Toll-like Receptor3Inhibits Pancreatic β-cell Proliferation Through Cell Cycle Arrest

Objective: Toll-like receptors (TLRs) are involved in the development of chronicinflammation-related diseases, including type2diabetes. Recent studies suggest thatTLRs are in close contact with insulin resistance of peripheral tissue in type2diabetes. However, the role of TLRs, especially Toll-like receptor3(TLR3), inregulation of β-cell overall function remains to be elucidated. The aim of this studywas to investigate the mechanism of TLR3on β-cell proliferation.Methods: Both rat pancreatic β-cell line INS-1and mice pancreatic β-cell line NIT-1were treated with high glucose (16.7mM) and high fatty acid (0-0.4mM palmiticacid), and in islets were isolated from diabetic db/db mice, to construct rodentdiabetic models. Real-time RT-PCR and Western blot were applied to detect TLR3mRNA and protein level of pancreatic β-cells with glucolipotoxicity, as well as itsdownstream signal molecular IRF-3protein. Polyinosinic-polycytidylic acid (poly (I:C)) was used to induce TLR3activation. MTT, Hoechst33258staining, EdU labelingand flow cytometry analysis were used to detect β-cell viability, proliferation,apoptosis and cell cycle, respectively. TLR3knockdown was achieved using specificsmall interfering RNA (siRNA). MG132and SP600125were used to inhibitproteasome and JNK activity, respectively. Real-time RT-PCR and Western blotanalysis were employed to detect the mRNA and protein levels of cyclin D1, D2, D3and E, p18and p27.Results: TLR3and its downstream signal molecular IRF-3were found to bemarkedly up-regulated in pancreatic β-cell with glucolipitoxicity. In addition,activation of TLR3significantly decreased β-cell vitality, induced cell apoptosis and inhibited cell proliferation, with cell cycle arrest at the G1phase. Knockdown ofTLR3reversed poly (I: C)-induced cell cycle arrest. Moreover, TLR3activationelicited significant downregulation of cyclin D1and D2protein levels, withoutobvious alteration of cyclin D1, D2, D3and E, p18and p27mRNA levels. Inhibitionof the proteasome with MG132rescued cyclin D1and D2protein from degradation.Furthermore, inhibition of c-Jun N-terminal kinase (JNK) activated by poly (I: C)with SP600125did not contribute to TLR3agonist-induced growth inhibition ofβ-cell.Conclusion: Glucolipitoxicity can significantly activate innate immunity signalpathways of TLR3in pancreatic β-cell. TLR3activation can not only promote β-cellapoptosis, but also inhibit proliferation through cell cycle arrest at the G1phase in aJNK-independent manner. Cyclin D1and D2are downregulated through thedegradation of protein, but not inhibition of their gene expression that mediates theeffects of TLR3on β-cell growth inhibition. These observations suggest a role forTLR3in β-cell mass inadequacy from promoting β-cell damage and inhibiting cellproliferation, which is involved in overall functional disorder of β-cell, providing anew way and drug targets for clinical treatment of type2diabetes.