Glycogen Synthase Kinase-3 and Mammalian Target of Rapamycin Pathways Contribute to DNA Synthesis, Cell Cycle Progression, and Proliferation in Human Islets

Abstract

OBJECTIVE-Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/␤-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human ␤-cell mass relevant to type 1 and 2 diabetes. RESEARCH DESIGN AND METHODS-Primary adult humanand rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of ␤-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed. RESULTS-Human islets treated for 4 days withLiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of ␤-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human ␤-cells. Nuclear translocation of ␤-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin.CONCLUSIONS-A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human ␤-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation.

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

“…To measure individual band intensity, background was subtracted for each protein band followed by normalization against the control protein (tubulin). (E) a-e, isolated adult human islets from four independent batches (two [HI-1 and HI-2] from low BMI [26][27] and two [HI-3 and HI-4] from high BMI [40][41][42][43][44][45][46][47][48][49][50] cadaveric donors) with high purity (80-90% for low BMI islets) and low purity (35-40% for high BMI islets) were cultured and equal amounts (50 μg) of human islet lysates or INS-1 CE were fractionated in SDS-PAGE and the transferred peptides were incubated with specific primary antibodies as shown. *p < 0.05; NS, not significant.…”

“…43 Recently a study has demonstrated increased nuclear translocation of β-catenin in human islets/bcells following GSK-3 inhibition via LiCl or 1-AKP, which is rapamycin sensitive and the same study has also shown the importance of synergistic effects of glucose and GSK-3 inhibition in enhancing human b-cell proliferation. 44 GSK-3 inhibitors are known to mimic some actions of insulin and GSK-3 is thus considered as a potential drug target for T2DM. 27 Furthermore, GSK-3 inhibitors, due to their proliferation-promoting abilities, may be suitable in combination with immunosuppressive agents for treatment of T1DM patients because studies suggest that in most cases, even those with long-standing T1DM, some b-cells persist and are continually destroyed and also because of enhanced b-cell formation in T1DM.…”

mTORC1 signaling and regulation of pancreatic β-cell mass

“…mTORC1 controls growth (cell size), proliferation (cell number) and metabolism directly, by modulating eukaryotic initiation factor 4E-binding proteins (4E-BP 1, 2 and 3) and ribosomal protein S6 kinases (S6K 1 and 2), and indirectly, by attenuating AKT signaling via an mTORC1/ S6K-mediated negative feedback loop. [1][2][3][4][5][6][7][8][9][10] How TSC/mTOR signaling regulates β-cell mass expansion is not completely understood. A better understanding of how mTORC1 regulates β-cell mass and the role of interactions between mTORC1 and other signaling pathways in this process are critical to discovering how β-cell mass adapts to insulin resistance and autoimmune injury and, ultimately, how it impacts diabetes management.…”

Fibrin supports human fetal islet-epithelial cell differentiation via p70s6k and promotes vascular formation during transplantation

“…[42][43][44] Also, the mTOR pathway has different effects on the islet cell cycle depending on the animal species. 45,46 In particular, stimulation of rodent islets with palmitate led to increased DNA synthesis (in an mTORdependent manner) in rodent islets, but not those isolated from humans. 46 Therefore, mTOR pathway activity may have different effects on islet cell differentiation based on whether When human fetal islet-epithelial cell clusters were cultured with fibrin, we observed significant increases in VEGF-A expression (Figure 2d).…”