Molecular Mechanism Of Pancreatic β-cell Adaptive Proliferation: Studies During Pregnancy In Rats And In Vitro

Objectives:1. To investigate the profiles of islet gene expressions at various stages of pregnancy in rats, to explore differential gene expressions during pregnancy, and to screen the key genes relevant to islet proliferation.2. To elucidate the expression and significance of key genes in pancreatic isletβ-cells of pregnant rats and in prolactin treated INS-1 cells in vitro.Methods:1. Pancreatic islets of SD rats during pregnancy at day 10.5, 14.5 and non-pregnant female rats were isolated and purified, respectively.2. Gene chips from Affymetrix company were applied to explore gene expression profiles of islet at various stages of pregnancy in rats. Data, especially genes related to islet proliferation, were then analyzed by bioinformatical methods.3. Expressions of genes related to islet proliferation were studied by RT-PCR, real-time PCR and Western blotting to verify the results of gene chips.4. INS-1 cells proliferation was assessed by MTT colorimetric assay.5. Expressions of key genes in INS-1 cells cultured with PRL for various periods were studied by Western blotting.Results:1. There were 31099 known or unknown genes included in the Affymetrix Rat Genome 230 2.0 Array. The detection rates in each group such as normal female rats(N), pregnant rats at day 10.5(P10.5) and day 14.5(P14.5) were 45.3%, 38.2% and 37.2%, respectively. Compared with normal control group, expressions of hundreds of genes were changed during pregnancy. The differentially expressed genes identified were distributed into 8 main categories:⑴genes involved in apoptosis or tumor;⑵g enes related to binding;⑶g enes involved in metabolism;⑷genes related to cell cycle;⑸genes for signal transducer activity;⑹genes related to structural molecule activity;⑺genes involved in transcription regulator activity;⑻genes for transporter activity.2. Expressions of genes related to isletβ-cells development and differentiation such as Foxa2, Pdx1, Nkx6.1, Nkx2.2, Pax6, Pax4 and Neurod1 were not changed.3. The differentially expressed genes related to islet proliferation were mainly distributed in three groups:⑴genes involved in transcription regulator activity;⑵genes involved in apoptosis or tumor;⑶genes for Wnt signaling pathway. Nupr1, Atf3, Btg2,β-catenin and c-Myc were screened by bioinformatics methods for further study.4. Compared with normal control group, expressions of five genes were all up-regulated during pregnancy. Among them, Nupr1, Atf3 and Btg2 had a peak in P10.5, whileβ-catenin and c-Myc in P14.5. Expression of Nupr1 and Atf3 protein were also increased during pregnancy, with peak in P10.5.5. After cultured with PRL(50~1000ng/ml), the proliferation of INS-1 cells was increased in a dose-dependent manner. Expressions of Atf3 and Nupr1 protein were also increased significantly followed the cell proliferation.Conclusions:1. Isletβ-cell replication rather than stem-cell or progenitor-cell differentiation is the primary mechanism for adaptation of pancreatic islets during pregnancy.2. Expressions of transcription factor Nupr1, Atf3, and Wnt pathway related genes were increased during pregnancy, suggesting that they may participate in regulating adaptive proliferation of pancreatic islets during pregnancy in rats. 3. Atf3 and Nupr1 may play an important role in oPRL-induced pancreaticβ-cell growth in vitro.