Lorella Marselli

Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta -cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation.

CONTEXT Elevated glucose levels impair islet function and survival, and it has been proposed that intraislet expression of IL-1beta contributes to glucotoxicity. OBJECTIVE The objective was to investigate IL-1beta mRNA expression in near-pure beta-cells of patients with type 2 diabetes (T2DM) and study the regulation of IL-1beta by glucose in isolated human islets. METHODS Laser capture microdissection was performed to isolate beta-cells from pancreas sections of 10 type 2 diabetic donors and nine controls, and IL-1beta mRNA expression was analyzed using gene arrays and PCR. Cultured human islets and fluorescence-activated cell sorter-purified human beta-cells were used to study the regulation of IL-1beta expression by glucose and IL-1beta. RESULTS Gene array analysis of RNA from beta-cells of individuals with T2DM revealed increased expression of IL-1beta mRNA. Real-time PCR confirmed increased IL-1beta expression in six of 10 T2DM samples, with minimal or no expression in nine control samples. In cultured human islets, IL-1beta mRNA and protein expression was induced by high glucose and IL-1beta autostimulation and decreased by the IL-1 receptor antagonist IL-1Ra. The glucose response was negatively correlated with basal IL-1beta expression levels. Autostimulation was transient and nuclear factor-kappaB dependent. Glucose-induced IL-1beta was biologically active and stimulated IL-8 release. Low picogram per milliliter concentrations of IL-1beta up-regulated inflammatory factors IL-8 and IL-6. CONCLUSION Evidence that IL-1beta mRNA expression is up-regulated in beta-cells of patients with T2DM is presented, and glucose-promoted IL-1beta autostimulation may be a possible contributor.

Autophagy in human type 2 diabetes pancreatic beta cells

Aims/hypothesisBeta cell loss contributes to type 2 diabetes, with increased apoptosis representing an underlying mechanism. Autophagy, i.e. the physiological degradation of damaged organelles and proteins, may, if altered, be associated with a distinct form of cell death. We studied several features of autophagy in beta cells from type 2 diabetic patients and assessed the role of metabolic perturbation and pharmacological intervention.MethodsPancreatic samples were obtained from organ donors and isolated islets prepared both by collagenase digestion and density gradient centrifugation. Beta cell morphology and morphometry were studied by electron microscopy. Gene expression studies were performed by quantitative RT-PCR.ResultsUsing electron microscopy, we observed more dead beta cells in diabetic (2.24 ± 0.53%) than control (0.66 ± 0.52%) samples (p < 0.01). Massive vacuole overload (suggesting altered autophagy) was associated with 1.18 ± 0.54% dead beta cells in type 2 diabetic samples and with 0.36 ± 0.26% in control samples (p < 0.05). Density volume of autophagic vacuoles and autophagosomes was significantly higher in diabetic beta cells. Unchanged gene expression of beclin-1 and ATG1 (also known as ULK1), and reduced transcription of LAMP2 and cathepsin B and D was observed in type 2 diabetic islets. Exposure of non-diabetic islets to increased NEFA concentration led to a marked increase of vacuole accumulation, together with enhanced beta cell death, which was associated with decreased LAMP2 expression. Metformin ameliorated autophagy alterations in diabetic beta cells and beta cells exposed to NEFA, a process associated with normalisation of LAMP2 expression.Conclusions/interpretationBeta cells in human type 2 diabetes have signs of altered autophagy, which may contribute to loss of beta cell mass. To preserve beta cell mass in diabetic patients, it may be necessary to target multiple cell-death pathways.

Gene Expression Profiles of Beta-Cell Enriched Tissue Obtained by Laser Capture Microdissection from Subjects with Type 2 Diabetes

Background Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D) should provide insights into their abnormal insulin secretion and turnover. Methodology/Principal Findings Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM). RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip) and Gene Set Enrichment Analysis (GSEA) software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG) family and metalloproteinase 7 (MMP7). Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2) were upregulated while JAZF1 and SLC30A8 were downregulated. Conclusions/Significance This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

Mutations at the BLK locus linked to maturity onset diabetes of the young and β-cell dysfunction

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency <1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK—a nonreceptor tyrosine-kinase of the src family of proto-oncogenes—is expressed in β-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of β-cell function, the deficit of which may lead to the development of diabetes.

Towards better understanding of the contributions of overwork and glucotoxicity to the β‐cell inadequacy of type 2 diabetes

Type 2 diabetes (T2D) is characterized by reduction of β‐cell mass and dysfunctional insulin secretion. Understanding β‐cell phenotype changes as T2D progresses should help explain these abnormalities. The normal phenotype should differ from the state of overwork when β‐cells compensate for insulin resistance to keep glucose levels normal. When only mild hyperglycaemia develops, β‐cells are subjected to glucotoxicity. As hyperglycaemia becomes more severe, so does glucotoxicity. β‐Cells in all four of these situations should have separate phenotypes. When assessing phenotype with gene expression, isolated islets have artefacts resulting from the trauma of isolation and hypoxia of islet cores. An advantage comes from laser capture microdissection (LCM), which obtains β‐cell‐rich tissue from pancreatic frozen sections. Valuable data can be obtained from animal models, but the real goal is human β‐cells. Our experience with LCM and gene arrays on frozen pancreatic sections from cadaver donors with T2D and controls is described. Although valuable data was obtained, we predict that the approach of taking fresh samples at the time of surgery is an even greater opportunity to markedly advance our understanding of how β‐cell phenotype evolves as T2D develops and progresses.

C/EBP homologous protein contributes to cytokine-induced pro-inflammatory responses and apoptosis in β-cells

Induction of the C/EBP homologous protein (CHOP) is considered a key event for endoplasmic reticulum (ER) stress-mediated apoptosis. Type 1 diabetes (T1D) is characterized by an autoimmune destruction of the pancreatic β-cells. Pro-inflammatory cytokines are early mediators of β-cell death in T1D. Cytokines induce ER stress and CHOP overexpression in β-cells, but the role for CHOP overexpression in cytokine-induced β-cell apoptosis remains controversial. We presently observed that CHOP knockdown (KD) prevents cytokine-mediated degradation of the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia sequence 1 (Mcl-1), thereby decreasing the cleavage of executioner caspases 9 and 3, and apoptosis. Nuclear factor-κB (NF-κB) is a crucial transcription factor regulating β-cell apoptosis and inflammation. CHOP KD resulted in reduced cytokine-induced NF-κB activity and expression of key NF-κB target genes involved in apoptosis and inflammation, including iNOS, FAS, IRF-7, IL-15, CCL5 and CXCL10. This was due to decreased IκB degradation and p65 translocation to the nucleus. The present data suggest that CHOP has a dual role in promoting β-cell death: (1) CHOP directly contributes to cytokine-induced β-cell apoptosis by promoting cytokine-induced mitochondrial pathways of apoptosis; and (2) by supporting the NF-κB activation and subsequent cytokine/chemokine expression, CHOP may contribute to apoptosis and the chemo attraction of mononuclear cells to the islets during insulitis.

Protective Unfolded Protein Response in Human Pancreatic Beta Cells Transplanted into Mice

Background There is great interest about the possible contribution of ER stress to the apoptosis of pancreatic beta cells in the diabetic state and with islet transplantation. Methods and Findings Expression of genes involved in ER stress were examined in beta cell enriched tissue obtained with laser capture microdissection (LCM) from frozen sections of pancreases obtained from non-diabetic subjects at surgery and from human islets transplanted into ICR-SCID mice for 4 wk. Because mice have higher glucose levels than humans, the transplanted beta cells were exposed to mild hyperglycemia and the abnormal environment of the transplant site. RNA was extracted from the LCM specimens, amplified and then subjected to microarray analysis. The transplanted beta cells showed an unfolded protein response (UPR). There was activation of many genes of the IRE-1 pathway that provide protection against the deleterious effects of ER stress, increased expression of ER chaperones and ERAD (ER-associated protein degradation) proteins. The other two arms of ER stress, PERK and ATF-6, had many down regulated genes. Downregulation of EIF2A could protect by inhibiting protein synthesis. Two genes known to contribute to apoptosis, CHOP and JNK, were downregulated. Conclusions Human beta cells in a transplant site had UPR changes in gene expression that protect against the proapoptotic effects of unfolded proteins.