Young Taek Oh

Activation of adenosine A3 receptor suppresses lipopolysaccharide-induced TNF-α production through inhibition of PI 3-kinase/Akt and NF-κB activation in murine BV2 microglial cells

Adenosine is an endogenous nucleoside that regulates many processes, including inflammatory responses, through activation of its receptors. Adenosine receptors have been reported to be expressed in microglia, which are major immune cells of brain, yet little is known about the role of adenosine receptors in microglial cytokine production. Thus, we investigated the effect of adenosine and adenosine A3 receptor ligands on LPS-induced tumor necrosis factor (TNF-alpha) production and its molecular mechanism in mouse BV2 microglial cells. Adenosine and Cl-IB-MECA, a specific adenosine A3 receptor agonist, suppressed LPS-induced TNF-alpha protein and mRNA levels. Moreover, MRS1523, a selective A3 receptor antagonist, blocked suppressive effects of both adenosine and Cl-IB-MECA on TNF-alpha. We further examined the effect of adenosine on signaling molecules, such as PI 3-kinase, Akt, p38, ERK1/2, and NF-kappaB, which are involved in the regulation of inflammatory responses. Adenosine inhibited LPS-induced phosphatidylinositol (PI) 3-kinase activation and Akt phosphorylation, whereas it had no effect on the phosphorylation of p38 and ERK1/2. We also found that adenosine as well as Cl-IB-MECA inhibited LPS-induced NF-kappaB DNA binding and luciferase reporter activity. Taken together, these results suggest that adenosine A3 receptor activation suppresses TNF-alpha production by inhibiting PI 3-kinase/Akt and NF-kappaB activation in LPS-treated BV2 microglial cells.

Oleic acid reduces lipopolysaccharide-induced expression of iNOS and COX-2 in BV2 murine microglial cells: Possible involvement of reactive oxygen species, p38 MAPK, and IKK/NF-κB signaling pathways

Microglia are the major cells involved in neuroinflammation resulting in brain tissue damage during infection and neurodegenerative diseases. In this study, we examined the effects of the monounsaturated fatty acid oleic acid (OA) on LPS-induced proinflammatory mediators production and the mechanisms involved in BV2 microglia. OA inhibited LPS-induced expression of iNOS and COX-2 as well as production of NO and prostaglandin E2. We showed that OA blocked LPS-induced NF-kappaB activation and phosphorylation of inhibitor kappaB kinase (IKK). We also showed that OA inhibited LPS-induced phosphorylation of Akt and p38 MAPK, but not that of ERK. Finally, we showed that OA reduced reactive oxygen species (ROS) accumulation and an anti-oxidant N-acetylcysteine inhibited NF-kappaB transactivation and phosphorylation of IKK and Akt in LPS-stimulated BV2 cells. Taken together, our results suggest that OA shows an anti-inflammatory effect by inhibiting ROS, p38 MAPK, and Akt/IKK/NF-kappaB signaling pathways in LPS-stimulated BV2 microglia.

Melatonin attenuates amyloid beta25–35-induced apoptosis in mouse microglial BV2 cells

Melatonin has been reported to possess strong antioxidant actions, and is able to directly scavenge a variety of reactive oxygen species (ROS). The present study investigated whether melatonin possesses protective effects against Abeta-induced cytotoxicity in microglial cells. Cells treated with Abeta exhibited several characteristic features of apoptosis, while cells pre-treated with melatonin prior to exposure to Abeta showed a decrease in the occurrence of such apoptotic features. Several previous studies have demonstrated the involvement of ROS in Abeta-induced neurotoxicity, and ROS generated by Abeta have been reported to lead to the activation of nuclear factor-kappa B (NF-kappaB), a transcription factor; pre-treatment with melatonin in the present study reduced the level of Abeta-induced intracellular ROS generation, inhibited NF-kappaB activation, and suppressed the Abeta-induced increase in caspase-3 enzyme activity. In addition, it was found that pre-treatment with melatonin inhibits Abeta-induced increase in the levels of bax mRNA and that it enhances the level of bcl-2 expression. Based on these findings, the authors speculate that melatonin may provide an effective means of treatment for Alzheimers disease through attenuation of Abeta-induced apoptosis.

Increasing plasma [K+] by intravenous potassium infusion reduces NCC phosphorylation and drives kaliuresis and natriuresis

Dietary potassium loading results in rapid kaliuresis, natriuresis, and diuresis associated with reduced phosphorylation (p) of the distal tubule Na(+)-Cl(-) cotransporter (NCC). Decreased NCC-p inhibits NCC-mediated Na(+) reabsorption and shifts Na(+) downstream for reabsorption by epithelial Na(+) channels (ENaC), which can drive K(+) secretion. Whether the signal is initiated by ingesting potassium or a rise in plasma K(+) concentration ([K(+)]) is not understood. We tested the hypothesis, in male rats, that an increase in plasma [K(+)] is sufficient to reduce NCC-p and drive kaliuresis. After an overnight fast, a single 3-h 2% potassium (2%K) containing meal increased plasma [K(+)] from 4.0 ± 0.1 to 5.2 ± 0.2 mM; increased urinary K(+), Na(+), and volume excretion; decreased NCC-p by 60%; and marginally reduced cortical Na(+)-K(+)-2Cl(-) cotransporter (NKCC) phosphorylation 25% (P = 0.055). When plasma [K(+)] was increased by tail vein infusion of KCl to 5.5 ± 0.1 mM over 3 h, significant kaliuresis and natriuresis ensued, NCC-p decreased by 60%, and STE20/SPS1-related proline alanine-rich kinase (SPAK) phosphorylation was marginally reduced 35% (P = 0.052). The following were unchanged at 3 h by either the potassium-rich meal or KCl infusion: Na(+)/H(+) exchanger 3 (NHE3), NHE3-p, NKCC, ENaC subunits, and renal outer medullary K(+) channel. In summary, raising plasma [K(+)] by intravenous infusion to a level equivalent to that observed after a single potassium-rich meal triggers renal kaliuretic and natriuretic responses, independent of K(+) ingestion, likely driven by decreased NCC-p and activity sufficient to shift sodium reabsorption downstream to where Na(+) reabsorption and flow drive K(+) secretion.

Lipopolysaccharide induces hypoxia-inducible factor-1 alpha mRNA expression and activation via NADPH oxidase and Sp1-dependent pathway in BV2 murine microglial cells.

Hypoxia-inducible factor-1 (HIF-1), the key transcription factor of hypoxia-inducible genes, is known to be involved in inflammation and immune response, but little is known about the regulation of HIF-1 during microglial activation. Thus, we examined effect of lipopolysaccharide (LPS) on HIF-1 activation and its signaling mechanism in BV2 microglial cells. LPS induced HIF-1alpha mRNA and protein expression as well as HIF-1 transcriptional activation. Moreover, HIF-1alpha knockdown by small interfering RNA (siRNA) decreased LPS-induced expression of hypoxia responsive genes, VEGF, iNOS, and COX-2. We then showed that LPS-induced HIF-1alpha mRNA expression was blocked by an antioxidant, NADPH oxidase inhibitors, and siRNA of gp91phox, a subunit of NADPH oxidase. In addition, we showed that specific pharmacological inhibitors of PI 3-kinase and protein kinase C decreased LPS-induced HIF-1alpha mRNA expression. Finally, we showed that inhibition of transcription factor Sp1 by mithramycin A or Sp1 siRNA decreased LPS-induced HIF-1alpha mRNA and protein expression. Consistently, LPS increased Sp1 DNA binding and its transcriptional activity. Taken together, these results suggest that LPS induces HIF-1alpha mRNA expression and activation via NADPH oxidase and Sp1 in BV2 microglia.

Baicalein suppresses hypoxia-induced HIF-1α protein accumulation and activation through inhibition of reactive oxygen species and PI 3-kinase/Akt pathway in BV2 murine microglial cells

Hypoxia induces an inflammatory activation of microglia during cerebral ischemia. The transcription factor of hypoxia-inducible genes hypoxia-inducible factor-1 (HIF-1) is known to be involved in inflammation and immune response. Although baicalein (BE), a flavonoid, is shown to have anti-inflammatory effects and attenuate ischemic injury, its action mechanism is not understood well. Thus, we examined effect of BE on hypoxia-induced HIF-1 activation and its signaling mechanism in BV2 microglial cells. BE inhibited hypoxia-induced HIF-1alpha protein accumulation and HIF-1 transcriptional activation. Consistently, BE suppressed hypoxia-induced expression of hypoxia responsive genes, iNOS, COX-2, and VEGF. We then showed that BE inhibited hypoxia-induced phosphorylation of Akt but not that of ERK and p38. Moreover, BE inhibited hypoxia-induced PI 3-kinase activation. Finally, we showed that BE inhibited hypoxia-induced ROS generation, and an antioxidant N-acetylcysteine reduced hypoxia-induced HIF-1alpha and iNOS protein expression and PI 3-kinase/Akt activation in BV2 microglia. Taken together, these results suggest that BE suppresses hypoxia-induced HIF-1alpha protein and activation as well as expression of hypoxia responsive genes by inhibiting ROS and PI 3-kinase/Akt pathway in BV2 microglia.

Oleamide suppresses lipopolysaccharide-induced expression of iNOS and COX-2 through inhibition of NF-κB activation in BV2 murine microglial cells

Oleamide (cis-9-octadecenamide) is an endogenous sleep-inducing fatty acid amide that accumulates in the cerebrospinal fluid of the sleep-deprived animals. Microglia are the major immune cells involved in neuroinflammation causing brain damage during infection, ischemia, and neurodegenerative disease. In this study, we examined the effects of oleamide on LPS-induced production of proinflammatory mediators and the mechanisms involved in BV2 microglia. Oleamide inhibited LPS-induced production of NO and prostaglandin E2 as well as expression of iNOS and COX-2. We showed that oleamide blocked LPS-induced NF-kappaB activation and phosphorylation of inhibitor kappaB kinase (IKK). We also showed that oleamide inhibited LPS-induced phosphorylation of Akt, p38 MAPK, and ERK, activation of PI 3-kinase, and accumulation of reactive oxygen species (ROS). Finally, we showed that a specific antagonist of the CB2 receptor, AM630, blocked the inhibitory effects of oleamide on LPS-induced production of proinflammatory mediators and activation of NF-kappaB. Taken together, our results suggest that oleamide shows an anti-inflammatory effect through inhibition of NF-kappaB activation in LPS-stimulated BV2 microglia.

Gut sensing of dietary K⁺ intake increases renal K⁺excretion.

Dietary K(+) intake may increase renal K(+) excretion via increasing plasma [K(+)] and/or activating a mechanism independent of plasma [K(+)]. We evaluated these mechanisms during normal dietary K(+) intake. After an overnight fast, [K(+)] and renal K(+) excretion were measured in rats fed either 0% K(+) or the normal 1% K(+) diet. In a third group, rats were fed with the 0% K(+) diet, and KCl was infused to match plasma [K(+)] profile to that of the 1% K(+) diet group. The 1% K(+) feeding significantly increased renal K(+) excretion, associated with slight increases in plasma [K(+)], whereas the 0% K(+) diet decreased K(+) excretion, associated with decreases in plasma [K(+)]. In the KCl-infused 0% K(+) diet group, renal K(+) excretion was significantly less than that of the 1% K(+) group, despite matched plasma [K(+)] profiles. We also examined whether dietary K(+) alters plasma profiles of gut peptides, such as guanylin, uroguanylin, glucagon-like peptide 1, and glucose-dependent insulinotropic polypeptide, pituitary peptides, such as AVP, α-MSH, and γ-MSH, or aldosterone. Our data do not support a role for these hormones in the stimulation of renal K(+) excretion during normal K(+) intake. In conclusion, postprandial increases in renal K(+) excretion cannot be fully accounted for by changes in plasma [K(+)] and that gut sensing of dietary K(+) is an important component of the regulation of renal K(+) excretion. Our studies on gut and pituitary peptide hormones suggest that there may be previously unknown humoral factors that stimulate renal K(+) excretion during dietary K(+) intake.

Activation of AMP-activated protein kinase by kainic acid mediates brain-derived neurotrophic factor expression through a NF-kappaB dependent mechanism in C6 glioma cells

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. Kainic acid (KA), a prototype excitotoxin is known to induce brain-derived neurotrophic factor (BDNF) in brain. In this study, we examined the role of AMPK in KA-induced BDNF expression in C6 glioma cells. We showed that KA and KA receptor agonist induced activation of AMPK and KA-induced AMPK activation was blocked by inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) beta. We then showed that inhibition of AMPK by compound C, a selective inhibitor of AMPK, or small interfering RNA of AMPKalpha1 blocked KA-induced BDNF mRNA and protein expression. Inhibition of AMPK blocked KA-induced phosphorylation of CaMKII and I kappaB kinase (IKK) in C6 cells. Finally, we showed that inhibition of AMPK reduced DNA binding and transcriptional activation of nuclear factor-kappaB (NF-kappaB) in KA-treated cells. These results suggest that AMPK mediates KA-induced BDNF expression by regulating NF-kappaB activation.

Widespread effects of nicotinic acid on gene expression in insulin-sensitive tissues: implications for unwanted effects of nicotinic acid treatment.

Nicotinic acid (NA; or niacin) has been used as a hypolipidemic agent for more than 4 decades. However, the mechanisms underlying the effects of NA treatment (wanted and unwanted) are still poorly understood. In the present study, we discovered that NA infusion in rats resulted in dephosphorylation (ie, activation) of the forkhead transcription factor FOXO1 in insulin-sensitive tissues such as skeletal and cardiac muscles, liver, and adipose tissue. These NA effects were opposite to the effects of insulin to increase FOXO1 phosphorylation. To test whether NA alters gene expression in these tissues, rats were infused for 7 hours with NA (30 μmol/h) and/or insulin (5 mU/[kg min]); and gene expression was evaluated using a microarray analysis. Nicotinic acid had widespread effects on gene expression in all of the tissues studied, and the number of genes affected by NA greatly exceeded that of genes affected by insulin. A systematic (or strategic) analysis of the microarray data revealed that there were numerous genes whose expression was regulated inversely by insulin and NA in correlation with FOXO1 phosphorylation, representing potential FOXO1 target genes. We also identified a group of genes whose expression was altered by NA exclusively in adipose tissue, presumably because of stimulation of the NA receptor in this tissue. Finally, there were genes whose expression was altered by both NA and insulin, likely via lowering plasma free fatty acid levels, including lipoprotein lipase and adenosine triphosphate-binding cassette A1, which play a major role in the regulation of circulating lipids. Thus, our data suggest that NA alters gene expression in insulin-sensitive tissues by various mechanisms. Some of the NA-induced changes in gene expression are discussed as potential mechanisms underlying wanted and unwanted effects of NA treatment.