization was associated with decreased Akt phosphorylation protein levels, suggesting an involvement of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the process. In line with this, PI3K inhibition by a 100-µM LY294002 treatment in human and clonal ß-cells caused the transporter relocalization in intracellular compartments and significantly reduced the glutamate uptake compared to control conditions, suggesting that hyperglycemia changes the trafficking of the transporter to the plasma membrane. Upregulation of the glutamate transporter upon treatment with the antibiotic ceftriaxone rescued hyperglycemia-induced ß-cells dysfunction and death. Our data underscore the significance of EAAT2 in regulating islet physiology and provide a rationale for potential therapeutic targeting of this transporter to preserve ß-cell survival and function in diabetes.NEW & NOTEWORTHY The glutamate transporter SLC1A2/excitatory amino acid transporter 2 (EAAT2) is expressed on the plasma membrane of pancreatic ß-cells and controls islet glutamate clearance and ß-cells survival. We found that the EAAT2 membrane expression is lost in the islets of Langerhans from type 2 diabetes mellitus (T2DM) patients due to hyperglycemia-induced downregulation of the phosphoinositide 3-kinase/Akt pathway and modification of its intracellular trafficking. Pharmacological rescue of EAAT2 expression prevents ß-cell dysfunction and death, suggesting EAAT2 as a new potential target of intervention in T2DM.

Mechanisms of action of incretin receptor based dual- and tri-agonists in pancreatic islets.

Simultaneous activation of the incretin G-protein-coupled receptors (GPCRs) via unimolecular dual-receptor agonists (UDRA) has emerged as a new therapeutic approach for type 2 diabetes. Recent studies also advocate triple agonism with molecules also capable of binding the glucagon receptor. In this scoping review, we discuss the cellular mechanisms of action (MOA) underlying the actions of these novel and therapeutically important classes of peptide receptor agonists. Clinical efficacy studies of several UDRAs have demonstrated favorable results both as monotherapies and when combined with approved hypoglycemics. Although the additive insulinotropic effects of dual glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic peptide receptor (GIPR) agonism were anticipated based on the known actions of either glucagon-like peptide-1 (GLP-1) or glucose-dependent insulinotropic peptide (GIP) alone, the additional benefits from GCGR were largely unexpected. Whether additional synergistic or antagonistic interactions among these G-protein receptor signaling pathways arise from simultaneous stimulation is not known. The signaling pathways affected by dual- and tri-agonism require more trenchant investigation before a comprehensive understanding of the cellular MOA. This knowledge will be essential for understanding the chronic efficacy and safety of these treatments.

Trafficking of the glutamate transporter is impaired in LRRK2-related Parkinson's disease.

The Excitatory Amino Acid Transporter 2 (EAAT2) accounts for 80% of brain glutamate clearance and is mainly expressed in astrocytic perisynaptic processes. EAAT2 function is finely regulated by endocytic events, recycling to the plasma membrane and degradation. Noteworthy, deficits in EAAT2 have been associated with neuronal excitotoxicity and neurodegeneration. In this study, we show that EAAT2 trafficking is impaired by the leucine-rich repeat kinase 2 (LRRK2) pathogenic variant G2019S, a common cause of late-onset familial Parkinson's disease (PD). In LRRK2 G2019S human brains and experimental animal models, EAAT2 protein levels are significantly decreased, which is associated with elevated gliosis. The decreased expression of the transporter correlates with its reduced functionality in mouse LRRK2 G2019S purified astrocytic terminals and in Xenopus laevis oocytes expressing human LRRK2 G2019S. In LRRK2 G2019S knock-in mouse brain, the correct surface localization of the endogenous transporter is impaired, resulting in its interaction with a plethora of endo-vesicular proteins. Mechanistically, we report that pathogenic LRRK2 kinase activity delays the recycling of the transporter to the plasma membrane via Rabs inactivation, causing its intracellular re-localization and degradation. Taken together, our results demonstrate that pathogenic LRRK2 interferes with the physiology of EAAT2, pointing to extracellular glutamate overload as a possible contributor to neurodegeneration in PD.

Autoantibodies against the glial glutamate transporter GLT1/EAAT2 in Type 1 diabetes mellitus-Clues to novel immunological and non-immunological therapies.

Islet cell surface autoantibodies were previously found in subjects with type 1 diabetes mellitus (T1DM), but their target antigens and pathogenic mechanisms remain elusive. The glutamate transporter solute carrier family 1, member 2 (GLT1/EAAT2) is expressed on the membrane of pancreatic ß-cells and physiologically controls extracellular glutamate concentrations thus preventing glutamate-induced ß-cell death. We hypothesized that GLT1 could be an immunological target in T1DM and that autoantibodies against GLT1 could be pathogenic. Immunoprecipitation and ELISA experiments showed that sera from T1DM subjects recognized GLT1 expressed in brain, pancreatic islets, and GLT1-transfected COS7-cell extracts. We validated these findings in two cohorts of T1DM patients by quantitative immunofluorescence assays. Analysis of the combined data sets indicated the presence of autoantibodies against GLT1 in 32 of the 87 (37%) T1DM subjects and in none of healthy controls (n = 64) (p < 0.0001). Exposure of pancreatic ßTC3 cells and human islets to purified IgGs from anti-GLT1 positive sera supplemented with complement resulted in plasma membrane ruffling, cell lysis and death. The cytotoxic effect was prevented when sera were depleted from IgGs. Furthermore, in the absence of complement, 6 out of 16 (37%) anti-GLT1 positive sera markedly reduced GLT1 transport activity in ßTC3 cells by inducing GLT1 internalization, also resulting in ß-cell death. In conclusion, we provide evidence that GLT1 is a novel T1DM autoantigen and that anti-GLT1 autoantibodies cause ß-cell death through complement-dependent and independent mechanisms. GLT1 seems an attractive novel therapeutic target for the prevention of ß-cell death in individuals with diabetes and prediabetes.

PCSK9 deficiency reduces insulin secretion and promotes glucose intolerance: the role of the low-density lipoprotein receptor.

Aims: PCSK9 loss of function genetic variants are associated with lower low-density lipoprotein cholesterol but also with higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Here, we investigated the molecular mechanisms underlying this association. Methods and results: Pcsk9 KO, WT, Pcsk9/Ldlr double KO (DKO), Ldlr KO, albumin AlbCre+/Pcsk9LoxP/LoxP (liver-selective Pcsk9 knock-out mice), and AlbCre-/Pcsk9LoxP/LoxP mice were used. GTT, ITT, insulin and C-peptide plasma levels, pancreas morphology, and cholesterol accumulation in pancreatic islets were studied in the different animal models. Glucose clearance was significantly impaired in Pcsk9 KO mice fed with a standard or a high-fat diet for 20 weeks compared with WT animals; insulin sensitivity, however, was not affected. A detailed analysis of pancreas morphology of Pcsk9 KO mice vs. controls revealed larger islets with increased accumulation of cholesteryl esters, paralleled by increased insulin intracellular levels and decreased plasma insulin, and C-peptide levels. This phenotype was completely reverted in Pcsk9/Ldlr DKO mice implying the low-density lipoprotein receptor (LDLR) as the proprotein convertase subtilisin/kexin Type 9 (PCSK9) target responsible for the phenotype observed. Further studies in albumin AlbCre+/Pcsk9LoxP/LoxP mice, which lack detectable circulating PCSK9, also showed a complete recovery of the phenotype, thus indicating that circulating, liver-derived PCSK9, the principal target of monoclonal antibodies, does not impact beta-cell function and insulin secretion. Conclusion: PCSK9 critically controls LDLR expression in pancreas perhaps contributing to the maintenance of a proper physiological balance to limit cholesterol overload in beta cells. This effect is independent of circulating PCSK9 and is probably related to locally produced PCSK9.

Malaria pigment accelerates MTT - formazan exocytosis in human endothelial cells.

Haemozoin is a by-product of haemoglobin digestion by intraerythrocytic malaria parasites, which induces immunologic responses on different tissues, including endothelial cells. In the present paper, the incubation of human microvascular endothelial cells with haemozoin significantly inhibited MTT reduction, a measure of cytotoxicity, without increasing the release of cytoplasmic lactate dehydrogenase. Moreover, haemozoin did not induce apoptosis or cell cycle arrest nor decreased the number of live cells, suggesting that cells viability itself was not affected and that the inhibition of MTT reduction was only apparent and probably due to accelerated MTT-formazan exocytosis. After 30 min of MTT addition, a significant increase in the % of cells exocytosing MTT formazan crystals was observed in haemozoin-treated cells compared with control cells. Such an effect was partially reversed by the addition of genistein, an inhibitor of MTT-formazan exocytosis. The rapid release of CXCL-8, a preformed chemokine contained in Weibel-Palade bodies, confirmed that haemozoin induces a perturbation of the intracellular endothelial trafficking, including the exocytosis of MTT-formazan containing vesicles. The haem moiety of haemozoin is responsible for the observed effect. Moreover, this work underlines that MTT assay should not be used to measure cytotoxicity induced by haemozoin and other methods should be preferred.

Pancreatic islet of Langerhans' cytoarchitecture and ultrastructure in normal glucose tolerance and in type 2 diabetes mellitus.

While a number of structural and cellular abnormalities occur in the islet of Langerhans in diabetes, and in particular in type 2 diabetes, the focus has been mostly on the insulin producing ß-cells and only more recently on glucagon producing α- and δ-cells. There is ample evidence that in type 2 diabetes mellitus (T2DM), in addition to a progressive decline in ß-cell function and associated insulin resistance in a number of insulin-sensitive tissues, alterations in glucagon secretion are also present and may play an important role in the pathogenesis of hyperglycemia both in the fasting and in the postprandial state. Recently, a number of studies have showed that there are also functional and structural alterations in glucagon-producing α-cells and somatostatin-producing δ-cells. Thus, it is becoming increasingly clear that multiple cellular alterations of multiple cell types occur, which adds even more complexity to our understanding of the pathophysiology of this common and severe disease. We believe that persistent efforts to increase the understanding of the pathophysiology of hormone secretion in the islets of Langerhans will also improve our capability to better prevent and treat diabetes mellitus.

Neurotransmitters and Neuropeptides: New Players in the Control of Islet of Langerhans' Cell Mass and Function.

Islets of Langerhans control whole body glucose homeostasis, as they respond, releasing hormones, to changes in nutrient concentrations in the blood stream. The regulation of hormone secretion has been the focus of attention for a long time because it is related to many metabolic disorders, including diabetes mellitus. Endocrine cells of the islet use a sophisticate system of endocrine, paracrine and autocrine signals to synchronize their activities. These signals provide a fast and accurate control not only for hormone release but also for cell differentiation and survival, key aspects in islet physiology and pathology. Among the different categories of paracrine/autocrine signals, this review highlights the role of neurotransmitters and neuropeptides. In a manner similar to neurons, endocrine cells synthesize, accumulate, release neurotransmitters in the islet milieu, and possess receptors able to decode these signals. In this review, we provide a comprehensive description of neurotransmitter/neuropetide signaling pathways present within the islet. Then, we focus on evidence supporting the concept that neurotransmitters/neuropeptides and their receptors are interesting new targets to preserve ß-cell function and mass. A greater understanding of how this network of signals works in physiological and pathological conditions would advance our knowledge of islet biology and physiology and uncover potentially new areas of pharmacological intervention. J. Cell. Physiol. 231: 756-767, 2016. © 2015 Wiley Periodicals, Inc.

Chronic continuous exenatide infusion does not cause pancreatic inflammation and ductal hyperplasia in non-human primates.

In this study, we aimed to evaluate the effects of exenatide (EXE) treatment on exocrine pancreas of nonhuman primates. To this end, 52 baboons (Papio hamadryas) underwent partial pancreatectomy, followed by continuous infusion of EXE or saline (SAL) for 14 weeks. Histological analysis, immunohistochemistry, Computer Assisted Stereology Toolbox morphometry, and immunofluorescence staining were performed at baseline and after treatment. The EXE treatment did not induce pancreatitis, parenchymal or periductal inflammatory cell accumulation, ductal hyperplasia, or dysplastic lesions/pancreatic intraepithelial neoplasia. At study end, Ki-67-positive (proliferating) acinar cell number did not change, compared with baseline, in either group. Ki-67-positive ductal cells increased after EXE treatment (P = 0.04). However, the change in Ki-67-positive ductal cell number did not differ significantly between the EXE and SAL groups (P = 0.13). M-30-positive (apoptotic) acinar and ductal cell number did not change after SAL or EXE treatment. No changes in ductal density and volume were observed after EXE or SAL. Interestingly, by triple-immunofluorescence staining, we detected c-kit (a marker of cell transdifferentiation) positive ductal cells co-expressing insulin in ducts only in the EXE group at study end, suggesting that EXE may promote the differentiation of ductal cells toward a ß-cell phenotype. In conclusion, 14 weeks of EXE treatment did not exert any negative effect on exocrine pancreas, by inducing either pancreatic inflammation or hyperplasia/dysplasia in nonhuman primates.

Delta cell death in the islet of Langerhans and the progression from normal glucose tolerance to type 2 diabetes in non-human primates (baboon, Papio hamadryas).

AIMS/HYPOTHESIS: The cellular composition of the islet of Langerhans is essential to ensure its physiological function. Morphophysiological islet abnormalities are present in type 2 diabetes but the relationship between fasting plasma glucose (FPG) and islet cell composition, particularly the role of delta cells, is unknown. We explored these questions in pancreases from baboons (Papio hamadryas) with FPG ranging from normal to type 2 diabetic values. METHODS: We measured the volumes of alpha, beta and delta cells and amyloid in pancreatic islets of 40 baboons (Group 1 [G1]: FPG < 4.44 mmol/l [n = 10]; G2: FPG = 4.44-5.26 mmol/l [n = 9]; G3: FPG = 5.27-6.94 mmol/l [n = 9]; G4: FPG > 6.94 mmol/l [n = 12]) and correlated islet composition with metabolic and hormonal variables. We also performed confocal microscopy including TUNEL, caspase-3, and anti-caspase cleavage product of cytokeratin 18 (M30) immunostaining, electron microscopy, and immuno-electron microscopy with anti-somatostatin antibodies in baboon pancreases. RESULTS: Amyloidosis preceded the decrease in beta cell volume. Alpha cell volume increased ∼ 50% in G3 and G4 (p < 0.05), while delta cell volume decreased in these groups by 31% and 39%, respectively (p < 0.05). In G4, glucagon levels were higher, while insulin and HOMA index of beta cell function were lower than in the other groups. Immunostaining of G4 pancreatic sections with TUNEL, caspase-3 and M30 showed apoptosis of beta and delta cells, which was also confirmed by immuno-electron microscopy with anti-somatostatin antibodies. CONCLUSIONS/INTERPRETATION: In diabetic baboons, changes in islet composition correlate with amyloid deposition, with increased alpha cell and decreased beta and delta cell volume and number due to apoptosis. These data argue for an important role of delta cells in type 2 diabetes.

Cholesterol Redistribution in Pancreatic ß-Cells: A Flexible Path to Regulate Insulin Secretion.

Pancreatic ß-cells, by secreting insulin, play a key role in the control of glucose homeostasis, and their dysfunction is the basis of diabetes development. The metabolic milieu created by high blood glucose and lipids is known to play a role in this process. In the last decades, cholesterol has attracted significant attention, not only because it critically controls ß-cell function but also because it is the target of lipid-lowering therapies proposed for preventing the cardiovascular complications in diabetes. Despite the remarkable progress, understanding the molecular mechanisms responsible for cholesterol-mediated ß-cell function remains an open and attractive area of investigation. Studies indicate that ß-cells not only regulate the total cholesterol level but also its redistribution within organelles, a process mediated by vesicular and non-vesicular transport. The aim of this review is to summarize the most current view of how cholesterol homeostasis is maintained in pancreatic ß-cells and to provide new insights on the mechanisms by which cholesterol is dynamically distributed among organelles to preserve their functionality. While cholesterol may affect virtually any activity of the ß-cell, the intent of this review is to focus on early steps of insulin synthesis and secretion, an area still largely unexplored.

The surface density of the glutamate transporter EAAC1 is controlled by interactions with PDZK1 and AP2 adaptor complexes.

The glutamate transporter excitatory amino acid carrier (EAAC1/EAAT3) mediates the absorption of dicarboxylic amino acids in epithelial cells as well as the uptake of glutamate from the synaptic cleft. Its cell-surface density is regulated by interaction with accessory proteins which remain to be identified. We detected a consensus sequence for interaction with post-synaptic density-95/Discs large/Zonula occludens (PDZ) proteins (-SQF) and a tyrosine-based internalization signal (-YVNG-) in the C-terminus of EAAC1, and investigated their role in the transporter localization. We demonstrated that PDZ interactions are required for the efficient delivery to and the retention in the plasma membrane of EAAC1 and we identified PDZK1/NHERF3 (Na+/H+-exchanger regulatory factor 3) as a novel EAAC1 interacting protein. Expression of PDZK1 in Madin-Darby canine kidney (MDCK) cells tethered EAAC1 to filopodia and increased its surface activity. Removal of the PDZ-target motif promoted the EAAC1 binding to α-adaptin and clathrin and the transporter internalization in endocytic/degradative compartments. This defect was largely prevented by hypertonic treatment or overexpression of the dominant-negative µ2-W421A-subunit of AP-2 clathrin-adaptor. The rate of transporter endocytosis was attenuated following tyrosine mutagenesis in the internalization signal, thus indicating that this motif can regulate the transporter endocytosis. We suggest that EAAC1 density is controlled by balanced interactions with PDZK1 and adaptor protein 2 (AP2): the former promotes the transporter expression at the cell surface, and the latter mediates its constitutive endocytosis.

The glial glutamate transporter 1 (GLT1) is expressed by pancreatic beta-cells and prevents glutamate-induced beta-cell death.

Glutamate is the major excitatory neurotransmitter of the central nervous system (CNS) and may induce cytotoxicity through persistent activation of glutamate receptors and oxidative stress. Its extracellular concentration is maintained at physiological concentrations by high affinity glutamate transporters of the solute carrier 1 family (SLC1). Glutamate is also present in islet of Langerhans where it is secreted by the α-cells and acts as a signaling molecule to modulate hormone secretion. Whether glutamate plays a role in islet cell viability is presently unknown. We demonstrate that chronic exposure to glutamate exerts a cytotoxic effect in clonal ß-cell lines and human islet ß-cells but not in α-cells. In human islets, glutamate-induced ß-cell cytotoxicity was associated with increased oxidative stress and led to apoptosis and autophagy. We also provide evidence that the key regulator of extracellular islet glutamate concentration is the glial glutamate transporter 1 (GLT1). GLT1 localizes to the plasma membrane of ß-cells, modulates hormone secretion, and prevents glutamate-induced cytotoxicity as shown by the fact that its down-regulation induced ß-cell death, whereas GLT1 up-regulation promoted ß-cell survival. In conclusion, the present study identifies GLT1 as a new player in glutamate homeostasis and signaling in the islet of Langerhans and demonstrates that ß-cells critically depend on its activity to control extracellular glutamate levels and cellular integrity.

Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes in Schwann cells.

Recent evidence shows that neurotransmitters (e.g., GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelin sheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatory neurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve the integrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished by the high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We first demonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments of the endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, we demonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesterone metabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve protein neo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation, promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, and modifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control the allopregnanolone-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamate uptake system, which can be dynamically modulated by allopregnanolone in Schwann cells. Glutamate transporters may represent a potential therapeutic target to control Schwann cell physiology.

Pancreatic islet amyloidosis, beta-cell apoptosis, and alpha-cell proliferation are determinants of islet remodeling in type-2 diabetic baboons.

beta-Cell dysfunction is an important factor in the development of hyperglycemia of type-2 diabetes mellitus, and pancreatic islet amyloidosis (IA) has been postulated to be one of the main contributors to impaired insulin secretion. The aim of this study was to evaluate the correlation of IA with metabolic parameters and its effect on islets of Langerhans remodeling and relative endocrine-cell volume in baboons. We sequenced the amylin peptide, determined the fibrillogenic propensities, and evaluated pancreatic histology, clinical and biochemical characteristics, and endocrine cell proliferation and apoptosis in 150 baboons with different metabolic status. Amylin sequence in the baboon was 92% similar to humans and showed superimposable fibrillogenic propensities. IA severity correlated with fasting plasma glucose (FPG) (r = 0.662, P < 0.001) and HbA1c (r = 0.726, P < 0.001), as well as with free fatty acid, glucagon values, decreased homeostasis model assessment (HOMA) insulin resistance, and HOMA-B. IA severity was associated with a decreased relative beta-cell volume, and increased relative alpha-cell volume and hyperglucagonemia. These results strongly support the concept that IA and beta-cell apoptosis in concert with alpha-cell proliferation and hypertrophy are key determinants of islets of Langerhans "dysfunctional remodeling" and hyperglycemia in the baboon, a nonhuman primate model of type-2 diabetes mellitus. The most important determinants of IA were age and FPG (R(2) = 0.519, P < 0.0001), and different FPG levels were sensitive and specific to predict IA severity. Finally, a predictive model for islet amyloid severity was generated with age and FPG as required variables.

Pancreatic PCSK9 controls the organization of the ß-cell secretory pathway via LDLR-cholesterol axis.

BACKGROUND: Cholesterol is central to pancreatic ß-cell physiology and alterations of its homeostasis contribute to ß-cell dysfunction and diabetes. Proper intracellular cholesterol levels are maintained by different mechanisms including uptake via the low-density lipoprotein receptor (LDLR). In the liver, the proprotein convertase subtilisin/kexin type 9 (PCSK9) routes the LDLR to lysosomes for degradation, thus limiting its recycling to the membrane. PCSK9 is also expressed in the pancreas and loss of function mutations of PCSK9 result in higher plasma glucose levels and increased risk of Type 2 diabetes mellitus. Aim of this study was to investigate whether PCSK9 also impacts ß-cells function. METHODS: Pancreas-specific Pcsk9 null mice (Pdx1Cre/Pcsk9 fl/fl) were generated and characterized for glucose tolerance, insulin release and islet morphology. Isolated Pcsk9-deficient islets and clonal ß-cells (INS1E) were employed to characterize the molecular mechanisms of PCSK9 action. RESULTS: Pdx1Cre/Pcsk9 fl/fl mice exhibited normal blood PCSK9 and cholesterol levels but were glucose intolerant and had defective insulin secretion in vivo. Analysis of PCSK9-deficient islets revealed comparable ß-cell mass and insulin content but impaired stimulated secretion. Increased proinsulin/insulin ratio, modifications of SNARE proteins expression and decreased stimulated­calcium dynamics were detected in PCSK9-deficient ß-cells. Mechanistically, pancreatic PCSK9 silencing impacts ß-cell LDLR expression and cholesterol content, both in vivo and in vitro. The key role of LDLR is confirmed by the demonstration that LDLR downregulation rescued the phenotype. CONCLUSIONS: These findings establish pancreatic PCSK9 as a novel critical regulator of the functional maturation of the ß-cell secretory pathway, via modulation of cholesterol homeostasis.

LIN7 mediates the recruitment of IRSp53 to tight junctions.

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (DeltaL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Delta5) or fused to the L27 domain of LIN7 (L27-IRSp53Delta5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.

Iron Metabolism in Pancreatic Beta-Cell Function and Dysfunction.

Iron is an essential element involved in a variety of physiological functions. In the pancreatic beta-cells, being part of Fe-S cluster proteins, it is necessary for the correct insulin synthesis and processing. In the mitochondria, as a component of the respiratory chain, it allows the production of ATP and reactive oxygen species (ROS) that trigger beta-cell depolarization and potentiate the calcium-dependent insulin release. Iron cellular content must be finely tuned to ensure the normal supply but also to prevent overloading. Indeed, due to the high reactivity with oxygen and the formation of free radicals, iron excess may cause oxidative damage of cells that are extremely vulnerable to this condition because the normal elevated ROS production and the paucity in antioxidant enzyme activities. The aim of the present review is to provide insights into the mechanisms responsible for iron homeostasis in beta-cells, describing how alteration of these processes has been related to beta-cell damage and failure. Defects in iron-storing or -chaperoning proteins have been detected in diabetic conditions; therefore, the control of iron metabolism in these cells deserves further investigation as a promising target for the development of new disease treatments.

Verbascoside Protects Pancreatic ß-Cells against ER-Stress.

Substantial epidemiological evidence indicates that a diet rich in polyphenols protects against developing type 2 diabetes. The phenylethanoid glycoside verbascoside/acteoside, a widespread polyphenolic plant compound, has several biological properties including strong antioxidant, anti-inflammatory and neuroprotective activities. The aim of this research was to test the possible effects of verbascoside on pancreatic ß-cells, a target never tested before. Mouse and human ß-cells were incubated with verbascoside (0.8-16 µM) for up to five days and a combination of biochemical and imaging techniques were used to assess the ß-cell survival and function under normal or endoplasmic reticulum (ER)-stress inducing conditions. We found a dose-dependent protective effect of verbascoside against oxidative stress in clonal and human ß-cells. Mechanistic studies revealed that the polyphenol protects ß-cells against ER-stress mediated dysfunctions, modulating the activation of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) branch of the unfolded protein response and promoting mitochondrial dynamics. As a result, increased viability, mitochondrial function and insulin content were detected in these cells. These studies provide the evidence that verbascoside boosts the ability of ß-cells to cope with ER-stress, an important contributor of ß-cell dysfunction and failure in diabetic conditions and support the therapeutic potential of verbascoside in diabetes.

Shaping Pancreatic ß-Cell Differentiation and Functioning: The Influence of Mechanotransduction.

Embryonic and pluripotent stem cells hold great promise in generating ß-cells for both replacing medicine and novel therapeutic discoveries in diabetes mellitus. However, their differentiation in vitro is still inefficient, and functional studies reveal that most of these ß-like cells still fail to fully mirror the adult ß-cell physiology. For their proper growth and functioning, ß-cells require a very specific environment, the islet niche, which provides a myriad of chemical and physical signals. While the nature and effects of chemical stimuli have been widely characterized, less is known about the mechanical signals. We here review the current status of knowledge of biophysical cues provided by the niche where ß-cells normally live and differentiate, and we underline the possible machinery designated for mechanotransduction in ß-cells. Although the regulatory mechanisms remain poorly understood, the analysis reveals that ß-cells are equipped with all mechanosensors and signaling proteins actively involved in mechanotransduction in other cell types, and they respond to mechanical cues by changing their behavior. By engineering microenvironments mirroring the biophysical niche properties it is possible to elucidate the ß-cell mechanotransductive-regulatory mechanisms and to harness them for the promotion of ß-cell differentiation capacity in vitro.