Determination of secretory granule maturation times in pancreatic islet ß-cells by serial block-face electron microscopy.

It is shown how serial block-face electron microscopy (SBEM) of insulin-secreting ß-cells in wild-type mouse pancreatic islets of Langerhans can be used to determine maturation times of secretory granules. Although SBEM captures the ß-cell structure at a snapshot in time, the observed ultrastructure can be considered representative of a dynamic equilibrium state of the cells since the pancreatic islets are maintained in culture in approximate homeostasis. It was found that 7.2 ± 1.2% (±st. dev.) of the ß-cell volume is composed of secretory granule dense-cores exhibiting angular shapes surrounded by wide (typically ≳100 nm) electron-lucent halos. These organelles are identified as mature granules that store insulin for regulated release through the plasma membrane, with a release time of 96 ± 12 h, as previously obtained from pulsed 35S-radiolabeling of cysteine and methionine. Analysis of ß-cell 3D volumes reveals a subpopulation of secretory organelles without electron-lucent halos, identified as immature secretory granules. Another subpopulation of secretory granules is found with thin (typically ≲30 nm) electron-lucent halos, which are attributed to immature granules that are transforming from proinsulin to insulin by action of prohormone convertases. From the volume ratio of proinsulin in the immature granules to insulin in the mature granules, we estimate that the newly formed immature granules remain in morphologically-defined immature states for an average time of 135 ± 14 min, and the immature transforming granules for an average time of 130 ± 17 min.

Combining quantitative 2D and 3D image analysis in the serial block face SEM: application to secretory organelles of pancreatic islet cells.

A combination of two-dimensional (2D) and three-dimensional (3D) analyses of tissue volume ultrastructure acquired by serial block face scanning electron microscopy can greatly shorten the time required to obtain quantitative information from big data sets that contain many billions of voxels. Thus, to analyse the number of organelles of a specific type, or the total volume enclosed by a population of organelles within a cell, it is possible to estimate the number density or volume fraction of that organelle using a stereological approach to analyse randomly selected 2D block face views through the cells, and to combine such estimates with precise measurement of 3D cell volumes by delineating the plasma membrane in successive block face images. The validity of such an approach can be easily tested since the entire 3D tissue volume is available in the serial block face scanning electron microscopy data set. We have applied this hybrid 3D/2D technique to determine the number of secretory granules in the endocrine α and ß cells of mouse pancreatic islets of Langerhans, and have been able to estimate the total insulin content of a ß cell.

Quantitative analysis of mouse pancreatic islet architecture by serial block-face SEM.

We have applied serial block-face scanning electron microscopy (SBF-SEM) to measure parameters that describe the architecture of pancreatic islets of Langerhans, microscopic endocrine organs that secrete insulin and glucagon for control of blood glucose. By analyzing entire mouse islets, we show that it is possible to determine (1) the distributions of alpha and beta cells, (2) the organization of blood vessels and pericapillary spaces, and (3) the ultrastructure of the individual secretory cells. Our results show that the average volume of a beta cell is nearly twice that of an alpha cell, and the total mitochondrial volume is about four times larger. In contrast, nuclear volumes in the two cell types are found to be approximately equal. Although the cores of alpha and beta secretory granules have similar diameters, the beta granules have prominent halos resulting in overall diameters that are twice those of alpha granules. Visualization of the blood vessels revealed that every secretory cell in the islet is in contact with the pericapillary space, with an average contact area of 9±5% of the cell surface area. Our data show that consistent results can be obtained by analyzing small numbers of islets. Due to the complicated architecture of pancreatic islets, such precision cannot easily be achieved by using TEM of thin sections.

The dense core vesicle protein IA-2, but not IA-2ß, is required for active avoidance learning.

The islet-antigens IA-2 and IA-2ß are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2ß alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2ß SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2ß SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2ß SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired.

Co-regulation of intragenic microRNA miR-153 and its host gene Ia-2 ß: identification of miR-153 target genes with functions related to IA-2ß in pancreas and brain.

AIMS/HYPOTHESIS: We analysed the genomic organisation of miR-153, a microRNA embedded in genes that encode two of the major type 1 diabetes autoantigens, islet-associated protein (IA)-2 and IA-2ß. We also identified miR-153 target genes that correlated with IA-2ß localisation and function. METHODS: A bioinformatics approach was used to identify miR-153's genomic organisation. To analyse the co-regulation of miR-153 and IA-2ß, quantitative PCR analysis of miR-153 and Ia-2ß (also known as Ptprn2) was performed after a glucose stimulation assay in MIN6B cells and isolated murine pancreatic islets, and also in wild-type Ia-2 (also known as Ptprn), Ia-2ß single knockout and Ia-2/Ia-2ß double knockout mouse brain and pancreatic islets. Bioinformatics identification of miR-153 target genes and validation via luciferase reporter assays, western blotting and quantitative PCR were also carried out. RESULTS: Two copies of miR-153, miR-153-1 and miR-153-2, are localised in intron 19 of Ia-2 and Ia-2ß, respectively. In rodents, only miR-153-2 is conserved. We demonstrated that expression of miR-153-2 and Ia-2ß in rodents is partially co-regulated as demonstrated by a strong reduction of miR-153 expression levels in Ia-2ß knockout and Ia-2/Ia-2ß double knockout mice. miR-153 levels were unaffected in Ia-2 knockout mice. In addition, glucose stimulation, which increases Ia-2 and Ia-2ß expression, also significantly increased expression of miR-153. Several predicted targets of miR-153 were reduced after glucose stimulation in vitro, correlating with the increase in miR-153 levels. CONCLUSIONS/INTERPRETATION: This study suggests the involvement of miR-153, IA-2ß and miR-153 target genes in a regulatory network, which is potentially relevant to insulin and neurotransmitter release.

Deletion of Ia-2 and/or Ia-2ß in mice decreases insulin secretion by reducing the number of dense core vesicles.

AIMS/HYPOTHESIS: Islet antigen 2 (IA-2) and IA-2ß are dense core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes. The present experiments were initiated to test the hypothesis that the knockout of the genes encoding these proteins impairs the secretion of insulin by reducing the number of DCV. METHODS: Insulin secretion, content and DCV number were evaluated in islets from single knockout (Ia-2 [also known as Ptprn] KO, Ia-2ß [also known as Ptprn2] KO) and double knockout (DKO) mice by a variety of techniques including electron and two-photon microscopy, membrane capacitance, Ca(2+) currents, DCV half-life, lysosome number and size and autophagy. RESULTS: Islets from single and DKO mice all showed a significant decrease in insulin content, insulin secretion and the number and half-life of DCV (p < 0.05 to 0.001). Exocytosis as evaluated by two-photon microscopy, membrane capacitance and Ca(2+) currents supports these findings. Electron microscopy of islets from KO mice revealed a marked increase (p < 0.05 to 0.001) in the number and size of lysosomes and enzymatic studies showed an increase in cathepsin D activity (p < 0.01). LC3 protein, an indicator of autophagy, also was increased in islets of KO compared with wild-type mice (p < 0.05 to 0.01) suggesting that autophagy might be involved in the deletion of DCV. CONCLUSIONS/INTERPRETATION: We conclude that the decrease in insulin content and secretion, resulting from the deletion of Ia-2 and/or Ia-2ß, is due to a decrease in the number of DCV.

Disturbances in the secretion of neurotransmitters in IA-2/IA-2beta null mice: changes in behavior, learning and lifespan.

Islet-associated protein 2 (IA-2) and IA-2beta are major autoantigens in type 1 diabetes and transmembrane proteins in dense core secretory vesicles (DCV) of neuroendocrine cells. The deletion of these genes results in a decrease in insulin secretion. The present study was initiated to test the hypothesis that this deletion not only affects the secretion of insulin, but has a more global effect on neuroendocrine secretion that leads to disturbances in behavior and learning. Measurement of neurotransmitters showed that norepinephrine, dopamine and 5-HT were significantly decreased in the brain of double knockout (DKO) mice (P<0.05 to <0.001). In tests evaluating anxiety-like behavior and conditioned-learning, the DKO mice showed a highly significant increase in anxiety-like behavior (P<0.01 to <0.001) and impairment of conditioned learning (P<0.01) as compared to WT mice. The DKO mice also displayed an increase in spontaneous and induced seizures (P<0.01) and age-related death. Contrary to the generally held view that IA-2 and IA-2beta are expressed exclusively in DCV, subcellular fractionation studies revealed that IA-2beta, but not IA-2, co-purifies with fractions rich in synaptic vesicles (SV), and that the secretion of dopamine, GABA and glutamate from the synaptosomes of the DKO mice was significantly decreased as was the number of SV (P<0.01). Taken together, these findings show that IA-2beta is present in both DCV and SV, and that the deletion of IA-2/IA-2beta has a global effect on the secretion of neurotransmitters. The impairment of secretion leads to behavioral and learning disturbances, seizures and reduced lifespan.

Insulin secretion in islets from mice with a double knockout for the dense core vesicle proteins islet antigen-2 (IA-2) and IA-2beta.

Islet antigen-2 (IA-2 or ICA 512) and IA-2beta (or phogrin) are major autoantigens in type 1 diabetes. They are located in dense core secretory vesicles including insulin granules, but their role in beta-cell function is unclear. Targeted disruption of either IA-2 or IA-2beta, or both, impaired glucose tolerance, an effect attributed to diminution of insulin secretion. In this study, we therefore characterized the dynamic changes in cytosolic Ca2+([Ca2+](c)) and insulin secretion in islets from IA-2/IA-2beta double knockout (KO) mice. High glucose (15 mM) induced biphasic insulin secretion in IA-2/IA-2beta KO islets, with a similar first phase and smaller second phase compared with controls. Since the insulin content of IA-2/IA-2beta KO islets was approximately 45% less than that of controls, fractional insulin secretion (relative to content) was thus increased during first phase and unaffected during second phase. This peculiar response occurred in spite of a slightly smaller rise in [Ca2+](c), could not be attributed to an alteration of glucose metabolism (NADPH fluorescence) and also was observed with tolbutamide. The dual control of insulin secretion via the K(ATP) channel-dependent triggering pathway and K(ATP) channel-independent amplifying pathway was unaltered in IA-2/IA-2beta KO islets, and so were the potentiations by acetylcholine or cAMP (forskolin). Intriguingly, amino acids, in particular the cationic arginine and lysine, induced larger fractional insulin secretion in IA-2/IA-2beta KO than control islets. In conclusion, IA-2 and IA-2beta are dispensable for exocytosis of insulin granules, but are probably more important for cargo loading and/or stability of dense core vesicles.

Overexpression of the autoantigen IA-2 puts beta cells into a pre-apoptotic state: autoantigen-induced, but non-autoimmune-mediated, tissue destruction.

IA-2 is a major autoantigen in type 1 diabetes and autoantibodies to it have become important diagnostic and predictive markers. IA-2 also is an intrinsic transmembrane component of dense core secretory vesicles and knock-out studies showed that IA-2 is a regulator of insulin secretion. Here we show that overexpression of IA-2 puts mouse insulinoma MIN-6 beta cells into a pre-apoptotic state and that exposure to high glucose results in G2/M arrest and apoptosis. Molecular study revealed a decrease in phosphoinositide-dependent kinase (PDK)-1 and Akt/protein kinase B (PKB) phosphorylation. Treatment of IA-2-transfected cells with IA-2 siRNA prevented both G2/M arrest and apoptosis and increased Akt/PKB phosphorylation. A search for IA-2 interacting proteins revealed that IA-2 interacts with sorting nexin (SNX)19 and that SNX19, but not IA-2, inhibits the conversion of PtdIns(4,5)P2 to PtdIns(3,4,5)P3 and thereby suppresses the phosphorylation of proteins in the Akt signalling pathway resulting in apoptosis. We conclude that IA-2 acts through SNX19 to initiate the pre-apoptotic state. Our findings point to the possibility that in autoimmune diseases, tissue destruction may be autoantigen-induced, but not necessarily immunologically mediated.

Human chorionic gonadotropin is an immune modulator and can prevent autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Expression of T helper (Th)1 cytokine mRNA in pregnant women is known to be inversely correlated with serum human chorionic gonadotropin (hCG). Type 1 diabetes is a Th1-mediated autoimmune disease, in which intervention at an early stage of the autoimmune process can prevent disease progression. We hypothesised that immune modulation by treating young NOD mice with hCG may prevent diabetes. METHODS: Female NOD mice were treated with hCG or recombinant hCG from 3 to 15 weeks of age and the incidence of diabetes and development of insulitis was determined. CD4(+) and CD8(+) T cell populations, T cell proliferation, cytokine production and CD4(+)CD25(+) regulatory T cells were examined and adoptive transfer experiments were performed. RESULTS: Both purified and recombinant hCG prevented development of diabetes in NOD mice. hCG decreased the proportion and number of CD4(+) and CD8(+) T cells and inhibited T cell proliferative responses against beta cell antigens. hCG treatment suppressed IFN-gamma production, but increased IL-10 and TGF-beta production in splenocytes stimulated with anti-CD3 antibody. hCG treatment also suppressed TNF-alpha production in splenocytes stimulated with lipopolysaccharide. Furthermore, hCG treatment increased the CD4(+)CD25(+)/CD4(+) T cell ratio in spleen and pancreatic lymph nodes. Depletion of CD4(+)CD25(+) T cells from splenocytes of hCG-treated NOD mice abolished their preventive effect on diabetes transfer. CONCLUSIONS/INTERPRETATION: We conclude that hCG has an immunomodulatory effect by downregulating effector cells, including Th1 cells, CD8(+) T cells and macrophages, and increasing the CD4(+)CD25(+)/CD4(+) T cell ratio, thus preventing autoimmune diabetes in NOD mice.

The IA-2 interactome.

AIMS/HYPOTHESIS: Islet antigen-2 (IA-2), a major autoantigen in type 1 diabetes, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase (PTP) family. IA-2 is located in dense-core secretory vesicles and is involved in the regulation of insulin secretion. The present experiments were initiated to identify those proteins that interact with IA-2 (i.e. the IA-2 interactome) as a first step towards elucidating the mechanism(s) by which IA-2 influences insulin secretion and serves as an autoantigen. MATERIALS AND METHODS: To determine the proteins with which IA-2 interacts, a yeast two-hybrid system was used to screen a human foetal library, and deletion mutants were used to determine the binding sites. Positive interactions were confirmed by immunoprecipitation pull-down experiments using cell lysate from transfected mammalian cell lines. RESULTS: Six new interacting proteins were identified by this approach: mitogen-activated protein kinase-activating death domain (MADD), the MADD isoform IG20, PTPrho, PTPsigma, sorting nexin 19 (SNX19) and cyclophilin A. Using a series of IA-2 deletion mutants, we identified the regions on the IA-2 molecule to which five of the interacting proteins bound. Amino acids 744-979 of IA-2 were required for the maximum binding of MADD, IG20 and SNX19, whereas amino acids 602-907 of IA-2 were required for the maximum binding of PTPrho and PTPsigma. Pull-down experiments with cell lysate from transfected mammalian cells confirmed the binding of the interacting proteins to IA-2. CONCLUSIONS/INTERPRETATION: The IA-2 interactome based on, pull-down experiments, currently consists of 12 proteins. The identification of these interacting proteins provides clues as to how IA-2 exerts its biological functions.

Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes.

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.

HLA-DQ8 transgenic and NOD mice recognize different epitopes within the cytoplasmic region of the tyrosine phosphatase-like molecule, IA-2.

Type 1 diabetes mellitus is strongly associated with HLA-DQ8 in humans and I-A(g7) in the NOD mouse. The disease is characterized by loss of tolerance to auto-antigens such as GAD, insulin, and the protein tyrosine phosphatase-like molecule, IA-2. We identified T cell epitopes on the intracytoplasmic region of IA-2 by immunizing DQ8/NOD, DQ8/B10, and NOD mice with overlapping 18 mer peptides in CFA. We identified four peptides presented both by DQ8 and NOD, five DQ8 specific peptides, and six NOD specific peptides. Both mouse lines failed to respond to ten peptides. We demonstrated MHC class II and CD4 restriction of proliferative responses using appropriate blocking antibodies. To understand the role of non-MHC genes in the generation of immune response to the islet auto-antigen, we evaluated cytokine secretion following immunization of DQ8 transgenic mice with strongly immunogenic peptides. The NOD background resulted in increased secretion of cytokines. In conclusion, we have identified IA-2 peptides that induce lymphoproliferative responses in DQ8 transgenic and NOD mice and shown that these peptides stimulate production of Th1 and Th2 cytokines.

Analysis of the coding and promoter regions of the autoantigen IA-2 in subjects with and without autoantibodies to IA-2.

Despite extensive studies on HLA polymorphism, there have been few, if any, studies on allelic forms or mutations in proteins that serve as autoantigens. The present experiments were designed to look for alterations in the coding and promoter regions of the autoantigen IA-2 in type one (insulin-dependent) diabetic patients with autoantibodies to IA-2 as compared with siblings without diabetes or autoantibodies to IA-2. Genomic DNA was used as a template and was amplified by polymerase chain reaction, with pairs of primers encompassing the promoter region and the 23 exons of the coding region of IA-2. A total of nine nucleotide changes were found in the coding region of the six type 1 diabetic patients; four were silent and five were missense changes, but all occurred in the extracellular domain of IA-2 to which autoantibodies are not directed. Few, if any, changes were found in the 5' upstream (-706 to +135) promoter region. The results of the experiments support the null hypothesis that differences among individuals in the nucleotide and amino acid sequences of the promoter and coding regions of IA-2, respectively, do not account for why some individuals develop autoantibodies to IA-2 and others do not.