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Cell-to-cell interactions play an important role in insulin secretion. Compared with intact islets, dispersed pancreatic beta-cells show increased basal and decreased glucose-stimulated insulin secretion. In this study, we used mouse MIN6B1 cells to investigate the mechanisms that control insulin secretion when cells are in contact with each other or not. RNAi-mediated silencing of the adhesion molecule E-cadherin in confluent cells reduced glucose-stimulated secretion to the levels observed in isolated cells but had no impact on basal secretion. Dispersed cells presented high cytosolic Ca(2+) activity, depolymerized cytoskeleton and ERK1/2 activation in low glucose conditions. Both the increased basal secretion and the spontaneous Ca(2+) activity were corrected by transient removal of Ca(2+) or prolonged incubation of cells in low glucose, a procedure that restored the ability of dispersed cells to respond to glucose (11-fold stimulation). In conclusion, we show that dispersed pancreatic beta-cells can respond robustly to glucose once their elevated basal secretion has been corrected. The increased basal insulin secretion of dispersed cells is due to spontaneous Ca(2+) transients that activate downstream Ca(2+) effectors, whereas engagement of cell adhesion molecules including E-cadherin contributes to the greater secretory response to glucose seen in cells with normal intercellular contacts.

Studies on the pathophysiology of type 2 diabetes mellitus (T2DM) have linked the accumulation of lipid metabolites to the development of beta-cell dysfunction and impaired insulin secretion. In most in vitro models of T2DM, rodent islets or beta-cell lines are used and typically focus is on specific cellular pathways or organs. Our aim was to, firstly, develop a combined lipidomics and proteomics approach for lipotoxicity in isolated human islets and, secondly, investigate if the approach could delineate novel and/ or confirm reported mechanisms of lipotoxicity. To this end isolated human pancreatic islets, exposed to chronically elevated palmitate concentrations for 0, 2 and 7 days, were functionally characterized and their levels of multiple targeted lipid and untargeted protein species determined. Glucose-stimulated insulin secretion from the islets increased on day 2 and decreased on day 7. At day 7 islet insulin content decreased and the proinsulin to insulin content ratio doubled. Amounts of cholesterol, stearic acid, C16 dihydroceramide and C24:1 sphingomyelin, obtained from the lipidomic screen, increased time-dependently in the palmitate-exposed islets. The proteomic screen identified matching changes in proteins involved in lipid biosynthesis indicating up-regulated cholesterol and lipid biosynthesis in the islets. Furthermore, proteins associated with immature secretory granules were decreased when palmitate exposure time was increased despite their high affinity for cholesterol. Proteins associated with mature secretory granules remained unchanged. Pathway analysis based on the protein and lipid expression profiles implicated autocrine effects of insulin in lipotoxicity. Taken together the study demonstrates that combining different omics approaches has potential in mapping of multiple simultaneous cellular events. However, it also shows that challenges exist for effectively combining lipidomics and proteomics in primary cells. Our findings provide insight into how saturated fatty acids contribute to islet cell dysfunction by affecting the granule maturation process and confirmation in human islets of some previous findings from rodent islet and cell-line studies.

Proinsulin is packaged into nascent (immature, clathrin-coated) secretory granules in the trans-Golgi network (TGN) of the beta -cell along with other granular constituents including the proinsulin conversion enzymes. It is assumed that such packaging is dependent on an active sorting process, separating granular proteins from other secretory or membrane proteins, but the mechanism remains elusive. As granules mature, the clathrin coat is lost, the intragranular milieu is progressively acidified, and proinsulin is converted to insulin and C-peptide. Loss of clathrin is believed to arise by budding of clathrin-coated vesicles from maturing granules, carrying with them any inappropriate or unnecessary products and providing an additional means for refinement of granular content.

Insulin is both produced and degraded within the pancreatic Beta cell. Production involves the synthesis of the initial insulin precursor preproinsulin, which is converted to proinsulin shortly after (or during) translocation into the lumen of the rough endoplasmic reticulum. Proinsulin is then transported to the trans-cisternae of the Golgi complex where it is directed towards nascent secretory granules. Conversion of proinsulin to insulin and C-peptide arises within secretory granules, and is dependent upon their acidification. Granule contents are discharged by exocytosis in response to an appropriate stimulus. This represents the regulated secretory pathway to which more than 99% of proinsulin is directed in Beta cells of a healthy individual. An alternative route also exists in the Beta cell, the constitutive secretory pathway. It involves the rapid transfer of products from the Golgi complex to the plasma membrane for immediate release, with, it is supposed, little occasion for prohormone conversion. Even if delivered appropriately to secretory granules, not all insulin is released; some is degraded by fusion of granules with lysosomes (crinophagy). Each event in the molecular lifestyles of insulin and its precursors in the Beta cell will be seen to be governed by their own discrete functional domains. The identification and characterisation of these protein domains will help elucidate the steps responsible for delivery of proinsulin to secretory granules and conversion to insulin. Understanding the molecular mechanism of these steps may, in turn, help to explain defective insulin production in certain disease states including diabetes mellitus.

BACKGROUND: Fasting hyperglycemia has been associated with HIV protease inhibitor (PI) therapy. OBJECTIVE: To determine whether absolute insulin deficiency or insulin resistance with relative insulin deficiency and an elevated body mass index (BMI) contribute to HIV PI-associated diabetes. DESIGN: Cross-sectional evaluation. PATIENTS: 8 healthy seronegative men, 10 nondiabetic HIV-positive patients naive to PI, 15 nondiabetic HIV-positive patients receiving PI (BMI = 26 kg/m2), 6 nondiabetic HIV-positive patients receiving PI (BMI = 31 kg/m2), and 8 HIV-positive patients with diabetes receiving PI (BMI = 34 kg/m2). All patients on PI received indinavir. MEASUREMENTS: Fasting concentrations of glucoregulatory hormones. Direct effects of indinavir (20 microM) on rat pancreatic beta-cell function in vitro. RESULTS: In hyperglycemic HIV-positive subjects, circulating concentrations of insulin, C-peptide, proinsulin, glucagon, and the proinsulin/insulin ratio were increased when compared with those of the other 4 groups (p

Rat pancreatic islets were maintained in tissue culture (8.3 mM glucose) for 3 days in the presence of [3H]leucine in order to label insulin stores. Alternatively, islets were labeled for 1 h at the end of a 3-day maintenance period in order to label only newly synthesized insulin. During a 24-h chase incubation, 13% of the prelabeled insulin stores (3-day label) had been released. By contrast, 30% of newly synthesized (1-h label) insulin was released during a similar chase period. Since islets were handled identically aside from the respective time of exposure to [3H]leucine, the total immunoreactive islet insulin content and that released into the medium was similar for both groups of islets. The specific radioactivity of the labeled insulin in the medium at the end of the chase period was thus lower than that remaining in the islets for islets prelabeled for 3 days but was higher after the labeling of newly synthesized insulin for 1 h. When the rate of newly synthesized insulin was followed during a 4-...

Islets of Langerhans isolated from adult rats were maintained in tissue culture for 3 days in the continued presence of [3H]leucine. Labelled proinsulin, C-peptide and insulin were measured by quantitative h.p.l.c., a method which also allowed for resolution of C-peptide I and II, and of insulin I and II (the products of the two rat insulin genes). The results showed that: (1) at early times, proinsulin was the major radiolabelled product; with progressive time in culture, intra-islet levels of [3H]proinsulin decreased, despite continuous labelling with [3H]leucine, indicating that the combined rates of proinsulin conversion into insulin and of proinsulin release, exceeded the rate of synthesis; (2) insulin I levels were always greater than those of insulin II, both in the islets and for products released to the medium; (3) the molar ratio of [3H]insulin I and II to their respective 3H-labelled C-peptides increased with time for products retained within islets, reaching a value close to 3:1 by 3 days; by contrast, for products released to the medium during the culture period, the ratio was always close to unity; (4) when islets were incubated with [3H]leucine for 2 days, and then left for a further 1 day without label (chase period), the intra-islet [3H]insulin/[3H]C-peptide ratios rose to values as high as 9:1. Again, for material released to the medium, the values were close to 1:1; (5) it is concluded that C-peptide is degraded more rapidly than insulin within islet cells, thereby accounting for the elevated insulin/C-peptide ratios. The difference between the ratios observed in the islets and those for material released to the medium is taken to indicate that degradation occurs in a discrete cellular compartment and not in the secretory granule itself.

HLA-DQA1*0102-DQB1*0602 is associated with protection against type 1 diabetes (T1D). A similar allele, HLA-DQA1*0102-DQB1*0604, contributes to T1D susceptibility in certain populations but differs only at seven amino acids from HLA-DQA1*0102-DQB1*0602. Five of these polymorphisms are found within the peptide-binding groove, suggesting that differences in peptide binding contribute to the mechanism of their association with T1D. In this study, we determine the peptide-binding motif for HLA-DQA1*0102-DQB1*0604 allelic protein (DQ0604) in comparison to the established HLA-DQA1*0102-DQB1*0602 (DQ0602) motif using binding assays with model peptides from T1D autoantigens and homology modeling using the coordinates of the DQ0602-hypocretin 1-13 crystal structure. The peptide binding preferences were deduced with a peptide from insulin that bound both with a 2- to 3-fold difference in avidity using the same amino acids in the peptide as anchors. Peptide binding differences directly influenced by the polymorphisms in or nearby pockets 1, 6, and 9 were observed. In pocket 1, DQ0604 was better able to accommodate aromatic residues due to the beta86 and beta87 polymorphisms. A negatively charged amino acid was preferred by DQ0604 in pocket 6 due to the positively charged beta30His. In pocket 9, DQ0604 preferred aromatic amino acids due to the beta9 and beta30 polymorphisms and had low tolerance of acidic residues. beta57Val in DQ0604 functions differently than beta57Ala, in that it pushes alpha76Arg outside of the pocket, preventing the formation of a salt bridge with an acidic amino acid in the peptide. This study furthers our understanding of the structure-function relationships of MHC class II polymorphisms. J Immunol