Effects of immunosuppression on alpha and beta cell renewal in transplanted mouse islets.

AIMS/HYPOTHESIS: Immunosuppressive drugs used in human islet transplantation interfere with the balance between beta cell renewal and death, and thus may contribute to progressive graft dysfunction. We analysed the influence of immunosuppressants on the proliferation of transplanted alpha and beta cells after syngeneic islet transplantation in streptozotocin-induced diabetic mice. METHODS: C57BL/6 diabetic mice were transplanted with syngeneic islets in the liver and simultaneously abdominally implanted with a mini-osmotic pump delivering BrdU alone or together with an immunosuppressant (tacrolimus, sirolimus, everolimus or mycophenolate mofetil [MMF]). Glycaemic control was assessed for 4 weeks. The area and proliferation of transplanted alpha and beta cells were subsequently quantified. RESULTS: After 4 weeks, glycaemia was significantly higher in treated mice than in controls. Insulinaemia was significantly lower in mice treated with everolimus, tacrolimus and sirolimus. MMF was the only immunosuppressant that did not significantly reduce beta cell area or proliferation, albeit its levels were in a lower range than those used in clinical settings. CONCLUSIONS/INTERPRETATION: After transplantation in diabetic mice, syngeneic beta cells have a strong capacity for self-renewal. In contrast to other immunosuppressants, MMF neither impaired beta cell proliferation nor adversely affected the fractional beta cell area. Although human beta cells are less prone to proliferate compared with rodent beta cells, the use of MMF may improve the long-term outcome of islet transplantation.

Novel standards in the measurement of rat insulin granules combining electron microscopy, high-content image analysis and in silico modelling.

AIMS/HYPOTHESIS: Knowledge of number, size and content of insulin secretory granules is pivotal for understanding the physiology of pancreatic beta cells. Here we re-evaluated key structural features of rat beta cells, including insulin granule size, number and distribution as well as cell size. METHODS: Electron micrographs of rat beta cells fixed either chemically or by high-pressure freezing were compared using a high-content analysis approach. These data were used to develop three-dimensional in silico beta cell models, the slicing of which would reproduce the experimental datasets. RESULTS: As previously reported, chemically fixed insulin secretory granules appeared as hollow spheres with a mean diameter of ∼350 nm. Remarkably, most granules fixed by high-pressure freezing lacked the characteristic halo between the dense core and the limiting membrane and were smaller than their chemically fixed counterparts. Based on our analyses, we conclude that the mean diameter of rat insulin secretory granules is 243 nm, corresponding to a surface area of 0.19 µm(2). Rat beta cells have a mean volume of 763 µm(3) and contain 5,000-6,000 granules. CONCLUSIONS/INTERPRETATION: A major reason for the lower mean granule number/rat beta cell relative to previous accounts is a reduced estimation of the mean beta cell volume. These findings imply that each granule contains about twofold more insulin, while its exocytosis increases membrane capacitance about twofold less than assumed previously. Our integrated approach defines new standards for quantitative image analysis of beta cells and could be applied to other cellular systems.

An update on preventive and regenerative therapies in diabetes mellitus.

Type 1A (immune-mediated) and type 2 diabetes mellitus are two of the most common severe chronic illnesses, affecting over 230 million people worldwide with an estimated global prevalence of 5.1%. Although type 1 and type 2 diabetes differ greatly in modes of pathogenesis, these illnesses share a common pathology and consequences characterized by loss of functional beta-cell mass and subsequent dysregulation of carbohydrate and lipid metabolism. Since therapy for diabetes and the associated complications poses enormous public health and economic burdens, novel preventive and regenerative therapies have emerged in the past decade with the aim to preserve beta-cell mass and delay the onset of diabetes. The goal of this review is to provide a comprehensive overview of current efforts in the fight against diabetes, and attempts to document all strategies that have emerged in clinical studies within the past 25 years. First, strategies to identify individuals at risk, ranging from whole-genome scans to autoantibody screening, will be discussed. Second, novel approaches to prevent or delay the onset of disease will be covered. Particular focus is given on emerging strategies for individuals at risk for type 1 diabetes that target T-cell regulation and induction of tolerance, while new pharmaceutical concepts in combination with lifestyle interventions are discussed within the scope of type 2 diabetes prevention. Lastly, important efforts to halt disease progression with emphasis on beta-cell regeneration are presented.

Proteomic profiling of beta-cells using a classical approach - two-dimensional gel electrophoresis.

Proteomics has rapidly become a major field of research in biology and medicine. Its main aim is to obtain a global overview of the expression pattern of proteins and their relationship in any given condition of a biological system. This knowledge is of particular interest to elucidate the pathogenesis of complex disorders, such as diabetes. Separation of proteins by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for their identification is perhaps the most widely used proteomic approach. In this review we have focused our attention on studies that have taken advantage of these methodologies to investigate the proteome of pancreatic islets, beta-cells and insulinoma cells in different conditions. As beta-cells of the pancreatic islets produce and secrete insulin, the main hormone for control of glucose homeostasis, these analyses may help to elucidate the mechanisms regulating insulin secretion and the development of various forms of diabetes, as well as to identify drug targets for therapeutic approaches.

Pancreatic disorders and diabetes mellitus.

Diabetes mellitus is a common disease among patients with pancreatic cancer and chronic pancreatitis, disorders of the exocrine pancreas. Different clinical features of diabetes are associated with these two conditions: hyperinsulinemia and peripheral insulin resistance are the prevailing diabetic traits in pancreatic cancer, whereas reduced islet cell mass and impaired insulin secretion are typically observed in chronic pancreatitis. Whether or not a causal relationship exists between diabetes and pancreatic carcinoma is an intriguing but unanswered question. Diabetes often precedes pancreatic cancer and is thus regarded as a potential risk factor for malignancy. Conversely, pancreatic cancer may secrete diabetogenic factors. Given these findings, there is increasing interest in whether close monitoring of the glycemic profile may aid early detection of pancreatic tumor lesions.

Emerging role of SUMO in pancreatic beta-cells.

Post-translational attachment of small ubiquitin-like modifier (SUMO), defined as SUMOylation, can affect the localization, interactions, stability and/or activity of substrate proteins, and thus can participate in a large variety of cellular processes. Most SUMO substrates are involved in transcriptional regulation. Hence, SUMOylation can either activate or, more commonly, repress gene transcription. The modulation of gene expression by SUMO through diverse mechanisms and specifically the recent findings concerning SUMOylation in pancreatic beta-cells are reviewed.

Markedly elevated GAD antibodies in SPS: effects of age and illness duration.

Five hundred seventy-six patients with suspected stiff-person syndrome (SPS) underwent immunocytochemistry (ICC). Of these, 286 underwent radioimmunoassay (RIA) for glutamic acid decarboxylase (GAD) antibodies; 116 were GAD antibody positive by one or both tests. Ninety-six percent of those positive by ICC had RIA values several standard deviations above normal. RIA did not correlate with age or illness duration. Marked elevations of RIA for GAD antibodies were characteristic of ICC-confirmed SPS, and modest elevations were not.

Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion.

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-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 Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-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 beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.

Antibodies against glutamic acid decarboxylase: prevalence in neurological diseases.

UNLABELLED: High prevalence of autoantibodies against glutamic acid decarboxylase (GAD-Ab) in stiff man syndrome (SMS) not only helps diagnosis, but also suggests immune mediated impairment of GABAergic functions. However, the presence of GAD-Ab has also been reported in other neurological syndromes. Therefore the prevalence of GAD-Ab was investigated in SMS, progressive encephalomyelitis with rigidity and myoclonus (PERM), and in other neurological diseases (OND). Serum antibodies against the GAD isoforms, GAD65 and GAD67, were investigated with radioimmunoassays in 13 patients with SMS, nine with PERM, 279 consecutive patients with OND, and in 100 normal controls. RESULTS: Prevalence of GAD65Ab was around 80% in patients with SMS/PERM compared with 5% in patients with OND and 1% in normal controls. Prevalence of GAD67Ab was 60% in SMS/PERM, 2% in patients with OND, and 1% in normal controls. Raised GAD-Ab clustered in an OND subgroup with sporadic progressive ataxia, but not in OND subgroups with recognised neuroimmunological diseases. In conclusion, increased GAD-Ab is neither a non-specific epiphenomenon of neuronal damage nor a common feature of recognised neuroimmunological disorders. In neurological diseases, GAD-Ab may be a pathogenetic agent or a marker for an ongoing autoimmune process, or both.

Autoimmunity to beta IV spectrin in paraneoplastic lower motor neuron syndrome.

Paraneoplastic neurological disorders may result from autoimmunity directed against antigens shared by the affected neurons and the associated cancer cells. We have recently reported the case of a woman with breast cancer and paraneoplastic lower motor neuron syndrome whose serum contained autoantibodies directed against axon initial segments and nodes of Ranvier of myelinated axons, including the axons of motoneurons. Here, we show that major targets of the autoantibodies of this patient are betaIVSigma1 spectrin and betaIV spectrin 140, two isoforms of the novel betaIV spectrin gene, as well as a neuronal surface epitope yet to be identified. Partial improvement of the neurological symptoms following cancer removal was associated with a drastic reduction in the titer of the autoantibodies against betaIV spectrin and nodal antigens in general, consistent with the autoimmune pathogenesis of the paraneoplastic lower motor neuron syndrome. The identification of betaIV spectrin isoforms and surface nodal antigens as novel autoimmune targets in lower motor neuron syndrome provide new insights into the pathogenesis of this severe neurological disease.

betaIV spectrin, a new spectrin localized at axon initial segments and nodes of ranvier in the central and peripheral nervous system.

We report the identification of betaIV spectrin, a novel spectrin isolated as an interactor of the receptor tyrosine phosphatase-like protein ICA512. The betaIV spectrin gene is located on human and mouse chromosomes 19q13.13 and 7b2, respectively. Alternative splicing of betaIV spectrin generates at least four distinct isoforms, numbered betaIVSigma1-betaIVSigma4 spectrin. The longest isoform (betaIVSigma1 spectrin) includes an actin-binding domain, followed by 17 spectrin repeats, a specific domain in which the amino acid sequence ERQES is repeated four times, several putative SH3-binding sites and a pleckstrin homology domain. betaIVSigma2 and betaIVSigma3 spectrin encompass the NH(2)- and COOH-terminal halves of betaIVSigma1 spectrin, respectively, while betaIVSigma4 spectrin lacks the ERQES and the pleckstrin homology domain. Northern blots revealed an abundant expression of betaIV spectrin transcripts in brain and pancreatic islets. By immunoblotting, betaIVSigma1 spectrin is recognized as a protein of 250 kD. Anti-betaIV spectrin antibodies also react with two additional isoforms of 160 and 140 kD. These isoforms differ from betaIVSigma1 spectrin in terms of their distribution on subcellular fractionation, detergent extractability, and phosphorylation. In islets, the immunoreactivity for betaIV spectrin is more prominent in alpha than in beta cells. In brain, betaIV spectrin is enriched in myelinated neurons, where it colocalizes with ankyrin(G) 480/270-kD at axon initial segments and nodes of Ranvier. Likewise, betaIV spectrin is concentrated at the nodes of Ranvier in the rat sciatic nerve. In the rat hippocampus, betaIVSigma1 spectrin is detectable from embryonic day 19, concomitantly with the appearance of immunoreactivity at the initial segments. Thus, we suggest that betaIVSigma1 spectrin interacts with ankyrin(G) 480/270-kD and participates in the clustering of voltage-gated Na(+) channels and cell-adhesion molecules at initial segments and nodes of Ranvier.

The receptor tyrosine phosphatase-like protein ICA512 binds the PDZ domains of beta2-syntrophin and nNOS in pancreatic beta-cells.

Islet cell autoantigen (ICA) 512 of type I diabetes is a receptor tyrosine phosphatase-like protein associated with the secretory granules of neurons and endocrine cells including insulin-secreting beta-cells of the pancreas. Here we show that in a yeast two-hybrid assay its cytoplasmic domain binds beta2-syntrophin, a modular adapter which in muscle cells interacts with members of the dystrophin family including utrophin, as well as the signaling molecule neuronal nitric oxide synthase (nNOS). The cDNA isolated by two-hybrid screening corresponded to a novel beta2-syntrophin isoform with a predicted molecular mass of 28 kDa. This isoform included the PDZ domain, but not the C-terminal region, which in full-length beta2-syntrophin is responsible for binding dystrophin-related proteins. In vitro binding of the beta2-syntrophin PDZ domain to ICA512 required both ICA512's C-terminal region and an internal polypeptide preceding its tyrosine phosphatase-like domain. Immunomicroscopy and co-immunoprecipitations from insulinoma INS-1 cells confirmed the occurrence of ICA512-beta2-syntrophin complexes in vivo. ICA512 also interacted in vitro with the PDZ domain of nNOS and ICA512-nNOS complexes were co-immunoprecipitated from INS-1 cells. Finally, we show that INS-1 cells, like muscle cells, contain beta2-syntrophin-utrophin oligomers. Thus, we propose that ICA512, through beta2-syntrophin and nNOS, links secretory granules with the actin cytoskeleton and signaling pathways involving nitric oxide.

Autoimmunity to gephyrin in Stiff-Man syndrome.

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by chronic rigidity, spasms, and autoimmunity directed against synaptic antigens, most often the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). In a subset of cases, SMS has an autoimmune paraneoplastic origin. We report here the identification of high-titer autoantibodies directed against gephyrin in a patient with clinical features of SMS and mediastinal cancer. Gephyrin is a cytosolic protein selectively concentrated at the postsynaptic membrane of inhibitory synapses, where it is associated with GABA(A) and glycine receptors. Our findings provide new evidence for a close link between autoimmunity directed against components of inhibitory synapses and neurological conditions characterized by chronic rigidity and spasms.

A novel antineuronal antibody in a motor neuron syndrome associated with breast cancer.

A 72-year-old woman developed a lower motor neuron syndrome (MNS) 4 months before the appearance of breast cancer. Monoparesis progressed to quadriparesis despite high-dose IV immunoglobulins, plasma exchange, and azathioprine, and high-dose IV methylprednisolone. The patient improved only after the removal of the tumor. MRI demonstrated hyperintensities in the cervical spinal cord. The patient had antibodies that reacted with axonal initial segments and nodes of Ranvier. The findings suggest that in this patient lower MNS may be a paraneoplastic condition associated with breast cancer.

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis.

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.

Post-translational modifications of ICA512, a receptor tyrosine phosphatase-like protein of secretory granules.

The autoantigen of type I diabetes ICA512 is a receptor tyrosine phosphatase-like protein enriched in the secretory granule membranes of neurons and peptide secreting endocrine cells. While the function of ICA512 remains unknown, it is thought to link regulated neuropeptide and peptide hormone secretion with signal transduction pathways involving tyrosine phosphorylation/dephosphorylation. To characterize further its biochemical properties, we conducted studies in the bovine pituitary, an abundant source of native ICA512, as well as in fibroblasts transfected with various human ICA512 cDNA constructs. Based on these studies we have established that the signal peptide of ICA512 encompasses residues 1-34 and that the ectodomain of ICA512 undergoes multiple post-translation modifications, including N-glycosylation. Newly synthesized ICA512 appears first as a pro-protein of 110 kDa that is then converted by post-translational modifications into a 130-kDa species. Cleavage of pro-ICA512 at a consensus for furin-like convertases generates a 60-66-kDa ICA512 transmembrane fragment (amino acids 449-979). Such processing ICA512 is not restricted to neuroendocrine cells, as it can also occur in transfected fibroblasts. Finally, the predicted N-terminal fragment of ICA512 resulting from this cleavage (amino acids 35-448) or parts thereof are present in the neurosecretosomes of posterior pituitary, raising the possibility that they may be secreted upon exocytosis of secretory granules.

Sequence analysis of the diabetes-protective human leukocyte antigen-DQB1*0602 allele in unaffected, islet cell antibody-positive first degree relatives and in rare patients with type 1 diabetes.

The human leukocyte antigen (HLA)-DQA1*0102/DQB1*0602/DRB1*1501 (DR2) haplotype confers strong protection from type 1 diabetes. Growing evidence suggests that such protection may be mostly encoded by the DQB1*0602 allele, and we reported that even first degree relatives with islet cell antibodies (ICA) have an extremely low diabetes risk if they carry DQB1*0602. Recently, novel variants of the DQB1*0602 and *0603 alleles were reported in four patients with type 1 diabetes originally typed as DQB1*0602 with conventional techniques. One inference from this observation is that DQB1*0602 may confer absolute protection and may never occur in type 1 diabetes. By this hypothesis, all patients typed as DQB1*0602 positive with conventional techniques should carry one of the above diabetes-permissive variants instead of the protective DQB1*0602. Such variants could also occur in ICA/DQB1*0602-positive relatives, with the implication that their diabetes risk could be significantly higher than previously estimated. We therefore sequenced the DQB1*0602 and DQA1*0102 alleles in all ICA/DQB1*0602-positive relatives (n = 8) previously described and in six rare patients with type 1 diabetes and DQB1*0602. We found that all relatives and patients carry the known DQB1*0602 and DQA1*0102 sequences, and none of them has the mtDNA A3243G mutation associated with late-onset diabetes in ICA-positive individuals. These findings suggest that diabetes-permissive DQB1*0602/3 variants may be very rare. Thus, although the protective effect associated with DQB1*0602 is extremely powerful, it is not absolute. Nonetheless, the development of diabetes in individuals with DQB1*0602 remains extremely unlikely, even in the presence of ICA, as confirmed by our further evaluation of ICA/DQB1*0602-positive relatives, none of whom has yet developed diabetes.