Molecular mechanism of db gene-induced diabetes mellitus – effect of db gene load on pancreatic β cell specific gene expression – *
Background and Aims:The db gene is a splicing variant caused by a point mutation of the leptin receptor gene. Diabetes develops in db gene homozygous BKS. Cg-+Leprdb/+Leprdb/Jcl（db/db）mice and is characterized with a marked obesity, severe insulin resistance, and a limited capacity for insulin secretion. The diabetes phenotype is not demonstrated in heterozygous mice（db/＋）. To clarify the molecular mechanism of pancreaticβcell dysfunction related to the db gene, the cell function and gene expression profile of pancreatic islets were analyzed in db gene homozygous db/db, heterozygous db/＋, and lean littermates（＋/＋）. Laser Capture Microdissection（LCM）and real-time RT-PCR were applied to analyze islet cell specific gene expression. Materials and Methods:Male db/db, db/＋and＋/＋were provided free access to standard feed. Body weight（BW）, fasted blood glucose（FBG）and insulin（FIRI）were measured at the age of ８ and 12 weeks. To analyze the gene expression profile in β cells, the islet core area was selectively obtained by LCM. Primer pairs encoding genes associated with pancreatic hormones, cell proliferation, cell differentiation, apoptosis, cell cycle, and organization construction were prepared, and a real-time RT-PCR with Sybr Green was applied. Each gene expression was relatively quantified by the comparative Ct method with each result in the＋/＋mice as a control. Results:BW, FBG and FIRI in ８ and 12 week-old db/db mice increased significantly compared with those in the other groups. At 12 weeks of age, however, insulin content in the islet was markedly reduced in db/db mice and had increased significantly in db/＋ mice as compared with＋/＋mice（5.1±3.3, 58.0±31.7, 24.4±15.4ng/islet, respectively, p＜0.05）. InsulinⅡ gene expression had significantly increased in db/＋mice compared with db/db mice at 8 weeks of age（db/＋:9.57 ±3.96, db/db:1.72±0.96, p＜0.05), but did not differ between the two groups at 12 weeks of age（db/＋:7.86±3.56, db/db:6.97±3.78）. Gene expression involved in cell proliferation（cyclin E & ERK1）and apoptosis（CAD）was accelerated in 8-week-old db/db mice. However, cyclin E & ERK1 gene expression was down-regulated and CAD gene expression was up-regulated at 12 weeks of age. On the other hand, a significant increase in bcl-2 gene expression was observed in db/＋ but not in db/db mice. Conclusion:The present results demonstrated that diabetes development in db/db mice was associated with pancreatic β cell dysfunction induced by acceleration of apoptosis and reduction of cell proliferation. The db gene heterozygous mice have a protective mechanism against the β cell damage observed in db gene homozygous mice. The increases in islet insulin content and gene expression suggest the existence of a compensatory mechanism in db gene heterozygous mice to reduce susceptibility to diabetes. (Accepted on August 30，2006)
