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The effects of prolonged exposure of pancreatic β-cells to high saturated fatty acids on glucagon-like peptide-1 (GLP-1) action were investigated. Murine islets, human pancreatic 1.1B4 cells, and rat INS-1E cells were exposed to palmitate for 24 hours. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting, respectively. Specific short interfering RNAs were used to knockdown expression of the GLP-1 receptor (Glp1r) and Srebf1. Insulin release was assessed with a specific ELISA. Exposure of murine islets, as well as of human and INS-1E β-cells, to palmitate reduced the ability of exendin-4 to augment insulin mRNA levels, protein content, and release. In addition, palmitate blocked exendin-4-stimulated cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, whereas phosphorylation of MAPK-ERK kinase-1/2 and ERK-1/2 was not altered. Similarly, RNA interference-mediated suppression of Glp1r expression prevented exendin-4-induced cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, but did not impair exendin-4 stimulation of MAPK-ERK kinase-1/2 and ERK-1/2. Both islets from mice fed a high fat diet and human and INS-1E β-cells exposed to palmitate showed reduced GLP-1 receptor and pancreatic duodenal homeobox-1 (PDX-1) and increased sterol regulatory element-binding protein (SREBP-1C) mRNA and protein levels. Furthermore, suppression of SREBP-1C protein expression prevented the reduction of PDX-1 and GLP-1 receptor levels and restored exendin-4 signaling and action. Finally, treatment of INS-1E cells with metformin for 24 h resulted in inhibition of SREBP-1C expression, increased PDX-1 and GLP-1 receptor levels, consequently, enhancement of exendin-4-induced insulin release. Palmitate impairs exendin-4 effects on β-cells by reducing PDX-1 and GLP-1 receptor expression and signaling in a SREBP-1C-dependent manner. Metformin counteracts the impairment of GLP-1 receptor signaling induced by palmitate.

The mechanisms of the protective effects of exendin-4 on NEFA-induced beta cell apoptosis were investigated. The effects of exendin-4 and palmitate were evaluated in human and murine islets, rat insulin-secreting INS-1E cells and murine glucagon-secreting alpha-TC1-6 cells. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting or immunofluorescence, respectively. Small interfering (si)RNAs for Ib1 and Gpr40 were used. Cell apoptosis was quantified by two independent assays. Insulin release was assessed with an insulin ELISA. Exposure of human and murine primary islets and INS-1E cells, but not alpha-TC1-6 cells, to exendin-4 inhibited phosphorylation of the stress kinases, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and prevented apoptosis in response to palmitate. Exendin-4 increased the protein content of islet-brain 1 (IB1), an endogenous JNK blocker; however, siRNA-mediated reduction of IB1 did not impair the ability of exendin-4 to inhibit JNK and prevent apoptosis. Exendin-4 reduced G-protein-coupled receptor 40 (GPR40) expression and inhibited palmitate-induced phosphorylation of mitogen-activated kinase kinase (MKK)4 and MKK7. The effects of exendin-4 were abrogated in the presence of the protein kinase A (PKA) inhibitors, H89 and KT5720. Knockdown of GPR40, as well as use of a specific GPR40 antagonist, resulted in diminished palmitate-induced JNK and p38 MAPK phosphorylation and apoptosis. Furthermore, inhibition of JNK and p38 MAPK activity prevented palmitate-induced apoptosis. Exendin-4 counteracts the proapoptotic effects of palmitate in beta cells by reducing GPR40 expression and inhibiting MKK7- and MKK4-dependent phosphorylation of the stress kinases, JNK and p38 MAPK, in a PKA-dependent manner.

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc) acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc) in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc) on Ser(36) and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66(Shc) in HUVEC resulted in enhanced p66(Shc) phosphorylation on Ser(36), increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc) protein, in which Ser(36) was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc) resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc) acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc) on Ser(36).