The negative cell cycle regulators, p27(Kip1), p18(Ink4c), and GSK-3, play critical role in maintaining quiescence of adult human pancreatic ß-cells and restrict their ability to proliferate.

Adult human pancreatic ß-cells are primarily quiescent (G0) yet the mechanisms controlling their quiescence are poorly understood. Here, we demonstrate, by immunofluorescence and confocal microscopy, abundant levels of the critical negative cell cycle regulators, p27(Kip1) and p18(Ink4c), 2 key members of cyclin-dependent kinase (CDK) inhibitor family, and glycogen synthase kinase-3 (GSK-3), a serine-threonine protein kinase, in islet ß-cells of adult human pancreatic tissue. Our data show that p27(Kip1) localizes primarily in ß-cell nuclei, whereas, p18(Ink4c) is mostly present in ß-cell cytosol. Additionally, p-p27(S10), a phosphorylated form of p27(Kip1), which was shown to interact with and to sequester cyclinD-CDK4/6 in the cytoplasm, is present in substantial amounts in ß-cell cytosol. Our immunofluorescence analysis displays similar distribution pattern of p27(Kip1), p-p27(S10), p18(Ink4c) and GSK-3 in islet ß-cells of adult mouse pancreatic tissue. We demonstrate marked interaction of p27(Kip1) with cyclin D3, an abundant D-type cyclin in adult human islets, and vice versa as well as with its cognate kinase partners, CDK4 and CDK6. Likewise, we show marked interaction of p18(Ink4c) with CDK4. The data collectively suggest that inhibition of CDK function by p27(Kip1) and p18(Ink4c) contributes to human ß-cell quiescence. Consistent with this, we have found by BrdU incorporation assay that combined treatments of small molecule GSK-3 inhibitor and mitogen/s lead to elevated proliferation of human ß-cells, which is caused partly due to p27(Kip1) downregulation. The results altogether suggest that ex vivo expansion of human ß-cells is achievable via increased proliferation for ß-cell replacement therapy in diabetes.

GSK-3 inactivation or depletion promotes ß-cell replication via down regulation of the CDK inhibitor, p27 (Kip1).

Diabetes (T1DM and T2DM) is characterized by a deficit in ß-cell mass. A broader understanding of human ß-cell replication mechanism is thus important to increase ß-cell proliferation for future therapeutic interventions. Here, we show that p27 (Kip1), a CDK inhibitor, is expressed abundantly in isolated adult human islets and interacts with various positive cell cycle regulatory proteins including D-type cyclins (D1, D2 and D3) and their kinase partners, CDK4 and CDK6. Also, we see interaction of cyclin E and its kinase partner, CDK2, with p27 suggesting a critical role of p27 as a negative cell cycle regulator in human islets. Our data demonstrate interaction of p27 with GSK-3 in ß-cells and show, employing rodent ß-cells (INS-1), isolated human islets and purified ß-cells derived from human islets, that siRNA-mediated depletion of GSK-3 or p27 or 1-AKP / BIO - mediated GSK-3 inhibition results in increased ß-cell proliferation. We also see reduction of p27 levels following GSK-3 inactivation or depletion. Our data show that serum induction of quiescent INS-1 cells leads to sequential phosphorylation of p27 on its S10 and T187 residues with faster kinetics for S10 corresponding with the decreased levels of p27. Altogether our findings indicate that p27 levels in ß-cells are stabilized by GSK-3 and thus p27 down regulation following GSK-3 depletion / inactivation plays a critical role in promoting ß-cell replication.

Generation of embryonic stem cells from mouse insulin I promoter-green fluorescent protein transgenic mice and characterization in a teratoma model.

Insulin-secreting pancreatic beta cells play a key role in the pathogenesis of diabetes mellitus. Potential new treatments for this disease include cell-replacement therapies using embryonic stem cells (ESCs). We have generated ESCs from a transgenic mouse model, mouse insulin 1 promoter (MIP) green fluorescent protein (GFP) mice, in which embryonic and adult beta cells are genetically tagged with GFP. The aim of the present study is to examine the differentiation potential of MIP-GFP ESCs in the microenvironment of the kidney capsule. The ESCs grew rapidly and formed a teratoma with GFP-expressing beta-like cells present in clusters that formed a cord-like structure similar to what is seen in the embryonic pancreas. These structures also included glucagon-expressing alpha cells and amylase-expressing acinar cells. Electron microscopic analysis showed insulin-like granules in columnar epithelium with microvilli adjacent to exocrine-like granule-containing cells. The MIP-GFP ESCs should be a useful research tool to study the differentiation capacity of ESCs toward pancreatic lineages.