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Toll-like receptor 4 signal transduction pathways in pancreatic beta cells and their effects on insulin production and secretion.

Grant number: 09/06547-6
Support type:Scholarships in Brazil - Master
Effective date (Start): September 01, 2009
Effective date (End): August 31, 2011
Field of knowledge:Biological Sciences - Immunology - Cellular Immunology
Principal Investigator:Anna Carla Renata Krepel Goldberg
Grantee:Fernanda Vieira Paladino
Home Institution: Instituto Israelita de Ensino e Pesquisa Albert Einstein (IIEPAE). Sociedade Beneficente Israelita Brasileira Albert Einstein (SBIBAE). São Paulo , SP, Brazil

Abstract

Langerhans islets present a mixture of several cell types, but most are insulin-producing beta cells. These beta cells are responsive to pro-inflammatory cytokines, activating the NF-kB pathway that leads to dysfunction and cell death. To obtain islets for transplantation of type 1 diabetic patients pancreata from brain-dead donors are selected and the harvested and purified islets are infused in the portal vein of these patients. During this procedure the islets are submitted to stressful conditions and an inflammatory milieu that can easily induce the expression of danger signaling molecules in beta cells known to be highly susceptible to these stimuli. The list of molecules produced by islets includes IL-1 beta, MCP-1, TNF-alpha and Toll-like receptors (TLR). TLR4, associated with CD14, is responsible for the intracellular signal transduction induced by LPS. TLR4 has been identified in non-immune cells from several tissues and was shown to respond to a variety of factors arising during tissue damage and ischemia. We have previously shown that in practically all human islet samples isolated, TLR4 is present on the surface of beta cells but not on CD11b macrophages. Upon incubation with LPS, we also observed increase in mRNA for TLR4 and CD14, but insulin mRNA and cell viability were lower. We have confirmed the same TLR4 expression on MIN6 cells (mouse insulinoma), the response to LPS, and the inhibition of insulin transcription and increased cell death. This project aims to characterize further the biological function of TLR4 in mouse beta cells, in order to identify which signaling pathways are activated by LPS and the relation to insulin secretion and cell death. We will: a) analyze LPS-activated TLR4 transduction pathways by tracking the components of the different signaling cascades, b) measure insulin intracellular production and secretion, and test capacity to respond to hyperglycemia to correlate glucose level, insulin production and secretion with TLR4 expression; c) measure pro-apoptotic potential of TLR4 activation; and d) identify TLR4 signal transduction when activated with endogenous ligands, such as heme, saturated fatty acids and Hsp60.