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- Title
Single Beta Cells from Adult and Neonatal Mice Differ in Co-Expression of Other Islet Hormones as Determined with Nested PCR.
- Authors
Katsuta, Hitoshi; Akashi, Tomoyuki; Morita, Rina; Nagaya, Masaki; Kim, Dohoon; Lock, Jennifer; Sharma, Arun; Hara, Manami; Bonner-Weir, Susan; Akashi, Koich; Weir, Gordon C.
- Abstract
To investigate islet hormone gene expression in single beta cells of adult and neonatal mice, we performed nested PCR on FACS-sorted GFP+ beta cells from mouse insulin 1 promoter (MIP)-GFP mice. Islets isolated from MIP-GFP mice (16-24 weeks old) or whole pancreatic tissue from MIP-GFP neonates (1.5 day-old) were digested with Liberase, dispersed by trypsin and then double sorted for GFP to obtain pure high expression GFP+ beta cells. Sorted cells were directly plated singly into wells containing cell lysis mixture, RT buffer, and gene specific reverse primers (insulin1, insulin2, glucagon, somatostatin, pancreatic polypeptide (PP), pdx-1, neuroD and HPRT). After RT, forward primers were added for multiplex PCR. After 1st PCR, 2nd round nested PCR was performed using single cell sample primers set within the 1st primers. By single cell nested PCR analysis, every GFP+ cell from adult or neonate expressed insulin 1, insulin 2, pdx-1 and neuroD. In adult mice, 60-70% of GFP+cells were positive for only insulin, while 30-40% of GFP+cells expressed two or more islet hormones. 3% of GFP+ cells expressed all 4 islets hormones. Single neonatal GFP+ cells also expressed multiple islet hormones, hut, notably, 80% of GFP+cells expressed PP in addition to insulin (versus about 30% in adult). Of these, 64% expressed just PP and insulin, and no other islet hormones (versus 10% in the adult). Only 1-2% of neonatal GFP +cells expressed all 4 islets hormones. For both adult and neonatal cells, expression of these other islet hormones was at a low level, and protein was not found with immunostaining. In conclusion, single beta cells express not only insulin but low levels of mRNA for other islet hormones. The pattern of expression in adult and neonatal beta cells differs; neonatal cells express PP much more frequently. These findings support the hypothesis that expression of small amounts of PP mRNA is a marker of young beta cells.
- Subjects
PANCREATIC beta cells; ISLANDS of Langerhans; DIABETES; GENE expression; INSULIN; MESSENGER RNA; LABORATORY mice
- Publication
Diabetes, 2007, Vol 56, pA420
- ISSN
0012-1797
- Publication type
Article