Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated GRP78 and reduced spontaneous NETosis of bone marrow-derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate, characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of IFNγ-producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes

Calsenilin is required for endocrine pancreas development in zebrafish.

peer reviewedCalsenilin/DREAM/Kchip3 is a neuronal calcium-binding protein. It is a multifunctional protein, mainly expressed in neural tissues and implicated in regulation of presenilin processing, repression of transcription, and modulation of A-type potassium channels. Here, we performed a search for new genes expressed during pancreatic development and have studied the spatiotemporal expression pattern and possible role of calsenilin in pancreatic development in zebrafish. We detected calsenilin transcripts in the pancreas from 21 somites to 39 hours postfertilization stages. Using double in situ hybridization, we found that the calsenilin gene was expressed in pancreatic endocrine cells. Loss-of-function experiments with anti-calsenilin morpholinos demonstrated that injected morphants have a significant decrease in the number of pancreatic endocrine cells. Furthermore, the knockdown of calsenilin leads to perturbation in islet morphogenesis, suggesting that calsenilin is required for early islet cell migration. Taken together, our results show that zebrafish calsenilin is involved in endocrine cell differentiation and morphogenesis within the pancreas

Calsenilin is required for endocrine pancreas development in zebrafish.

peer reviewedCalsenilin/DREAM/Kchip3 is a neuronal calcium-binding protein. It is a multifunctional protein, mainly expressed in neural tissues and implicated in regulation of presenilin processing, repression of transcription, and modulation of A-type potassium channels. Here, we performed a search for new genes expressed during pancreatic development and have studied the spatiotemporal expression pattern and possible role of calsenilin in pancreatic development in zebrafish. We detected calsenilin transcripts in the pancreas from 21 somites to 39 hours postfertilization stages. Using double in situ hybridization, we found that the calsenilin gene was expressed in pancreatic endocrine cells. Loss-of-function experiments with anti-calsenilin morpholinos demonstrated that injected morphants have a significant decrease in the number of pancreatic endocrine cells. Furthermore, the knockdown of calsenilin leads to perturbation in islet morphogenesis, suggesting that calsenilin is required for early islet cell migration. Taken together, our results show that zebrafish calsenilin is involved in endocrine cell differentiation and morphogenesis within the pancreas

Molecular phenotyping of multiple mouse strains under metabolic challenge uncovers a role for <i>Elovl2</i> in glucose-induced insulin secretion.

In type 2 diabetes (T2D), pancreatic β cells become progressively dysfunctional, leading to a decline in insulin secretion over time. In this study, we aimed to identify key genes involved in pancreatic beta cell dysfunction by analyzing multiple mouse strains in parallel under metabolic stress. Male mice from six commonly used non-diabetic mouse strains were fed a high fat or regular chow diet for three months. Pancreatic islets were extracted and phenotypic measurements were recorded at 2 days, 10 days, 30 days, and 90 days to assess diabetes progression. RNA-Seq was performed on islet tissue at each time-point and integrated with the phenotypic data in a network-based analysis. A module of co-expressed genes was selected for further investigation as it showed the strongest correlation to insulin secretion and oral glucose tolerance phenotypes. One of the predicted network hub genes was &lt;i&gt;Elovl2&lt;/i&gt; , encoding Elongase of very long chain fatty acids 2. &lt;i&gt;Elovl2&lt;/i&gt; silencing decreased glucose-stimulated insulin secretion in mouse and human β cell lines. Our results suggest a role for &lt;i&gt;Elovl2&lt;/i&gt; in ensuring normal insulin secretory responses to glucose. Moreover, the large comprehensive dataset and integrative network-based approach provides a new resource to dissect the molecular etiology of β cell failure under metabolic stress

A New Strategy to Generate Functional Insulin-Producing Cell Lines by Somatic Gene Transfer into Pancreatic Progenitors

BACKGROUND: There is increasing interest in developing human cell lines to be used to better understand cell biology, but also for drug screening, toxicology analysis and future cell therapy. In the endocrine pancreatic field, functional human beta cell lines are extremely scarce. On the other hand, rodent insulin producing beta cells have been generated during the past years with great success. Many of such cell lines were produced by using transgenic mice expressing SV40T antigen under the control of the insulin promoter, an approach clearly inadequate in human. Our objective was to develop and validate in rodent an alternative transgenic-like approach, applicable to human tissue, by performing somatic gene transfer into pancreatic progenitors that will develop into beta cells. METHODS AND FINDINGS: In this study, rat embryonic pancreases were transduced with recombinant lentiviral vector expressing the SV40T antigen under the control of the insulin promoter. Transduced tissues were next transplanted under the kidney capsule of immuno-incompetent mice allowing insulinoma development from which beta cell lines were established. Gene expression profile, insulin content and glucose dependent secretion, normalization of glycemia upon transplantation into diabetic mice validated the approach to generate beta cell lines. CONCLUSIONS: Somatic gene transfer into pancreatic progenitors represents an alternative strategy to generate functional beta cell lines in rodent. Moreover, this approach can be generalized to derive cells lines from various tissues and most importantly from tissues of human origin

Beta Cells within Single Human Islets Originate from Multiple Progenitors

BACKGROUND: In both humans and rodents, glucose homeostasis is controlled by micro-organs called islets of Langerhans composed of beta cells, associated with other endocrine cell types. Most of our understanding of islet cell differentiation and morphogenesis is derived from rodent developmental studies. However, little is known about human islet formation. The lack of adequate experimental models has restricted the study of human pancreatic development to the histological analysis of different stages of pancreatic development. Our objective was to develop a new experimental model to (i) transfer genes into developing human pancreatic cells and (ii) validate gene transfer by defining the clonality of developing human islets. METHODS AND FINDINGS: In this study, a unique model was developed combining ex vivo organogenesis from human fetal pancreatic tissue and cell type-specific lentivirus-mediated gene transfer. Human pancreatic progenitors were transduced with lentiviruses expressing GFP under the control of an insulin promoter and grafted to severe combined immunodeficient mice, allowing human beta cell differentiation and islet morphogenesis. By performing gene transfer at low multiplicity of infection, we created a chimeric graft with a subpopulation of human beta cells expressing GFP and found both GFP-positive and GFP-negative beta cells within single islets. CONCLUSION: The detection of both labeled and unlabeled beta cells in single islets demonstrates that beta cells present in a human islet are derived from multiple progenitors thus providing the first dynamic analysis of human islet formation during development. This human transgenic-like tool can be widely used to elucidate dynamic genetic processes in human tissue formation

Oxygen Tension Regulates Pancreatic β-Cell Differentiation Through Hypoxia-Inducible Factor 1α

International audienceThese data demonstrate that beta-cell differentiation is controlled by pO2 through HIF1alpha. Modifying pO2 should now be tested in protocols aiming to differentiate beta-cells from embryonic stem cells

LDHB contributes to the regulation of lactate levels and basal insulin secretion in human pancreatic β cells

Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning β cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and β cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human β cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in β cells to maintain appropriate insulin release.</p

Insulin Storage and Glucose Homeostasis in Mice Null for the Granule Zinc Transporter ZnT8 and Studies of the Type 2 Diabetes–Associated Variants

International audienceObjective. Zinc ions are essential for the formation of hexameric insulin and hormone crystallisation. Correspondingly, a non-synonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. Here, we describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. Research Design and Methods. Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection, or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles, were undertaken using standard protocols. Results. ZnT8(-/-) mice displayed age, sex and diet-dependent abnormalities in glucose tolerance, insulin secretion and body weight. Islets isolated from null mice had reduced granule zinc content, and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion and insulin crystal dissolution, as assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modelling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. Discussion and conclusions. ZnT8 is required for normal insulin crystallisation and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risk