Masao Miwa

Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions.

Although paracellin-1 (PCLN-1) is known to have a crucial role in the control of Mg2+ reabsorption in the kidney, the molecular pathways involved in the regulation of PCLN-1 have not been clarified. We used FLAG-tagged PCLN-1 to investigate these pathways further, and found that PCLN-1 is phosphorylated at Ser217 by protein kinase A (PKA) under physiological conditions in Madin-Darby canine kidney (MDCK) cells. PCLN-1 expression decreased Na+ permeability, resulting in a decrease in the transepithelial electrical resistance (TER). By contrast, PCLN-1 enhanced transepithelial Mg2+ transport. PKA inhibitors, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) and myristoylated protein kinase A inhibitor 14-22 amide PKI, and an adenylate cyclase inhibitor, 2′,5′-dideoxy adenosine (DDA), reduced the phosphoserine level of PCLN-1. The inhibitory effect of DDA was rescued by 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). PKA and adenylate cyclase inhibitors decreased transepithelial Mg2+ transport and TER. Dephosphorylated PCLN-1 moved from detergent-insoluble to soluble fractions and was dissociated from ZO-1. A fusion protein of PCLN-1 with glutathione-S-transferase revealed that Ser217 was phosphorylated by PKA. Phosphorylated PCLN-1 was localized in the tight junction (TJ) along with ZO-1, whereas dephosphorylated PCLN-1 and the S217A mutant were translocated into the lysosome. The degradation of dephosphorylated PCLN-1 and S217A mutant was inhibited by chloroquine, a specific lysosome inhibitor. Thus, the PKA-dependent phosphorylation of Ser217 in PCLN-1 is essential for its localization in the TJ and transepithelial Mg2+ transport.

All ApoB-Containing Lipoproteins Induce Monocyte Chemotaxis and Adhesion When Minimally Modified Modulation of Lipoprotein Bioactivity by Platelet-Activating Factor Acetylhydrolase

Mildly oxidized LDL has many proinflammatory properties, including the stimulation of monocyte chemotaxis and adhesion, that are important in the development of atherosclerosis. Although ApoB-containing lipoproteins other than LDL may enter the artery wall and undergo oxidation, very little is known regarding their proinflammatory potential. LDL, IDL, VLDL, postprandial remnant particles, and chylomicrons were mildly oxidized by fibroblasts overexpressing 15-lipoxygenase (15-LO) and tested for their ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. When conditioned on 15-LO cells, LDL, IDL, but not VLDL increased monocyte chemotaxis and adhesion approximately 4-fold. Chylomicrons and postprandial remnant particles were also bioactive. Although chylomicrons had a high 18:1/18:2 ratio, similar to that of VLDL, and should presumably be less susceptible to oxidation, they contained (in contrast to VLDL) essentially no platelet-activating factor acetylhydrolase (PAF-AH) activity. Because PAF-AH activity of lipoproteins may be reduced in vivo by oxidation or glycation, LDL, IDL, and VLDL were treated in vitro to reduce PAF-AH activity and then conditioned on 15-lipoxygenase cells. All 3 PAF-AH-depleted lipoproteins, including VLDL, exhibited increased stimulation of monocyte chemotaxis and adhesion. In a similar manner, lipoproteins from Japanese subjects with a deficiency of plasma PAF-AH activity were also markedly more bioactive, and stimulated monocyte adhesion nearly 2-fold compared with lipoproteins from Japanese control subjects with normal plasma PAF-AH. For each lipoprotein, bioactivity resided in the lipid fraction and monocyte adhesion could be blocked by PAF-receptor antagonists. These data suggest that the susceptibility of plasma lipoproteins to develop proinflammatory activity is in part related to their 18:1/18:2 ratio and PAF-AH activity, and that bioactive phospholipids similar to PAF are generated during oxidation of each lipoprotein. Moreover, LDL, IDL, postprandial remnant particles, and chylomicrons and PAF-AH-depleted VLDL all give rise to proinflammatory lipids when mildly oxidized.

Expression of Hepatic UDP-Glucuronosyltransferase 1A1 and 1A6 Correlated with Increased Expression of the Nuclear Constitutive Androstane Receptor and Peroxisome Proliferator-Activated Receptor α in Male Rats Fed a High-Fat and High-Sucrose Diet

Rats that consumed a high-fat and high-sucrose (HF1) diet or a high-fat (HF2) diet developed hepatic steatosis. The alteration in nutritional status affected hepatic cytochrome P450 and UDP-glucuronosyltransferase (UGT) levels. Messenger RNA and protein levels of UGT1A1 and UGT1A6 in the liver but not the jejunum were increased in male rats fed the HF1 diet. These protein levels did not increase in HF2-fed male rats or HF1-fed female rats. In contrast, the CYP1A2 protein level was decreased in the HF1 but not HF2 diet group, whereas CYP2E1 and CYP4A protein levels were elevated in the HF2 but not HF1 diet group. No significant difference in the organic anion transporter polypeptide (Oatp) 1, Oatp2, multidrug resistance-associated protein (Mrp) 2, or Mrp3 protein levels was found between the standard and HF1 diet groups of male rats. Consumption of the HF1 diet affected the in vivo metabolism of acetaminophen (APAP) such that the area under the APAP-glucuronide plasma concentration-time curve was elevated 2.1-fold in male rats but not female rats. In liver cell nuclei of male rats but not female rats, constitutive androstane receptor (CAR) and proliferator-activated receptor α (PPARα) protein levels were significantly enhanced by intake of the HF1 diet. Additionally, administration of the PPARα agonist clofibrate to male rats up-regulated UGT1A1 and UGT1A6 and down-regulated CYP1A2 in the liver. Taken together, these results indicate that nutritional status may gender-specifically influence the expression and activation of CAR and PPARα in liver cell nuclei, and this effect appears to be associated with alterations in UGT1A1 and UGT1A6 expression.

High-density lipoprotein inhibits the synthesis of platelet-activating factor in human vascular endothelial cells

The regulation of platelet-activating factor (PAF) synthesis by serum lipoproteins was investigated in human umbilical vein endothelial cells. High-density lipoprotein (HDL) inhibited PAF synthesis in agonist (thrombin, histamine, and A23187)-stimulated endothelial cells, that was determined by incorporation of [3H]acetate into PAF and by bioassay. The inhibition by HDL was increased in a concentration-dependent manner, but was reversed as the concentration of thrombin increased. HDL did not affect the time course of PAF production. HDL lipids suppressed the PAF production to a lesser extent than HDL. The reduction of PAF accumulation in HDL, did not result from degradation of PAF but inhibition of PAF synthesis, which was mainly mediated via the blockade of acetyl-CoA:1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase activation. HDL did not prevent the release of [3H]arachidonic acid in thrombin-stimulated endothelial cells. The binding of 125I-HDL to endothelial cells and its uptake were not enhanced by thrombin stimulation. These results demonstrate that HDL may inhibit the activation of acetyltransferase by thrombin at the cell surface. This observation may explain a part of mechanism of HDL action.

Hepatic CYP3A Expression is Attenuated in Obese Mice Fed a High-Fat Diet

PurposeChanges in physiological, pathophysiological, and/or nutritional conditions often alter the expression of drug-metabolizing enzymes. In this study, we investigated obesity-induced changes in hepatic cytochrome P450 (P450) levels using nutritionally obese mice.MethodsTo induce obesity, mice were fed a high-fat diet or treated with gold thioglucose, which impairs ventromedial hypothalamus. Total RNAs and microsomal and nuclear proteins were prepared from the liver of these mice, and mRNA and protein levels of P450s and transcription factors were determined.ResultsAmong P450s examined, the constitutive expression of CYP3As was drastically reduced at both mRNA and protein levels by nutrition-induced obesity. One-week administration of a high-fat diet also reduced hepatic CYP3As. However, changes in nuclear receptors involved in the transcriptional regulation of CYP3A genes were not correlated with that of CYP3As. Obese mice induced by gold thioglucose exhibited a different expression profile of hepatic P450s with no significant change in CYP3As.ConclusionHigh-fat diet-induced changes in energy metabolism, which eventually result in obesity, modulate the hepatic expression profile of P450s, particularly CYP3As. Alternatively, the accumulation of a certain component in a high-fat diet may directly attenuate the CYP3A expression, suggesting a clinically important drug–diet interaction.

Role of lecithin-cholesterol acyltransferase in the metabolism of oxidized phospholipids in plasma: Studies with platelet-activating factor-acetyl hydrolase-deficient plasma

To determine the relative importance of platelet-activating factor-acetylhydrolase (PAF-AH) and lecithin-cholesterol acyltransferase (LCAT) in the hydrolysis of oxidized phosphatidylcholines (OXPCs) to lyso-phosphatidylcholine (lyso-PC), we studied the formation and metabolism of OXPCs in the plasma of normal and PAF-AH-deficient subjects. Whereas the loss of PC following oxidation was similar in the deficient and normal plasmas, the formation of lyso-PC was significantly lower, and the accumulation of OXPC was higher in the deficient plasma. Isolated LDL from the PAF-AH-deficient subjects was more susceptible to oxidation, and stimulated adhesion molecule synthesis in endothelial cells, more than the normal LDL. Oxidation of 16:0-[1-14C]-18:2 PC, equilibrated with plasma PC, resulted in the accumulation of labeled short- and long-chain OXPCs, in addition to the labeled aqueous products. The formation of the aqueous products decreased by 80%, and the accumulation of short-chain OXPC increased by 110% in the deficient plasma, compared to the normal plasma, showing that PAF-AH is predominantly involved in the hydrolysis of the truncated OXPCs. Labeled sn-2-acyl group from the long-chain OXPC was not only hydrolyzed to free fatty acid, but was preferentially transferred to diacylglycerol, in both the normal and deficient plasmas. In contrast, the acyl group from unoxidized PC was transferred only to cholesterol, showing that the specificity of LCAT is altered by OXPC. It is concluded that, while PAF-AH carries out the hydrolysis of mainly truncated OXPCs, LCAT hydrolyzes and transesterifies the long-chain OXPCs.

Dietary inulin alleviates hepatic steatosis and xenobiotics-induced liver injury in rats fed a high-fat and high-sucrose diet : Association with the suppression of hepatic cytochrome p450 and hepatocyte nuclear factor 4α expression

Inulin enzymatically synthesized from sucrose is a dietary component that completely escapes glucide digestion. Supplementing inulin to a high-fat and high-sucrose diet (HF) ameliorated hypertriglycemia and hepatic steatosis in 8-week-fed rats by suppressing elevated levels of serum triacylglycerols, fatty acids, and glucose, and the accumulation of hepatic triacylglycerols and fatty acids. Inulin intake prevented phenobarbital (PB)- and dexamethasone-induced liver injuries in the HF group. No significant alteration in the baseline expression of CYP2B, CYP2C11, CYP3A, and NADPH-cytochrome P450 (P450) reductase mRNAs and proteins was found. In contrast, baseline and PB-treated expressions of CYP2E1 mRNA were reduced in HF-fed rats. The induction of P450s in response to PB was affected by the nutritional status of the rats; mRNA levels of CYP2B1 and CYP3A1 after PB treatment, as assessed by quantitative real-time polymerase chain reaction analysis were reduced in the inulin-supplemented HF (HF+I) group, compared with those in the HF group. Western blot analysis detected the corresponding changes of CYP2B and CYP3A proteins. These alterations were correlated with changes in hepatic thiobarbituric acid-reactive substances. Furthermore, no significant difference in the expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and retinoid X receptor α and coactivator peroxisome proliferator-activated receptor-γ coactivator 1α proteins was found in the hepatic nucleus between the HF and HF+I groups, but the expression of hepatocyte nuclear factor α (HNF4α) protein was significantly reduced in the HF+I group. Taken together, these results indicate that inulin intake ameliorates PB-induced liver injury, associated with a decline in lipid accumulation and PB-induced expression of CYP2B and CYP3A, which may be related by a reduction in the nuclear expression of HNF4α.

Regulation of the human UGT1A1 gene by nuclear receptors constitutive active/androstane receptor, pregnane X receptor, and glucocorticoid receptor.

Human UDP-glucuronosyltransferase (UGT) 1A1 is the enzyme that detoxifies neurotoxic bilirubin by conjugating it with glucuronic acid. In addition to bilirubin, UGT1A1 conjugates various endogenous and exogenous lipophilic compounds such as estrogens and the active metabolite of the anticancer drug irinotecan SN-38. Thus, activation by specific inducers of the UGT1A1 gene is critical in treating patients with unconjugated hyperbili-rubinemia and in preventing side effects of drug treatment such as SN-38-induced toxicity. This chapter describes the experimental processes used to identify the 290-bp distal enhancer module at -3499/-3210 of the UGT1A1 gene and to characterize its regulation by nuclear receptors: constitutive active/androstane receptor, pregnane X receptor, and glucocorticoid receptor.

Study on the new phospholipid, n-acyl-1-alkyl glycerophosphorylethanolamine, from bovine erythrocytes

Abstract 1. 1. Isolation and characterization of a new phospholipid from bovine erythrocytes are described. It is confirmed on thin-layer chromatography that the bovine erythrocyte ethanolamine phospholipid fraction contains, in addition to the significant amounts of ethanolamine glycerophospholipids, a new type of phospholipid, “N-acyl ethanolamine phospholipid”, which gives negative reaction with ninhydrin reagent. 2. 2. Characterization of the chemical degradation products has shown that the phospholipid is N-acyl-1-alkyl-2-acyl glycerophosphorylethanolamine. The content of the phospholipid is 5 mole % in bovine erythrocyte ethanolamine phospholipid fraction. 3. 3. The compositions of alkyl glycerol prepared from 1-alkyl glycerophosphorylethanolamine and N-acyl-1-alkyl glycerophosphorylethanolamine, which were obtained from bovine erythrocyte ethanolamine phospholipid fraction by alkaline hydrolysis, were analyzed by gas-liquid chromatography, and were found similar to each other; therefore, it is suggested that the metabolism of these phospholipids are closely related. 4. 4. Analysis by gas-liquid chromatography of the relative compositions of fatty acids linked to the nitrogen atom as amide in the molecule of N-acyl-1-alkyl glycerophosphorylethanolamine and those found at the 1- and 2-positions of bovine erythrocyte ethanolamine phospholipids gave a result that the fatty acids of the N-acyl amide are more unsaturated than those of O-acyl ester. 5. 5. Hemolytic activity of N-acyl-1-alkyl glycerophosphorylethanolamine was compared with that of 1-alkyl glycerophosphorylethanolamine, and the minimal hemolytic effects of those two lysophospholipids on sheep red cells were found identical (1.5μg/ml).

Transcriptional regulation of human UGT1A1 gene expression through distal and proximal promoter motifs : implication of defects in the UGT1A1 gene promoter

Human UDP-glucuronosyltransferase (UGT)1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds, such as potentially neurotoxic bilirubin and the anticancer drug irinotecan SN-38, via conjugation with glucuronic acid. A 290-bp distal enhancer module, phenobarbital-responsive enhancer module of UGT1A1 (gtPBREM), fully accounts for constitutive androstane receptor (CAR)-, pregnane X receptor (PXR)-, glucocorticoid receptor (GR)-, and aryl hydrocarbon receptor (AhR)-mediated activation of the UGT1A1 gene. This study indicates that hepatocyte nuclear factor 1α (HNF1α) bound to the proximal promoter motif not only enhances the basal reporter activity of UGT1A1, including the distal (−3570/−3180) and proximal (−165/−1) regions, but also influences the transcriptional regulation of UGT1A1 by CAR, PXR, GR, and AhR to markedly enhance reporter activities. Moreover, we assessed the influence of the TA repeat polymorphism and gtPBREM T-3279G mutation on transcriptional activation of UGT1A1 by CAR, PXR, GR, and AhR. Transcriptional activation of the A(TA)7TAA mutant by CAR, the PXR activator rifampicin, the GR activator dexamethasone, and the AhR activator benzo[a]pyrene was more reduced than that of the T-3279G variant, and the activity of the UGT1A1 promoter with both T-3279G and A(TA)7TAA mutations was still lower. Thus, UGT1A1 gene promoter variations, including the TA repeat polymorphism and T-3279G gtPBREM, have important clinical implications.