wedelolactone and Hyperglycemia

Islet inflammation and cytokine production are implicated in pancreatic β-cell dysfunction and diabetes pathogenesis. However, we lack therapeutics to protect the insulin-producing β-cells from inflammatory damage. Closing this clinical gap requires the establishment of new disease models of islet inflammation to facilitate screening efforts aimed at identifying new protective agents. Here, we have developed a genetic model of Interleukin-1β (Il-1β)-driven islet inflammation in zebrafish, a vertebrate that allows for non-invasive imaging of β-cells and

Topics: Animals; Animals, Genetically Modified; Apoptosis; Biological Products; Calcium; Coumarins; Cytokines; Disease Models, Animal; Down-Regulation; Glucose; Humans; Hyperglycemia; Inflammation; Insulin-Secreting Cells; Interleukin-1beta; Larva; Macrophages; Mice; Models, Genetic; Time-Lapse Imaging; Transcription, Genetic; Zebrafish

A decline in the bioavailability of nitric oxide (NO) that causes endothelial dysfunction is a hallmark of diabetes. The availability of NO to the vasculature is regulated by endothelial nitric oxide synthase (eNOS) activity and the involvement of heat shock protein-90 (Hsp-90) in the regulation of eNOS activity has been demonstrated. Hsp-90 has been shown to interact with upstream kinases [inhibitor kappaB kinases (IKK)alpha, beta, and gamma] in nonvascular cells. In this study, we have investigated the interaction of Hsp-90-IKKbeta in endothelial cells under conditions of high glucose (HG) as a possible mechanism that diminishes Hsp-90-eNOS interaction, which could contribute to reduced bioavailability of NO. We report for the first time that IKKbeta interacts with Hsp-90, and this interaction is augmented by HG in vascular endothelial cells. HG also augments transcriptional (3.5 +/- 0.65-fold) and translational (1.97 +/- 0.17-fold) expression as well as the catalytic activity of IKKbeta (2.45 +/- 0.4-fold). Both IKKbeta and eNOS could be coimmunoprecipitated with Hsp-90. Inhibition of Hsp-90 with geldanamycin (2 microM) or Radicicol (20 microM) mitigated (0.45 +/- 0.04-fold and 0.93 +/- 0.16-fold, respectively) HG induced-IKKbeta activity (2.5 +/- 0.42-fold). Blocking of IKKbeta expression by IKK inhibitor II (15 microM wedelolactone) or small interferring RNA (siRNA) improved Hsp-90-eNOS interaction and NO production under conditions of HG. These results illuminate a possible mechanism for the declining eNOS activity reported under conditions of HG.

Topics: Animals; Benzoquinones; Cattle; Cells, Cultured; Coumarins; Endothelial Cells; Endothelium, Vascular; Enzyme Induction; Fluorescence Resonance Energy Transfer; Glucose; HSP90 Heat-Shock Proteins; Hyperglycemia; I-kappa B Kinase; Immunoprecipitation; Lactams, Macrocyclic; Macrolides; Nitric Oxide; Nitric Oxide Synthase Type III; Recombinant Fusion Proteins; RNA Interference; RNA, Small Interfering; Time Factors; Transcription, Genetic; Transfection; Two-Hybrid System Techniques