Abstract
Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.
Highlights
Inadequate b-cell mass is a defect common to both types 1 & 2 diabetes (T1DM, T2DM)
We found that the L-WRN+ medium stimulates a striking level of DNA synthesis in islet cells cultured in 5 mM glucose
H-89, an inhibitor of protein kinase A (PKA), significantly blocked increases in DNA synthesis in response to LiCl (Figure 6C) and L-WRN+ (Figure 6D). These results suggest that L-WRN+ promotes crosstalk between PKA, Wnt/b-catenin and Akt signaling in human islets to enhance proliferative processes
Summary
Adult human b-cells have very low proliferation rates in vivo, significant levels of proliferation occur, for example during pregnancy and conditions of insulin resistance, indicating the existence of regulatory mechanisms. Neilson et al observed that intact isolated human islets remained functional for months, but did not proliferate under the culture conditions used [5]. Based on this in vitro proliferation barrier, there is a compelling need to identify the regulatory mechanisms and strategies that will unmask the proliferative capacity of pre-existing differentiated adult human b-cells in intact islets, and may lead to the identification of new drug targets [6]
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