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Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

ABSTRACT: Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to approximately 5% in the culture conditions. Analysis by the Cre/loxP-based direct cell lineage tracing system indicates that these newly made cells originate from amylase/elastase-expressing pancreatic acinar cells. Insulin secretion is stimulated by glucose, sulfonylurea, and carbachol, and potentiation by glucagon-like peptide-1 also occurs. Insulin-containing secretory granules are present in these cells. In addition, we found that the enzymatic dissociation of pancreatic acini itself leads to activation of EGF signaling, and that inhibition of EGF receptor kinase blocks the transdifferentiation. These data demonstrate that pancreatic acinar cells can transdifferentiate into insulin-secreting cells with secretory properties similar to those of native pancreatic beta cells, and that activation of EGF signaling is required in such transdifferentiation.

SUBMITTER: Minami K

PROVIDER: S-EPMC1257737 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

Minami Kohtaro K Okuno Masaaki M Miyawaki Kazumasa K Okumachi Akinori A Ishizaki Katsuhiko K Oyama Kazunobu K Kawaguchi Miho M Ishizuka Nobuko N Iwanaga Toshihiko T Seino Susumu S

Proceedings of the National Academy of Sciences of the United States of America 20051006 42

Although several studies have suggested that insulin-secreting cells can be generated in vitro from cells residing in adult exocrine pancreas, neither the origin of these cells nor their precise insulin secretory properties was obtained. We show here that insulin-secreting cells can be derived from adult mouse pancreatic exocrine cells by suspension culture in the presence of EGF and nicotinamide. The frequency of insulin-positive cells was only 0.01% in the initial preparation and increased to ...[more]

PMID: 16210247

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Project description:Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosy1[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that the methyltransferase was co-localized, with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5'-[gamma-thio]-triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins. Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12-myristate 13-acetate did not alter enzyme activity. In addition, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.

Project description:BackgroundExpansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells.Methodology/principal findingRedifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation.Conclusions/significanceThese findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.

Dataset Information

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

Publications

Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells.

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