Nitric Oxide-Induced Activation of the AMP-Activated Protein Kinase α2 Subunit Attenuates IκB Kinase Activity and Inflammatory Responses in Endothelial Cells

BACKGROUND: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2(-/-) mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2(-/-) mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2(+/+) versus AMPKα2(-/-) mice. CONCLUSIONS: These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK

Impact of short-term dietary modification on postprandial oxidative stress

<p>Abstract</p> <p>Background</p> <p>We have recently reported that short-term (21-day) dietary modification in accordance with a stringent vegan diet (i.e., a Daniel Fast) lowers blood lipids as well as biomarkers of oxidative stress. However, this work only involved measurements obtained in a fasted state. In the present study, we determined the postprandial response to a high-fat milkshake with regards to blood triglycerides (TAG), biomarkers of oxidative stress, and hemodynamic variables before and following a 21-day Daniel Fast.</p> <p>Methods</p> <p>Twenty-two subjects (10 men and 12 women; aged 35 ± 3 years) completed a 21-day Daniel Fast. To induce oxidative stress, a milkshake (fat = 0.8 g·kg^-1; carbohydrate = 1.0 g·kg^-1; protein = 0.25 g·kg^-1) was consumed by subjects on day one and day 22 in a rested and 12-hour fasted state. Before and at 2 and 4 h after consumption of the milkshake, heart rate (HR) and blood pressure were measured. Blood samples were also collected at these times and analyzed for TAG, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), advanced oxidation protein products (AOPP), nitrate/nitrite (NOx), and Trolox Equivalent Antioxidant Capacity (TEAC).</p> <p>Results</p> <p>A time effect was noted for HR (<it>p </it>= 0.006), with values higher at 2 hr post intake of the milkshake as compared to pre intake (<it>p </it>< 0.05). Diastolic blood pressure was lower post fast as compared to pre fast (<it>p </it>= 0.02), and a trend for lower systolic blood pressure was noted (<it>p </it>= 0.07). Time effects were noted for TAG (<it>p </it>= 0.001), MDA (<it>p </it>< 0.0001), H₂O₂(<it>p </it>< 0.0001), AOPP (<it>p </it>< 0.0001), and TEAC (<it>p </it>< 0.0001); all concentrations were higher at 2 h and 4 h post intake compared to pre intake, except for TEAC, which was lower at these times (<it>p </it>< 0.05). A condition effect was noted for NOx (<it>p </it>= 0.02), which was higher post fast as compared to pre fast. No pre/post fast × time interactions were noted (<it>p </it>> 0.05), with the area under the curve from pre to post fast reduced only slightly for TAG (11%), MDA (11%), H₂O₂(8%), and AOPP (12%), with a 37% increase noted for NOx.</p> <p>Conclusion</p> <p>Partaking in a 21-day Daniel Fast does not result in a statistically significant reduction in postprandial oxidative stress. It is possible that a longer time course of adherence to the Daniel Fast eating plan may be needed to observe significant findings.</p

Weekly Intra-Amniotic IGF-1 Treatment Increases Growth of Growth-Restricted Ovine Fetuses and Up-Regulates Placental Amino Acid Transporters

Frequent treatment of the growth-restricted (IUGR) ovine fetus with intra-amniotic IGF-1 increases fetal growth. We aimed to determine whether increased growth was maintained with an extended dosing interval and to examine possible mechanisms. Pregnant ewes were allocated to three groups: Control, and two IUGR groups (induced by placental embolization) treated with weekly intra-amniotic injections of either saline (IUGR) or 360 µg IGF-1 (IGF1). IUGR fetuses were hypoxic, hyperuremic, hypoglycemic, and grew more slowly than controls. Placental glucose uptake and SLC2A1 (GLUT2) mRNA levels decreased in IUGR fetuses, but SLC2A3 (GLUT3) and SLC2A4 (GLUT4) levels were unaffected. IGF-1 treatment increased fetal growth rate, did not alter uterine blood flow or placental glucose uptake, and increased placental SLC2A1 and SLC2A4 (but not SLC2A3) mRNA levels compared with saline-treated IUGR animals. Following IGF-1 treatment, placental mRNA levels of isoforms of the system A, y+, and L amino acid transporters increased 1.3 to 5.0 fold, while the ratio of phosphorylated-mTOR to total mTOR also tended to increase. Weekly intra-amniotic IGF-1 treatment provides a promising avenue for intra-uterine treatment of IUGR babies, and may act via increased fetal substrate supply, up-regulating placental transporters for neutral, cationic, and branched-chain amino acids, possibly via increased activation of the mTOR pathway