Sialylated form of the neural cell adhesion molecule (NCAM): a new tool for the identification and sorting of beta-cell subpopulations with different functional activity.

Bernard-Kargar, Catherine; Kassis, Nadim; Berthault, Marie‐France; Pralong, William; Ktorza, Alain

doi:10.2337/diabetes.50.2007.s125

Cited by 38 publications

(32 citation statements)

References 24 publications

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“…No PSA-NCAM signal was detected in CK19-positive duct cells (Fig 1d). These results are different from a previous report [7], where PSA-NCAM expression was restricted to beta cells only in rats. This is another example of interspecies variation between rodent and human islets.…”

Section: Resultscontrasting

confidence: 99%

“…It thus appears that although PSA-NCAM immunoreactivity is detected in all islet cell types, the affinity of the antibody is much higher against beta cells. In the rat, high PSA-NCAM specifically characterises a subpopulation of functionally active beta cells [7]. It is thus possible that the observed enrichment is due to the selection of a population of beta cells with high PSA-NCAM expression.…”

Section: Resultsmentioning

confidence: 99%

“…Attachment of sialic acid polymers through α-2,8 binding to NCAM leads to formation of a polysialylated form of the molecule, namely polysialic acid-NCAM (PSA-NCAM) with modified adhesive properties [6]. PSA-NCAM is expressed in pancreatic beta cells of adult rats and has been used to sort rat beta cells [7].…”

Section: Introductionmentioning

confidence: 99%

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A simple two-step protocol for the purification of human pancreatic beta cells

Banerjee

Otonkoski

2009

Diabetologia

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Aims/hypothesis Isolated pure human beta cells would be helpful for a number of research purposes. However, lack of beta cell-specific surface antigens has been a major problem. We aimed to develop a simple method for human beta cell isolation based on the initial elimination of ductal cells by their expression of carbohydrate antigen 19-9 (CA19-9), followed by positive selection of beta cells by their expression of polysialic acid-neural cell adhesion molecule (PSA-NCAM). Methods Cell type-specific expression of CA19-9, NCAM and PSA-NCAM was studied in sections of adult human pancreas and in cultured primary endocrine and exocrine cells. Dispersed human islet cells were purified in two steps, after 4 days of suspension culture, by binding to magnetic microbeads coupled to antibodies against CA19-9 and PSA-NCAM. Results NCAM expression was detected in ducts and islets in the human pancreas. In contrast, PSA-NCAM immunoreactivity was detected only in islets. PSA-NCAM staining in dispersed cells revealed that the marker is expressed in all endocrine cell types, but not in duct cells. Purification of dispersed islet cells using PSA-NCAM microbeads alone did not completely eliminate contaminating duct cells. However, elimination of the duct cells by CA19-9 microbeads followed by positive sorting of the PSA-NCAM-positive cells in five consecutive islet preparations resulted in 90 to 98% pure endocrine cells, of which 89 to 97% were beta cells. Conclusions/interpretation We describe a simple and reproducible method for purification of viable human pancreatic beta cells devoid of exocrine acini and ducts.

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Section: Resultscontrasting

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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A simple two-step protocol for the purification of human pancreatic beta cells

Banerjee

Otonkoski

2009

Diabetologia

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“…Mice were killed, and islets of Langerhans were isolated after collagenase digestion of the pancreas as previously described (47). Briefly, the pancreatic duct was ligated distally and injected with an ice-chilled solution of 630 units/ml collagenase (Type V collagenase; SigmaAldrich) and 5.5 mM glucose in Hank's balanced salt solution (HBSS).…”

Section: Methodsmentioning

confidence: 99%

Role of uncoupling protein UCP2 in cell-mediated immunity: How macrophage-mediated insulitis is accelerated in a model of autoimmune diabetes

Emre

Hurtaud

Karaca

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Infiltration of inflammatory cells into pancreatic islets of Langerhans and selective destruction of insulin-secreting ␤-cells are characteristics of type 1 diabetes. Uncoupling protein 2 (UCP2) is a mitochondrial protein expressed in immune cells. UCP2 controls macrophage activation by modulating the production of mitochondrial reactive oxygen species (ROS) and MAPK signaling. We investigated the role of UCP2 on immune cell activity in type 1 diabetes in Ucp2-deficient mice. Using the model of multiple low-dose streptozotocin (STZ)-induced diabetes, we found that autoimmune diabetes was strongly accelerated in Ucp2-KO mice, compared with Ucp2-WT mice with increased intraislet lymphocytic infiltration. Macrophages from STZ-treated Ucp2-KO mice had increased IL-1␤ and nitric oxide (NO) production, compared with WT macrophages. Moreover, more macrophages were recruited in islets of STZ-treated Ucp2-KO mice, compared with Ucp2-WT mice. This finding also was accompanied by increased NO/ROS-induced damage. Altogether, our data show that inflammation is stronger in Ucp2-KO mice and islets, leading to the exacerbated disease in these mice. Our results highlight the mitochondrial protein UCP2 as a new player in autoimmune diabetes.inflammation ͉ nitric oxide M itochondria are important organelles for cellular functions, including ATP synthesis. Oxidative glucose metabolism in pancreatic ␤-cells increases the ATP/ADP ratio, leading to insulin release. Uncoupling protein 2 (UCP2) is a mitochondrial carrier protein expressed in pancreatic ␤-cells (1, 2) and immune cells (3,4).In pancreatic ␤-cells, UCP2 was reported to alter the yield of ATP synthesis from glucose, and it has been proposed as a negative regulator of glucose-stimulated insulin secretion (2). In other respects, there is much evidence for the influence of UCP2 on immune responses. First, Ucp2-KO mice exhibit increased resistance to a Toxoplasma gondii or Listeria monocytogenes infection (4, 5). Second, in an LDL receptor-null background, Ucp2-KO mice develop greater and more unstable atherosclerotic plaques than WT mice (6). Third, in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis, Ucp2-KO mice develop higher disease scores than Ucp2-WT mice (7). We showed that UCP2 regulates LPS-induced reactive oxygen species (ROS) signaling in macrophages (8). MAPK activation in Ucp2-KO macrophages is quicker and stronger than in WT macrophages (8), leading to increased nitric oxide (NO), cytokine production, and migration ability (8, 9). Consistent with these findings, overexpression of UCP2 in macrophages is associated with diminished NO production (10) and decreased migration capacity (11).Given the evidence for a role of UCP2 in the immune system, in macrophages, and in ␤-cell function, we investigated whether UCP2 affects the development of autoimmune diabetes by using the model of multiple low-dose of streptozotocin (STZ). In this report, we show that the absence of UCP2 renders animals more sensitive to the onset of diabetes in mice....

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“…In some studies mainly the NCAM-140 isoform is reported [7,25,28,29] whereas others report that mainly NCAM-120 is expressed in adult islets and expression of NCAM-140 is found only during development and in tumours [26,27]. In beta cells, NCAM has been suggested to be involved in the organisation of biosociology in the islets of Langerhans [26], in beta cell functional activity measured as insulin release [30], and also in natural killer T cell-induced beta cell death [31]. Furthermore, in mouse pancreatic tumour beta cells, NCAM has been shown to activate a Src-kinase and to bind and activate FGFR [32].…”

Section: Introductionmentioning

confidence: 99%

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

et al. 2006

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Aims/hypothesis: IL-1β released from immune cells induces beta cell pro-apoptotic signalling via mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In neurons, the neural cell adhesion molecule (NCAM) signals to several elements involved in IL-1β-induced pro-apoptotic signalling in beta cells. Pancreatic beta cells express NCAM, but its biological effects in these cells are unclear. The aim of this study was to investigate whether there is cross-talk between NCAM signalling and cytokine-induced pro-apoptotic signalling. Materials and methods: Western blotting was used to investigate levels of NCAM and inducible nitric oxide synthase, phosphorylation of Src and MAPKs, and cleavage of caspase-3. MAPK activity was investigated with an in vitro kinase assay. Apoptosis was detected by cleaved caspase-3 and a Cell Death Detection ELISA plus assay. NCAM-induced fibroblast growth factor receptor (FGFR) activation was investigated in NCAM −/− Trex293 cells where FGFR phosphorylation was measured by Western blotting after NCAM transfection. Results: Preexposure of INS-1E cells to the FGFR-inhibitor SU5402, but not to the Src-inhibitor PP2, dose-dependently inhibited IL-1β-mediated MAPK activity. A synthetic peptide, C3d, reported to bind NCAM, did not activate MAPK or Akt as reported in neurons but inhibited IL-1β-induced MAPK activity, thereby mimicking the effect of SU5402. Furthermore, C3d inhibited NCAM-induced FGFR phosphorylation and apoptosis induced by IL-1β plus IFN-γ, but did not affect IL-1β-induced NF-κB signalling. Conclusions/interpretation: We suggest that NCAM signalling through FGFR is required for efficient IL-1β pro-apoptotic signalling by facilitating IL-1β-induced MAPK activation downstream of the NF-κB-MAPK branching point. Further, these data identify a novel function of C3d as an inhibitor of NCAM-induced FGFR activity and of IL-1β-induced MAPK activation in beta cells.

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Sialylated form of the neural cell adhesion molecule (NCAM): a new tool for the identification and sorting of beta-cell subpopulations with different functional activity.

Cited by 38 publications

References 24 publications

A simple two-step protocol for the purification of human pancreatic beta cells

A simple two-step protocol for the purification of human pancreatic beta cells

Role of uncoupling protein UCP2 in cell-mediated immunity: How macrophage-mediated insulitis is accelerated in a model of autoimmune diabetes

IL-1β-induced pro-apoptotic signalling is facilitated by NCAM/FGF receptor signalling and inhibited by the C3d ligand in the INS-1E rat beta cell line

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