Insulin augments tumor necrosis factor-alpha stimulated expression of vascular cell adhesion molecule-1 in vascular endothelial cells

<p>Abstract</p> <p>Background</p> <p>Atherosclerosis is an inflammatory disease that is marked by increased presence of Tumor Necrosis Factor-alpha (TNFα), increased expression of Vascular Cell Adhesion Molecule-1 (VCAM-1), increased presence of serum monocytes and activation of the canonical inflammatory molecule, Nuclear Factor Kappa-B (NFκB). Hyperinsulinemia is a hallmark of insulin resistance and may play a key role in this inflammatory process.</p> <p>Methods</p> <p>Using Western blot analysis, immunocytochemistry, flow cytometry and biochemical inhibitors, we measured changes in VCAM-1 protein expression and NFκB translocation in vascular endothelial cells in the presence of TNFα and/or hyperinsulinemia and in the absence or presence of kinase pathway inhibitors.</p> <p>Results</p> <p>We report that hyperinsulinemia augmented TNFα stimulated increases in VCAM-1 protein greater than seen with TNFα alone and decreased the time in which VCAM-1 translocated to the cell surface. We also observed that in the presence of Wortmannin, a biochemical inhibitor of phosphatidylinositol 3-kinase (a hallmark of insulin resistance), VCAM-1 expression was greater in the presence of TNFα plus insulin as compared to that seen with insulin or TNFα alone. Additionally, nuclear import of NFκB occurred sooner in the presence of insulin and TNFα together as compared to each alone, and in the presence of Wortmannin, nuclear import of NFκB was greater than that seen with insulin and TNFα alone.</p> <p>Conclusions</p> <p>hyperinsulinemia and insulin resistance appear to augment the inflammatory effects of TNFα on VCAM-1 expression and NFκB translocation, both of which are markers of inflammation in the vasculature.</p

Metabolic Stress Induces Caspase-3 Mediated Degradation and Inactivation of Farnesyl and Geranylgeranyl Transferase Activities in Pancreatic β-Cells

Background/Aims: At least 300 prenylated proteins are identified in the human genome; the majority of which partake in a variety of cellular processes including growth, differentiation, cytoskeletal organization/dynamics and vesicle trafficking. Aberrant prenylation of proteins is implicated in human pathologies including cancer; neurodegenerative diseases, retinitis pigmentosa, and premature ageing syndromes. Original observations from our laboratory have demonstrated that prenylation of proteins [small G-proteins and &#x03B3;-subunits of trimeric G-proteins] is requisite for physiological insulin secretion. Herein, we assessed the impact of metabolic stress [gluco-, lipotoxicity and ER-stress] on the functional status of protein prenylation pathway in pancreatic β-cells. Methods: Farnesyltransferase [FTase] and geranylgeranyltransferase [GGTase] activities were quantified by radioisotopic methods. Caspase-3 activation and FTase/GGTase-α subunit degradation were determined by Western blotting. Results: We observed that metabolic stress activates caspase-3 and induces degradation of the common α-subunit of FTase and GGTase-I in INS-1 832/13 cells, normal rodent islets and human islets leading to functional defects [inactivation] in FTase and GGTase activities. Caspase-3 activation and FTase/GGTase-α degradation were also seen in islets from the Zucker diabetic fatty [ZDF] rat, a model for Type 2 diabetes. Consequential to defects in FTase/GGTase-α signaling, we observed significant accumulation of unprenylated proteins [Rap1] in β-cells exposed to glucotoxic conditions. These findings were replicated in β-cells following pharmacological inhibition of generation of prenylpyrophosphate substrates [Simvastatin] or catalytic activity of prenylating enzymes [GGTI-2147]. Conclusions: Our findings provide the first evidence to suggest that metabolic stress induced dysfunction of the islet β-cell may, in part, be due to defective protein prenylation signaling pathway